Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by ∼30%. The death of motor neurons was confirmed using the terminal transferase‐mediated deoxyuridine triphosphate‐biotin nick‐end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl‐modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 185–201, 1999     

Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis.

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by approximately 30%. The death of motor neurons was confirmed using the terminal transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl-modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord.     

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.     

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...     

Upregulation of Gem relates to retinal ganglion cells apoptosis after optic nerve crush in adult rats

GTP-binding protein Gem, a member protein of the Ras superfamily, can regulate actin cytoskeleton reorganization mediated by Rho-associated coiled-coil-containing protein kinase (ROCK). One attractive activity of the ROCK is playing a potential role in physiological and pathological process in retinal ganglion cells (RGCs) apoptosis. However, the function of Gem in retina is still with limited understanding. To investigate whether Gem is involved in optic nerve injury, we performed an optic nerve crush (ONC) model in adult rats. Western blot analysis indicated that Gem was significantly increased in the retina at the 3rd day after ONC. Meanwhile, double-immunofluorescent staining showed that Gem expression was mainly up-regulated in ganglion cell layer and co-localized with NeuN (a marker of RGCs). Additionally, the co-localizations of Gem/active-caspase-3 and Gem/TUNEL-positive cells were detected in RGCs. Furthermore, the expression of active-caspase-3 and TUNEL-positive cells was parallel with that of Gem. Finally, expression pattern of ROCK family (only ROCK2 but not ROCK1) was increased in the differentiated process, which was collected with the expression of GEM and active-caspase-3. Based on the present results, it is suggested that Gem might play a crucial role in RGCs apoptosis after ONC, which might be involved in ROCK pathway.     

Axotomy-induced apoptosis in adult rat primary sensory neurons

Neuronal death following unilateral axotomy of a sensory nerve has long been inferred from neuronal counts of dorsal root ganglion neurons, using the contralateral ganglia as a control. The counting methods used usually involved the counting of neuronal nucleoli and made assumptions about them which could conceivably be flawed. Very few studies have used direct observations of dying or degenerating neurons to address questions concerning the duration of the period of neuronal death or the mechanisms involved in this process. Here we describe a morphological, morphometric and histochemical study into the nature and duration of sensory neuron death following transection and ligation of the sciatic nerve at mid-thigh level in the adult rat. We show that at least some of this neuronal loss occurs by apoptosis as defined by morphological criteria and in situ end-labelling of damaged DNA. Absolute numbers of apoptotic neurons were counted from serial paraffin sections of ganglia and estimates of neuronal numbers obtained by disector analysis at 1, 2, 3 and 6 months after axotomy. Using this approach we show that axotomy-induced apoptosis begins at around 1 week and continues up to at least 6 months after axotomy.     

Effect of sciatic nerve crush on enzyme activities of skeletal muscles of the rat.

Weight loss and reduction in specific activity of cytochrome oxidase of both red (soleus) and white muscles (gastrocnemius and plantaris) of the rat was greatest 5 days after sciatic nerve crush (p less than 0.001) and then became minimal. In neither was there a significant, concomitant loss of protein. By 19 days, the specific activity of cytochrome oxidase was the same in both muscle types. The specific activity of lactate dehydrogenase was reduced significantly (p less than 0.001) in the white muscles, to a value approaching that of the red soleus by 19 days postoperatively, but remained unaltered in the soleus. Nerve crush is proposed as a model experimental system for studying neural regulation of skeletal muscle metabolism.     

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.     

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.     

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.     

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.     

(−)-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.     

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.     

Synthesis and localization of ciliary neurotrophic factor in the sciatic nerve of the adult rat after lesion and during regeneration

Ciliary neurotrophic factor (CNTF) is expressed in high quantities in Schwann cells of peripheral nerves during postnatal development of the rat. The absence of a hydrophobic leader sequence and the immunohistochemical localization of CNTF within the cytoplasm of these cells indicate that the factor might not be available to responsive neurons under physiological conditions. However, CNTF supports the survival of a variety of embryonic neurons, including spinal motoneurons in culture. Moreover we have recently demonstrated that the exogenous application of CNTF protein to the lesioned facial nerve of the newborn rat rescued these motoneurons from cell death. These results indicate that CNTF might indeed play a major role in assisting the survival of lesioned neurons in the adult peripheral nervous system. Here we demonstrate that the CNTF mRNA and protein levels and the manner in which they are regulated are compatible with such a function in lesioned peripheral neurons. In particular, immunohistochemical analysis showed significant quantities of CNTF at extracellular sites after sciatic nerve lesion. Western blots and determination of CNTF biological activity of the same nerve segments indicate that extracellular CNTF seems to be biologically active. After nerve lesion CNTF mRNA levels were reduced to less than 5% in distal regions of the sciatic nerve whereas CNTF bioactivity decreased to only one third of the original before-lesion levels. A gradual reincrease in Schwann cells occurred concomitant with regeneration.     

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.