Sanderson Thomas H, Wider Joseph M
Department of Emergency Medicine, Wayne State University School of Medicine.
J Vis Exp. 2013 Jun 22(76):50173. doi: 10.3791/50173.
Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia (1). Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion (2). The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) (2), as first presented in its current form in Sanderson, et al. (2008) (3), which produces reproducible injury to selectively vulnerable brain regions (3-6). The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time. In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.
心脏骤停后继以复苏常常会导致由脑缺血及随后的脑再灌注引起的严重脑损伤。全脑缺血会对特定的脑区造成损伤，这些脑区对缺血高度敏感(1)。与其他细胞群体相比，海马神经元对缺血性损伤更为敏感，具体而言，海马的CA1区对缺血/再灌注尤为脆弱(2)。治疗干预措施的设计或对脑损伤相关机制的研究需要一个能产生与临床情况相似且可重复的损伤的模型。双侧颈动脉血管闭塞合并低血压(2VOH)是一种能产生可逆性前脑缺血的模型，模拟了心脏骤停和复苏过程中可能发生的脑部事件。我们描述了一种对Smith等人(1984年)(2)的模型进行改良的模型，该模型最初以当前形式由Sanderson等人(2008年)(3)提出，能对选择性易损脑区产生可重复的损伤(3 - 6)。该模型的可靠性取决于在低血压期间对全身血压的精确控制、缺血持续时间、严格的温度控制、特定的麻醉方案以及精心的术后护理。8分钟的缺血性损伤会导致CA1海马神经元在再灌注6至24小时的过程中发生细胞死亡，而较不易受损的脑区则得以幸免。这种进行性细胞死亡在再灌注7 - 14天后很容易量化，因为此时CA1神经元几乎完全丧失是显而易见的。除了这种脑损伤模型，我们还介绍了一种使用简单但全面的方法对CA1损伤进行量化的方法。重要的是，使用一个简单的带摄像头显微镜和免费的ImageJ(美国国立卫生研究院)软件插件就可以完成量化，无需使用成本高昂的体视学软件程序和用于损伤评估的电动显微镜载物台。
