Global ischemia is the resulting effect of a cardiopulmonary arrest (CPA). Presently there is no effective treatment to address neurological deficits in patients who survived a CPA. Granulocyte-colony stimulating factor is a growth factor (G-CSF) with a plethora of beneficial effects, including neuroprotection. Clinical application of human G-CSF (hG-CSF) is limited due to its plasma half-life of 4 h. Therefore, novel approaches need to be investigated that would (1) enable prolonged manifestation of hG-CSF and (2) demonstrate G-CSF efficacy from studying the underlying protective mechanisms of hG-CSF. In our previous work, we used the self-complementary adeno-associated virus (stereotype2: scAAV2) as a vector to transfect the hG-CSF gene into the global ischemic brain of a mouse. As an extension of that work, we now seek to elucidate the protective mechanisms of hG-CSF gene therapy against endoplasmic reticulum induced stress, mitochondrial dynamics and autophagy in global ischemia. A single drop of either AAV-CMV-hG-CSF or AAV-CMV-GFP was dropped into the conjunctival sac of the Swiss Webster mouse’s left eye, 30–60 min after bilateral common artery occlusion (BCAO). The efficacy of the expressed hG-CSF gene product was analyzed by monitoring the expression levels of endoplasmic reticulum stress (ER), mitochondrial dynamics and autophagic proteins over 4- and 7-days post-BCAO in vulnerable brain regions including the striatum, overlying cortex (frontal brain regions) and the hippocampus (middle brain regions). Statistical analysis was performed using mostly One-Way Analysis of variance (ANOVA), except for behavioral analysis, which used Repeated Measures Two-Way ANOVA, post hoc analysis was performed using the Tukey test. Several biomarkers that facilitated cellular death, including CHOP and GRP78 (ER stress) DRP1 (mitochondrial dynamics) and Beclin 1, p62 and LC3-ll (autophagy) were significantly downregulated by hG-CSF gene transfer. hG-CSF gene therapy also significantly upregulated antiapoptotic Bcl2 while downregulating pro-apoptotic Bax. The beneficial effects of hG-CSF gene therapy resulted in an overall improvement in functional behavior. Taken together, this study has substantiated the approach of sustaining the protein expression of hG-CSF by eye drop administration of the hG-CSF gene. In addition, the study has validated the efficacy of using hG-CSF gene therapy against endoplasmic reticulum induced stress, mitochondrial dynamics and autophagy in global ischemia.

中文翻译：

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粒细胞集落刺激因子基因疗法作为小鼠模型中风的新疗法

整体缺血是心肺骤停（CPA）的结果。目前，尚无有效的方法来解决CPA幸存者的神经功能缺损。粒细胞集落刺激因子是一种生长因子（G-CSF），具有多种有益作用，包括神经保护作用。人类G-CSF（hG-CSF）的临床应用因其血浆半衰期为4小时而受到限制。因此，需要研究新颖的方法，该方法将（1）能够延长hG-CSF的表现，以及（2）通过研究hG-CSF的潜在保护机制来证明G-CSF的功效。在我们以前的工作中，我们使用自我互补腺伴随病毒（stereotype2：scAAV2）作为载体将hG-CSF基因转染到小鼠的整体缺血性脑中。作为这项工作的延伸，我们现在寻求阐明hG-CSF基因疗法对内质网诱导的应激，线粒体动力学和自噬在全球缺血中的保护机制。在双侧常见动脉闭塞30-60分钟后，将一滴AAV-CMV-hG-CSF或AAV-CMV-GFP滴入Swiss Webster小鼠左眼的结膜囊中。通过监测BCAO后4天和7天，在包括纹状体，上皮层在内的脆弱大脑区域内质网应激（ER），线粒体动力学和自噬蛋白的表达水平，分析了表达的hG-CSF基因产物的功效。 （额叶脑区）和海马区（中脑区）。除行为分析外，大多数使用单向方差分析（ANOVA）进行统计分析，使用重复测量双向方差分析，使用Tukey测试进行事后分析。hG-CSF基因转移显着下调了一些促进细胞死亡的生物标志物，包括CHOP和GRP78（ER应激）DRP1（线粒体动力学）和Beclin 1，p62和LC3-II（自噬）。hG-CSF基因治疗还显着上调抗凋亡Bcl2，同时下调促凋亡Bax。hG-CSF基因治疗的有益作用导致功能行为的全面改善。两者合计，这项研究证实了通过滴眼液hG-CSF基因维持hG-CSF蛋白表达的方法。此外，这项研究已验证了使用hG-CSF基因疗法抗内质网诱导的应激，