CatWalk gait analysis in assessment of functional changes of the hind paw after SD rats being established the artificial bladder reflex arc / 中华神经医学杂志

Objective To assess the functional changes of the hind paw after SD rats being cut offthe L5 spinal nerve by CatWalk gait analysis.Methods Eleven male SD rats were chosen; the right side of these rats,as the experimental side,was cut the ventral root and dorsal root of L5 spinal nerve,while the left side was without any operation.One day after the operation,whether the models were successfully constructed was verified by tracer experiment with wheat germ agglutinin horseradish peroxidase (WGA-HRP) in 3 rats; 1 day before the operation and 3 months after the operation,the changes of such parameters as general parameters,individual paw parameters,pain-related parameters and coordination-related parameters were assessed by using CatWalk gait analysis.Results The WGA-HRP-labeled motoneurons and fibers were not found in posterior horn and anterior horn of the experimental side of L5 spinal gray matter,but both located in the left side,indicating that the models were constructed successfully.General parameters,individual paw parameters and pain-related gait parameters could be recorded 3 months after the operation,but no statistically significant difference was noted as compared with those 1 day before the operation; however,significant decrease of values of regularity index (RI) and coupling diagonal right after-left front in rats 3 months after the operation was noted as compared with those 1 day before the operation (P＜0.05).Conclusion Only a part of coordination-related gait parameters (the lower limbs) is influenced after being cut L5 spinal nerve;therefore,L5 spinal nerve can be used for constructing of a bladder reflex arc.

Early physical training improves long-lasting prognosis following hypoxic-ischemic brain damage in neonatal rats / 中华神经医学杂志

Objective To investigate the effect of early physical training on long-lasting neurological function,pathology changes,spatial learning and memory abilities,sensorimotor function and ultrastructure changes in neonatal rats with hypoxic-ischemic brain damage (HIBD).Methods Ninety 7-day-old sprague-dawley rats were randomly divided into three groups:a group that was subjected to left carotid ligation followed by 2 hours hypoxic stress (vehicle group),a group that received physical trainings (grabbing,rotation,walking and balance) 1 weeks after HIBD event (trained group) and a sham-operated group that was subjected to a sham-operation without ligation and hypoxic stress (n=30).Following four weeks of physical trainings,neurological scale was performed on all the rats; the number of neurons in the CA1 region of hippocampus and cortex forehead was measured by Nissl count; the expression levels of synaptophysin and c-fos were examined; Morris water maze tests and cortex sensorimotor tests were performed to detect the spatial learning and memory abilities and sensory functions; transmission electron microscope was employed to observe the synapses and neuronal ultrastructure.Results As compared with those in the vehicle group,the neurological scale scores were significantly increased in the trained group and the sham-operated group 14,21 and 28 days after trainings (P＜0.05).As compared with those in the vehicle group,the number of neurons in the left hippocampal CA1 area and cortex increased in the trained group and sham-operated group (P＜0.05).The expressions of synaptophysin and c-los in the trained group increased significantly as compared with those in the vehicle group (P＜0.05).As compared with those in the vehicle group,the spatial learning and memory abilities and sensorimotor functions in the trained group were significantly increased (P＜0.05).The ultrastructure of the left hippocampus and cortex was remarkably abnormal in the vehicle group by the transmission electron microscopy,while no obvious abnormality was observed in the trained group.Conclusion Early physical training can restrain neuron damage in the hippocampus and cortex and enhance synaptic plasticity to ameliorate the long-term neurological function after HIBD; the strong expression of synaptophysin and c-fos in the hippocampus and cortex caused by early physical training may be one of the improvement mechanisms.

Long-term behavioral and ultrastructural alterations following hypoxic-ischemic brain damage in neonatal rats / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study long-term behavioral and ultrastructural alterations in a hypoxic-ischemic brain damage (HIBD) model of neonatal rats.</p><p><b>METHODS</b>Sixty seven-day-old Sprague-Dawley rats were randomly subjected to unilateral carotid artery ligation followed by hypoxic exposure (HIBD group) or sham operation (n=30 each). A battery of behavioral tests, including Morris water maze test and sensorimotor tests, were performed at a postnatal age of 5 weeks. Nissl staining was used for counting neurons. Transmission electron microscopy was used for observing synapse structures and measuring the thickness of the postsynaptic density area and the length of the postsynaptic active area. The correlations of histological changes with the results of behavioral tests were evaluated.</p><p><b>RESULTS</b>The HIBD group showed a significantly longer escape latency (P<0.05) and a lower frequency of original platform crossing (P<0.05) in the Morris water maze test compared with the sham operation group. The sensorimotor function test showed that the sensorimotor function in the HIBD group was worse than in the sham operation group. Nissl staining showed that the number of neurons in the HIBD group was significantly reduced (P<0.01) compared with the sham operation group. Transmission electron microscopy showed that synapses were significantly reduced in number, and that the thickness of the postsynaptic density area and the length of the postsynaptic active area were reduced in the HIBD group. The thickness of the postsynaptic density area was negatively correlated with escape latency in the Morris water maze test (r=-0.861, P<0.01), and also negatively correlated with the total score of sensorimotor function tests (r=-0.758, P<0.05) in the HIBD group.</p><p><b>CONCLUSIONS</b>Hypoxia ischemia can lead to neuron loss and ultrastructure damage, resulting in long-term deficit of behavioral functions in neonatal rats.</p>

Affection of embolism on rat aneurysm model with new platinum coils modified by VEGF / 中华神经医学杂志

Objective To explore the embolization effect of new platinum coils coated with [4COOH-P (DLLA-co-TMC)] biodegradable polymer and released vascular endothelial growth factor (VEGF) into intracranial aneurysms on rat intracranial aneurysms. Methods A total of 54 adult healthy female SD rats were randomly divided into Group Ⅰ with general platinum coils, Group Ⅱ with polymer-coated platinum coils and Group Ⅲ with platinum coils modified with VEGF (n=18).The right common carotid arteries (CCA) of rats in each group were exposed; and the 8 mm lengths of platinum coil segments were inserted into the ligated right CCA of rats. The distal right CCA was performed ligation and restored the blood flow; 6 rats each time at 15,30 and 90 d after the surgery were chosen;and the distal right CCA was used as aneurysm models,and the left CCA without the coil placement or surgical disruption in Group I with general platinum coil was chosen as normal control.The proliferation and fibrosis of endothelial cells were observed by HE staining; von Willebrand Factor (vWF) expression was detected by immunohistochemical staining; and VEGF expression was examined by Western blotting. Results Cellular proliferation and fibrosis in Group Ⅲ with platinum coils modified with VEGF enjoyed significantly higher grade than those in Group Ⅰ with general platinum coils 10,60 and 90d after the surgery (P＜0.05); Cellular proliferation and fibrosis in Group Ⅲ with platinum coils modified with VEGF enjoyed significantly higher grades than those in Group Ⅱ with polymer-coated platinum coils 30 d after the surgery (P＜0.05).Pathological observations showed that the massive intimal hyperplasia and substantial clot completely occluded the aneurysm lumen in Group Ⅲ with platinum coils modified with VEGF; New small blood vessels having vwf-positive expression were noted in the fiberized tissues;the thrombosis in Group Ⅰ with general platinum coils and Group Ⅱ with polymer-coat platinum coils were not fully organized and showed loose hyperplasia structure with a large number of internal spaces.Western blotting indicated that the VEGF level in Group Ⅲ with platinum coils modified with VEGF were significantly higher than that in other groups 15 and 30 d after the operation,however,the VEGF level in Group Ⅲ with platinum coils modified with VEGF 90 d after the surgery was decreased because the lumen completed fibration and degradation of 4COOH-P (DLLA-co-TMC). Conclusion The VEGF-eontaining biodegradable polymer,by slowly releasing VEGF to modify the surface of platinum coils, could enhance the cellular proliferation, thrombosis and formation of dense fibrous tissue in aneurysm lumen; as compared with general platinum coils,these new platinum coils could occlude the rat aneurysm faster and more completely.

The photodynamic effects of a novel photosensitizer ZnPcS4-BSA on human U251 glioma cells in vitro / 中华神经医学杂志

Objective To report the phototoxicity effects of a novel photosensitizer ZnPcS4-BSA on photodynamic therapy (PDT) towards human U251 glioma cells in vitro. Methods The cellular uptake of ZnPcS4-BSA by U251 glioma cells was quantified by UV-spectra to determine the optimal incubation time. Human U251 glioma cells were incubated with ZnPcS4-BSA of various concentrations and received laser irradiation of different energy densities. Cell survival rates were measured by CCK-8 assay.Flow cytometer was used to detect apoptosis.Gene expressions of vascular endothelial growth factor (VEGF) were detected by Real-Time PCR in the U251 cells after PDT and β-actin was used as an internal standard. The normal U251 cells severed as controls. Results The uptake of ZnPcS4-BSA by U251 glioma cells reached the maximum after incubation for 4 hours.ZnPcS4-BSA of different concentrations without laser irradiation had no significant effects on cell survival rates (P＞0.05).Without ZnPcS4-BSA incubation,compared with 0,25,50,100,200 J/cm2 groups, the cell survival rate of the 400 J/cm2 group was significantly lower (P＜0.05), whereas no significant difference was found between any other two groups. When the U251 glioma cells incubated with 30 μ mol/L ZnPcS4-BSA for 4 hours underwent laser irradiations of 25,50,100,200 J/cm2,the cellular survival rates significantly decreased with the increased energy densities (P＜0.05). When the U251 glioma cells incubated with ZnPcS4-BSA of 20,40,60,80,100 μ mol/L for 4 hours underwent laser irradiation of 200 J/cm2, the cellular inhibition rates significantly increased with the increased concentrations (P ＜0.05). Compared with controls, the cellular apoptosis and VEGF expression significantly increased in the U251 glioma cells incubated with ZnPcS4-BSA of 20 μmol/L after laser irradiation of 100 J/cm2 (P＜0.05). Conclusion The novel ZnPcS4-BSA is a good photosensitizer for PDT towards U251 glioma cells,because the ZnPcS4-BSA-mediated PDT can induce effective apoptosis of the targeted cells.

Quantitative CatWalk analysis of gaits in rats with 6-hydroxydopamine-induced PD / 中华神经医学杂志

Objective To investigate the probability of quantitative analysis of parkinsonian gaits in unilateral 6-hydroxyl dopamine-lesioned rats. Methods A total of 24 male Wistar rats were assigned to control group (n=6),sham-operated group (n=6) and 6-OHDA inducement group (n=12).All subjects received anesthetization and the latter 2 groups further processed with injection of equivalent volumes of normal saline and 6-OHDA into the left medial longitudinal fasciculus on the operation day,respectively. The cylinder test and Catwalk analysis were applied successively 1 week before the operation for baseline value,and 3 d, 1 and 2 weeks after the operation. Results The cylinder test indicated that the proportion of using the right forelimbs was significantly decreased as compared with that using the left forelimbs in the 6-OHDA inducement group 3 d, 1 and 2 weeks after the operation (P＜0.05), but no significant differences of using the right forelimbs were shown in the control and sham-operated counterpart (P＞0.05). Catwalk analysis showed no significant differences either on the girdle comparisons of former/hinder limbs or on pre- and post-operation comparisons of individual limbs between control and sham-operated groups (P＞0.05). In the 6-OHDA inducement group,max contact area (MCA),paw length (PL),paw width (PW) and paw area (PA) for fore girdle comparisons and MCA for hinder ones showed significant differences (P＜0.05); all the 4 parameters of right hinder limb,the PL,PW and PA of left fore limb,the PA,PL and MCA of right fore limb,the PW of left hinder limb after the operation significantly decreased as compared with those before the operation (P＜0.05). Conclusion It is applicable to detect changes in gaits of Parkinson's disease quantitatively with Catwalk analysis,especially in terms of MCA,adding new tool for further study.

Proliferation characteristics of adipose-derived stem cells from neonatal suckling rats and adult ones / 中华神经医学杂志

Objective To investigate the proliferative differences of adipose-derived stem cells (ADSCs) from neonatal suckling SD rats (5-d-old) and adult ones under the same culture condition.Methods ADSCs were isolated from the subcutaneous adipose tissues of neonatal suckling SD rats and adult ones,and then,type Ⅰ collagenase digestion was employed to obtain the ADSCs; the morphology of these cells was detected.The expressions of such cell surface markers as CD45,CD29 and CD90 were observed. The number of ADSCs on the 4th d of culture under the same condition and with the same planted density was compared between the neonate and adult rats. In vitro culture of the second generation of ADSCs was performed in the 96-well plates, and CCK-8 and alamar blue kit were employed to compare and quantitate the proliferative differences; optical density was observed by microplate reader. Results The ADSCs from neonatal SD rats and adult ones expressed the stem cell biomarkers: the expression of CD45 was negative, and that of CD29 was 98.04％ and 93.17％,respectively,and that of CD90 was 94.92％ and 93.3％,respectively,for neonate SD rat and adult ones.The cell counting results indicated that the number of ADSCs from neonatal rats ([8.87±0.13]×105 cells) was larger than that of adult ones ([4.51±0.36]×105 cells) after being cultured under the same condition and at the same planted density. The optical density value of ADSCs in neonatal rats was significantly higher than that in adult ones on the 6th and 7th d of culturing detected by CCK-8 kit and on the 2nd-7th d of culturing by alamar blue assay. Conclusion The proliferative ability of ADSCs from neonatal rats is greater than that of adult ones.

Hospital first aid effect on patients with traffic accident severe craniocerebral injury: a report of 1107 cases / 中华神经医学杂志

Objective To make reference for relevant departments of our country to develop acute craniocerebral trauma prevention and control measures based on the general rule of hospital first aid by changing the links as signs of life, consciousness, pupil and time for hospital emergency in patients with traffic accident severe traumatic brain injury. Methods The clinical data of 1107 patients with severe traumatic brain injury,collected from April 1,2008 to March 30,2009,were analyzed; these patients were chosen according to the severe traumatic brain injury statistical table made by Department of Neurosurgery of Zhujiang Hospital and authorized by committee of experts from 71 other hospitals.The consciousness,pupil changes,GCS scores,blood pressure, respiration,pulse index changes on admission and at time out of emergency department were analyzed and compared; according to these results,function projections was performed; development tendency of the disease and rescue effect were judged, and the next treatment measure step was determined. Results According to the hospital emergency time,most patients got to the Emergency at 10-60 min after the injury,mostly at 10-30 min after the injury (38.9％).The consciousness,pupil changes,GCS scores,blood pressure,respiration,pulse index changes of most patients gradually developed to good trends after hospital first aid; before the patients received further specific treatment, the vital signs and indicators of them were gradually stabilized. Function projections supported these results. Conclusion Hospital first aid and effective treatment,timely controlling indicators,correct diagnosis and all reasonable measures improving hospital first aid can help to improve the first aid quality of patients with traffic accident severe traumatic brain injury.

Blockage of transforming growth factor-β signaling pathway inhibits vasculogenic mimicry in gliomas / 中华神经医学杂志

Objective To study the effect of transforming growth factor-β (TGF-β) signaling pathway blockage on vasculogenic mimicry (VM) in gliomas and explore its possible mechanism.Methods Three-dimentional culture was performed on the glioma cell lines U251 and SHG44; the effects of U251 culture supematant and TGF-β on VM formation of SHG44 cells were observed; the capability of VM formation of U251 and SHG44 cells after being treated with 0 μg/mL (PBS group),15 μg/mL TGF-β neutralizing antibody (Ab15 group) and 30 μg/mL TGF-β neutralizing antibody (Ab30 group) was evaluated.ELISA was used to detect the concentrations of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in the supematant of U251 cells from the blank group,PBS group,Ab 15 group and Ab30 group and the concentrations of VEGF and PDGF in the supernatant of SHG44 cells from the blank group,TGF-β treatment group,PBS group,Ab15 group and Ab30 group.Results VM was formed in the U251 cells while not in the SHG44 cells during the three-dimentional culture; SHG44 cells could only gather into colonies of different sizes.U251 culture supernatant could induce SHG44 cells to form VM,enjoying the most obvious effect at 24-48 h of culture; TGF-β could not induce SHG44 cells to form VM.The number of U251 cells annulation in PBS group,Ab15 group and Ab30 group decreased in sequence with significant difference (P＜0.05).The number of U251 cells armulation in SHG44 cells cultured in U251 culture supematant from the PBS group,Ab15 group and Ab30 group decreased in sequence after being added TGF-β antibody with significant difference (P＜0.05).As compared with that in the blank group and PBS group,significant decrease of VEGF and PDGF concentrations in the U251 cells from Ab15 group and Ab30 group was noted (P＜0.05); as compared with that in the blank group and TGF-β treatment group,significant increase of VEGF and PDGF concentrations in the SHG44 cells from PBS group,Ab15 group and Ab30 group was noted.Conclusion Blockage of TGF-β signaling pathways inhibits VM in glioma,and it maybe probably due to the decrease of VEGF and PDGF expressions..

Pre-hospital emergency treatment and prognosis analysis of patients with severe brain injury accidents: a report of 1107 cases / 中华神经医学杂志

Objective To analyze the importance of pre-hospital emergency treatment in patients with severe head injury in traffic accidents in terms of time and explore the general rule of severe head injury in traffic accidents to provide some evidence for relevant authority making the prevention and control measures of acute traumatic brain injury. Methods The patients were selected according to "statistical table of severe head injury in a traffic accident" established by our department; their correlated data were statistically analyzed. Based on the time intervals between receiving telephone time asking for first-aid and arriving to location of incidence, and time intervals between time of giving first-aid and injured time, the regularity of prognosis was analyzed.Results With the prolonged time intervals between time of giving first-aid and injured time, the mortality and disability rates gradually increased,followed by poor prognosis, which indicated that the prognosis was negatively correlated to the delayed time on the way and the on-site rescue time (rs=-0.10,P=0.002; rs=-0.06,P=0.034). Conclusion Prevention and control center for acute brain injury should be built and the roles for pre-hospital emergency treatment and transportation should be established to decrease the injured time (the delayed time on the way and the on-site rescue time) to improve the quality of care, and to reduce the death and disability in patients with severe head injury in traffic accidents.

CatWalk-assisted gait test in evaluating motor funtion of rat models of traumatic brain injury / 中华神经医学杂志

Objective To explore the value of CatWalk-assisted gait test in evaluating the changes of motor function in the rat models of traumatic brain injury.Methods Improved Feeney tree fall brain damage device was employed to establish the craniocerebral injury rat models; injured loci were located in the right side of the brain cortex; CatWalk-assisted gait test and mNSS were performed on the 3rd,7th,14th and 28th d of injury.SPSS 13.0 software was used to compare the scores of the above tests.Results Significant differences on each parameter of the left hind limb were noted between the day before injury and the day of 7 d after injury(P＜0.05); and significant differences were also noted between left and right hind limbs(P＜0.05).Significant differences on such motion parameters as maximum contact area,contact strength,claw length and walking cycle in the left and right fore limbs were noted between the 14th d of injury and before injury(P＜0.05); and the walking cycle,average intensity and time of claws leaving the flat between two steps in these rats were signficantly different between the 14th d of injury and before injury(P＜0.05.No significant differences on each motion parameter were found between the 3rdand 28th d of injury(P＞0.05).Significant differences on mNSS scores were noted between the day before injury and the 3rd,7th 14th,and 28th d of injury(P＜0.05); and the mNSS scores on the 3rd and 7th d of injury were obviously higher than those before injury.Conclusion Catwalk-assisted gait test can accurately detect the changes of motor function,enjoying an important significance in the study of behavior of rat with traumatic brain injury.

Catwalk-assisted behavioral analysis of rat models of middle cerebral artery occlusion / 中华神经医学杂志

Objective To investigate the feasibility of Catwalk-assisted system in analyzing the behavioral features of rat models of middle cerebral artery occlusion(MCAO).Methods Thirty male SD rats were randomly assigned to experimental group and control group.Suture-occluded method was used to establish the focal cerebral ischemia/reperfusion models in the experimental group.Traditional modified neurological severity scale(mNSS),vibrissae-evoked forelimb placing test and cylinder test were employed to analyze the behavioral characteristics of the SD rats 1,3,7,14 and 28 dafter establishment of rat models.Besides that,Catwalk-assisted gait analysis system was employed at the same time points.Results Catwalk-assisted gait analysis results suggest rats from experimental group had smaller intensity,print area and slower walk speed than rats from control group.Besides that,interlimb coordination also changed on the 3rd d of operation and existed until the 28th d of operation.Rotate step patterns like Ra(1.5％)and Rb(2.3％)were detected in experimental group but never seen in control group.Parameters as cadence,stance,swing and other regularity index had no significant changes between the 2 groups.Conclusion Catwalk analysis system,by analyzing such movement parameters of MCAO rats as intensity,speed,print area,step pattern and interlimb coordination,can commendably reflect the damage of brain ischemia and its changes crossing time,which can comprehensively analyze many behavioral features of the models.

Role of Na(+)-K(+)-ATPase in lipopolysaccharide-induced cardiomyocyte hypertrophy in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the possible mechanism of lipopolysaccharide (LPS)-induced cardiomyocyte hypertrophy in rats.</p><p><b>METHODS</b>Neonatal rat cardiomyocytes cultured in vitro were stimulated with 100 µg/L LPS for 1, 4 or 8 h and scanned by atomic force microscopy (AFM) for measurement of the two-dimensional area, three-dimensional surface area and volume of each cell. The total proteins and Na(+)-K(+)-ATPase activity in the cardiomyocytes were determined. The same measurements were also carried out in neonatal rat cardiomyocyte cultures stimulated by 0.5 µmol/L ouabain for 8 h and the total protein levels were measured.</p><p><b>RESULTS</b>Following a 8-hour stimulation with LPS, the two-dimensional area, three-dimensional surface area and volume of the single cardiomyocyte became enlarged and the total cellular proteins increased significantly as compared with those in the normal control cells (P < 0.05). LPS treatment for 4 and 8 h resulted in significantly decreased activity of Na(+)-K(+)-ATPase in the cardiomyocytes (P < 0.05). In the cells treated with ouabain for 8 h, the two-dimensional area, three-dimensional surface area, volume of the single cardiomyocyte and the total cellular proteins increased significantly in comparison with the normal control group (P < 0.05).</p><p><b>CONCLUSION</b>LPS can result in cardiomyocyte hypertrophy in rats possibly in relation to lowered Na(+)-K(+)-ATPase activity in the cardiomyocytes after LPS exposure.</p>

Immunogenicity of recombinant DNA vaccine that coded for the neurite growth inhibitors / 中华神经医学杂志

Objective To detect the immunogenicity of the recombinant DNA vaccine that encoded for neurite growth inhibitors: Nogo-A, oligodendrocyte myelin glycoprotein (OMgp), tenascin-R (TN-R) and myelin-associated glycoprotein (MAG) after the nerve injury under the help of pAdEasy, a kind of adenovirus plasmid being the vector of the DNA. Methods Sixteen 5-w-old Lewis rats were randomized into DNA vaccination group (vaccine group) and pAdEasy group. Rats in the vaccine group were immunized once weekly for a consecutive 8 w by bilateral injection of the recombinant plasmid into the musculus tibialis. The immunized animals in the 2 groups were exsanguinated each time before the vaccination for sera collection, and the qualitation and quantitation of the antibodies in the serum were detected by Dot-blot analysis and ELISA. Results The vaccine group could produce fusion-protein antibodies against Nogo-A, MAG, OMgp and TN-R at the 6th w of vaccine injection, while pAdEasy group could not. The valency of antiserum was shown by ELISA as 1:1 000 000 at the 6th w of vaccine injection and kept this level stably. Conclusion The DNA vaccine exclusively induces the generation of the fusion-protein antibodies against Nogo-A, MAG, OMgp and TN-R in vivo, which controls the favorable immunogenicity.

Comparison of biological characteristics among adult neural stem cells derived from different origins in vitro / 中华神经医学杂志

Objective To evaluate and compare the adult neural stem cells (NSCs) derived from the subventricular zone (SVZ), adipose tissue (AD) and bone marrow (BM) in SD rat in terms of their morphologies, their potential of neural differentiation and their ability to secrete neurotrophins (NTs).Methods Tissues from the suventricular zone, adipose tissue and bone marrow in the same SD rat were chosen and induced in vitro into SVZ-NSCs, AD-NSCs and BM-NSCs, respectively. The abilities of proliferation and differentiation of these 3 NSCs were compared. Immunocytochemistry and Western blotting were employed to detect the expressions of surface markers of neurons and neuroglia, including nestin,βtubulin, galactocerebroside C (GalC) and glial fibrillary acidic protein (GFAP). The secretions of BDNF and NGF were detected by ELIS A. Results No obvious differences of morphology between SVZ-NSCs and both BM-NSCs and AD-NSCs were found (P>0.05). The proliferation ability of SVZ-NSCs was stronger than that of BM-NSCs and AD-NSCs. The percentage of nestin-positive cells in the SVZ-NSCs was significantly higher than that in the BM-NSCs or AD-NSCs (P<0.05). No obvious differences in the expressions of βtubulin, GFAP, and GalC among the 3 groups were found (P>0.05).The secretions of BDNF and NGF in all the 3 groups could be noted; those in the SVZ-NSCs was significantly higher than those in the BM-NSCs and AD-NSCs (P<0.05); those in the AD-NSCs was slightly higher than those in the BM-NSCs. Conclusions SVZ-NSCs, AD-NSCs and BM-NSCs show similar morphological and phenotypic characteristics; however, SVZ-NSCs present more powerful proliferation, differentiation and secretion abilities than AD-NSCs and BM-NSCs. Considering about such problems as immuno-repulsion, ethic and the origins, AD-NSCs appear to be the better choice than BSVZ-NSCs and M-NSCs.

Effect of collagen scaffold loaded with collagen-targeting neurotrophin-3 on the extension of cellular processes of dorsal root ganglion / 中华神经医学杂志

Objective To investigate the effect of collagen scaffold loaded with collagen-binding domain neurotrophin-3 (CBD-NT3) on the extension of cellular processes of dorsal root ganglions (DRGs), and explore the significance of this kind of combinatorial strategies in the spinal cord injury repair. Methods The tail tendons of SD neonatal rats were performed the removal of cellular components to prepare the collagen scaffold; HE staining was employed to evaluate whether the cells were completely removed from the collagen scaffold. The collagen scaffold was loaded with CBD-NT3,and then, they were co-cultured with primary DRGs for 1, 3 and 5 d, respectively. NT3 and PBS were also co-cultured with primary DRGs for 1, 3 and 5 d, respectively, as controls. Cells on the scaffold were stained by fluorescein diacetate (FDA) for morphology observation and the lengths and angles of the processes in each group were also quantitatively analyzed. Scanning electron microcopy (SEM) was employed to observe the topography of scaffold and the ultrastructure of DRGs 3 d after the co-culture.Results HE staining indicated that the cellular components in the scaffold were removed completely.The length of processes elongation in CBD-NT3 treatment group was significantly longer than that in the controls 3 d after the co-culture (P＜0.05). The 95% confidence interval of the angle between the line which the process emerged from the cell soma to the growing tip of the process and the long axis of fiber was 18.8-20.7 degrees. The results of SEM showed that cells could rely on the topography of the scaffold to anchor and grow. Conclusion The combinatorial strategies of collagen scaffold with CBD-NT3 can play a double function for oriented guiding and inducing extension of cellular processes effectively,which may provide a better therapeutic approach for spinal cord injury repair.

Prokaryotic expression, purification of human LINGO-1(aa76-319) and preparation of its polyclonal antibody / 南方医科大学学报

<p><b>OBJECTIVE</b>To express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb).</p><p><b>METHODS</b>The 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting.</p><p><b>RESULTS</b>The prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity.</p><p><b>CONCLUSION</b>The fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.</p>

Comparison of the effect of adult neural stem cells derived from different origins in treatment of rat spinal cord injury / 中华神经医学杂志

Objective To evaluate the therapeutic effects of adult neural stem cells derived from the brain, adipose tissue and bone marrow on spinal cord injuries in adult rats. Methods The brain subventricular zone (SVZ), adipose tissue and bone marrow from the same rat were obtained to induce the neural stem cells (NSCs). In 72 SD rats with spinal contusive injury, the NSCs from the 3 origins were grafted into the injured spinal cord one week after the injury, with 24 rats as the saline control group and another 24 as the sham-operated group. The locomotor recovery of the rats was evaluated according to the BBB scores, and the cell survival, distribution, migration and differentiation in the injured spinal cord were evaluated by immunohistochemistry. Results Compared with the rats in sham-operated and saline groups, the rats receiving transplantation of NSCs of different origins all showed significantly increased BBB scores. At 9 weeks after the transplantation, the rats receiving brain SVZ-derived NSCs (SVZ-NSs) exhibited significantly improved locomotor function compared with those grafted with the other two NSCs (P<0.05). The SVZ-NSs showed significantly higher Brdu/nestin+cell percentage than bone marrow-derived NSCs(BM-NSs) and adipose-derived NSCs(AD-NSs) at 4 weeks after grafting, but till 8 week after the grafting, only a few positive cells were found in the injured spinal cord in the 3 groups, without significant difference between them (P>0.05). Conclusion Grafting of the NSCs derived form the brain SVZ, adipose tissue and bone marrow all help improve the locomotor recovery of the rats following spinal cord injury, and the SVZ-NSs has the most obvious effect. But AD-NSs may seem a better option than those of other origins for repairing the injured spinal cord due to their abundant sources and strong proliferation ability.s

Feasibility of local LINGO-1 polyclonal antibody treatment for spinal cord injury in adult rats / 中华神经医学杂志

Objective To analyze the feasibility of local LINGO-1 polyclonal antibody administration for treatment of spinal cord injury in adult rats. Methods Twenty-four adult female SD rats were randomized into sham-operated group, rabbit IgG group and LINGO-1 antibody group. In the latter two groups, partial transaction of the T9 segment of the spinal cord was performed to completely sever the dorsal eorticospinal tract, followed immediately by administration of rabbit IgG and anti-LINGO polyclonal antibody via a mini-osmotic pump, respectively. At 3 and 28 days after the operation, the T8～10 segments of the spinal cord were harvested to prepare cryosections, and immunofluorescence staining was used to analyze the penetration of LINGO-1 polyclonal antibody into the spinal cord tissue and its specific binding to LINGO-1 molecules. Results In LINGO-1 antibody group, the presence of rabbit antibodies was detected at the injured sites of the spinal cord at 3 and 28 days after the operation. The mean immunofluorescence density was significantly lower in L1NGO-1 antibody group than in rabbit IgG group at 3 days after the operation (P<0.05). In rabbit IgG group, the mean immunofluorescence density for LINGO-1 in the crysections pre-treated with LINGO-1 polyclonal antibody was significantly lower than that in sections pre-treated with rabbit IgG(P<0.05). Conclusion Locally administered LINGO-1 polyclonal antibody can penetrate into the injured sites in the spinal cord in a wide time window and recognizes LINGO-1 molecule specifically, suggesting the feasibility of passive immunotherapy for spinal cord injury.

Genetic analysis of adult human bone marrow-derived neural stem cells with strong migration potential / 中华神经医学杂志

Objective To analyze the gene expression profiles in relation to the migration ability of adult human bone marrow-derived neural stem cells (Md-NSCs), and identify the genetic basis of the high migration potential of Md-NSCs in the central nervous system (CNS). Methods Adult human bone marrow stromal celIs(BMSCs) obtained from adult healthy volunteers were induced to differentiate into Md-NSCs in vitro, and the expressions of the genes related to cell invasiveness and metastasis were investigated by microarray analysis. Quantitative real-time PCR (RT-PCR) was used to verify the microarray results. Results The results of microarray analysis revealed significant overexpressions of the genes MMP1, MMP2, MMP17, ITGA3, RhoB, RhoC and RhoD in the Md-NSCs as compared with the expressions in fresh normal human adult bone marrow cells depleted of red blood cells. Quantitative RT-PCR verified the overexpression ofMMP2 gene by 2.84×100 folds, ITGA3 gene by 2.22×102folds, and RhoC gene by 4.92×100 folds. Conclusion The high migration potential of the Md-NSCs in the CNS is probably associated with the overexpression of the genes that promote cell invasiveness and metastasis. These overexpressed genes are also important oncogenes, and therefore the tumorigenicity of the Md-NSCs warrants further investigation.