Effects of vitamin D administration on nociception and spinal cord pro-oxidant and antioxidant markers in a rat model of neuropathic pain

Reactive oxygen species (ROS) are involved in neuropathic pain, a complicated condition after nerve tissue lesion. Vitamin D appears to improve symptoms of pain and exhibits antioxidant properties. We investigated the effects of oral administration of vitamin D3, the active form of vitamin D, on nociception, the sciatic functional index (SFI), and spinal cord pro-oxidant and antioxidant markers in rats with chronic constriction injury (CCI) of the sciatic nerve, a model of neuropathic pain. Vitamin D3 (500 IU/kg per day) attenuated the CCI-induced decrease in mechanical withdrawal threshold and thermal withdrawal latency (indicators of antinociception) and SFI. The vitamin prevented increased lipid hydroperoxide levels in injured sciatic nerve without change to total antioxidant capacity (TAC). Vitamin D3 prevented increased lipid hydroperoxide, superoxide anion generation (SAG), and hydrogen peroxide (H2O2) levels in the spinal cord, which were found in rats without treatment at 7 and 28 days post-CCI. A significant negative correlation was found between mechanical threshold and SAG and between mechanical threshold and H2O2 at day 7. Vitamin D3 also prevented decreased spinal cord total thiols content. There was an increase in TAC in the spinal cord of vitamin-treated CCI rats, compared to CCI rats without treatment only at 28 days. No significant changes were found in body weight and blood parameters of hepatic and renal function. These findings demonstrated, for first time, that vitamin D modulated pro-oxidant and antioxidant markers in the spinal cord. Since antinociception occurred in parallel with oxidative changes in the spinal cord, the oxidative changes may have contributed to vitamin D-induced antinociception.

The effect of time course of hydrogen peroxide solution flush on the recovery of peripheral nerve injury / 医学研究生学报

Objective Currently, the effect of hydrogen peroxide solution( H2 O2 ) flush on the recovery of peripheral nerve injury are still undetermined. This study aims to explore the effects of different H2 O2 flushing time course on recovery of peripheral nerve injury during surgical debridement. Methods 18 New Zealand male white rabbits were randomly divided into isotonic saline group( isotonic saline flush) , H2 O21 min group ( hydrogen peroxide solution flush 1 min + isotonic saline flush) , H2 O23 min group ( H2 O2 flush 3 min + isotonic saline flush. Each group has 6 rabbits. We established rabbit sciatic nerve injury model. After 12 weeks, we executed the animals, drew materials at the suture nerve, detected motor nerve conduction velocity and observed pathological HE staining, as well as measuring the sciatic nerve function indexes( SFI) of rabbits at the 8th and 12th week, respectively. Results Nerve conduction velocity in H2O21min group and isotonic saline group[(25.89±1.42), (26.46±1.67) m/s] were higher than that in the H2O23min group[(23.80±2.91)m/s](P<0.05). Pathological section shows that the isotonic saline group has a better sheathstructure, nerve fibers structure damage is not obvious and myelin degeneration degree is mild in H2 O21 min group. blurred sheath structure and myelin axons obvious deformation in H2 O23 min group. 8 weeks and 12 weeks after operation, SFI in isotonic saline group [(-43.00±0.82), (-39.17±0.91) m/s] was significant higher than H2O21 min group [ (-49.79±1.47), (-46.60±0.79) m/s] and H2O23min group [(-51.66±1.27), (-46.39±0.94) m/s], thedifference was statistically significant (P<0.05). SFI in group of H2O21min [(-49.79±1.47)m/s] was higher than group og H2O23 min [(-51.66±1.27) m/s][P<0.05]. Conclusion H2O2 solution has side effect on the recovery of damaged peripheral nerve inju-ry and with prolonging the H2 O2 flushing time, the damage of peripheral nerve can be aggravated. So in the process of clinical surgical debridement, under the premise of guaranteed sterilization the time of H2 O2 flush should be shortened as far as possible.

N-acetylcysteine downregulates phosphorylated p-38 expression but does not reverse the increased superoxide anion levels in the spinal cord of rats with neuropathic pain

We determined the effect of N-acetylcysteine (NAC) on the expression of the phosphorylated p38 (p-p38) protein and superoxide anion generation (SAG), two important players in the processing of neuropathic pain, in the lumbosacral spinal cord of rats with chronic constriction injury (CCI)-induced neuropathic pain. The sciatic functional index (SFI) was also measured to assess the functional recovery post-nerve lesion. Thirty-six male Wistar rats were divided equally into the following groups: Naive (rats did not undergo surgical manipulation); Sham (rats in which all surgical procedures involved in CCI were used except the ligature), and CCI (rats in which four ligatures were tied loosely around the right common sciatic nerve), which received 2, 4, or 8 intraperitoneal injections of NAC (150 mg·kg-1·day-1) or saline beginning 4 h after CCI. Rats were sacrificed 1, 3, and 7 days after CCI. The SFI was measured on these days and the lumbosacral spinal cord was used for analysis of p-p38 expression and SAG. CCI induced a decrease in SFI as well as an increase in p-p38 expression and SAG in the spinal cord. The SFI showed a partial recovery at day 7 in saline-treated CCI rats, but recovery was improved in NAC-treated CCI rats. NAC induced a downregulation in p-p38 expression at all time-points evaluated, but did not reverse the increased SAG induced by CCI. Since p-p38 is a mediator in neuropathic pain and/or nerve regeneration, modulation of this protein may play a role in NAC-induced effects in CCI rats.

Effect of Cluster Needling of Scalp Acupuncture Combined with Treadmill Training on Axonal Regeneration and Expression of S-100 Protein in Rats with Sciatic Nerve Injury / 中国康复理论与实践

Objective To explore the effect of cluster needling of scalp acupuncture combined with treadmill training on axonal regener-ation and expression of S-100 protein in rats with sciatic nerve injury. Methods Ninety male Sprague-Dawley rats were randomly divided in-to blank group, sham group, model group, treadmill group and cluster needling of scalp point combined with treadmill group (combination group), each group was further divided into 7, 14 and 21 days subgroups, with 6 rats in each subgroup. The sciatic nerve of rats was clamped in the model group, the treadmill group and the combination group. The sham group was subjected to the same surgical procedure with sciat-ic nerve exposure but without crush injury. Three days after modeling, the treadmill group was treated with treadmill training, and the combi-nation group was treated with cluster needling of scalp acupuncture and treadmill training, while the other groups were grabbed regularly, with no other intervention. The sciatic functional index (SFI) was tested, the condition of axon growth was observed with HE staining, and the expression of S-100 protein was examined with immunohistochemistry at each time point. Results At each time point, SFI was higher, the axon grew better, and the S-100 protein was higher expressed in the treadmill group and the combination group than in the model group (P<0.05), especially in the combination group (P<0.05). Conclusion Cluster needling of scalp acupuncture combined with treadmill training can accelerate the regeneration of axons and increase the expression of S-100 protein and improve sciatic function after sciatic nerve injury.

Repercussões morfológicas e funcionais do exercício sobre a regeneração nervosa periférica / Morphological and functional repercussion on peripheral nerve regeneration after exercises

INTRODUÇÃO: A indicação de exercícios físicos como abordagem terapêutica após lesões nervosas periféricas, apesar de resultados clínicos favoráveis, ainda não é consenso na literatura pertinente. Objetivos: Este estudo buscou avaliar a interferência do exercício em roda motorizada no processo de regeneração nervosa periférica. MATERIAIS E MÉTODOS: Foram utilizados 48 ratos Wistar divididos proporcionalmente em quatro grupos de 12 ratos cada (seis controles e seis experimentais). Após treinamento, os ratos foram submetidos à axonotmese do nervo ciático direito. Todos os grupos iniciaram o regime de exercícios diários 24 horas após a lesão, porém com durações diferenciadas (G1 por 28 dias; G2 por 21 dias, G3 por 14 dias e G4 por sete dias). O exercício teve tempo e velocidade ajustados de acordo com o tempo de tratamento para cada grupo. RESULTADOS: Por meio do cálculo do Índice Funcional do Ciático, os animais submetidos ao exercício apresentaram sinais de atraso na recuperação funcional da marcha, em comparação a animais controles. Na análise histológica dos nervos lesionados, observou-se que animais exercitados apresentaram sinais de preservação dos axônios no coto proximal e de degeneração no coto distal, e os animais exercitados por somente sete dias não apresentaram a mesma característica degenerativa no coto distal. A aplicação de exercícios ativos e sem carga em animais que sofreram axonotmese interfere negativamente no processo de recuperação funcional de alguns parâmetros da marcha. CONCLUSÃO: Apesar de o exercício ativo não ter interferido na manutenção da viabilidade neuronal nos sítios proximais à lesão, sua continuidade prejudicou a viabilidade dos cotos neurais distais, provável causa do atraso na recuperação funcional da marcha.

INTRODUCTION: This study aimed to investigate if exercise in motorized wheel can affect the process of peripheral nerve regeneration in a rat model of axonotmesis. MATERIAL AND METHODS: Forty-eight Wistar rats were divided equally into four groups containing six experimental and six control subjects. After proper training, the rats underwent right sciatic nerve axonotmesis. All groups started the exercises, in a daily basis, 24 hours after nerve injury, but at different durations (28 days for G1, 21 days for G2, 14 days for G3 and seven days for G4). Both duration and speed of the exercises were gradually adjusted during the experiments. RESULTS: The Sciatic Function Index revealed that exercised animals showed clear signs of delayed functional gait recovery. The histological analysis of injured nerves revealed that exercised animals showed signs of proximal stump preservation and degenerative signs in the distal stump. In other hand, exercised animals during seven days only showed axonal preservation in the distal stump. CONCLUSION: Based on these results, we believe that active exercises should be avoided, at least during the peripheral nerve regeneration process, i.e., during first 21 days after axonotmesis.".

Avaliação de método digital para análise do índice funcional do ciático em ratos / Evaluation of digital method for analysis of the sciatic functional index in rats

Este trabalho teve como objetivo o estudo da reprodução e descrição de técnica para colheita digital de imagens durante a marcha de ratos e determinação do Índice Funcional do Ciático (IFC), através de uma passarela de vidro e obtenção de imagens com uma câmera digital. Após lesão por estrangulamento controlado de nervo ciático, na extensão de 3mm, durante 30 segundos, utilizando pinça hemostática, um grupo de 32 ratos foram amostrados 24h antes da lesão que serviu de controle e 24h, 7, 14 e 21 dias após a compressão do nervo ciático. Estes ensaios consistiam na filmagem da marcha de cada animal em vista inferior (através de um espelho a 45º), e posteriormente analisadas através do programa IMAGE-J. As medidas retiradas foram os comprimentos das patas (direita e esquerda posteriores), bem como, a distância entre os artelhos. Na análise do IFC, valores próximos do "zero" (0) sugerem que a função do nervo Ciático mantém-se preservada e valores próximos de "menos cem" (-100) indicam perda total de função. Verificou-se neste estudo que 24horas antes da cirurgia causadora da lesão, a média do IFC foi de -7,07 ± 7,88 e 24 horas após a lesão estes valores passaram para uma média de -77,95 ± 13,81, sendo cerca de 10 vezes maiores, em que 78% dos animais apresentavam de 60 a 100% de perda funcional na atividade motora que demonstrou recuperação gradativa ao longo dos dias analisados, comprovando a acuidade e eficácia da metodologia proposta. Tais resultados sugerem que estudos possam ser conduzidos de forma mais simples e barata com a técnica de obtenção e manipulação de imagens digitais das pegadas, durante a marcha de ratos em laboratório.

This work aimed to study the reproduction and description of technique for digital sampling images during rats gait and determination of the sciatic functional index (SFI), through a glass walkway to obtain shoots with a digital camera. After controlled injury by strangulation of sciatic nerve, with 3mm of length, during 30 seconds, using hemostatic forceps, a group of 32 rats was performed 24 hours before the lesion which served as control and 24 hours, 7, 14 and 21 days after injury. The tests consisted in the filming and shooting each animal in order to observe the view from below (by a mirror to 45°) and subsequently analyzed using the IMAGE-J program. Measures were taken from the lengths of the legs (right and left), and the distance between the ankle. In the analysis of IFC, values close to zero (0) suggest that the function of the sciatic nerve is still preserved and values coming from "less one hundred" (-100) indicate total loss of function. It was verified in this study that 24 hours before surgery the average SFI was -7.07 ± 7.88 and 24 hours after injury these values rose to an average of -77.95 ± 13.81, being about 10 times larger, where 78% of the animals showed 60 to 100% of functional loss in motor activity, demonstrated by the gradual recovery over the days analyzed, confirming the accuracy and effectiveness of the proposed methodology. These results suggest that studies can be conducted to simplify and reduce costs using the technique for digital images of footprints during rats gait in the laboratory.

Exercício imediato versus tardio na regeneração do nervo isquiático de ratos após axoniotmese: análise histomorfométrica e funcional / Immediate versus later exercises for rat sciatic nerve regeneration after axonotmesis: histomorphometric and functional analyses

OBJETIVO: Devido à controvérsia sobre o melhor momento para iniciar o exercício físico, bem como sua influência sobre a regeneração nervosa periférica, este estudo realizou uma análise histomorfométrica e funcional para avaliar a influência do exercício físico em esteira, aplicado nas fases imediata e tardia da regeneração do nervo isquiático de ratos, após axoniotmese. MÉTODOS: Vinte ratos Wistar machos (229,05±18,02g) foram divididos nos grupos: controle (CON); desnervado (D); desnervado+exercício+gaiola (DEG) e desnervado+ gaiola+exercício (DGE). Após 24 horas da axoniotmese, o grupo DEG iniciou o exercício, enquanto o grupo DGE iniciou no 14º dia, com o seguinte protocolo: velocidade=8m/min, inclinação=0 por cento, 30min/dia, durante 14 dias. Em seguida, a porção distal do nervo isquiático foi retirada para análise histomorfométrica. Realizou-se o registro da marcha (pré-operatório e 7º, 14º, 21º, 28º dias pós-operatório (PO)), através do índice funcional do ciático (IFC). RESULTADOS: O número de axônios regenerados nos grupos D foi maior que no CON (p<0,05), não havendo diferença intergrupos D. O diâmetro do axônio do grupo DGE foi maior que do grupo D, enquanto os demais parâmetros morfométricos apenas apresentaram diferença significativa com o grupo CON. Não houve diferença nos valores de IFC intergrupos, enquanto na comparação intragrupos, o 7º e o 14º dias diferem do pré-operatório, 21º e o 28º dias PO. CONCLUSÕES: O protocolo de exercício em esteira aplicado nas fases imediata e tardia, não influenciou o brotamento axonal, o grau de maturação das fibras regeneradas e nem a funcionalidade dos músculos reinervados.

OBJECTIVE: Considering the controversies regarding the best period to begin physical exercise in relation to peripheral nerve regeneration, along with its influence on regeneration, this study accomplished a histomorphometric and functional analysis to evaluate the influence of physical exercise on a treadmill, applied to the immediate and late stages of sciatic nerve regeneration in rats following crushing injury. METHODS: Twenty male Wistar rats (229.05±18.02g) were divided into the following groups: control (CON); denervated (D); denervated+exercise+cage (DEC) and denervated+cage+exercise (DCE). The DEC group started the exercise 24 hours after the nerve injury, while the DCE group started on the 14th day after the injury, with the following protocol: speed=8m/min, inclination=0 percent, 30min/day, for 14 days. The distal segment of the sciatic nerve was then removed for histomorphometric analysis. The gait was recorded before the operation and on the 7th, 14th, 21st and 28th days after the operation, using the sciatic functional index (SFI). RESULTS: The number of regenerated axons in the D groups was greater than in the CON group (p<0.05), without differences between the D groups. The axon diameter in the DCE group was greater than the diameter in the D group, whereas the other morphometric parameters only showed significant differences with the CON group. There was no difference in SFI values between the groups, whereas within the groups, the 7th and 14th days differed from the values before the operation and on the 21st and 28th days after the operation. CONCLUSIONS: The treadmill exercise protocol that was applied to the immediate and late stages of nerve regeneration did not influence the axonal budding, the degree of maturation of the regenerated nerve fibers or the functional performance of the reinnervated muscles.

The Effect of Low Energy Laser Irradiation on the Sciatic Nerve Regeneration of the Rat

OBJECTIVE: To verify the effect of low energy laser irradiation (LELI) on the regeneration of injured sciatic nerve of the rat by showing the functional improvement and the elevated immunoreactivities (IRs) of growth-associated protein 43 (GAP-43). METHOD: Twenty rats, which had standardized compression injuries to the sciatic nerves, received the calculated LELI therapy immediately after the nerve injury and four consecutive days. The functional status was evaluated by sciatic functional index (SFI), and GAP-43-IRs was evaluated by immunohistochemistry and RT-PCR. RESULTS: The SFI was recovered in LELI rats faster than in the control group. Although expression of GAP-43 in the injured sciatic nerve was increased both in the LELI and control groups, the intensities of GAP-43-IRs were much greater in LELI treated group at 1 and 3 weeks after nerve injury. Both SFI and GAP-43-IRs reached the same level at 5 weeks after the nerve injury. CONCLUSION: LELI enhanced the neural regeneration after experimentally induced sciatic nerve injury at the early stage of recovery. Considering the effect of LELI on nerve regeneration was not fully explained until now, this study could suggest the meaningful explanation on the mechanism of LELI effectiveness on neural regeneration.