PURPOSE: Wallerian degeneration (WD) is an antegrade degenerative process distal to peripheral nerve injury. Numerous genes are differentially regulated in response to the process. However, the underlying mechanism is unclear, especially the early response. We aimed at investigating the effects of sciatic nerve injury on WD via CLDN 14/15 interactions in vivo and in vitro. METHODS: Using the methods of molecular biology and bioinformatics analysis, we investigated the molecular mechanism by which claudin 14/15 participate in WD. Our previous study showed that claudins 14 and 15 trigger the early signal flow and pathway in damaged sciatic nerves. Here, we report the effects of the interaction between claudin 14 and claudin 15 on nerve degeneration and regeneration during early WD. RESULTS: It was found that claudin 14/15 were upregulated in the sciatic nerve in WD. Claudin 14/15 promoted Schwann cell proliferation, migration and anti-apoptosis in vitro. PKCα, NT3, NF2, and bFGF were significantly upregulated in transfected Schwann cells. Moreover, the expression levels of the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK signaling pathways were also significantly altered. CONCLUSION Claudin 14/15 affect Schwann cell proliferation, migration, and anti-apoptosis via the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK pathways in vitro and in vivo. The results of this study may help elucidate the molecular mechanisms of the tight junction signaling pathway underlying peripheral nerve degeneration.
Animals ; Claudins ; Nerve Regeneration ; Peripheral Nerve Injuries ; Rats ; Schwann Cells/pathology* ; Sciatic Nerve ; Wallerian Degeneration/pathology*

To explore the value of conventional ultrasound combined with shear-wave elastography in the quantitative evaluation of sciatic nerve crush injury in rabbit models. Forty healthy male New Zealand white rabbits were randomly divided into four groups (=10 in each group):three crush injury (CI) groups (2,4,and 8 weeks after crush) and control group (without injury). The thickness and stiffness of the crushed sciatic nerves and denervated triceps surae muscles were measured at different time points and compared with histopathologic parameters. Inter-reader variability was assessed with intraclass correlation coefficients. Compared with the control group,the inner diameters of the sciatic nerves significantly increased in the 2-week CI group [(1.65±0.34) mm (0.97±0.15) mm,=0.00] but recovered to the nearly normal level in the 8-week CI group [(1.12±0.18) mm (0.97±0.15) mm,=0.06];however,compared with control group [(8.75±1.02)kPa],the elastic modulus of the nerves increased significantly in all the CI groups [2-week:(14.77±2.53) kPa;4-week:(19.12±3.46) kPa;and 8-week:(28.39±5.26) kPa;all =0.00];pathologically,massive hyperplasia of collagen fibers were found in the nerve tissues. The thickness of denervated triceps surae muscle decreased gradually,and the elastic modulus decreased 2 weeks after injury but increased gradually in the following 6 weeks;pathologically,massive hyperplasia of collagen fibers and adipocytes infiltration were visible,along with decreased muscle wet-weight ratio and muscle fiber cross-sectional area. The inter-reader agreements were good. Conventional ultrasound combined with shear-wave elastography is feasible for the quantitative evaluation of the morphological and mechanical properties of crushed nerves and denervated muscles.
Animals ; Crush Injuries ; diagnostic imaging ; Elastic Modulus ; Elasticity Imaging Techniques ; Male ; Muscle, Skeletal ; innervation ; pathology ; Rabbits ; Random Allocation ; Sciatic Nerve ; injuries ; Ultrasonography

Biopsy ; Humans ; Male ; Middle Aged ; Muscle Neoplasms ; diagnosis ; pathology ; Neoplasm Metastasis ; Nerve Sheath Neoplasms ; diagnosis ; pathology ; Recurrence ; Sciatic Nerve ; pathology ; Tomography, X-Ray Computed

OBJECTIVETo observe the deregulation of autophagy in diabetic peripheral neuropathy (DPN) and investigate whether Jinmaitong ( JMT) alleviates DPN by inducing autophagy.

METHODSDPN models were established by streptozotocin-induced diabetic rats and Schwann cells (SCs) cultured in high glucose medium. The pathological morphology was observed by the improved Bielschowsky's nerve fiber axonal staining and the Luxol fast blue-neutral red myelin staining. The ultrastructure was observed by the transmission electron microscopy. Beclin1 level was detected by immunohistochemistry and Western blot. The proliferation of cultured SCs was detected by methylthiazolyldiphenyl-tetrazolium bromide.

RESULTSDiabetic peripheral nerve tissues demonstrated pathological morphology and reduced autophagic structure, accompanied with down-regulation of Beclin1. JMT apparently alleviated the pathological morphology change and increased the autophagy [in vivo, Beclin1 integral optical density (IOD) value of the control group 86.6±17.7, DM 43.9±8.8, JMT 73.3 ±17.8, P<0.01 or P<0.05, in vitro Beclin1 IOD value of the glucose group 0.47±0.25 vs the control group 0.88±0.29, P<0.05]. Consequently, inhibition of autophagy by 3-methyladenine resulted in a time- and concentration-dependent decrease of the proliferation of SCs (P<0.05, P<0.01).

CONCLUSIONSDown-regulation of autophagy in SCs might contribute to the pathogenesis of DPN. JMT alleviates diabetic peripheral nerve injury at least in part by inducing autophagy.

Animals ; Autophagy ; drug effects ; Axons ; drug effects ; pathology ; Beclin-1 ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Diabetes Mellitus, Experimental ; complications ; drug therapy ; pathology ; Diabetic Neuropathies ; complications ; drug therapy ; pathology ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Glucose ; pharmacology ; Immunohistochemistry ; Male ; Rats, Wistar ; Schwann Cells ; drug effects ; pathology ; Sciatic Nerve ; drug effects ; pathology ; ultrastructure ; Staining and Labeling

OBJECTIVETo observe the effects of mecobalamin on the expression of apoptosis-related proteins, Bax and Bcl-2, in neurons after peripheral nerve injury, and to explore the role of neuron apoptosis in peripheral nerve regeneration after injury.

METHODSThirty healthy adult male wistar rats were randomly divided into sham-operation group, model group, and mecobalamin group, with 10 rats in each group. A rat model of left sciatic nerve semi-injury was established using forceps. Rats in the mecobalamin group were fed mecobalamin, while rats in the sham-operation group and model group were given the same dose of normal saline. The protein expression of Bax and Bcl-2 in neurons was measured at 14 days after operation. A semi-quantitative analysis of Bax and Bcl-2 proteins was performed by image analysis technology.

RESULTSThe model group had significantly increased Bax protein expression and significantly reduced Bcl-2 protein expression in spinal cord anterior horn motor neurons and ganglion sensory neurons compared with the sham-operation group (P<0.05). Compared with the model group and sham-operation group, the mecobalamin group had significantly reduced Bax protein expression and significantly increased Bcl-2 protein expression in spinal cord anterior horn motor neurons and ganglion sensory neurons (P<0.05).

CONCLUSIONMecobalamin has anti-apoptotic effect, and it contributes to neurological function recovery possibly by inhibiting the death of injured neurons.

Animals ; Apoptosis ; Male ; Neurons ; cytology ; drug effects ; Peripheral Nerve Injuries ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; Rats ; Rats, Wistar ; Sciatic Nerve ; pathology ; Spinal Cord ; cytology ; Vitamin B 12 ; analogs & derivatives ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo observe the effects of adipose-derived mesenchymal stem cells (ADSCs) with continous over-expression of glial cell line-derived neurotrophic factor (GDNF) on the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

METHODSFive SD rats were collected to prepare ADSCs with over-expression of GDNF. One hundred and fifty SD rats were divided into normal control group (N), GDNF-ADSCs group (GA), ADSCs group (A), GDNF group (G), and physiological saline group (P) according to the random number table, with 30 rats in each group. Rats in group N were routinely fed without treatment, and rats in the other 4 groups were inflicted with electrical injury on sciatic nerve of thigh of the right hind leg. Rats in groups GA, A, G, and P were respectively injected with 100 µL suspension of ADSCs with over-expression of GDNF (1 x 10(7) cells per mL), 100 [µL ADSCs suspension (1 x 10(7) cells per mL), 100 µL GDNF solution (100 mg/L) , and 100 µL physiological saline to the surface of the injured nerves immediately after injury. Six rats of each group were collected for measuring hind limb stride from post injury week (PIW) 1 to 8, and morphology of the sciatic nerves was observed in PIW 8. In PIW 4, the protein expression of GDNF of sciatic nerves of the rest rats in each group was determined with Western blotting. Data were processed with one-way analysis of variance, analysis of variance of repeated measurement, and SNK test.

RESULTSCompared with that of group N, the hind limb stride values in groups GA, A, G, and P were significantly lower at each time point (with P values below 0.05). Compared with those of group P, the hind limb stride values in group GA from PIW 3 to 8, in group A in PIW 3, 5, and 7, and in group G in PIW 3, 5, 7, and 8 were significantly longer (with P values below 0.05). The hind limb stride values in group GA from PIW 4 to 8 were respectively (10.83 ± 0.97), (13.25 ± 1.40), (12.86 ± 1.42), (14.06 ± 1.50), and (15.09 ± 1.17) cm, which were significantly longer than those in group A [(8.90 ± 0.82), (9.03 ± 0.57), (9.27 ± 0.36), (9.86 ± 0.36), and (9.52 ± 0.58) cm] and group G [(8.87 ± 0.69), (8.51 ± 1.18), (9.34 ± 0.87), (9.76 ± 0.67), and (9.50 ± 1.22) cm], with P values below 0.05. Compared with that of group N, the number of myelinated nerve fibers of sciatic nerves was obviously decreased in group P but obviously increased in groups GA, A, and G; the diameter of axons was obviously shorter, and the myelin thickness was obviously increased in groups GA, A, G, and P in PIW 8 (with P values below 0.05). The number of myelinated nerve fibers in group GA was 31.2 ± 0.8, which was significantly higher than that in group A (23.7 ± 2.7), group G (22.3 ± 2.7), or group P (9.3 ± 2.8), with P values below 0.05. The diameter values of axons among groups P, A, G, and GA were similar (with P values above 0.05). The myelin thickness of rats in group GA was (3.41 ± 0.34) µm, which was significantly thicker than that in group A [(2.64 ± 0.37) µm] or group G [(2.41 ± 0.34) µm], with P values below 0.05. In PIW 4, the protein expression of GDNF of sciatic nerves was significantly higher in groups P, A, G, and GA than in group N (with P values below 0.05), and the protein expression of GDNF in group GA was significantly higher than that in group P, A, or G (with P values below 0.05).

CONCLUSIONSADSCs over-expressing GDNF protein can obviously promote the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

Adipose Tissue ; Animals ; Electrophysiology ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; metabolism ; Nerve Crush ; Nerve Regeneration ; physiology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; pathology ; physiology

Neural crest stem cells originated from hair follicle (epidermal neural crest stem cell, EPI-NCSC) are easy to obtain and have potentials to differentiate into various tissues, which make them eminent seed cells for tissue engineering. EPI-NCSC is now used to repair nerve injury, especially, the spinal cord injury. To investigate their effects on repairing peripheral nerve injury, EPI-NCSC from a GFP-SD rat were primarily cultured on coated dishes and on a poly lactic acid coglycolic acid copolymer (PLGA) membrane. Methyl thiazolyl tetrazolium (MTT) assay showed that the initial adhesion rate of EPI-NCSC was 89.7% on PLGA membrane, and the relative growth rates were 89.3%, 87.6%, 85.6%, and 96.6% on the 1st, 3rd, 5th, 7th day respectively. Cell cycles and DNA ploidy analysis demonstrated that cell cycles and proliferation indexes of cultured EPI-NCSC had the same variation pattern on coated dishes and PLGA membrane. Then cultured EPI-NCSC were mixed with equal amount of extracellular matrix and injected into a PLGA conduit to connect a 10 mm surgery excision gap of rat sciatic nerve, Dulbecco's Modified Eagle's medium (DMEM) was used to substitute EPI-NCSC in the control group. After four weeks of transplantation, the defected sciatic nerve achieved a histological restoration, the sensory function of rat hind limb was partly recovered and the sciatic nerve index was also improved. The above results showed that a PLGA conduit filled with EPI-NCSC has a good repair effect on the peripheral nerve injury.
Animals ; Cells, Cultured ; Neural Crest ; cytology ; Neural Stem Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; pathology ; Spinal Cord Injuries ; Stem Cell Transplantation ; Tissue Engineering

mRNAs of alpha-adrenoceptor (α-AR) subtypes are found in neurons in dorsal root ganglion (DRG) and change after peripheral nerve injury. In this study, the distribution of α-AR subtype proteins was studied in L5 DRG of normal rats and rats with chronic constriction injury of sciatic nerve (CCI). Using immunofluorescence technique, it was found that α1A-, α1B-, and α2A-AR proteins were expressed in large, medium, and small size neurons in normal DRG, and significantly increased in all size neurons 14 days after CCI. α1D- and α2C-AR was also expressed in all size neurons in normal DRG. However, α1D-AR was significantly increased and α2C-AR was decreased in small size neurons 14 days post CCI. α2B-AR neurons were not detectable in normal and CCI DRG. Co-expression of α1A- and α2A-AR in the same neuron was observed in normal DRG and increased post CCI. Collectively, these results indicated that there is distinct distribution of α-AR subtypes in DRG neurons, and the distribution and levels of expression of α-AR subtypes change differently after CCI. The up-regulation of α-AR subtypes in DRG neurons may play an important role in the process of generating and transmitting neuropathic pain.
Animals ; Cell Size ; Chronic Disease ; Constriction, Pathologic ; Fluorescent Antibody Technique ; Ganglia, Spinal ; metabolism ; pathology ; Male ; Microscopy, Confocal ; Neurons ; metabolism ; pathology ; Pain Measurement ; methods ; Pain Threshold ; Protein Isoforms ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, alpha-1 ; metabolism ; Receptors, Adrenergic, alpha-2 ; metabolism ; Sciatic Nerve ; injuries ; surgery

OBJECTIVETo explore the effect of electroacupuncture (EA) on the pathomorphology of the sciatic nerve and the role of P2X3 receptors in EA analgesia.

METHODSThe chronic constriction injury (CCI) model was adopted in this study. A total of 32 rats were randomly divided into four groups: sham CCI, CCI, CCI plus contralateral EA (CCI + conEA) and CCI plus ipsilateral EA (CCI + ipsEA). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured. EA began at day 7 after the CCI operation and was applied to the Zusanli (ST 36) and Yanglingquan acupoints (GB 34). At day 14, the pathomorphologic changes of the operated sciatic nerve were demonstrated by hematoxylin and eosin staining. In addition, dorsal root ganglion (DRG) neurons isolated from rats were examined by electrophysiological recording to determine if the P2X3 receptor agonists, adenosine 5'-triphosphate disodium (ATP) and α,β-methylen-ATP (α,β-meATP) evoked inward currents.

RESULTSPain thresholds in the CCI group were obviously decreased post CCI surgery (P<0.01). In the EA groups, thermal and mechanical threshold values were increased after the last EA treatment (P<0.05, P<0.01). There was no significant difference in light microscopic examination among the four groups (P>0.05). Current amplitude after application of ATP and α,β-meATP in DRG neurons were much larger in the CCI group compared to those obtained in sham CCI (P<0.05). ATP and α, β-meATP invoked amplitudes in the CCI + EA groups were reduced. There was no signififi cant difference between the CCI + conEA group and the CCI + ipsEA group (P>0.05).

CONCLUSIONEA analgesia may be mediated by decreasing the response of P2X3 receptors to the agonists ATP and α,β-meATP in the DRG of rats with CCI. No pathological changes of the sciatic nerve of rats were observed after EA treatment.

Adenosine Triphosphate ; analogs & derivatives ; pharmacology ; Animals ; Constriction, Pathologic ; Electroacupuncture ; Ganglia, Spinal ; drug effects ; metabolism ; pathology ; Hyperalgesia ; pathology ; Ion Channel Gating ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Reaction Time ; drug effects ; Receptors, Purinergic P2X3 ; metabolism ; Sciatic Nerve ; injuries ; metabolism ; pathology ; Staining and Labeling

OBJECTIVETo study the effects of the Chinese medicine Jinmaitong Capsule (, JMT) on the pathomorphology of sciatic nerves, ciliary neurotrophic factor (CNTF), and the mRNA expressions of CNTF in rats with streptozotocin-induced diabetes mellitus (STZ-DM).

METHODSThe animal model was established by one time intraperitoneal injection of streptozotocin. The rats were simply divided by random into 5 groups including model group, low-dose JMT group (JL), medium-dose JMT group (JM), high-dose JMT group (JH) and neurotropin group. For each of the above 5 groups, a group of 10 normal Wistar rats matched in body weight, age and gender were set as normal group. Intragastric administrations were started after the animal model established. The JL group were administered with five times the JMT dose recommended for a human adult; the JM group were administered with ten times the JMT dose recommended for a human adult; the JH group were administered with twenty times the JMT dose recommended for a human adult. The neurotropin group was administered with ten times the neurotropin dose recommended for a human adult. All rats were given intragastric administration for 16 weeks and then killed. In the 4th, 8th, 12th, 16th week, body weight and blood glucose level were detected before and after the intervention. The morphologic changes of the sciatic nerves were observed by optical microscope and transmission electron microscope. The CNTFmRNA expressions were detected by real-time fluorescent quantitative polymerase chain protein, and the CNTF protein expressions were detected by immunohistochemical method.

RESULTSThe blood glucose levels of the STZ-DM rats were much higher than normal group (P<0.01), and there was no apparent difference between any treatment groups and the model group (P>0.05). Before and after the intervention in the 4th, 8th, 12th, 16th week, there were no significant differences in the body weight among all the groups (P>0.05). The sciatic nerves of STZ-DM rats might have pathomorphological changes in axons, myelin sheaths, and interstitium. The levels of CNTF and CNTF-mRNA expressions in the STZ-DM rats were both significantly decreased (P<0.01). The sciatic nerves of STZ-DM rats might have pathomorphological changes in axons, myelin sheaths, and interstitium.

CONCLUSIONJMT could improve the pathomorphology of sciatic nerves by increasing CNTF's and CNTF-mRNA expressions in sciatic nerve tissues, and promote the repair and regeneration of damaged nerve fibers.

Animals ; Blood Glucose ; drug effects ; Body Weight ; drug effects ; Ciliary Neurotrophic Factor ; genetics ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; pathology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Gene Expression Regulation ; drug effects ; Humans ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Sciatic Nerve ; drug effects ; pathology ; ultrastructure