腦脊髓液與培養基成份對神經幹細胞/前驅細胞的影響與中風功能性恢復之研究

Abstract

過去文獻指出，動物體內之內源性神經幹細胞會分化成新生神經元，將有助於中風後的功能性恢復；然而，並無相關文獻證實神經新生與患者的癒後之間的關聯性。因此本研究收集了36位蜘蛛網膜下腔出血患者的腦脊髓液，並將收集到的腦脊髓液與大鼠的神經幹細胞共同培養，觀察對神經幹細胞增生之影響(增生指數[PI]為有表現Ki-67的細胞比例)。後續使用失能評估量表(mRS)評估蜘蛛網膜下腔出血患者三個月後的癒後情形，並找出癒後結果與PI值之相關性。實驗結果顯示，相對於常壓性水腦症患者或是並未加入腦脊髓液的組別，由蜘蛛網膜下腔出血患者收集到的腦脊髓液表現了較高的增生比例(8.2±5.1 與 7.8±3.0 vs. 20.3±8.8, P<0.001)，得知促進神經幹細胞增生的因子確實存在於中風後的腦脊髓液中，且PI值與蜘蛛網膜下腔出血患者的腦出血量呈現正相關 (p=0.025)。接著依出血體積將蜘蛛網膜下腔出血患者分兩群，分別為低出血量與高出血量，可發現於低出血量且PI值較高的組別患者癒後較良好 (20.8±6.9 vs. 14.6±4.3, p=0.047)。而使用多變量邏輯回歸分析，發現PI值是具有良好癒後的決定性因素 (優勢比例, 1.17; 95% 信賴區間, 1.00 到 1.36)，當腦脊髓液中具有越多的促進細胞增生的因子，有助於蜘蛛網膜下腔出血患者癒後之功能性恢復。由上述結果可知，若提高內生性神經幹細胞大量增生與爬行，將有助於中風後的癒後功能性之恢復。在過去我們實驗研究中證實培養基B (表皮生長因子與纖維連接蛋白)有助於神經幹/前驅細胞(NSPC)增生與遷移。而本研究實驗結果顯示，在三維結構下培養基B於幾丁聚醣與去細胞後的腦支架上也具有促進神經幹細胞增生與遷移之效果。以體外中風模型試驗，將大鼠皮質神經元培養於氧氣與葡萄糖缺少(OGD)的環境下，發現與神經幹/前驅細胞共培養且於培養基B的環境下可以增加神經元的活性並減少細胞凋亡；在動物實驗，本研究使用短暫性大腦中動脈閉塞(MCAO)的大鼠模型，並於動物誘發中風後注射培養基B於腦室內，結果顯示培養基B增強了神經幹細胞增生、遷移與神經元分化並且減少梗塞腦中的細胞凋亡，進一步深入研究相關機制得知，使用培養基 B 治療後均可增加Bcl-xL、Bcl-2、phospho-Akt、phospho-GSK-3β與β-catenin這些蛋白質的表現量，並且降低cleaved caspase-3蛋白質表現量，顯示培養基B在抗細胞凋亡扮演決定性的腳色。另外值得注意，注射培養基B於腦室內可增強神經元的細胞數，改善了運動功能並且減少了梗塞面積，因此可證實以培養基B治療後，不管在細胞實驗或是動物實驗上，均具有促進神經細胞的新生並減少了中風後細胞凋亡且使得癒後的功能性表現變好。

Neurogenesis from endogenous neural stem/precursor cell (NSPCs) might contribute to functional recovery after stroke based on animal studies; however, the relationship between neurogenesis and post-stroke outcome has rarely been demonstrated in humans. We prospectively collected cerebrospinal fluid (CSF) from 36 patients with subarachnoid hemorrhage (SAH). The CSF was added to the culture medium of the rat NSPCs to test the effects on proliferation (proliferation index [PI], percentage of Ki-67 immunoreactive cells). We correlated the PI with functional outcome based on the modified Rankin Scale at 3 months post-SAH. Treatment with the CSF samples collected from SAH patients showed a higher PI compared with those collected from patients with normal pressure hydrocephalus and untreated controls (20.3±8.8 vs. 8.2±5.1 and 7.8±3.0, P<0.001), indicating proliferation-promoting factors in CSF after SAH. The PI was positively correlated with SAH volume (p=0.025). For patients with lower SAH volume, patients with favorable outcome had a higher PI than those with poor outcome (20.8±6.9 vs. 14.6±4.3, p=0.047). Using multivariable logistic regression analysis, the PI was a positive determinant for favorable outcome (odds ratio, 1.17; 95% confidence interval, 1.00 to 1.36) that more proliferation-promoting factors in CSF was associated with better functional outcome in SAH patients. Enhancement of endogenous neurogenesis after ischemic stroke may improve functional recovery. We have previously demonstrated that medium B, which is a combination with epidermal growth factor and fibronectin, is able to promote neural stem/precursor cell (NSPC) proliferation and migration. Here, we showed that medium B promoted proliferation and migration of cultured NSPCs on the chitosan scaffold and decellularized brain structure. When rat cortical neurons with oxygen glucose deprivation (OGD) was co-cultured with NSPCs, medium B treatment increased neuronal viability and reduced cell apoptosis. In a rat model with transient middle cerebral artery occlusion (MCAO), post-insult intraventricular medium B treatment enhanced proliferation, migration, and neuronal differentiation of NSPCs and diminished cell apoptosis in the infarct brain. In cultured post-OGD neuronal cells and the infarct brain from MCAO rats, medium B treatment increased protein levels of Bcl-xL, Bcl-2, phospho-Akt, phospho-GSK-3β, and β-catenin and decreased levels of cleaved caspase-3, which may be associated with the effects of anti-apoptosis. Notably, intraventricular medium B treatment increased neuronal density, improved motor function and reduced infarct size of MCAO rats. In summary, medium B treatment promotes neurogenesis and reduces apoptosis, leading to less neuronal death and better functional outcome in both cellular and rodent models of ischemic stroke.