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>

Human umbilical cord-derived Schwann-like cell transplantation combined with neurotrophin-3 administration in dyskinesia of rats with spinal cord injury.

Mesenchymal stem cells are capable of differentiating into Schwann-like cells. In this study, we induced human umbilical-cord mesenchymal stem cells (HUMSCs) in vitro into neurospheres constituted by neural stem-like cells, and further into cells bearing strong morphological, phenotypic and functional resemblances with Schwann-like cells. These HUMSC-derived Schwann-like cells, after grafting into the injured area of the rats' spinal cord injury (SCI), showed a partial therapeutic effect in terms of improving the motor function. Neurotrophin-3 (NT-3) was reported to improve the local microenvironment of the grafted cells, and we, therefore, further tested the effect of Schwann-like cell grafting combined with NT-3 administration at the site of cell transplantation. The results showed that NT-3 administration significantly promoted the survival of the grafted cells in the host-injured area. Significant improvement in rats treated by Schwann-like cell grafting combined with NT-3 administration was demonstrated in the behavioral test as compared with that in animal models received the cell grafting only. These results suggest that transplantation of the Schwann-like cells combined with NT-3 administration may represent a new strategy of stem cell therapy for spinal cord injury.

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>

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>

Effect of N-acetyl-cysteine and depakine pretreatment on ferrous chloride-induced membrane potential and peroxidate changes in rat cortex neurons / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of N-acetyl-cysteine (NAC) and depakine (DP) on the changes of membrane potential and peroxidate in rat cortex neurons exposed to ferrous chloride (FeCl(2)).</p><p><b>METHODS</b>Cultured cortex neurons of newly born SD rats were randomly divided into control group (PBS group), model group (FeCl(2) group), NAC pretreatment group (NAC group), DP pretreatment group (DP group) and NAC+DP pretreatment group (NAC+DP group). In the latter three groups, NAC (0.08 mg/ml) and DP (0.1 mg/ml) were added in the cell culture 2 and 3 h before FeCl(2) (1 mmol/L) exposure, respectively. After exposure to FeCl(2), the membrane potential of the neurons was detected with fluorescent dye DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol), and the peroxidate level with 2,7-dichlorofluorescin diacetate (H(2)DCF) by laser confocal scanning microscope (LCSM) and nuclear factor-KappaB (NF-KappaB) level with immunocytochemistry.</p><p><b>RESULTS</b>Compared with FeCl(2) group, the expression of NF-KappaB and peroxidate level in the neurons were decreased significantly in NAC and NAC+DP groups (P<0.01), but not in DP group (P>0.05). FeCl(2) depolarized the membrane potential and increased the expression of NF-KappaB in the neurons. Compared with FeCl(2) group, significant changes in the membrane potential were observed in DP and NAC+DP groups (P<0.01) but not in NAC or PBS group (P>0.05).</p><p><b>CONCLUSION</b>Both NAC and DP can protect the neurons from FeCl(2)-induced damage but through different pathways, and their combined use can significantly alleviate neuronal damages due to FeCl(2) exposure. Antioxidants such as NAC in combination with antiepileptic drugs may produce favorable effect in prevention and treatment of posttraumatic epilepsy.</p>