Diagnostic validity of epidermal nerve fiber densities in painful sensory neuropathies

In this prospective study, intraepidermal nerve fiber densities (IENFD) and subepidermal nerve plexus densities (SENPD) were quantified by immunostaining in skin punch biopsies from the distal calf in 99 patients with clinical symptoms of painful sensory neuropathy and from 37 age-matched healthy volunteers. The clinical diagnosis was based on history and abnormal thermal thresholds on quantitative sensory testing (QST). In patients with neuropathy, IENFD and SENPD were reduced to about 50% of controls. Elevated warm detection thresholds on QST correlated with IENFD but not with SENPD. Using receiver-operating characteristic (ROC) curve analysis of IENFD values, the diagnostic sensitivity for detecting neuropathy was 0.80 and the specificity 0.82. For SENPD, sensitivity was 0.81 and specificity 0.88. With ROC analysis of both IENFD and SENPD together, the diagnostic sensitivity was further improved to 0.92. The combined examination of IENFD and SENPD is a highly sensitive and specific diagnostic tool in patients suspected to suffer from painful sensory neuropathies but with normal values on clinical neurophysiological studies.     

Broadening the spectrum of controls for skin biopsy in painful neuropathies

Epidermal nerve fiber density (ENFD) is useful in the evaluation of small‐fiber neuropathies (SFSNs). A recent evidence‐based review highlighted the need to broaden the spectrum of ENFD controls to include lower limb pain states other than polyneuropathy. We studied epidermal innervation in multiple sclerosis (MS) and distal lower limb burning pain (DLLBP). Distal‐leg ENFD/morphology in MS DLLBP patients did not differ significantly from that of healthy controls. This study extends the range of ENFD controls and further supports use of ENFD assessment in SFSN. Muscle Nerve, 2010     

Etanercept reduces hyperalgesia in experimental painful neuropathy

Etanercept, a recombinant tumor necrosis factor receptor (p75)-Fc fusion protein competitively inhibits tumor necrosis factor-alpha (TNF). Etanercept has been successfully used in patients with rheumatoid arthritis, where it reduces pain and inflammation. Because locally produced proinflammatory cytokines play a role in pain after nerve injury, we investigated whether etanercept can reduce pain and hyperalgesia in an animal model of painful neuropathy, the chronic constriction injury of the sciatic nerve. C57BL/6 mice received etanercept or sham treatment by local near-nerve injection to the injured nerve or by systemic application. Treatment with etanercept reduced thermal hyperalgesia and mechanical allodynia significantly in both modes of application. The effect of etanercept was present in animals that were treated from the time of surgery and in those that were treated from day 6, when hyperalgesia was already present. These results suggest the potential of etanercept as a treatment option for patients with neuropathic pain.     

Endoneurial remodeling by TNFalph- and TNFalpha-releasing proteases. A spatial and temporal co-localization study in painful neuropathy

Peripheral nerve injury causing Wallerian degeneration results in endoneurial remodeling initiated by an increase in tumor necrosis factor-alpha (TNF), which is activated from its precursor by extracellular proteases of the matrix metalloproteinase (MMP) family. We used immunohistochemistry to analyze the distribution of TNF, TNF-releasing MMPs, including gelatinases (MMP-2 and MMP-9), and TNF-alpha converting enzyme (TACE) in painful neuropathy caused by chronic constriction injury of rat sciatic nerve. Tissue was analyzed at the injury site and in the corresponding L4 and L5 dorsal root ganglia (DRG) throughout the time-course of the neuropathy. Using confocal laser scanning microscopy, we co-localized TNF with each MMP, and observed spatial and temporal distinction in their distribution. TNF co-localized in vessel endothelium with MMP-2 and in macrophages with MMP-9 and TACE at the period of active immune cell migration. TNF co-localized with myelin degrading MMP-9 within Schwann cells during demyelination, and intraaxonally during remyelination. These studies were performed to explore the role of basal-lamina degrading gelatinases and other TNF-releasing MMPs in TNF-mediated Wallerian degeneration. The data provided in this study may be useful in designing selective therapy for painful neuropathy using synthetic hydroxamate MMP inhibitors.     

Regional Differences in Epidermal Thickness and Behavioral Response Following Partial Denervation of the Rat Paw

In order to investigate the relation between changes in epidermal thickness and pain-related behavior in different rat hind paw areas we performed partial denervation using two experimental models of neuropathic pain injury: spinal nerve ligation (SNL) and chronic constriction injury (CCI). Denervation produced significant epidermal thinning in both models. The most pronounced thinning was observed in the lateral paw region of the SNL group. Both models showed increase in hyperalgesic response during the postoperative period and no behavioral differences between testing regions. Our study showed that partial denervation induces regional differences in epidermal thinning but not in pain-related behavior.     

Adrenergic innervation of blood vessels in rat tibial nerve during Wallerian degeneration

Summary Adrenergic innervation of blood vessels in rat tibial nerve during Wallerian degeneration was examined, using the formaldehyde-induced histo-fluorescence method. The left sciatic nerve was transected at the level of the sciatic notch, whereas the right sciatic nerve was left intact and used as control. At 1, 3, 7, 14, 42, 56 or 84 days after transection, the tibial nerves of the transected and contralateral sides were exposed. Pieces of each nerve were used for light microscopy or for examination of adrenergic innervation with the fluorescence microscope. One day after transection, no adrenergic nerve fiber was observed in the endoneurium of the transected nerve. After 3 days, adrenergic innervation of small-and medium-sized arterioles in the epi-perineurium was absent, and after 7 days no fibers were visible around large arterioles. Fluorescent fibers were not detected even at 84 days post-surgery. It is concluded that adrenergic innervation of blood vessels in the rat tibial nerve is irreversibly lost after permanent axotomy, and that adrenergic regulation of nerve blood flow may also be lost.     

Involvement of astroglial glutamate–glutamine shuttle in modulation of the jaw‐opening reflex following infraorbital nerve injury

To evaluate the mechanisms underlying orofacial motor dysfunction associated with trigeminal nerve injury, we studied the astroglial cell activation following chronic constriction injury (CCI) of the infraorbital nerve (ION) immunohistochemically, nocifensive behavior in ION‐CCI rats, and the effect of the glutamine synthase (GS) blocker methionine sulfoximine (MSO) on the jaw‐opening reflex (JOR), and also studied whether glutamate–glutamine shuttle mechanism is involved in orofacial motor dysfunction. GFAP‐immunoreactive (IR) cells were observed in the trigeminal motor nucleus (motV) 3 and 14 days after ION‐CCI, and the nocifensive behavior and JOR amplitude were also strongly enhanced at these times. The number of GS‐ and GFAP‐IR cells was also significantly higher in ION‐CCI rats on day 7. The amplitude and duration of the JOR were strongly suppressed after MSO microinjection (m.i.) into the motV compared with that before MSO administration in ION‐CCI rats. After MSO administration, the JOR amplitude was strongly suppressed, and the duration of the JOR was shortened. Forty minutes after m.i. of glutamine, the JOR amplitude was gradually returned to the control level and the strongest attenuation of the suppressive effect of MSO was observed at 180 min after glutamine m.i. In addition, glutamine also attenuated the MSO effect on the JOR duration, and the JOR duration was extended and returned to the control level thereafter. The present findings suggest that astroglial glutamate–glutamine shuttle in the motV is involved in the modulation of excitability of the trigeminal motoneurons affecting the enhancement of various jaw reflexes associated with trigeminal nerve injury.     

Neuropeptide expression following constriction or section of the inferior alveolar nerve in the ferret

Some of the sensory abnormalities that follow peripheral nerve injury may result from the development of ectopic discharge from the damaged axons. Previous studies in our laboratory have shown that, following tight ligation of the inferior alveolar nerve (IAN), there is a close association between the time-course of this neural activity and the accumulation of neuropeptides at the injury site. In this study we investigated whether the type of injury has any effect on the time-course or level of neuropeptide expression. In 36 adult ferrets, the IAN was either loosely constricted or sectioned, and the animals left to recover for 3 days, 3 weeks, or 3 months. The tissue was processed using indirect immunofluorescence and image analysis was used to quantify levels of substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, enkephalin, galanin, and neuropeptide Y. Immunoreactivity to all of the neuropeptides was present within the injured nerve 3 days after both types of injury, and decreased to lower levels by 3 weeks and 3 months. Comparisons between the levels of neuropeptide immunoreactivity in each group revealed that the pattern of accumulation was similar following loose constriction or section, and also similar to that found in our previous study on tight ligation. For each injury the time-course of neuropeptide expression was similar to that of the spontaneous activity we had previously recorded. These data support the suggestion that neuropeptide accumulation may be linked to the development of ectopic neural activity but indicate that the type of injury has little effect on the extent of expression.     

Altered pain behavior and regeneration after nerve injury in TNF receptor deficient mice

The pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha is an important mediator in hyperalgesia, nerve injury, and regeneration. Here, we used mice deficient of TNF receptor (TNFR) 1 or 2 to investigate the role of TNF signaling via receptor in each pain behavior and nerve de- and regeneration after chronic constriction injury (CCI) of the sciatic nerve. We found an absence of thermal hyperalgesia in mice deficient of TNFR1 and a reduction in mechanical and cold allodynia in mice deficient of TNFR1 or TNFR2 compared with wild-type mice. Nerve conduction studies and nerve pathology did not reveal major differences between genotypes in the temporal course of de- and regeneration of the nerve. We propose that the functional effects of the TNFRs on pain symptoms are independent of effects on nerve regeneration. Furthermore, the differential action of TNF via each of its receptors should be taken into account when considering clinical trials with TNF inhibitors for pain.     

Intraepidermal nerve fiber density in rat foot pad: neuropathologic-neurophysiologic correlation

Quantification of cutaneous innervation in rat footpad is a useful tool to investigate sensory small-diameter nerve fibers, which are affected early in peripheral neuropathies. The aim of this work was to provide normative reference data on the density of intraepidermal nerve fibers (IENFs) and Langerhans cells in the hindpaw footpad of Sprague-Dawley and Wistar rats. We also evaluated the sensibility of IENF density by comparing neuropathologic findings with neurophysiologic examination and the presence of peripheral neuropathy in two well-characterized animal models of neuropathy. IENF density was quantified in 22 Sprague-Dawley rats and 13 Wistar rats and compared with 19 age-matched Sprague-Dawley rats with streptozotocin-induced diabetic neuropathy and 30 age-matched Wistar rats with cisplatin- or paclitaxel-induced neuropathy. Antidromic tail sensory nerve conduction velocity (SNCV) was assessed in all animals. IENF and Langerhans cell densities were constant in healthy Sprague-Dawley rats at any age, and they were similar to those observed in healthy Wistar rats. In neuropathic rats, both SNCV and IENF density were significantly reduced with respect to controls. Quantification of IENF density was significantly correlated with changes in conduction velocity. Diabetic neuropathy rats alone showed a significantly higher density of Langerhans cells compared with controls. Our study demonstrated that IENF density quantification correlates with SNCV changes and suggests that this might represent a useful outcome measurement in experimental neuropathies.     

Ascorbic acid accelerates Wallerian degeneration after peripheral nerve injury

Wallerian degeneration occurs after peripheral nerve injury and provides a beneficial microenvironment for nerve regeneration. Our previous study demonstrated that ascorbic acid promotes peripheral nerve regeneration, possibly through promoting Schwann cell proliferation and phagocytosis and enhancing macrophage proliferation, migration, and phagocytosis. Because Schwann cells and macrophages are the main cells involved in Wallerian degeneration, we speculated that ascorbic acid may accelerate this degenerative process. To test this hypothesis, 400 mg/kg ascorbic acid was administered intragastrically immediately after sciatic nerve transection, and 200 mg/kg ascorbic acid was then administered intragastrically every day. In addition, rat sciatic nerve explants were treated with 200 μM ascorbic acid. Ascorbic acid significantly accelerated the degradation of myelin basic protein-positive myelin and neurofilament 200-positive axons in both the transected nerves and nerve explants. Furthermore, ascorbic acid inhibited myelin-associated glycoprotein expression, increased c-Jun expression in Schwann cells, and increased both the number of macrophages and the amount of myelin fragments in the macrophages. These findings suggest that ascorbic acid accelerates Wallerian degeneration by accelerating the degeneration of axons and myelin in the injured nerve, promoting the dedifferentiation of Schwann cells, and enhancing macrophage recruitment and phagocytosis. The study was approved by the Southern Medical University Animal Care and Use Committee(approval No. SMU-L2015081) on October 15, 2015.     

Short‐ and intermediate‐term efficacy of buprenorphine TDS in chronic painful neuropathies

Abstract Buprenorphine is a potent opioid available as a transdermal delivery system (TDS) formulation. This open‐label study investigated its safety, tolerability, and efficacy in 30 patients with chronic painful neuropathy. Subjects with visual analogue scale (VAS) score ≥5 under stable analgesic treatment were entered. The starting dosage of 35 μg/h was increased up to 70.0 μg/h in case of unsatisfactory pain control as assessed by fortnightly visits. The primary endpoint was the number of patients achieving at least 30% pain relief at day 42 visit. Treatment was safe over the study period. Nine patients dropped out for side effects, mostly nausea and daily sleepiness. Buprenorphine TDS was well tolerated in 21 patients. Thirteen patients achieved >30% of pain relief at day 42 visit. Five patients needed to increase the dosage to 52.5 μg/h. Eight patients did not meet the primary outcome, but none allowed increasing the dosage to 70 μg/h, and four patients withdrew consent to continue the study before day 42 visit because of a ‘fear to become addicted,’ although 40% had obtained VAS reduction. In our study, which needs to be confirmed by a controlled trial, buprenorphine TDS induced clinically meaningful pain relief in about 40% of patients with chronic painful neuropathy, suggesting its use as a third‐line treatment.     

Comparison of sildenafil,fluoxetine and its co-administration against chronic constriction injury induced neuropathic pain in rats: An influential additive effect

ABSTRACT

Current drug treatment available for neuropathic pain (NP) provides meager and partial pain relief due to incomplete efficacy and dose-dependent adverse effect. Hence, combination therapy can provide prolongation in analgesic effect with milder side effects. The present investigation aimed at observing the effects of sildenafil (SD) on Fluoxetine (FLX) in attenuation of chronic constriction injury (CCI) induced NP in rats. CCI was achieved in rats by placing four loose ligations around the sciatic nerve and rats were received respective treatments on SD and FLX till 14 days further behaviors parameters like heat hyperalgesia and allodynia, pin prick and acetone drop test were executed in order to access thermal, mechanical and cold allodynia, respectively, on a predetermined time interval. On the 21st day the animals were sacrificed for determination of total protein, myeloperoxidase activity in the adjoining muscular tissues while glutathione and TNF-α in the sciatic nerve. Co-administration of SD + FLX + CCI gave the pronounced effect that was superior over individual responses of SD and FLX in all behavioral as well as biochemical parameters. It was observed that attenuation in the altered behavioral pattern of CCI induced rats was modified prominently from 3rd day only in a group of rats treated with SD + FLX + CCI. The whole study was finally supported by histopathological results. Finally, it was concluded that SD produces an additive effect when given with FLX in attenuation of NP may be due to elevation in the level of intracellular concentrations of cyclic guanosine monophosphate which further causes downregulation of calcium channel.     

Changes in proteins related to early nerve repair in a rat model of sciatic nerve injury

Peripheral nerves have a limited capacity for self-repair and those that are severely damaged or have significant defects are challenging to repair. Investigating the pathophysiology of peripheral nerve repair is important for the clinical treatment of peripheral nerve repair and regeneration. In this study, rat models of right sciatic nerve injury were established by a clamping method. Protein chip assay was performed to quantify the levels of neurotrophic, inflammation-related, chemotaxis-related and cell generation-related factors in the sciatic nerve within 7 days after injury. The results revealed that the expression levels of neurotrophic factors (ciliary neurotrophic factor) and inflammation-related factors (intercellular cell adhesion molecule-1, interferon γ, interleukin-1α, interleukin-2, interleukin-4, interleukin-6, monocyte chemoattractant protein-1, prolactin R, receptor of advanced glycation end products and tumor necrosis factor-α), chemotaxis-related factors (cytokine-induced neutrophil chemoattractant-1, L-selectin and platelet-derived growth factor-AA) and cell generation-related factors (granulocyte-macrophage colony-stimulating factor) followed different trajectories. These findings will help clarify the pathophysiology of sciatic nerve injury repair and develop clinical treatments of peripheral nerve injury. This study was approved by the Ethics Committee of Peking University People’s Hospital of China (approval No. 2015-50) on December 9, 2015.

Chinese Library Classification No. R446; R745; R363     

Skin biopsy and quantitative sensory testing do not predict response to lidocaine patch in painful neuropathies

Predictors of response to neuropathic pain treatment in patients with painful distal sensory neuropathies are lacking. The 5% lidocaine patch is believed to exert its effects on neuropathic pain via a local stabilizing effect on cutaneous sensory afferents. As such, it provides a model to assess whether the status of epidermal innervation as determined by skin biopsy or quantitative sensory testing (QST) of small- and large-diameter sensory afferents might serve as predictors of response to topical, locally active treatment. In this study we assessed associations between epidermal nerve fiber (ENF) densities, sensory nerve conduction studies (NCS), QST, and response to a 5% lidocaine patch in patients with painful distal sensory neuropathies. We observed no association between distal leg epidermal and subepidermal innervation and response to the lidocaine patch. Several patients with complete loss of distal leg ENF showed a response to the lidocaine patch. Similarly we observed no consistent association between treatment response and QST for vibration, cooling, warm, heat-pain, and cold-pain thresholds, or distal sensory NCS. Thus, distal-leg skin biopsy, QST, and sensory NCS cannot be used to identify patients with painful polyneuropathy likely to respond to a lidocaine patch in clinical practice. Further studies are required to clarify precisely the mechanism and site of action of the lidocaine patch in patients with peripheral neuropathic pain.     

Ca2+/calmodulin-dependent protein kinase II in the spinal cord contributes to neuropathic pain in a rat model of mononeuropathy

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to subserve activity-dependent neuronal plasticity in the central nervous system. To examine in vivo the implication of spinal CaMKII activity in the generation and development of neuropathic pain after peripheral nerve injury, we used an animal model of mononeuropathy, the chronic constriction injury (CCI) model, in the rat. We found that, 3 days after CCI, the total CaMKII (tCaMKII) immunoreactivity increased in the superficial laminae of the spinal cord and this increase continued for up to 14 days. The immunoreactivity of phosphorylated CaMKII showed an increase from 1 day after CCI, which preceded the up-regulation of tCaMKII. A non-selective N-methyl-d-aspartate receptor antagonist, MK801, significantly attenuated the increase of tCaMKII and phosphorylated CaMKII. Moreover, intrathecal administration of an inhibitor of CaMKII, KN93, before the CCI surgery attenuated the development of thermal hyperalgesia and mechanical allodynia. In addition, KN93 significantly reduced the nociceptive behavior in phase II of the formalin test. These findings demonstrate that the activity of CaMKII in spinal neurons is elevated after peripheral nerve injury and may be involved in central sensitization. The alteration of CaMKII is considered to be a neuroplastic change that occurs in spinal neurons that contributes to neuropathic pain, suggesting the potential for the development of novel therapeutics for neuropathic pain that target CaMKII.