Broadwell R D, Charlton H M, Balin B J, Salcman M
J Comp Neurol. 1987 Jun 1;260(1):47-62. doi: 10.1002/cne.902600105.
Blood vessels of the fetal, neonatal, and adult subprimate and primate CNS, including circumventricular organs (e.g., median eminence, pituitary gland, etc.), and of solid CNS and nonneural (anterior pituitary gland) allografts placed within brains of adult mammalian hosts were visualized with peroxidase cytochemistry applied in three ways: to tissues from animals injected systemically with native horseradish peroxidase (HRP) or peroxidase conjugated to the lectin wheat germ agglutinin (WGA) prior to perfusion fixation; to tissues from animals infused with native HRP into the aorta subsequent to perfusion fixation; and to tissues from animals fixed by immersion and incubated for endogenous peroxidase activity in red cells retained within blood vessels. In neonatal and adult animals receiving native HRP intravascularly, non-fenestrated vessels contributing to a blood-brain barrier were outlined with HRP reaction product when tetramethylbenzidine (TMB) as opposed to diaminobenzidine (DAB) was used as the chromogen; fenestrated vessels of circumventricular organs were not discernible due to the density of extravascular reaction product. Fenestrated and non-fenestrated cerebral and extracerebral blood vessels exposed to bloodborne WGA-HRP were visible when incubated in TMB and DAB solutions. Native HRP infused into the aorta of fixed animals likewise labeled non- fenestrated vessels throughout the brain upon exposure to TMB or DAB but obscured fenestrated vessels of the circumventricular organs. Endogenous peroxidase activity of red cells, seen equally well with TMB and DAB, outlined blood vessels throughout the cerebral gray and white matter and all circumventricular organs in fetal, neonatal, and adult animals. Application of the three peroxidase cytochemical approaches to study the development or absence of a blood-brain barrier in intracerebral allografts demonstrated that the vascularization of day 16-19 fetal/1 day neonatal CNS allografts is not well defined prior to 7 days following intracerebral placement of the grafts. CNS allografts secured from donor sites expected to possess a blood-brain barrier exhibited blood vessels that were not leaky to HRP injected intravenously in the host. Fenestrated blood vessels associated with anterior pituitary allografts were evident prior to 3 days posttransplantation within the host brain and permitted blood-borne HRP in the host to enter the graft and surrounding host brain parenchyma.
采用三种方法应用过氧化物酶细胞化学技术，对胎儿、新生儿以及成年灵长类和非灵长类动物中枢神经系统（CNS）的血管进行可视化观察，这些血管包括室周器官（如正中隆起、垂体等），以及植入成年哺乳动物宿主脑内的实体CNS和非神经（垂体前叶）同种异体移植物的血管：对在灌注固定前经全身注射天然辣根过氧化物酶（HRP）或与凝集素麦胚凝集素（WGA）偶联的过氧化物酶的动物的组织进行观察；对在灌注固定后经主动脉注入天然HRP的动物的组织进行观察；对经浸入固定并针对血管内留存红细胞的内源性过氧化物酶活性进行孵育的动物的组织进行观察。在新生和成年动物血管内注射天然HRP后，当使用四甲基联苯胺（TMB）而非二氨基联苯胺（DAB）作为显色剂时，构成血脑屏障的非窗孔血管会被HRP反应产物勾勒出来；由于血管外反应产物的密度，室周器官的窗孔血管无法辨别。当在TMB和DAB溶液中孵育时，暴露于血源WGA-HRP的脑和脑外窗孔血管及非窗孔血管可见。在固定动物的主动脉中注入天然HRP，在暴露于TMB或DAB时同样会标记全脑的非窗孔血管，但会掩盖室周器官的窗孔血管。红细胞的内源性过氧化物酶活性在TMB和DAB下观察效果相同，勾勒出胎儿、新生儿和成年动物脑灰质和白质以及所有室周器官中的血管。应用这三种过氧化物酶细胞化学方法研究脑内同种异体移植物血脑屏障的发育或缺失情况表明，在将第16 - 19天胎儿/ 1天新生儿CNS同种异体移植物脑内植入后7天之前，其血管化情况尚不明确。从预期具有血脑屏障的供体部位获取的CNS同种异体移植物，其血管对宿主静脉注射的HRP不渗漏。与垂体前叶同种异体移植物相关的窗孔血管在移植后3天内在宿主脑内明显可见，并允许宿主血源HRP进入移植物及周围宿主脑实质。
