Brain stem lesion size determined by DEAD red or conjugation of neurotoxin to fluorescent beads.
Brain stem lesion size determined by DEAD red or conjugation of neurotoxin to fluorescent beads.
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通过 DEAD red 或神经毒素与荧光珠的结合确定脑干病变大小。
DOI：
10.1152/jappl.1998.85.6.2370
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Li,A
中科院分区：
文献类型：
--
作者：
Nattie,EE;Erlichman,JS;Li,A
将神经毒素显微注射到麻醉大鼠的梯形后核中可降低膈活动并消除对 CO2 的反应。在未麻醉的大鼠中，这种治疗对清醒、静息呼吸没有影响，并且使 CO2 敏感性降低 40%（M. Akilesh、M. Kamper、A. Li 和 E. E. Nattie.J. Appl. Physiol.82: 469–479, 1997）。解释这些不同结果的一个重要因素是解剖病变的实际大小。在本研究中，我们将鹅膏蕈酸注射到麻醉大鼠的梯形后核中，并使用两种新方法评估病变大小：1）DEAD red，一种荧光探针，通过渗漏膜进入受损细胞并与核酸结合，2）将毒素与荧光珠结合。使用 DEAD red 后，包含标记死亡细胞的区域为 313 ± 104 nl (n= 4)，比初始注射体积大六倍，并且对膈振幅、CO2 反应和血压的生理影响在几分钟内就开始出现，而且是显着的。理论上，对于结合毒素，神经元损伤将仅限于可检测到的荧光区域（49 ± 10 nl；n= 4）。对膈振幅、CO2 敏感性和血压的影响直到注射后约 2 小时才出现。对照实验，在体外孵育神经毒素-微珠缀合物 2 小时并在离心后注射上清液，显示出类似的结果，表明缀合的神经毒素的释放。我们得出的结论是，DEAD red 提供了一种在体内急性研究中监测神经元损伤的有用方法。在这方面，神经毒素与微珠的结合可能不太可靠。
Neurotoxin microinjected into the retrotrapezoid nucleus of anesthetized rats decreases phrenic activity and eliminates the response to CO2. In unanesthetized rats, such treatment has no effect on awake, resting breathing and decreases CO2sensitivity by 40% (M. Akilesh, M. Kamper, A. Li, and E. E. Nattie.J. Appl. Physiol.82: 469–479, 1997). One important factor in explaining these disparate results is the actual size of the anatomic lesion. In the present study, we injected ibotenic acid into the retrotrapezoid nucleus of anesthetized rats and evaluated lesion size by using two new approaches:1) DEAD red, a fluorescent probe that enters impaired cells through leaky membranes and binds to nucleic acids, and2) conjugation of toxin to fluorescent beads. With the use of DEAD red, the region containing labeled dying cells was 313 ± 104 nl (n= 4), six times larger than the initial injected volume, and the physiological effects on phrenic amplitude, the CO2response, and blood pressure began within minutes and were substantial. With conjugated toxin, in theory, neuronal damage would be limited to the region of detectable fluorescence (49 ± 10 nl;n= 4). Effects on phrenic amplitude, CO2sensitivity, and blood pressure were absent until ∼2 h postinjection. Control experiments, with 2 h of in vitro incubation of the neurotoxin-microbead conjugate and injection of the supernatant after centrifugation, showed similar results that suggest release of conjugated neurotoxin. We conclude that DEAD red provides a useful means to monitor neuronal impairment in acute studies in vivo. Conjugation of neurotoxin to microbeads may be less reliable in this regard.