Adaptive changes following crush injury of brachial plexus terminal branches in adult rats

Abstracts

Objective To investigate whether mild hypothermia can promote neurogenesis in the dentate gyrus of hippocampus and cognitive function recovery after traumatic brain injury (TBI) through inhibiting apoptosis of hippocampal neurons. Methods A total of 66 healthy adult Sprague-Dawley rats were randomly divided into sham group, TBI group and TBI+ hypothermia group, with 22 rats in each group. The rat TBI model was established using the fluid percussion device. The rats in TBI+ hypothermia group received 4-hour hypothermia therapy immediately after injury, with the target temperature of 33.5℃. Bromodeoxyuridine (BrdU) was injected into the rats' abdominal cavity to label the mitotic cells. The test of Morris water maze was used to evaluate the rats' spatial learning and memory capabilities. Immunofluorescence staining was used to observe the expression levels of BrdU, doublecortin (DCX), neuron specific nuclear protein (NeuN), cysteinyl aspartate specific proteinase 3 (caspase-3) and cleaved caspase-3 expressions in dentate gyrus of hippocampus at 7 days and 28 days after injury. Expressions apoptosis-related proteins including the factor associated suicide (FAS)/factor associated suicide ligand (FASL), B-cell lymphoma-2 (Bcl-2), caspase-3 and cleaved caspase-3 expressions were detected by Western blot assay. Results The water maze tests at 28 days after injury showed that compared with TBI group, the escape latency in TBI+ hypothermia group was significantly shorter [(24.2±5.9)s∶(18±4.1)s], and both the time in the target quadrant and the number of platform crossing were increased significantly [(24.9±6.5)s∶(31.7±5.2)s; (1.9±0.8) times∶(3.5±1.2)times](P<0.05). Compared with the sham group, in TBI group and TBI+ hypothermia group, the BrdU+ new-born cells in the dentate gyrus of hippocampus were significantly increased at 7 days after injury [(9.4±4.1)∶(33.4±3.8); (9.4±4.1)∶(45.8±5.6)], the BrdU+ /DCX+ new-born neurons were increased at 7 days after injury [(2.0±0.6)∶(9.6±1.6); (2.0±0.6)∶(19.2±3.7)], and the BrdU+ /NeuN+ mature neurons were increased at 28 days after injury [(2.6±1.0)∶(17.2±3.9); (2.6±1.0)∶(33.6±9.1)] (P<0.01). TBI group showed more obvious increase than the TBI+ hypothermia group (P<0.01). Moreover, compared with 7 days after injury, the number of BrdU+ cells at 28 days after injury was further increased in TBI+ hypothermia group but decreased in TBI group [(45.8±5.6)∶(58.8±9.2); (33.4±3.8)∶(22.0±3.5)](P<0.05 or <0.01). Compared with the sham group, the caspase-3+ NeuN+ and caspase-3+ NeuN+ apoptotic neurons were significantly increased at 7 days after injury in TBI group [(2.0±0.9)∶(11.6±2.6); (2.6±1.0)∶(10.2±2.9)] (P<0.05). Compared with the TBI group, the cleaved caspase-3+ NeuN+ apoptotic neurons were decreased in TBI+ hypothermia group [(6.6±2.0)∶(11.6±2.6)](P<0.05). Furthermore, compared with the TBI group, mild hypothermia might down-regulate the expression of FAS, FASL, cleaved caspase-3 and caspase-3 and up-regulate the expression of Bcl-2 in the hippocampus [(1.54±0.15) ∶(1.14±0.12); (1.06±0.04)∶(0.80±0.09); (0.84±0.03)∶(0.62±0.08); (0.93±0.06)∶(0.86±0.09); (0.71±0.01)∶(1.58±0.18)](P<0.05). Conclusions Mild hypothermia might inhibit apoptosis of hippocampal neurons through cleaved caspase-3, FAS/FASL and Bcl-2 pathways, thus improving the neurogenesis and maturation of neurons in the dentate gyrus of hippocampus and facilitating cognitive function recovery in rats. It indicates that the function of hypothermia in anti-apoptosis and neurogenesis and maturity of hippocampal neurons may have a potential role in predicting the prognosis of TBI patients. Key words: Hypothermia; Brain injuries; Hippocampus; Neuve regeneration; Apoptosis

Mutation of the acid sphingomyelinase (ASM) gene and its reduced enzymatic activity is the main cause of the Type A Niemann-Pick disease. Recent advances demonstrated that ASM is necessary and sufficient to control the formation and release of microvesiscles containing the proinflammatory cytokine interleukin-1β (IL-1β) by glial cells [1]. Since IL-1β modulates the events caused by nerve damage and repair and seems to act as a neuro-modulator between activated glia and neurons [2], the control of its production and secretion might represent a new strategy in nerve regeneration and in the control of neuropathic pain. In this study we used a well-characterized ASM knockout mouse (ASMKO, [3]) to evaluate, through a multimodal approach, the onset and the course of the morphological and functional nerve damage and of neuropathic pain after sciatic nerve crush. Adult (1 and 5 month-old) male ASMKO and age-matched wild-type (WT) mice underwent sciatic nerve crush lesion. Nerve conduction velocity (NCV), walking track analysis followed by ultra-structural and morphometric analysis of sciatic nerves were performed to evaluate the features of nerve damage. Thermal (Plantar test) and mechanical sensitivity (Dynamic Plantar Aesthesiometer apparatus) were used to measure the severity of neuropathic pain. Moreover, the rotarod test completed the analysis as an indicator of motor impairment. One or two months after the nerve crush motor functional recovery was similar in WT and KO mice and the NCV measures performed in the sciatic nerve demonstrated a moderate and progressive improvement of nerve function. The results of the morphological examination confirmed the expected course of nerve recovery, but also demonstrated defective nerve regeneration, particularly evident in older, but already present in younger ASMKO mice. Behavioral tests suggested that the mutated phenotype in ASMKO might have an effect on the onset and development of mechanical and thermal hyperalgesia after nerve crush in both 1 - month and 5 - months - old groups. In conclusion, these data suggest a possible role for ASM-related microvesicles in nerve regeneration and suggest that targeting the IL-1β production and release may represent a new therapeutic strategy for the treatment of nerve damage and neuropathic pain.

Objective To investigate the effects of exogenous basic fibroblast growth factor (bFGF) on neurogenesis in the subventricular zone (SVZ) of 3-day-old rats following brain ischemia. Methods Seventy-two brain ischemia models of 3-day-old SD rats were established by ligating bilateral common carotid artery. The models were divided into treatment group with 10 ng/g bFGF injection and the control group with saline. Another 36 rats underwent operation without ischemia served as sham-operate group. Proliferating cells were labeled by bromodeoxyuridine (BrdU) through intraperitoneal injection in a cumulative protocol. Rats were killed at 4, 7 and 14 days after ischemic injury. Immunofluoreseence assays, Western blot and real-time PCR were used to observe cellular changes, including the protein expression of nestin, Tuj1, glial fibrillary acidic protein (GFAP) and NG2, and also their mRNA expression in SVZ, respectively. Results (1) Immunofluorescence assay showed that the number of BrdU1/nestin+ cells in SVZ of treatment group was the highest at day 7 after operation [(48. 7±5.9)/field], higher than those in control [(32. 2±3. 1)/field] and sham-operate group [(17.3±1.6)/field] (P<0. 01). The number of BrdU+/Tuj1+ cells, BrdU+/ GFAP+ cells and BrdU+/NG2+ cells in SVZ of treatment group were highest at day 14 after brain ischemia [(92.6±9.7) ,(58.2±6.1) ,(57.3±5.4)/field, respectively], higher than those in control [(65. 8±7. 1), (42. 1 ± 4. 4), (37.8 ± 3. 2)/field, respectively] and sham-operate group [(35.3± 3. 1), (33.6± 3.4), (22.4 ± 2. 1)/field, respectively] (P<0. 01 ). (2) Western blot and real-time PCR analysis found that the expressions of nestin protein and mRNA peaked on day 7, and the expressions of Tuj1, GFAP, and NG2 protein and mRNA peaked on day 14 after operation. The highest expression of nestin, Tuj1, GFAP, and NG2 protein and mRNA were detected in the treatment group. Conclusions bFGF not only increases the proliferation of neural stem cells in SVZ, but also stimulates the differentiation of these cells into neurons, astrocytes, and oligodendroeytes after ischemic injury, bFGF helps repair neonatal ischemic brain injury in rats. Key words: Fibroblast growth factor 2; Brain ischemia; Nerve regeneration; Rats

Management of peripheral nerve injury

Objective To explore the effect of bexarotene (BEX) on spatial cognition,impairment in memory and learning and hippocampal nerve regeneration of Aβ25-35 mice.Methods Eighty healthy male ICR mice were randomly divided into control group,model group,low-dosage BEX treatment group and high-dosage BEX treatment group (n=20); AD mouse models in the later three groups were induced by intracerebroventricular injection of Aβ25-35; and mice in the low-dosage BEX treatment group and high-dosage BEX treatment group treated with BEX (50 and 100 mg/kg·d) by gavage 7 days after Aβ25-35 injection for consecutive 14 days.Morris water maze and Y-maze tests were used to test the spatial cognitive function and abilities of learning and memory,respectively,28 days after Aβ25-35 injection.Intraperitoneal injection of 5-bromodeoxyuridine (BrdU) three times daily for 28 days was performed,and then,immumohistochemical staining was employed to detect the BrdU expression in the hippocampus; dual cortex protein (DCX) staining and Hoechst staining were,respectively,used to evaluate the proliferation and migration,and apoptosis of neuronal precursor cells; toluidine blue staining was employed to evaluate the mature neurons in the hippocampus.The levels of hippocampal brain-derived neurotrophic factor (BDNF) and the expressions of hippocampal glial fibrillary acidic protein (GFAP) and growth associated protein-43 (GAP-43) were measured by ELISA and Western blotting,respectively.Results Morris water maze test showed that the latency of crossing the platform was gradually increased in the control group,high-dosage BEX treatment group,low-dosage BEX treatment group and model group on the 3nd,4th,5th and 6th day,and the duration of stay was gradually decreased on the 6th day; increased learning frequency and reduced memory frequency was noted in Y-maze test; number of BrdU-positive cells,length and density of DCX neurite,and number of granule cells in the hippocampus decreased gradually,while Hoechst-positive cells decreased gradually; hippocampal levels of GFAP,GAP-43 and BDNF decreased gradually; the differences in the above levels were statistically significant (P＜0.05).Conclusion BEX can improve the spatial cognition and the impairment in memory and learning and promote their hippocampal neurogenesis of Aβ25-35 mice. Key words: Bexarotene; Aβ; Spatial cognition; Impairment in memory and learning; Hippocampal neurogenesis

The POU domain transcription factor Oct-6 is a major regulator of Schwann cell differentiation and myelination. During nerve development and regeneration, expression of Oct-6 is under the control of axonal signals. Identification of the cis-acting elements necessary for Oct-6 gene regulation is an important step in deciphering the complex signalling between Schwann cells and axons governing myelination. Here we show that a fragment distal to the Oct-6 gene, containing two DNase I-hypersensitive sites, acts as the Oct-6 Schwann cell-specific enhancer (SCE). The SCE is sufficient to drive spatially and temporally correct expression, during both normal peripheral nerve development and regeneration. We further demonstrate that a tagged version of Oct-6, driven by the SCE, rescues the peripheral nerve phenotype of Oct-6-deficient mice. Thus, our isolation and characterization of the Oct-6 SCE provides the first description of a cis-acting genetic element that responds to converging signalling pathways to drive myelination in the peripheral nervous system.

Applications of Proteomics to Nerve Regeneration Research

The neurotrophic peptide Org 2766 does not influence the expression of the immediate early gene c-fos following sciatic nerve crush in the rat

Objective To find out the topographic location of radial nerve deep branch in the main trunk of radial nerve at the upper arm level,and to investigate the results of transferring full length phrenic nerve to the deep branch of the radial nerve for restoration of wrist and finger extension.Methods Thirtytwo sides of 16 cadavers were dissected.Microsurgical retrograde dissection of the radial nerve was carried out to delineate the fascicular features of the deep and superficial branches of the radial nerve at the upper arm.The topographic location of the deep branch in the main trunk of radial nerve at the level of latissimus dorsi insertion was also observed.Clinically full length phrenic nerve was transferred to the deep branch of the radial nerve at the level of latissimns dorsi insertion in 2 cases.Postoperatively the results of the tansfer were evaluated.Results There were two large fascicular groups in the antebrachial portion of the radial nerve in the arm.The deep branch of the radial nerve was mostly in the medial portion at the level of latissimns dorsi insertion.One of the 2 cases was followed for 1 year and 10 months.The patient had 80°of shoulder abduction,90°of elbow flexion,0°of elbow extension,10°of wrist flexion,full wrist and finger extension but no finger flexion.Muscle power of wrist extensor and finger extensor was M4 and Ms respectively.The other patient had 40°of shoulder abduction,30°of elbow flexion,0° of elbow extension,full wrist extension,slightly impaired finger extension but no wrist and finger flexion at 2-yesx follow-up.Muscle power of wrist extensor and finger extensor was M3 and M3.respectively.Condnsion Targeted transfer of full lengh phrenic nerve to the medial fascicles of the antebrachial portion of the radial nerve at the level of latissimus dorsi insertion could be an effective procedure to recover wrist and finger extension in total brachial plexus injury patients. Key words: Phrenic nerve; Radial nerve; Anatomy,regional; Open chest

Perioperative Nerve Injury: A Silent Scream?

Objective To observe the anatomical characteristics of the radial nerve and explore the functional evaluation methods after repairing radial nerve defect in primates.Methods Three adult rhesus monkeys were selected for dissecting radial nerve from axilla to the elbow,and their position and branches were recorded.Five healthy adult rhesus monkeys were selected to establish the model of bilateral radial nerve 25 mm long defect,and nerve defects were bridged with autologous nerve (group A) and acellular porcine nerve (group B) respectively.Wrist extension and general conditions were observed after operation.Postoperative cumulative maximum wrist angle (DEmax) and wrist angle recovery rate (R) were measured and calculated to evaluate the effect of restoration of neurological function by a special fixed chair before operation,1 week and 5 months after operation respectively.Results The first muscular branch of extensor carpi radialis longus (ECRL) in rhesus monkeys was issued 9-14 mm proximal to the lateral condyle of the humerus.The radial nerve could be exposed proximal to the issued point with the length of 52-62 mm,diameter of 3.3-4.1 mm,which could create 25 mm radial nerve defect.Wristdrop and fingerdrop were observed for rhesus monkeys after surgery,with good general conditions and wound healing.After 5 months,the mean of DEmax in group A and group B was 120° and 96° respectively,and R was 99％ and 69％ respectively.Conclusion The radial nerve defect model in the lower arm with safe operation,dynamic observation and quantitative description of the recovery of neurological function,can be used as the standard model of peripheral nerve repair in primate. Key words: Peripheral nerve injuries ; Nerve regeneration ; Rhesus monkeys ; Model, animal

Background: Nerve conduction studies (NCS) are electrodiagnostic tests which are used to evaluate the ability of the conduction of the motor and the sensory nerves. NCS tests are used in the diagnosis of polyneuropathies, mononeuropathies, radicular lesions, tunnel syndromes and nerve damages caused by trauma and compression. Objective: The objective of this study was to evaluate the normative data of nerve conduction study (NCS) parameters among healthy young adults and effect of gender on nerve conduction study parameters.. Materials and methods: This study was conducted in Kathmandu University School of Medical Science, Dhulikhel from January to November, 2015. The study was done in total 40 out of which 20 were males and 20 were females consenting healthy adults. The compound muscle action potential (CMAP) and sensory nerve action potential (SNAP) were recorded. Result: The mean age of participants was 19.50 1.56, height was 167.24 7.32, weight was 60.22 5.47 with mean BMI 20.12 3.24 . The motor nerve conduction duration is minimum in median nerve while maximum in radial nerve. Amplitude is higher in median nerve while lower in radial nerve. The motor nerve conduction duration is minimum in median nerve while maximum in radial nerve. Amplitude is higher in median nerve while lower in radial nerve. The sensory nerve conduction duration, amplitude and latency are more in median nerve than in ulnar nerve. Conclusion: Nerve conduction study variables are within the normal range compared to previous studies.

Objective To investigate the differentiation of neural stem cells in the subventrieular zone(SVZ)of the 3-day-old neonatal rats with ischemic brain injury and to provide the experimental evidences on endogenous repair mechanism of premature brain injury after ischemia. Methods Thirty-two 3-day-old rats were divided into experimental group and the control group.Rats in the experimental group were subjected to bilateral common arteries occlusion.while those in control group were not.All rats were administrated 5-bromodeoxyuridine(BrdU)50 mg/kg by intraperitoneal injection twice daily during 5 to 7 days after the operation.At 14 days and 28 days after operation,rats were sacrificed and their brains were collected.The changes of newborn cells in the SVZ of brain were observed by marking the BrdU,neuronal class Ⅲ β-tubulin(TuJ1),oligodendrocyte O antigen-4 (O4)and glial fibrillary acidic protein(GFAP)with immunofluorescence. Results Compared with the control group,the number(7800±800 vs 4200±700,10 700±1400 vs 4600±600)of newborn neuron(BrdU+/TuJl+)and the number(6100±1000 vs 2600±500,7300±1400 vs 2800±800)of newborn oligodendrocytes(BrdU+/O4+)per field increased significantly at 14 days and 28 days after operation in the experimental group,so did the number of newborn astrocytes per field(4500±700 vs 2700±500,6700±1100 vs 3000±600)respectively(P<0.01). Conclusions The number of neurons,oligodendroeytes and atrocytes in the SVZ increased after brain ischemia,which suggested that SVZ might have the function of repair after brain ischemia injury. Key words: Brain ischemia; Cerebral ventricles; Nerve regeneration; Rats

No matter what treatment is used after nerve transection, a complete cure is impossible, so basic and clinical research is underway to find a cure. As part of this research, autophagy is being investigated for its role in nerve regeneration. Here, we review the existing literature regarding the involvement and significance of autophagy in peripheral nerve injury and regeneration. A comprehensive literature review was conducted to assess the induction and role of autophagy in peripheral nerve injury and subsequent regeneration. Studies were included if they were prospective or retrospective investigations of autophagy and facial or peripheral nerves. Articles not mentioning autophagy or the facial or peripheral nerves, review articles, off-topic articles, and those not written in English were excluded. A total of 14 peripheral nerve studies that met these criteria, including 11 involving sciatic nerves, 2 involving facial nerves, and 1 involving the inferior alveolar nerve, were included in this review. Studies conducted on rats and mice have demonstrated activation of autophagy and expression of related factors in peripheral nerves with or without stimulation of autophagy-inducing factors such as rapamycin, curcumin, three-dimensional melatonin nerve scaffolds, CXCL12, resveratrol, nerve growth factor, lentinan, adipose-derived stem cells and melatonin, basic fibroblast growth factor, and epothilone B. Among the most studied of these factors in relation to degeneration and regeneration of facial and sciatic nerves are LC3II/I, PI3K, mTOR, Beclin-1, ATG3, ATG5, ATG7, ATG9, and ATG12. This analysis indicates that autophagy is involved in the process of nerve regeneration following facial and sciatic nerve damage. Inadequate autophagy induction or failure of autophagy responses can result in regeneration issues after peripheral nerve damage. Animal studies suggest that autophagy plays an important role in peripheral nerve degeneration and regeneration.