Effects of Whole-Body Vibration and Manually Assisted Locomotor Therapy on Neurotrophin-3 Expression and Microglia/Macrophage Mobilization Following Thoracic Spinal Cord Injury in Rats

Round 1

Reviewer 1 Report

The study aimed to investigate whether whole-body vibration (WBV) and assisted locomotor therapy such as passive flexion-extension (PFE) therapy can induce overall functional recovery with a focus on changes in NT-3 expression and on microglia/macrophage reaction because it is believed that they play a major role in the reconstitution of CNS integrity after injury. The topic is relevant considering the pathophysiology of spinal cord injury (SCI). However, some major corrections are required as below.

Major corrections:

1. Table 1 & 2: Authors showed a comparatively higher number of NT3-positive cells on day 14 than on day 28 after WBV.  However, Iba1 expression is higher on day 28 than that on day 14 after WBV. The authors should provide more evidence about it. For example, is microglia regulate neurogenesis and gliogenesis later time point?  
2. Figures 5 & 6: Quantification is not enough. They should use different methods including qPCR to quantify neurogenesis, and microglia. In addition,  they should stain different subtypes of microglia rather than just staining with iba1 to see if any specific subtypes of microglia are active in SCI recovery after WBV.
3. Figure 7:  Authors need to analyze the expression of NT-3  using a real-time quantitative method. They should also perform a co-staining with Iba1 and NT-3 antibodies.
4. Figures 5, 6 & 7: Image quality is poor. It can be improved.

Author Response

Table 1 & 2: Authors showed a comparatively higher number of NT3-positive cells on day 14 than on day 28 after WBV. However, Iba1 expression is higher on day 28 than that on day 14 after WBV. The authors should provide more evidence about it. For example, is microglia regulate neurogenesis and gliogenesis later time point?

We thank the reviewer for the valuable comment. Based on our current findings, we propose that the increased expression of NT-3 at day 14 is what drives the locomotor recovery that was identified on the WBD-treated animals. On the other hand, we determine that Iba-1 expression simultaneously drops within the same day examined. Reviewer finds it rather intriguing that Iba-1 expression increases within the next 2 weeks without having a clinically relevant phenotype in these same animals. It is important to state here that microglia have two specific phenotypes, namely M1 and M2; the phenotype of microglia seems to have shifted from a pro inflammatory M1 state to an anti-inflammatory M2 one, promoting and sustaining the beneficial effect of WBV. It is now briefly argued in the discussion section.

            In addition to that, we have included an extended comment about the blood brain barrier and the way it may also affect the levels of microglial cells in the central nervous system.

 

 

Figures 5 & 6: Quantification is not enough. They should use different methods including qPCR to quantify neurogenesis, and microglia. In addition, they should stain different subtypes of microglia rather than just staining with iba1 to see if any specific subtypes of microglia are active in SCI recovery after WBV.

Figure 7: Authors need to analyze the expression of NT-3 using a real-time quantitative method. They should also perform a co-staining with Iba1 and NT-3 antibodies.

We do appreciate reviewer’s comment. The analyses of microglia and NT3 have been performed in paraformaldehyde fixed spinal cord tissue a very long time ago. In this way no further, molecular biological assessments are possible. The microglia which we observed shows typical morphology of a resting ramified microglia and we feel confident that it belongs to the subtype that does not promote inflammation. This is also logical because the sections which we evaluated are from the intact lumbar intumescence, which is rather remote from the lesion site of the spinal cord.

Nevertheless, these remarks which have come to our attention, led us to perform comparisons between the present data on IBA and NT3 and Synaptophysin, GFAP, bladder function. Correlation- and principal components analysis revealed complex relationships which have been presented and discussed (Manthou et al., 2017)

Whole body vibration (WBV) following spinal cord injury (SCI) in rats: Timing of intervention. Restorative neurology and neuroscience, (2017), 185-216, 35(2)

 

 

Figures 5, 6 & 7: Image quality is poor. It can be improved.

Thank you for your suggestion. We have improved the existing photos and included additional ones as complete immunopanels within groups.

 

 

 

Reviewer 2 Report

In the present study, the authors WBV therapy was initiated at day 7, 14, and 28 and PFE therapy was initiated at day 14. Why did not the authors compare the effects after that PFE therapy was also initiated at 7, 14, and 28? Why and/or how did the author judge "WBV therapy appeared to be superior to PFE therapy in terms of recovery" based on the different condition of the therapies?

Line 27, WBV therapy.....resulted in a moderate increase of Iba1 and the highest increase in NT-3; increase of Iba1 showing microglial activation might cause not just recovery but injure as the authors described in Introduction. Please discuss about it.

In the present study, the authors used female rats. Did the authors have any reasons?

Table 3 was time schedule of this study. It was not result. It should be put on Materials and Methods 2.1.

In addition, the result of foot stepping angle (FSA) scores should be shown in Results 3.1.

The names of authors and the numbers of the references were mismatches in line 118, 124-125, and 210. Therefore, the authors should check all of the references and renumber or reorganize.

In review version, the position of Figure 4 was wrong.

Table 1 and Table 2 were results. They should be put in not Materials and Method but Results section.

Moreover, in Materials and Methods, Animal groups demonstrated Intact (n=3) and other groups (n=10). Why was n number in each group different in Table 1 and Table 2? Why did n number reduce?

In Figure 5, 6, and 7, the images belonging to WBV28 or WBV14 were shown. If the authors wrote in Results, the images of immunostaining from all experimental groups should be shown in Figure 5, 6, and 7.

Author Response

In the present study, the authors WBV therapy was initiated at day 7, 14, and 28 and PFE therapy was initiated at day 14. Why did not the authors compare the effects after that PFE therapy was also initiated at 7, 14, and 28? Why and/or how did the author judge "WBV therapy appeared to be superior to PFE therapy in terms of recovery" based on the different condition of the therapies?

Thank you for your important remark. It is true that PFE14 can only be compared with WBV14, which was not very clear in our text. The additional onsets of WBV should be considered and compared separately. This is now clear both within the text, and in the abstract.

 

 

Line 27, WBV therapy.....resulted in a moderate increase of Iba1 and the highest increase in NT-3; increase of Iba1 showing microglial activation might cause not just recovery but injure as the authors described in Introduction. Please discuss about it.

We thank the reviewer for the valuable comment. The microglia which we observed showed typical morphology of a resting ramified microglia and we feel confident that it belonged to the subtype that does not promote inflammation. This is also logical because the sections which we evaluated are from the intact lumbar intumescence, which is rather remote from the lesion site of the spinal cord. We have included a relevant comment in our discussion.

 

 

In the present study, the authors used female rats. Did the authors have any reasons?

We are using in our work only female rats, because earlier work has shown that testosterone has a neuroprotective effect (Bia3ek et al., 2004; Fargo et al., 2009; Karegar & Mohammadi, 2015)

 

Bia³ek M, Zaremba P, Borowicz KK, Czuczwar SJ (2004) Neuroprotective role of testosterone in the nervous system. Pol J Pharmacol 56:509–518.

Fargo KN, Foster AM, Sengelaub DR (2009) Neuroprotective Role of Testosterone. Neurosci Lett 465:123–127.

Karegar M, Mohammadi R (2015) Assessment of neuroregenerative effect of dihydrotestosterone, on peripheral nerve regeneration using allografts: a rat sciatic nerve model. Neurol Res 37:908–915.

 

 

Table 3 was time schedule of this study. It was not result. It should be put on Materials and Methods 2.1.

Thank you for this observation which actually slipped our attention. Table 3 has been moved in the materials and methods section.

 

 

In addition, the result of foot stepping angle (FSA) scores should be shown in Results 3.1.

Thank you for your suggestion. A table with all foot stepping angle results was added.

 

 

The names of authors and the numbers of the references were mismatches in line 118, 124-125, and 210. Therefore, the authors should check all of the references and renumber or reorganize.

Thank you for noticing. The list of references has been renewed so that they correspond to the citations.

 

 

In review version, the position of Figure 4 was wrong.

We have remedied the position in the revised manuscript. Thank you for the thorough scrutiny and paying attention to relevant inconsistencies.

 

Table 1 and Table 2 were results. They should be put in not Materials and Method but Results section.

Thank you for the comment. Tables 1 and 2 have been moved to the result section.

 

 

Moreover, in Materials and Methods, Animal groups demonstrated Intact (n=3) and other groups (n=10). Why was number in each group different in Table 1 and Table 2? Why did number reduce?

Thank you again for picking up all these details. However, we do wish to let you know that some of the spinal cord sections could not be used for calculations, because they were ruined during the process, or during observation in the microscope there was too much artefact, interfering with the fluorescence reading. If we could not collect at least 9 readings from each animal to get a reliable mean value, the animal was excluded from the study. The indications within the table refer to the number of animals actually used for measurements. This has now been clarified in our text.

 

 

In Figure 5, 6, and 7, the images belonging to WBV28 or WBV14 were shown. If the authors wrote in Results, the images of immunostaining from all experimental groups should be shown in Figure 5, 6, and 7.

Thank you for your suggestion. We have now included a complete panel of photos for the 2 stainings in addition to our measurements from the 2 tables.

 

Round 2

Reviewer 2 Report

The authors addressed for the reviewer's comments.

One point was not completely improved. In Figure 5, 6, and 7, why did not the authors show the images of immunostaining in all experimental groups, Intact, SCI-noEx, WBA7, WBV14, WBV28, and PFE?