Protective role of neuronal and lymphoid cannabinoid CB2 receptors in neuropathic pain
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Abstract
Cannabinoid CB 2 receptor (CB 2 ) agonists are potential analgesics void of psychotropic effects. Peripheral immune cells, neurons and glia express CB 2 ; however, the involvement of CB 2 from these cells in neuropathic pain remains unresolved. We explored spontaneous neuropathic pain through on-demand self-administration of the selective CB 2 agonist JWH133 in wild-type and knockout mice lacking CB 2 in neurons, monocytes or constitutively. Operant self-administration reflected drug-taking to alleviate spontaneous pain, nociceptive and affective manifestations. While constitutive deletion of CB 2 disrupted JWH133-taking behavior, this behavior was not modified in monocyte-specific CB 2 knockouts and was increased in mice defective in neuronal CB 2 knockouts suggestive of increased spontaneous pain. Interestingly, CB 2 -positive lymphocytes infiltrated the injured nerve and possible CB 2 transfer from immune cells to neurons was found. Lymphocyte CB 2 depletion also exacerbated JWH133 self-administration and inhibited antinociception. This work identifies a simultaneous activity of neuronal and lymphoid CB 2 that protects against spontaneous and evoked neuropathic pain.
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###Reviewer #2
This is an interesting paper that addresses and important and timely subject, namely identification of novel non opioid approaches to pain management. The authors direct their attention the CB2 receptor. Using perhaps the best characterized, and quite selective CB2 ligand, the authors implicated CB2 receptors in both neuronal and non neuronal cells in the spontaneous pain that occurs in a partial nerve injury model in the mouse. The studies largely used either mice in which the CB2 receptor was deleted in all cells only in neurons, or selectively in monocyte derived cells.
There are many intriguing findings in the paper, however, one is left with the feeling that there are hints at mechanisms, but nothing definitive is established. And major questions, which I believe could have been addressed with more selective …
###Reviewer #2
This is an interesting paper that addresses and important and timely subject, namely identification of novel non opioid approaches to pain management. The authors direct their attention the CB2 receptor. Using perhaps the best characterized, and quite selective CB2 ligand, the authors implicated CB2 receptors in both neuronal and non neuronal cells in the spontaneous pain that occurs in a partial nerve injury model in the mouse. The studies largely used either mice in which the CB2 receptor was deleted in all cells only in neurons, or selectively in monocyte derived cells.
There are many intriguing findings in the paper, however, one is left with the feeling that there are hints at mechanisms, but nothing definitive is established. And major questions, which I believe could have been addressed with more selective Cre-mediated deletion of the receptor, are never answered. Hints here and there, but nothing definitive.
Major questions
The authors focus on sensory neurons and the non neuronal cells that surround the neuronal cell bodies in the DRG. How might the neurons in the DRG, which they appear to presume must be mediating the input that drives spontaneous pain, not be relevant to acute pain processing? The previous report of Soethoudt et al., 2017, which defined the specificity of JWH133, found that this compound is without effect on acute pain even at doses up to 100mg/kg. I am not sure how to translate that dose to the iv administration in the present paper, but my assumption is that the 100mg/kg dose is at least equivalent. Granted JWH133 is not potent, but then how does it affect spontaneous pain?
The most straightforward test of the DRG neurons is to delete the CB2 receptor from neurons, using one of several selective Cre lines (e.g. NaV1.8-Cre). This is particularly important as the authors highlight the apparent translocation of the CB2 receptor from non neuronal cells around the DRG to neurons. But they only used peripherin to mark the neurons, so that any change in myelinated afferents would be missed. As neurons are the only structure that can get information into the spinal cord, do the authors propose that it is this small 4% of DRG neurons that is key?
Perhaps the most glaring piece missing as to mechanism is the fact, acknowledged by the authors, that JWH133 has a significant action at TRPA1, which is expressed by sensory neurons. Most importantly, the authors found that the CB2R null only had 50% reduced nose poke for JWH133. Clearly, JWH133 must exert its effect, at least in part, on another target. In their Abstract, the authors are very careful concerning this finding, writing that ""While constitutive deletion of CB2r disrupted JWH133-taking behavior....". In other words JWH133 disrupted, it did not prevent or eliminate the behavior. So clearly, there is something else mediating JWH133's effects. Studying JWH133 effects in the TRPA1 mutant, and ideally in the mouse in which TRPA1 is selectively deleted from sensory neurons, is critical to understanding this drug's actions. Results from that study would add greatly to the authors study.
Also, as TRPA1 is expressed in sensory neurons, are they now proposing that TRPA1 only contributes to spontaneous pain? That is certainly not the case. In fact, a previous study did report that JWH133 blocked pain behaviors provoked by AITC, a TRPA1 agonist. A simple experiment would be to test the effect of JWH133 against 0.5% formalin evoked nocifensive behaviors. As for AITC, 0.5% formalin evoked behaviors are lost in the TRPA1 ko.
Another simple experiments that would get at mechanism is to examine the effect of JWH133 on nerve injury provoked microglial activation in the dorsal horn. If a decreased activation were demonstrated, the case would be much stronger that the drug is acting on sensory neurons.
Concerns about the immunohistochemistry: First, the images presented are not all that convincing, and particularly difficult to read given that the percentage of double labeled neurons is small. It is also very odd that the authors had to use TSA amplification. Also there is no mention of controls for antibody specificity.
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###Reviewer #1
This is a very interesting paper. While demonstration of CB2 receptor agonist self-administration in rodent models of chronic pain is not in itself novel, there is a sufficient body of additional novel and exciting work in this paper to set it apart from previously published work. In particular, the mechanistic dissection using tissue-specific KO mice, coupled with the demonstration of that CB2 receptor-expressing lymphocytes infiltrate peripheral neurons, and to a greater degree in nerve-injured versus sham mice. The anxiety-related results are also very interesting. The paper is well-written and the results, for the most part, are clear.
Major comments
While there are significant novel results and important conclusions that can be and have been drawn from the work, the mechanism underlying the increased …
###Reviewer #1
This is a very interesting paper. While demonstration of CB2 receptor agonist self-administration in rodent models of chronic pain is not in itself novel, there is a sufficient body of additional novel and exciting work in this paper to set it apart from previously published work. In particular, the mechanistic dissection using tissue-specific KO mice, coupled with the demonstration of that CB2 receptor-expressing lymphocytes infiltrate peripheral neurons, and to a greater degree in nerve-injured versus sham mice. The anxiety-related results are also very interesting. The paper is well-written and the results, for the most part, are clear.
Major comments
While there are significant novel results and important conclusions that can be and have been drawn from the work, the mechanism underlying the increased self-administration of JWH133 in PSNL mice has still not been fully elucidated. The authors have shown it is CB¬2 receptor-dependent, but not due to CB2 receptors in neurons or monocyte-derived cells. Neither does it appear to be due to CB2 receptors on infiltrating lymphocytes. So the question still remains as to what mechanism or target is mediating the effect. I think the Discussion should address this limitation in more detail, and put forward some potential mechanisms.
Is it possible that there could be some involvement for CB1 receptors? JWH133 is relatively selective for CB2 over CB¬1, but to my knowledge it does still have some affinity for (and potential activity at) CB1. How can the authors rule out a potential involvement of CB1 in the self-administration of JWH133 after PSNL.
Given the anxiety-related aspect (and indeed the self-administration/pain aspect), why did the authors not look at whether lymphocytes expressing CB2 also infiltrate brain neurons? It would be very interesting to know if they infiltrate neurons in brain regions such as the amygdala, PFC, PAG and other regions known to be important in pain, anxiety and drug self-administration in pain models.
Statistics: The authors should determine whether they need to adjust for multiple comparisons when doing repeated Mann-Whitney U tests, e.g. perhaps do a Bonferroni-Holm correction to control for alpha when doing multiple MW U test comparisons. Alternatively, they could perform a Dunn's test instead of the MW U test.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on May 1, 2020, follows.
Summary
Both reviewers found many positive aspects to your manuscript. However, although Reviewer #1 believes that the concerns could be addressed without providing additional experimental data, Reviewer 2 has identified some significant concerns that do need additional information. The specific experiments that are indicated address the underlying mechanism, including the extent to which receptor expression on sensory neurons is involved and most importantly, the contribution of TRPA1. We appreciate that the present COVID-19 pandemic will make it impossible to complete the requested studies within the normal two-month period, which will involve new mouse crosses, so we are willing to accept a revised manuscript …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on May 1, 2020, follows.
Summary
Both reviewers found many positive aspects to your manuscript. However, although Reviewer #1 believes that the concerns could be addressed without providing additional experimental data, Reviewer 2 has identified some significant concerns that do need additional information. The specific experiments that are indicated address the underlying mechanism, including the extent to which receptor expression on sensory neurons is involved and most importantly, the contribution of TRPA1. We appreciate that the present COVID-19 pandemic will make it impossible to complete the requested studies within the normal two-month period, which will involve new mouse crosses, so we are willing to accept a revised manuscript when you are able to return to the laboratory and complete the studies.
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