| 摘要: | 血腦屏障主要是由特化的內皮細胞排列而成，可保護及維持腦中環境的衡定。腦組織受傷後會誘發腦水腫並釋放大量的麩胺酸。研究顯示麩胺酸經由活化NMDA受體，會引起神經的興奮性毒性，進而促使神經細胞的死亡。然而麩胺酸是否也能破壞血腦屏障進而引起腦水腫，目前尚未明瞭。本實驗以初代培養的小鼠腦內皮細胞為細胞研究模型，發現NMDA受體的次單元GluN1及GLUN2B蛋白存在於小鼠腦內皮細胞。活化NMDA受體後可使細胞內鈣離子增加，降低腦內皮細胞所形成的胞膜電阻，並且抑制封閉蛋白的表現量。其分子機制是透過磷酸化MAPK激酶連鎖反應來進行，同時活化NMDA受體也增加細胞內基質金屬蛋白酶-2/9的表現量。因此，初步細胞實驗證實了內皮細胞上NMDA受體的活化會破壞小鼠腦內皮細胞所形成緊密連結屏障。
臨床上腦膠質瘤常引起腦水腫，且腫瘤組織常分泌高量的麩胺酸。因此利用小鼠腦膠質瘤為動物實驗的模型，首先以免疫組織化學染色來分析膠質原纖維酸性蛋白及表皮生長因子受體的表現量，證實成功建立了小鼠腦膠質瘤組織。檢測結果顯示膠質瘤組織分泌大量麩胺酸，利用螢光抗原抗體實驗，發現膠質瘤組織的血管內有大量NMDA受體的表現，高於正常腦組織。實驗發現膠質瘤組織內的封閉蛋白表現量較少，在共軛焦顯微鏡觀察下，血瘤屏障呈現較不連續的封閉蛋白聚合結構，我們的實驗提供了膠質瘤所引起腦水腫的另一種可能原因。
在腦部損傷的過程中會產生大量的緩激肽，導致血管通透性的改變，因而增加了腦水腫的嚴重度。氯胺酮為NMDA受體的拮抗劑。利用初代培養的小鼠腦內皮細胞模式，發現氯胺酮能抑制緩激肽所導致細胞內鈣離子濃度增加，且減少緩激肽刺激後胞內游離性封閉蛋白增加，進而保護了內皮細胞所建構的胞膜電阻值。本實驗證實了氯胺酮可能經由抑制緩激肽所引起內皮細胞內鈣離子濃度的改變，進而穩定了細胞緊密連結中封閉蛋白的結構。經由這一連串實驗結果，我們認為在腦內皮細胞內的NMDA受體參與了調控血腦屏障通透性的機制。
The blood–brain barrier (BBB) is formed by specialized endothelial cells that is essential to maintain neuronal function. Following brain injury, cerebral edema occurred and large amounts of glutamate are released. Accumulating evidence suggests that high concentrations of glutamate activate N-methyl-D-aspartate receptors (NMDARs) that account for neuronal excitotoxicity. However, the toxic effects of NMDARs activation on BBB integrity and its possible mechanisms remain unclear.
The primary culture of mouse cerebrovascular endothelial cell (MCEC) was used as in vitro model to demonstrate the expressions of NMDAR subunits, GluN1, in membrane portion of MCECs. GluN1 mRNA was also detected in MCECs and brain tissue. Functional assays showed that exposure of MCECs to NMDA increased Ca2+ influx. Therefore, we confirmed the existence of functional NMDARs in MCECs. Activation of NMDARs suppressed electrical resistance of MCEC-constructed monolayer and tight junction (TJ) occludin protein levels. As to the mechanism, NMDA stimulated sequential phosphorylation of ERK1/2 and MEK1/2. The NMDA-induced alterations in ERK1/2 phosphorylation and occludin levels were reversed by pretreatment with a MEK inhibitor and a NMDAR antagonist, respectively. Furthermore, amounts of MMP-2/-9 in MCECs were augmented by exposure to NMDA. Therefore, the first study shows NMDAR activation disrupts the MCEC-constructed TJ barrier via activation of the MEK1/2-ERK1/2 signaling pathway and upregulation of MMP-2/9 expressions.
Malignant glioma often releases glutamate and causes cerebral edema, which indicates an incomplete BBB. Therefore, the murine GL261 glioma was used as an in vivo experimental model to evaluate effects of NMDAR activation on the BBB and blood-tumor barrier (BTB). With IHC analysis of GFAP and EGFR, we first identified and confirmed glioma in mouse brain. Glioma releases higher levels of glutamate than brain tissue. After labelling with GluN1 and factor VIII antibodies, analysis by TissueFaxs imaging system revealed higher GluN1 expression in tumor microvasculature than in normal brain tissue. Occludin levels were downregulated in tumor tissue. Confocal microscopic analysis showed irregular occludin TJ structure in tumor tissue, which might partially explain the leaky characteristics of the BTB.
Following neuronal death, large amounts of bradykinin were produced and subsequently increase BBB permeability. Previously our lab has demonstrated that ketamine blocked intracellular Ca2+ influx after exposure to bradykinin in HUVECs. In this study, pretreatment of MCECs with ketamine time- and concentration-dependently lowered the bradykinin-induced Ca2+ influx. As to the mechanisms, ketamine did not affect levels of B2R, receptor, a major receptor that responds to bradykinin stimulation. Bradykinin increased amounts of soluble occludin in MCECs, which was mitigated by pretreatment of ketamine. Consequently, pretreatment of ketamine also alleviated bradykinin-induced disruption of MCEC-constructed barrier. Therefore, ketamine at a therapeutic concentration can protect against bradykinin-induced breakage of the BBB via suppressing Ca2+-dependent redistribution of occludin TJ. Taken together, our results suggest that endothelial NMDARs play a role in modulation of the BBB integrity. |
