The aim of this study was to propose new more reliable peripheral nerve transection model to overcome the defect of the traditional sciatic axotomy model by specifically transecting L5 spinal nerve just after emerging from the intervertebral foramen and confining analysis area to the L5 spinal segment. The adult male Sprague-Dawley rats, weighing 300~350 g at the time of surgery, were used for the experiments. Four different experimental groups were used. 1. Sciatic nerve transection (Sc-Tx) group: transect the sciatic nerve in the popliteal fossa where it divided into the common peroneal nerve and tibial nerve. 2. L5 spinal nerve transection (L5-Tx) group: L5 spinal nerve was specifically transected. 3. Suture (Su) group: L5 spinal nerve was transected and immediately sutured. 4. Control group: the same surgical procedure with L5 spinal nerve transection group was performed except for the excision of L5 spinal nerve. To distinguish L5 motoneurons from the other level ones, the animals were received the retrograde tracer, FluoroGold into the axotomized proximal nerve stump. Serial coronal frozen sections at 40 microm thick through the L4 to L6 spinal segment was performed and the resultant total number of sections was about 180. Approximate serial 50 sections (approximately 2 mm) could be considered as the L5 segment based on the number of the fluorescent signals (above 20). L5 spinal segment could be differentiated from L4 and L6 segment based on their morphological characteristics under Cresyl violet stain. In L5-Tx group, at 2 and 4 weeks post-transection, the number of L5 spinal motoneurons was reduced by 8%. Meanwhile, Sc-Tx and Su groups showed no statistically notable changes. In this study, the authors could propose more reliable peripheral nerve axotomy model than the conventional sciatic nerve axotomy model by specifically transecting L5 spinal nerve and confining the investigating area within the L5 spinal segment.
Adult ; Animals ; Axotomy ; Benzoxazines ; Frozen Sections ; Humans ; Male ; Peripheral Nerve Injuries ; Peripheral Nerves ; Peroneal Nerve ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; Spinal Nerves ; Sutures ; Tibial Nerve ; Viola

The aim of this study was to propose new more reliable peripheral nerve transection model to overcome the defect of the traditional sciatic axotomy model by specifically transecting L5 spinal nerve just after emerging from the intervertebral foramen and confining analysis area to the L5 spinal segment. The adult male Sprague-Dawley rats, weighing 300~350 g at the time of surgery, were used for the experiments. Four different experimental groups were used. 1. Sciatic nerve transection (Sc-Tx) group: transect the sciatic nerve in the popliteal fossa where it divided into the common peroneal nerve and tibial nerve. 2. L5 spinal nerve transection (L5-Tx) group: L5 spinal nerve was specifically transected. 3. Suture (Su) group: L5 spinal nerve was transected and immediately sutured. 4. Control group: the same surgical procedure with L5 spinal nerve transection group was performed except for the excision of L5 spinal nerve. To distinguish L5 motoneurons from the other level ones, the animals were received the retrograde tracer, FluoroGold into the axotomized proximal nerve stump. Serial coronal frozen sections at 40 microm thick through the L4 to L6 spinal segment was performed and the resultant total number of sections was about 180. Approximate serial 50 sections (approximately 2 mm) could be considered as the L5 segment based on the number of the fluorescent signals (above 20). L5 spinal segment could be differentiated from L4 and L6 segment based on their morphological characteristics under Cresyl violet stain. In L5-Tx group, at 2 and 4 weeks post-transection, the number of L5 spinal motoneurons was reduced by 8%. Meanwhile, Sc-Tx and Su groups showed no statistically notable changes. In this study, the authors could propose more reliable peripheral nerve axotomy model than the conventional sciatic nerve axotomy model by specifically transecting L5 spinal nerve and confining the investigating area within the L5 spinal segment.
Adult ; Animals ; Axotomy ; Benzoxazines ; Frozen Sections ; Humans ; Male ; Peripheral Nerve Injuries ; Peripheral Nerves ; Peroneal Nerve ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; Spinal Nerves ; Sutures ; Tibial Nerve ; Viola

Activating transcription factor 3 (ATF3) and c-Jun play key roles in either cell death or cell survival, depending on the cellular background. To evaluate the functional significance of ATF3/c-Jun in the peripheral nervous system, we examined neuronal cell death, activation of ATF3/c-Jun, and microglial responses in facial motor nuclei up to 24 weeks after an extracranial facial nerve axotomy in adult rats. Following the axotomy, neuronal survival rate was progressively but significantly reduced to 79.1% at 16 weeks post-lesion (wpl) and to 65.2% at 24 wpl. ATF3 and phosphorylated c-Jun (pc-Jun) were detected in the majority of ipsilateral facial motoneurons with normal size and morphology during the early stage of degeneration (1-2 wpl). Thereafter, the number of facial motoneurons decreased gradually, and both ATF3 and pc-Jun were identified in degenerating neurons only. ATF3 and pc-Jun were co-localized in most cases. Additionally, a large number of activated microglia, recognized by OX6 (rat MHC II marker) and ED1 (phagocytic marker), gathered in the ipsilateral facial motor nuclei. Importantly, numerous OX6- and ED1-positive, phagocytic microglia closely surrounded and ingested pc-Jun-positive, degenerating neurons. Taken together, our results indicate that long-lasting co-localization of ATF3 and pc-Jun in axotomized facial motoneurons may be related to degenerative cascades provoked by an extracranial facial nerve axotomy.
Activating Transcription Factor 3 ; Adult ; Animals ; Axotomy ; Cell Death ; Cell Survival ; Facial Nerve ; Humans ; Microglia ; Neurons ; Peripheral Nervous System ; Rats ; Survival Rate

BACKGROUNDBy unbiased stereological methods, we have observed preferential dorsal root ganglion (DRG) B-cell loss in rodents after nerve injury, and caspase-3 activation and cell loss were related to the present of p75 receptor (p75(NTR)). We hypothesized that DRG B-cells express higher levels of pro-apoptotic proteins as compared to A-cells and the expressions of pro-apoptotic proteins can be reduced by depletion of p75(NTR). This study aimed to identify the p75(NTR) involved apoptotic pathway in DRG neurons after nerve injury.

METHODSThe p75(NTR) knockout mice (p75-/-) and wildtype Balb/C mice (p75+/+) were used in this study. The expressions of pro-apoptotic proteins, c-Jun-N-terminal kinase (JNK), c-jun and p38 in DRG were evaluated with immunohistochemistry 2 and 7 days following unilateral sciatic nerve transection. In addition, extra-cellular related kinase (ERK), a transducer of survival signals, was also tested with immunohistochemistry and Western blotting methods in these animal models.

RESULTSPhosphorylated JNK (P-JNK) and phosphorylated p38 (P-p38) were mainly located in small B-cells, whereas phosphorylated c-jun (P-c-jun) was located in both A- and B-cells. Phosphorylated ERK (P-ERK) was located in both B-cells and satellite cells. Axotomy dramatically increased the expressions of P-JNK and P-c-jun (paired t-test), with no influence on the expressions of P-p38 and P-ERK. Furthermore, the increase of P-JNK in p75+/+ mice 2 days after nerve axotomy was approximately 2.2-folds of that in p75-/- mice (P = 0.001, unpaired t-test).

CONCLUSIONp75(NTR)-dependent JNK-caspase-3 pathway is involved in DRG B-cell loss after nerve injury and JNK is not the unique upstream of c-jun activation.

Animals ; Apoptosis ; genetics ; physiology ; Axotomy ; adverse effects ; Blotting, Western ; Ganglia, Spinal ; cytology ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Knockout ; Neurons ; cytology ; metabolism ; Receptors, Nerve Growth Factor ; genetics ; metabolism ; Sciatic Nerve ; surgery

We previously reported that nidogen is an extracellular matrix protein regulating Schwann cell proliferation and migration. Since Schwann cells play a critical role in peripheral nerve regeneration, nidogen may play a role in it via regulation of Schwann cells. Here, we demonstrate direct evidence that nidogen induces elongation of regenerative axon growth of adult sensory neurons, and that the effect is Schwann cell dependent. Continuous infusion of recombinant ectodomain of tumor endothelial marker 7, which specifically blocks nidogen function in Schwann cells, suppressed regenerative neurite growth in a sciatic nerve axotomy model. Taken together, it is likely that nidogen is required for proper regeneration of peripheral nerves after injury.
Animals ; Axotomy ; Cell Movement ; Cell Proliferation ; Male ; Membrane Glycoproteins/*physiology ; Membrane Proteins/pharmacology ; *Nerve Regeneration ; Nerve Tissue Proteins/pharmacology ; Neurites/drug effects/*physiology/ultrastructure ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/pharmacology ; Schwann Cells/cytology/*physiology ; Sensory Receptor Cells/*physiology

OBJECTIVETo investigate the effect of exogenous glial cell line-derived neurotrophic factor (GDNF) infused into the cavitas subarachnoidealis on cornu anterius medullae spinalis motor neurons after sciatic nerve axotomy.

METHODSForty-eight healthy SD rats were divided into 2 groups randomly: GDNF group and NS group. The left sciatic nerve in rats were cut off. And then 0.9% saline (6 microl) and GDNF solution (6 microl) were injected into cavitas subarachnoidealis at L4-L6 in NS group and GDNF group,respectively. The rats were sacrificed on postoperative 1, 2, 4 and 8 weeks respectively. Their specimen of L4-L6 spinal cord were taken at different time and sectioned. The HE staining, Nissl staining and cholinesterase (ChE) staining in motor neurons were used for counting of motor neurons.

RESULTSIn GDNF group the number of motor neurons in cornu anterius medullae spinalis and the ChE activity were higher than that of NS group.

CONCLUSIONThe exogenous GDNF infused into the cavitas subarachnoidealis are supposed to protect the degenerated spinal motor neuron from death after sciatic nerve injury.

Animals ; Axotomy ; Cholinesterases ; metabolism ; Glial Cell Line-Derived Neurotrophic Factor ; pharmacology ; Male ; Motor Neurons ; drug effects ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; cytology ; drug effects ; metabolism ; Spinal Cord ; cytology ; drug effects ; metabolism ; surgery

BACKGROUND AND OBJECTIVES: The object of this study was to evaluate the effect of platelet rich plasma (PRP) on facial nerve regeneration from an axotomy injury in the guinea pig model. MATERIALS AND METHOD: Experiments involved the transection and repair of right facial nerve. The right facial nerve of 14 albino guinea pigs were completely transected and immediately sutured, followed by fibrin glue only (control group) or fibrin glue +PRP (PRP group). Western blot assay was used to detect neurotrophic factors secreted by PRP. Nerve regeneration was assessed by motor function, electrophysiology, and histology studies. RESULTS: High levels of neurotrophin-3, angiopoietin-1, glial cell line derived neurotrophic factors, nerve growth factors and brain derived neurotrophic factors were demonstrated in PRP. Motor function recovery, compound motor action potentials, and axon count showed significant improvement in guinea pig treated with PRP. CONCLUSION: There was an improved functional outcome with the use of PRP in comparison with control. The increased nerve regeneration found in this study may be due to the neurotrophic factors secreted by PRP.
Action Potentials ; Angiopoietin-1 ; Animals ; Axons ; Axotomy ; Blood Platelets ; Blotting, Western ; Brain-Derived Neurotrophic Factor ; Electrophysiology ; Facial Nerve ; Fibrin Tissue Adhesive ; Glial Cell Line-Derived Neurotrophic Factor ; Glial Cell Line-Derived Neurotrophic Factors ; Guinea Pigs ; Nerve Growth Factor ; Nerve Growth Factors ; Nerve Regeneration ; Platelet-Rich Plasma ; Recovery of Function ; Regeneration

Traumatic injury or inflammatory irritation of the peripheral nervous system often leads to persistent pathophysiological pain states. It has been well-documented that, after peripheral nerve injury or inflammation, functional and anatomical alterations sweep over the entire peripheral nervous system including the peripheral nerve endings, the injured or inflamed afferent fibers, the dorsal root ganglion (DRG), and the central afferent terminals in the spinal cord. Among all the changes, ectopic discharge or spontaneous activity of primary sensory neurons is of great clinical interest, as such discharges doubtless contribute to the development of pathological pain states such as neuropathic pain. Two key sources of abnormal spontaneous activity have been identified following peripheral nerve injury: the injured afferent fibers (neuroma) leading to the DRG, and the DRG somata. The purpose of this review is to provide a global account of the abnormal spontaneous activity in various animal models of pain. Particular attention is focused on the consequence of peripheral nerve injury and localized inflammation. Further, mechanisms involved in the generation of spontaneous activity are also reviewed; evidence of spontaneous activity in contributing to abnormal sympathetic sprouting in the axotomized DRG and to the initiation of neuropathic pain based on new findings from our research group are discussed. An improved understanding of the causes of spontaneous activity and the origins of neuropathic pain should facilitate the development of novel strategies for effective treatment of pathological pain.
Animals ; Axotomy ; Ganglia, Spinal ; cytology ; Humans ; Neuralgia ; physiopathology ; Neurons, Afferent ; cytology ; Peripheral Nerve Injuries ; physiopathology ; Spinal Cord ; cytology

Objective A calcium-activated chloride current (IClCa) has been observed in medium-sized sensory neurons of the dorsal root ganglion (DRG). Axotomy of the sciatic nerve induces a similar current in the majority of medium and large diameter neurons. Our aim is to identify the molecule(s) underlying this current. Methods Using conventional and quantitative RT-PCR, we examined the expression in DRG of members of three families of genes, which have been shown to have IClCa current inducing properties. Results We showed the detection of transcripts representing several members of these families, i.e. chloride channel calcium-activated (CLCA), Bestrophin and Tweety gene families in adult DRG, in the normal state and 3 d after sciatic nerve section, a model for peripheral nerve injury. Conclusion Our analysis revealed that that mBest1 and Tweety2 appear as the best candidates to play a role in the injury-induced IClCa in DRG neurons.
Animals ; Axotomy ; Chloride Channels ; biosynthesis ; genetics ; DNA Primers ; Ganglia, Spinal ; metabolism ; Gene Expression ; Mice ; Neurons, Afferent ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sciatic Nerve ; physiology

This study demonstrated that xenogenic human marrow mesenchymal stem cells (hMSCs) could elicit the regeneration of the sensory nerve after axotomy in the adult rats'infraorbital nerves without immunosuppression. For this, we evaluated the behavioral testing for functional recovery of the nerve and histological findings at weeks 3 and 5 compared to controls. Xenogenic hMSCs did not evoke any significant inflammatory or immunologic reaction after systemic and local administrations. HMSCs-treated rats exhibited significant improvement on sensory recovery tested with von Frey monofilaments. At 5 postoperative weeks, in the hMSCs treated nerve, expression of myelin basic protein (MBP), neurofilament (NF) at the site of axotomy was higher than control. And mRNA expression of neurotropin receptor Trk precursor (TrkPre), nerve growth factor receptor (NGFR) and neuropeptide (NPY) in trigeminal ganglion were also higher. The number of myelinated nerve at distal stump and cells in trigeminal ganglion were higher in hMSC treated rats. So it was supposed that transplanted MSCs contributed to reducing post-traumatic degeneration and production of neurotrophic factors. Immunofluorescence labeling showed small portion of hMSCs(<10%) expressed a phenotypic marker of Schwann cell (S-100). Xenogenic or allogenic mesenchymal stem cells might have immune privileged characteristics and useful tool for cell based nerve repair.
Adult ; Animals ; Axotomy ; Bone Marrow* ; Fluorescent Antibody Technique ; Humans* ; Immunosuppression ; Mesenchymal Stromal Cells* ; Myelin Basic Protein ; Myelin Sheath ; Nerve Growth Factor ; Nerve Growth Factors ; Nerve Regeneration ; Neuropeptides ; Rats* ; Regeneration* ; RNA, Messenger ; Trigeminal Ganglion