Age-dependent loss of cholinergic neurons in learning and memory-related brain regions and impaired learning in SAMP8 mice with trigeminal nerve damage

The tooth belongs to the trigeminal sensory pathway. Dental damage has been associated with impairments in the central nervous system that may be mediated by injury to the trigeminal nerve. In the present study, we investigated the effects of damage to the inferior alveolar nerve, an important peripheral nerve in the trigeminal sensory pathway, on learning and memory be-haviors and structural changes in related brain regions, in a mouse model of Alzheimer’s disease. Inferior alveolar nerve transection or sham surgery was performed in middle-aged （4-month-old） or elderly （7-month-old） senescence-accelerated mouse prone 8 （SAMP8） mice. When the middle-aged mice reached 8 months （middle-aged group 1） or 11 months （middle-aged group 2）, and the elderly group reached 11 months, step-down passive avoidance and Y-maze tests of learn-ing and memory were performed, and the cholinergic system was examined in the hippocampus （Nissl staining and acetylcholinesterase histochemistry） and basal forebrain （choline acetyltrans-ferase immunohistochemistry）. In the elderly group, animals that underwent nerve transection had fewer pyramidal neurons in the hippocampal CA1 and CA3 regions, fewer cholinergic ifbers in the CA1 and dentate gyrus, and fewer cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band, compared with sham-operated animals, as well as showing impairments in learning and memory. Conversely, no signiifcant differences in histology or be-havior were observed between middle-aged group 1 or group 2 transected mice and age-matched sham-operated mice. The present ifndings suggest that trigeminal nerve damage in old age, but not middle age, can induce degeneration of the septal-hippocampal cholinergic system and loss of hippocampal pyramidal neurons, and ultimately impair learning ability. Our results highlight the importance of active treatment of trigeminal nerve damage in elderly patients and those with Alzheimer’s disease, and indicate that tooth extract     

丹参酮对大鼠脊髓缺血再灌注损伤NMDAR1蛋白表达的影响

BACKGROUND： Tanshinone has been previously shown to be involved in the prevention and treatment of cerebral ischemia/reperfusion injury. In addition, excitatory amino acid-mediated neu- rotoxicity may induce neuronal damage following spinal cord ischemia/reperfusion injury.
OBJECTIVE： To explore the interventional effect of tanshinone on N-methyl-D-aspartate receptor 1 （NMDAR1） protein expression in a rat model of spinal cord ischemia/reperfusion injury.
DESIGN, TIME AND SETTING： A randomized molecular biology experiment was conducted at the Traumatology ＆ Orthopedics Laboratory of Fujian Hospital of Traditional Chinese Medicine （Key Laboratory of State Administration of Traditional Chinese Medicine） between September 2007 and May 2008. MATERIALS： A total of 88 Sprague Dawley rats were randomly divided into a sham operation （n = 8）, model （n = 40）, and tanshinone （n = 40） groups. Thirty minutes after ischemia, rats in the model and tanshinone groups were observed at hour 0.5, 1, 4, 8, and 12 following perfusion, with eight rats for each time point. METHODS： Abdominal aorta occlusion was performed along the right renal arterial root using a Scoville-Lewis clamp to induce spinal cord ischemia. Blood flow was recovered 30 minutes following occlusion to establish models of spinal cord ischemia/reperfusion injury. Abdominal aorta occlusion was not performed in the sham operation group. An intraperitoneal injection of tanshinone ⅡA sulfonic sodium solution （0.2 L/g） was administered to rats in the tanshinone group, preoperatively. In addition, rats in the sham operation and model groups were treated with an intraperitoneal injection of the same concentration of saline, preoperatively.
MAIN OUTCOME MEASURES： NMDAR1 protein expression in the anterior horn of the spinal cord, accumulative absorbance, average absorbance, and area of positive cells were detected in the three groups through immunohistochemistry.
RESULTS： All 88 rats were included in the final analysis. （1） NMDAR1 protein expression increased following 30-minute ischemia/1-hour reperfusion injury to the spinal cord, and reached a peak 4 hours after reperfusion. （2） Accumulative absorbance and average absorbance of NMDAR1, as well as area of positive cells in the model group, were significantly greater than the sham operation group at each time point （P 〈 0.05）. However, values in the tanshinone group were significantly less than the model group （P 〈 0.05）.
CONCLUSION： NMDAR1 protein expression was rapidly increased following spinal cord ischemia/reperfusion injury and reached a peak 4 hours following reperfusion. In addition, tanshinone downregulated NMDAR1 protein expression in the anterior horn of the spinal cord.     

人参皂甙Rg1对去卵巢大鼠面神经核脑源性神经营养因子表达的影响：半定量分析

BACKGROUND： Estrogen is neuroprotective effects such as breast carcinoma, endometria but long-term estrogen treatment can induce side cancer, and stroke. However, phytoestrogen is neuroprotective without these side effects. OBJECTIVE： To study the effects of Ginsenoside Rgl on facial neurons and brain-derived neurotrophic factor （BDNF） expression in the facial nucleus in ovariectomized rats. DESIGN, TIME AND SETTING： The randomized, controlled animal experiments were performed at the Ultrasonic Institute, Second Affiliated Hospital, Chongqing Medical University, China, from September 2007 to September 2008. MATERIALS： Ginsenoside Rgl （Sigma, USA）, rabbit anti-rat BDNF, Bcl-2, Bax antibodies, biotin-labeled goat anti-rabbit IgG （Boster, China）, and a TUNEL kit （Roche, Germany） were used in this study. METHODS： A total of 48 adult Sprague Dawley rats undergoing ovariectomy were randomly assigned into sham operation （n = 8）, model （n = 20）, and Ginsenoside Rgl （n = 20） groups. Facial nerve damage was induced by bilateral clamping of the facial nerve trunk. The bilateral facial nerve trunk was exposed in the sham operation group, with no clamping. Rats in the Ginsenoside Rgl group were intraperitoneally injected with 10 mg/kg per day Ginsenoside Rgl; other groups received 2 mL saline, once a day, for 14 days. MAIN OUTCOME MEASURES： Morphologic changes in neurons of the facial nucleus were observed following hematoxylin-eosin staining. Neuronal apoptosis was detected by TUNEL. Changes in ultrastructure of the facial nerve fibers were observed with a transmission electron microscope. Expression of BDNF, Bcl-2, and Bax protein was quantified by semiquantitative immunohistochemistry. RESULTS： At 3-14 days following facial nerve damage, Ginsenoside Rgl increased BDNF expression and the number of regenerated nerve fibers, and produced thicker myelin sheaths （P 〈 0.05）. Ginsenoside Rgl also gradually increased Bcl-2 protein expression and decreased Bax protein expression （P 〈 0.05）. By day 7, apoptosis was observed in facial neurons, but Ginsenoside Rgl reduced the number of apoptotic neurons. Sham animals did not show any changes in BDNF, Bcl-2, or Bax expression or facial neuron morphology. CONCLUSION： Ginsenoside Rgl can substantially inhibit facial neuronal apoptosis by increasing endogenous BDNF and Bcl-2 expression and by decreasing Bax expression in ovariectomized rats after facial nerve damage.     

Effects of exogenous ganglioside-1 on learning and memory in a neonatal rat model of hypoxia-ischemia brain injury★

BACKGROUND: Exogenous ganglioside-1 (GM1) can cross the blood-brain barrier and play a protective role against hypoxia-ischemia-induced brain damage. OBJECTIVE: To examine the possible mechanisms of exogenous GM1 protection in hypoxia-ischemia-induced brain damage in a neonatal rat model by measuring changes in brain mass, pathological morphology, growth-associated protein-43 expression, and neurobehavioral manifestations. DESIGN, TIME AND SETTING: A randomized block-design study was performed at the Immunohistochemistry Laboratory of the Pediatric Research Institute, Children’s Hospital of Chongqing Medical University from August 2005 to August 2006. MATERIALS: A total of 36 neonatal, 7-day-old, Sprague Dawley rats were used in this experiment. The hypoxia-ischemia-induced brain damage model was established by permanently occluding the right carotid artery, followed by oxygen inhalation at a low concentration (8% O2, 92% N2) for 2 hours. METHODS: All rats were randomly divided into the following groups: GM1, model, and sham operation, with 12 rats each group. Rats in the GM1 and model groups received hypoxic/ischemic-induced brain damage. Rats in the GM1 group received injections of GM1 (i.p., 20 mg/kg) at 0, 24, 48, 72, 96, 120, and 144 hours following models established, and rats in the model group were administered (i.p.) the same amount of saline. The right carotid artery was separated, but not ligated, in the sham operation group rats. MAIN OUTCOME MEASURES: At 1 week after surgery, expression of growth-associated protein-43, a marker of neural development and plasticity, was detected in the hippocampal CA3 region by immunohistochemistry. Brain mass was measured, and the pathological morphology was observed. At 4 weeks after surgery, behavioral changes in the remaining rats were tested by Morris water maze, and growth-associated protein-43 expression was measured. RESULTS: (1) In the GM1 and sham operation groups, growth-associated protein-43 expression was greater in the hippocampal CA3 region compared to the model group 1 week after surgery (P < 0.05). In all three groups, brain weight of the right hemisphere was significantly less than the left hemisphere, in particular in the model group (P < 0.05). In the GM1 group, the weight difference between two hemispheres, as well as the extent of damage in the right hemisphere, was less than the model group (P < 0.01). In the sham operation group, brain tissue consisted of integrated structures and ordered cells. In the model group, the cerebral cortex layers of the right hemisphere were not defined, neurons were damaged, and neurons were disarranged in the hippocampal area. In the GM1 group, neurons were dense in the right cerebral cortex and hippocampal area, with no significant change in glial proliferation. (2) The average time of escape latency in the GM1 group was shortened 4 weeks after surgery, and significantly less than the model group (P < 0.05). In addition, the frequency platform passing in the GM1 group was significantly greater than the model group (P < 0.01). CONCLUSION: Exogenous GM1 may reduce brain injury and improve learning and memory in hypoxia-ischemia-induced brain damage rats. This protection may be associated with increased growth-associated protein-43 expression, which is involved in neuronal remodeling processes. Key Words: ganglioside; growth-associated protein-43; hypoxia-ischemia brain damage; Morris water maze     

Effects of exogenous ganglioside-1 on learning and memory in a neonatal rat model of hypoxia-ischemia brain injury★

BACKGROUND: Exogenous ganglioside-1 (GM1) can cross the blood-brain barrier and play a protective role against hypoxia-ischemia-induced brain damage. OBJECTIVE: To examine the possible mechanisms of exogenous GM1 protection in hypoxia-ischemia-induced brain damage in a neonatal rat model by measuring changes in brain mass, pathological morphology, growth-associated protein-43 expression, and neurobehavioral manifestations. DESIGN, TIME AND SETTING: A randomized block-design study was performed at the Immunohistochemistry Laboratory of the Pediatric Research Institute, Children’s Hospital of Chongqing Medical University from August 2005 to August 2006. MATERIALS: A total of 36 neonatal, 7-day-old, Sprague Dawley rats were used in this experiment. The hypoxia-ischemia-induced brain damage model was established by permanently occluding the right carotid artery, followed by oxygen inhalation at a low concentration (8% O2, 92% N2) for 2 hours. METHODS: All rats were randomly divided into the following groups: GM1, model, and sham operation, with 12 rats each group. Rats in the GM1 and model groups received hypoxic/ischemic-induced brain damage. Rats in the GM1 group received injections of GM1 (i.p., 20 mg/kg) at 0, 24, 48, 72, 96, 120, and 144 hours following models established, and rats in the model group were administered (i.p.) the same amount of saline. The right carotid artery was separated, but not ligated, in the sham operation group rats. MAIN OUTCOME MEASURES: At 1 week after surgery, expression of growth-associated protein-43, a marker of neural development and plasticity, was detected in the hippocampal CA3 region by immunohistochemistry. Brain mass was measured, and the pathological morphology was observed. At 4 weeks after surgery, behavioral changes in the remaining rats were tested by Morris water maze, and growth-associated protein-43 expression was measured. RESULTS: (1) In the GM1 and sham operation groups, growth-associated protein-43 expression was greater in the hippocampal CA3 region compared to the model group 1 week after surgery (P < 0.05). In all three groups, brain weight of the right hemisphere was significantly less than the left hemisphere, in particular in the model group (P < 0.05). In the GM1 group, the weight difference between two hemispheres, as well as the extent of damage in the right hemisphere, was less than the model group (P < 0.01). In the sham operation group, brain tissue consisted of integrated structures and ordered cells. In the model group, the cerebral cortex layers of the right hemisphere were not defined, neurons were damaged, and neurons were disarranged in the hippocampal area. In the GM1 group, neurons were dense in the right cerebral cortex and hippocampal area, with no significant change in glial proliferation. (2) The average time of escape latency in the GM1 group was shortened 4 weeks after surgery, and significantly less than the model group (P < 0.05). In addition, the frequency platform passing in the GM1 group was significantly greater than the model group (P < 0.01). CONCLUSION: Exogenous GM1 may reduce brain injury and improve learning and memory in hypoxia-ischemia-induced brain damage rats. This protection may be associated with increased growth-associated protein-43 expression, which is involved in neuronal remodeling processes. Key Words: ganglioside; growth-associated protein-43; hypoxia-ischemia brain damage; Morris water maze     

Effects of tacrolimus （FK506） on sciatic nerve regeneration in rats

BACKGROUND： Tacrolimus （FK506） protects peripheral nerves located in damaged regions by inhibiting T lymphocyte proliferation and activation. OBJECTIVE： To evaluate the effect of FK506 on promoting regeneration of rat sciatic nerve. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Laboratory of the Department of Orthopedic Surgery, Dalian Medical University, China, from September 2007 to September 2008. MATERIALS： A total of 60 adult, male, Sprague-Dawley rats were equally and randomly divided into model, local administration and systemic administration groups. All rats received a neurotomy of bilateral sciatic nerves to establish models of nerve regeneration chambers. The powder and injection of FK506 were supplied by Fujisawa Pharmaceutical, Japan. METHODS： The regeneration chambers of the model group were infused with 0.2 mL saline. The systemic group were injected with 0.2 mL saline, followed by daily subcutaneous injections of FK506 （1 mg/kg）, for 14 days. The local administration group was infused with 0.2 mL FK506 （1 μg/mL）. MAIN OUTCOME MEASURES： Local immune response was observed using hematoxylin-eosin staining. Myelinated nerve fiber number, myelin sheath and nerve fiber thickness were observed using toluidine blue staining. Wet weight of gastrocnemius was evaluated. Compound muscle action potential amplitude, latency, and conduction time were recorded, and motor nerve conduction velocity was calculated using electrophysiology. RESULTS： The total number of myelinated nerve fibers in the local and systemic administration groups was significantly higher than in the model group. The density of myelinated nerve fibers, myelin sheath thickness and mean axon diameter were significantly increased in the systemic administration group compared with the model group （P 〈 0.05）. Lymphocyte infiltration was decreased in the local and systemic administration groups compared with the model group. The wet weight of rat gastrocnemius in the local and systemic administration groups were significantly greater compared with the model group （P 〈 0.05）. Motor nerve conduction velocity was the fastest in the systemic administration group, and the slowest in the model group. Compound muscle action potential amplitude was larger in the systemic administration group compared with the local administration and model groups （P 〈 0.05）. CONCLUSION： Systemic administration of FK506 can promote regeneration of rat sciatic nerve and recovery of neural function. Systemic administration produced better regeneration and recovery of function than local administration of FK506.     

Skeletal muscle-derived cells repair peripheral nerve defects in mice

Skeletal muscle-derived cells have strong secretory function,while skeletal muscle-derived stem cells,which are included in muscle-derived cells,can differentiate into Schwann cell-like cells and other cell types.However,the effect of muscle-derived cells on peripheral nerve defects has not been reported.In this study,5-mm-long nerve defects were created in the right sciatic nerves of mice to construct a peripheral nerve defect model.Adult female C57BL/6 mice were randomly divided into four groups.For the muscle-derived cell group,muscle-derived cells were injected into the catheter after the cut nerve ends were bridged with a polyurethane catheter.For external oblique muscle-fabricated nerve conduit and polyurethane groups,an external oblique muscle-fabricated nerve conduit or polyurethane catheter was used to bridge the cut nerve ends,respectively.For the sham group,the sciatic nerves on the right side were separated but not excised.At 8 and 12 weeks post-surgery,distributions of axons and myelin sheaths were observed,and the nerve diameter was calculated using immunofluorescence staining.The number,diameter,and thickness of myelinated nerve fibers were detected by toluidine blue staining and transmission electron microscopy.Muscle fiber area ratios were calculated by Masson’s trichrome staining of gastrocnemius muscle sections.Sciatic functional index was recorded using walking footprint analysis at 4,8,and 12 weeks after operation.The results showed that,at 8 and 12 weeks after surgery,myelin sheaths and axons of regenerating nerves were evenly distributed in the muscle-derived cell group.The number,diameter,and myelin sheath thickness of myelinated nerve fibers,as well as gastrocnemius muscle wet weight and muscle area ratio,were significantly higher in the muscle-derived cell group compared with the polyurethane group.At 4,8,and 12 weeks post-surgery,sciatic functional index was notably increased in the muscle-derived cell group compared with the polyurethane group.These criteria of the muscle-derived cell group were not significantly different from the external oblique muscle-fabricated nerve conduit group.Collectively,these data suggest that muscle-derived cells effectively accelerated peripheral nerve regeneration.This study was approved by the Animal Ethics Committee of Plastic Surgery Hospital,Chinese Academy of Medical Sciences(approval No.040)on September 28,2016.     

温针灸对腰神经根受压大鼠模型一氧化氮合酶和降钙素相关基因肽的影响

BACKGROUND： Varying degrees of inflammatory responses occur during lumbar nerve root compression. Studies have shown that nitric oxide synthase （NOS） and calcitonin gene-related peptide （CGRP） are involved in secondary disc inflammation. OBJECTIVE： To observe the effects of warm acupuncture on the ultrastructure of inflammatory mediators in a rat model of lumbar nerve root compression, including NOS and CGRP contents. DESIGN, TIME AND SETTING： Randomized, controlled study, with molecular biological analysis, was performed at the Experimental Center, Sixth People＇s Hospital Affiliated to Shanghai Jiao Tong University, between September 2006 and April 2007. MATERIALS： Acupuncture needles and refined Moxa grains were purchased from Shanghai Taicheng Technology Development Co., Ltd., China; Mobic tablets were purchased from Shanghai Boehringer Ingelheim Pharmaceuticals Co., Ltd., China; enzyme linked immunosorbent assay （ELISA） kits for NOS and CGRP were purchased from ADL Biotechnology, Inc., USA. METHODS： A total of 50, healthy, adult Sprague-Dawley rats, were randomly divided into five groups normal, model, warm acupuncture, acupuncture, and drug, with 10 rats in each group. Rats in the four groups, excluding the normal group, were used to establish models of lumbar nerve root compression. After 3 days, Jiaji points were set using reinforcing-reducing manipulation in the warm acupuncture group. Moxa grains were burned on each needle, with 2 grains each daily. The acupuncture group was the same as the warm acupuncture group, with the exception of non-moxibustion. Mobic suspension （3.75 mg/kg） was used in the oral drug group, once a day. Treatment of each group lasted for 14 consecutive days. Modeling and medication were not performed in the normal group. MAIN OUTCOME MEASURES： The ultrastructure of damaged nerve roots was observed with transmission electron microscopy; NOS and CGRP contents were measured using ELISA. RESULTS： The changes of the radicular ultramicrostructure were characterized by Wallerian degeneration; nerve fibers were clearly demyelinated; axons collapsed or degenerated; outer Schwann cell cytoplasm was swollen and its nucleus was compacted. Compared with the normal group, NOS and CGRP contents in the nerve root compression zone in the model group were significantly increased （P 〈 0.01）. Nerve root edema was improved in the drug, acupuncture and the warm acupuncture groups over the model group. NOS and CGRP expressions were also decreased with the warm acupuncture group having the lowest concentration （P 〈 0.01）. CONCLUSION： In comparison to the known effects of Mobic drug and acupuncture treatments, the warm acupuncture significantly decreased NOS and CGRP expression which helped improve the ultrastructure of the compressed nerve root.     

补阳还五汤对老龄大鼠脑缺血再灌注海马齿状回细胞增殖分化的影响

BACKGROUND： The mobilization of endogenous stem cells is an effective way to promote repair following ischemic brain damage. Buyang Huanwu decoction （BHD） can effectively improve cerebral blood flow and protect against cerebral ischemia/reperfusion damage. OBJECTIVE： To study the effects of BHD on cell proliferation and differentiation in the hippocampal dentate gyrus of rats following cerebral infarction, to investigate the protective effects of BHD against cerebral infarction, and to analyze the dose-effect relationship. DESIGN, TIME AND SETTING： This randomized, controlled, animal study was performed at the Laboratory of Department of Physiology, Henan College of Traditional Chinese Medicine, China from June 2007 to February 2008. MATERIALS： A total of 36 male, Sprague Dawley rats, aged 20-21 months, were equally and randomly assigned to the following groups： sham operation, model control, and nimodipine, as well as high-dose, moderate-dose, and low-dose BHD. BHD was composed of milkvetch root, Chinese angelica, red peony root, earthworm, peach seed, safflower, and Szechwan Iovage rhizome, which were provided by the Outpatient Department, Henan College of Traditional Chinese Medicine, China. METHODS： The Chinese medicinal ingredients described above were decocted. The external carotid artery was ligated in rats from the sham operation group. Rat models of focal cerebral infarction were established by middle cerebral artery occlusion in the model control and nimodipine groups, as well as the high-dose, moderate-dose, and low-dose BHD groups. The drugs were administered by gavage 5 days, as well as 2 hours, prior to model induction. Rats in the nimodipine group were daily administered a 6 mg/kg nimodipine suspension by gavage. Rats in the high-dose, moderate-dose, and low-dose BHD groups were administered daily 26, 13, and 6.5 g/kg BHD, respectively. Rats in the sham operation and model control groups were treated with an equal volume of saline. MAIN OUTCOME MEASURES： The effects of BHD on neurological dysfunction score, brain water content, cell proliferation and differentiation in the hippocampal dentate gyrus, and pathological changes in the ischemic brain hemisphere were measured in cerebral infarction rats. RESULTS： Compared with the sham operation group, the neurological dysfunction score, brain water content, number of BrdU-positive cells, BrdU/NeuN-positive cells, and BrdU/GFAP-positive cells in the hippocampal dentate gyrus significantly increased in the model control group （P 〈 0.01 ）. Compared with the model control group, neurological dysfunction score and brain water content were significantly decreased （P 〈 0.01 or 0.05）, as were the number of BrdU-positive and BrdU/NeuN-positive cells （P 〈 0.01 or 0.05）. The number of BrdU/GFAP-positive cells was significantly reduced （P 〈 0.05） in the nimodipine group, high-dose, moderate-dose, and low-dose BHD groups. Compared with the nimodipine group, the neurological dysfunction score was significantly reduced in the moderate-dose BHD group （P 〈 0.05）. However, the number of BrdU-positive cells was significantly increased in the rat hippocampal dentate gyrus in the high-dose and moderate-dose BHD groups （P 〈 0.01 or 0.05）. The following was determined by microscopy： slightly disarranged neural cells, mild vascular dilatation, inflammatory cell infiltration, and light tissue edema were observed in the nimodipine group; inflammatory celt infiltration was reduced in the low-dose BHD group; cerebral edema and inflammatory cell infiltration were significantly reduced in the high-dose and in the moderate-dose BHD group. Electron microscopy revealed lipofuscin, slightly swollen mitochondria, and normal rough endoplasmic reticulum in the high-dose and moderate-dose BHD groups. Improvement was best in the moderate-dose BHD group. CONCLUSION： Cerebral ischemia activated proliferation of neural stem cells in the rat hippocampal dentate gyrus. The actions of BHD against cerebral ischemia/reperfusion damage correlated with proliferation and differentiation of neural stem cells in the hippocampal dentate gyrus. A moderate-dose of BHD resulted in the most effective outcome.     

微囊化兔坐骨神经组织细胞移植对大鼠损伤脊髓核因子κB表达的影响

BACKGROUND： It has been reported that nuclear factor-kappa B （NF- κB）, activated after spinal cord injury in rats, plays a key role in inflammatory responses in the central nervous system. OBJECTIVE： To investigate the effects of transplantation of microencapsulated rabbit sciatic nerve on NF- κB expression and motor function after spinal cord injury in rats, and to compare the results with the transplantation of rabbit sciatic nerve alone. DESIGN, TIME AND SETTING： This completely randomized, controlled study was performed at the Department of Neurobiology, Medical College of Nanchang University between December 2007 and July 2008. MATERIALS： A rabbit anti-NF- κB P65 monoclonal antibody was made by the Santa Cruz Company, USA and a streptavidin peroxidase immunohistochemical kit was provided by the Sequoia Company, China. METHODS： Eight rabbits were used to prepare a sciatic nerve cell suspension that was divided into two parts： one stored for transplantation, and the other mixed with a 1.5% sodium alginate solution. One hundred and twenty adult Sprague Dawley rats weighing 220-250 g were randomly divided into four groups： the microencapsulated cell group （n = 36）, the non-encapsulated cell group （n = 36）, the saline group （n = 36） and the sham operation group （n = 12）. The first three groups underwent a right hemisection injury of the spinal cord at the T10 level, into which was transplanted a gelatin sponge soaked with 10 μL of a microencapsulated nerve tissue/cell suspension （microencapsulated cell group）, a tissue/cell suspension （non-encapsulated cell group） or physiological saline （saline group）. In the sham operation group the vertebrae were exposed, but the spinal cord was not injured, and no implantation was given. MAIN OUTCOME MEASURES： Pathological changes were detected using hematoxylin-eosin staining; NF- κB expression was quantified using immunohistochemical staining; motor function was assessed using the Basso, Beattie and Bresnahan （BBB） scale. RESULTS： Spinal cord injuries, such as neuronal death and inflammatory cell infiltration, were found in the microencapsulated cell group, the non-encapsulated cell group and the saline group. However, the damage in the microencapsulated cell group was milder than in the non-encapsulated cell or saline groups. NF- κB expression in the microencapsulated cell group, the non-encapsulated cell group and the saline group was increased after spinal cord injury; it reached a peak after 24 hours, gradually decreased after 3 days, and was close to normal levels after 7 days. NF- κB expression in the microencapsulated cell group was significantly lower than in the saline group and the non-encapsulated cell group （P 〈 0.05）. With time, the motor function of the animals in each group improved to a certain extent, but did not reach normal levels. There were no significant differences in BBB scores between the different groups on post-operative day 3; however, the BBB scores for the microencapsulated cell group and the non-encapsulated cell group were significantly higher than the saline group on post-operative day 7 （P 〈 0.05）. In addition, the motor function recovered better in the microencapsulated cell group than in the non-encapsulated cell group （P 〈 0.05）. CONCLUSION： The transplantation of microencapsulated rabbit sciatic nerve can inhibit NF- κB expression and inflammatory reactions and promote recovery of motor function after spinal cord injury in rats. The effects of microencapsulated cell transplantation are superior to those of transplantation of cells alone.     

M1 muscarinic acetylcholine receptor in Alzheimer＇s disease

The degeneration of cholinergic neurons and cholinergic hypofunction are pathologies associated with Alzheimer＇s disease （AD）. Muscarinic acetylcholine receptors （mAChRs） mediate acetylcholine-induced neurotransmission and five mAChR subtypes （M1-M5） have been identified. Among them, M1 mAChR is widely expressed in the central nervous system and has been implicated in many physiological and pathological brain functions. In addition, M1 mAChR is postulated to be an important therapeutic target for AD and several other neurodegenerative diseases. In this article, we review recent progress in understanding the functional involvement of M1 mAChR in AD pathology and in developing M1 mAChR agonists forAD treatment.     

神经生长因子促再生坐骨神经血管生成的作用

BACKGROUND： It remains to be determined whether nerve growth factor （NGF） can promote angiogenesis in regenerating peripheral nerves during repairing peripheral nerve injury.
OBJECTIVE： To evaluate the effects of NGF on angiogenesis, and to analyze the influencing mechanisms of NGF, according to the expression patterns of CD34, von Willebrand factor （vWF）, vascular endothelial cell growth factor （VEGF）, and the NGF receptor TrkA in proliferating vascular endothelial cells from a rat model of sciatic nerve injury.
DESIGN, TIME AND SETTING： Randomized, controlled study performed at the Research Institute of Field Surgery, Daping Hospital affiliated to the Third Military Medical University of Chinese PLA, between October 2003 and July 2005.
MATERIALS： Forty-five healthy, adult, Wistar rats underwent sciatic nerve injury. The rats were randomly divided into four groups： NGF ＋ chitosan （n = 15）, NGF ＋ chitosan ＋ anti-VEGF （n = 10）, chitosan （n = 10）, and physiological saline （n = 10）. METHODS： A 1 -cm defected sciatic nerve was bridged with a silica gel conduit. NGF ＋ chitosan group： 100 μ L chitosan and 5 μ L NGF （20 mg/L） were injected into the silica gel conduit; NGF ＋ chitosan ＋ anti-VEGF group： an additional 5μ L anti-VEGF monoclonal antibody （1 g/L） was injected into the silica gel conduit; chitosan group： 100μL chitosan and 5 μL physiological saline were injected into the silica gel conduit; physiological saline group： only 5μL physiological saline was injected into the silica gel conduit.
MAIN OUTCOME MEASURES： CD34 and vWf were used to label blood capillaries and large-diameter blood vessels in the regenerating peripheral nerves, respectively. At day 14 following surgery, immunohistochemistry was used to detect and semi-quantitatively analyze expressions of CD34, vWf, VEGF, and TrkA in proliferating vascular endothelial cells in the regenerating sciatic nerve. A confocal laser microscope was used to determine co-expression. RESULTS： Expressions of TrkA, CD34, vWf, and VEGF in the NGF ＋ chitosan group were significantly greater than the physiological saline and chitosan groups （P 〈 0.05-0.01）. Expressions of CD34 and VEGF in the NGF ＋ chitosan ＋ anti-VEGF group were completely inhibited, while expressions of vWf and TrkA gradually decreased, compared with the NGF ＋ chitosan group （P 〈 0.01）. Confocal microscopy revealed strong co-expression of VEGF and CD34 in the regenerating sciatic nerve, and CD34 expression positively correlated with VEGF expression. In addition, VEGF expression was greater than CD34 expression, and coexpression of VEGF and vWf was also strong.
CONCLUSION： VEGF was expressed in blood capillaries and large-diameter blood vessels, while exogenous NGF promoted VEGF expression in regenerating sciatic nerves, thereby increasing angiogenesis.     

Environmental factors in the development and progression of late-onset Alzheimer＇s disease

Late-onset Alzheimer＇s disease （LOAD） is an age-related neurodegenerative disorder characterized by gradual loss of synapses and neurons, but its pathogenesis remains to be clarified. Neurons live in an environment constituted by neurons themselves and glial cells. In this review, we propose that the neuronal degeneration in the AD brain is partially caused by diverse environmental factors. We first discuss various environmental stresses and the corresponding responses at different levels. Then we propose some mechanisms underlying the specific pathological changes, in particular, hypothalamic-pituitary adrenal axis dysfunction at the systemic level; cerebrovascular dysfunction, metal toxicity, glial activation, and Aβ toxicity at the intercellular level; and kinase-phosphatase imbalance and epigenetic modification at the intracellular level. Finally, we discuss the possibility of developing new strategies for the prevention and treatment of LOAD from the perspective of environmental stress. We conclude that environmental factors play a significant role in the development of LOAD through multiple pathological mechanisms.     

左旋丁苯酞对Aβ1-42诱导原代培养大鼠基底前脑和海马神经元Caspase-3及NF-κB表达的影响

BACKGROUND： L-3-n-butylphthalide （L-NBP） can inhibit phosphorylation of tau protein and reduce the neurotoxicity of beta-amyloid peptide 1-42 （Aβ1-42）. OBJECTIVE： To observe the neuroprotective effects of L-NBP on caspase-3 and nuclear factor kappa-B （NF- K B） expression in a rat model of Alzheimer＇s disease. DESIGN, TIME AND SETTING： A cell experiment was performed at the Central Laboratory of Provincial Hospital affiliated to Shandong University between January 2008 and August 2008. MATERIALS： L-NBP （purity 〉 98%） was provided by Shijiazhuang Pharma Group NBP Pharmaceutical Company Limited. Aβ1-42, 3-[4,5-dimethylthiazolo-2]-2,5 iphenyltetrazolium bromide （MTT）, and rabbit anti-Caspase-3 polyclonal antibody were provided by Cell Signaling, USA; goat anti-choactase and rabbit anti-NF- kB antibodies were provided by Santa Cruz, USA. METHODS： Primary cultures were generated from rat basal forebrain and hippocampal neurons at 17 or 19 days of gestation. The cells were assigned into five groups： the control group, the Aβ1-42 group （2 μmol/L）, the Aβ1-42 ＋ 0.1 μmol/L L-NBP group, the Aβ1-42 ＋ 1 μ mol/L L-NBP group, and the Aβ1-42 ＋ 10μmol/L L-NBP group. The neurons were treated with Aβ1-42 （2 μmol/L） alone or in combination with L-NBP （0.1, 1, 10 μmol/L） for 48 hours. Cells in the control group were incubated in PBS. MAIN OUTCOME MEASURES： Morphologic changes were evaluated using inverted microscopy, viability using the M-I-I- method, and the changes in caspase-3 and NF- k B expression using Western blot. RESULTS： Induction with Aβ1-42 for 48 hours caused cell death and soma atrophy, and increased caspase-3 and NF- K B expression （P 〈 0.05）. L-NBP blocked these changes in cell morphology, decreased caspase-3 and NF- k B expression （P 〈 0.05）, and improved cell viability, especially at the high dose （P 〈 0.05）. CONCLUSION： AI3~-42 is toxic to basal forebrain and hippocampal primary neurons; L-NBP protects against this toxicity and inhibits the induction of caspase-3 and NF- K B expression.     

Cryoanalgesia of intercostal nerves following thoracotomy Clinical trial based on animal experiment

BACKGROUND： Cryoanalgesia at -50 ℃ for 90 seconds yields effective pain relief following thoracotomy. In China, -50 ℃ is a common temperature for intercostal cryoanalgesia following thoracotomy. However, experimental results vary. OBJECTIVE： To explore intercostal nerve pathological changes at -70 ℃ for various freezing times by studying canines, and to evaluate long-term clinical efficacy of intercostal nerve cryoanalgesia for postoperative pain relief based on the animal experiments. DESIGN, TIME AND SETTING： A comparative animal study was performed at the Animal Experimental Center of the General Hospital of the People＇s Liberation Army. Based on results from the animal study, a randomized, controlled, clinical trial was performed at the Department of Thoracic Surgery of the General Hospital of the People＇s Liberation Army between October 2006 and October 2008. PARTICIPANTS： A total of 120 patients undergoing posterolateral single incision Iobectomy at the Department of Thoracic Surgery of PLA General Hospital between October 2006 and October 2008 were selected. Nervous system diseases were excluded. METHODS： Animal experiment： 8 anaesthetized, mixed-breed dogs were used. The intercostal nerves （costal bone 6-10） were frozen at -70 ℃ for varying times （30, 60, 90, 120, and 180 seconds）. Clinical study： 120 patients were randomly assigned to 2 groups （n = 60）. In the cryoanalgesia group, the intercostal nerves were frozen prior to chest closure, and 4 costal nerves （1 at incision level, 2 above and below incision, and 1 at drainage tube level） were frozen for 90 seconds at-70 ℃, respectively. Intercostal nerves were not frozen in the control group patients. Dolantin was used to relieve postoperative pain in patients from both groups. MAIN OUTCOME MEASURES： Pathological changes in frozen intercostal nerves were examined at days 1, 10, 30, and 60 following freezing. Following surgery, the degree of postoperative pain in all patients was evaluated by visual analogue scale at days 1,3, 5, 9, 30, 60, 90, and 180. Dolantin doses at days 1, 3, 5, 9 post-surgery and postoperative complications were noted. RESULTS： Nerve damage progressively increased with length of freezing time at -70 ℃, and recovery time from damage was gradually increased. After freezing for 90 seconds, the nerves exhibited obvious histopathological damage, and then completely recovered. In addition, after freezing for 180 seconds, the histopathological changes in nerves were reversible. In the clinical study, visual analogue scale scores were significantly less in the cryoanalgesia group compared with the control group （P 〈 0.01）, which was maintained over 30 days. In the cryoanalgesia group, the mean dolantin dose administered and postoperative complications were significantly reduced compared with the control group （P 〈 0.01）. CONCLUSION： Freezing of the intercostal nerve at -70 ℃ for 90 seconds is a safe and long-term effective method for relieving post-thoracotomy pain.     

骨髓神经组织定向干细胞化组织工程神经桥接坐骨神经缺损

BACKGROUND： Schwann cells are the most commonly used cells for tissue-engineered nerves. However, autologous Schwann cells are of limited use in a clinical context, and allogeneic Schwann cells induce immunological rejections. Cells that do not induce immunological rejections and that are relatively easy to acquire are urgently needed for transplantation. OBJECTIVE： To bridge sciatic nerve defects using tissue engineered nerves constructed with neural tissue-committed stem cells （NTCSCs） derived from bone marrow; to observe morphology and function of rat nerves following bridging; to determine the effect of autologous nerve transplantation, which serves as the gold standard for evaluating efficacy of tissue-engineered nerves. DESIGN, TIME AND SETTING： This randomized, controlled, animal experiment was performed in the Anatomical Laboratory and Biomedical Institute of the Second Military Medical University of Chinese PLA between September 2004 and April 2006. MATERIALS： Five Sprague Dawley rats, aged 1 month and weighing 100-150 g, were used for cell culture. Sixty Sprague Dawley rats aged 3 months and weighing 220-250 g, were used to establish neurological defect models. Nestin, neuron-specific enolase （NSE）, glial fibrillary acidic protein （GFAP）, and S-100 antibodies were provided by Santa Cruz Biotechnology, Inc., USA. Acellular nerve grafts were derived from dogs. METHODS： All rats, each with 1-cm gap created in the right sciatic nerve, were randomly assigned to three groups. Each group comprised 20 rats. Autograft nerve transplantation group： the severed 1-cm length nerve segment was reverted, but with the two ends exchanged; the proximal segment was sutured to the distal sciatic nerve stump and the distal segment to the proximal stump. Blank nerve scaffold transplantation group： a 1-cm length acellular nerve graft was used to bridge the sciatic nerve gap. NTCSC engineered nerve transplantation group： a 1-cm length acellular nerve graft, in which NTCSCs were inoculated, was used to bridge the sciatic nerve gap. MAIN OUTCOME MEASURES： Following surgery, sciatic nerve functional index and electrophysiology functions were evaluated for nerve conduction function, including conduction latency, conduction velocity, and action potential peak. Horseradish peroxidase （HRP, 20%） was injected into the gastrocnemius muscle to retrogradely label the 1-4 and L5 nerve ganglions, as well as neurons in the anterior horn of the spinal cord, in the three groups. Positive expression of nestin, NSE, GFAP, and S-100 were determined using an immunofluorescence double-labeling method. RESULTS： NTCSCs differentiated into neuronal-like cells and glial-like cells within 12 weeks after NTCSC engineered nerve transplantation. HRP retrograde tracing displayed a large amount of HRP-labeled neurons in I-45 nerve ganglions, as well as the anterior horn of the spinal cord, in both the autograft nerve transplantation and the NTCSC engineered nerve transplantation groups. However, few HRP-labeled neurons were detected in the blank nerve scaffold transplantation group. Nerve bridges in the autograft nerve transplantation and NTCSC engineered nerve transplantation groups exhibited similar morphology to normal nerves. Neither fractures or broken nerve bridges nor neuromas were found after bridging the sciatic nerve gap with NTCSCs-inoculated acellular nerve graft, indicating repair. Conduction latency, action potential, and conduction velocity in the NTCSC engineered nerve transplantation group were identical to the autograft nerve transplantation group （P 〉 0.05）, but significantly different from the blank nerve scaffold transplantation group （P 〈 0.05）. CONCLUSION＂ NTCSC tissue-engineered nerves were able to repair injured nerves and facilitated restoration of nerve conduction function, similar to autograft nerve transplantation. ＂     

Express,on of estrogen receptor alpha,nerve growth factor,interleukin-2, and androgen receptor in the cerebellum of ovariectomized rats following soybean isoflavone treatment

BACKGROUND： Studies have shown that estrogen receptor alpha （ERα）, nerve growth factor （NGF）, interleukin-2 （IL-2）, and androgen receptor （AR） expression in the cerebellum decreases when estrogen levels decrease in vivo. Soybean isoflavone, a type of non-steroid estrogen with similar molecular structure and function to estradiol, exhibits estrogen-like characteristics. OBJECTIVE： To investigate the effects of various doses of soybean isoflavone on expression of ERa, NGF, IL-2, and AR in the cerebellum of ovariectomized rat, and to determine whether there is a dose-dependent effect.DESIGN, TIME AND SETTING： Controlled trial at the cellular and molecular level. The study was performed at the Experimental Animal Engineering Center, College of Veterinary Medicine, Sichuan Agricultural University from July 2006 to May 2008. MATERIALS： Soybean isoflavone, comprised of daidzin, genistein and isoflavone, was provided by Taiyuan Yuantai Biochemical Industry, China. The ERα, NGF, IL-2, and AR in situ hybridization kit, rabbit anti-rat ERa, NGF, IL-2, and AR monoclonal antibodies, and SABC kit were purchased from Wuhan Boster Biological Technology, China. METHODS： A total of 50 female, Sprague Dawley rats, aged 3 months, were randomly assigned to 5 groups, with 10 animals in each group. With the exception of the sham-operation group （abdominal cavity opening alone）, all rats underwent bilateral ovariectomy. At 14 days after surgery, rats in the high-, middle-, and low-dose soybean isoflavone groups were subcutaneously injected with 1.5, 1.0, and 0.5 mg/kg soybean isoflavone, respectively, every 2 days for 6 consecutive weeks. Rats in the sham-operation and ovariectomized groups were subcutaneously injected with absolute alcohol （0.5 mL/kg）. MAIN OUTCOME MEASURES： Expression levels and distribution of ERα, NGF, IL-2, and AR in the cerebellum were detected by immunohistochemistry and in situ hybridization. RESULTS： Compared with the sham-operation group, immunoreactive products and hybridization signals of ERa, NGF, IL-2, and AR were significantly decreased in the cerebellar cortex and nuclei of ovariectomized rats （P 〈 0.05 or P 〈 0.01）, but increased following soybean isoflavone treatment. In particular, levels of the high-dose soybean isoflavone group were almost restored to levels of the sham-operation group （P 〉 0.05）. The immunoreactive products were primarily located in the cytoplasm and neurites, and rarely in the cell membrane and nuclei. However, the hybridization signals were predominantly located in the nuclei, but rarely in the cytoplasm, cell membrane, or neurites. CONCLUSION： Soybean isoflavone upregulated ERα, NGF, IL-2, and AR protein and gene expression in a dose-dependent manner, and played an important role in sustaining and protecting structure and function of cerebellar neurons. Moreover, the similarity of expression patterns of these molecules indicated that they were mutually interactive during the regulation of soybean isoflavone to the cerebellum.     

Hippocampal CA1 pyramidal cells and neurotrophic factors in a rat model of vascular dementia following Xiongma drop pill versus Ginkgo leaf tablets

BACKGROUND： Previous studies have demonstrated the neuroprotective effects of Xiongma drop pill （XMDP） in a mouse model of vascular dementia. Neurotrophic factors play an important role in repair and regeneration of injured neurons. OBJECTIVE： To compare the effects of XMDP and Ginkgo leaf tablets on the appearance and number of hippocampal CA1 pyramidal neurons, as well as neurotrophic factor content in brain tissues, during vascular dementia formation to explore the neuroprotective mechanisms of XMDP. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Pharmacology, College of Pharmacy, Harbin University of Commerce between April 2007 and December 2008. MATERIALS： XMDP was prepared by the College of Pharmacy, Harbin University of Commerce, with each 40 mg pill containing ferulic acid （≥ 0.149 mg） and gastrodin （≥ 0.171 mg）. Ginkgo leaf tablets were purchased from Taiyuan Qianyuan Pharmacy, China. METHODS： Healthy, adult, male, Wistar rats were randomly assigned to 6 groups： sham-operation, model, XMDP （high-, middle-, and low- dose）, and Ginkgo leaf tablets. The 6 groups were subdivided into two subgroups according to administration days, i.e., 30 and 60 days, with 8 animals in each subgroup. Rats in the model, XMDP, and Ginkgo leaf tablets groups were subjected to permanent bilateral ligation of the common carotid artery to establish a vascular dementia model. At 8 days after model establishment, all groups received intragastric administration once daily of the following： 10 mL/kg normal saline in the sham-operation and model groups; 0.4, 0.2, and 0.1 g/kg XMDP in the high-, middle-, and low-dose XMDP groups, respectively; and 50 mg/kg Ginkgo leaf tablets in the Ginkgo leaf tablets group. MAIN OUTCOME MEASURES： Hematoxylin-eosin staining was used to observe appearance and to quantify the number of hippocampal CA1 pyramidal neurons. Brain-derived neurotrophic factor and nerve growth factor concentrations in brain tissues were detected by enzyme-linked immunosorbent assay. RESULTS： Following model establishment, hippocampal CA1 neurons exhibited pathological changes. Compared with the sham-operation group, the number of pyramidal neurons significantly decreased （P 〈 0.05 or P 〈 0.01）, and neurotrophic factor concentration increased in the model rats （P 〈 0.05 or P 〈 0.01）. XMDP attenuated neuronal injury in a dose-dependent manner： the number of pyramidal neurons and neurotrophic factor concentrations were significantly increased compared with the model group （P〈 0.05 or P〈 0.01）. High- and middle-dose XMDP resulted in equivalent effects to Ginkgo leaf tablets. In addition, neurotrophic factor concentrations in all XMDP groups, after 60 days of administration, were remarkably greater than corresponding concentrations at 30 days （P 〈 0.05 or P 〈 0.01 ）. CONCLUSION： Hippocampal CA1 pyramidal cells exhibited pathological injury following establishment of the vascular dementia model. Middle- and high-dose XMDP increased neurotrophic factor expression in the brain of vascular dementia rats, which suggested neuroprotection equivalent to Ginkgo leaf tablets.     

高血压脑出血的显微外科治疗进展

高血压脑出血（Hypertensive intrac-rebral hemorrhage,HICH）是具有高发病率、高病死率、高致残率的急性脑血管疾病,占所有脑卒中患者的10%-20%,早期病死率可高达49.4%。随着人口老龄化,其发病率逐年提高;而外科手术的干预,使其病死率有所下降,但致残率居高不下。如何提高手术疗效和患者生存质量,一直是神经外科医师努力的方向。微侵袭血肿清除术因其手术创伤小,恢复快,是目前国内治疗高血压脑出血的重要手段。