以免疫組織化學染色法及原位雜合試驗偵測促腎上腺皮質激素釋放激素對評估貓隻瀕死壓力之應用

Abstract

在法醫學中，動物在死前承受痛苦的程度是一項重要的評估因素。死亡的過程可能持續一段時間，稱為瀕死期（agonal phase），而瀕死期的持續時間可能反映動物受苦的程度。儘管有少數研究探討了能夠評估瀕死壓力的免疫組織化學（immunohistochemistry, IHC）標記，但在獸醫法醫學中仍缺乏一個有效的標記物。促腎上腺皮質激素釋放激素（corticotropin-releasing factor, CRF）是一種由腦室旁核（paraventricular nucleus, PVN）產生並釋放到腦下垂體門脈系統（hypothalamo-hypophyseal portal system）的神經胜肽，是調節Hypothalamus-pituatary-adrenal axis（HPA軸）的重要因子，在壓力反應中扮演重要作用。本研究旨在通過分析貓腦中CRF胜肽和信使核糖核酸（messenger ribonucleic acid, mRNA）的表達，建立一個可靠的免疫組織化學標記以評估瀕死期。 透過回溯性搜尋的方式，本研究共蒐集79例貓法醫解剖病例，對包含下視丘與視丘的兩個腦切面（4A與4B）進行IHC染色，其中12例包含原位雜合試驗（in-situ hybridization, ISH）染色，所有結果以影像數位化和全片分析技術進行定量或半定量。 本研究以半定量方式量化陽性控制組基因探針的ISH陽性訊號，發現受組織在固定前冷凍解凍以及隨死後變化程度影響，ISH染色所偵測的表現量會明顯減少。CRF mRNA基因探針染色後，在4A切面，CRF mRNA訊號主要可見於視丘、下視丘與新皮質，在海馬迴、杏仁核也可見少量陽性訊號，其中下視丘的陽性神經元尤其集中於第三腦室周圍。透過半定量的方式，本研究比較了CRF mRNA表現量與不同瀕死期的關係，然而，受限於樣本數較少，短瀕死期（1小時以內）與長瀕死期（1小時以上）之間無顯著差異。 CRF染色結果發現在冷藏保存和冷凍保存組織製作的切片中，貓腦中皆可偵測到CRF 胜肽。CRF胜肽在4A與4B腦切面的分布區域包含視丘、下視丘、新皮質、大腦白質、海馬迴、及杏仁核皆可見陽性訊號。透過西方墨點分析法（Western bolt），可見貓下視丘樣本中有與CRF前激素原大小相似的條帶。以統計方式分析比較CRF胜肽表現量後，本研究發現冷藏保存的組織相較冷凍保存的組織有顯著較高的CRF胜肽表現量，也發現體態評分（body condition score, BCS）在6至9之間的解剖病例相較BCS在1至3之間以及4至5之間的有顯著較高CRF胜肽表現量。最後，本研究發現CRF胜肽在短瀕死期以及在嚴重痛苦的死因下有顯著較高表現量。這些發現證明屍體保存方式及體態可能與CRF的IHC表現量有關，且CRF可作為獸醫法醫學中評估瀕死期和瀕死壓力的標記。

In forensic science, the degree of suffering endured by animals before death is a critical evaluation factor. The death process, known as the agonal phase, may continue for a varying length, reflecting the extent of the animal's agonal stress and suffering. Although some researchers have investigated immunohistochemical markers for evaluating the length of the agonal phase, there is still a lack of a well-established marker in veterinary forensics. Corticotropin-releasing factor (CRF) is a neuropeptide produced in the paraventricular nucleus (PVN) and released into the hypothalamo-hypophyseal portal system, playing an important role in regulating the hypothalamus-pituitary-adrenal (HPA) axis and stress responses. This study aims to establish a reliable immunohistochemical marker to evaluate the agonal phase by analyzing the expression of CRF polypeptide and messenger ribonucleic acid (mRNA) in cat brains. Through a retrospective study, selected brain sections of 79 forensic necropsy cases were collected for immunohistochemistry (IHC), including 12 cases with in-situ hybridization (ISH). The selected brain sections were 4A and 4B sections, which contain the hypothalamus and thalamus. All results were quantitatively or semi-quantitatively analyzed by whole-slide imaging techniques. The ISH positive signals of the positive control group gene probe were quantified by semi-quantification, finding that the expression detected by ISH staining markedly decreased due to tissue freezing and thawing before fixation and postmortem changes. After CRF mRNA gene probe staining in the 4A sections, CRF mRNA signals were mainly observed in the thalamus, hypothalamus, and neocortex, particularly concentrated around the third ventricle in the hypothalamus, with a small number of positive signals also seen in the hippocampus and amygdala. Through semi-quantitative methods, the current study compared the CRF mRNA expression between different agonal phases. However, due to the limited number of samples, no significant differences were found between short agonal phases (less than one hour) and long agonal phases (more than one hour). IHC staining against CRF showed detectable CRF peptides in cat brain sections made from both refrigerated and frozen-preserved tissues. In the 4A and 4B sections, positive signals were observed in the thalamus, hypothalamus, neocortex, cerebral white matter, hippocampus, and amygdala. Western blot analysis revealed a band in cat hypothalamic samples similar to the size of CRF preprohormone. Statistical analysis revealed that tissues preserved by refrigeration exhibited significantly higher CRF peptide expression compared to those preserved by freezing. It also found cases with body condition score (BCS) between 6 and 9 showed significantly higher CRF peptide expression levels compared to those with BCS between 1 and 3, and 4 and 5. Last but not least, the study found significantly higher CRF peptide expression in cases with a short agonal period and in those with causes of death involving severe distress. These findings suggest that the methods of tissue preservation and body condition may be associated with CRF expression levels in immunohistochemistry (IHC). Moreover, these findings demonstrate that CRF can serve as a marker for assessing the agonal phase and stress in veterinary forensics.