Fibrinogen triggers intial perivascular fibroblast activation in a mouse model of cortical ischemic stroke

Perivacular fibroblasts (PVFs) in the perivascular space delaminate from blood vessels, expand, express and deposit an excess of collagen I (Col I) and become an integral component of the fibrotic scar after central nervous system (CNS) disease. Here, we show that blood-derived fibrinogen deposition in the perivascular space initially induces PVF activation after photothrombosis, a mouse model of ischemic stroke. Pharmacological fibrinogen depletion reduces PVF activation and their delamination from blood vessels to build up the fibrotic scar. Fibrinogen induces beta1 integrin signaling in PVF to induce Col I expression and secretion. Single-cell RNA sequencing (scRNA-Seq) and genetic approches revealed a contribution of fibrinogen-induced perivascular macrophages to PVF activation. Finally, fibrinogen depletion abrogates PVF-astrocyte signaling and astrocyte lesion border formation, promoting neuronal survival and plasticity. Therefore, we propose that fibrinogen is a critical trigger for PVF activation and fibrotic scar formation, inhibiting neuronal regeneration after stroke. Overall design: C57Bl/6J mice were implanted with subcutaneous pumps filled with saline or Ancrod (2.4 U) 2 days before receiving photothrombotic stroke using rose bengal and a cold light illuminator over 4-mm diameter of the right sensorimotor cortex. After 6 days, the lesion and 1-mm of surrounding spared tissue (not including the corpus callosum) was microdissected and dissociated. No FACS or beads were used to filter the cells or to enrich for a specific cell type.

血管周成纤维细胞（Perivascular fibroblasts, PVFs）定位于血管周间隙，在中枢神经系统（CNS）疾病发生后，可从血管壁脱离、增殖活化，表达并沉积过量I型胶原蛋白（Collagen I, Col I），最终成为纤维化瘢痕的核心组成部分。本研究证实，在光血栓性脑卒中（缺血性脑卒中小鼠模型）造模后，血管周间隙内血液来源的纤维蛋白原沉积可初始诱导PVF活化。药物性纤维蛋白原耗竭可降低PVF活化程度，并减少其从血管壁脱离以形成纤维化瘢痕的过程。纤维蛋白原可在PVF中激活β1整合素信号通路，进而诱导Col I的表达与分泌。单细胞RNA测序（scRNA-Seq）与遗传实验手段揭示，纤维蛋白原诱导的血管周巨噬细胞同样参与了PVF的活化过程。最终，纤维蛋白原耗竭可阻断PVF-星形胶质细胞信号通路与星形胶质细胞病变边界的形成，从而促进神经元存活与突触可塑性。据此，本研究提出：纤维蛋白原是PVF活化与纤维化瘢痕形成的关键触发因子，可抑制脑卒中后的神经元再生。实验整体设计：于通过孟加拉玫瑰红与冷光源照射右侧体感皮层4mm直径区域构建光血栓性脑卒中模型的2天前，向C57Bl/6J小鼠皮下植入填充生理盐水或Ancrod（2.4 U）的皮下缓释泵。造模6天后，对梗死病灶及周围1mm范围的存活组织（不包含胼胝体）进行显微解剖与细胞解离。本研究未使用荧光激活细胞分选（FACS）或磁珠进行细胞筛选，亦未对特定细胞类型进行富集。