Bipartite left-right sided endocrine system: processing of contralateral effects of brain injury
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Abstract
The crossed descending neural tracts set a basis for contralateral effects of brain injury. In addition, the left-right side-specific effects of the unilateral brain lesions may be mediated by neurohormones through the humoral pathway as discovered in animals with disabled descending motor tracts. We here examined if counterparts of the endocrine system that convey signals from the left and right brain injuries differ in neural and molecular mechanisms. In rats with completely transected cervical spinal cords a unilateral injury of the hindlimb sensorimotor cortex produced hindlimb postural asymmetry with contralateral hindlimb flexion, a proxy for neurological deficit. The effects of the left and right side brain lesions were differently inhibited by antagonists of the δ-, κ- and µ-opioid receptors suggesting differential neuroendocrine control of the left-right side-specific hormonal signaling. Bilateral deafferentation of the lumbar spinal cord eliminated hormone-mediated effects of the left-side brain injury but not the right-side lesion suggesting their afferent and efferent mechanisms, respectively. Analysis of gene-gene co-expression patterns identified the left and right side-specific gene regulatory networks that were coordinated across the hypothalamus and lumbar spinal cord through the humoral pathway. The coordination was ipsilateral and perturbed by brain injury. These findings suggest that the neuroendocrine system that conveys left-right side-specific hormonal messages from injured brain is bipartite, contributes to contralateral neurological deficits through asymmetric neural mechanisms, and enables ipsilateral coordination of molecular processes across neural areas along the neuraxis.
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Reply to the reviewers
__Points raised by both reviewers in their cross-comments __
- “emphasizing the acute nature of the study is important as well as the use of only male rats” RESPONSE: Thank you for pointing this out. It has been clarified throughout the manuscript, including the abstract, limitations section, and conclusions.
“The need for improvement of the presentation cannot be stressed enough”.
RESPONSE: The manuscript has undergone extensive revisions to enhance the clarity of data presentation and discussion, and to highlight its novelty in comparison to our prior studies. We have reduced the use of technical terminology and abbreviations, and when they do appear, …
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
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Reply to the reviewers
__Points raised by both reviewers in their cross-comments __
- “emphasizing the acute nature of the study is important as well as the use of only male rats” RESPONSE: Thank you for pointing this out. It has been clarified throughout the manuscript, including the abstract, limitations section, and conclusions.
“The need for improvement of the presentation cannot be stressed enough”.
RESPONSE: The manuscript has undergone extensive revisions to enhance the clarity of data presentation and discussion, and to highlight its novelty in comparison to our prior studies. We have reduced the use of technical terminology and abbreviations, and when they do appear, they are explained with their first use and in the new Glossary section. The manuscript has been better organized, ensuring a logical flow of data and conclusions.
Reviewer #1
Major comments
“the extensive statistical analysis done for the gene expression would require assistance unless the in-house expertise already existed. If these are in place the work could be reproduced with the details provided.”
RESPONSE: Terms and abbreviations used in statistical and correlation analyses are thoroughly explained in the text and in the newly added Glossary section in the revised manuscript to the extent acceptable in a biological paper.
All statistical codes are accessible in the public GitHub repository at https://github.com/YaromirKo/biostatistics-nms. These codes may be utilized for the purpose of replicating studies.
Minor comments
“It is not clear how the genes that were studied here were picked. It is clearly stated what groups the genes fall into and their relevance to the study but it isn't clear how these were decided upon. Clarifying this would be helpful.”
RESPONSE: There is currently no consensus regarding the classification of genes as related to neuroplasticity. In particular, there is no agreement on lists of genes consistently associated with neuroplasticity across studies, and providers of mRNA analysis platforms do not offer panels of neuroplasticity-related genes. Most companies, such as Thermofisher, Illumina, and Nanostring, provide "Neurological" or "Neuropathology Research" panels that contain genes related to neuroplasticity. However, these panels are not specifically designed for targeted analysis of neuroplasticity-related genes.
The gene selection is arbitrary, and the chosen genes may vary across different studies depending on their objectives. In the present study, genes were selected based on several significant works having determined that these genes were likely related to neuroplasticity. Each gene's selection is justified by citing these works in the "Materials and Methods" section and we made every effort to avoid any bias. We do not assert that the gene set is all-encompassing. This matter is addressed in the Limitations section of the revised manuscript.
“It is not always clear what had been done in the previous work and what is completely new in this work, that could be addressed better.”
RESPONSE: Thank you for emphasizing that. It has been thoroughly addressed in the revised manuscript. While our previous study has discovered a left-sided neuroendocrine system, the current work delves into its organizational principles, which are equally crucial. We have shown that this system is bipartite and mirror asymmetric, and that its left and right counterparts can be targeted differently by pharmacological means. Additionally, we have revealed the left-right side-specific gene regulatory networks that operate in the neuroendocrine system and which activities are laterally coordinated by this system along the neuraxis.
“The text and figures are quite complex and require thorough reading the knowledge of the background to understand, therefore not making this work for a general audience.”
“Given the complexity of the work the reading of the results is quite dense and difficult to maneuver unless you have some prior understanding. My suggestion would be to try to simplify this but I wouldn't know exactly how to go about this.”
RESPONSE: We appreciate the Reviewer’s comments here, and agree that this is a complex work. We have endeavored to find a balance between a comprehensive presentation of the methods and results while also providing a level of simplification that will allow the reader who is not versed in this field to still appreciate this work. However, because of the nature of the experimental designs and of the findings that we report, we believe it to be important to provide a comprehensive explanation of the work and results. We believe that we have struck a balance between simplification and comprehensiveness with this revision. We have simplified the presentation of the results, their statistical analysis, and the analysis of gene regulatory networks for easier understanding. We also provide detailed explanations of technical terms in the newly added Glossary section. Please also refer to our response to point 2.
We believe that the revised manuscript has a level of complexity in data presentation and density similar to that of most combined physiological and molecular studies, complemented with advanced statistical and bioinformatics analysis. See please, for example papers published in Plos Biology (doi.org/10.1371/journal.pbio.3002328; doi.org/10.1371/journal.pbio.3002282; doi.org/10.1371/journal.pbio.3001465) and eLIFE (doi.org/10.7554/eLife.85756; https://doi.org/10.7554/eLife.90511.1).
General assessment
“The limitation would be understanding exactly what was done before and how this work expands on that, often it required the reader to look up references and prior work.”
RESPONSE: The introduction and discussion have been modified accordingly in order to comply with this comment. We have clarified how this study expands upon our previous work. In addition, please see the response to Comment 5 that also addresses this issue.
“The audience would be rather specialized, although it does gear towards clinical translation, this aspect could be highlighted better in the introduction and discussion.”
RESPONSE: Clinical aspects of the findings have been further highlighted in the revised manuscript. In the introduction, we note that the discovered phenomenon could contribute to asymmetrical neurological deficits following stroke and TBI. In the discussion section, we examine mechanical similarities between hindlimb asymmetry in rats and spastic dystonia in patients and hypothesize that the rat asymmetries may model this human neuropathology. In the concluding remarks, we state that it is crucial to examine the balance between neural and endocrine pathways in their contribution to neurological impairments, and to establish pharmacological approaches targeting the neuroendocrine system to restore the disturbed neurohormonal equilibrium.
“Those interested in brain injury/neurodegeneration as well as how signaling of motor control could be affected by not just damage to electrical descending motor tracts but to neuroendocrine signaling would be the specific audience.”
RESPONSE: We agree that the experts in neurotrauma, stroke and motor control may be interested in this study. However, the left-right side-specific neuroendocrine signaling may be a general biological phenomenon essential for regulation of lateralized brain functions, and, in a broader biological perspective, regulation of the body plan along the left-right axis.
Furthermore, the study presents what, to the best of our knowledge, is the first evidence for the existence of the left and right side-specific gene regulatory networks in the CNS. They operate in the neuroendocrine system and its peripheral target, and are coordinated across them via the humoral pathway. This is a novel molecular dimension in asymmetric organization of the generally mirror-symmetric CNS.
We are confident that experts in the establishment of the body plan and functional and molecular brain asymmetries will be interested in the concept formulated in this study.
Reviewer #2
Major comments:
“It should be made clear in the introduction that an acute complete cervical SCI is used and the discussion should be extended to include advantages and disadvantages of the used model and the alternatives.”
RESPONSE: Thank you for your suggestions. The introduction and discussion have been supplemented with the requested information. Specifically, we have noted that hindlimb postural asymmetry, a proxy model for neurological deficits, has enabled the discovery and characterization of the left-right side-specific neuroendocrine system. It is a binary model with two qualitatively different responses generated on either the left or right side. On the other hand, it cannot be used to analyze awake animals, and knowledge of its mechanisms is limited. A role for the neuroendocrine phenomenon in the persistent left-right specific biological and pathophysiological processes requires further investigation. This can be addressed by analyzing the effects of unilateral TBI in subchronic experiments with awake animals whose spinal cords are completely transected to disable neural pathways. The methodology could involve an integrated evaluation of hindlimb function during body weight-supported stepping, utilizing behavioral, electrophysiological, and biomechanical measures.
“A similar concern poses the use of pentobarbital and the interpretation of the results of the deafferentation. Were timing of the application and dosage strictly controlled between the different groups? It's effects on somatosensory afferent transmission through presynaptic inhibition are a concern.”
RESPONSE: Thank you for the remark. We have paid special attention to this issue. The rats were deeply anesthetized with the same dose and timing of anesthesia. These parameters were thoroughly controlled in all of the experiments. The depth of pentobarbital anesthesia was characterized by a barely perceptible corneal reflex and a lack of overall muscle tone. Of note, the side and magnitude of postural asymmetry do not apparently depend on anesthesia and its type; the asymmetry was virtually the same after brain injury in rats under deep pentobarbital or isoflurane anesthesia (this study and Lukoyanov et al., 2021; Watanabe et al., 2020; Watanabe et al., 2021; Zhang et al., 2020) and also in decerebrate unanesthetized rats (Zhang et al., 2020). Similar left-right differences were observed in the rats with left and right brain injury which were deafferentated 3 days later, and then analyzed under isoflurane anesthesia (Zhang et al., 2020). This is discussed in the revised manuscript.
Furthermore, no nociceptive stimulation was applied and tactile stimulation was negligible in the course of the asymmetry analysis; the legs were stretched by pulling the threads glued to nails of the toes. The application of lidocaine to the toes, which were pulled during stretching, had no impact on the formation of asymmetry. After all, the stretch and postural limb reflexes are immediately abolished and remain so for several days, and markedly decreased under anesthesia as it was firmly established in many studies. As these reflexes likely do not play a role in the formation of the asymmetric hindlimb posture, their afferent mechanisms could not be a cause of variations in our experiments.
In summary, three main arguments speak against an interference of pentobarbital with asymmetry formation in rats after rhizotomy. First, a similar asymmetry phenomenon developed in pentobarbital anesthetized rats, isoflurane anesthetized rats, and decerebrate un-anesthetized rats. Second, in rats that underwent rhizotomy, the primary sensory nerve fibers were entirely severed. Thus, the hypothetical link between pentobarbital's impact on asymmetry through its effect on presynaptic inhibition could be eliminated. Third, although there may be some variability in the depth of anesthesia among animals, the probability of such strong and statistically significant differences in the effects of brain injury and deafferentation arising from bias in the depth of anesthesia among groups of animals likely to be negligible.
*“Only two test for the asymmetry of spinal processing were used and the two tests are likely measuring very similar phenomena (tonic flexor over activation). Additional reflex tests could shed light onto underlying mechanisms.” *
RESPONSE: We agree. In previous studies, we also analyzed asymmetry in withdrawal reflexes between the left and right hindlimbs as an indicator of the effects of brain injury (Lukoyanov et al., 2021; Watanabe et al., 2021; Zhang et al., 2020). In the present study, we do not focus on the neurophysiological mechanisms of postural asymmetry. We instead prioritize characterizing the phenomenology and organizational principle of the left-right side-specific neuroendocrine system using the postural asymmetry model as a "black box" and as a robust and reliable readout.
Of note, there are several other equally important issues that remain to be addressed, including the identification of signaling pathways from the injured cortex to the hypothalamic-pituitary system, the identification of signaling molecules in the blood that convey information about the side of the brain injury, and the dissection of encoding and decoding mechanisms in the hypothalamus and spinal cord, respectively. No single study could investigate all of these mechanisms.
Minor comments:
“Figure 3 shows only the magnitude of the postural asymmetry in response to the different opioid receptor antagonists, yet the directionality is of interest, especially in case of the control animals. Pre2 values are missing too.”
RESPONSE: We appreciate the reviewer's comment and apologize for any errors in our previous version. The legend for Figure 3 has been revised and simplified. It is unnecessary to include PAS (Postural Asymmetry Size) in addition to MPA as the direction of PAS in all animals in each group was the same. This is stated in the revised manuscript's Legend for Figure 3. MPA was used to compare the left and right UBI groups, which had positive and negative PAS values, respectively. This comparison could not be carried out with PAS.
“Too many abbreviations are used which makes the text and figures very difficult to read at times.” “Terminology is sometimes inconsistent (e.g., delta vs contrast).”
RESPONSE: The manuscript now features a reduced amount of abbreviations. Technical terms and abbreviations are defined upon their first use and are also included in the newly added Glossary section. Corrections have been made to the use of the term "contrast" and its abbreviation "delta" in Figures. Additionally, the term "deltaW" as the left-right difference is no longer utilized within the manuscript.
“The section "correlation patterns in the hypothalamus and spinal cord" was almost impossible for me to understand and could use rephrasing.”
RESPONSE: We apologize for the previous version, and have simplified the presentation of molecular data. We believe that the level of complexity in the revised manuscript's statistics and data presentation is now comparable to that of many other molecular studies featuring system-level analyses; please see also response to Comment # 6 of the first reviewer.
“Only male rats are used.”
RESPONSE: This limitation has been addressed in the Limitation section. It is important to investigate whether identical or distinct neurohormones are responsible for the outcomes of left and right brain injury in male and female rats. However, this requires prior identification of most hypothalamic neurohormones and neuropeptides that regulate the asymmetric processes. Their number may be considerable, given the constellation of left and right gene regulatory networks in the hypothalamus.
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Referee #2
Evidence, reproducibility and clarity
Watanabe et al. build on their previous work to show that the left-right side specific effect of unilateral brain injury after acute complete spinal transection is indeed mediated by side-specific endocrine signaling. This is done by looking at a cervical spinal transection as opposed to a thoracic as in previous work. They further characterize the side-specific humoral hypothalamus-lumbar spinal cord pathways using gene expression patterns, application of opioid receptor antagonists, and dorsal root rhizotomy. Overall the evidence is very convincing and excludes mediation through the sympathetic system in addition to central …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #2
Evidence, reproducibility and clarity
Watanabe et al. build on their previous work to show that the left-right side specific effect of unilateral brain injury after acute complete spinal transection is indeed mediated by side-specific endocrine signaling. This is done by looking at a cervical spinal transection as opposed to a thoracic as in previous work. They further characterize the side-specific humoral hypothalamus-lumbar spinal cord pathways using gene expression patterns, application of opioid receptor antagonists, and dorsal root rhizotomy. Overall the evidence is very convincing and excludes mediation through the sympathetic system in addition to central descending tracts. Curiously the deafferentation, while having an effect on both sides only reversed the postural asymmetry caused by left-sided brain injury, and gene-gene co-expression revealed ipsilateral coordination.
Major comments:
- It is possible that many of the observations in the paper are dependent on the acute state of the spinal cord injury. This is mentioned in the limitations section and it is clear that the presented experiments are important and advance our understanding of this curious phenomenon. Yet, it should be made clear in the introduction that an acute complete cervical SCI is used and the discussion should be extended to include advantages and disadvantages of the used model and the alternatives.
- A similar concern poses the use of pentobarbital and the interpretation of the results of the deafferentation. Were timing of the application and dosage strictly controlled between the different groups? It's effects on somatosensory afferent transmission through presynaptic inhibition are a concern.
- Only two test for the asymmetry of spinal processing were used and the two tests are likely measuring very similar phenomena (tonic flexor over activation). Additional reflex tests could shed light onto underlying mechanisms.
- All major comments shouldn't be seen as a request for additional data but only require discussion.
Minor comments:
- Figure 3 shows only the magnitude of the postural asymmetry in response to the different opioid receptor antagonists, yet the directionality is of interest, especially in case of the control animals. Pre2 values are missing too.
- Too many abbreviations are used which makes the text and figures very difficult to read at times.
- Terminology is sometimes inconsistent (e.g., delta vs contrast).
- The section "correlation patterns in the hypothalamus and spinal cord" was almost impossible for me to understand and could use rephrasing.
- Only male rats are used.
Referees cross-commenting
I agree with reviewer #1's comments; most of them are in line with mine. The need for improvement of the presentation cannot be stressed enough. This is excellent and important work, which makes it even more important to convey it in an accessible way (be clear about prior work and what the novel results add, reduce number of abbreviations, guide the reader in how to interpret the figures, etc.). Otherwise, the audience will be limited.
Significance
General assessment: The manuscript provides clear evidence that there is a side-specific effect of UBI that is mediated by humoral signaling. Specifically, the present work excludes the sympathetic system. This is a very important finding that was missing in previous work. Further characterization of this recently discovered non-neuronal component of UBI is of very high importance as the potential for clinical implications are high.
Advance: The study provides a clear advance of our understanding of side-specific endocrine signaling to the spinal cord.
Audience: This study should be of interest to a wide audience, particularly for neuroscientists and neurologists who deal with the motor system.
My field of expertise: Neural control of locomotion, spinal cord injury, motor control, sensorimotor integration.
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Referee #1
Evidence, reproducibility and clarity
Summary
Watanabe et al. show in "Bipartite left-right sided endocrine system: processing contralateral effects of brain injury" a continuation of previously published work, that hindlimb postural asymmetry (HL-PA) is due to the neuroendocrine signaling and not the cervical parasympathetic pathways in anesthetized spinal C6-C7 fully transected unilateral brain injured (UBI) rats. Further, this differential neuroendocrine control of the left-right side-specific hormonal signaling is affected differently by either right or left unilateral hindlimb sensorimotor cortex brain injuries. However, bilateral deafferentation (L1-S1) showed …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary
Watanabe et al. show in "Bipartite left-right sided endocrine system: processing contralateral effects of brain injury" a continuation of previously published work, that hindlimb postural asymmetry (HL-PA) is due to the neuroendocrine signaling and not the cervical parasympathetic pathways in anesthetized spinal C6-C7 fully transected unilateral brain injured (UBI) rats. Further, this differential neuroendocrine control of the left-right side-specific hormonal signaling is affected differently by either right or left unilateral hindlimb sensorimotor cortex brain injuries. However, bilateral deafferentation (L1-S1) showed that only left-side brain injury was altered, indicating differing inputs. Adding to the previous finding that blocking opioid signaling in UBI non-injured spinal rats leads HL-PA, here they demonstrated this finding holds with right-left differences following a spinal transection. Furthering the previous findings of left-right lumbar spinal gene expression differences, this time they found hypothalmus and lumbar spinal cord gene expression differences that were ipsilaterally coordinated and affected by brain injury.
Major comments
- Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them? Yes, the claims are supported by the data presented in this manuscript.
- Are the data and the methods presented in such a way that they can be reproduced? The data and methods have been presented in a way that could be reproduced, however given the expertise of this laboratory in developing new systems not for purchase it is likely it would take a given expertise to replicate the data. Additionally, the extensive statistical analysis done for the gene expression would require assistance unless the in-house expertise already existed. If these are in place the work could be reproduced with the details provided.
- Are the experiments adequately replicated and statistical analysis adequate? Yes, the experiments have been adequately replicated and statistical analysis to my understanding is adequate.
Minor comments
- Specific experimental issues that are easily addressable. It is not clear how the genes that were studied here were picked. It is clearly stated what groups the genes fall into and their relevance to the study but it isn't clear how these were decided upon. Clarifying this would be helpful.
- Are prior studies referenced appropriately? It is not always clear what had been done in the previous work and what is completely new in this work, that could be addressed better. The references themselves are extensive and well-used throughout the work.
- Are the text and figures clear and accurate? The text and figures are quite complex and require thorough reading the knowledge of the background to understand, therefore not making this work for a general audience.
- Do you have suggestions that would help the authors improve the presentation of their data and conclusions? Given the complexity of the work the reading of the results is quite dense and difficult to maneuver unless you have some prior understanding. My suggestion would be to try to simplify this but I wouldn't know exactly how to go about this.
Referees cross-commenting
Reviewer #2 makes a crucial point that emphasizing the acute nature of the study is important as well as the use of only male rats. Otherwise, reviewer #2's comments overlap partially with my own in increasing the accessibility of the work. Neither recommended changes would require new experimental data.
Significance
General assessment:
I would this topic quite intriguing and a novel understanding of motor control. The multiple experiments that were performed that addressed various contingencies of HL-PA may occur after UBI were addressed here (ie. parasympathetic and sensory input). Further experiments expanded on previous findings of the involvement of opioids, the pituitary gland, and spinal gene networks. The limitation would be understanding exactly what was done before and how this work expands on that, often it required the reader to look up references and prior work.
Advance:
Although this is my first encounter with the work, it is a follow-up study on work that was published previously in eLife in 2021. Therefore, given some of the overlap it wouldn't be entirely conceptually new but it would be addressing open questions which arose from that work and further add to our understanding of the mechanism involved in this phenomenon.
Audience:
The audience would be rather specialized, although it does gear towards clinical translation, this aspect could be highlighted better in the introduction and discussion. Those interested in brain injury/ neurodegeneration as well as how signaling of motor control could be affected by not just damage to electrical descending motor tracts but to neuroendocrine signaling would be the specific audience. My expertise is in spinal cord injury, sensorimotor coordination of hindlimbs and gene expression. Although not an expert in brain injury or neuroendocrine signaling, my background allows me to understand the experiments performed here and the relevance of the work.
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