Altered Expression of Inducible Nitric Oxide Synthase After Sciatic Nerve Injury in Rat

Nitric oxide is known to contribute to neuronal damage as well as to peripheral neuronal regeneration following injury. Sciatic nerve injury is a common and serious complication of intramuscular injections. In order to ascertain the role of inducible nitric oxide synthase (iNOS) in the injured sciatic nerve, we studied the expression of this enzyme by RT-PCR and immunohistochemistry, in a rat model of sciatic nerve injury. In sham-operated control rats iNOS expression was undetectable by immunohistochemistry and its mRNA level was also very low. In contrast, in the experimental group that was subjected to sciatic nerve injury, both mRNA and protein of iNOS were found to be significantly elevated. The protein level of iNOS, as revealed by positive immunostaining, peaked at 7 days post-surgery followed by a decrease. Similarly, the iNOS mRNA levels remained elevated at 1, 3, 7 days but declined to very low level by day 21, after surgery. This study indicates that the increased expression of iNOS after sciatic nerve injury in rats may contribute to nerve regeneration. Thus our results suggest that excessive expression of iNOS after nerve injury is not conducive to nerve regeneration.     

Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.     

大鼠坐骨神经挤压损伤导致脊髓单突触反射回返性抑制的长时程减弱

坐骨神经损伤是临床常见的周围神经疾病。神经损伤后再生肌肉和运动神经元会出现各种功能障碍,虽然其中一部分因素已被阐明,但多局限于受损神经局部,而对于再生后脊髓运动神经元的回返性抑制(recurrent inhibition,RI)通路的功能变化却很少被报道。本文研究大鼠短暂坐骨神经损伤后,恢复神经再支配(reinnervation)情况下,脊髓RI通路的功能变化。在正常或坐骨神经挤压(crush)受损后的成年大鼠上,通过刺激离断的脊髓背根(L5),在外侧腓肠肌-比目鱼肌(lateral gas-trocnemius-soleus,LG-S)神经或内侧腓肠肌(medial gastrocnemius,MG)神经记录单突触反射(monosynaptic reflex,MSR),并同时在另一神经给予条件性刺激,以检测LG-S和MG运动神经元间RI的变化。结果显示:(1)脊髓运动神经元的RI在坐骨神经挤压受损后即基本丢失(<5周),至损伤6周后部分恢复至正常的50%,并至少维持至损伤14周后;(2)一侧的坐骨神经损伤对对侧的RI没有影响;(3)外周神经损伤后,免疫组织化学方法显示脊髓运动神经元数目本身并不发生减少。以上...     

Down‐regulation of long non‐coding RNA MEG3 promotes Schwann cell proliferation and migration and repairs sciatic nerve injury in rats

Peripheral nerve injury and regeneration are complex processes and involve multiple molecular and signalling components. However, the involvement of long non‐coding RNA (lncRNA) in this process is not fully clarified. In this study, we evaluated the expression of the lncRNA maternally expressed gene 3 (MEG3) in rats after sciatic nerve transection and explored its potential mechanisms. The expression of lncRNA MEG3 was up‐regulated following sciatic nerve injury and observed in Schwann cells (SCs). The down‐regulation of lncRNA MEG3 in SCs enhanced the proliferation and migration of SCs via the PTEN/PI3K/AKT pathway. The silencing of lncRNA MEG3 promoted the migration of SCs and axon outgrowth in rats after sciatic nerve transection and facilitated rat nerve regeneration and functional recovery. Our findings indicated that lncRNA MEG3 may be involved in nerve injury and injured nerve regeneration in rats with sciatic nerve defects by regulating the proliferation and migration of SCs. This gene may provide a potential therapeutic target for improving peripheral nerve injury.     

Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression

Minocycline, a tetracycline antibiotic, is known to exert vasculoprotective effects independent of its anti-bacterial properties; however the underlying molecular mechanisms are not completely understood. Reversion Inducing Cysteine Rich Protein with Kazal Motifs (RECK) is a cell surface expressed, membrane anchored protein, and its overexpression inhibits cancer cell migration. We hypothesized that minocycline inhibits platelet-derived growth factor (PDGF)-induced human aortic smooth muscle cell (SMC) proliferation and migration via RECK upregulation. Our data show that the BB homodimer of recombinant PDGF (PDGF-BB) induced SMC migration and proliferation, effects significantly blunted by pre-treatment with minocycline. Further investigations revealed that PDGF-BB induced PI3K-dependent AKT activation, ERK activation, reactive oxygen species generation, Nuclear Factor-κB and Activator Protein-1 activation, microRNA (miR)-221 and miR-222 induction, RECK suppression, and matrix metalloproteinase (MMP2 and 9) activation, effects that were reversed by minocycline. Notably, minocycline induced RECK expression dose-dependently within the therapeutic dose of 1–100 μM, and silencing RECK partially reversed the inhibitory effects of minocycline on PDGF-BB-induced MMP activation, and SMC proliferation and migration. Further, targeting MMP2 and MMP9 blunted PDGF-BB-induced SMC migration. Together, these results demonstrate that minocycline inhibits PDGF-BB-induced SMC proliferation and migration by restoring RECK, an MMP inhibitor. These results indicate that the induction of RECK is one of the mechanisms by which minocycline exerts vasculoprotective effects.     

Inhibition of Ras extracellular-signal-regulated kinase (ERK) mediated signaling promotes ciliary neurotrophic factor (CNTF) expression in Schwann cells

Ciliary neurotrophic factor (CNTF) can prevent injury-induced motor neuron death. However, it is also evident that expression of CNTF in Schwann cells is suppressed during nerve regeneration. In this report, we have addressed the mechanism underlying the down-regulation of CNTF expression in injured nerves using a mouse Schwann cell line IMS32 and mouse sciatic nerve. In IMS32 cells, activation of the Ras extracellular-signal-regulated kinase (ERK) pathway by adenoviral vector-mediated expression of dominant active MEK1 did not alter a basal level of CNTF expression, whereas inhibition of the Ras-ERK pathway by using adenoviral vectors resulted in a marked increase in CNTF expression. This inverse relation between before and after axotomy was also observed in mouse sciatic nerve. In the axotomized sciatic nerve, the phosphorylated ERK was markedly increased; in contrast, the expression of CNTF was markedly decreased. These findings suggest that an inactive state of ERK is crucial for the CNTF expression in Schwann cells, and that activation of ERK following nerve injury critically influences the expression of CNTF. This might well explain why CNTF is highly expressed in quiescent Schwann cells in the peripheral nervous system, and also why CNTF is not abundant in axotomized nerves or cultured Schwann cells in which the proliferation signal is obviously active.     

Molecular approaches to nerve regeneration.

Current research into regeneration of the nervous system has focused on defining the molecular events that occur during regeneration. One well-characterized system for studying nerve regeneration is the sciatic nerve of rat. Numerous studies have characterized the sequence of events that occur after a crush injury to the sciatic nerve (Cajal 1928; Hall 1989). These events include axon and myelin breakdown, changes in the permeability of the blood vessels, proliferation of Schwann cells, invasion of macrophages, and the phagocytosis of myelin fragments by Schwann cells and macrophages. The distal segment of the injured sciatic nerve provides a supportive environment for the regeneration of the nerve fibres (Cajal 1928; David & Aguayo 1981). Within a period of weeks, the injured sciatic nerve is able to regrow and successfully reinnervate the appropriate targets. Some of the molecules that provide trophic support for the regrowing nerve fibres have been identified, including nerve growth factor (NGF) (Heumann et al. 1987) and glial maturation factor beta (Bosch et al. 1989). Another class of molecules show changes in their rates of synthesis during regeneration, including both proteins (Skene & Shooter 1983; Muller et al. 1986) and mRNA species (Trapp et al. 1988; Meier et al. 1989). To better understand nerve regeneration, we have taken two, parallel molecular approaches to study the events associated with regeneration. The first of these is to study in detail the mechanism of action of a molecule that has been implicated in the regeneration process, nerve growth factor. The second approach is to identify novel gene sequences which are regulated during regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deletion of p75NTR impairs regeneration of peripheral nerves in mice

AimsAfter peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves.Main methodsIn p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry.Key findingsThe results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice.SignificanceOur data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves.     

In vivo USPIO magnetic resonance imaging shows that minocycline mitigates macrophage recruitment to a peripheral nerve injury

ABSTRACT: BACKGROUND: Minocycline has proven anti-nociceptive effects, and delays the development of allodynia/hyperalgesia after peripheral nerve injury. However, the mechanism by which this occurs remains unclear. Inflammatory cells, in particular macrophages, are critical components of the response to nerve injury. Using ultrasmall superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI) to monitor macrophage trafficking, the purpose of this project is to determine whether minocycline modulates macrophage trafficking to the site of nerve injury in vivo and, in turn, results in altered pain thresholds. RESULTS: Animal experiments were approved by Stanford IACUC. A model of neuropathic pain was created using the Spared Nerve Injury (SNI) model that involves ligation of the left sciatic nerve in the left thigh of adult Sprague-Dawley rats. Animals with SNI and uninjured animals (control) were then injected with/without USPIOs (300umol/kg IV) and with/without minocycline (50mg/kg IP). Bilateral sciatic nerves were scanned with a volume coil in a 7T magnet 7 days after USPIO administration. Fluid-sensitive MR images were obtained, and ROIs were placed on bilateral sciatic nerves to quantify signal intensity. Pain behavior modulation by minocycline was measured using the Von Frey filament test. Sciatic nerves were ultimately harvested at day 7, fixed in 10% buffered formalin and stained for the presence of iron oxide-laden macrophages. Behavioral measurements confirmed the presence of allodynia in the neuropathic pain model while the uninjured and minocycline-treated injured group had significantly higher paw withdrawal thresholds (p<0.011). Decreased MR signal is observed in the SNI group that received USPIOs (3.3+/-0.5%) compared to the minocycline-treated SNI group that received USPIOs (15.2+/-4.5%) and minocycline-treated group (no USPIOs; 41.2+/-2.3%) (p<0.04). Histology of harvested sciatic nerve specimens confirmed the presence USPIOs at the nerve injury site in the SNI group without minocycline treatment. CONCLUSION: Animals with neuropathic pain in the left hindpaw show increased trafficking of USPIO-laden macrophages to the site of sciatic nerve injury. Minocycline appears to retard the migration of macrophages to the nerve injury site, which may partly explain its anti-nociceptive effects. USPIO-MRI is an effective in vivo imaging tool to study the role of macrophages in the development of neuropathic pain.     

Regenerating Sciatic Nerve Does Not Utilize Circulating Cholesterol

The rapid accumulation of myelin in the peripheral nervous system during the early postnatal period requires large amounts of cholesterol, a major myelin lipid. All of the cholesterol accumulating in the developing rat sciatic nerve is synthesized locally within the nerve, rather than being derived from the supply in lipoproteins in the systemic circulation (Jurevics and Morell, J. Lipid Res. 5:112–120; 1994). Since this lack of utilization of circulating cholesterol may relate to exclusion by the blood-nerve barrier, we examined the sources of cholesterol needed for regeneration following nerve injury, when the blood-nerve barrier is breached. One sciatic nerve was crushed or transected, and at various times later, the rate of cholesterol accumulation was compared with the rate of local in vivo synthesis of cholesterol within the nerve, utilizing intraperitoneally injected ³H₂O as precursor. The accumulation of additional cholesterol in nerve during regeneration and remyelination could all be accounted for by that locally synthesized within the nerve. There was also an increase in cholesterol esters in injured nerve segments; in crushed nerves, these levels decreased during regeneration and remyelination, consistent with reutilization of cholesterol originally salvaged by phagocytic macrophages and Schwann cells. Thus, regeneration and remyelination following injury in sciatic nerve utilizes both salvaged cholesterol and cholesterol synthesized locally within the nerve, but not cholesterol from the circulation.     

Therapeutic Effect of Vinorine on Sciatic Nerve Injured Rat

Vinorine is a monoterpenoid indole alkaloid, a type of natural alkaloids. Growing reports exhibited the numerous pharmacology activities of vinorine such as anti-inflammation, anti-bacterial and anti-tumor. In this study, the effect of vinorine injection (7.5, 15 and 30 mg/kg) on motor function, sensation and nerve regeneration in sciatic nerve crush injury rat was investigated. The results of behavioral analysis, electrophysiological analysis and muscle histological analysis suggested that vinorine promoted the motor function recovery after sciatic nerve injury. The results of mechanical withdrawal thresholds assay and hot plate test demonstrated that vinorine improved the sensation recovery after sciatic nerve injury. The results of Fluoro-gold retrograde labeling, transmission electron microscope assay, toluidine blue and HE staining showed that vinorine attenuated the nerve damage caused by sciatic nerve injury and promoted the nerve regeneration. Furthermore, nerve growth factor (NGF) and its downstream extracellular signal-regulated kinase (ERK) signaling pathway participated in the neuro-recovery effect of vinorine after crush. In conclusion, vinorine treatment accelerated the sciatic nerve regeneration, motor function recovery and sensation recovery after crush injury via regulation of NGF and ERK activity. These results suggested that vinorine is a promising agent for never injury therapy.     

Human Amniotic Fluid Mesenchymal Stem Cells in Combination with Hyperbaric Oxygen Augment Peripheral Nerve Regeneration

Purpose Attenuation of pro-inflammatory cytokines and associated inflammatory cell deposits rescues human amniotic fluid mesenchymal stem cells (AFS) from apoptosis. Hyperbaric oxygen (HBO) suppressed stimulus-induced pro-inflammatory cytokine production in blood-derived monocyte-macrophages. Herein, we evaluate the beneficial effect of hyperbaric oxygen on transplanted AFS in a sciatic nerve injury model. Methods Peripheral nerve injury was produced in Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. The AFS were embedded in fibrin glue and delivered to the injured site. Hyperbaric oxygen (100% oxygen, 2 ATA, 60 min/day) was administered 12 h after operation for seven consecutive days. Transplanted cell apoptosis, oxidative stress, inflammatory cell deposits and associated chemokines, pro-inflammatory cytokines, motor function, and nerve regeneration were evaluated 7 and 28 days after injury. Results Crush injury induced an inflammatory response, disrupted nerve integrity, and impaired nerve function in the sciatic nerve. However, crush injury-provoked inflammatory cytokines, deposits of inflammatory cytokines, and associated macrophage migration chemokines were attenuated in groups receiving hyperbaric oxygen but not in the AFS-only group. No significant increase in oxidative stress was observed after administration of HBO. In transplanted AFS, marked apoptosis was detected and this event was reduced by HBO treatment. Increased nerve myelination and improved motor function were observed in AFS-transplant, HBO-administrated, and AFS/HBO-combined treatment groups. Significantly, the AFS/HBO combined treatment showed the most beneficial effect. Conclusion AFS in combination with HBO augment peripheral nerve regeneration, which may involve the suppression of apoptotic death in implanted AFS and the attenuation of an inflammatory response detrimental to peripheral nerve regeneration.     

Axonal Regeneration after Sciatic Nerve Lesion Is Delayed but Complete in GFAP- and Vimentin-Deficient Mice

Peripheral axotomy of motoneurons triggers Wallerian degeneration of injured axons distal to the lesion, followed by axon regeneration. Centrally, axotomy induces loss of synapses (synaptic stripping) from the surface of lesioned motoneurons in the spinal cord. At the lesion site, reactive Schwann cells provide trophic support and guidance for outgrowing axons. The mechanisms of synaptic stripping remain elusive, but reactive astrocytes and microglia appear to be important in this process. We studied axonal regeneration and synaptic stripping of motoneurons after a sciatic nerve lesion in mice lacking the intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP) and vimentin, which are upregulated in reactive astrocytes and Schwann cells. Seven days after sciatic nerve transection, ultrastructural analysis of synaptic density on the somata of injured motoneurons revealed more remaining boutons covering injured somata in GFAP^–/–Vim^–/– mice. After sciatic nerve crush in GFAP^–/–Vim^–/– mice, the fraction of reinnervated motor endplates on muscle fibers of the gastrocnemius muscle was reduced 13 days after the injury, and axonal regeneration and functional recovery were delayed but complete. Thus, the absence of GFAP and vimentin in glial cells does not seem to affect the outcome after peripheral motoneuron injury but may have an important effect on the response dynamics.     

c-fos Expression in Gracilothalamic Tract Neurons after Electrical Stimulation of the Injured Sciatic Nerve in the Adult Rat

The number of c-fos protein-like immunoreactive (Fos-LI) cells in the gracile nucleus was determined after electrical stimulation at Aα/Aβ-fiber strength of the normal and of the previously injured sciatic nerve in adult rats. No Fos-LI cells were seen after electrical stimulation of the noninjured sciatic nerve, or after sciatic nerve injury without electrical stimulation. However, stimulation 21 days after sciatic nerve transection resulted in numerous Fos-LI cells in the ipsilateral gracile nucleus. Combined Fos immunocytochemistry and retrograde labeling from the thalamus showed that the majority (76%; range = 70–80%) of the cells in the gracile nucleus that expressed Fos-LI after nerve injury projected to the thalamus. The results indicate that morphological, biochemical, and physiological alterations in primary sensory central endings and second-order neurons, which have earlier been demonstrated in the dorsal column nuclei after peripheral nerve injury, are accompanied by changes in the c-fos gene activation pattern after stimulation of the injured sciatic nerve. A substantial number of the c-fos-expressing neurons project to the thalamus.     

Urokinase-Type Plasminogen Activator Induces BV-2 Microglial Cell Migration Through Activation of Matrix Metalloproteinase-9

In response to brain injury, microglia migrate and accumulate in the affected sites, which is an important step in the regulation of inflammation and neuronal degeneration/regeneration. In this study, we investigated the effect of urokinase-type plasminogen activator (uPA) on the BV-2 microglial cell migration. At resting state, BV-2 microglial cells secreted uPA and the release of uPA was increased by ATP, a chemoattractant released from injured neuron. The migration of BV-2 cell was significantly induced by uPA and inhibited by uPA inhibitors. In this condition, uPA increased the activity of matrix metalloproteinase (MMP-9) and the inhibition of MMP activity with pharmacological inhibitors against either uPA (amiloride) or MMP (phenanthrolene and SB-3CT) effectively prevented BV2 cell migration. Interestingly, the level of MMP-9 protein and mRNA in the cell were not changed by uPA. These results suggest that the increase of MMP-9 activity by uPA is regulated at the post-translational level, possibly via increased activation of the enzyme. Unlike the uPA inhibitor, plasmin inhibitor PAI-1 only partially inhibited uPA-induced cell migration and MMP-9 activation. The incubation of recombinant MMP-9 with uPA resulted in the activation of MMP-9. These results suggest that uPA plays a critical role in BV-2 microglial cell migration by activating pro-MMP-9, in part by its direct action on MMP-9 and also in part by the activation of plasminogen/plasmin cascade.     

Combination of G-CSF Administration and Human Amniotic Fluid Mesenchymal Stem Cell Transplantation Promotes Peripheral Nerve Regeneration

Amniotic fluid mesenchymal stem cells (AFS) harbor the potential to improve peripheral nerve injury by inherited neurotrophic factor secretion, but present the drawback of the short-term survival after transplantation. Granulocyte-colony stimulating factor (G-CSF) has a diversity of functions, including anti-inflammatory and anti-apoptotic effects. This study was conducted to evaluate whether G-CSF could augment the neuroprotective effect of transplanted AFS against peripheral nerve injury. The potential involvement of anti-inflammation/anti-apoptosis effect was also investigated. Peripheral nerve injury was produced in Sprauge-Dawley rats by crushing left sciatic nerve using a vessel clamp. The AFS were embedded in fibrin glue and delivered to the injured site. G-CSF (50 μg/kg) was administrated by intra-peritoneal injection for 7 consecutive days. Cell apoptosis, inflammatory cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. Crush injury induced inflammatory response, disrupted nerve integrity, and impaired nerve function in sciatic nerve. Crush injury-provoked inflammation was attenuated in groups receiving G-CSF but not in AFS only group. In transplanted AFS, marked apoptosis was detected and this event was reduced by G-CSF treatment. Increased nerve myelination and improved motor function were observed in AFS transplanted, G-CSF administrated, and AFS/G-CSF combined treatment groups. Significantly, the combined treatment showed the most beneficial effect. In conclusion, the concomitant treatment of AFS with G-CSF augments peripheral nerve regeneration which may involve the suppression of apoptotic death in implanted AFS and the attenuation of inflammatory response.