Ceccatelli S, Hökfelt T, Hallman H, Nylander I, Terenius L, Elde R, Brownstein M
Peptides. 1987 Mar-Apr;8(2):371-84. doi: 10.1016/0196-9781(87)90114-8.
The brain and spinal cord of untreated and cysteamine-treated rats were analyzed with immunohistochemistry using antisera raised against somatostatin (SOM)-28(1-14) and SOM-28(15-28). Sections incubated with increasing dilutions of antiserum were evaluated subjectively on coded slides and with computer-assisted image analysis. For control experiments, antisera raised against methionine-enkephalin, neuropeptide Y (NPY) and dynorphin (DYN)(1-13) were used. The latter antiserum does not visualize the conventional DYN systems in the brain, but reacts with an unknown epitope, which here could be shown to be present in SOM neurons. In cysteamine-treated rats a marked decrease in SOM-28(15-28)-like immunoreactivity (1.1) could be recorded subjectively at all antibody concentrations in fibers in several brain areas, including nucleus accumbens, tuberculum olfactorium and the hypothalamic ventromedial and arcuate nuclei. In these areas SOM-LI is fairly weak in untreated rats. In SOM-rich regions such as the median eminence and the dorsal horn of the spinal cord, the depleting effect of cysteamine could be recorded subjectively only when diluted antisera were used. Image analysis confirmed the subjective analysis, and, in addition, differences between controls and cysteamine-treated rats could be shown also at high antiserum concentrations. SOM-28(15-28)-immunoreactive cell bodies could be seen in the brains of either control or drug-treated rats. No effect of cysteamine could be observed when antiserum raised to SOM-28(1-14) was used. Cysteamine did not seem to affect enkephalin-LI, NPY-LI or an epitope in SOM neurons reacting with DYN(1-13) antiserum. After preabsorption of SOM-28(15-28) antiserum with SOM-28(15-28) peptide, the staining patterns described above disappeared completely. However, if the SOM-28(15-28) peptide was pretreated with a high concentration (1 M) of cysteamine before being used for absorption with SOM antiserum, no blocking effect could be observed. The present results demonstrate with immunohistochemistry that cysteamine causes depletion of SOM-28(15-28) in fibers but apparently not in cell bodies. No effects on SOM-28(1-14)-LI were observed. This supports earlier evidence that cysteamine interacts with the disulphide bond in the SOM-28(15-28) molecule. The present results also emphasize that when analyzing drug effects on peptide neurons with immunohistochemical techniques, it is important to use dilution series of antibodies and preferably to carry out the analysis with objective image analysis methods.
使用针对生长抑素（SOM）-28（1-14）和SOM-28（15-28）产生的抗血清，通过免疫组织化学方法分析未处理和半胱胺处理大鼠的脑和脊髓。用递增稀释度的抗血清孵育的切片在编码玻片上进行主观评估，并使用计算机辅助图像分析。作为对照实验，使用针对甲硫氨酸脑啡肽、神经肽Y（NPY）和强啡肽（DYN）（1-13）产生的抗血清。后一种抗血清不能使脑中的传统DYN系统显色，但与一个未知表位反应，在此处可以证明该表位存在于SOM神经元中。在半胱胺处理的大鼠中，在几个脑区，包括伏隔核、嗅结节以及下丘脑腹内侧核和弓状核的纤维中，在所有抗体浓度下主观上都可记录到SOM-28（15-28）样免疫反应性（LI）明显降低。在这些区域，未处理大鼠的SOM-LI相当弱。在富含SOM的区域，如正中隆起和脊髓背角，仅当使用稀释的抗血清时才能主观记录到半胱胺的耗竭作用。图像分析证实了主观分析，此外，在高抗血清浓度下也可显示对照大鼠和半胱胺处理大鼠之间的差异。在对照或药物处理大鼠的脑中均可看到SOM-28（15-28）免疫反应性细胞体。当使用针对SOM-28（1-14）产生的抗血清时，未观察到半胱胺的作用。半胱胺似乎不影响脑啡肽-LI、NPY-LI或SOM神经元中与DYN（1-13）抗血清反应的表位。用SOM-28（15-28）肽预吸收SOM-28（15-28）抗血清后，上述染色模式完全消失。然而，如果SOM-28（15-28）肽在用于与SOM抗血清吸收之前用高浓度（1M）的半胱胺预处理，则未观察到阻断作用。本研究结果通过免疫组织化学证明，半胱胺导致纤维中SOM-28（15-28）耗竭，但显然不影响细胞体。未观察到对SOM-28（1-14）-LI的影响。这支持了早期的证据，即半胱胺与SOM-28（15-28）分子中的二硫键相互作用。本研究结果还强调，当用免疫组织化学技术分析药物对肽能神经元的作用时，使用抗体稀释系列并最好采用客观图像分析方法进行分析很重要。
