Treadmill Stepping after Epidural Stimulation Cessation in Decerebrated Cats
Round 1
Reviewer 1 Report
The author has replicated the model of after-stepping post-cessation of electrical stimulation in cats. Based on the literature cited by the authors, the novelty of this study is low. At several instances the authors themselves mentioned that the results obtained here are already know. For example, "several peculiarities for it compared to the one-joint muscles were obtained previously [17]". And, "Previously it was also shown that the total duration of the after-stepping increased after spinalization [9]." Therefore, the novelty of this paper is not clear.
As a control condition, it would be beneficial to present data when the cat is on the treadmill and stimulation if off. Does the EMG/GRF/POT signals register just under the influence of gravity or is it really after-stepping?
The discussion section merely talk about the differences between the different muscles activation and/or differences between GRF and kinematics, which is again not novel. More insights into the cause for the differences should be presented. It is also advisable to avoid speculative statements such as "Under these facts we can suppose a different influence of the supraspinal input on the fast flexor and fast extensor muscles during after-stepping."
A picture or sketch for the experiment setup must be included to clearly understand the placement of sensors, for e.g. POT
Author Response
Reviewer 1.
The author has replicated the model of after-stepping post-cessation of electrical stimulation in cats. Based on the literature cited by the authors, the novelty of this study is low. At several instances the authors themselves mentioned that the results obtained here are already know. For example, "several peculiarities for it compared to the one-joint muscles were obtained previously [17]". And, "Previously it was also shown that the total duration of the after-stepping increased after spinalization [9]." Therefore, the novelty of this paper is not clear.
We change the main text. “This so-called after-stepping reflects the maintenance level for the activation of locomotor neuronal circuitry, but only scarce information devoted to the peculiarities of the after-stepping. We provide a comparative investigation of the after-stepping and stepping under epidural stimulation, using electromyographic and kinematic signals, and ground reaction forces in 16 decerebrate cats. Our principal findings are: (1) the ground reaction forces are more decreased after epidural stimulation cessation compared with anterior-posterior limb movements; (2) step cycle duration is longer in after-steps; (3) the electromyographic signal of extensor gastrocnemius lateralis muscle during after-stepping decreased faster compared to signal from flexors iliopsoas and tibialis anterior, and to an extensor soleus muscle; and (4) electromyographic stability is reduced after epidural stimulation cessation. We suppose that different levels of the spinal central pattern generator can be differently attenuated after external trigger cessation. These data can be important in elaboration for the locomotor models and in rehabilitation techniques”.
As a control condition, it would be beneficial to present data when the cat is on the treadmill and stimulation if off. Does the EMG/GRF/POT signals register just under the influence of gravity or is it really after-stepping?
We add this information in Figure 1 and Figure legends.
The discussion section merely talk about the differences between the different muscles activation and/or differences between GRF and kinematics, which is again not novel. More insights into the cause for the differences should be presented. It is also advisable to avoid speculative statements such as "Under these facts we can suppose a different influence of the supraspinal input on the fast flexor and fast extensor muscles during after-stepping."
We change the Discussion part.
A picture or sketch for the experiment setup must be included to clearly understand the placement of sensors, for e.g. POT
We include it.
Reviewer 2 Report
The present study investigates the persistent stepping manifested after the end of epidural stimulation of the spinal locomotor region in decerebrated cats with the aim of describing the unique characteristics of this behaviour and in so doing contributing to a better understanding of the organization of supraspinal and spinal locomotor centres. In a rather large group of decerebrated adult cats, the authors collected kinematic data of hind limb stepping movements during supported treadmill walking, ground reaction forces and recorded electromyographic (EMG) activity from hip flexors (iliopsoas), and ankle extensors (soleus and lateral gastrocnemius) and flexors (tibialis anterior). In all animals, hind limb stepping during supported treadmill locomotion continued after cessation of epidural stimulation with such after-steps progressively decreasing in amplitude to vanish past a variable number of steps. Two key observations are reported about post-stimulation stepping: a faster decline in ground reaction forces than in limb kinematics and a faster drop in EMG bursts of the lateral gastrocnemius in comparison with other hip ankle muscles. Although only descriptive, these observations are of interest and call attention to a phenomenon less studied. The methods are sound and the results are nicely presented. However, there are some issues that must be addressed. Although the paper is reasonably well written, it would profit from an overall English revision to improve its clarity. This is particularly important in the Discussion section. In the following, I’ll highlight some of these issues.
Abstract and New Findings
· Abstract’s and New Findings’ texts are a bit confusing and should be improved. In particular, I suggest replacing ambiguous statements such as “To systematize the peculiarity…” with clear statements about the aim of the study, the methods, the main results and conclusions. Be aware that addressing the findings of the study as related to “peculiarities of the spinal and supraspinal control…” is unsatisfactory. Please provide a clear and reasonable explanation for the findings, even if hypothetical and speculative due to the descriptive character of the study.
Introduction
· The Introduction is brief but informative. However, the scientific reason(s) for conducting this experiment must be stated more clearly. On line 42, one can read “In general, the data presented are weakly systematised…”. This is too unspecific to be considered a valid reason justifying conducting the study. Which data do authors refer to? What do they mean by “weakly systematised”? Please address these issues and clearly describe what are the gaps in knowledge regarding after-stepping that need to be addressed.
Materials & Methods
· Please provide further details about the method employed to assess EMG stability and particularly how this method specifically applies to EMG data. Also, indicate what measurements were derived from EMG recordings other than stability.
· The details about the protocol for data collection are missing. In particular, the information about the duration of the stepping recording sessions, number of trials, and minimum number of steps during ES is lacking. Please, provide this information in the Material & Methods section.
· For clarity, it would be helpful to describe the statistical methods in a separate subsection. Also, a clear description of which pairwise comparisons were undertaken should be added to this section (i.e., ES versus AS, across muscles, etc). Also missing in the text is a description of how data was reduced to obtain the final measures to enter the pairwise comparisons. (The mean of all step cycles during ES and AS?)
Results
· Lines 154-161. Please, explain what it means “burst duration” or “EMG bursting activity” within the context of this study.
· Figure 1. Please add to the caption the meaning of the abbreviation “AS”.
· Figure 2. The caption could be revised for improved clarity. For instance, it should be made clear that per cent ratios during AS are in relation to ES steps. Also, “inter-columnar comparisons” should be altered to something like “comparisons between stepping variables”.
Discussion
The Discussion should be improved. I recommend:
· Include a statement of principal findings.
· Address study findings other than lateral gastrocnemius changes during AS.
· Provide a further discussion of the implications of the study including unanswered questions and further research.
Please acknowledge the limitations of the study, in particular the descriptive nature of the study and the lack of mechanistic evidence.
Author Response
Reviewer 2
The present study investigates the persistent stepping manifested after the end of epidural stimulation of the spinal locomotor region in decerebrated cats with the aim of describing the unique characteristics of this behaviour and in so doing contributing to a better understanding of the organization of supraspinal and spinal locomotor centres. In a rather large group of decerebrated adult cats, the authors collected kinematic data of hind limb stepping movements during supported treadmill walking, ground reaction forces and recorded electromyographic (EMG) activity from hip flexors (iliopsoas), and ankle extensors (soleus and lateral gastrocnemius) and flexors (tibialis anterior). In all animals, hind limb stepping during supported treadmill locomotion continued after cessation of epidural stimulation with such after-steps progressively decreasing in amplitude to vanish past a variable number of steps. Two key observations are reported about post-stimulation stepping: a faster decline in ground reaction forces than in limb kinematics and a faster drop in EMG bursts of the lateral gastrocnemius in comparison with other hip ankle muscles. Although only descriptive, these observations are of interest and call attention to a phenomenon less studied. The methods are sound and the results are nicely presented. However, there are some issues that must be addressed.
Thank You for your review!
Although the paper is reasonably well written, it would profit from an overall English revision to improve its clarity. This is particularly important in the Discussion section. In the following, I’ll highlight some of these issues.
We change the Discussion part, and corrected English.
Abstract and New Findings
Abstract’s and New Findings’ texts are a bit confusing and should be improved. In particular, I suggest replacing ambiguous statements such as “To systematize the peculiarity…” with clear statements about the aim of the study, the methods, the main results and conclusions. Be aware that addressing the findings of the study as related to “peculiarities of the spinal and supraspinal control…” is unsatisfactory. Please provide a clear and reasonable explanation for the findings, even if hypothetical and speculative due to the descriptive character of the study.
Summary. “It was repeatedly shown that locomotion evoked by the epidural electrical stimulation can last for time after stimulation cessation, in decerebrated and spinal animals. This so-called after-stepping reflects the maintenance level for the activation of locomotor neuronal circuitry, but only scarce information devoted to the peculiarities of the after-stepping. We provide a comparative investigation of the after-stepping and stepping under epidural stimulation, using electromyographic and kinematic signals, and ground reaction forces in 16 decerebrate cats. Our principal findings are: (1) the ground reaction forces are more decreased after epidural stimulation cessation compared with anterior-posterior limb movements; (2) step cycle duration is longer in after-steps; (3) the electromyographic signal of extensor gastrocnemius lateralis muscle during after-stepping decreased faster compared to signal from flexors iliopsoas and tibialis anterior, and to an extensor soleus muscle; and (4) electromyographic stability is reduced after epidural stimulation cessation. We suppose that different levels of the spinal central pattern generator can be differently attenuated after external trigger cessation. These data can be important in elaboration for the locomotor models and in rehabilitation techniques”.
New Findings. “Our new findings of comparative investigations of the so-called after-stepping (locomotion after electrical stimulation cessation) and locomotion during epidural electrical stimulation. Our new findings are: after epidural stimulation cessation: (1) the ground reaction forces are decreased faster compared with anterior-posterior limb movements; (2) the electromyographic signal of extensor gastrocnemius lateralis muscle decreased faster compared to that from flexors iliopsoas and tibialis anterior, and to an extensor soleus muscle; and (3) electromyographic stability is reduced.”
Introduction
The Introduction is brief but informative. However, the scientific reason(s) for conducting this experiment must be stated more clearly. On line 42, one can read “In general, the data presented are weakly systematised…”. This is too unspecific to be considered a valid reason justifying conducting the study. Which data do authors refer to? What do they mean by “weakly systematised”? Please address these issues and clearly describe what are the gaps in knowledge regarding after-stepping that need to be addressed.
We change the text: “Since after-stepping reflects the long-lasted recruitability of the locomotor neuronal networks, we believe that after-stepping is not a simple experimental curiosity but can be a marker for the rhythmogenic status of the spinal CPGs. In general, many questions related to the after-stepping phenomenon are still unresolved; and its peculiarities like stepping cycle structure and duration, and activity of flexor / extensor muscles are scarce. In present work we investigated the after-stepping in decerebrated cats after locomotion evoked by the electrical epidural stimulation (ES), using kinematic (for the step cycle analysis) and electromyographic recording (for the analysis of activity of flexor and extensor muscles to reveal the possible dissimilarity of their ES-cessation activity; such dissimilarity may be due to different control schemes performed by CPG to flexor and extensor muscles).”
Materials & Methods
Please provide further details about the method employed to assess EMG stability and particularly how this method specifically applies to EMG data. Also, indicate what measurements were derived from EMG recordings other than stability.
We include information in Figure 1 and into the main text: “The stability of EMG of individual muscles was estimated using the self-similarity coefficient (the amplitude of the second peak of autocorrelation functions [14] of EMG smoothed by a moving average with a window of 50 ms) (Figure 1B). This coefficient reflects the similarity between an EMG activity for steps series”.
The details about the protocol for data collection are missing. In particular, the information about the duration of the stepping recording sessions, number of trials, and minimum number of steps during ES is lacking. Please, provide this information in the Material & Methods section.
Each step cycle was divided into 20 bins, where 10 bins - in swing, and 10 bins - in stance phase. An integrated rectified EMG was calculated in bins corresponding to the stance or swing phase, according to the potentiometric signal of the hindlimb. We also should note that in our conditions, GL was active only during the stance phase (like it was previously shown [15]). The selection of trials for the EMG analysis was performed using the following criteria: the trial should include at least 10 steps - for ES-stepping, or at least 6 steps - for AS-stepping, and no rude artefacts should be at EMG channels. In every cat, one trial was used for every condition (ES-stepping and AS-stepping).
For clarity, it would be helpful to describe the statistical methods in a separate subsection. Also, a clear description of which pairwise comparisons were undertaken should be added to this section (i.e., ES versus AS, across muscles, etc). Also missing in the text is a description of how data was reduced to obtain the final measures to enter the pairwise comparisons. (The mean of all step cycles during ES and AS?)
We include a separate “Statistical Methods” section. Data presented as mean±SD. Mean values for kinematic and EMG parameters were obtained after averaging for 10 to 15 ES steps and from 6 to 10 after-steps. Since a non-normal distribution of data (Shapiro-Wilk test), and since only paired comparisons (ES vs AS, inter-muscle comparisons, inter-parameters comparisons) were analysed, a non-parametric Wilcoxon matched-pairs signed rank test (W test) was used (pairing effectiveness was assessed by the Spearman criterion).
Results
Lines 154-161. Please, explain what it means “burst duration” or “EMG bursting activity” within the context of this study.
We change the text: “We also assessed the duration of EMG activity having bursts corresponding to the hindlimb steps during after-stepping”.
Figure 1. Please add to the caption the meaning of the abbreviation “AS”.
We add it.
Figure 2. The caption could be revised for improved clarity. For instance, it should be made clear that per cent ratios during AS are in relation to ES steps. Also, “inter-columnar comparisons” should be altered to something like “comparisons between stepping variables”.
We change the capture: (1) include the phrase “C-G - kinematic, electromyographic, GRF data presented as a percent ratio of the AS variables to the ES ones”, and (2) replace the phrase “inter-columnar comparisons” onto the “comparisons between stepping variables”.
Discussion
The Discussion should be improved. I recommend:
Include a statement of principal findings.
Our principal findings are: (1) the ground reaction forces are more decreased after ES cessation compared with anterior-posterior limb movements; (2) step cycle duration is longer in after-steps; (3) the EMG signal of extensor gastrocnemius lateralis muscle was more decreased and has the shortest bursting duration during after-stepping compared to flexors, and to an extensor soleus muscle; and (4) EMG stability is well saved after ES cessation.
Address study findings other than lateral gastrocnemius changes during AS.
We change the Discussion text.
Provide a further discussion of the implications of the study including unanswered questions and further research.
We change the Discussion text.
Please acknowledge the limitations of the study, in particular the descriptive nature of the study and the lack of mechanistic evidence.
We include this part. “The main limitation of our study is its descriptive nature. We expand the knowledge about the after-stepping phenomenon, describing its peculiarities; but new studies devoted to the elaboration of algorithms for after-stepping analisis are needed since many questions are still unresolved. In particular, what about other muscles, especially extensors (knee and hip extensors)? Are any muscle synergies saved longer compared to others? Is postural control saved longer compared to locomotor ability?”
Reviewer 3 Report
Authors attempt to explain or to bring some new explanations to the mechanism of the fictive locomotion or so called after-stepping following the epidural electrical stimulation (even when the stimulation is ceased) in decerebrated cats. The article is original from the field of basic neuroscience with the possibility of future introducing the results to the clinical practice by means of rehabilitation of patients after the incomplete spinal cord injury.
The article is well written.
The paper seems to develop the old works of Grillner et al, Orlovsky et al and Rossignol (as well findings of Slawinska et al, Gorska et al and Kasickiet al – not cited), authors admit that the mentioned phenomenon was repeatedly shown. However the methodology in the current study represents the modern advances in the techniques of poly-EMG recordings and the interpretation of neurophysiological findings, since the results “sound”. The novelty is to compare the after-stepping compared with stepping during epidural stimulation during electromyographic recordings and kinematic signals, as well as the ground reaction forces in 16 decerebrate cats. The number of experiments and recordings is satisfactory to obtain the reliable results.
Nevertheless , the final conclusion has been generalized, and authors (lines 19-21) …”suppose that data obtained can be related to the peculiarities of the spinal and supraspinal control upon the flexor and extensor muscle activity, and activity of postural muscle soleus”… Does it require the further studies? It has been not indicated. Moreover they state (lines 28-29): …”This data can be important in elaboration for the locomotion models and in rehabilitation techniques”… - yes, it can be, but should authors suggest how, do they mean by the modification of the kiesiotherapeutic procedures, for example during locomotion re-learning of patients after the incomplete spinal cord injury? Authors detected the phenomenon that the EMG signal of extensor gastrocnemius lateralis muscle was more decreased and has the shortest bursting duration during after-stepping compared to flexors, and to an extensor soleus muscle. It has not been discussed taking into account the possibility of central and peripheral electrostimulation for the treatment purposes, nor the study limitation was mentioned.
The “state-of-art” in Introduction is good enough as well as and the aim.
M&M section is perfect.
Results section is very concise but clearly presented.
Indications how to improve Discussion section are presented above.
Author Response
Reviewer 3
Authors attempt to explain or to bring some new explanations to the mechanism of the fictive locomotion or so called after-stepping following the epidural electrical stimulation (even when the stimulation is ceased) in decerebrated cats. The article is original from the field of basic neuroscience with the possibility of future introducing the results to the clinical practice by means of rehabilitation of patients after the incomplete spinal cord injury.
The article is well written.
Thank You for your review!
The paper seems to develop the old works of Grillner et al, Orlovsky et al and Rossignol (as well findings of Slawinska et al, Gorska et al and Kasickiet al – not cited), authors admit that the mentioned phenomenon was repeatedly shown. However the methodology in the current study represents the modern advances in the techniques of poly-EMG recordings and the interpretation of neurophysiological findings, since the results “sound”. The novelty is to compare the after-stepping compared with stepping during epidural stimulation during electromyographic recordings and kinematic signals, as well as the ground reaction forces in 16 decerebrate cats. The number of experiments and recordings is satisfactory to obtain the reliable results.
Thank You for new references. We include them into the main text.
Nevertheless , the final conclusion has been generalized, and authors (lines 19-21) …”suppose that data obtained can be related to the peculiarities of the spinal and supraspinal control upon the flexor and extensor muscle activity, and activity of postural muscle soleus”… Does it require the further studies? It has been not indicated. Moreover they state (lines 28-29): …”This data can be important in elaboration for the locomotion models and in rehabilitation techniques”… - yes, it can be, but should authors suggest how, do they mean by the modification of the kiesiotherapeutic procedures, for example during locomotion re-learning of patients after the incomplete spinal cord injury? Authors detected the phenomenon that the EMG signal of extensor gastrocnemius lateralis muscle was more decreased and has the shortest bursting duration during after-stepping compared to flexors, and to an extensor soleus muscle. It has not been discussed taking into account the possibility of central and peripheral electrostimulation for the treatment purposes, nor the study limitation was mentioned.
We changed the Discussion part, and included “Limitations” part.
The “state-of-art” in Introduction is good enough as well as and the aim.
M&M section is perfect.
Results section is very concise but clearly presented.
Indications how to improve Discussion section are presented above.
Round 2
Reviewer 1 Report
All major comments have been addressed. A minor comment is that the authors could improve the discussion by adding future implications of this work.
Author Response
We have improved an English.
