Optical measurement of brain oxygen tension during cerebral ischemia and reperfusion

脑内氧分压(PO_2)的测量在缺血性疾病的研究中具有十分重要的意义。到目前为止，大多数PO_2测量都是用氧微电极进行的。然而，微电极并不总是适合研究脑氧合作用的工具，因为电极插入脑内本身会导致出血，从而导致错误的PO2测量。此外，利用氧敏荧光膜技术和适当的修饰，微电极只测量脑表面的PO_2作为荧光显微图案。氧猝灭荧光染料三(1，10-菲咯啉)六水合Ru(II)被固定在直径3微米的硅胶小球中，染色的头部被嵌入在显微镜盖片上形成的高度透气(~20微米厚)的不透明硅橡胶膜中。与最初用于透明肠系膜组织的方法不同，…采用了不透明的硅橡胶将脑组织氧化还原状态对PO2光学测量的影响降至最低。用Pulsinelli‘s四血管阻断大鼠评价了该技术用于脑微循环研究的可行性。戊巴比妥钠麻醉(35 mg/kg)。雄性Wistar大鼠(300-350g，n=8)行双侧椎动脉电灼术。苏醒24小时后，在麻醉(15%乌拉坦，10ml/kg，S.C.)下于颅骨上开出两个颅窗。氧敏膜被放置在脑上，氧敏感侧通过一个颅窗与脑表面非常温和地接触。在人工通气下，阻断颈总动脉造成可逆性脑缺血，激光多普勒血流仪通过另一窗口监测。在缺血/再灌流期间，连续记录氧敏感膜上的荧光图案，并进行回放分析和转换为PO_2值。本方法使我们能够在不造成皮质损伤的情况下，显示大鼠脑表面的PO_2梯度。在脑缺血/再灌流模型中，脑表面PO2分布的异质性被检测到，特别是在动脉周围区域和其他区域之间，并且这种异质性随着缺血的减少而增加

The measurement of oxygen tension (PO_2) in the brain is quite important in the study of ischemic diseases. To date, most PO_2 measurements have been performed using oxygen microelectrodes. However, the microelectrode is not always a suitable tool for studying brain oxygenation because insertion of the electrode into the brain per se causes hemorrhage and hence leads to erroneous PO_2 measurement. Furthermore, microelectrodes only measure PO_2 in the brain surface as epifluorescent microscopic patterns, using the oxygen-sensitive fluorescent membrane technique with proper modificationsAn oxygen-quenching fluorescence dye, tris (1,10-phenanthroline) ruthenium (II) chloride hexahydrate, was immobilized in silica-gel beads 3 um in diameter and the dyed heads were embedded in a highly gas-permeable (〜20 um thick), opaque silicone-rubber film formed on a microscope coverslip. Unlike the original method, intended for transparent mesenteric tissue, he opaque silicone elastomer was adopted to … More minimize the effect of the redox state of the brain tissue on the optical measurement of PO_2. The feasibility of this technique for use in brain microcirculatory studies was assessed using Pulsinelli's four-vessel occlusion rat. Under pentobarbital anesthesia (35 mg/Kg. I.p.), male wistar rats (300-350g, n=8) underwent bilateral electrocauterization of the vertebral arteries. After 24 hours recovery, two cranial windows were made in the skull under anesthesia (15% urethane, 10 ml/Kg, s.c.) and the oxygen-sensitive membrane was placed on the brain, with the PO_2-sensing side in very gentle contactwith the brain surface through one cranial window. Under artificial ventilation, the animals were subjected to occlusion of the common carotid arteries, which resulted in reversible brain ischemia as monitored by a Laser-Doppler flowmeter through the other window. During the ischemia/reperfusion periods, the fluorescence pattern on the oxygen-sensitive membrane was video-recorded continuously and subjected to subsequent playback analysis and conversion to PO_2 valuesThe present method enabled us to visualize the PO_2 gradient on the rat brain surface without causing cortical injury. In ischemia/reperfusion protocol, heterogeneity in the PO_2 distribution on the brain surface was detected, in particular between the periarteiolar region and other regions, and this heterogeneity was observed to increase in association with ischemia Less