Therapeutic potential of astrocytes via in vivo expression of reprogramming factor in mouse models of cerebral ischemia

Abstract

Cerebral ischemia and stroke can lead incurable brain damage or death. Astrocytes, one of the most abundant cells in the brain, are activated by injury such as ischemia. Reactive astrocytes, in particular, play a crucial role in recovery from brain injury. Four reprogramming factors (Pou5f1, Sox2, Myc, and Klf4) expression have been used to convert cell types, but it has rarely been studied promoting functional recovery by expression of reprogramming factors in ischemic injury. The aim of this study is therefore to determine whether in vivo transient expression of reprogramming factor can improve neurobehavioral function. Cerebral ischemia was induced by two methods—transient bilateral common carotid artery occlusion (BCCAO) and permanent unilateral common carotid artery ligation with hypoxia (8% O2)—after which brains were treated (in the lateral ventricle or striatum) with doxycycline (DOX) in transgenic mice in which the four reprogramming factors were expressed by doxycycline. Either doxycycline (DOX-1; 1 μg/ml, DOX-100; 100 μg/ml) or phosphate-buffered saline (PBS) was then infused into the brain via an osmotic pump for 7 days. In the BCCAO model, histologic evaluation showed that this transient expression of reprogramming factor induced the proliferation of astrocytes and neural progenitors, while neurons and glial scar formation was not observed. Furthermore, in vivo expression of reprogramming factor caused neuroprotective effects and angiogenesis in the striatum. Tumor formation was not observed in any group. Importantly, the rotarod and ladder walking tests showed to promote functional restoration from ischemic damage via the expression of the four reprogramming factors. To elucidate the therapeutic mechanisms associated with astrogliosis, RNA sequencing analysis was performed in order to identify a transcriptome that was significantly changed in the DOX-100 group compared to PBS group. Among downregulated genes (complement C3, C4a, C4b, C1qa, C1qb and C1qc) and astrocyte markers (GFAP, Vimentin and S100β) were validated using qRT-PCR. Because C3 is a detrimental astrocyte marker, C3 in particular was measured using histologic analysis. The results showed that C3 was significantly reduced in DOX-100 treated mice compared with the DOX-1 and PBS groups. A hypoxic-ischemic (HI) model, a stroke model used in another experimental group, was used to compare efficacy of reprogrammed expression in two areas of the brain, the ventricle and striatum. That results showed that the astrocytes and neural progenitors were significantly proliferated, but not neurons or glial scar, and the condition of blood vessels in the injured brain was improved following in vivo reprogramming factor expression. Furthermore, in vivo reprogramming factor expression was protective of neurons under hypoxic ischemic conditions. Notably, neurobehavioral evaluations such as the grip strength, cylinder, ladder walking and open field tests showed functional recovery was dramatically improved via the expression of the four reprogramming factors in the lateral ventricle. Interestingly, the expression of the reprogramming factors in the striatum did not lead to changes in neurological function in any group. In the rotarod test, there were no significant differences in both the lateral ventricular-targeted and striatum-targeted group. Tumor development was not observed in the lateral ventricle-targeted group, but the striatum-targeted group showed that abnormal cell proliferation in DOX-100 treated mice brain. Furthermore, treatment with DOX-1,000 (doxycycline; 1,000 μg/ml) lead to tumor formation in the striatum-targeted group. Taken together, newly generated astrocytes are essential for protecting neurons from damage following cerebral ischemia (BCCAO and HI mouse model) and for enhancing blood vessels. These results show that a therapeutic potential of methods that aim to improve functional recovery by reducing reactive astrocytes (harmful A1 astrocytes) in BCCAO mouse models. In the HI mouse model, we noted a recovery effect in the group in which the lateral ventricle was targeted with the expression of reprogramming factors, but not in the striatum-targeted group. Therefore, targeting the lateral ventricle for expression of reprogramming factors may offer better therapeutic results.