背景:单纯疱疹病毒(HSV)脑炎(HSE)是一种严重且可能危及生命的疾病,影响成人和新生儿。目前的大脑模型的局限性阻碍了对新生儿HSE涉及的病毒和宿主因素的理解,这些模型不能完全概括健康和疾病中发育中的人脑的组织结构和细胞组成。这里,我们开发了人胎儿器官型脑切片培养(hfOBSC)模型,并确定了其在体外模拟HSE神经病理学中的价值。
方法:通过上清液中的乳酸脱氢酶释放和免疫组织学(IHC)分析确定细胞活力和组织完整性。用表达HSV-1和HSV-2的绿色荧光蛋白(GFP-)感染脑切片。通过共聚焦显微镜确定病毒的复制和传播,PCR和病毒培养。通过PCR检测促炎细胞因子和趋化因子的表达。通过IHC分析确定细胞嗜性和HSV诱导的神经病理学。最后,将HSV感染的hfOBSC的原位数据与人类HSE脑切片中检测到的神经病理学进行比较.
结果:优化切片和无血清培养条件以在37°C下在CO2培养箱中维持离体人胎儿脑切片的活力和组织结构至少14天。hfOBSC支持生产性HSV-1和HSV-2感染,主要涉及神经元和星形胶质细胞的感染,导致促炎细胞因子和趋化因子的表达。两种病毒在感染后的较晚时间点在感染的脑切片中诱导程序性细胞死亡-尤其是坏死。病毒传播,细胞嗜性和程序性细胞死亡在HSV诱导的细胞死亡中的作用类似于HSE的神经病理学。
结论:我们开发了一种新型的人脑培养模型,其中包括神经元在内的主要大脑驻留细胞的活力,小胶质细胞,星形胶质细胞和少突胶质细胞-并且在无血清培养条件下在体外维持组织结构至少2周。细胞嗜性非常相似,HSV-1和HSV-2在具有人类HSE病例的神经病理学特征的hfOBSC模型中的传播和神经毒力强调了其在详述其他嗜神经病毒的病理生理学方面的潜力,以及作为测试新型治疗干预措施的临床前模型的潜力.
BACKGROUND: Herpes simplex virus (HSV) encephalitis (HSE) is a serious and potentially life-threatening disease, affecting both adults and newborns. Progress in understanding the virus and host factors involved in neonatal HSE has been hampered by the limitations of current brain models that do not fully recapitulate the tissue structure and cell composition of the developing human brain in health and disease. Here, we developed a human fetal organotypic brain slice culture (hfOBSC) model and determined its value in mimicking the HSE neuropathology in vitro.
METHODS: Cell viability and tissues integrity were determined by lactate dehydrogenase release in supernatant and immunohistological (IHC) analyses. Brain slices were infected with green fluorescent protein (GFP-) expressing HSV-1 and HSV-2. Virus replication and spread were determined by confocal microscopy, PCR and virus culture. Expression of pro-inflammatory cytokines and chemokines were detected by PCR. Cell tropism and HSV-induced neuropathology were determined by IHC analysis. Finally, the in situ data of HSV-infected hfOBSC were compared to the neuropathology detected in human HSE brain sections.
RESULTS: Slicing and serum-free culture conditions were optimized to maintain the viability and tissue architecture of ex vivo human fetal brain slices for at least 14 days at 37 °C in a CO2 incubator. The hfOBSC supported productive HSV-1 and HSV-2 infection, involving predominantly infection of neurons and astrocytes, leading to expression of pro-inflammatory cytokines and chemokines. Both viruses induced programmed cell death-especially necroptosis-in infected brain slices at later time points after infection. The virus spread, cell tropism and role of programmed cell death in HSV-induced cell death resembled the neuropathology of HSE.
CONCLUSIONS: We developed a novel human brain culture model in which the viability of the major brain-resident cells-including neurons, microglia, astrocytes and oligodendrocytes-and the tissue architecture is maintained for at least 2 weeks in vitro under serum-free culture conditions. The close resemblance of cell tropism, spread and neurovirulence of HSV-1 and HSV-2 in the hfOBSC model with the neuropathological features of human HSE cases underscores its potential to detail the pathophysiology of other neurotropic viruses and as preclinical model to test novel therapeutic interventions.