Expression Of TGF-β1 With Its Two Type Ⅰ Receptors And Variations Of Microvascular Density Induced By Transient Focal Cerebral Ischemia In Rats

Objective: Based on the previous studies of the effect of endostatin and ratio of E/V in rats subjected to cerebral ischemia-reperfusion injury, we studied the expression of transforming growth factor-β1 (TGF-β1) and its two typeⅠreceptors- Activin receptor-like kinase 1 (ALK1) and Activin receptor-like kinase 5 (ALK5) and variations of microvessels densty induced by transient focal cerebral ischemia/reperfusion.Methods: The models of focal brain ischemia/reperfusion injury by middle cerebral artery occlusion (MCAO) were established in Wistar rats according to the advanced Zea Longa's method. Rats were randomly divided into two groups: sham-operated group and cerebral ischemia/reperfusion injury group. The sham group (n=8) underwent the same surgical procedure but without the introduction of the filament into the MCA. The model group (n=48) was divided into 6 subgroups (n=8 for each) and the subgroups were anesthetized and euthanized by decapitation at 6hours, 1 day, 2 days, 3 days, 4 days and 7 days after reperfusion.1) Cerebral slices were stained with triphenyl tetrazolium choride (TTC).2) Morphological study was performed on paraffin sections stained with hematoylin and eosin.3) Immunohistochemitry techniques were used to detect protein localization of CD105, TGF-β1 with its two typeⅠreceptors -ALK1 and ALK5 in cerebral tissue.4) Microvascular density (MVD) was quantified by visually counting CD105 staining microvessels in 5 high-power fields per section.5) Levels of TGF-β1 mRNA, ALK1mRNA and ALK5mRNA in brain tissue were quantified with real time polymerase chain reaction (RT-PCR).6) Pearson's test was used to analyze the correlations between ALK1mRNA, ALK5mRNA and MVD. Results:1) Six hours after reperfusion, many neurons were slightly shrunken and surrounded by swollen cellular processes, while the nuclei of the shrunken neurons were often triangular and even darkly stained. Some microvacuolation was evident in the cytoplasm of these injured neurons. One day after reperfusion, neurons in which nissle body lightly stained or disappeared were severely shrunken, scalloped in appearance and some had pyknotic nuclei and eosinophilic cytoplasm, both signs of irreversible injury. Vacuolation of the neuropil, which was possibly the result of both cytoxic and vasogenic edema, indicative of swelling of dendrites, was evident. Cell layers in hippocampus decreased sharply. 2d after reperfusion, the injury was progressing and the infract area appeared like sponge. Liquefaction necrosis could be noted, inflammatory cells infiltrated the parenchyma, neuropil disappeared. Three days after reperfusion, pyramidal neurons decreased significantly in the amount and many of them became degenerated or necrotic. The infract area was enlarging and glial cells began to proliferate. Gial cells continue to be proliferated and the penumbral region (the tissue surrounding the infarction) was developing at 4d to 7d after reperfusion.2) Cells intensively marked by CD105-immunochrmistry were found in all types of vessels but were most abundant in capillaries. Immunostaining for TGF-β1 was barely detected by immunohistochemistry in the brains of sham-operated animals. After ischemic reperfusion, immunostaining for TGF-β1 protein was obviously increased in the cytoplasm of ischemic neurons and glia cells. Immunostaining for ALK1 was detected in samples of both sham group and model groups. Immunostaining for ALK1 protein was observed attenuated in neuronal cell and some endothelial cells after ischemic reperfusion, while the intense of expression became lower. Immunostaining for ALK5 was hardly detected in the sections of sham-operated group. ALK5 protein immunostaining became evident not only in neurons, glia cells but also in a few endothelial cells.3) Microvascular density (MVD) was 34.23±1.45 in the sham group. Six hours after reperfusion, the number of labeled microvessels considerably increased and peaked at 1 day with 44.1±18.30, but the difference was not statistically significant. It was then declined gradually at 2 days after reperfusion with a level of 31.76±15.06. The lowest level of MVD was 15.50±5.72, which appeared at 3 days after reperfusion and t Test revealed a significant difference (p<0.05) contrast to the sham group. 4 days after reperfusion, the microvascular density began to up-regulate and reached a level of 38.85±11.55 at 7 days after reperfusion, but the difference revealed no statistical significance.4) The quantity of TGF-β1 mRNA levels in sham group was set as 1.TGF-β1 mRNA expression showed a statistically significant upregulation after reperfusion. At 2d time point, the up-regulation of TGF-β1 mRNA began to shrink, but the level (2.01±0.49) was still significantly higher compared to sham-operated group (p<0.05). Then until 7d post reperfusion, the expression up-regulated again and reached peak (4.90±1.42, P<0.01), approached a 5-fold greater amount of transcript. The level of ALK1 mRNA in sham group was set as 1. Compared to the sham group, ALK1 mRNA levels down-regulated as early as 6 hours after reperfusion and reached a lowest level after 2d of reperfusion (p<0.01), only expressed 44% of that in the sham group. At 3d time point and 4d time point, the down-regulation of ALK1 mRNA reduced. At 7d time point, another significantly lower level was detected (0.46±0.12, p<0.01). For ALK5 mRNA, at 6h time point, the expression (0.96±0.19) stayed at almost the same level with the sham group whose ALK5 mRNA level was set as 1. Then, ALK5 mRNA up-regulated from 1 days (1.12±0.25) and the peak value was distinguished at 4d time point where approximately 4-fold greater amount of transcript was measured (3.97±1.03, p<0.01). It remained a higher level of nearly 3-fold of that in sham group at 7 days after reperfusion (p<0.01).5) The correlation between microvascular density and the levels of ALK1 mRNA was positive with r=0.370 (p <0.05), but there was no correlation observed between microvascular density and the level of ALK5 mRNA with r=0.182.Conclusions: After ischemic reperfusion, immunostaining for TGF-β1 was mostly localized in the cytoplasm of ischemic neurons and glia cells; immunostaining for ALK1 was observed in neuronal cells, glia cells and some endothelial cells; immunostaining for ALK5 was evident not in single bodies of neurons, glia cells and a few endothelial cells. After ischemic reperfusion, microvascular density increased at earlier time followed by a tendency of down-regulation and the lowest level appeared at 3d. Both of the expressions of TGF-β1 mRNA and ALK5 mRNA showed an increasingly up-regulation tendency after reperfusion; the former reached to the highest level at 7d time point, but experienced a down fluctuating at 2d and 3d time points while the later got to peak earlier at 4d without any fluctuation. The expression of ALK1 mRNA kept down-regulating after reperfusion, especially at 2d time point and 3d time point. The expression of ALK1 mRNA was correlated with the variations of microvascular density. Before ischemia, ALK1 may be predominant in TGF-β1 signal, but ALK5 became more activated after ischemia reperfusion.The immunohistochemical distributions of TGF-β1 protein and its two type I receptors were not restricted in cells of one type, suggesting the signal of TGF-β1 correlate with angiogenesis through a framework crosstalk among different cells after cerebral ischemia and reperfusion injury.