Study On The Mechanism Of Vascular Cell Proliferation Induced By Vascular Proliferation In Ischemic Brain Injury

Neurogenesis appears in the non-neurogenic regions, such as, the striatum and cerebral cortex, under experimental pathological condition. Many studies have shown that cerebral ischemia could induce angiogenesis and neurogenesis in the peri-infarct area. Overproduction or administration of VEGF, an angiogenic protein, in rat brains could simultaneously increase angiogenesis and neurogenesis in ischemic injured brains. To investigate the role of angiogenesis in the ischemia-induced neurogenesis, in the present study, we injected a pTIE2-GFP plasmid to target the differentiation of GFP positive (GFP*) endothelial cells and intraventricularly injected VEGF-expressive plasmid (phVEGF-DsRed) or control plasmid (pDsRed) to observe effects of VEGF overproduction on the angiogenesis and neurogenesis in ischemic striatum of rat brains after MCAO. We used multiple immunofluorescent staining combined with confocal laser scanning to investigate the changes of angiogenesis, neurogenesis and astrogliosis in the brain following a transient MCAO. Then, we further applied multiple immunostaining and whole cell patch clamp recording to study morphological differentiation and electrophysiological property of GFP+cells in the ischemic brains. We interestingly found that ischemic induced angiogenic endothelial (GFP*) cells could express different lineage neuronal makers and showed action potentials. The results were summarized below:1.Time-dependent changes of endothelial and different lineage neuronal expressive proteins in the ischemic injured rat brains.Immunohistochemical staining was used to study the time-dependent expression of vWF、nestin、DCX and Tuj-1in the ischemic striatum of rat brains from3days to2weeks after MCAO. The results showed that, in the striatum, vWF+cells significantly increased at3days, then reduced at1week and last to2weeks after reperfusion, which the positive cell number was still higher than that of sham operated group. Ischemic injury also induced a markedly increase of nestin+cells at3days followed by the increase of DCX+and Tuj-1+cells at1weeks after MCAO. Immunofluorecent triple staining results showed that the active astrocytes, together with endothelial cells, support the surrounding neurons in the ischemic brain. These results suggest existence of some relationship among astrocytes, neurogenesis and angiogenesis, main components of neurovascular units, in the pathophysiological process of the brain following ischemic injury.2.The transdifferentiation of GFP+endothelial cells into neurons in the ischemic brains.Following injection of pTIE2-GFP plasmids into the striatum of rat brains, we detected green fluorescence cells in the ischemic striatum and frontal cortex at3days, remained at1,2,4and8weeks after MCAO under the fluorescent microscopy. Multiple immunofluorescent staining was used to confirm the cell type of GFP-labeled cells. The results showed that, at3days after reperfusion, a great proportion of GFP+cells (75%) could express vWF, while a small percentage of GFP+cells expressed CD34+(17%) and CD133+(9%), two endothelial progenitor cell (EPC) markers.Under this condition, we did multiple staining of GFP with many neural markers to determine whether these endothelial-derived cells could be local precursors or neural progenitors in the ischemic striatum. The results showed that GFP+-Sox2+or GFP+-nestin+neural stem cells and GFP+-Pax6+neural progenitor cells could be observed in the striatum at3days after MCAO operation. The number of GFP+-nestin+neural stem cells and GFP+-Pax6+neural progenitor cells reached a peak at3days, and significantly declined at1and2weeks after reperfusion. However, neither GFP+-Oligo2+nor GFP+-GFAP+cells was found in the ischemic brain. These results suggested that endothelial-derived cells might be partly become neural stem cells/progenitors for generating new neurons in the ischemic injured regions.Further study interestingly found that GFP+cells could further express DCX (GFP+-DCX+neuroblasts), Tuj-1(GFP+-Tuj-1+immature neurons) and MAP-2(GFP+-MAP-2+mature neurons) in the ischemic striatum in time-dependent manners. Furthermore, part of GFP+cells became GFP+-GAD67+GABAergic neurons and a small portion of them were GFP+-ChAT+newborn cholinergic neurons in the striatum at4weeks after MCAO operation. These results revealed that angiogenic endothelial cells could differentiate into mature neurons in the brains after ischemic injury.To explore whether those endothelial-derived neurons can be neural functional, we used whole cell patch clamp techniques to record electrical physiological activity of GFP+labeled cells in the brain slides of rat brains after MCAO. The results showed that16of26GFP+cells displayed typical characteristics of mature neuronal membrane potential, capacitance (Cm) and input resistance (Rin). Moreover, these cells showed that the membrane could be elicited to depolarize and fire action potential (AP). Those cells also showed spontaneous post-synaptic currents (PSC), indicating that these newborn neurons could receive inputs. These results indicated that part of the endothelial-derived cells could finally differentiate into mature neurons and behave electrophsiological characteristics of neurons.3.Enhancement of the endothelial-derived neurogenesis in ischemic injured striatum by VEGF overexpressionImmunofluorescent staining results showed that the numbers of GFP+cells were significantly increased in the phVEGF-DsRed group compared with that in ihepDsRed control group at2weeks after MCAO. Moreover, VEGF overexpression could markedly increase the numbers of GFP+-MAP-2+cells in the ischemic striatum and also increased the proportion of GFP+-MAP-2+cells in GFP+cells. These results suggested that VEGF overexpression could enhance neurogenesis via raising the transdifferentiation rate of GFP+cells.4. Direct regulating effects of VEGF on neuronal activity via phosphorylation of synapsinTo study the effects of VEGF on neuronal activity, we used primary cultured cortical and hippocampal neurons combined with immnocytochemistry and western blotting analysis. The results showed that neurons could express VEGF mRNA and its protein. High concentration of potassium ions (K4) was added in the culture medium of neurons or synaptosomes to induce membrane depolarization. Interestingly, K+treatment could significantly increase the level of VEGF in the culture medium, suggesting VEGF release from the neurons. Moreover, administration of VEGF into the cultured hippocampal neurons could increase the level of phosphorylated synapsin in a SU1498antagonized manner. Putting together, VEGF may play an important modulator role in the neurotransmission at both pre-and post-synaptic levels. Conclusion:Present results provided the first evidence that angiogenic endothelial cells might be one of neural progenitors and further became local functional mature neurons in the injured striatum, indicating angiogenesis plays important role in the reconstruction of neurovascular units in the ischemic injured brains.