Autotomy Scores Research Articles

Nerve Defect Treatment with a Capping Hydroxyethyl Cellulose/Soy Protein Isolate Sponge Conduit for Painful Neuroma Prevention.

Painful neuroma, as one of the complications of nerve injury from disease or trauma, results in instinctive neuropathic pain that adversely affects a patient's quality of life. To intercept neuroma development, capping strategies have been performed as effective therapies. Nonetheless, the most appropriate biocompatible material to shield the nerves is an urgent clinical requirement. Herein, a compatible hydroxyethyl cellulose (HEC)/soy protein isolate (SPI) sponge capping conduit (HSSC) is used to prevent neuroma in vivo. Following capping on the sciatic nerve stump in vivo, the behavior of the rats and the structure of tissues are compared through histological assessment and autotomy scoring. The HSSCs gained a dismal autotomy score and enhanced the amelioration, where inflammatory invasions and overdeposition of collagen are defeated. The expression of myelin growth linked genes (Krox20, MPZ, and MAG) in the HSSC group at the eighth week was almost 2 times higher than that of the no capping group. The HSSC conduit served as a physical barrier to repress the infiltration of inflammation as well as provided an optimum microenvironment for facilitating nerve rejuvenation and intercepting neuroma development during nerve amelioration.

Preventive Effect of Local Lidocaine Administration on the Formation of Traumatic Neuroma

Traumatic neuroma is a common sequela of peripheral nerve injury or amputation, which often leads to severe neuropathic pain. The present study investigated the effect of local lidocaine administration on preventing the formation of traumatic neuroma. Forty-eight male Sprague-Dawley rats were randomly assigned to two groups. The lidocaine group underwent sciatic nerve transection, followed by an injection of lidocaine (0.5%) around the proximal of a severed sciatic nerve under ultrasound-guidance 2-7 days after neurectomy. In the control group, rats received an injection of saline following neurectomy. The autotomy score, mechanical allodynia, thermal hyperalgesia, histological assessment, expression of neuroma, and pain-related markers were detected. Lidocaine treatment reduced the autotomy score and attenuated mechanical allodynia and thermal hyperalgesia. The mRNA expression of α-SMA, NGF, TNF-α, and IL-1β all significantly decreased in the lidocaine group in comparison to those in the saline control group. The histological results showed nerve fibers, demyelination, and collagen hyperplasia in the proximal nerve stump in the saline control group, which were significantly inhibited in the lidocaine group. The present study demonstrated that local lidocaine administration could inhibit the formation of painful neuroma due to traumatic nerve injury.

Nerve-End Capping Treatment with a Polyglycolic Acid Conduit for Rat Sciatic Neuroma: A Preliminary Report.

The treatment of painful neuroma remains challenging. Recently, a nerve-end capping technique using a bioabsorbable nerve conduit was newly introduced to treat amputation neuroma. A collagen-coated polyglycolic acid (PGA) conduit has been commercially available for the reconstruction of peripheral nerve defects, yielding successful clinical outcomes. However, no experimental research has been conducted using this PGA nerve conduit as capping device for treating amputation neuroma. The purpose of this study was to investigate nerve-end capping treatment with the PGA conduit in the rat sciatic nerve amputation model, focusing on histological scar formation and neuroinflammation. Forty-seven rats were divided into two groups: no capping (transected nerve stump without capping; n = 25) and capping (nerve-end capping with collagen-coated PGA nerve conduit; n = 22). Twelve weeks after sciatic neurectomy, neuropathic pain was evaluated using the autotomy score. Stump neuromas were histologically evaluated or perineural scar and neuroinflammation. While autotomy scores gradually exacerbated in both groups, they were consistently lower in the capping group at 4, 8, and 12 weeks postprocedure. Twelve weeks after surgery, the transected nerve stumps in the no-capping group had formed macroscopic bulbous neuromas strongly adhering to surrounding tissues, whereas they remained wrapped with the PGA nerve conduits loosely adhering to surrounding tissues in the capping group. Histologically, distal axonal fibers were expanded radially and formed neuromas in the no-capping group, while they were terminated within the PGA conduit in the capping group. Perineural scars and neuroinflammation were widely found surrounding the randomly sprouting nerve end in the no-capping group. In capped counterparts, scars and inflammation were limited to closely around the terminated nerve end. Nerve-end capping with a collagen-coated PGA conduit after rat sciatic neurectomy might prevent neuroma formation by suppressing perineural scar formation and neuroinflammation around the nerve stump, potentially relieving neuropathic pain.

Exploring the Correlation Between the Regulation of Macrophages by Regulatory T Cells and Peripheral Neuropathic Pain.

ObjectiveIntractable pain after peripheral nerve injury has become a major concern in the field of pain. Current evidence shows that routine medications or surgical treatment is associated with inconsistent results and different curative effects. Stable and effective treatment methods in clinical practice are also lacking. To date, there is no consensus on the pathophysiological mechanisms of pain. The present study investigates the potential regulatory role of regulatory T cells in the differentiation of macrophages on dorsal root ganglion (DRG) and explores the mechanism of nociceptive signals in the signal transfer station. The findings are expected to guide the prevention of various types of peripheral neuropathic pain.MethodsThirty-six male Sprague Dawley (SD) rats and 18 male Nude rats, of equal weight (250–300g), were used in this study. The rats were divided into 3 groups: SD rat sciatic nerve transection group (SNT group, n = 18), SD rat nerve transection experimental group (SNT/RAPA group, n = 18) and Nude rat nerve transection experimental group (SNT/NUDE group, n = 18). The behavior related to neuropathic pain of animals were comprehensively evaluated in all groups. Furthermore, we analyzed the degree of neuroma development, histology, gene, and protein expression, and compared their correlation with the ultrastructural changes of M1/M2 type differentiation of macrophages in DRG.ResultsSciatic nerve transection (SNT), induced the aggregation of several types of macrophages in lumbar DRG of SD rats leading to a higher ratio of M1/M2. Following the inhibition of the M1 type polarization of macrophages, axon outgrowth increased significantly. A significantly lower average autotomy score was reported in the SNT/NUDE group (*p < 0.05) and the SNT/RAPA group (@p < 0.05) as compared to that of the SNT group. The SNT/NUDE group showed no noticeable neuroma formation 30 days after the nerve transection. However, bulbous neuromas were observed in the nerve stumps of both the SNT control and SNT/RAPA groups. Immunofluorescence staining revealed a significant decrease in the proportion of M1/M2 macrophages in lumbar DRG of the SNT/NUDE group (**p < 0.001) and the SNT/RAPA group (@p < 0.05) compared to the SNT group. The expression of pain-related proteins was also decreased (@p < 0.05, *p < 0.05,**p < 0.001). Also, the expression of alpha-smooth muscle actin (α-SMA), neurofilament 200 (NF-200), and nerve growth factor low-affinity receptor p75 were significantly down-regulated in the nerve tissue (@p < 0.05, @@p < 0.001, **p < 0.001).ConclusionM1/M2 type differentiation of macrophages on DRG plays a significant role in the formation of traumatic painful neuroma after neurotomy. In combination with our previous study, the results of this study suggest that regulatory T cells reduce the ratio of M1/M2 macrophages and alleviate the pain of neuroma by regulating the polarization direction of macrophages on neuroma. These findings provide key insights into developing new strategies to manage painful neuroma.

Improving Nerve Coaptation Outcomes in Targeted Muscle Reinnervation: A Novel Bioengineered Device

PURPOSE: Despite extensive research efforts, neuroma formation remains a persistent challenge in the treatment of peripheral nerve injuries. Targeted muscle reinnervation (TMR) has emerged as a promising approach for prevention and treatment of painful neuromas. However, the significant size-mismatch inherent to TMR contributes to substantial axonal escape from the coaptation site and consequently patients rarely achieve full resolution of symptoms. To address this limitation, we developed a funnel shaped conduit to mechanically guide the regenerating axons across the repair site and thereby prevent axonal escape. The conduit is modified from a biodegradable nerve wrap that has demonstrated excellent biocompatibility and anti-inflammatory properties in prior studies by our group. Given the limited capacity of the distal nerve stump to accept the axons regenerating from the larger proximal nerve, we incorporated chondroitin sulfate proteoglycans (CSPGs) within the lumen of the conduits to inhibit the majority of the regenerating axons. We applied the funnel conduit with and without CSPGs in a TMR model to assess the impact on functional recovery and neuroma formation at the repair site. METHODS: A conduit device composed of nonwoven poly-ε-caprolactone (PCL) was developed by electrospinning. The conduit walls prevent intraneural macrophage infiltration and inflammation, which limits scarring and fibrosis at the coaptation site. Within the conduit, CSPGs incorporated into a nanofiber hydrogel form an interpenetrating network. Using a TMR rodent hindlimb model, we tested the effects of this device on neuroma formation, axonal growth, muscle reinnervation, pain behaviors, and functional recovery. RESULTS: Qualitative assessment of the coaptation site showed that the significant size mismatch between the sciatic nerve and tibial branch resulted in neuroma formation in the TMR and neuroma groups, while the use of the conduit resulted in tapered reinnervation of the sciatic nerve, demonstrating the effectiveness of this device in mechanically guiding axonal growth. Neuroma and TMR groups demonstrated more co-labelling of Substance P and SCG10 (regeneration marker) than conduit groups. No significant differences were observed between the Positive Control and CSPG-Conduit groups in gastrocnemius muscle mass, myofibril cross-sectional area, and neuromuscular junction reinnervation. However, the Positive Control group exhibited significantly greater gastrocnemius mass than the TMR and the Negative Control groups, suggesting better axonal guidance and muscle reinnervation was enabled by the conduit. By Week 5, mechanical stimulation of the coaptation site elicited significantly less pain behavior response scores in the CSPG-Conduit group compared with the Neuroma group. Autotomy scores of CSPG-Conduit scores were similar to Positive Control scores, suggesting successful prevention of neuroma formation. CONCLUSIONS: We introduce a novel engineered device in which mechanical guidance of axons is combined with inhibition of axonal regeneration to prevent neuroma formation. This conduit presents a biologically compatible, noninvasive means by which we could optimize postoperative care of peripheral nerve injury. Built from materials and components currently used in FDA-approved devices, the device is poised for clinical translation. This therapeutic approach has high potential for success as a reliable technique to prevent neuromas and facilitate prosthetic use.

Nerve capping treatment using a bioabsorbable nerve conduit with open or closed end for rat sciatic neuroma

Background and aimsNerve capping treatment using bioabsorbable nerve conduits has recently been introduced for painful amputation neuroma. However, no clinical or experimental data are available for comparing nerve conduits with open distal ends and closed distal ends. Here, we investigated the nerve conduit with open or closed distal ends as the superior capping device, using a commercially available polyglycolic acid (PGA) nerve conduit in a rat sciatic nerve amputation model. MethodsNinety-one rats were assigned to three groups: no-capping (n = 30), capping the resected nerve stump with open ends (n = 31), and closed-end nerve conduits (n = 30). Twelve weeks after sciatic neurectomy, with or without capping, the evaluation of neuropathic pain using the autotomy score was performed. Stump neuromas with perineural scars and neuroinflammation were evaluated histologically. ResultsThe mean autotomy scores in the closed-end nerve conduit group were significantly lower than those in the no-capping group. However, the difference between the open-end nerve conduit and the closed-end nerve conduit groups was insignificant. Histologically, distal axonal fibers expanded radially and formed neuromas in the no-capping group while they were terminated within the PGA conduit in both capping groups. In particular, the closed-end version of the PGA nerve conduit blocked scarring from intruding through the open end and protected the nerve stump with less neuroinflammation. Nerve capping with the closed-end version of the PGA nerve conduit most effectively suppressed perineural neuroinflammation and scar formation around the resected nerve stump. InterpretationNerve capping with the PGA nerve conduit, particularly those with closed ends, after rat sciatic neurectomy prevented amputation neuroma and relieved neuropathic pain.

Experimental study of the effect of the sciatic nerve elongation on pain in rats

To investigate the effect of the sciatic nerve elongation on pain in rats. Thirty-six adult male Wistar rats of SPF grade, weighing 250-300 g. Eighteen of them were randomly divided into 3 groups, 6 rats in each group. They were sciatic nerve elongation group (group A), nerve no-elongation group (group B), and nerve ligation group (group C). The model of 10-mm sciatic nerve defect was established in all 3 groups. The sciatic nerve was extended at a speed of 1 mm/d for 14 days in group A. The group B was only installed with external fixation. The nerve stumps were ligated in the group C. At 3, 7, 10, and 14 days after operation, the foot injury was evaluated by the autotomy scoring scale. At 14 days after operation, the dorsal root ganglia (DRG) of L 4-S 1 spinal cord of rats in each group was observed by tumor necrosis factor α (TNF-α) immunohistochemical staining, and the primary antibodies were replaced by pure serum as negative control group. Another 18 rats were randomly divided into 3 groups, 6 rats in each group. They were sciatic nerve elongation group (group A1), nerve no-elongation group (group B1), positive control group (group C1). In groups A1 and B1, the 10-mm long sciatic nerve defect model was established by the same method as groups A and B, and then fixed with external fixation. Nerve elongation was done or not done without anesthesia at 3 days after operation. In group C1, no modeling was done and 20 μL 2.5% formaldehyde was injected into the toes. After 90 minutes, the dorsal horn of spinal cord of L 4-S 1 segment of rats was cutting for c-Fos immunohistochemical staining and the number of positive cells was counted. Primary antibodies were replaced with pure serum as negative control group. The autotomy scores of rats in groups B and C gradually increased postoperatively, and group A remained stable at 0.25±0.50. The scores of group C were significantly higher than those of group A and group B at each time point postoperatively ( P<0.05). The scores of group A were significantly lower than those of group B at 10 and 14 days postoperatively ( P<0.05). TNF-α immunohistochemical staining showed that the TNF-α expression in group A was weak, slightly positive (+/-); in group B was positive (+); in group C was strongly positive (++); and the negative control group had no TNF-α expression (-). c-Fos immunohistochemical staining showed that the c-Fos expressions in groups A1 and B1 were weak positive, in group C1 was strong positive, and negative control group had no c-Fos positive expression. The number of c-Fos positive cells in groups A1, B1, C1, and negative control group were (21.5±6.6), (19.3±8.1), (95.6±7.4), and 0 cells/field, respectively, and group C1 was significantly higher than groups A1 and B1 ( P<0.05), there was no significant difference between group A1 and group B1 ( P>0.05). Nerve elongation does not cause obvious pain neither during the operation of elongation nor throughout the whole elongation.

Nerve capping with a nerve conduit for the treatment of painful neuroma in the rat sciatic nerve.

Treatment of painful neuroma remains difficult, despite the availability of numerous surgical procedures. Recently, nerve capping treatment for painful neuroma using artificial nerve conduits has been introduced in clinical and basic research. However, the appropriate length of the nerve conduit and the pain relief mechanism have not been determined. In this study the authors aimed to investigate nerve capping treatment with a bioabsorbable nerve conduit using the rat sciatic nerve amputation model. Using histological analysis, the authors focused on the nerve conduit length and pain relief mechanism. Sixteen Sprague Dawley rats were evaluated for neuropathic pain using an autotomy (self-amputation) score and gross and histological changes of the nerve stump 2, 4, 8, and 12 weeks after sciatic nerve neurectomy without capping. Forty-five rats were divided into 3 experimental groups, no capping (control; n = 15), capping with a 3-mm nerve conduit (n = 15), and capping with a 6-mm nerve conduit (n = 15). All rats were evaluated using an autotomy score and nerve stump histology 12 weeks after neurectomy. The nerve conduit was approximately 0.5 mm larger than the 1.5-mm diameter of the rat sciatic nerves to prevent nerve constriction. The autotomy scores gradually exacerbated with time. Without capping, a typical bulbous neuroma was formed due to random axonal regeneration 2 weeks after neurectomy. Subsequently, the adhesion surrounding the neuroma expanded over time for 12 weeks, and at the 12-week time point, the highest average autotomy scores were observed in the no-capping (control) group, followed by the 3- and the 6-mm nerve conduit groups. Histologically, the distal axonal fibers became thinner and terminated within the 6-mm nerve conduit, whereas they were elongated and protruded across the 3-mm nerve conduit. Minimal perineural scar formation was present around the terminated axonal fibers in the 6-mm nerve conduit group. Expressions of anti-α smooth muscle actin and anti-sigma-1 receptor antibodies in the nerve stump significantly decreased in the 6-mm nerve conduit group. In the rat sciatic nerve amputation model, nerve capping treatment with a bioabsorbable nerve conduit provided relief from neuroma-induced neuropathic pain and prevented perineural scar formation and neuroinflammation around the nerve stump. The appropriate nerve conduit length was determined to be more than 4 times the diameter of the original nerve.

An aligned nanofiber nerve conduit that inhibits painful traumatic neuroma formation through regulation of the RhoA/ROCK signaling pathway.

Traumatic neuromas represent a prevalent source of neuropathic pain. As of yet, there has been no single treatment method that can guarantee permanent relief of symptoms. Although nerve-capping techniques have shown promise, their exact mechanisms remain elusive. The authors' aim was to examine the role of the RhoA/ROCK signaling pathway in the prevention of neuroma formation after neurectomy utilizing a nerve-capping technique. An aligned nanofiber tube was fabricated to cap the sciatic nerve in Sprague Dawley rats. The rats (n = 60) were randomly divided into the aligned SF/P (LLA-CL) capping group (capping group, n = 20), the capping and Y-27632 (ROCK pathway inhibitor) intervention group (intervention group, n = 20), and the no-capping group (control group, n = 20). The authors undertook a comprehensive assessment of the capping group, examining the animals' behavior, the extent of neuroma development, histology, gene and protein expression, and ultrastructural changes associated with the RhoA/ROCK signaling pathway. These findings were compared with those in the intervention and control groups. The inciting injury resulted in the expression of the RhoA/ROCK signaling pathway, as well as its further upregulation in peripheral neurons. Axon outgrowth was significantly increased when RhoA/ROCK signaling pathway was suppressed. The average autotomy score in the capping group was observed to be much lower than that of the intervention and control groups. At 30 days postneurectomy, the capping group displayed no obvious neuroma formation, while a bulbous neuroma was found in the nerve stumps of both the control and intervention groups. Quantitative real-time polymerase chain reaction and the Western blot analysis demonstrated that the expression of myelin-associated glycoprotein was substantially upregulated in the capping group; in contrast, the expression of NF-200 was significantly downregulated. The expression of myosin light chain was notably lower in the intervention group, but there was no significant difference when compared with the control group (p > 0.05). The RhoA/ROCK signaling pathway has emerged as a critical player in the process of traumatic neuroma formation after neurectomy. It is possible that the nerve-capping technique could generate a "regenerative brake" based on the regulation of the RhoA/ROCK signaling pathway in this event. These findings may provide concrete evidence that could help develop new strategies for the management of painful neuromas.

Painful Terminal Neuroma Prevention by Capping PRGD/PDLLA Conduit in Rat Sciatic Nerves.

Neuroma formation after amputation as a long‐term deficiency leads to spontaneous neuropathic pain that reduces quality of life of patients. To prevent neuroma formation, capping techniques are implemented as effective treatments. However, an ideal, biocompatible material covering the nerves is an unmet clinical need. In this study, biocompatible characteristics presented by the poly(D,L‐lactic acid)/arginylglycylaspartic acid (RGD peptide) modification of poly{(lactic acid)‐co‐ [(glycolic acid)‐alt‐(L‐lysine)]} (PRGD/PDLLA) are evaluated as a nerve conduit. After being capped on the rat sciatic nerve stump in vivo, rodent behaviors and tissue structures are compared via autotomy scoring and histological analyses. The PRGD/PDLLA capped group gains lower autotomy score and improves the recovery, where inflammatory infiltrations and excessive collagen deposition are defeated. Transmission electron microscopy images of the regeneration of myelin sheath in both groups show that abnormal myelination is only present in the uncapped rats. Changes in related genes (MPZ, MBP, MAG, and Krox20) are monitored quantitative real‐time polymerase chain reaction (qRT‐PCR) for mechanism investigation. The PRGD/PDLLA capping conduits not only act as physical barriers to inhibit the invasion of inflammatory infiltration in the scar tissue but also provide a suitable microenvironment for promoting nerve repairing and avoiding neuroma formation during nerve recovery.

Significance of alpha smooth muscle actin expression in traumatic painful neuromas: a pilot study in rats.

Treatment of painful neuromas remains a challenge and the mechanism of neuroma-associated pain is not yet fully understood. In this study, we aimed to observe the expression of alpha smooth muscle actin (α-SMA) in traumatic neuromas and to investigate its possible roles in the cause of neuropathic pain in a rat model. The rat sciatic nerve was used and the experiment was divided into two parts. In part I, our results showed significantly higher levels of α-SMA and the pain marker c-fos in the autotomy group than in the no-autotomy group. In part II, the expression of α-SMA in neuromas was down- and up-regulated using SB-431542 and GW9662, respectively. A significant correlation between autotomy scores and the expression level of α-SMA was found (R = 0.957; p < 0.001) and the expression level of α-SMA was positively related to the autotomy scores (R2 = 0.915, p < 0.001). We concluded that the expression of α-SMA plays certain roles in the neuroma-associated pain, either as a direct cause of pain or as an indirect marker of existence of local mechanical stimuli. Our findings may provide new insights into the development of new treatment modalities for the management of intractable painful neuromas.

The effects of picric acid (2,4,6-trinitrophenol) and a bite-deterrent chemical (denatonium benzoate) on autotomy in rats after peripheral nerve lesion.

Denervation of the hind limb is a technique used to study peripheral nerve regeneration. Autotomy or autophagia is an undesirable response to denervation in such studies. Application of a commercially available lotion used to deter nail biting in humans reduced autotomy in rats after denervation but did not completely prevent it. In this study, this authors evaluated the application of picric acid to prevent autotomy in rats in peripheral nerve experiments. They carried out sciatic nerve transection in 41 adult female Wistar rats and then applied either bite-deterrent lotion (n = 26) or saturated picric acid solution (n = 15) topically to the affected hind limb immediately after surgery and every day for 1 month. Autotomy scores were lower for rats treated with picric acid than for rats treated with bite-deterrent lotion 1 week and 2 weeks after surgery but were not different between the two groups 4 weeks after surgery. The authors conclude that application of picric acid could be used as an alternative strategy to prevent autotomy in peripheral nerve studies.

Transplantation of Schwann cells in a collagen tube for the repair of large, segmental peripheral nerve defects in rats

Segmental nerve defects pose a daunting clinical challenge, as peripheral nerve injury studies have established that there is a critical nerve gap length for which the distance cannot be successfully bridged with current techniques. Construction of a neural prosthesis filled with Schwann cells (SCs) could provide an alternative treatment to successfully repair these long segmental gaps in the peripheral nervous system. The object of this study was to evaluate the ability of autologous SCs to increase the length at which segmental nerve defects can be bridged using a collagen tube. The authors studied the use of absorbable collagen conduits in combination with autologous SCs (200,000 cells/μl) to promote axonal growth across a critical size defect (13 mm) in the sciatic nerve of male Fischer rats. Control groups were treated with serum only-filled conduits of reversed sciatic nerve autografts. Animals were assessed for survival of the transplanted SCs as well as the quantity of myelinated axons in the proximal, middle, and distal portions of the channel. Schwann cell survival was confirmed at 4 and 16 weeks postsurgery by the presence of prelabeled green fluorescent protein-positive SCs within the regenerated cable. The addition of SCs to the nerve guide significantly enhanced the regeneration of myelinated axons from the nerve stump into the proximal (p < 0.001) and middle points (p < 0.01) of the tube at 4 weeks. The regeneration of myelinated axons at 16 weeks was significantly enhanced throughout the entire length of the nerve guide (p < 0.001) as compared with their number in a serum-only filled tube and was similar in number compared with the reversed autograft. Autotomy scores were significantly lower in the animals whose sciatic nerve was repaired with a collagen conduit either without (p < 0.01) or with SCs (p < 0.001) when compared with a reversed autograft. The technique of adding SCs to a guidance channel significantly enhanced the gap distance that can be repaired after peripheral nerve injury with long segmental defects and holds promise in humans. Most importantly, this study represents some of the first essential steps in bringing autologous SC-based therapies to the domain of peripheral nerve injuries with long segmental defects.

Chondroitinase ABC improves recovery of long sciatic nerve defects.

Sciatic nerves from allogeneic Sprague-Dawley rats were pretreated with chondroitinase ABC and were used to bridge damaged sciatic nerves in Wistar rats. Chondroitin sulfate proteoglycans were removed from the chemically extracted acellular nerves. At 3 months after grafting, the footplate pinch test result was positive in the Wistar rats. Autotomy scores decreased, and increased muscular contraction tension appeared when triceps surae muscles were stimulated. In addition, the recovery rate of wet triceps surae muscle weight increased, and the distal segment of the chondroitinase ABC-treated graft exhibited Schwann cells next to the nerve fibers. These results suggested that chondroitinase ABC pretreatment enhanced repair of long nerve defects via acellular nerve grafting.

Stump Rotation in a Nerve Autograft by 2 Factors

The aim of this study was to determine the influence of stump rotation (fascicular cross-connection) on nerve autograft functional recovery. Thirty rat sciatic nerves were transected and repaired by autograft. The rats were divided into 3 groups according to the number of stump rotations of the autograft: (1) plain graft (PG-g, with no change in fascicular orientation, no difference in diameter), (2) graft rotation (GR-g, with no change in fascicular orientation, and diameter difference between fascicles), (3) stump rotation (SR-g, with cross-connection and diameter difference between fascicles). Relative gastrocnemius muscle weight and nerve fiber count were examined as anatomic recovery indices, and autotomy score (ATS) and toe-out angle as functional recovery indices. The results showed that SR-g had a significantly low functional recovery compared with PG-g and GR-g. However, there was no difference found in the anatomic recovery. These findings suggest that the stump rotation in nerve autograft had no effect on neural regeneration and muscle reinnervation; however, it had a negative effect on functional recovery. Because GR-g was similar to PG-g rather than SR-g in functional recovery, the diameter difference between fascicles appears to have had little effect on the functional recovery. The results of this study suggest that a novel approach to ATS interpretation is needed. It is recommended that (1) ATS 2 be considered the onset of autotomy instead of ATS 1; and (2) the frequency of "ATS 2 and above" should be considered for the comparison of the autotomy rather than the mean ATS.

Expression of the Dopaminergic D1 and D2 Receptors in the Anterior Cingulate Cortex in a Model of Neuropathic Pain

BackgroundThe anterior cingulate cortex (ACC) has been related to the affective component of pain. Dopaminergic mesocortical circuits, including the ACC, are able to inhibit neuropathic nociception measured as autotomy behaviour. We determined the changes in dopamine D1 and D2 (D1R and D2R) receptor expression in the ACC (cg1 and cg2) in an animal model of neuropathic pain. The neuropathic group had noxious heat applied in the right hind paw followed 30 min. later by right sciatic denervation. Autotomy score (AS) was recorded for eight days and subsequently classified in low, medium and high AS groups. The control consisted of naïve animals.A semiquantitative RT-PCR procedure was done to determine mRNA levels for D1R and D2R in cg1 and cg2, and protein levels were measured by Western Blot.ResultsThe results of D1R mRNA in cg1 showed a decrease in all groups. D2R mRNA levels in cg1 decreased in low AS and increased in medium and high AS. Regarding D1R in cg2, there was an increase in all groups. D2R expression levels in cg2 decreased in all groups. In cg1, the D2R mRNA correlated positively with autotomy behaviour. Protein levels of D2R in cg1 increased in all groups but to a higher degree in low AS. In cg2 D2R protein only decreased discretely. D1R protein was not found in either ACC region.ConclusionsThis is the first evidence of an increase of inhibitory dopaminergic receptor (D2R) mRNA and protein in cg1 in correlation with nociceptive behaviour in a neuropathic model of pain in the rat.

Chronic blockade of interleukin-1 (IL-1) prevents and attenuates neuropathic pain behavior and spontaneous ectopic neuronal activity following nerve injury

Neuropathic pain is a chronic pain state resulting from peripheral nerve injury, characterized by hyperalgesia and allodynia. We have reported that mice with genetic impairment of IL-1 signaling display attenuated neuropathic pain behavior and ectopic neuronal activity. In order to substantiate the role of IL-1 in neuropathic pain, WT mice were implanted subcutaneously with osmotic micropumps containing either IL-1ra or vehicle. Two days following the implantation, two models of neuropathic pain were used; partial nerve injury (spinal nerve transection, SNT), or complete nerve cut (spinal neuroma model). Mechanosensitivity was assessed seven consecutive days following SNT, and on day 7 recordings of spontaneous ectopic activity were performed. In the spinal nerve neuroma model, autotomy scores were recorded up to 35 days. Vehicle-treated mice developed significant allodynia and autotomy, and clear ectopic activity (4.1 ± 1.1% of the axons); whereas IL-1ra-treated mice did not display allodynic response, displayed delayed onset of autotomy and markedly reduced severity of autotomy scores, and displayed reduced spontaneous activity (0.8 ± 0.4% of the axons). To test whether IL-1 is involved in maintenance of mechanical allodynia, a separate group of WT mice was treated with a single injection of either saline or IL-1ra four days following SNT, after the allodynic response was already manifested. Whereas saline-treated mice displayed robust allodynia, acute IL-1ra treatment induced long-lasting attenuation of the allodynic response. The results support our hypothesis that IL-1 signaling plays an important role in neuropathic pain and in the ectopic neuronal activity that underling its development.

Insular cortex lesion diminishes neuropathic and inflammatory pain-like behaviours

Injury to the insular cortex in humans produces a lack of appropriate response to pain. Also, there is controversial evidence on the lateralization of pain modulation. The aim of this study was to test the effect of insular cortex lesions in three models of pain in the rat. An ipsilateral, contralateral or bilateral radiofrequency lesion of the rostral agranular insular cortex (RAIC) was performed 48 h prior to acute, inflammatory or neuropathic pain models in all the experimental groups. Acute pain was tested with paw withdrawal latency (PWL) after thermal stimulation. Inflammation was induced with carrageenan injected in the paw and PWL was tested 1 h and 24 h afterwards. Neuropathic pain was tested after ligature of the sciatic nerve by measuring mechanical nociceptive response after stimulation with the von Frey filaments. Another model of neuropathy consisted of thermo stimulation followed by right sciatic neurectomy prior to the recording of autotomy behaviour. Acute pain was not modified by the RAIC lesion. All the RAIC lesion groups showed diminished pain-related behaviours in inflammatory (increased PWL) and neuropathic models (diminished mechanical nociceptive response and autotomy score). The lesion of the RAIC produces a significant decrease in pain-related behaviours, regardless of the side of the lesion. This is a clear evidence that the RAIC plays an important role in the modulation of both inflammatory and neuropathic – but not acute – pain.

Expression of muscarinic M1 and M2 receptors in the anterior cingulate cortex associated with neuropathic pain

The anterior cingulate cortex (ACC) and muscarinic receptors modulate pain. This study investigates changes in the expression of muscarinic-1 and -2 receptors (M1R, M2R) in rats’ ACC (cg1-rostral- and cg2-caudal) using a model of neuropathic pain by denervation, measured as autotomy score (AS) for 8 days. Changes were analysed with painful stimuli and with scopolamine into the ACC prior to this scheme. We used reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence to determine M1R and M2R’s mRNA and protein levels, respectively. Animals were divided in low, medium and high AS groups. Cg1 showed decreased mRNA levels for both M1R and M2R in the low AS group, as opposed to an increased expression in the medium and high AS groups. Both receptors correlated positively with AS in these groups. In the scopolamine-treated animals there was an increase in mRNA levels for both receptors in cg1, whereas in cg2, mRNA levels of M1R decreased in all the AS and scopolamine groups. The increased M2R mRNA in cg2 correlated with AS in the low, medium and high AS groups whereas all the scopolamine groups showed an increase. Immunoreactivity of the M2R in cg1 decreased in the medium AS group in comparison to controls but scopolamine treatment produced an increase in the medium scopolamine AS group compared to the medium AS group. The M1R in cg1 and both receptors in cg2 showed no immunoreactivity changes. These results highlight the role of the M2R in cg1 related to the degree of autotomy.

Ralfinamide administered orally before hindpaw neurectomy or postoperatively provided long-lasting suppression of spontaneous neuropathic pain-related behavior in the rat

Ralfinamide is analgesic when applied as a single dose in rodent models of stimulus-evoked chronic pain. However, it is unknown whether its chronic application after nerve injury can suppress spontaneous chronic pain, the main symptom driving patients to seek treatment. In this study ralfinamide was administered to rats at doses producing plasma levels similar to those causing analgesia in pain patients. The analgesic effect was tested on autotomy, a behavior of self-mutilation of a denervated paw that models spontaneous neuropathic pain. Sprague–Dawley male rats ( N = 10–20/group) underwent transection of the sciatic and saphenous nerves unilaterally. Ralfinamide or its vehicle were administered per os for 7 days preoperatively (80 mg/kg; bid), followed by the vehicle or Ralfinamide, until postoperative d42. Autotomy was scored daily until d63. Lasting ‘preemptive analgesia’ was found in rats treated with ralfinamide preoperatively, expressed by delayed autotomy onset ( P = 0.009) and reduced scores on d63 ( P = 0.01). Rats treated with ralfinamide (30 or 60 mg/kg; bid) from the operation till d42, but not preoperatively, also showed delayed autotomy ( P = 0.05, P = 0.006), and reduced autotomy scores lasting till d63 ( P = 0.02, P = 0.01), for the two doses, respectively. Combining ralfinamide treatments for 7 days preoperatively and 42 days postoperatively also resulted in significantly suppressed scores on d42 and d63 ( P = 0.005, P = 0.001, respectively). Suppression of neuropathic pain-related behavior was likely caused by a combination of mechanisms reported for ralfinamide, including inhibition of Na + and Ca ++ currents in Nav1.3, Nav1.7, Nav1.8, and Cav2.2 channels in rat DRG neurons, inhibition of substance P release from spinal cord synaptosomes, NMDA receptor antagonism and neuroprotection.