ABSTRACT
<p><b>OBJECTIVE</b>To study the effects of immunization with the fusion protein CAC (a product of prokaryotic expression of recombinant HBcAg and β-amyloid peptide fusion gene) against the toxicity induced by intrahippocampal injection of aggregated β-amyloid peptide (Aβ) in rats.</p><p><b>METHODS</b>SD rats were immunized intraperitoneally with the fusion protein CAC, and the titer of anti-Aβ antibody was evaluated by ELISA. When the titers of the anti-Aβ antibody reached 1:3 000, aggregated Aβ was injected into the CA1 region of the rat hippocampus. Two weeks after Aβ injection, the rats underwent morris water maze test before sacrificed to prepare the brain slices with Congo red and haematoxylin staining.</p><p><b>RESULTS</b>The titer of anti-Aβ antibody reached 1:3 000 after 5 immunizations with the fusion protein. After Aβ injection, the saline-immunized rats showed a reduced cognitive behavior in the Morris water maze test compared to the CAC-immunized rats. In the saline-immunized rats, the neurons around the site of Aβ injection exhibited obvious cell damages with Aβ deposits and glial infiltration, whereas in CAC-immunized rats, Aβ deposits were significantly reduced or even absent.</p><p><b>CONCLUSION</b>Immunization with the fusion protein CAC can inhibit the toxicity induced by intrahippocampal aggregated Aβ injection.</p>
Subject(s)
Animals , Male , Rats , Amyloid beta-Peptides , Genetics , Allergy and Immunology , Antibodies , Blood , Hepatitis B Core Antigens , Genetics , Hippocampus , Metabolism , Immunization , Injections , Peptide Fragments , Allergy and Immunology , Rats, Sprague-Dawley , Recombinant Fusion Proteins , Genetics , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of vasoactive intestinal peptide (VIP) on angiogenesis after focal cerebral ischemia.</p><p><b>METHODS</b>Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 120 min in adult SD rats with intracerebroventricular VIP administration at the beginning of reperfusion. Immunohistochemistry was performed to assay BrdU immunoreactive endothelial cells, expressions of VEGF, flt-1 and flk-1 in the ischemic zone, and the protein expressions of vascular endothelial growth factor (VEGF) in the brain was measured using Western blotting.</p><p><b>RESULTS</b>Immunohistochemical staining revealed significantly increased BrdU immunoreactive endothelial cells on the margins of the ischemic lesion in rats treated with VIP as compared with that in the control rats (P<0.05). VIP significantly increased the number of VEGF immunoreactive cells and flt-1- and flk-1-positive endothelial cells in comparison with the control group (P<0.01). Western blotting showed that VIP treatment resulted in significantly increased VEGF protein level in the ipsilateral hemisphere (P<0.05).</p><p><b>CONCLUSIONS</b>VIP enhances angiogenesis in the ischemic brain by increasing the expressions of VEGF in the brain tissue and its receptors flt-1 and flk-1 in the endothelial cells.</p>
Subject(s)
Animals , Male , Rats , Brain , Metabolism , Brain Ischemia , Allergy and Immunology , Metabolism , Pathology , Endothelial Cells , Metabolism , Gene Expression Regulation , Neovascularization, Physiologic , Rats, Sprague-Dawley , Vascular Endothelial Growth Factor A , Metabolism , Vascular Endothelial Growth Factor Receptor-1 , Metabolism , Vascular Endothelial Growth Factor Receptor-2 , Metabolism , Vasoactive Intestinal Peptide , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To observe the cellular uptake of beta amyloid protein (Abeta) by cultured human neuroblastoma (SH-SY5Y) cells and the location of Abeta in the subcellular structures.</p><p><b>METHODS</b>The time course of cellular uptake of Abeta1-42-fluo in the SH-SY5Y cells was observed directly under laser scanning confocal microscope (LSCM). Image analysis was conducted to compare the differences of cellular Abeta uptake after treatment of the cells with different concentrations of extracellular Abeta for 24 h. Multiple immunofluorescence staining was employed to identify the location of Abeta in the subcellular structures.</p><p><b>RESULTS</b>SH-SY5Y cells showed Abeta internalization after incubation with Abeta1-42-fluo (200 nmol/L) for 1 h, and the quantity of Abeta uptake was time-dependent. A higher concentration of extracellular Abeta1-42-fluo resulted in increased Abeta uptake, which differed significantly between the 3 groups with treatment at different concentrations (P<0.01 or 0.05). Immunofluorescence staining revealed a co-localization of part of the Abeta and Lamp-1 (a lysosome marker) in the cytosome.</p><p><b>CONCLUSION</b>SH-SY5Y cells can clear Abeta through a time- and dose-dependent cellular uptake mechanism. Part of the Abeta uptaken in the cytoplasm is located in the lysosome .</p>
Subject(s)
Humans , Alzheimer Disease , Metabolism , Amyloid beta-Peptides , Metabolism , Cell Line, Tumor , Neuroblastoma , Metabolism , Pathology , Peptide Fragments , MetabolismABSTRACT
OBJECTIVE@#To observe the internalization of beta-amyloid protein (Abeta) in primary cultured neurons and effect of astrocyte on it.@*METHODS@#The purified cortical neurons of mouse were cultured for 14 d, and were divided into a control group and an Abeta group. Each group was further divided into 3 subgroups. The neurons and 3 different concentration fluorescein or Abeta1-42-fluo were co-incubated for 24 h. The internalization of Abeta and the location of Abeta in subcellular structure were examined by the laser scanning confocal microscope combined with the image analysis method directly or after immunofluorescence staining. Neurons and astrocytes were co-cultured for 14 d. The cultured neurons and astrocytes were divided into a control group and a Abeta group. The cultures were treated with 200 nmol/L fluorescein or 200 nmol/L Abeta1-42-fluo for 24 h respectively. The effect of astrocyte on the internalization was analyzed by the above method.@*RESULTS@#There were no fluorescent granules within neurons in every fluorescein group. The purified cortical neurons could internalize 100 nmol/L, or 200 nmol/L Abeta1-42-fluo in 24 h. The fluorescent granules of Abeta1-42 distributed within perikaryon and processes. The internalization was related to the concentration of Abeta. The part of Abeta was located in the lysosome of neurons indicated by immunofluorescence staining. Compared with the purified neurons, the neurons co-cultured with the astrocytes internalized Abeta increased in the internalization of Abeta. There was significant difference between the purified neurons and the co-cultured neurons with astrocytes (P<0.05).@*CONCLUSION@#Neurons could internalize the proper concentration of Abeta. Astrocyte might facilitate the internalization of Abeta in neurons.
Subject(s)
Animals , Mice , Alzheimer Disease , Metabolism , Amyloid beta-Peptides , Metabolism , Amyloid beta-Protein Precursor , Metabolism , Animals, Newborn , Astrocytes , Cell Biology , Cells, Cultured , Cerebral Cortex , Cell Biology , Metabolism , Coculture Techniques , Endocytosis , Genetics , Neurons , Cell Biology , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study the effect of ligustrazine on the migration of neuronal precursors (NPs) after focal cerebral ischemia in adult rats and explore its acting mechanism on recovery of function.</p><p><b>METHODS</b>Rat model of left middle cerebral artery occlusion (MCAO) was established by thread ligation. Ligustrazine 40 mg/kg was injected peritoneally once a day 2 h after modeling. On the 3rd, 7th, 14th and 21st day after operation, the migration of Doublecortin (DCX, the marker of NPs) in subventricular zone (SVZ) and the rostral migratory stream (RMS) were observed with immunohistochemistry.</p><p><b>RESULTS</b>The migration of DCX-positive cells in SVZ (abbrev. as migration below) through RMS into the olfactory bulb started from the 3rd day after ischemia, and lasted to the 21st day; the migration directly or through RMS into the ischemic penumbra of the adjacent striatum started on the 7th day, and increased significantly on the 14th day; and a few of DCX positive cells migrated through corpus callosum into the ischemic cortex on the 21st day. The migration was similar in the two groups in its pathway, but the extent in the ligustrazine group was more intensive.</p><p><b>CONCLUSION</b>Ligustrazine could promote direct migration of NPs into the ischemic cerebral cortex and striatum, suggesting that it might play an important role in promoting self-recovery of brain function after ischemia through accelerating the migration of NPs.</p>
Subject(s)
Animals , Male , Rats , Anti-Inflammatory Agents, Non-Steroidal , Pharmacology , Brain Ischemia , Cell Movement , Immunohistochemistry , Microtubule-Associated Proteins , Neurons , Metabolism , Pathology , Neuropeptides , Pyrazines , Pharmacology , Rats, Sprague-DawleyABSTRACT
<p><b>OBJECTIVE</b>To explore the neuroprotective effect of vasoactive intestinal peptide (VIP) in rat ischemic brain injury.</p><p><b>METHODS</b>VIP was administered via intracerebroventricular injection in SD rats prior to focal cerebral ischemia by intraluminal occlusion of the middle cerebral artery. The infarct volume was assessed with TTC staining, and immunohistochemistry was performed to analyze the S100beta expression in the cerebral tissue, with the serum concentrations of S100beta detected by double-antibody sandwich enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>After VIP injection, the relative infarct volume in the rats with cerebral ischemia was significantly reduced by 32.3% as compared with the volume in the control group on day 1 (P<0.05), and the number of S100beta-positive cells was significantly decreased in the cerebral tissue (P<0.05). The injection also resulted in significantly decreased serum S100beta concentrations in the rats (P<0.05).</p><p><b>CONCLUSION</b>VIP injection can reduce the infarct volume in rats with focal cerebral ischemia, suggesting the neuroprotective effect of VIP in brain ischemia possibly by reducing S100beta overexpression.</p>