Argonaute 2 restored erectile function and corpus cavernosum mitochondrial function by reducing apoptosis in a mouse model of cavernous nerve injury.

PURPOSE: To determine whether the overexpression of the Argonaute RNA-induced silencing complex catalytic component 2 (Ago2) improves erectile function in mice after cavernous nerve injury (CNI). MATERIALS AND METHODS: Lentiviruses containing Ago2 open reading frame (ORF) mouse clone (Ago2 O/E) were used to overexpress Ago2, and lentiviruses ORF negative control particles (NC) were used as a negative control. Three days before preparing the CNI model, we injected lentiviruses into the penises of 8-week-old male C57BL/6 mice. Animals were then divided into four groups: the sham operation control group and the CNI+phosphate-buffered saline, CNI+NC, and CNI+Ago2 O/E groups. One week later, erectile function was assessed by electrically stimulating cavernous nerves bilaterally and obtaining intracavernous pressure parameters. Penile tissue was also collected for molecular mechanism studies. RESULTS: Ago2 overexpression improved erectile function in mice after CNI-induced erectile dysfunction (ED). Immunofluorescence staining and Western blot analysis showed that under Ago2 overexpressing conditions, the contents of endothelial cells, pericytes, and neuronal cells increased in the penile tissues of CNI mice, and this was attributed to reduced apoptosis and ROS production. In addition, we also found that Ago2 overexpression could restore penile mitochondrial function, thereby improving erectile function in CNI-induced ED mice. CONCLUSIONS: Our findings demonstrate that Ago2 overexpression can reduce penile cell apoptosis, restore penile mitochondrial function, and improve erectile function in CNI-induced ED mice.

CBP Expression Contributes to Neuropathic Pain via CREB and MeCP2 Regulation in the Spared Nerve Injury Rat Model.

Background and Objectives: This study aimed to investigate the relationship between neuropathic pain and CREB-binding protein (CBP) and methyl-CpG-binding protein 2 (MeCP2) expression levels in a rat model with spared nerve injury (SNI). Materials and Methods: Rat (male Sprague-Dawley white rats) models with surgical SNI (n = 6) were prepared, and naive rats (n = 5) were used as controls. The expression levels of CBP and MeCP2 in the spinal cord and dorsal root ganglion (DRG) were compared through immunohistochemistry at 7 and 14 days after surgery. The relationship between neuropathic pain and CBP/MeCP2 was also analyzed through intrathecal siRNA administration. Results: SNI induced a significant increase in the number of CBPs in L4 compared with contralateral DRG as well as with naive rats. The number of MeCP2 cells in the dorsal horn on the ipsilateral side decreased significantly compared with the contralateral dorsal horn and the control group. SNI induced a significant decrease in the number of MeCP2 neurons in the L4 ipsilateral DRG compared with the contralateral DRG and naive rats. The intrathecal injection of CBP siRNA significantly inhibited mechanical allodynia induced by SNI compared with non-targeting siRNA treatment. MeCP2 siRNA injection showed no significant effect on mechanical allodynia. Conclusions: The results suggest that CBP and MeCP2 may play an important role in the generation of neuropathic pain following peripheral nerve injury.

Minocycline Abrogates Individual Differences in Nerve Injury-Evoked Affective Disturbances in Male Rats and Prevents Associated Supraspinal Neuroinflammation.

Chronic neuropathic pain precipitates a complex range of affective and behavioural disturbances that differ markedly between individuals. While the reasons for differences in pain-related disability are not well understood, supraspinal neuroimmune interactions are implicated. Minocycline has antidepressant effects in humans and attenuates affective disturbances in rodent models of pain, and acts by reducing neuroinflammation in both the spinal cord and brain. Previous studies, however, tend not to investigate how minocycline modulates individual affective responses to nerve injury, or rely on non-naturalistic behavioural paradigms that fail to capture the complexity of rodent behaviour. We investigated the development and resolution of pain-related affective disturbances in nerve-injured male rats by measuring multiple spontaneous ethological endpoints on a longitudinal naturalistic foraging paradigm, and the effect of chronic oral minocycline administration on these changes. Disrupted foraging behaviours appeared in 22% of nerve-injured rats - termed 'affected' rats - and were present at day 14 but partially resolved by day 21 post-injury. Minocycline completely prevented the emergence of an affected subgroup while only partly attenuating mechanical allodynia, dissociating the relationship between pain and affect. This was associated with a lasting downregulation of ΔFosB expression in ventral hippocampal neurons at day 21 post-injury. Markers of microglia-mediated neuroinflammation were not present by day 21, however proinflammatory microglial polarisation was apparent in the medial prefrontal cortex of affected rats and not in CCI minocycline rats. Individual differences in affective disturbances following nerve injury are therefore temporally related to altered microglial morphology and hippocampal neuronal activation, and are abrogated by minocycline.

Neuropathic pain and mood disorders in earthquake survivors with peripheral nerve injuries.

Background and purpose:

        Natural disasters, such as earthquakes, frequently result in mood disorders among affected individuals. It is established that neuropathic pain arising from traumatic neuropathies is also linked to mood disorders. This study investigates the influence of neuropathic pain on the development of mood disorders in earthquake survivors with peripheral nerve injuries, following the earthquake centered in Kahramanmaras on February 6, 2023. Additionally, we aim to assess the electro­physiological aspects of neuropathic injuries in these survivors.

The study comprised 46 earth-quake survivors with electrophysiologically confirmed peripheral nerve injuries, with 39 trauma-free survivors serving as the control group. Neuropathic pain, anxiety and depression were assessed using the Douleur Neuropathique 4 (DN4) questionnaire and the Hospital Anxiety and Depression Scale (HADS).

Our findings revealed that the ulnar and peroneal nerves were the most commonly injured structures. Among the survivors with peripheral nerve injury, 31 out of 46 (67%) were found to experience neuropathic pain. Furthermore, plexopathy and multiple extremity injuries were associated with more severe neuropathic pain. However, there was no significant difference in anxiety and depression scores between the two groups and neuropathic pain was found to have no independent effect.

The study indicates that the intensity of neuropathic pain varies based on the localization and distribution of peripheral nerve injuries. However, the presence of peripheral nerve damage or neuropathic pain was not directly associated with HADS scores, suggesting that mood disorders following disasters may have multifactorial causes beyond physical trauma.

Sexually dimorphic effects of pexidartinib on nerve injury-induced neuropathic pain in mice.

It is well-established that spinal microglia and peripheral macrophages play critical roles in the etiology of neuropathic pain; however, growing evidence suggests sex differences in pain hypersensitivity owing to microglia and macrophages. Therefore, it is crucial to understand sex- and androgen-dependent characteristics of pain-related myeloid cells in mice with nerve injury-induced neuropathic pain. To deplete microglia and macrophages, pexidartinib (PLX3397), an inhibitor of the colony-stimulating factor 1 receptor, was orally administered, and mice were subjected to partial sciatic nerve ligation (PSL). Following PSL induction, healthy male and female mice and male gonadectomized (GDX) mice exhibited similar levels of spinal microglial activation, peripheral macrophage accumulation, and mechanical allodynia. Treatment with PLX3397 significantly suppressed mechanical allodynia in normal males; this was not observed in female and GDX male mice. Sex- and androgen-dependent differences in the PLX3397-mediated preventive effects were observed on spinal microglia and dorsal root ganglia (DRG) macrophages, as well as in expression patterns of pain-related inflammatory mediators in these cells. Conversely, no sex- or androgen-dependent differences were detected in sciatic nerve macrophages, and inhibition of peripheral CC-chemokine receptor 5 prevented neuropathic pain in both sexes. Collectively, these findings demonstrate the presence of considerable sex- and androgen-dependent differences in the etiology of neuropathic pain in spinal microglia and DRG macrophages but not in sciatic nerve macrophages. Given that the mechanisms of neuropathic pain may differ among experimental models and clinical conditions, accumulating several lines of evidence is crucial to comprehensively clarifying the sex-dependent regulatory mechanisms of pain.

Contribution of pudendal nerve injury to stress urinary incontinence in a male rat model.

Urinary incontinence is a common complication following radical prostatectomy, as the surgery disturbs critical anatomical structures. This study explored how pudendal nerve (PN) injury affects urinary continence in male rats. In an acute study, leak point pressure (LPP) and external urethral sphincter electromyography (EMG) were performed on six male rats with an intact urethra, the urethra exposed (UE), the PN exposed (NE), and after PN transection (PNT). In a chronic study, LPP and EMG were tested in 67 rats 4 days, 3 weeks, or 6 weeks after sham PN injury, PN crush (PNC), or PNT. Urethras were assessed histologically. Acute PNT caused a significant decrease in LPP and EMG amplitude and firing rate compared to other groups. PNC resulted in a significant reduction in LPP and EMG firing rate 4 days, 3 weeks, and 6 weeks later. EMG amplitude was also significantly reduced 4 days and 6 weeks after PNC. Neuromuscular junctions were less organized and less innervated after PNC or PNT at all timepoints compared to sham injured animals. Collagen infiltration was significantly increased after PNC and PNT compared to sham at all timepoints. This rat model could facilitate preclinical testing of neuroregenerative therapies for post-prostatectomy incontinence.

Rbfox1 regulates alternative splicing of Nrcam in primary sensory neurons to mediate peripheral nerve injury-induced neuropathic pain.

The primary sensory neurons of the dorsal root ganglia (DRG) are subject to transcriptional alterations following peripheral nerve injury. These alterations are believed to play a pivotal role in the genesis of neuropathic pain. Alternative RNA splicing is a process that generates multiple transcript variants from a single gene, significantly contributing to the complexity of the transcriptome. However, little is known about the functional significance and control of alternative RNA splicing in injured DRG after spinal nerve ligation (SNL). In our study, we conducted a comprehensive transcriptome profiling and bioinformatic analysis to approach and identified a neuron-specific isoform of an RNA splicing regulator, RNA-binding Fox1 (Rbfox1, also known as A2BP1), as a crucial regulator of alternative RNA splicing in injured DRG after SNL. Notably, Rbfox1 expression is markedly reduced in injured DRG following peripheral nerve injury. Restoring this reduction effectively mitigates nociceptive hypersensitivity. Conversely, mimicking the downregulation of Rbfox1 expression generates neuropathic pain symptoms. Mechanistically, we uncovered that Rbfox1 may be a key factor influencing alternative RNA splicing of neuron-glial related cell adhesion molecule (NrCAM), a key neuronal cell adhesion molecule. In injured DRG after SNL, the downregulation of Rbfox1amplifies the insertion of exon 10 in Nrcam transcripts, leading to an increase in long Nrcam variants (L-Nrcam) and a corresponding decrease in short Nrcam variants (S-Nrcam) within injured DRG. In summary, our study supports the essential role of Rbfox1 in neuropathic pain within DRG, probably via the regulation of Nrcam splicing. These findings suggest that Rbfox1 could be a potential target for neuropathic pain therapy.

Modulatory role of neurosteroidogenesis in the spinal cord during peripheral nerve injury-induced chronic pain.

The brain and spinal cord (SC) are both targeted by various hormones, including steroid hormones. However, investigations of the modulatory role of hormones on neurobiological functions usually focus only on the brain. The SC received little attention although this structure pivotally controls motor and sensory functions. Here, we critically reviewed key data showing that the process of neurosteroid biosynthesis or neurosteroidogenesis occurring in the SC plays a pivotal role in the modulation of peripheral nerve injury-induced chronic pain (PNICP) or neuropathic pain. Indeed, several active steroidogenic enzymes expressed in the SC produce endogenous neurosteroids that interact with receptors of neurotransmitters controlling pain. The spinal neurosteroidogenesis is differentially regulated during PNICP condition and its blockade modifies painful sensations. The paper suggests that future investigations aiming to develop effective strategies against PNICP or neuropathic pain must integrate in a gender or sex dependent manner the regulatory effects exerted by spinal neurosteroidogenesis.

Microglial STING activation alleviates nerve injury-induced neuropathic pain in male but not female mice.

Microglia, resident immune cells in the central nervous system, play a role in neuroinflammation and the development of neuropathic pain. We found that the stimulator of interferon genes (STING) is predominantly expressed in spinal microglia and upregulated after peripheral nerve injury. However, mechanical allodynia, as a marker of neuropathic pain following peripheral nerve injury, did not require microglial STING expression. In contrast, STING activation by specific agonists (ADU-S100, 35 nmol) significantly alleviated neuropathic pain in male mice, but not female mice. STING activation in female mice leads to increase in proinflammatory cytokines that may counteract the analgesic effect of ADU-S100. Microglial STING expression and type I interferon-ß (IFN-ß) signaling were required for the analgesic effects of STING agonists in male mice. Mechanistically, downstream activation of TANK-binding kinase 1 (TBK1) and the production of IFN-ß, may partly account for the analgesic effect observed. These findings suggest that STING activation in spinal microglia could be a potential therapeutic intervention for neuropathic pain, particularly in males.

Deactivation of dorsal CA1 pyramidal neurons projecting to medial prefrontal cortex contributes to neuropathic pain and short-term memory impairment.

ABSTRACT: Neuropathic pain after peripheral nerve injury is a multidimensional experience that includes sensory, affective, and cognitive components that interact with one another. Hypoexcitation of the medial prefrontal cortex (mPFC) was observed in mice with peripheral nerve injury, but the changes in neural inputs onto the mPFC have not been completely explored. Here, we report that the neural terminals from the dorsal hippocampus CA1 (dCA1) form excitatory connection with layer 5 pyramidal neurons in the prelimbic area (PrL) of the mPFC. Spared nerve injury (SNI) induced a reduction in the intrinsic excitability of dCA1 pyramidal neurons innervating the PrL and impairment in excitatory synaptic transmission onto dCA1 pyramidal cells. Specifically, activating the neural circuit from dCA1 to mPFC alleviated neuropathic pain behaviors and improved novel object recognition ability in SNI mice, whereas deactivating this pathway in naïve animals recapitulated tactile allodynia and memory deficits. These results indicated that hypoactivity in dCA1 pyramidal cells after SNI in turn deactivated layer 5 pyramidal neurons in PrL and ultimately caused pain hypersensitivity and memory deficits.

Voluntary wheel running prevents formation of membrane attack complexes and myelin degradation after peripheral nerve injury.

Regular aerobic activity is associated with a reduced risk of chronic pain in humans and rodents. Our previous studies in rodents have shown that prior voluntary wheel running can normalize redox signaling at the site of peripheral nerve injury, attenuating subsequent neuropathic pain. However, the full extent of neuroprotection offered by voluntary wheel running after peripheral nerve injury is unknown. Here, we show that six weeks of voluntary wheel running prior to chronic constriction injury (CCI) reduced the terminal complement membrane attack complex (MAC) at the sciatic nerve injury site. This was associated with increased expression of the MAC inhibitor CD59. The levels of upstream complement components (C3) and their inhibitors (CD55, CR1 and CFH) were altered by CCI, but not increased by voluntary wheel running. Since MAC can degrade myelin, which in turn contributes to neuropathic pain, we evaluated myelin integrity at the sciatic nerve injury site. We found that the loss of myelinated fibers and decreased myelin protein which occurs in sedentary rats following CCI was not observed in rats with prior running. Substitution of prior voluntary wheel running with exogenous CD59 also attenuated mechanical allodynia and reduced MAC deposition at the nerve injury site, pointing to CD59 as a critical effector of the neuroprotective and antinociceptive actions of prior voluntary wheel running. This study links attenuation of neuropathic pain by prior voluntary wheel running with inhibition of MAC and preservation of myelin integrity at the sciatic nerve injury site.

[Diagnostics and surgical treatment of painful neuromas]. / Diagnostik und chirurgische Therapie schmerzhafter Neurome.

BACKGROUND: Painful neuromas that often develop after peripheral nerve injury require adequate diagnosis and treatment because of the suffering they cause. The scientific basis for the development of painful neuromas has not yet been sufficiently investigated. In addition to conservative procedures, a larger number of surgical techniques are available for treatment of painful neuromas. OBJECTIVE: A review of the basic principles, diagnostic and treatment options for painful neuromas. MATERIAL AND METHODS: Presentation of the scientific basis regarding the development of painful neuromas. Illustration and discussion of the most common diagnostic and treatment procedures. RESULTS: The scientific basis regarding the development of painful neuromas after peripheral nerve injury has not yet been adequately developed. In order to be able to make a correct diagnosis, the use of standardized diagnostic criteria and adequate imaging techniques are recommended. In the sense of a paradigm shift, the use of the formerly neuroma-bearing nerve for reinnervation of target organs is to be preferred over mere burying in adjacent tissue. CONCLUSION: In addition to standardized diagnostics the management of painful neuromas often requires a surgical intervention after all conservative therapeutic measures have been exhausted. As an alternative to restoring the continuity of the injured nerve, targeted reinnervation of electively denervated target organs by the formerly neuroma-bearing nerve is preferable over other techniques.

A porcine model of postoperative hemi-diaphragmatic paresis to evaluate a unilateral diaphragmatic pacemaker.

Unilateral phrenic nerve damage is a dreaded complication in congenital heart surgery. It has deleterious effects in neonates and children with uni-ventricular circulation. Diaphragmatic palsy, caused by phrenic nerve damage, impairs respiratory function, especially in new-borns, because their respiration depends on diaphragmatic contractions. Furthermore, Fontan patients with passive pulmonary perfusion are seriously affected by phrenic nerve injury, because diaphragmatic contraction augments pulmonary blood flow. Diaphragmatic plication is currently employed to ameliorate the negative effects of diaphragmatic palsy on pulmonary perfusion and respiratory mechanics. This procedure attenuates pulmonary compression by the abdominal contents. However, there is no contraction of the plicated diaphragm and consequently no contribution to the pulmonary blood flow. Hence, we developed a porcine model of unilateral diaphragmatic palsy in order to evaluate a diaphragmatic pacemaker. Our illustrated step-by-step description of the model generation enables others to replicate and use our model for future studies. Thereby, it might contribute to investigation and advancement of potential improvements for these patients.

Managing Complications Related to Peripheral Nerve Surgery: Selected Illustrative Cases.

Peripheral nerve surgery mostly involves elective procedures; thus, the associated complications are of great clinical, social, and medicolegal importance. Apart from the general perioperative morbidity, complications during interventions on peripheral nerves are extremely rare. However, iatrogenic peripheral nerve injuries during unrelated surgical procedures performed by those not specialised in peripheral nerve surgery remain the most significant group of complications, accounting for up to approximately 17% of all cases. The aims of this review are to provide better insight into the multifaceted nature of complications related to peripheral nerve surgery-from the perspective of their causes, treatment, and outcome-and to raise surgeons' awareness of the risks of such morbidity. It should be emphasized that intraoperative complications in peripheral nerve surgery are largely "surgeon-related" rather than "surgery-related"; therefore, they have great potential to be avoided.

Astrocyte senescence-like response related to peripheral nerve injury-induced neuropathic pain.

BACKGROUND: Peripheral nerve damage causes neuroinflammation, which plays a critical role in establishing and maintaining neuropathic pain (NeP). The mechanisms contributing to neuroinflammation remain poorly elucidated, and pharmacological strategies for NeP are limited. Thus, in this study, we planned to explore the possible link between astrocyte senescence and NeP disorders following chronic sciatic nerve injury. METHODS: An NeP animal model was established by inducing chronic constrictive injury (CCI) to the sciatic nerve in adult rats. A senolytic drug combination of dasatinib and quercetin was gavaged daily from the first postoperative day until the end of the study. Paw mechanical withdrawal threshold (PMWT) and paw thermal withdrawal latency (PTWL) were evaluated to assess behaviors in response to pain in the experimental rats. Senescence-associated ß-galactosidase staining, western blot analysis, and immunofluorescence were applied to examine the levels of proinflammatory factors and severity of the senescence-like response in the spinal cord. Lipopolysaccharide (LPS) was administered to induce senescence of spinal astrocytes in primary cultures in vitro, to explore the potential impacts of senescence on the secretion of proinflammatory factors. Furthermore, single-cell RNA sequencing (scRNA-seq) was conducted to identify senescence-related molecular responses in spinal astrocytes under neuropathic pain. RESULTS: Following sciatic nerve CCI, rats exhibited reduced PMWT and PTWL, increased levels of spinal proinflammatory factors, and an enhanced degree of senescence in spinal astrocytes. Treatment with dasatinib and quercetin effectively attenuated spinal neuroinflammation and mitigated the hypersensitivities of the rats subjected to sciatic nerve CCI. Mechanistically, the dasatinib-quercetin combination reversed senescence in LPS-stimulated primary cultured astrocytes and decreased the levels of proinflammatory factors. The scRNA-seq data revealed four potential senescence-related genes in the spinal astrocyte population, and the expression of clusterin (CLU) protein was validated via in vitro experiments. CONCLUSION: The findings indicate the potential role of astrocyte senescence in neuroinflammation following peripheral nerve injury, and suggest that targeting CLU activation in astrocytes might provide a novel therapeutic strategy to treat NeP.

HBO treatment enhances motor function and modulates pain development after sciatic nerve injury via protection the mitochondrial function.

BACKGROUND: Peripheral nerve injury can cause neuroinflammation and neuromodulation that lead to mitochondrial dysfunction and neuronal apoptosis in the dorsal root ganglion (DRG) and spinal cord, contributing to neuropathic pain and motor dysfunction. Hyperbaric oxygen therapy (HBOT) has been suggested as a potential therapeutic tool for neuropathic pain and nerve injury. However, the specific cellular and molecular mechanism by which HBOT modulates the development of neuropathic pain and motor dysfunction through mitochondrial protection is still unclear. METHODS: Mechanical and thermal allodynia and motor function were measured in rats following sciatic nerve crush (SNC). The HBO treatment (2.5 ATA) was performed 4 h after SNC and twice daily (12 h intervals) for seven consecutive days. To assess mitochondrial function in the spinal cord (L2-L6), high-resolution respirometry was measured on day 7 using the OROBOROS-O2k. In addition, RT-PCR and Immunohistochemistry were performed at the end of the experiment to assess neuroinflammation, neuromodulation, and apoptosis in the DRG (L3-L6) and spinal cord (L2-L6). RESULTS: HBOT during the early phase of the SNC alleviates mechanical and thermal hypersensitivity and motor dysfunction. Moreover, HBOT modulates neuroinflammation, neuromodulation, mitochondrial stress, and apoptosis in the DRG and spinal cord. Thus, we found a significant reduction in the presence of macrophages/microglia and MMP-9 expression, as well as the transcription of pro-inflammatory cytokines (TNFa, IL-6, IL-1b) in the DRG and (IL6) in the spinal cord of the SNC group that was treated with HBOT compared to the untreated group. Notable, the overexpression of the TRPV1 channel, which has a high Ca2+ permeability, was reduced along with the apoptosis marker (cleaved-Caspase3) and mitochondrial stress marker (TSPO) in the DRG and spinal cord of the HBOT group. Additionally, HBOT prevents the reduction in mitochondrial respiration, including non-phosphorylation state, ATP-linked respiration, and maximal mitochondrial respiration in the spinal cord after SNC. CONCLUSION: Mitochondrial dysfunction in peripheral neuropathic pain was found to be mediated by neuroinflammation and neuromodulation. Strikingly, our findings indicate that HBOT during the critical period of the nerve injury modulates the transition from acute to chronic pain via reducing neuroinflammation and protecting mitochondrial function, consequently preventing neuronal apoptosis in the DRG and spinal cord.

Activation of the N-methyl-D-aspartate receptor and calcium/calmodulin-dependent protein kinase IIα signal in the rostral anterior cingulate cortex is involved in pain-related aversion in rats with peripheral nerve injury.

The rostral anterior cingulate cortex (rACC) of rat brain is associated with pain-related emotions. However, the underlying molecular mechanism remains unclear. Here, we investigated the effects of the N-methyl-D-aspartate (NMDA) receptor and Ca2+/Calmodulin-dependent protein kinase type II (CaMKII)α signal on pain-related aversion in the rACC of a rat model of neuropathic pain (NP). Mechanical and thermal hyperalgesia were examined using von Frey and hot plate tests in a rat model of NP induced by spared nerve injury (SNI) of the unilateral sciatic nerve. Bilateral rACC pretreatment with the CaMKII inhibitor tat-CN21 (derived from the cell-penetrating tat sequence and CaM-KIIN amino acids 43-63) or tat-Ctrl (the tat sequence and the scrambled sequence of CN21) was performed on postoperative days 29-35 in Sham rats or rats with SNI. Spatial memory performance was tested using an eight-arm radial maze on postoperative days 34-35. Pain-related negative emotions (aversions) were evaluated using the place escape/avoidance paradigm on postoperative day 35 following the spatial memory performance test. The percentage of time spent in the light area was used to assess pain-related negative emotions (i.e., aversion). The expression levels of the NMDA receptor GluN2B subunit, CaMKIIα, and CaMKII-Threonine at position 286 (Thr286) phosphorylation in contralateral rACC specimens were detected by Western blot or real time PCR following the aversion test. Our data showed that pretreatment of the rACC with tat-CN21 increased determinate behavior but did not alter hyperalgesia or spatial memory performance in rats with SNI. In addition, tat-CN21 reversed the enhanced CaMKII-Thr286 phosphorylation and had no effect on the upregulated expression of GluN2B, CaMKIIα protein, and mRNA. Our data suggested that activation of the NMDA receptor-CaMKIIα signal in rACC is associated with pain-related aversion in rats with NP. These data may provide a new approach for the development of drugs that modulate cognitive and emotional pain aspects.

The potential role of T-cell metabolism-related molecules in chronic neuropathic pain after nerve injury: a narrative review.

Neuropathic pain is a common type of chronic pain, primarily caused by peripheral nerve injury. Different T-cell subtypes play various roles in neuropathic pain caused by peripheral nerve damage. Peripheral nerve damage can lead to co-infiltration of neurons and other inflammatory cells, thereby altering the cellular microenvironment and affecting cellular metabolism. By elaborating on the above, we first relate chronic pain to T-cell energy metabolism. Then we summarize the molecules that have affected T-cell energy metabolism in the past five years and divide them into two categories. The first category could play a role in neuropathic pain, and we explain their roles in T-cell function and chronic pain, respectively. The second category has not yet been involved in neuropathic pain, and we focus on how they affect T-cell function by influencing T-cell metabolism. By discussing the above content, this review provides a reference for studying the direct relationship between chronic pain and T-cell metabolism and searching for potential therapeutic targets for the treatment of chronic pain on the level of T-cell energy metabolism.

Neuron-associated macrophage proliferation in the sensory ganglia is associated with peripheral nerve injury-induced neuropathic pain involving CX3CR1 signaling.

Resident macrophages are distributed across all tissues and are highly heterogeneous due to adaptation to different tissue-specific environments. The resident macrophages of the sensory ganglia (sensory neuron-associated macrophages, sNAMs) are in close contact with the cell body of primary sensory neurons and might play physiological and pathophysiological roles. After peripheral nerve injury, there is an increase in the population of macrophages in the sensory ganglia, which have been implicated in different conditions, including neuropathic pain development. However, it is still under debate whether macrophage accumulation in the sensory ganglia after peripheral nerve injury is due to the local proliferation of resident macrophages or a result of blood monocyte infiltration. Here, we confirmed that the number of macrophages increased in the sensory ganglia after the spared nerve injury (SNI) model in mice. Using different approaches, we found that the increase in the number of macrophages in the sensory ganglia after SNI is a consequence of the proliferation of resident CX3CR1+ macrophages, which participate in the development of neuropathic pain, but not due to infiltration of peripheral blood monocytes. These proliferating macrophages are the source of pro-inflammatory cytokines such as TNF and IL-1b. In addition, we found that CX3CR1 signaling is involved in the sNAMs proliferation and neuropathic pain development after peripheral nerve injury. In summary, these results indicated that peripheral nerve injury leads to sNAMs proliferation in the sensory ganglia in a CX3CR1-dependent manner accounting for neuropathic pain development. In conclusion, sNAMs proliferation could be modulated to change pathophysiological conditions such as chronic neuropathic pain.

Preparation of Primary Mixed Glial Cell Cultures from Adult Mouse Spinal Cord Tissue.

Central nervous system glial cells are known to mediate many neurocognitive/neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Similar glial responses have been recognized as critical factors contributing to the development of diseases in the peripheral nervous system, including various types of peripheral neuropathies, such as peripheral nerve injury-induced neuropathic pain, diabetic neuropathy, and HIV-associated sensory neuropathy. Investigation of the central mechanisms of these peripherally-manifested diseases often requires the examination of spinal cord glial cells at cellular/molecular levels in vitro. When using rodent models to study these diseases, many investigators have chosen to use neonatal cerebral cortices to prepare glial cultures or immortalized cell lines in order to obtain sufficient numbers of cells for assessment. However, differences in responses between cell lines versus primary cultures, neonatal vs. adult cells, and brain vs. spinal cord cells may result in misleading data. Here, we describe a protocol for preparing mixed glial cells from adult mouse spinal cord that can be used for direct in vitro evaluations or further preparation of microglia-enriched and microglia-depleted cells. In this protocol, spinal cord tissue is enzymatically dissociated and adult mixed glial cells are ready to be used between 12 and 14 days after the establishment of the culture. This protocol may be further refined to prepare spinal cord glial cells from spinal cord tissues of adult rats and potentially other species. Mixed glial cultures can be prepared from animals of different strains or post-in vivo manipulations and therefore are suitable for studying a variety of diseases/disorders that involve spinal cord pathological changes, such as amyotrophic lateral sclerosis and multiple sclerosis, as well as toxin-induced changes. © 2023 Wiley Periodicals LLC. Basic Protocol: Preparation of primary mixed glial cell cultures from adult mouse spinal cord tissue.