紅藻胺酸處理後對大白鼠視網膜內神經元及神經膠細胞的影響

Abstract

本研究主要的目的在探討經由紅藻胺酸(kainate)所引發的興奮性神經毒性(excitotoxicity)反應後，視網膜內神經元與神經膠細胞相互作用之反應。首先，在給予紅藻胺酸一天後，具有FJB (Fluoro Jade B)免疫反應主要會出現在神經節細胞層(ganglion cell layer; GCL)和內核層(inner nuclear layer; INL)的內緣。此時，具有NR1 (NMDA receptor)和GluR2/3 (AMPA receptor)免疫反應的細胞開始減少，但在第七天上升。然而，具有GluR5/6/7 (KA receptor)和EAAT1 (excitatory amino acid transporters 1)免疫反應的細胞則在給予紅藻胺酸後會增加。雙重免疫螢光反應結果指出，在給予紅藻胺酸三天後，在Müller氏細胞和星狀神經膠細胞會同時表現GFAP和NR1 (NR1+/GFAP+)、GFAP和GluR2/3 (GluR2/3+/GFAP+)以及GFAP和GluR5/6/7 (GluR5/6/7+/GFAP+)。給予紅藻胺酸後，在神經節細胞(ganglion cells)和水平細胞(horizontal cells)上具有calbindin (CB)免疫反應的細胞沒有明顯的改變。相反的是，具有parvalbumin (PV)免疫反應的神經節細胞，有減少的情形。但仍可發現具有CB和PV點狀免疫染色的產物散佈在變薄的視網膜內。本實驗結果顯示，這些不同的離子性麩氨酸鹽接受器之次單位體，在紅藻胺酸所引發的興奮性神經毒性反應中各自扮演不同的角色。在Müller氏細胞和星狀神經膠細胞上增加興奮性胺基酸運輸者與GFAP/iGluRs的表現，代表他們在紅藻胺酸所引發的類似興奮性神經毒性反應過程中，與麩氨酸鹽代謝系統障礙所引發的神經退化有密切關係。此外，具有CB及PV免疫反應的細胞與鈣離子的緩衝無關。 第二，在給予紅藻胺酸後，增加的complement receptor type 3 (CR3)、the major histocompatibility complex (MHC) class II和ED-1的免疫反應細胞主要是出現在內核層內緣。有趣的是，可在不同的時程裏，在光受器層的外節(OSPRL)發現具有這些免疫反應的細胞。根據定量分析數據，CR3的免疫數值是在給予紅藻胺酸七天後急速的達到最高峰，然後消退。相對的，MHC class II或ED-1的免疫數值則在給予紅藻胺酸三天前，和緩的達到高峰，然後緩慢的消退。雙重免疫螢光反應進一步顯示，微小膠細胞/巨噬細胞會同時表現CR3和ED1 (OX42+/ED1+)或MHC class II molecules (OX-42+/OX-6+)，並在給予紅藻胺酸早期，維持分枝狀的型態。電子顯微鏡結果顯示，具有CR3的微小膠細胞/巨噬細胞，呈現豐富的細胞質包含著許多具有小泡和噬菌體。同時，有些類似Müller氏細胞和星狀神經膠細胞，也可吞噬外來物質。 第三，在給予紅藻胺酸後，Müller氏細胞和星狀神經膠細胞會活化並增加nestin、glial fibrillary acidic protein (GFAP)和glutamine synthetase (GS)的表現。主要的發現是給予紅藻胺酸一天開始，GFAP合併著nestin的表現有明顯增加。根據西方點墨法的統計數據顯示，nestin、GFAP和GS的表現相較於正常的視網膜有明顯增加的情形。而雙重免疫螢光反應顯示，Müller氏細胞和星狀神經膠細胞會表現放射狀神經膠細胞的標誌，並且會共同表現nestin和bromodeoxyuridine (BrdU)，暗示其可能再進入細胞週期裏。在Müller氏細胞和星狀神經膠細胞增加這些蛋白質的表現，可能代表著在給予紅藻胺酸後所引發的受傷，會增加神經膠細胞的反應，並引發與再生成果相關的去分化反應。另外，在紅藻胺酸後所造成的傷害中，增加在Müller氏細胞和星狀神經膠細胞內BLBP和DCX的表現，暗示著這些細胞具有去分化的潛力，並在視網膜神經退化的過程中，再次獲得這些不成熟的分子。

The present study aimed to investigate the interaction between glial cells and neurons in the adult rat retina after kainic acid (KA)-induced excitotoxicity. First, prominent cell death revealed by Fluoro Jade B (FJB) staining appeared in the ganglion cell layer (GCL) and inner border of the inner nuclear layer (INL) at 1 day post-injection. The immunoreactivity of NR1 (NMDA receptor) and GluR2/3 (AMPA receptor) was decreased initially at the same time intervals but elevated at 7 days. However, the expression of GluR5/6/7 (KA receptor) and EAAT1 (excitatory amino acid transporters 1) were increased at 1 day after KA treatment. Double labeling study has revealed that the retinal Müller glial cells expressed concurrently GFAP and NR1 antigens (GFAP+/NR1+), GluR2/3 (GFAP+/GluR2/3+) or GluR5/6/7 (GFAP+/GluR5/6/7+) after 3 days KA challenge. Calbindin (CB) labelled ganglion cells and horizontal cells showed no significant changes throughout the excitotoxic lesion. On the contrary, parvalbumin (PV) immunoreactivity in ganglion cells was decreased. Remarkably, KA-induced neurotoxicity also resulted in the expression of CB or PV in cells scattered throughout the shrunk retina. Present results suggest that different iGluRs subunits probably play different roles in mediating kainate-induced excitotoxicity. That the retinal Müller glial cells and astrocytes were induced to express EAAT1 and GFAP/ iGluRs further indicates that they are linked to neuronal degeneration in the glutamatergic system in this KA-induced excitotoxicity. Second, an increased expression of the complement receptor type 3 (CR3), the major histocompatibility complex (MHC) class II and ED-1 antigens were mainly observed in the inner retina after kainate injection. Interestingly, some immunoreactive cells appeared in the outer segment of photoreceptor layer (OSPRL) at different time intervals. Our quantitative analysis further showed that CR3 immunoreactivity was drastically increased peaking at 7 days but subsided thereafter. MHC class II and ED-1 immunoreactivities showed a moderate but steady increase peaking at 3 days and declined thereafter. Double labeling study further revealed that retinal microglia/macrophages expressed concurrently CR3 and ED-1 antigens (OX-42+/ED-1+) or MHC class II molecules (OX-42+/OX-6+) and remained branched in shape at early stage of kainate challenge. By electron microscopy, microglia/macrophages with CR3 immunoreactivity displayed abundant cytoplasm containing a few vesicles and phagosomes. Other cells ultrastructurally similar to Müller cells or astrocytes could also engulf exogenous substances. Third, both the astrocytes and Müller glial cells reacted vigorously to kainate injection as shown by their up-regulated expression of nestin, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). A major finding was the induced expression of nestin together with GFAP beginning at 1 day post-injection of kainate. Western blotting analysis confirmed a marked increase in expression of nestin, GFAP and GS when compared to untreated/normal retina. Double labeling study has revealed that astrocytes and Müller glial cells expressed radial glia marker, nestin, and incorporated bromodeoxyuridine (BrdU) to re-enter into their cell cycle. The induced expression of these proteins in astrocytes and Müller glial cells indicated an induction of gliotic responses and de-differentiation that may be associated with regenerative efforts after kainate-induced injury. Additionally, induced expression of brain lipid-binding protein (BLBP) and doublecortin (DCX) in kainate-impacted astrocytes and Müller glial cells re-strengthened the potentiality of these cells to de-differentiate and reacquire an immature molecular profile in retinal neurodegeneration.