Ultrastructural Remodeling of the Blood–Brain Barrier and Neurovascular Unit by Lipopolysaccharide-Induced Neuroinflammation
Round 1
Reviewer 1 Report
Well written paper on a relevant topic
Author Response
Response to reviewer #1 round one:
Regarding Manuscript ID
ijms-2156752
The authors would like to thank reviewer #1 round 1 for the time and effort required to review our submitted manuscript.
Sincerely,
Melvin R Hayden
Submitting author
Response to reviewer #1 round one:
Regarding Manuscript ID
ijms-2156752
The authors would like to thank reviewer #1 round 1 for the time and effort required to review our submitted manuscript.
Sincerely,
Melvin R Hayden
Submitting authorcc
Response to reviewer #1 round one:
Regarding Manuscript ID
ijms-2156752
The authors would like to thank reviewer #1 round 1 for the time and effort required to review our submitted manuscript.
Sincerely,
Melvin R Hayden
Submitting author
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The current study “Ultrastructural Remodeling of the Blood-Brain Barrier and Neurovascular Unit by Lipopolysaccharide-Induced Neuroinflammation” by Michelle A. Erickson and the team focuses on the morphological/structural perturbations followed by the LPS administration in BBB. While the manuscript demonstrates the information on mechanisms of BBB dysfunction with the required experimental support and literature shreds of evidence, the following are points that should be addressed.
My comments on the study:
1. As mentioned by the authors that the study is limited by the number of experimental subjects and functional evidence. How do the authors convincingly support the findings? Functional aspects make more sense than mere morphological ones as the former decides the fate of pathological consequences. I think the current finding must be supported with a minimum functional assay with required biological replicates.
2. Were the control mice arm given vehicle control to rule out background effects of LPS injections?
3. How do the author’s findings fill the gaps in current knowledge and its significance in pathological conditions? Authors may cite the relevant literature here.
Author Response
Response to reviewer # 2 round 1:
Re: Manuscript ID ijms-2156752
Author’s would like to thank reviewer # 2 for the time, effort, and sharing of knowledge required to review our submitted manuscript.
Comments and Suggestions for Authors
The current study “Ultrastructural Remodeling of the Blood-Brain Barrier and Neurovascular Unit by Lipopolysaccharide-Induced Neuroinflammation” by Michelle A. Erickson and the team focuses on the morphological/structural perturbations followed by the LPS administration in BBB. While the manuscript demonstrates the information on mechanisms of BBB dysfunction with the required experimental support and literature shreds of evidence, the following are points that should be addressed
My comments on the study:
- As mentioned by the authors that the study is limited by the number of experimental subjects and functional evidence. How do the authors convincingly support the findings? Functional aspects make more sense than mere morphological ones as the former decides the fate of pathological consequences. I think the current finding must be supported with a minimum functional assay with required biological replicates.
Response to Reviewer:
Responses to reviewer #2 and the changes made to the revised manuscript will appear in blue lettering.
Authors wish to thank the reviewer for pointing out the need for functional studies to support the ultrastructural remodeling changes identified in this experiment. As pointed out by authors in the title, abstract, and introduction, this experimental study was designed to be an ultrastructural examination of the tissues from LPS-treated models utilizing ultrastructural TEM. The decision to perform this ultrastructural cytoarchitectural remodeling to the neurovascular BBB with LPS-induced neuroinflammation was made largely because there exists a paucity of ultrastructural studies in this field of research
In regards to biochemical endpoints/neuroinflammation: We did not measure biochemical endpoints in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, as we cited in the bibliography, the 3mg/kg LPS injection regimen used here has been extensively validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in Iba-1, F4/80, and GFAP that were compatible with neuroinflammation and reactive astrogliosis.
Please see the revised manuscript in the Discussion section: paragraph 3 as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
- Were the control mice arm given vehicle control to rule out background effects of LPS injections?
Response to Reviewer:
Yes, please see the added sentence in 4.1 lines 7, 8, and 9 as follows in the revised manuscript in
blue lettering as follows:
All control CD-1 male models received saline (the vehicle for LPS) injections without LPS at the same time intervals and sacrificed in parallel with LPS-treated mice.
- How do the author’s findings fill the gaps in current knowledge and its significance in
pathological conditions? Authors may cite the relevant literature here.
Please see Discussion Section 3. Last paragraph of the revised manuscript as follows:
To date, the majority of work investigating BBB disruption has focused on disturbances in tight and adherens junction proteins, which regulate paracellular leakage. Whereas, ultrastructural studies of vesicular/transcellular routes of BBB disruption have been less frequently studied. Our experimental study has filled in some of our gaps in knowledge in regard to increased permeability and neuroinflammation due to LPS administration. For example, and to our current knowledge, our findings are among the first to clearly identify and quantify the different sizes and types of vesicles that appear in BEC capillaries following a sublethal regimen of LPS that has been well-characterized for its effects on BBB functions [9,23,50]. Indeed, these findings highlight a need to further investigate the contribution of vesicular pathways to aspects of inflammation-induced BBB dysfunction.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer # 2 round 1:
Re: Manuscript ID ijms-2156752
Author’s would like to thank reviewer # 2 for the time, effort, and sharing of knowledge required to review our submitted manuscript.
Comments and Suggestions for Authors
The current study “Ultrastructural Remodeling of the Blood-Brain Barrier and Neurovascular Unit by Lipopolysaccharide-Induced Neuroinflammation” by Michelle A. Erickson and the team focuses on the morphological/structural perturbations followed by the LPS administration in BBB. While the manuscript demonstrates the information on mechanisms of BBB dysfunction with the required experimental support and literature shreds of evidence, the following are points that should be addressed
My comments on the study:
- As mentioned by the authors that the study is limited by the number of experimental subjects and functional evidence. How do the authors convincingly support the findings? Functional aspects make more sense than mere morphological ones as the former decides the fate of pathological consequences. I think the current finding must be supported with a minimum functional assay with required biological replicates.
Response to Reviewer:
Responses to reviewer #2 and the changes made to the revised manuscript will appear in blue lettering.
Authors wish to thank the reviewer for pointing out the need for functional studies to support the ultrastructural remodeling changes identified in this experiment. As pointed out by authors in the title, abstract, and introduction, this experimental study was designed to be an ultrastructural examination of the tissues from LPS-treated models utilizing ultrastructural TEM. The decision to perform this ultrastructural cytoarchitectural remodeling to the neurovascular BBB with LPS-induced neuroinflammation was made largely because there exists a paucity of ultrastructural studies in this field of research
In regards to biochemical endpoints/neuroinflammation: We did not measure biochemical endpoints in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, as we cited in the bibliography, the 3mg/kg LPS injection regimen used here has been extensively validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in Iba-1, F4/80, and GFAP that were compatible with neuroinflammation and reactive astrogliosis.
Please see the revised manuscript in the Discussion section: paragraph 3 as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
- Were the control mice arm given vehicle control to rule out background effects of LPS injections?
Response to Reviewer:
Yes, please see the added sentence in 4.1 lines 7, 8, and 9 as follows in the revised manuscript in
blue lettering as follows:
All control CD-1 male models received saline (the vehicle for LPS) injections without LPS at the same time intervals and sacrificed in parallel with LPS-treated mice.
- How do the author’s findings fill the gaps in current knowledge and its significance in
pathological conditions? Authors may cite the relevant literature here.
Please see Discussion Section 3. Last paragraph of the revised manuscript as follows:
To date, the majority of work investigating BBB disruption has focused on disturbances in tight and adherens junction proteins, which regulate paracellular leakage. Whereas, ultrastructural studies of vesicular/transcellular routes of BBB disruption have been less frequently studied. Our experimental study has filled in some of our gaps in knowledge in regard to increased permeability and neuroinflammation due to LPS administration. For example, and to our current knowledge, our findings are among the first to clearly identify and quantify the different sizes and types of vesicles that appear in BEC capillaries following a sublethal regimen of LPS that has been well-characterized for its effects on BBB functions [9,23,50]. Indeed, these findings highlight a need to further investigate the contribution of vesicular pathways to aspects of inflammation-induced BBB dysfunction.
Respectfully submitted,
Melvin R Hayden
Submitting author
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Author Response
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Response to reviewer #3 round 1:
Re: Manuscript ID ijms-2156752
Authors would like to thank reviewer # 3 for the time, effort, and sharing of knowledge required to review our submitted manuscript. Response to reviewer and revised manuscript will appear in blue lettering.
Comments and Suggestions for Authors
The authors present a histological assessment of the in vivo effects on blood brain barrier of mice of a substance (lipopolysaccharide from Salmonella typhimurium) typically used in neuroinflammation studies. The authors report ultrastructural remodelling of brain endothelial cells, astrocytes, pericytes, microglia and perivascular space. Surprisingly, the authors do not find alterations paracellular junctions.
The study seems to be well designed and performed. The main limitation is clearly stated by the authors in the manuscript (only 2 animals per group). I would add to this limitation that there is no biochemical demonstration of the level of neuroinflammation reached by the dose of lipopolysaccharide. Nevertheless, I would have only minor suggestions for modifying the manuscript before suggest publication.
Response to reviewer
Authors have also responded to reviewer # 2 in a similar regard please see Discussion section 3. in the 3rd paragraph of the revised manuscript as follows:
Here, it is important to note that we did not measure functional biochemical endpoints of neuroinflammation in this particular mouse cohort because this is an ultrastructural study. It is difficult to do in tandem functional studies with TEM studies since brain fixation methods are specialized and require perfusion with EM fixatives. Therefore, these fixed tissues are not compatible with biochemical/functional methodologies and are not feasible in the same mouse models. However, the 3mg/kg LPS injection regimen used in this experiment has been previously validated by authors to cause BBB disruption to 14C-sucrose, 99mTc-DTPA, and 99mTc-albumin and cytokine/chemokines elevations (including RANTES) in the brain and blood of adult male CD-1 mice [9, 23]. Notably, these publications were in separate cohorts of mice and conducted over many years, thus highlighting the reproducibility of our findings that this particular injection regimen of LPS induces BBB disruption and systemic inflammation and importantly neuroinflammation. Therefore, we did not conduct duplicitous experiments in this mouse cohort in this experiment. Further, these previous experiments in the same mouse model, with the same LPS dose (3mg/kg), and the same injection times demonstrated significant elevations in ionized calcium-binding adapter molecule 1 (Iba-1), F4/80, and glial fibrillary acid protein (GFAP) that were compatible with neuroinflammation and reactive astrogliosis.
The authors describe in section 4.1 that the dosage of the lipopolysaccharide induces robust cytokine response, signs of sickness, weight loss and blood brain barrier disruption and no mortalities. This is supported by bibliography. As I stated above, we do not have real probes of the level of neuroinflammation reached in the two dosed animals. I would suggest to enlarge the description of the adverse effects reported in the literature of this dose regime, with specially emphasis on the molecular proofs of neuroinflammation.
Please see the previous response above to the reviewer above with the changes made in the revised manuscript note above.
OTHER MINOR POINTS
Please, be careful with abbreviations in abstract. The abbreviations EC, TJ/AJ and NVU were not defined.
Response to reviewer:
Please see changes in blue lettering to the abstract in the revised manuscript as follows: EC was changed to BEC previously called out; TJ/AJ was spelled out in the revised manuscript; NVU was spelled out in the revised manuscript.
Section 2.1 in page 2. “… defined as being within 20 µM of a brain vessel…” Twenty micromolar? Shouldn’t be twenty micrometres?
Response to reviewer
Please see 2.1 line 5 in the revised manuscript as follows:
… defined as being within 20 micrometers of a brain vessel which is consistent with definitions of vasculature-associated microglia established by other groups [18].
Not very sure about the meaning of the sentence (section 3, page 8): ”but it could have been derived from NVU cell secretions of CCL5 or from CCL5 that is increased in the circulation and crosses the BBB”. I suggest to rewrite this sentence.
Authror’s agree and please see Section 3. Paragraph 4 in the revised manuscript as follows:
Since LPS does not cross the BBB [17], we assume that LPS initially activates the BECs with ensuing changes to the NVU. However, LPS can exert its effects on the NVU by other mechanisms as well, such as an increased passage across the BBB of cytokines and immune cells [8]. Recently, it was shown that the chemokine CCL5 was a mediator of microglial recruitment to the brain vasculature during inflammation [16]. The source of CCL5 in vivo could not be identified in this ultrastructure study, however, prior work has shown that CCL5 secretion by brain endothelial cells and other cell types of the NVU as well as CCL5 transport across the BBB in the brain-to-blood direction are increased following LPS treatment [24,26]. The CNS chemokine CCL5/RANTES is thus a possible chemotactic mediator of MGCs to the NVU during systemic inflammation [15, 16].
Is the abbreviation “NVU” wide enough extended to be used as such in the key words?
Response to review:
No this is not wide enough and note that in the revised manuscript that NVU has been spelled out to the following: neurovascular unit in the Keywords section of the revised manuscript.
Respectfully submitted,
Melvin R Hayden
Submitting author
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
I am satisfied with the responses by the authors. Good job!
