Long-lasting neonatal inflammation enhances pain responses to subsequent inflammation,but not peripheral nerve injury in adult rats

The early postnatal period has been suggested to be the vulnerable time for structural and functional reorganization of sensory systems, and painful stimuli at this time may alter neuronal circuits, thereby leading to changes in an individual's response to pain later in life. In the present study, we examined whether inflammatory experience in the early life can affect pain responses to subsequent noxious insults later in life. The two groups of neonatal rats, treated with an inflammatory irritant and untreated, were subjected to inflammation and peripheral nerve injury in adulthood. Neonatal inflammation was induced by injection of complete Freund's adjuvant (CFA, 25 μl) into the hindpaw or tail of newborn rat pups. Adult rats which had suffered from neonatal paw inflammation at P0 were subjected to re-injection of CFA into the paw neonatally exposed to CFA or L5 spinal nerve ligation. Paw thickness and histology of inflamed paw were examined to assess the neonatal inflammation. Adult animals whose tail had been subjected to CFA injection on P3 received tail-innervating nerve injury. The results showed that the neonatal CFA-treated rats suffered from chronic inflammation, confirmed by persistent increase of paw thickness and histological result of inflamed paw. These animals showed enhanced pain responses to re-inflammatory challenge by injection of CFA (200 μl) into the neonatally inflamed paw 8 weeks after birth compared with the neonatally untreated animals. However, neuropathic pain on the hindpaw and the tail which had been induced by peripheral nerve injury in the neonatal CFA-treated group were not different from those of the untreated group. The present data suggest that early neonatal long-lasting inflammation differentially affects pain responses later in life, depending on the types of subsequent noxious insults.     

Cold intolerance and neuropathic pain after peripheral nerve injury in upper extremity

Cold intolerance and pain can be a substantial problem in patients with peripheral nerve injury. We aimed at investigating the relationships among sensory recovery, cold intolerance, and neuropathic pain in patients affected by upper limb peripheral nerve injury (Sunderland type V) treated with microsurgical repair, followed by early sensory re‐education. In a cross‐sectional clinical study, 100 patients (male/female 81/19; age 40.5 ± 14.8 years and follow‐up 17 ± 5 months, mean ± SD), with microsurgical nerve repair and reconstruction in the upper extremity and subsequent early sensory re‐education, were evaluated, using Cold Intolerance Symptoms Severity questionnaire‐Italian version (CISS‐it, cut‐off pathology >30/100 points), CISS questionnaire‐12 item version (CISS‐12, 0‐46 points‐grouping: healthy that means no cold intolerance [0‐14], mild [15‐24], moderate [25‐34], severe [35‐42], very severe [43‐46] cold intolerance), probability of neuropathic pain (DouleurNeuropathique‐4; [DN4] 4/10), deep and superficial sensibility, tactile threshold (monofilaments), and two‐point discrimination (cutoff S2; Medical Research Council scale for sensory function; [MRC‐scale]). A high CISS score is associated with possible neuropathic pain (DN4 ≥ 4). Both a low CISS‐it score (ie, < 30) and DN4 < 4 is associated with good sensory recovery (MRC ≥ 2). In conclusion patients affected by upper limb peripheral nerve injuries with higher CISS scores more often suffer from cold intolerance and neuropathic pain, and the better their sensory recovery is, the less likely they are to suffer from cold intolerance and neuropathic pain.     

Electroacupuncture attenuates neuropathic pain after brachial plexus injury

Electroacupuncture has traditionally been used to treat pain, but its effect on pain following brachial plexus injury is still unknown. In this study, rat models of an avulsion injury to the left brachial plexus root(associated with upper-limb chronic neuropathic pain) were given electroacupuncture stimulation at bilateral Quchi(LI11), Hegu(LI04), Zusanli(ST36) and Yanglingquan(GB34). After electroacupuncture therapy, chronic neuropathic pain in the rats' upper limbs was significantly attenuated. Immunofluorescence staining showed that the expression of β-endorphins in the arcuate nucleus was significantly increased after therapy. Thus, experimental findings indicate that electroacupuncture can attenuate neuropathic pain after brachial plexus injury through upregulating β-endorphin expression.     

Sex-related difference in collateral sprouting of nociceptive axons after peripheral nerve injury in the rat

Possible sex-related differences in the extent of collateral sprouting of noninjured nociceptive axons after peripheral nerve injury were examined. In the first experiment, peroneal, tibial, and saphenous nerves were transected and ligated in female and male rats. Eight weeks after nerve injury, skin pinch tests revealed that the nociceptive area of the noninjured sural nerve in the instep skin expanded faster in females; the final result was a 30% larger increase in females than in males. In the second experiment, the end-to-side nerve anastomosis was used as a model for axon sprouting. In addition to the previous procedure, the end of an excised peroneal nerve segment was sutured to the side of the intact sural nerve. Eight weeks later, collateral sprouting of nociceptive axons into the anastomosed peroneal nerve segment was assessed by the nerve pinch test and axon counting. There was no significant difference with respect to the percentages of male and female rats with a positive nerve pinch test. The number of myelinated axons in the anastomosed nerve segment was significantly larger in female (456 +/- 217) than in male (202 +/- 150) rats, but the numbers of unmyelinated axons were not significantly different. In normal sural nerves, the numbers of either all myelinated axons or thin myelinated axons did not significantly differ between the two sexes. Therefore, the more extensive collateral axon sprouting observed in female than in male rats is probably due to the higher sprouting capacity of thin myelinated sensory axons in females.     

Increased miR‐195 aggravates neuropathic pain by inhibiting autophagy following peripheral nerve injury

Following peripheral nerve injury (PNI) microglia proliferates and adopts inflammation that contributes to development and maintenance of neuropathic pain. miRNAs and autophagy are two important factors in the regulation of inflammation. However, little is known about whether miRNAs regulate neuroinflammation and neuropathic pain by controlling autophagy. In the study, we demonstrated that miR‐195 levels were markedly increased in rats subjected to L5 spinal nerve ligation (SNL). Upregulated miR‐195 was also found in spinal microglia of rats with SNL. The overexpression of miR‐195 contributed to lipopolysaccharide‐induced expression of proinflammatory cytokines IL‐1β, TNF‐α, and iNOS in cultured microglia. Upregulated miR‐195 also resulted in increased mechanical and cold hypersensitivity after PNI, whereas miR‐195 inhibition reduced mechanical and cold sensitivity. We further demonstrated that PNI significantly inhibited microglial autophagy activation, whereas miR‐195 inhibitor treatment increased autophagy activation and suppressed neuroinflammation in vivo and in vitro. More important, autophagy inhibition impaired miR‐195 inhibitor‐induced downregulation of neuroinflammation and neuropathic pain. Additionally, ATG14 was identified as the functional target of miR‐195. Conclusions: These data demonstrated that miR‐195/autophagy signaling represents a novel pathway regulating neuroinflammation and neuropathic pain, thus offering a new target for therapy of neuropathic pain.     

高压电烧伤后周围神经损害的调查研究

目的探讨高压电烧伤病例周围神经的损害情况。方法采用回顾性研究,收集2012-01—2016-10因"高压电烧伤"入我院治疗的病例资料,所有患者均接受过神经传导测试(NCSs),收集受伤侧及正常侧数据并进行分析。结果 30例患者(42侧肢体)纳入研究,其中56.7%的患者从事电工维修职业,其余主要为意外触电所致。52.5%的患者正中神经至少出现一项参数异常;44.7%的患者出现尺神经的异常,未发现脱髓鞘的证据;12例患者行正常侧的NCSs检测,复合肌肉动作电位及神经传导速度显著高于患侧(P0.05);25例随访显示复合肌肉动作电位及神经传导速度均显著改善(P0.05)。结论高压电烧伤后主要造成患者的周围神经损害而非脱髓鞘病变。虽伤后NCSs检测结果正常,患者仍可能存在外周神经损害;对侧肢体的对照及后续的随访对于周围神经损害的确切评价具有重要意义。     

Effects of injury discharge on the persistent expression of spinal cord fos-like immunoreactivity produced by sciatic nerve transection in the rat

We recently reported that peripheral nerve injury produced by sciatic nerve transection induces a persistent increase in the expression of the immunoreactive Fos protein product of the c-fos proto-oncogene, an indicator of neuronal activity, in the lumbar spinal cord of the rat and that local anesthetic blockade of the peripheral neuroma attenuates this long-term expression of Fos^6,7. In addition to the sustained activity of the injured afferents, the nerve transection itself results, acutely, in a massive injury-induced neural discharge. In this study we evaluated the effect of blocking this massive injury discharge on the persistence of Fos expression. Just prior to nerve transection we applied the short-acting local anesthetic, lidocaine, to the sciatic nerve. Control injections were made subcutaneously on the dorsum of the neck. We report that injection of the local anesthetic, by either route, significantly reduced the number of fos-like immunoreactive neurons at 2 days after nerve transection. The effect was only observed on neurons in the superficial dorsal horn. These results indicate that along with sustained activity of injured afferents and of reorganization of central circuits after injury, the initial brief discharge at the time of nerve injury contributes to a prolonged increase in the activity of spinal cord neurons.     

KDM6B epigenetically regulated-interleukin-6 expression in the dorsal root ganglia and spinal dorsal horn contributes to the development and maintenance of neuropathic pain following peripheral nerve injury in male rats

The lysine specific demethylase 6B (KDM6B) has been implicated as a coregulator in the expression of proinflammatory mediators, and in the pathogenesis of inflammatory and arthritic pain. However, the role of KDM6B in neuropathic pain has yet to be studied. In the current study, the neuropathic pain was determined by assessing the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) following lumbar 5 spinal nerve ligation (SNL) in male rats. Immunohistochemistry, Western blotting, qRT-PCR, and chromatin immunoprecipitation (ChIP)-PCR assays were performed to investigate the underlying mechanisms. Our results showed that SNL led to a significant increase in KDM6B mRNA and protein in the ipsilateral L4/5 dorsal root ganglia (DRG) and spinal dorsal horn; and this increase correlated a markedly reduction in the level of H3K27me3 methylation in the same tissue. Double immunofluorescence staining revealed that the KDM6B expressed in myelinated A- and unmyelinated C-fibers in the DRG; and located in neuronal cells, astrocytes, and microglia in the dorsal horn. Behavioral data showed that SNL-induced mechanical allodynia and thermal hyperalgesia were impaired by the treatment of prior to i.t. injection of GSK-J4, a specific inhibitor of KDM6B, or KDM6B siRNA. Both microinjection of AAV2-EGFP-KDM6B shRNA in the lumbar 5 dorsal horn and sciatic nerve, separately, alleviated the neuropathic pain following SNL. The established neuropathic pain was also partially attenuated by repeat i.t. injections of GSK-J4 or KDM6B siRNA, started on day 7 after SNL. SNL also resulted in a remarkable increased expression of interleukin-6 (IL-6) in the DRG and dorsal horn. But this increase was dramatically inhibited by i.t. injection of GSK-J4 and KDM6B siRNA; and suppressed by prior to microinjection of AAV2-EGFP-KDM6B shRNA in the dorsal horn and sciatic nerve. Results of ChIP-PCR assay showed that SNL-induced enhanced binding of STAT3 with IL-6 promoter was inhibited by prior to i.t. injection of GSK-J4. Meanwhile, the level of H3K27me3 methylation was also decreased by the treatment. Together, our results indicate that SNL-induced upregulation of KDM6B via demethylating H3K27me3 facilitates the binding of STAT3 with IL-6 promoter, and subsequently mediated-increase in the expression of IL-6 in the DRG and dorsal horn contributes to the development and maintenance of neuropathic pain. Targeting KDM6B might a promising therapeutic strategy to treatment of chronic pain.     

Differential pharmacological modulation of the spontaneous stimulus-independent activity in the rat spinal cord following peripheral nerve injury

Peripheral nerve injury is a significant clinical problem that is often difficult to treat. The major clinical symptoms are numbness, tactile and cooling allodynia, hyperalgesias as well as ongoing pain. In animal models of neuropathy, abnormal responses to applied (or evoked) stimuli can be gauged, but spontaneous pain, a major clinical issue, has proved very difficult to assess. In neuropathic animals, spinal neuronal hyperexcitability indicative of peripheral and central changes with high levels of spontaneous neuronal firing has been reported. This latter stimulus-independent firing of sensory neurones may be a measure related to ongoing pain. Two weeks after L5/6 spinal nerve ligation, deep dorsal horn neurones were recorded in halothane-anesthetized rats. The majority of neurones in neuropathic rats showed increased levels of spontaneous firing with irregular firing patterns. We examined and compared the effects of 5 centrally acting pharmacological agents: morphine (i.t. or i.v.), gabapentin, ketamine, memantine and mepyramine on stimulus-independent neuronal firing. This ongoing activity showed high sensitivity to gabapentin (s.c.) and morphine (i.t.) administration, being significantly reduced in a dose-dependent manner. Morphine administered via the systemic route produced modest but non-significant reductions of spontaneous activity. The two NMDA receptor antagonists, ketamine and memantine, and the histamine H1 receptor antagonist, mepyramine, produced minor effects at doses known to be effective on stimulus evoked measures of deep dorsal horn neurones. This may form an electrophysiological basis for the efficacy of gabapentin and spinal morphine on ongoing pain in patients with peripheral neuropathy.     

Adoptive transfer of peripheral immune cells potentiates allodynia in a graded chronic constriction injury model of neuropathic pain

Recent evidence demonstrates that peripheral immune cells contribute to the nociceptive hypersensitivity associated with neuropathic pain by infiltrating the central nervous system (CNS). We have recently developed a rat model of graded chronic constriction injury (CCI) by varying the exposure of the sciatic nerve and control non-nerve tissue to surgical placement of chromic gut. We demonstrate that splenocytes can contribute significantly to CCI-induced allodynia, as adoptive transfer of these cells from high pain donors to low pain recipients potentiates allodynia (P < 0.001). The phenomenon was replicated with peripheral blood mononuclear cells (P < 0.001). Adoptive transfer of allodynia was not achieved in sham recipients, indicating that peripheral immune cells are only capable of potentiating existing allodynia, rather than establishing allodynia. As adoptively transferred cells were found by flow cytometry to migrate to the spleen (P < 0.05) and potentiation of allodynia was prevented in splenectomised low pain recipients, adoptive transfer of high pain splenocytes may induce the migration of host-derived immune cells from the spleen to the CNS as observed by flow cytometry (P < 0.05). Importantly, intrathecal transfer of CD45⁺ cells prepared from spinal cords of high pain donors into low pain recipients led to potentiated allodynia (P < 0.001), confirming that infiltrating immune cells are not passive bystanders, but actively contribute to nociceptive hypersensitivity in the lumbar spinal cord.     

A subpopulation of rats show social and sleep-waking changes typical of chronic neuropathic pain following peripheral nerve injury

Neuropathic conditions for which treatment is sought, the so-called chronic pain syndrome, are characterized usually by complex behavioural disturbances as well as pain. In this study we evaluated whether social behavioural and sleep disruptions occurred after nerve injury. Before and after chronic constriction of the sciatic nerve, resident-intruder and sleep-wake cycles, as well as mechanical and thermal allodynia/hyperalgesia, were quantified. Sciatic nerve injury in all animals reduced withdrawal thresholds to tactile and thermal (cold) stimuli. Resident-intruder and sleep-waking behaviours were altered in some but not all animals. One group (30%, 'persistent change') had enduring reductions in dominant behaviour to an intruder and decreased slow-wave sleep and increased wakefulness during both light and dark cycles. Another group (25%, 'recovery') had a transient reduction in dominant behaviours and decreased slow-wave sleep and increased wakefulness during only the light cycle. In a third group (45%, 'no effect') resident-intruder and sleep-waking behaviours remained normal. Our finding that the degree of 'pain' as inferred from the allodynia/hyperalgesia was identical in all animals suggests that the alterations to resident-intruder and sleep-wake cycles were independent of the level of sensory disturbance. An absence of correlation between intensity of sensory disturbances and measures of disability (loss of sleep, familial/social problems) is also characteristic of human neuropathic pain. These data indicate that: (i) in a subpopulation of animals sciatic injury results in two of the major complex behavioural changes which are characteristic of neuropathic pain in humans; (ii) testing only for allodynia and hyperalgesia is not sufficient to detect this subpopulation.     

Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat

Peripheral nerve injury commonly leads to neuropathic pain states fostered, in part, by neuroimmunologic events. We used two models of neuropathic pain (L5 spinal nerve cryoneurolysis (SPCN) and chronic constriction injury (CCI)) to assess the role of spinal glial activation responses in producing pain behaviors. Scoring of glial responses subjectively encompassed changes in cell morphology, cell density and intensity of immunoreactivity with specific activation markers (OX-42 and anti-glial fibrillary acidic protein (GFAP) for microglia and astrocytes, respectively). Glial responses were compared with tactile sensitivity (mechanical allodynia) at 1, 3 or 10 days following SPCN and with thermal hyperalgesia at 10 days in the CCI group. Neuropathic pain behaviors preceded and did not closely correlate with microglial responses in either model. Perineural application of bupivacaine prior to SPCN prevented spinal microglial responses but not pain behaviors. Spinal astrocytic responses to SPCN were early, robust and not altered by bupivacaine. The current findings support the use of bupivacaine as a tool to suppress microglial activation and challenge the putative role of microglia in initiating or potentiating pain behaviors which result from nerve injury.     

Cortical plasticity and nerve regeneration after peripheral nerve injury

With the development of neuroscience, substantial advances have been achieved in peripheral nerve regeneration over the past decades. However, peripheral nerve injury remains a critical public health problem because of the subsequent impairment or absence of sensorimotor function. Uncomfortable complications of peripheral nerve injury, such as chronic pain, can also cause problems for families and society. A number of studies have demonstrated that the proper functioning of the nervous system depends not only on a complete connection from the central nervous system to the surrounding targets at an anatomical level, but also on the continuous bilateral communication between the two. After peripheral nerve injury, the interruption of afferent and efferent signals can cause complex pathophysiological changes, including neurochemical alterations, modifications in the adaptability of excitatory and inhibitory neurons, and the reorganization of somatosensory and motor regions. This review discusses the close relationship between the cerebral cortex and peripheral nerves. We also focus on common therapies for peripheral nerve injury and summarize their potential mechanisms in relation to cortical plasticity. It has been suggested that cortical plasticity may be important for improving functional recovery after peripheral nerve damage. Further understanding of the potential common mechanisms between cortical reorganization and nerve injury will help to elucidate the pathophysiological processes of nerve injury, and may allow for the reduction of adverse consequences during peripheral nerve injury recovery. We also review the role that regulating reorganization mechanisms plays in functional recovery, and conclude with a suggestion to target cortical plasticity along with therapeutic interventions to promote peripheral nerve injury recovery.     

Neuronal changes resulting in up-regulation of alpha-1 adrenoceptors after peripheral nerve injury

Under normal conditions, the sympathetic neurotransmitter noradrenaline inhibits the production and release of pro-inflammatory cytokines. However, after peripheral nerve and tissue injury, pro-inflammatory cytokines appear to induce the expression of the alphalA-adreno- ceptor subtype on immune cells and perhaps also on other cells in the injured tissue. In turn, noradrenaline may act on up-regulated alphal-adrenoceptors to increase the production of the pro-inflammatory cytokine interleukin-6. In addition, the release of inflammatory mediators and nerve growth factor from keratinocytes and other cells may augment the expression of alphal-adrenoceptors on peripheral nerve fibers. Consequently, nociceptive afferents acquire an abnormal excitability to adrenergic agents, and inflammatory processes build. These mechanisms could contribute to the development of sympathetically maintained pain in conditions such as post-herpetic neuralgia, cutaneous neuromas, amputation stump pain and complex regional pain syndrome.     

Aging and miR-155 in mice influence survival and neuropathic pain after spinal cord injury

Spinal cord injury (SCI) elicits chronic pain in 65% of individuals. In addition, SCI afflicts an increasing number of aged individuals, and those with SCI are predisposed to shorter lifespan. Our group previously identified that deletion of the microRNA miR-155 reduced neuroinflammation and locomotor deficits after SCI. Here, we hypothesized that aged mice would be more susceptible to pain symptoms and death soon after SCI, and that miR-155 deletion would reduce pain symptoms in adult and aged mice and improve survival. Adult (2 month-old) and aged (20 month-old) female wildtype (WT) and miR-155 knockout (KO) mice received T9 contusion SCI. Aged WT mice displayed reduced survival and increased autotomy – a symptom of spontaneous pain. In contrast, aged miR-155 KO mice after SCI were less susceptible to death or spontaneous pain. Evoked pain symptoms were tested using heat (Hargreaves test) and mechanical (von Frey) stimuli. At baseline, aged mice showed heightened heat sensitivity. After SCI, adult and aged WT and miR-155 KO mice all exhibited heat and mechanical hypersensitivity at all timepoints. miR-155 deletion in adult (but not aged) mice reduced mechanical hypersensitivity at 7 and 14 d post-SCI. Therefore, aging predisposes mice to SCI-elicited spontaneous pain and expedited mortality. miR-155 deletion in adult mice reduces evoked pain symptoms, and miR-155 deletion in aged mice reduces spontaneous pain and expedited mortality post-SCI. This study highlights the importance of studying geriatric models of SCI, and that inflammatory mediators such as miR-155 are promising targets after SCI for improving pain relief and longevity.     

脊神经结扎神经痛大鼠脊髓背角广动力范围神经元的电生理学特性(英文)

目的探讨腰5脊神经结扎(spinal nerve ligation, SNL)后,大鼠脊髓背角的广动力范围(wide dynamic range,WDR)神经元电生理学特性的改变。方法将健康雄性 Sprague-Dawley 大鼠分为正常组和 SNL 组,利用细胞外电生理学方法记录脊髓背角的 WDR 神经元放电。结果与正常大鼠相比,SNL 组大鼠 WDR 神经元兴奋性增加,表现为感受野扩大、有自发放电的神经元比例增加,以及 C 纤维诱发放电的阈值降低、潜伏期缩短、发放时程增加。此外,SNL组大鼠WDR神经元A和C纤维诱发放电数目较正常大鼠降低。结论大鼠腰5脊神经结扎后主要引起WDR神经元的兴奋性增加。WDR神经元的兴奋性增加可能参与神经病理痛的发生。     

Ca(2+)/calmodulin-protein kinase IIalpha in the trigeminal subnucleus caudalis contributes to neuropathic pain following inferior alveolar nerve transection

Calcium-calmodulin protein kinase IIalpha (CaMKIIalpha) is mainly found in brain cells, and the mRNA concentrates highly in the postsynaptic density. CaMKIIalpha is an effector of calcium and calmodulin mediated functions, and the phosphorylated CaMKIIalpha (pCaMKIIalpha) activates glutamate receptors, such as the AMPA receptor, and enhances its function. In the present study, we examined whether CaMKIIalpha in trigeminal brainstem neurons contributed to the neuropathic pain induced by inferior alveolar nerve (IAN) transection. Using immunohistochemistry and in situ hybridization, we found that the expression of CaMKIIalpha and pCaMKIIalpha increased in the trigeminal subnucleus caudalis (Vc) after IAN transection. The significant increase in the protein of CaMKIIalpha peaked at 30 min after IAN transection, and the mRNA of CaMKIIalpha increased from 2 to 14 days. Double immunofluorescent staining for CaMKIIalpha and MAP2, a marker of dendrite, revealed a significant increase in the overlapping area at 30 min after injury. This suggests that CaMKIIalpha protein is synthesized from the local mRNA pool in the dendrite 30 min after IAN transection and may quickly transmit information after nerve injury. In the behavioral test in which the escape threshold from mechanical stimulation to the lateral face was measured, intrathecal administration of KN-93, a CaMKII inhibitor, for 7 days significantly inhibited mechano-allodynia induced by IAN transection, as compared with administration of a control peptide. These data suggest that CaMKIIalpha in the trigeminal subnucleus caudalis may be involved in neuropathic pain caused by IAN transection.