Optimization of trypsin digestion intensity to obtain high-purity in vitro cultured astrocytes / 生理学报

The aim of the present study was to observe the effect of trypsin digestion on the purity of in vitro cultured astrocytes and optimize the culture methods. The cerebral cortical tissue from newborn Sprague Dawley (SD) rats was isolated and digested with 0.25% trypsin for 20, 30, or 40 min. The obtained single cell suspension was then cultured. Once reaching confluence, the cells were shaken at a constant temperature. Then, each of 20 and 30 min groups was subdivided into two groups, the control group with normal digestion and two-time-digestion group, and the cells were passaged and purified. Through inverted phase contrast microscope and MTT assay, cell growth and proliferation were observed, respectively. Immunofluorescence for glial fibrillary acidic protein (GFAP) was used to observe the morphology of astrocytes and to assess their purity in different stages. Flow cytometric analysis was used to detect the apoptotic rates of purified astrocytes. The results showed that, the cells being digested for 20 min usually reached confluence at 9 d after seeding. When the digestion time was extended to 30 min, the cells grew faster and reached confluence at 7 d after seeding, meanwhile the morphology of astrocytes was normal, GFAP positive rate (70.2 ± 4.0)% being much higher than that of the 20 min group (P < 0.05). Compared with 20 min group, 40 min group showed higher GFAP positive rate, whereas the cell proliferation was slower, and cell injury was more obvious. After shaking at constant temperature, two times of trypsin digestion could decrease the number of contaminated cells after passage. The GFAP positive rates of two-time-digestion groups in passage 1 (P1) were higher than those of corresponding control groups, and the GFAP positive rate of 30 min + two-time-digestion group in P1 reached (98.1 ± 1.7)%, which was equivalent to that of the 20 min + control group in P3. However, the apoptotic rate showed no significant difference between these two groups. Based on above mentioned results, we conclude that 30 min + two-time of trypsin digestion effectively improves the purity of astrocytes and shortens the time of primary culture and purification, suggesting that it is a rapid and effective method to obtain astrocytes with high purity in vitro.

Neurons in the corpus callosum of rats: expression of Cav2.2 and their connection / 中南大学学报(医学版)

OBJECTIVE@#To prove the existence neurons in the rat corpus callosum, the potential function of these neurons and their connection.@*METHODS@#Immunohistochemistry was used performed to examine the expressions of NeuN, a mature neuron marker,and N-type voltage-dependent valcium channel alpha1-subunit (Cav2.2)in the section of the rat corpus callosum. Horseradish peroxidase (HRP) normal sodium solution (30%), the retrograde tracer,was injected under the frontal forceps of corpus callousm and HRP absorbed by the process of neurons in the brain slices was stained with tetramethyl benzidine.@*RESULTS@#There were some NeuN positive cells in the rat corpus callosum and Cav2.2 was detected in some of these NeuN positive cells.Neurons with positive HRP were found in the rat corpus callosum and some of these neurons connected to the cortex or corpus striatum.@*CONCLUSION@#There are a few neurons in the corpus callosum of adult rats and some of them express Cav2.2. Neurons in the corpus callosum have connections with the brain cortex or corpora striatum.

Proliferation of neural progenitor cells in the subventricular zone and around the hematoma after intracerebral hemorrhage in adult rat / 南方医科大学学报

<p><b>OBJECTIVE</b>To examine the proliferation of the neural progenitor cells in the subventricular zone (SVZ) and around the hematoma after intracerebral hemorrhage (ICH) in adult rats.</p><p><b>METHODS</b>ICH was induced by stereotactic injection of type VII collagenase into the corpus striatum of adult rats, followed by pulse or continuous intrapenitoneal injection of bromodeoxyuridine (Brdu) to label the proliferating cells. The rats were sacrificed on days 2, 7, 14 and 28 following the ICH for immunohistochemistry of the tissues in the SVZ and around the hemotoma to determine the number of Brdu- immunoreactive cells.</p><p><b>RESULTS</b>With pulse Brdu labeling, a significant increase in the number of Brdu-immunoreactive cells in the ipsilateral and contralateral tissues in the SVZ and around the hematoma was observed 2-14 days, and the cell number reached the maximum on day 7 after ICH as compared with that of the sham-operated group. With continuous Brdu injection, the increase was observed on day 14 after ICH, and till day 28, the Brdu-immunoreactive cells in the SVZ decreased to the control level, but some positive cells still persisted in the tissues around the hematoma.</p><p><b>CONCLUSION</b>ICH induces transient and regional increase in the cell proliferation in the ipilateral and contraletral SVZ and tissues around the hematoma, and the proliferating cells in the SVZ may migrate towards the hematoma area.</p>

Effect of histamine on intracortical blood vessels of rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate histamine-induced changes of the intracortical vessels in the cortical slice of rat brain.</p><p><b>METHODS</b>Immunohistochemistry was employed to detect the expression of H1 and H2 receptors in the intracortical blood vessels of rats. Histamine-induced constriction of the intracortical blood vessels of the brain slices was observed with differential interference contrast microscope. Measurements of the luminal diameter were made on-line during the course of the experiment and confirmed off-line from the stored images. In order to observe whether histamine H1 and H2 receptors affected histamine-induced constriction, the intracortical blood vessels in the brain slices were pre-treated with H1 receptor antagonist diphenhydramine and H2 receptor antagonist cimetidine.</p><p><b>RESULTS</b>Expression of H1 and H2 receptors was detected in the intracortical blood vessels of the rat brain. Histamine (1-100 micromol/L) induced a concentration-dependent constriction from (1.48-/+0.67)% to (32.91-/+7.91)%. The reactions to each histamine concentration were significantly (P<0.01) different from each other, with the exception of the highest histamine concentrations (30 and 100 micromol/L) when maximal constriction due to histamine were observed (P>0.05). With pre-treatment of the slice with 10 micromol/L diphenhydramine, application of histamine did not elicit constriction. Pre-treatment of the slice with 10 micromol/L cimetidine did not completely inhibit but somehow significantly weakened vascular constriction in response to histamine treatment at 10 and 30 micromol/L (P<0.05).</p><p><b>CONCLUSION</b>Histamine can induce constriction of the intracortical blood vessels, which is mediated by H1 receptor.</p>