| 摘要: | 腦創傷是由外力撞擊所導致的腦組織損害，是造成慢性創傷腦病變的危險因子，會導致神經退化、行為以及認知功能缺失，在醫學研究上非常受到重視。創傷後的傷害機制有很多種，其中之一是膠細胞活化並分泌發炎性細胞激素，例如：Tumour Necrosis Factor-α (TNF-α)、Interleukin-6 (IL-6)、Interleukin-1β (IL-1β)，引起腦細胞發炎反應，造成腦創傷之續發性傷害的變嚴重與擴大。
Thalidomide (Thal) 是一種TNF-α抑制劑，可以降解TNF-α mRNA，進而抑制促發炎性的細胞激素產生，但是Thal具有強烈的致畸性副作用，Pomalidomide (Pom) 是Thal的類似物，在抑制TNF-α mRNA表現量上比Thal更有效力，且副作用較少，Pom已被美國FDA核准用以治療多發性骨髓瘤，但還未應用於腦傷研究，而另一種類似物3,6’-dithiothalidomide (3,6’-DT)則是在小鼠輕微腦創傷研究中，展現良好的治療效果，可以改善創傷所引起之行為缺失。因此本論文研究目的在於評估受傷後給予Pom或3,6’-DT對大鼠大腦組織、功能和發炎反應具有治療作用，以及使用藥物之時間窗。
本論文包括活體實驗及離體（細胞培養）實驗。活體實驗利用大鼠可控制皮質撞擊模式（CCI），在CCI後5或7小時給予Pom (0.5 mg/kg, i.v.)或3,6’-DT (28 mg/kg, i.p.)或vehicle，以神經行為測試：觸覺測試、擺動測試、平衡木步行測試和神經學損傷評估，包含運動、感覺，反射以及平衡測試，比較受傷前與受傷後24小時之行為功能；腦切片進行甲酚紫染色測量大腦受損體積以及Fluoro-Jade C染色測量受傷皮質區之退化性神經元數目；發炎性細胞激素TNF-α、IL-1β和IL-6在皮質損傷區之mRNA表現量和組織中濃度分別以反轉錄即時定量聚合酶連鎖反應和酵素免疫分析法分析；並以免疫螢光雙重染色觀察神經細胞凋亡與氧化傷害；在離體實驗中使用初代培養大鼠大腦皮質細胞，研究Pom在H2O2 (300μM)或麩胺酸 (100 mM)對腦細胞造成傷害模式中之治療效果。
我們發現腦創傷後5小時給予Pom或3,6’-DT 24小時後，可以顯著改善行為功能缺失，並且減少腦部創傷體積以及創傷區域周邊之退化神經細胞數目，也顯著減少皮質損傷區神經細胞凋亡，此外我們觀察到在腦創傷後24小時的效果比8小時顯著，代表受傷後5小時給予Pom或3,6’-DT能夠降低續發性傷害的擴大。除此之外兩種藥物在腦創傷中亦有降低氧化傷害的效果，減少被自由基攻擊而產生的脂質氧化產物4-Hydroxynonenal (4-HNE)與蛋白質氧化產物3-Nitrotyrosine (3-NT)。創傷後給予Pom與3,6’-DT在第8小時的受傷側皮質區，我們測量到由創傷所引起的發炎性細胞激素的基因表現與組織中的濃度都顯著下降。離體實驗中，Pom (50μM)在興奮性神經傳導物質-麩胺酸所引起的興奮性毒性及氧化性壓力，或是H2O2所導致的氧化傷害中，可減少神經細胞死亡，更能顯著降低TNF-α的釋放。
綜合以上結果，我們認為在腦創傷後給予Pom或3,6’-DT，可藉由多種機制阻斷細胞傷害訊息傳遞，如降低發炎反應與氧化傷害，達到減少神經細胞死亡之保護效果，此二種藥物將來可繼續研究以提供腦創傷一個有效的治療方式。
Traumatic brain injury (TBI) which is caused by external force to the head is a risk factor for chronic traumatic encephalopathy worldwide and resulted in neurondegenerative, behavioral and cognitive deficits. In response to acute brain trauma, glial cells become activated and secrete pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) to cause neuroinflammation which plays a crucial role in the secondary tissue damage. Thalidomide is a TNF-α inhibitor that enhances TNF-α mRNA degradation. However, it has teratogenic side effects. Its analog Pomalidomide (Pom) has been shown to be more potent as a TNF-α inhibitor and fewer side effects than Thalidomide. Pom has anti-tumor and anti-inflammatory effects and is used clinically for treatment of multiple myeloma. The other analog, 3,6’-dithiothalidomide (3,6’-DT), has show that it can reverses behavioral impairments induced by minimal TBI in mice. The aim of our study is to investigate the therapeutic effect and time window of Pom and 3,6’-DT on histological and functional outcomes and inflammatory responses after TBI. To evaluate whether these drugs have therapeutic effect in vivo, Sprague Dawley rats were subjected to controlled cortical impact (CCI). Pom (0.5mg/kg, i.v.) or 3,6’-DT (28mg/kg, i.p.) or vehicle was administered to rats at 5 hr or 7 hr after CCI. Neurobehavioral testing was performed before CCI and at 24 hr after CCI. The evaluation consisted of a tactile adhesive removal test, an elevated body swing test, a beam walk test and a modified neurological severity score (mNSS) assessment which is a composite of motor, sensory, reflex and balance tests. Degree of brain injury was evaluated by contusion volume and neuronal degeneration using cresyl violet and FluoroJade C staining. Levels of inflammatory cytokines: TNF-α, IL-1β and IL-6 mRNA in the cortical contusion region were measured by reverse transcription-qPCR. Tissue concentration of cytokine was measured by enzyme-linked immunosorbent assay. Neuronal apoptosis or damaged by ROS were indentified by double immunofluorescence staining. In vitro, primary rat cortical cultures were challenged with H2O2 (300μM) or glutamate (100mM) and post-treatment with various concentrations of Pom. We found that post-injury Pom or 3,6’-DT treatment at 5 hr, but not 7 hr, after TBI significantly improved functional recovery and decreased contusion volume at 24 hr post-injury. Pom treatment also significantly reduced the number of degenerating neurons at the contusion site. Treatment with Pom and 3,6’-DT reduced apoptotic neurons in the cortical contusion region at 24 hr after TBI. In addition, Pom and 3,6’-DT treatment decreased 4-Hydroxynonenal (4-HNE) and 3-Nitrotyrosine (3-NT) positive neurons in the cortical contusion region, implying that the drugs can protect neurons from oxidative stress. Post-treatment with Pom and 3,6’-DT at 5 hr after TBI significantly reduced injury-induced mRNA and protein expression of cytokines at 8 hr after TBI in the ipsilateral hemisphere cortex. In vitro, Pom (50 μM) enhanced neuronal survival against H2O2- induced oxidative stress and glutamate-induced cytotoxicity. Our data suggest that post-treatment with Pom and 3,6’-DT reduces neuroinflammatory responses and oxidative stress that improves histological and functional outcomes after TBI. Pom and 3,6’-DT have a potential to be developed as new therapies for TBI. |
