Loss and recovery of the blood–nerve barrier in the rat sciatic nerve after crush injury are associated with expression of intercellular junctional proteins

The blood–nerve barrier in peripheral nerves is important for maintaining the environment for axons. Breakdown of the barrier by nerve injury causes various pathologies. We hypothesized that the breakdown and recovery of the blood–nerve barrier after injury are associated with the changes in the expression of intercellular junctional proteins. To test this hypothesis, we induced crush injuries in the rat sciatic nerve by ligation and analyzed spatiotemporal changes of claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 by immunoconfocal microscopy and morphometry and compared them with changes in the permeability of the blood–nerve barrier by intravenous and local administration of Evans blue–albumin (EBA). On day 1 after removal of the ligature EBA leaked into the connective tissue in the endoneurium and then the leakage gradually decreased and disappeared on day 7. On day 1 claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 had totally disappeared from the perineurium and endoneurium. Thereafter, claudin-1, claudin-5, occludin, and VE-cadherin recovered from day 2, whereas connexin43 was redetected on day 5. These results indicate that the breakdown and following recovery of the blood–nerve barrier are closely associated with changes in the expression of claudins, occludin, VE-cadherin, and connexin43 and that the recovery time course is similar but nonidentical.     

Increased expression of Gem after rat sciatic nerve injury

Gem belongs to the Rad/Gem/Kir subfamily of Ras-related GTPases, whose expression is induced in several cell types upon activation by extracellular stimuli. Two functions of Gem have been demonstrated, including regulation of voltage-gated calcium channel activity and inhibition of Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction. Because of the essential relationship between actin reorganization and peripheral nerve regeneration, we investigated the spatiotemporal expression of Gem in a rat sciatic nerve crush (SNC) model. After never injury, we observed that Gem had a significant up-regulation from 1 day, peaked at day 5 and then gradually decreased to the normal level. At its peak expression, Gem expressed mainly in Schwann cells (SCs) and macrophages of the distal sciatic nerve segment, but had few colocalization in axons. In addition, the peak expression of Gem was in parallel with PCNA, and numerous SCs expressing Gem were PCNA positive. Thus, all of our findings suggested that Gem may be involved in the pathophysiology of sciatic nerve after SNC.     

Intraperitoneal Alpha-Lipoic Acid to prevent neural damage after crush injury to the rat sciatic nerve

The hypothesis is explored that CRPS I (the "new" RSD) persists due to undiagnosed injured joint afferents, and/or cutaneous neuromas, and/or nerve compressions, and is, therefore, a misdiagnosed form of CRPS II (the "new" causalgia). An IRB-approved, retrospective chart review on a series of 100 consecutive patients with "RSD" identified 40 upper and 30 lower extremity patients for surgery based upon their history, physical examination, neurosensory testing, and nerve blocks. Based upon decreased pain medication usage and recovery of function, outcome in the upper extremity, at a mean of 27.9 months follow-up (range of 9 to 81 months), gave results that were excellent in 40% (16 of 40 patients), good in 40% (16 of 40 patients) and failure 20% (8 of 40 patients). In the lower extremity, at a mean of 23.0 months follow-up (range of 9 to 69 months) the results were excellent in 47% (14 of 30 patients), good in 33% (10 of 30 patients) and failure 20% (6 of 30 patients). It is concluded that most patients referred with a diagnosis of CRPS I have continuing pain input from injured joint or cutaneous afferents, and/or nerve compressions, and, therefore, similar to a patient with CRPS II, they can be treated successfully with an appropriate peripheral nerve surgical strategy.     

Potentiation of angiogenesis and regeneration by G-CSF after sciatic nerve crush injury

Granulocyte colony-stimulating factor (G-CSF) demonstrates neuroprotective effects through different mechanisms, including mobilization of bone marrow cells. However, the influence of G-CSF-mediated mobilization of bone marrow-derived cells on injured sciatic nerves remains to be elucidated. The administration of G-CSF promoted a short-term functional recovery 7 days after crush injury in sciatic nerves. A double-immunofluorescence study using green fluorescent protein-chimeric mice revealed that bone marrow-derived CD34+ cells were predominantly mobilized and migrated into injured nerves after G-CSF treatment. G-CSF-mediated beneficial effects against sciatic nerve injury were associated with increased CD34+ cell deposition, vascular endothelial growth factor (VEGF) expression, and vascularization/angiogenesis as well as decreased CD68+ cell accumulation. However, cell differentiation and VEGF expression were not demonstrated in deposited cells. The results suggest that the promotion of short-term functional recovery in sciatic nerve crush injury by G-CSF involves a paracrine modulatory effect and a bone marrow-derived CD34+ cell mobilizing effect.     

Rat sciatic nerve crush injury and recovery tracked by plantar test and immunohistochemistry analysis

An experimental crush injury to the sciatic nerve, with a crush force of 49.2 N (pressure p=1.98x10(8) Pa), was inflicted in 30 male rats (Wistar). A control group (sham), with the same number of rats, was also operated upon exactly as the experimental group but without the crush injury. We tested the sensory and motor recovery of the sciatic nerve with Hargreaves method, using an apparatus from Ugo Basile, Italy. Testing was continued for both legs of each rat, injured and uninjured, starting preoperatively (0 day), and then 1, 7, 14, 21, and 28 days postoperatively. The same experiment was run simultaneously with the sham group. The Plantar test showed recovery of the sensory and motor function of the sciatic nerve, though not complete recovery, by 28 days. An immunohistochemical experiment was run in parallel with the plantar test on L3-L6 segments of the spinal cord from where the sciatic nerve extends. We used antibodies for Myelin-associated glycoprotein (MAG), and gangliosides GD1a and GT1b on the aforesaid part of the spinal cord. The immunohistochemical methods showed changes in sensory and motor axons in the spinal cord segment L3-L6 which suggest correspondence with the results of the Plantar test, in terms of recovery of the sensory and motor function after injury of the sciatic nerve. The immunohistochemical results also show ipsilateral and contralateral changes following injury. Results of the plantar test are suggestive that the rat shows compensation for an injury in its contralateral leg.     

Peripheral nerve pericytes originating from the blood-nerve barrier expresses tight junctional molecules and transporters as barrier-forming cells

The objective of this study was to establish pure blood-nerve barrier (BNB)-derived peripheral nerve pericyte cell lines and to investigate their unique properties as barrier-forming cells. We isolated peripheral nerve, brain, and lung pericytes from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines expressed several pericyte markers such as alpha-smooth muscle actin, NG2, osteopontin, and desmin, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, these cell lines expressed several tight junction molecules such as occludin, claudin-12, ZO-1, and ZO-2. In particular, the expression of occludin was detected in peripheral nerve and brain pericytes, although it was not detected in lung pericytes by a Western blot analysis. An immunocytochemical analysis confirmed that occludin and ZO-1 were localized at the cell-cell boundaries among the pericytes. Brain and peripheral nerve pericytes also showed significantly higher trans-pericyte electrical resistance values and lower inulin clearances than lung pericytes. We considered that occludin localized at the cell-cell boundaries among the pericytes might mechanically stabilize the microvessels of the BNB and the blood-brain barrier. Furthermore, we also showed that these cell lines expressed many barrier-related transporters. ABCG2, p-gp, MRP-1, and Glut-1 were detected by a Western blot analysis and were observed in the cytoplasm and outer membrane by an immunocytochemical analysis. These transporters on pericytes might facilitate the peripheral nerve-to-blood efflux and blood-to-peripheral nerve influx transport of substrates in cooperation with those on endothelial cells in order to maintain peripheral nerve homeostasis.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

Simvastatin对大鼠坐骨神经crush损伤修复作用研究

目的：探讨他汀类（statins）药物Simvastatin在大鼠坐骨神经损伤修复中的作用及可能的作用机制。方法：制作SD大鼠标准坐骨神经钳夹损伤（crush）模型后,分别予Simvastatin和溶媒对照干预2周。手术前后不同时间点进行趾展功能指数测定、神经电生理学、血脂水平、血清IL-6检测和组织学评价。结果：Simvastatin干预组与对照组比较,趾展功能指数在术后5d和8d显著增大（P〈0．05）,足趾展开速度快;2周肌肉复合动作电位幅度高,4周神经传导速度快;组织学显示有髓神经纤维数量多,髓鞘厚,排列相对整齐。各组手术前血脂水平无差异,手术后2周均有不同程度的降低,但Simvastatin干预组总胆固醇降低程度最轻,与对照组比较有显著差异（P〈0．05）;Simvastatin干预组手术后5d,血清IL-6水平明显低于对照组（P〈0．05）。结论：本研究发现,Simvastatin可能通过抑制免疫炎症反应,维持神经损伤后胆固醇的平衡,促进大鼠坐骨神经损伤的修复和再生。     

Profile of adult rat sensory neuron loss, apoptosis and replacement after sciatic nerve crush

Following permanent transection of the adult rat sciatic nerve, sensory neuron apoptosis in the contributing L4 and L5 dorsal root ganglia can be observed for at least 6 months afterwards. To establish the profile of any sensory neuron apoptosis and loss over time when axonal regeneration is allowed, serial sections of L4 and L5 ganglia were examined and the neurons counted using a stereological technique 1, 2 and 3 months after crushing the right sciatic nerve at mid-thigh level. Our results show that an identical degree of sensory neuron loss and apoptosis occurs 1 month after crush as at 1 month after permanent transection. However, at 3 months no neurons undergoing apoptosis could be observed and no significant loss could be detected in the ipsilateral ganglia when compared to unoperated controls. One explanation was a neuronal replacement mechanism, which was investigated by administering bromodeoxyuridine to rats for 1 month after sciatic nerve transection or crush, prior to detection using immunohistochemistry on sections of their ganglia after 2 months. The presence of bromodeoxyuridine in the nuclei of occasional cells that would be counted as neurons on the basis of size and morphology indicates that a process of apparent neurogenesis may underlie the profile of sensory neuron loss after axotomy.     

Neurturin enhances the recovery of erectile function following bilateral cavernous nerve crush injury in the rat

Peripheral sensory diabetic neuropathy is characterized by morphological, electrophysiological and neurochemical changes to a subpopulation of primary afferent neurons. Here, we utilized a transgenic mouse model of diabetes (OVE26) and age-matched controls to histologically examine the effect of chronic hyperglycemia on the activity or abundance of the enzymes acid phosphatase, cytochrome oxidase and NADPH-diaphorase in primary sensory neuron perikarya and the dorsal horn of the spinal cord. Quantitative densitometric characterization of enzyme reaction product revealed significant differences between diabetic, compared to control, animals for all three enzymes. Levels of acid phosphatase reaction product were found to be significantly reduced in both small diameter primary sensory somata and the dorsal horn. Cytochrome oxidase activity was found to be significantly lower in small primary sensory somata while NADPH-diaphorase labeling was found to be significantly higher in small primary sensory somata and significantly lower in the dorsal horn. In addition to these observed biochemical changes, ratiometric analysis of the number of small versus large diameter primary sensory perikarya in diabetic and control animals demonstrated a quantifiable decrease in the number of small diameter cells in the spinal ganglia of diabetic mice. These results suggest that the OVE26 model of diabetes mellitus produces an identifiable disturbance in specific metabolic pathways of select cells in the sensory nervous system and that this dysfunction may reflect the progression of a demonstrated cell loss.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

(−)-Epigallocatechin-3-gallate (EGCG) attenuates peripheral nerve degeneration in rat sciatic nerve crush injury

Recently, we have shown that green tea (GT) consumption improves both reflexes and sensation in unilateral chronic constriction injury to the sciatic nerve. Considering the substantial neuroprotective properties of GT polyphenols, we sought to investigate whether (−)-epigallocatechin-3-gallate (EGCG) could protect the sciatic nerve and improve functional impairments induced by a crushing injury. We also examined whether neuronal cell apoptosis induced by the crushing injury is affected by EGCG treatment.     

Changes in CLIP3 expression after sciatic nerve injury in adult rats

CLIP3 (cytoplasmic linker protein 3) is a 547 amino acid residue cytoplasmic protein that localises to Golgi stacks and tubulovesicular elements juxtaposed to Golgi cisternae. Composed of three Ank (ankyrin) repeats and two CAP-Gly (cytoskeleton-associated protein-glycine) domains, CLIP3 may function as a cytoplasmic linker protein that is involved in TGN–endosome dynamics. To define the expression and role of CLIP3 during peripheral nervous system degeneration and regeneration, we created an acute sciatic nerve injury (SNI) model in adult rats. Western blot analyses revealed prominent up-regulation of CLIP3 and PCNA (proliferating cell nuclear antigen) protein levels at 3?days after SNI. Immunohistochemistry displayed that the expression of CLIP3 was noticeably increased in the injured nerve. Immunofluorescence further revealed that the CLIP3 and PCNA proteins colocalised respectively with S100 in the cytoplasm of Schwann cells. The expression profile of the SC/neuron co-cultures demonstrated that CLIP3 and PCNA protein levels were markedly expressed during the early stage of myelination. These results suggest that CLIP3 is likely associated with the myelination of proliferating Schwann cells, and nerve tissue regeneration after peripheral nerve injury. CLIP3 and PCNA expression during early myelination may be related to the direct uptake and transport of lipids and cholesterol, which were derived from the degenerating myelin, by Schwann cells to prepare for the formation of myelin sheath-like structures around regenerated axons after SNI.     

Retrograde tracing and toe spreading after experimental autologous nerve transplantation and crush injury of the sciatic nerve: a descriptive methodological study

ABSTRACT: Evaluation of functional and structural recovery after peripheral nerve injury is crucial to determine the therapeutic effect of a nerve repair strategy. In the present study, we examined the relationship between the structural evaluation of regeneration by means of retrograde tracing and the functional evaluation analysis of toe spreading. Two standardized rat sciatic nerve injury models were used to address this relationship. As such, animals received either a 2 cm sciatic nerve defect (neurotmesis) followed by autologous nerve transplantation (ANT animals) or a crush injury with spontaneous recovery (axonotmesis; CI animals). Functional recovery of toe spreading was observed over an observation period of 84 days. In contrast to CI animals, ANT animals did not reach pre-surgical levels of toe spreading. After the observation period, the lipophilic dye DiI was applied to label sensory and motor neurons in dorsal root ganglia (DRG; sensory neurons) and spinal cord (motor neurons), respectively. No statistical difference in motor or sensory neuron counts could be detected between ANT and CI animals. In the present study we could indicate that there was no direct relationship between functional recovery (toe spreading) measured by SSI and the number of labelled (motor and sensory) neurons evaluated by retrograde tracing. The present findings demonstrate that a multimodal approach with a variety of independent evaluation tools is essential to understand and estimate the therapeutic benefit of a nerve repair strategy.     

Axoplasmic transport of microtubule-associated proteins in the rat sciatic nerve

32P-ATP was injected into the L5 dorsal root ganglion and axoplasmic transport of the phosphorylate MA proteins 2, microtubule-associated proteins 2, was observed. After the injection of 32P-ATP, the nerve was dissected out at prescribed time intervals and sliced into 5-mm pieces. Each segment was electrophoresed on an SDS-polyacrylamide slab gel and subjected to autoradiography. A protein of 310,000 dalton was transported at a velocity of 6.6-10.6 mm/day in the axon with the electrophoretic mobility identical to that of MA proteins 2, one of the key components associated with the microtubules.     

Altered neuronatin expression in the rat dorsal root ganglion after sciatic nerve transection

Background  

Several molecular changes occur following axotomy, such as gene up-regulation and down-regulation. In our previous study using Affymetrix arrays, it was found that after the axotomy of sciatic nerve, there were many novel genes with significant expression changes. Among them, neuronatin (Nnat) was the one which expression was significantly up-regulated. Nnat was identified as a gene selectively expressed in neonatal brains and markedly reduced in adult brains. The present study investigated whether the expression of Nnat correlates with symptoms of neuropathic pain in adult rats with transected sciatic nerve.     

GDNF-ADSCs-APG embedding enhances sciatic nerve regeneration after electrical injury in a rat model

The pluripotency of adipose-derived stem cells (ADSCs) makes them appropriate for tissue repair and wound healing. Owing to the repair properties of autologous platelet–rich gel (APG), which is based on easily accessible blood platelets, its clinical use has been increasingly recognized by physicians. The aim of this study was to investigate the effect of combined treatment with ADSCs and APG on sciatic nerve regeneration after electrical injury. To facilitate the differentiation of ADSCs, glial cell line–derived neurotrophic factor (GDNF) was overexpressed in ADSCs by lentivirus transfection. GDNF-ADSCs were mingled with APG gradient concentrations, and in vitro, cell proliferation and differentiation were examined with 5-ethynyl-2′-deoxyuridine staining and immunofluorescence. A rat model was established by exposing the sciatic nerve to an electrical current of 220 V for 3 seconds. Rat hind-limb motor function and sciatic nerve regeneration were subsequently evaluated. Rat ADSCs were characterized by high expression of CD90 and CD105, with scant expression of CD34 and CD45. We found that GDNF protein expression in ADSCs was elevated after Lenti-GDNF transfection. In GDNF-ADSCs-APG cultures, GDNF was increasingly produced while tissue growth factor-β was reduced as incubation time was increased. ADSC proliferation was augmented and neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) expression were upregulated in GDNF-ADSCs-APG. In addition, limb motor function and nerve axon growth were improved after GDNF-ADSCs-APG treatment. In conclusion, our study demonstrates the combined effect of ADSCs and APG in peripheral nerve regeneration and may lead to treatments that benefit patients with electrical injuries.