An Experimental Study Of The Mechanism Of Altered Endothelial Permeability And Its Relantionship With The Expression Of Associated Proteins In C6 Glioma In Vivo.

Objective: Increased permeability of blood brain tumor barrier (BBTB) has been observed in gliomas. This study was performed to investigate the change of BBB permeability in different rigeons of middle-late phase C6 glioma and to evaluate the mechanisms from multiple aspects, by which gliomas induce enhanced BBTB permeability, including ultrastructure of microvessle and functional signal proteins.Methods: (1) Male SDrats (weight 250³50g) were chosen as subjects. 10μl solution containing 1×10⁶ C₆ gliomaous cells was sterotactically implanted into the right caudate nuclei to establish an experimental model of brain glioma in SD rats. The rats with brain-bearing C6 glioma were sacrificed at the middle-late phase of glioma, and then the samples were chosen from tumor core, tumor periphery region, BAT (tissues within 2 mm of the border of the tumor), BDT (cortical tissues at least 2 mm away from tumor border but within the ipisilateral hemisphere) and NBT (cortical tissues on the opposite hemisphere of the brain) respectively. (2) By using immunohistochemical SP method , the semiquantitative analysis of extravasation of endogenous albumin was performed to analyse the integrity of endothelial barrier. (3)After the rats were infused intravenously (i.v.) withvarious-sized tracers lanthanum nitrate and HRP, the ultrastructure of BBTB in gliomas was observed by lanthanum nitrate-tracing and HRP-tracing transmission electronic microscope to demonstrate the anatomical routes of BBTB leakage. (4) By examining the expression of specific TJ proteins occludin with Western-blot analysis in blood vessels of glioma, the possible involvement of interendothelial TJPs was investigated. (5) MVD was assessed by enzymohistochemistry of alkaline phosphatase (ALP) . (6) The expression of MMP-9 and iNOS were detected immunohistochemiscaly in gliomas to examine the associations between their activity, expression of occludin and permeability of microvessels.Results: (1) After implantation with 1 X 106 C6 gliomaous cells into the right caudate nuclei, all the SD rats developed visible solid glioma during middle-late phase.(2) The intensity of Immunoreaction products of albumin varied among different sectors of the C6 tumor. Albumin positive staining located diffusely among the interstitial spaces of C6 glioma. Moderate and intensive positive reaction of albumin was found in interstitial spaces and necrosis region of tumor, respectively. In the normal brain tissues, the albumin staining was negtive. BAT showed weak positive reaction of albumin and the intensity decreased significantly with the increasing distance to tumor edge.(3) HRP-tracing light microscope results:? Endogenic activity of HRP was only found in the necrosis and bleeding regions.? For the non-tumorous tissues, localization of HRP was restricted in thecapillary lumen and a thin, linear staining along the lumen face of endothelial cells was seen.(3) In C6 glioma, HRP reaction products DAB was located both in the lumen and along the outer wall of capillary in a thick diffusion pattern, consistent with the results of electronic microscope. (4) Electronic microscope results:? In samples of nontumorous brain tissues, low permeability were consistently recorded. HRP or lanthanum is excluded from the tight junction. Both of them were not observed leaking out of the capillaries. (D In brain adjacent to tumor (BAT) , permeability and ultrastructure of tight junctions were comparable to normal brain capillaries. Occasionally, the tight junction was completely infiltrated with lanthanum salt granules or HRP and extravasation to the basement membrane was seen in certain sites of the capillaries.(3)The permeability and ultrastructure of tight junctions in both the periphery sector and core of glomas greatly altered. Lanthanum salt granules and HRP penetrated the cellular gaps of endothelia and seeped in the basement and interstitial spaces, esp for the latter, Lanthanum salt granules distributed extensively among the interstitial spaces and sometimes appeared in the cytoplasm of tumor cell. Moreover, there was continuity of La3+/ HRP deposit from capillary lumen through TJ to the basal lamina and then to the interstitial clefts of the surrounding brain.? There was a significant increase in the number of vesicles in the cytoplasm of tumor capillaries endothelial cells as compared with nontumorous brain tissues(P<0.05). However, very few Lanthanum-filled orHRP-filled vesicles were seen in the cytoplasm of cerebral microvessel endothelial cells both in tumor and normal brain tissues(P>0.05).(5) In the microvessels of glioma, the opened tight junction could be found. But the basement membrane was continued, compact and lineal. There were tumor cells arranging along the basement membrane, which acted as pericytes. No fenestration on the basement membrane was seen. In addition, there were no glioma cells found in the blood vessels.(5) Microvessel density (MVD) was significantly different between C6 glioma tissues and normal brain tissues (P<0.05) .(6)Positive immunohistochemical staining of iNOS and MMP-9 were all localized in the cytoplasm. In contrast with the non-tumor brain tissues, C6 glioma tissues showed overexpression of iNOS and MMP-9. The expression was remarkable at the edge of tumor tissue and undetectable in normal brian tissue. The positive rate of iNOS expression was 100% in C6 glioma tissues, significantly different from that of nontumorous brain tissues (PO.05), so was the positive rate of MMP-9 expression. Both the expression of iNOS and MMP-9 were positively correlated with the MVD of C6 glioma and negatively correlated with the exvasatation of albumin. The stronger MMP-9 and iNOS expressed, the more albumin appeared in the interstitial of C6 gliomas.(7) Western blot analyses showed occludin bands as broad signal in the normal brain tissues, while the tumor tissues narrowed to a more focused band (apparent size approximately 65 kDa). There was no additional band. Occludin band of both tumor tissues and BAT showed a significantdecrease (35.20% and 7.39%, respectively) compared with normal brain tissues (PO.05). BDT showed no significant change in comparison to normal brain tissues.Conclusion: In conclusion, these data suggest C6 gliomas are accompanied by alteration in BBB, including both functional and ultrastructural aspects. Tight junctions (TJ) between the cerebral microvessel endothelial cells are critical for maintaining brain homeostasis and low permeability of blood-brain barrier (BBB). Regulation of endothelial tight junction at the BBB of glioma is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins.(1) It's reliable to establish an experimental model of brain glioma in SD rats by implanting 1 X 106C6 gliomaous cells sterotactically into the right caudate nuclei.(2) In C6 gliomas, large-sized molecule albumin can pass through the disrupted BBTB and extravasate from microvessels to the local peri-vascular area and distribute diffusely in the interstitial spaces, which may result in peritumoral oedema.( 3 ) It's the intercellular tight junctions that completely restrict extravasation of HRP and lanthanum. Alterations in tight junctions is the major ultrastructural characteristic of tumor-induced capillary endothelium. The disruption of BBB tight junction in G6 glioma might provide the main route of transport of lanthanum nitrate and HRP through endothelial wall. La3+/ HRP probably don't pass through the endothelial cells by transcytosis.(4) The BBB integrity in brain adjacent to tumor (BAT) keeps relativelywell, whose permeability is greatly reduced in comprarison to the tumor. Unfortunately, BAT sometimes consists of invading single tumor cells, which limites the response of brain tumors to systemic chemotherapy.(5) The permeability of regional capillary greatly varied among different brain and among different sectors of the dissected tumor, which may be related to alteration of tight junctions in corresponding sectors.(6) The glioma cells show some atypia, however they still remain part of ability to induce the formation of the BBB. As a result, the glioma cells fail to enter the blood stream and form metastasis in distant areas.(7) The increased MMP-9 and iNOS activity may be correlated with the altered vascular permeability and decreased expression of the tight junction protein occludin in glioma.(8) Occludin changes correlates negatively with alterations in vascular permeability. TJ proteins occludin is involved in the regulation of TJ permeability. Loss of the permeability of BBB was accompanied by decreased expression of TJ proteins.(9) Altered tight junctions are the main structural feature of the disturbed BBB in C6 gliomas and in gliomas in general, which contributes to BBB disruption during gliomas in the rat.