Potential of Fibrin Glue and Mesenchymal Stem Cells (MSCs) to Regenerate Nerve Injuries: A Systematic Review.

Cell-based therapy is a promising treatment to favor tissue healing through less invasive strategies. Mesenchymal stem cells (MSCs) highlighted as potential candidates due to their angiogenic, anti-apoptotic and immunomodulatory properties, in addition to their ability to differentiate into several specialized cell lines. Cells can be carried through a biological delivery system, such as fibrin glue, which acts as a temporary matrix that favors cell-matrix interactions and allows local and paracrine functions of MSCs. Thus, the aim of this systematic review was to evaluate the potential of fibrin glue combined with MSCs in nerve regeneration. The bibliographic search was performed in the PubMed/MEDLINE, Web of Science and Embase databases, using the descriptors ("fibrin sealant" OR "fibrin glue") AND "stem cells" AND "nerve regeneration", considering articles published until 2021. To compose this review, 13 in vivo studies were selected, according to the eligibility criteria. MSCs favored axonal regeneration, remyelination of nerve fibers, as well as promoted an increase in the number of myelinated fibers, myelin sheath thickness, number of axons and expression of growth factors, with significant improvement in motor function recovery. This systematic review showed clear evidence that fibrin glue combined with MSCs has the potential to regenerate nervous system lesions.

Nerve recovery from treatment with a vascularized nerve graft compared to an autologous non-vascularized nerve graft in animal models: A systematic review and meta-analysis.

BACKGROUND: Treatment of nerve injuries proves to be a worldwide clinical challenge. Vascularized nerve grafts are suggested to be a promising alternative for bridging a nerve gap to the current gold standard, an autologous non-vascularized nerve graft. However, there is no adequate clinical evidence for the beneficial effect of vascularized nerve grafts and they are still disputed in clinical practice. OBJECTIVE: To systematically review whether vascularized nerve grafts give a superior nerve recovery compared to non-vascularized nerve autografts regarding histological and electrophysiological outcomes in animal models. MATERIAL AND METHODS: PubMed and Embase were systematically searched. The inclusion criteria were as follows: 1) the study was an original full paper which presented unique data; 2) a clear comparison between a vascularized and a non-vascularized autologous nerve transfer was made; 3) the population study were animals of all genders and ages. A standardized mean difference and 95% confidence intervals for each comparison was calculated to estimate the overall effect. Subgroup analyses were conducted on graft length, species and time frames. RESULTS: Fourteen articles were included in this review and all were included in the meta-analyses. A vascularized nerve graft resulted in a significantly larger diameter, higher nerve conduction velocity and axonal count compared to an autologous non-vascularized nerve graft. However, during sensitivity analysis the effect on axonal count disappeared. No significant difference was observed in muscle weight. CONCLUSION: Treating a nerve gap with a vascularized graft results in superior nerve recovery compared to non-vascularized nerve autografts in terms of axon count, diameter and nerve conduction velocity. No difference in muscle weight was seen. However, this conclusion needs to be taken with some caution due to the inherent limitations of this meta-analysis. We recommend future studies to be performed under conditions more closely resembling human circumstances and to use long nerve defects.

Exercise induced hypoalgesia profile in rats is associated with IL-10 and IL-1 ß levels and pain severity following nerve injury.

BACKGROUND: Pain may undergo modulation in the central nervous system prior to reaching the primary somatosensory cortex and being perceived as pain. Faulty pain modulation mechanisms have been linked to various chronic pain conditions. Cytokines such as IL-10 and IL-1beta, are known to be involved in initiation and maintenance of neuropathic pain. In this study, we investigated the association between pain modulation profile, pain intensity and cytokines (IL-10 and IL-1beta) levels in a rat model of neuropathic pain. METHODS: Exercise-Induced Hypoalgesia (EIH) was assessed by evaluating the percentage of responses to a train of 60g mechanical stimuli before and after 180 seconds of exercise on a rotating rod. The differences in the response rates before and after the exercise were used to divide the rats into low and high EIH responders. Rats from low and high EIH groups underwent constriction injury of the left sciatic nerve. Pain behavior (allodynia and hyperalgesia) were assessed by measuring responses to mechanical and thermal stimuli applied to the plantar surface of the foot. Serum, sciatic nerve and the related Dorsal Root Ganglia (DRG) levels of IL-10 and IL-1beta were determined by ELISA. The DRG mRNA levels of IL-10 and IL-1beta measured with PCR. A comparison between the low and high EIH rats of all measured parameters was made. RESULTS: The low EIH rats developed significantly more severe allodynia and hyperalgesia in the affected paw and allodynia in the contralateral paw compared to the high EIH rats, 7 days following the injury. The low EIH rats had higher IL-1beta protein levels in serum prior to and following injury, higher affected and contralateral sciatic nerve IL-1beta levels following injury and higher IL-1beta levels in the contralateral DRG (protein and mRNA) following injury when compared to high EIH rats. The high EIH rats had higher affected sciatic nerve IL-10 levels following nerve injury and higher IL-10 levels of both protein and mRNA in the affected and contralateral DRG at baseline and following injury. CONCLUSION: EIH profile was found to be predictive of pain behavior following nerve injury, low EIH rats developed more severe allodynia and hyperalgesia. IL-1beta may be associated with painful neuropathy developed in rats with low EIH while the anti-inflammatory cytokine IL-10 may have a protective role, inhibiting the development of painful.

Potassium Channels Kv1.3 and Kir2.1 But Not Kv1.5 Contribute to BV2 Cell Line and Primary Microglial Migration.

(1) Background: As membrane channels contribute to different cell functions, understanding the underlying mechanisms becomes extremely important. A large number of neuronal channels have been investigated, however, less studied are the channels expressed in the glia population, particularly in microglia. In the present study, we focused on the function of the Kv1.3, Kv1.5 and Kir2.1 potassium channels expressed in both BV2 cells and primary microglia cultures, which may impact the cellular migration process. (2) Methods: Using an immunocytochemical approach, we were able to show the presence of the investigated channels in BV2 microglial cells, record their currents using a patch clamp and their role in cell migration using the scratch assay. The migration of the primary microglial cells in culture was assessed using cell culture inserts. (3) Results: By blocking each potassium channel, we showed that Kv1.3 and Kir2.1 but not Kv1.5 are essential for BV2 cell migration. Further, primary microglial cultures were obtained from a line of transgenic CX3CR1-eGFP mice that express fluorescent labeled microglia. The mice were subjected to a spared nerve injury model of pain and we found that microglia motility in an 8 µm insert was reduced 2 days after spared nerve injury (SNI) compared with sham conditions. Additional investigations showed a further impact on cell motility by specifically blocking Kv1.3 and Kir2.1 but not Kv1.5; (4) Conclusions: Our study highlights the importance of the Kv1.3 and Kir2.1 but not Kv1.5 potassium channels on microglia migration both in BV2 and primary cell cultures.

The IL33 receptor ST2 contributes to mechanical hypersensitivity in mice with neuropathic pain.

Pathogen infection triggers pain via activation of the innate immune system. Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are the main components of innate immunity and have been implicated in pain signaling. We previously revealed that the TLR2-NLRP3-IL33 pathway mediates inflammatory pain responses during hyperactivity of innate immunity. However, their roles in neuropathic pain had remained unclear. Here we report that although knockout of TLR2 or NLRP3 does not affect spared nerve injury (SNI)-induced neuropathic pain, intrathecal inhibition of IL33/ST2 signaling with ST2 neutralizing antibodies reverses mechanical thresholds in SNI mice compared to PBS vehicle treated animals. This effect indicates a universal role of IL33 in both inflammatory and neuropathic pain states, and that targeting the IL33/ST2 axis could be a potential therapeutic approach for pain treatment.

Genomic Action of Sigma-1 Receptor Chaperone Relates to Neuropathic Pain.

Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) chaperones implicated in neuropathic pain. Here we examine if the Sig-1R may relate to neuropathic pain at the level of dorsal root ganglia (DRG). We focus on the neuronal excitability of DRG in a "spare nerve injury" (SNI) model of neuropathic pain in rats and find that Sig-1Rs likely contribute to the genesis of DRG neuronal excitability by decreasing the protein level of voltage-gated Cav2.2 as a translational inhibitor of mRNA. Specifically, during SNI, Sig-1Rs translocate from ER to the nuclear envelope via a trafficking protein Sec61ß. At the nucleus, the Sig-1R interacts with cFos and binds to the promoter of 4E-BP1, leading to an upregulation of 4E-BP1 that binds and prevents eIF4E from initiating the mRNA translation for Cav2.2. Interestingly, in Sig-1R knockout HEK cells, Cav2.2 is upregulated. In accordance with those findings, we find that intra-DRG injection of Sig-1R agonist (+)pentazocine increases frequency of action potentials via regulation of voltage-gated Ca2+ channels. Conversely, intra-DRG injection of Sig-1R antagonist BD1047 attenuates neuropathic pain. Hence, we discover that the Sig-1R chaperone causes neuropathic pain indirectly as a translational inhibitor.

Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation.

Leptin is an adipocytokine that is primarily secreted by white adipose tissue, and it contributes to the pathogenesis of neuropathic pain in collaboration with N-methyl-D-aspartate receptors (NMDARs). Functional NMDARs are a heteromeric complex that primarily comprise two NR1 subunits and two NR2 subunits. NR2A is preferentially located at synaptic sites, and NR2B is enriched at extrasynaptic sites. The roles of synaptic and extrasynaptic NMDARs in the contribution of leptin to neuropathic pain are not clear. The present study examined whether the important role of leptin in neuropathic pain was related to synaptic or extrasynaptic NMDARs. We used a rat model of spared nerve injury (SNI) and demonstrated that the intrathecal administration of the NR2A-selective antagonist NVP-AAM077 and the NR2B-selective antagonist Ro25-6981 prevented and reversed mechanical allodynia following SNI. Administration of exogenous leptin mimicked SNI-induced behavioral allodynia, which was also prevented by NVP-AAM077 and Ro25-6981. Mechanistic studies showed that leptin enhanced NR2B- but not NR2A-mediated currents in spinal lamina II neurons of naïve rats. Leptin also upregulated the expression of NR2B, which was blocked by the NR2B-selective antagonist Ro25-6981, in cultured dorsal root ganglion (DRG) neurons. Leptin enhanced neuronal nitric oxide synthase (nNOS) expression, which was also blocked by Ro25-6981, in cultured DRG cells. However, leptin did not change NR2A expression, and the NR2A-selective antagonist NVP-AAM077 had no effect on leptin-enhanced nNOS expression. Our data suggest an important cellular link between the spinal effects of leptin and the extrasynaptic NMDAR-nNOS-mediated cellular mechanism of neuropathic pain.

The influence of rat strain on the development of neuropathic pain and comorbid anxio-depressive behaviour after nerve injury.

Back-translating the clinical manifestations of human disease burden into animal models is increasingly recognized as an important facet of preclinical drug discovery. We hypothesized that inbred rat strains possessing stress hyper-reactive-, depressive- or anxiety-like phenotypes may possess more translational value than common outbred strains for modeling neuropathic pain. Rats (inbred: LEW, WKY, F344/ICO and F344/DU, outbred: Crl:SD) were exposed to Spared Nerve Injury (SNI) and evaluated routinely for 6 months on behaviours related to pain (von Frey stimulation and CatWalk-gait analysis), anxiety (elevated plus maze, EPM) and depression (sucrose preference test, SPT). Markers of stress reactivity together with spinal/brain opioid receptor expression were also measured. All strains variously developed mechanical allodynia after SNI with the exception of stress-hyporesponsive LEW rats, despite all strains displaying similar functional gait-deficits after injury. However, affective changes reflective of anxiety- and depressive-like behaviour were only observed for F344/DU in the EPM, and for Crl:SD in SPT. Although differences in stress reactivity and opioid receptor expression occurred, overall they were relatively unaffected by SNI. Thus, anxio-depressive behaviours did not develop in all strains after nerve injury, and correlated only modestly with degree of pain sensitivity or with genetic predisposition to stress and/or affective disturbances.

Orthodontic force induces nerve injury-like transcriptomic changes driven by TRPV1-expressing afferents in mouse trigeminal ganglia.

Orthodontic force produces mechanical irritation and localized inflammation in the periodontium, which causes pain in most patients. Nocifensive behaviors resulting from orthodontic force in mice can be substantially attenuated by intraganglionic injection of resiniferatoxin (RTX), a neurotoxin that specifically ablates a subset of neurons expressing transient receptor potential vanilloid 1 (TRPV1). In the current study, we determined changes in the transcriptomic profiles in the trigeminal ganglia (TG) following the application of orthodontic force, and assessed the roles of TRPV1-expressing afferents in these transcriptomic changes. RTX or vehicle was injected into the TG of mice a week before the placement of an orthodontic spring exerting 10 g of force. After 2 days, the TG were collected for RNA sequencing. The application of orthodontic force resulted in 1279 differentially expressed genes (DEGs) in the TG. Gene ontology analysis showed downregulation of gliogenesis and ion channel activities, especially of voltage-gated potassium channels. DEGs produced by orthodontic force correlated more strongly with DEGs resulting from nerve injury than from inflammation. Orthodontic force resulted in the differential expression of multiple genes involved in pain regulation, including upregulation of Atf3, Adcyap1, Bdnf, and Csf1, and downregulation of Scn10a, Kcna2, Kcnj10, and P2ry1. Orthodontic force-induced DEGs correlated with DEGs specific to multiple neuronal and non-neuronal subtypes following nerve injury. These transcriptomic changes were abolished in the mice that received the RTX injection. These results suggest that orthodontic force produces transcriptomic changes resembling nerve injury in the TG and that nociceptive inputs through TRPV1-expressing afferents leads to subsequent changes in gene expression not only in TRPV1-positive neurons, but also in TRPV1-negative neurons and non-neuronal cells throughout the ganglia. Orthodontic force-induced transcriptomic changes might be an active regenerative program of trigeminal ganglia in response to axonal injury following orthodontic force.

Tonic GABAergic Inhibition Is Essential for Nerve Injury-Induced Afferent Remodeling in the Somatosensory Thalamus and Ectopic Sensations.

Peripheral nerve injury induces functional and structural remodeling of neural circuits along the somatosensory pathways, forming the basis for somatotopic reorganization and ectopic sensations, such as referred phantom pain. However, the mechanisms underlying that remodeling remain largely unknown. Whisker sensory nerve injury drives functional remodeling in the somatosensory thalamus: the number of afferent inputs to each thalamic neuron increases from one to many. Here, we report that extrasynaptic γ-aminobutyric acid-type A receptor (GABAAR)-mediated tonic inhibition is necessary for that remodeling. Extrasynaptic GABAAR currents were potentiated rapidly after nerve injury in advance of remodeling. Pharmacological activation of the thalamic extrasynaptic GABAARs in intact mice induced similar remodeling. Notably, conditional deletion of extrasynaptic GABAARs in the thalamus rescued both the injury-induced remodeling and the ectopic mechanical hypersensitivity. Together, our results reveal a molecular basis for injury-induced remodeling of neural circuits and may provide a new pharmacological target for referred phantom sensations after peripheral nerve injury.

Dopamine Inputs from the Ventral Tegmental Area into the Medial Prefrontal Cortex Modulate Neuropathic Pain-Associated Behaviors in Mice.

The medial prefrontal cortex (mPFC) is a brain region involved in the affective components of pain and undergoes plasticity during the development of chronic pain. Dopamine (DA) is a key neuromodulator in the mesocortical circuit and modulates working memory and aversion. Although DA inputs into the mPFC are known to modulate plasticity, whether and how these inputs affect pain remains incompletely understood. By using optogenetics, we find that phasic activation of DA inputs from the ventral tegmental area (VTA) into the mPFC reduce mechanical hypersensitivity during neuropathic pain states. Mice with neuropathic pain exhibit a preference for contexts paired with photostimulation of DA terminals in the mPFC. Fiber photometry-based calcium imaging reveals that DA increases the activity of mPFC neurons projecting to the ventrolateral periaqueductal gray (vlPAG). Together, our findings indicate an important role of mPFC DA signaling in pain modulation.

Effect of Paroxetine on the Neuropathic Pain: A Molecular Study.

Background: Neuropathic pain, due to peripheral nerve damage, has influenced millions of people living all over the world. It has been shown that paroxetine can relieve neuropathic pain. Recently, the role of certain proteins like brain-derived neurotrophic factor (BDNF), GABAA receptor, and K+-Cl- cotransporter 2 (KCC2) transporter in the occurrence of neuropathic pain has been documented. In the current study, the expression of these proteins affected by paroxetine was evaluated. Methods: Male Wistar rats were allocated into two main groups of pre- and post-injury. Rats in each main group received paroxetine before nerve injury and at day seven after nerve damage till day 14, respectively. The lumbar spinal cord of animals was extracted to assess the expression of target genes and proteins. Results: In the preventive study, paroxetine decreased BDNF and increased KCC2 and GABAA gene and protein expression, while in the post-injury paradigm, it decreased BDNF and increased KCC2 genes and protein expression. In this regard, an increase in the protein expression of GABAA was observed. Conclusion: It seems that paroxetine with a change in the expression of three significant proteins involved in neuropathic pain could attenuate this type of chronic pain.

Remote ischemic postconditioning attenuates damage in rats with chronic cerebral ischemia by upregulating the autophagolysosome pathway via the activation of TFEB.

The transcription factor EB (TFEB) is known for its role in lysosomal biogenesis, and it coordinates this process by driving autophagy and lysosomal gene expression during ischemia. In the present study, we aimed to explore the role of the TFEB-regulated autophagolysosome pathway (ALP) in rats with chronic cerebral ischemia (CCI) that were treated with remote ischemic postconditioning (RIPC). A modified 2-vessel occlusion (2-VO) method was utilized to establish the CCI rat model, and the CCI rats were identified by the Morris water maze test and histological staining. After the CCI rats were treated with RIPC, the damage to the rat cortex and hippocampal tissues and the status of the ALP were determined. Western blot analysis and immunofluorescence assays were performed to observe the nuclear translocation of TFEB. The rats were injected with TFEB siRNA via the lateral ventricle to investigate the effect of TFEB siRNA on the RIPC-treated CCI rats. The results suggested that RIPC of the CCI rats alleviated nerve injury, induced TFEB translocation into the nucleus, upregulated autophagy-related protein expression, and activated ALP machinery. Furthermore, TFEB siRNA decreased the levels of TFEB and impaired the neuroprotective effects of RIPC on the CCI rats. Collectively, we highlighted that RIPC attenuates damage in CCI rats via the activation of the TFEB-mediated ALP.

Reference interval and preanalytical properties of serum neurofilament light chain in Scandinavian adults.

The neurofilament light chain (NfL) is a promising biomarker of neuronal injury which is approaching routine clinical use. With the development of ultra-sensitive technologies, NfL has become measurable in the peripheral blood but the reference interval for serum NfL remains to be established. NfL was measured by a single-molecule array (Simoa™) analysis under internal and external quality control which is established for routine clinical use. Serum samples from 342 reference subjects, 18-87 years were analyzed. The age-partitioned reference interval was established according to Clinical and Laboratory Standards Institute guidelines, an approximation of the upper reference interval limit per 10-year age-groups was performed, and key pre-analytical properties were examined. Serum NfL levels increased 2.9% per year. The non-parametric reference interval for the age groups 18-40, 41-65, and >65 years were 2.8-9.7 ng/L, 4.6 - 21.4 ng/L, and 7.5-53.8 ng/L, respectively. The estimated upper reference interval limits per 10-year intervals corresponded well with the 90% confidence limits of the non-parametric reference interval. The recovery of serum NfL after seven days at room temperature or three freeze-thaw cycles were 93% (95% CI: 89%-97%) and 92% (95% CI: 83%-102%) and levels in serum were only slightly higher than levels in plasma (p < .0001). The study establishes the serum NfL reference interval, provide estimated upper reference intevral limits in 10-year intervals to increase the clinical applicability and uncover pre-analytical properties that make serum NfL feasible for clinical use.

Histone methyltransferase G9a diminishes expression of cannabinoid CB1 receptors in primary sensory neurons in neuropathic pain.

Type 1 cannabinoid receptors (CB1Rs) are expressed in the dorsal root ganglion (DRG) and contribute to the analgesic effect of cannabinoids. However, the epigenetic mechanism regulating the expression of CB1Rs in neuropathic pain is unknown. G9a (encoded by the Ehmt2 gene), a histone 3 at lysine 9 methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined G9a's role in regulating CB1R expression in the DRG and in CB1R-mediated analgesic effects in an animal model of neuropathic pain. We show that nerve injury profoundly reduced mRNA levels of CB1Rs but increased the expression of CB2 receptors in the rat DRG. ChIP results indicated increased enrichment of histone 3 at lysine 9 dimethylation, a G9a-catalyzed repressive histone mark, at the promoter regions of the CB1R genes. G9a inhibition in nerve-injured rats not only up-regulated the CB1R expression level in the DRG but also potentiated the analgesic effect of a CB1R agonist on nerve injury-induced pain hypersensitivity. Furthermore, in mice lacking Ehmt2 in DRG neurons, nerve injury failed to reduce CB1R expression in the DRG and to decrease the analgesic effect of the CB1R agonist. Moreover, nerve injury diminished the inhibitory effect of the CB1R agonist on synaptic glutamate release from primary afferent nerves to spinal cord dorsal horn neurons in WT mice but not in mice lacking Ehmt2 in DRG neurons. Our findings reveal that nerve injury diminishes the analgesic effect of CB1R agonists through G9a-mediated CB1R down-regulation in primary sensory neurons.

Analgesic effects of optogenetic inhibition of basolateral amygdala inputs into the prefrontal cortex in nerve injured female mice.

Peripheral nerve injury can lead to remodeling of brain circuits, and this can cause chronification of pain. We have recently reported that male mice subjected to spared injury of the sciatic nerve undergo changes in the function of the medial prefrontal cortex (mPFC) that culminate in reduced output of layer 5 pyramidal cells. More recently, we have shown that this is mediated by alterations in synaptic inputs from the basolateral amygdala (BLA) into GABAergic interneurons in the mPFC. Optogenetic inhibition of these inputs reversed mechanical allodynia and thermal hyperalgesia in male mice. It is known that the processing of pain signals can exhibit marked sex differences. We therefore tested whether the dysregulation of BLA to mPFC signaling is equally altered in female mice. Injection of AAV-Arch3.0 constructs into the BLA followed by implantation of a fiberoptic cannula into the mPFC in sham and SNI operated female mice was carried out, and pain behavioral responses were measured in response to yellow light mediated activation of this inhibitory opsin. Our data reveal that Arch3.0 activation leads to a marked increase in paw withdrawal thresholds and latencies in response to mechanical and thermal stimuli, respectively. However, we did not observe nerve injury-induced changes in mPFC layer 5 pyramidal cell output in female mice. Hence, the observed light-induced analgesic effects may be due to compensation for dysregulated neuronal circuits downstream of the mPFC.

Dual and Opposing Functions of the Central Amygdala in the Modulation of Pain.

Pain perception is essential for survival and can be amplified or suppressed by expectations, experiences, and context. The neural mechanisms underlying bidirectional modulation of pain remain largely unknown. Here, we demonstrate that the central nucleus of the amygdala (CeA) functions as a pain rheostat, decreasing or increasing pain-related behaviors in mice. This dual and opposing function of the CeA is encoded by opposing changes in the excitability of two distinct subpopulations of GABAergic neurons that receive excitatory inputs from the parabrachial nucleus (PB). Thus, cells expressing protein kinase C-delta (CeA-PKCδ) are sensitized by nerve injury and increase pain-related responses. In contrast, cells expressing somatostatin (CeA-Som) are inhibited by nerve injury and their activity drives antinociception. Together, these results demonstrate that the CeA can amplify or suppress pain in a cell-type-specific manner, uncovering a previously unknown mechanism underlying bidirectional control of pain in the brain.

From injury to full repair: nerve regeneration and functional recovery in the common octopus, Octopus vulgaris.

Spontaneous nerve regeneration in cephalopod molluscs occurs in a relative short time after injury, achieving functional recovery of lost capacity. In particular, transection of the pallial nerve in the common octopus (Octopus vulgaris) determines the loss and subsequent restoration of two functions fundamental for survival, i.e. breathing and skin patterning, the latter involved in communication between animals and concealment. The phenomena occurring after lesion have been investigated in a series of previous studies, but a complete analysis of the changes taking place at the level of the axons and the effects on the animals' appearance during the whole regenerative process is still missing. Our goal was to determine the course of events following injury, from impairment to full recovery. Through imaging of the traced damaged nerves, we were able to characterize the pathways followed by fibres during regeneration and end-target re-innervation, while electrophysiology and behavioural observations highlighted the regaining of functional connections between the central brain and periphery, using the contralateral nerve in the same animal as an internal control. The final architecture of a fully regenerated pallial nerve does not exactly mirror the original structure; however, functionality returns to match the phenotype of an intact octopus with no observable impact on the behaviour of the animal. Our findings provide new important scenarios for the study of regeneration in cephalopods and highlight the octopus pallial nerve as a valuable 'model' among invertebrates.

Lysozyme elicits pain during nerve injury by neuronal Toll-like receptor 4 activation and has therapeutic potential in neuropathic pain.

The role of neuronal Toll-like receptor 4 (TLR4) in nerve injury is being pursued actively. However, the endogenous activation of neuronal TLR4 during neuroinflammation, in absence of the participation of glial TLR4, remains elusive. Here, we identified lysozyme as an endogenous activator of neuronal TLR4 signaling during nerve injury. Upon nerve injury, enhanced expression of lysozyme promoted neuronal hyperexcitability and neuropathic pain. Injections of lysozyme in healthy rats increased their mechanical and thermal pain sensitivity. Likewise, infusion of spinal cord slices with lysozyme increased neuronal excitability typical of neuropathic pain. Our results also showed that lysozyme activated excitability of both Aδ- and C-fibers. Thus, in addition to the discovery of lysozyme as an endogenous ligand for regulating neuronal TLR4 signaling, this study also lays the foundation of our understanding of its role in nervous system pathologies, providing multiple avenues for treating neuroinflammation.

Impaired AMPK­CGRP signaling in the central nervous system contributes to enhanced neuropathic pain in high­fat diet­induced obese rats, with or without nerve injury.

Obesity is associated with increased sensitivity to pain, including neuropathic pain, but the precise mechanisms are not fully understood. Recent evidence has revealed that AMP­activated protein kinase (AMPK) in the central nervous system (CNS) regulates the neuropeptide calcitonin gene­related peptide (CGRP), a principal neurotransmitter of the class C nerve fiber, which serves an important role in initiating and maintaining neuropathic pain. AMPK has been demonstrated to be downregulated in the CNS in obesity. The present study hypothesized that obesity may lead to increased sensitivity to neuropathic pain by downregulating AMPK and upregulating CGRP expression levels in the CNS. Sprague­Dawley rats consuming a high­fat diet (HF) for 12 weeks developed obesity; they exhibited significantly decreased levels of phospho (p)­AMPK and increased CGRP expression levels in the spinal cord (SC) and dorsal root ganglion (DRG), respectively, compared with rats consuming a low­fat (LF) diet. HF­fed rats that underwent spared nerve injury (SNI) also exhibited lower p­AMPK and higher CGRP expression levels in the SC and DRG, compared with the corresponding LF­diet rats. The 50% paw withdrawal threshold (PWT; as measured by Von Frey testing) was significantly lower in HF­fed compared with LF­fed rats, with or without SNI. Through intrathecal treatment, the AMPK activator 5­aminoimidazole­4­carboxamide riboside (AICAR) or the CGRP antagonist CGRP8­37 decreased CGRP expression levels and increased the 50% PWT; however, the AMPK inhibitor dorsomorphin augmented CGRP expression levels and further reduced the 50% PWT in HF­fed rats, but not LF­fed rats, with or without SNI. The results indicated that blocking the AMPK­CGRP pathway may enhance neuropathic pain in HF­induced obesity, with or without nerve injury. Targeting AMPK in the CNS may be a novel strategy for the prevention and treatment of obesity­associated neuropathic pain.