17β-Estradiol stimulates regeneration of sciatic nerve in female mice

In ovariectomized mice with and without estrogen replacement, regeneration of the sciatic nerve after crush injury was studied. Functional recovery, quantified with sciatic functional index was significantly accelerated in estrogen-treated mice throughout the regeneration. On semi-thin sections of sciatic nerves in estrogen-treated mice we registered a greater total number of regenerating nerve fibers at the first week, and a higher mean axonal area at the third week of regeneration. Our results demonstrated that estrogen treatment enhances regeneration of the sciatic nerve.     

Raloxifene analog LY117018 enhances the regeneration of sciatic nerve in ovariectomized female mice

The aim of this study was to examine the effects of LY117018, a selective estrogen receptor modulator, on peripheral nerve regeneration, using a model of sciatic nerve crush injury in mice. Sciatic functional index, an index of functional recovery, was significantly higher in LY117018 treated mice throughout regeneration. Analysis of semi-thin sections revealed a significant increase in both the total number of regenerating nerve fibers at day 7, and the mean axonal area of myelinated fibers at 7, 14, and 21 days after injury, in LY117018 treated mice. Analysis of axonal transport through retrograde labeling of motor neurons showed that LY117018 increased transport, and ICI 182,780 blocked the effects of LY117018, delineating estrogen receptors as its target. Our study suggests that LY117018 may markedly accelerate peripheral nerve regeneration and functional recovery through activation of estrogen receptors.     

Effect of 17 beta-estradiol on gene expression in lumbar spinal cord following sciatic nerve crush injury in ovariectomized mice

Previously, we observed that estrogen treatment enhances regeneration of the sciatic nerve after crush injury [Brain Res. 943 (2002) 283]. In this research, we studied expression of estrogen receptors and effects of estrogen on gene expression in the lumbar spinal cord, following sciatic nerve crush injury. Using the Atlas Mouse 1.2 Array, changes in the expression of 267 of 1176 genes were registered 4 days after nerve injury. Those genes that exhibited a change in signal intensity ratios of 2-fold or greater were selected as up-regulated (42) or down-regulated (21). In estrogen treated mice, we have observed up-regulation of the genes known to control apoptosis, cell proliferation, and growth, which might account for the positive effects of estrogen on the regeneration of motor neurons. Immunohistochemical staining revealed estrogen receptor-alpha and estrogen receptor-beta localized in the nucleus and cytoplasm of lumbar motor neurons, and in the regenerating neurites of the sciatic nerve. Expression of estrogen receptor-alpha and estrogen receptor-beta mRNA in lumbar spinal cord was shown by traditional RT-PCR. Using real-time quantitative RT-PCR, we demonstrated increased expression of estrogen receptors-alpha and -beta mRNA on the injured side of the lumbar spinal cord. Western blot analysis showed the accumulation of ERs in regenerating sciatic nerve, and revealed a 40% increase of activated ERK1/2 in estrogen treated mice, compared to placebo. Our findings indicate that: (i). axotomized motor neurons increase expression of estrogen receptors-alpha and -beta mRNA, (ii). estrogen mediates the expression of genes which accelerate the growth and maturation of axons, and (iii). estrogen receptors are transported from the perikaryon into regenerating neurites, and estrogen promotes regeneration locally through the non-genomic ERK-activated signaling pathway.     

A deficiency of axonal regeneration in C57BL/6J mice

Axonal regeneration within peripheral nerves and dorsal spinal roots was investigated in inbred strains of mice with known differences in macrophage recruitment and inflammatory functions. During the second week after sciatic nerve crush, counts of regenerating newly myelinated fibres were significantly lower in C57BL/6J mice than in 4 other strains. After dorsal root crush with or without concomitant sciatic nerve transection to enhance regeneration, fibre counts in roots of C57BL/6J were one-fifth of those in A/J mice. Axonal regeneration is subnormal in C57BL/6J mice but this defect appears not to be linked to known deficiencies in macrophage function.     

Estrogen effects on pain sensitivity and neuropeptide expression in rat sensory neurons

While a number of chronic pain conditions are much more prevalent in women than men, the role of estrogen in regulating nociception remains unclear. Estrogen receptors (ER) are known to be expressed in various parts of the nociceptive pathway, including in the small-sized primary sensory neurons of the dorsal root ganglion (DRG). This study evaluated the effects of long term estrogen replacement on pain sensitivity and neuropeptide expression in the DRG of female Sprague Dawley rats. The goal was to evaluate whether estrogen modulates nociceptive neuropeptides in the DRG in a manner consistent with its effects on pain sensitivity. Our results show that long term (28 days) ovariectomy (ovx) of adult rats induces a profound thermal and mechanical hyperalgesia of the hindpaw and tail compared to ovariectomized animals that were continuously estrogen-treated (ovx + E). Significant changes in the expression of two neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP), were observed using immunocytochemistry and in situ hybridization (ISH) in the small lumbar DRG neurons which contain ER. CGRP and SP were differentially regulated by estrogen, with SP showing a significant downregulation at both the peptide and mRNA levels while CGRP and its mRNA were increased in the DRG of estrogen-treated animals. We also evaluated the development of mechanical allodynia after partial sciatic nerve injury and found that both ovx and ovx + E animals developed significant allodynia within a week of the partial nerve injury, which continued for at least one month. The estrogen-treated animals showed a partial amelioration of the extent of the allodynia at 2 weeks post injury. Overall, the results suggest that estrogen has significant anti-nociceptive actions that can be directly correlated with changes in expression of two peptides in the small nociceptive ERα expressing neurons of the DRG.     

Comparative study of peripheral neuropathy and nerve regeneration in NOD and ICR diabetic mice

The non-obese diabetic (NOD) mouse was suggested as an adequate model for diabetic autonomic neuropathy. We evaluated sensory-motor neuropathy and nerve regeneration following sciatic nerve crush in NOD males rendered diabetic by multiple low doses of streptozotocin, in comparison with similarly treated Institute for Cancer Research (ICR) mice, a widely used model for type I diabetes. Neurophysiological values for both strains showed a decline in motor and sensory nerve conduction velocity at 7 and 8 weeks after induction of diabetes in the intact hindlimb. However, amplitudes of compound muscle and sensory action potentials (CMAPs and CNAPs) were significantly reduced in NOD but not in ICR diabetic mice. Morphometrical analysis showed myelinated fiber loss in highly hyperglycemic NOD mice, but no significant changes in fiber size. There was a reduction of intraepidermal nerve fibers, more pronounced in NOD than in ICR diabetic mice. Interestingly, aldose reductase and poly(ADP-ribose) polymerase (PARP) activities were increased already at 1 week of hyperglycemia, persisting until the end of the experiment in both strains. Muscle and nerve reinnervation was delayed in diabetic mice following sciatic nerve crush, being more marked in NOD mice. Thus, diabetes of mid-duration induces more severe peripheral neuropathy and slower nerve regeneration in NOD than in ICR mice.     

Impaired peripheral nerve regeneration in type‐2 diabetic mouse model

Peripheral neuropathy is one of the most common and serious complications of type‐2 diabetes. Diabetic neuropathy is characterized by a distal symmetrical sensorimotor polyneuropathy, and its incidence increases in patients 40 years of age or older. In spite of extensive research over decades, there are few effective treatments for diabetic neuropathy besides glucose control and improved lifestyle. The earliest changes in diabetic neuropathy occur in sensory nerve fibers, with initial degeneration and regeneration resulting in pain. To seek its effective treatment, here we prepared a type‐2 diabetic mouse model by giving mice 2 injections of streptozotocin and nicotinamide and examining the ability for nerve regeneration by using a sciatic nerve transection‐regeneration model previously established by us. Seventeen weeks after the last injection, the mice exhibited symptoms of type‐2 diabetes, that is, impaired glucose tolerance, decreased insulin level, mechanical hyperalgesia, and impaired sensory nerve fibers in the plantar skin. These mice showed delayed functional recovery and nerve regeneration by 2 weeks compared with young healthy mice and by 1 week compared with age‐matched non‐diabetic mice after axotomy. Furthermore, type‐2 diabetic mice displayed increased expression of PTEN in their DRG neurons. Administration of a PTEN inhibitor at the cutting site of the nerve for 4 weeks promoted the axonal transport and functional recovery remarkably. This study demonstrates that peripheral nerve regeneration was impaired in type‐2 diabetic model and that its combination with sciatic nerve transection is suitable for the study of the pathogenesis and treatment of early diabetic neuropathy.     

含神经生长因子的壳聚糖导管桥接周围神经缺损的实验研究

目的 应用含有神经生长因子(NGF)的壳聚糖神经导引管作为神经再生室桥接大鼠坐骨神经缺损，观察其对神经再生的作用。方法 选用Wistat大鼠60只，手术造成右后肢坐骨神经长约15mm的缺损，A组以含有NGF的壳聚糖神经导引管桥接神经缺损，B组则单纯采用壳聚糖导管，分别于术后4、12、24周进行大体及显微解剖观察、组织学检查、电镜观察和神经电生理测定。结果 A组在促进神经再生、加快血管化进程、再生神经纤维排列规律化、提高再生神经髓鞘化、加速再生神经功能重建等方面均优于B组。结论 壳聚糖神经导引管可以为大鼠坐骨神经再生提供一个良好的再生微环境，NGF对神经再生有显促进作用。     

Ursolic acid induces neural regeneration after sciatic nerve injury

In this study,we aimed to explore the role of ursolic acid in the neural regeneration of the injured sciatic nerve.BALB/c mice were used to establish models of sciatic nerve injury through unilateral sciatic nerve complete transection and microscopic anastomosis at 0.5 cm below the ischial tuberosity.The successfully generated model mice were treated with 10,5,or 2.5 mg/kg ursolic acid via intraperitoneal injection.Enzyme-linked immunosorbent assay results showed that serum S100 protein expression level gradually increased at 1-4 weeks after sciatic nerve injury,and significantly decreased at 8 weeks.As such,ursolic acid has the capacity to significantly increase S100 protein expression levels.Real-time quantitative PCR showed that S100 mRNA expression in the L4-6 segments on the injury side was increased after ursolic acid treatment.In addition,the muscular mass index in the soleus muscle was also increased in mice treated with ursolic acid.Toluidine blue staining revealed that the quantity and average diameter of myelinated nerve fibers in the injured sciatic nerve were significantly increased after treatment with ursolic acid.10 and 5 mg/kg of ursolic acid produced stronger effects than 2.5 mg/kg of ursolic acid.Our findings indicate that ursolic acid can dose-dependently increase S100 expression and promote neural regeneration in BALB/c mice following sciatic nerve injury.     

Valproic acid enhances axonal regeneration and recovery of motor function after sciatic nerve axotomy in adult rats

It has recently been demonstrated that valproic acid (VPA) robustly promotes neurite outgrowth, activates the extracellular signal regulated kinase pathway, and increases growth cone-associated protein 43 and bcl-2 levels in cultured human neuroblastoma SH-SY5Y cells. We hypothesized that VPA could also enhance peripheral nerve regeneration in adult animals. To test this hypothesis, we examined the effects of VPA (300 mg/kg daily for 16 weeks) on sciatic axonal regeneration following single or conditional axotomies in rats. The results showed that in VPA-treated rats there was a significant increase in the total numbers of regenerated myelinated nerve fibers and reinnervated muscle fibers in comparison with those rats not treated with VPA. As measured by sciatic function index and toe spread index, the motor function of the reinnervated hind limbs of rats receiving single axotomy without VPA treatment significantly improved at week 8 and reached plateau levels at about week 11, whereas the motor function of the reinnervated hind limbs of rats receiving single axotomy plus VPA and rats receiving conditional axotomy with or without VPA treatment significantly improved at week 4 and reached plateau levels at about week 8; there was no significant difference of the motor function among the three later groups. The results demonstrated that VPA is able to enhance sciatic nerve regeneration and recovery of motor function in adult rats, suggesting the potential clinical application of VPA for the treatment of peripheral nerve injury in humans.     

Lack of effects of transforming growth factor-alpha gene knockout on peripheral nerve regeneration may result from compensatory mechanisms.

Transforming growth factor-alpha (TGF-alpha), previously identified as a major member of the epidermal growth factor (EGF) family of growth factors, plays a role in proliferation, differentiation, and survival of neuronal and glial precursors and is implicated in development of the nervous system. However, its roles in nerve injury-induced responses remain obscure. The current study examined roles of endogenous TGF-alpha in peripheral nerve regeneration using sciatic nerve injury models with TGF-alpha knockout mice. Three weeks after a sciatic nerve crush, no significant differences were found between TGF-alpha wild-type and mutant mice in the number of retrogradely labeled L5 dorsal root ganglion (DRG) sensory neurons and L5 spinal cord motor neurons and in the morphology of myelinated regenerating nerve fibers, indicating that TGF-alpha is not essential for sensory and motor nerve regeneration. To assess a possible functional redundancy among TGF-alpha-related ligands in response to a nerve injury, mRNA expression of the EGF family was analyzed by RT-PCR in L4/L5 DRG pools and distal degenerating sciatic nerve segments after sciatic nerve ligation. Prior to and 1 day after ligation, there was a higher level of EGF-R mRNA in DRGs and in nerve in TGF-alpha null mice compared to wild types, and there was an induction of ligand amphiregulin mRNA in DRGs in mutant mice in place of the TGF-alpha upregulation present in wild types. These results indicate that TGF-alpha gene knockout does not affect peripheral nerve regeneration, probably due to a functional redundancy within the EGF family through a compensatory expression mechanism at both the receptor and ligand levels in TGF-alpha knockout mice.     

Targeted disruption of the FGF-2 gene affects the response to peripheral nerve injury

Basic fibroblast growth factor (FGF-2) is involved in the development, maintenance, and survival of the nervous system. To study the physiological role of endogenous FGF-2 during peripheral nerve regeneration, we analyzed sciatic nerves of FGF-2-deleted mice by using morphometric, morphological, and immunocytochemical methods. Quantification of number and size of myelinated axons in intact sciatic nerves revealed no difference between wild-type and FGF-2 knock-out (ko) animals. One week after nerve crush, FGF-2 ko mice showed about five times more regenerated myelinated axons with increased myelin and axon diameter in comparison to wild-types close to the injury site. In addition, quantitative distribution of macrophages and collapsed myelin profiles suggested faster Wallerian degeneration in FGF-2-deleted mice close to the lesion site. Our results suggest that endogenous FGF-2 is crucially involved in the early phase of peripheral nerve regeneration possibly by regulation of Schwann cell differentiation.     

Effect of age and maturation on sudomotor nerve regeneration in mice

This study demonstrates that regeneration of unmyelinated sudomotor axons in mice becomes progressively slower during aging. Identical lesions were made in mice aged 0, 2, 4, 6, 7, 24 and 60 weeks. The peroneal, sural and saphenous nerves were cut and tied to prevent regeneration. The sciatic nerve was then frozen at the thigh, leaving the hind paw completely denervated. By 7 days, sweat glands (SGs) of the paw had ceased sweating after pilocarpine injection. Subsequent regeneration of sudomotor axons was judged by the rate of return of pilocarpine sensitivity. SGs in the hind paws of normal newborn mice did not sweat at birth. Cholinergic stimulation first activated sweating at 13 days of life. The number of responsive SGs increased progressively to reach the adult level by 30 days. In one-week-old mice, whose sciatic nerve had been sectioned, the SG response to cholinergic stimulation was very delayed in time and reduced in number. Sweat glands of young mice, between 2 and 4 weeks of age, regained cholinergic sensitivity at a faster rate than mature animals and attained normal SG counts. Throughout a broad intermediate range of age in adulthood (7-24 weeks), the rate of sudomotor nerve regeneration was the same, but in older mice (60 weeks) it was slower and less complete.     

Reinnervation of the tibialis anterior following sciatic nerve crush injury: a confocal microscopic study in transgenic mice

Transgenic mice whose axons and Schwann cells express fluorescent chromophores enable new imaging techniques and augment concepts in developmental neurobiology. The utility of these tools in the study of traumatic nerve injury depends on employing nerve models that are amenable to microsurgical manipulation and gauging functional recovery. Motor recovery from sciatic nerve crush injury is studied here by evaluating motor endplates of the tibialis anterior muscle, which is innervated by the deep peroneal branch of the sciatic nerve. Following sciatic nerve crush, the deep surface of the tibialis anterior muscle is examined using whole mount confocal microscopy, and reinnervation is characterized by imaging fluorescent axons or Schwann cells (SCs). One week following sciatic crush injury, 100% of motor endplates are denervated with partial reinnervation at 2 weeks, hyperinnervation at 3 and 4 weeks, and restoration of a 1:1 axon to motor endplate relationship 6 weeks after injury. Walking track analysis reveals progressive recovery of sciatic nerve function by 6 weeks. SCs reveal reduced S100 expression within 2 weeks of denervation, correlating with regression to a more immature phenotype. Reinnervation of SCs restores S100 expression and a fully differentiated phenotype. Following denervation, there is altered morphology of circumscribed terminal Schwann cells demonstrating extensive process formation between adjacent motor endplates. The thin, uniformly innervated tibialis anterior muscle is well suited for studying motor reinnervation following sciatic nerve injury. Confocal microscopy may be performed coincident with other techniques of assessing nerve regeneration and functional recovery.     

Myelinated fiber regeneration after sciatic nerve crush: morphometric observations in young adult and aging mice and the effects of macrophage suppression and conditioning lesions.

To study myelinated nerve fiber regeneration during aging, the right sciatic nerves of 6- and 24-month-old mice were crushed at the sciatic notch. Two, 4, and 8 weeks later, both groups of mice were perfused. The sciatic nerves were processed so that the transverse sections of each nerve subsequently studied by light and electron microscopy included the entire posterior tibial fascicle 5 mm distal to the crush site. Two weeks after axotomy, fascicles of aging mice contained significantly fewer regenerated myelinated fibers than those of young adults. After 4 weeks, the difference in the number of myelinated fibers was less. However, measurements of myelinated fibers in fascicles of aging mice showed that areas of Schwann cell cytoplasm and myelin were significantly reduced at all intervals. In contrast, although axon diameters in aging mice were somewhat less 2 weeks after crushing, the difference decreased with time, suggesting that in nerves of aging mice, regenerative responses of Schwann cells were more affected than those of axons. Other experiments in young mice showed that myelinated fiber regeneration could be retarded by suppressing macrophage responses and was not significantly changed by conditioning lesions before crush injury.     

氧化巴西苏木素干预BALB/c小鼠坐骨神经损伤

氧化巴西苏木素已经被证实对中枢神经的再生具有免疫调节作用,但其对坐骨神经损伤的效应尚无共识。应用Western blot及Real-time PCR的检测显示,经16,8 g/kg氧化巴西苏木素干预后,坐骨神经损伤小鼠L4~6脊髓节段的S100蛋白和mRNA明显高于4g/kg氧化巴西木素干预的小鼠及模型组。髓鞘固兰染色显示,16,8 g/kg氧化巴西木素干预的神经再生情况也明显优于4g/kg氧化巴西木素干预及模型组。同时电生理检查和免疫组化检测进一步证实了氧化巴西苏木素对BALB/c小鼠坐骨神经损伤有修复作用。由此认为,氧化巴西苏木素对脊髓前角细胞中S100有活化作用,促进了坐骨神经再生与修复,且以高中剂量最为显著。     

Effects of neutralizing antibodies to TNF-alpha on pain-related behavior and nerve regeneration in mice with chronic constriction injury

Inhibition of proinflammatory cytokines reduces hyperalgesia in animal models of painful neuropathy. We set out to investigate the consequences of this treatment for nerve regeneration. Here we examined the sequels of epineurial application of neutralizing antibodies to tumor necrosis factor-alpha (TNF) in chronic constriction injury (CCI) of the sciatic nerve in C57/BL 6 mice. The mice were tested behaviorally for manifestations of thermal hyperalgesia and mechanical allodynia. Nerve regeneration was assessed by morphometry of myelinated nerve fibers in the sciatic nerve and of the epidermal innervation density in the glabrous skin of the hindpaws. Antibodies to TNF reduced thermal hyperalgesia and mechanical allodynia after CCI. Myelinated fiber density in the sciatic nerve was reduced to 30% of normal on day 7 after surgery, and reached 60% on day 45, with no difference between antibody-treated and untreated animals. Epidermal innervation density as shown by PGP 9.5 and CGRP immunohistochemistry was reduced to 25-47% at both time points after CCI, again without differences between antibody treated and untreated mice. Myelinated fiber density but not epidermal innervation density was correlated to thermal and mechanical withdrawal thresholds. We conclude that neutralization of endoneurial TNF attenuates pain related behavior but has no effect on nerve regeneration. Furthermore, the number of epidermal nerve fibers is not relevant to the magnitude of behavioral hyperalgesia in CCI.     

Beta secretase activity in peripheral nerve regeneration

While the peripheral nervous system has the capacity to regenerate following a nerve injury, it is often at a slow rate and results in unsatisfactory recovery, leaving patients with reduced function. Many regener-ation associated genes have been identified over the years, which may shed some insight into how we can manipulate this intrinsic regenerative ability to enhance repair following peripheral nerve injuries. Our lab has identified the membrane bound protease beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1), or beta secretase, as a potential negative regulator of peripheral nerve regeneration. When beta secretase activity levels are abolished via a null mutation in mice, peripheral regeneration is enhanced fol-lowing a sciatic nerve crush injury. Conversely, when activity levels are greatly increased by overexpressing beta secretase in mice, nerve regeneration and functional recovery are impaired after a sciatic nerve crush injury. In addition to our work, many substrates of beta secretase have been found to be involved in reg-ulating neurite outgrowth and some have even been identified as regeneration associated genes. In this review, we set out to discuss BACE1 and its substrates with respect to axonal regeneration and speculate on the possibility of utilizing BACE1 inhibitors to enhance regeneration following acute nerve injury and potential uses in peripheral neuropathies.     

Functional recovery after peripheral nerve injury is dependent on the pro-inflammatory cytokines IL-1β and TNF: implications for neuropathic pain

IL-1β and TNF are potential targets in the management of neuropathic pain after injury. However, the importance of the IL-1 and TNF systems for peripheral nerve regeneration and the mechanisms by which these cytokines mediate effects are to be fully elucidated. Here, we demonstrate that mRNA and protein levels of IL-1β and TNF are rapidly upregulated in the injured mouse sciatic nerve. Mice lacking both IL-1β and TNF, or both IL-1 type 1 receptor (IL-1R1) and TNF type 1 receptor (TNFR1), showed reduced nociceptive sensitivity (mechanical allodynia) compared with wild-type littermates after injury. Microinjecting recombinant IL-1β or TNF at the site of sciatic nerve injury in IL-1β- and TNF-knock-out mice restored mechanical pain thresholds back to levels observed in injured wild-type mice. Importantly, recovery of sciatic nerve function was impaired in IL-1β-, TNF-, and IL-1β/TNF-knock-out mice. Notably, the infiltration of neutrophils was almost completely prevented in the sciatic nerve distal stump of mice lacking both IL-1R1 and TNFR1. Systemic treatment of mice with an anti-Ly6G antibody to deplete neutrophils, cells that play an essential role in the genesis of neuropathic pain, did not affect recovery of neurological function and peripheral axon regeneration. Together, these results suggest that targeting specific IL-1β/TNF-dependent responses, such as neutrophil infiltration, is a better therapeutic strategy for treatment of neuropathic pain after peripheral nerve injury than complete blockage of cytokine production.     

The mechanism of astragaloside IV promoting sciatic nerve regeneration

3-O-beta-D-xylopyranosyl-6-O-beta-D-glucopyranosyl-cycloastragenol (astragaloside Ⅳ), the main active component of the traditional Chinese medicine astragalus membranaceus, has been shown to be neuroprotective. This study investigated whether astragaloside Ⅳ could promote the repair of injured sciatic nerve. Denervated sciatic nerve of mice was subjected to anastomosis. The mice were intraperitoneally injected with 10, 5, 2.5 mg/kg astragaloside Ⅳ per day for 8 consecutive days. Western blot assay and real-time PCR results demonstrated that growth-associated protein-43 expression was upregulated in mouse spinal cord segments L4-6 after intervention with 10, 5, 2.5 mg/kg astragaloside Ⅳ per day in a dose-dependent manner. Luxol fast blue staining and electrophysiological detection suggested that astragaloside Ⅳ elevated the number and diameter of myelinated nerve fibers, and simultaneously increased motor nerve conduction velocity and action potential amplitude in the sciatic nerve of mice. These results indicated that astragaloside Ⅳ contributed to sciatic nerve regeneration and functional recovery in mice. The mechanism underlying this effect may be associated with the upregulation of growth-associated protein-43 expression.