Study on Postmortem Changes of Multiple Organs in Rats under Different Temperatures

國內外對於動物福利及動物權日漸重視，使著許多動物醫療糾紛及虐待動物案件浮出檯面，因此動物法醫學研究開始盛興。評估死後經過時間（postmortem interval, PMI）與死後變化（postmortem changes）的觀察有密切的關係，然而，死後變化經常受到外源性或內源性因子的影響，導致PMI的錯誤評估。近期發表指出，死後變化的研究幾乎侷限於巨觀的研究，顯微鏡的觀察研究則很少。 本研究透過肉眼及組織學的觀察，配合免疫組織化學染色；嘗試於不同溫度下（25℃及35℃），進行為期0-7天的大鼠死後變化之研究，藉此了解死後經過時間與肉眼、組織學及免疫組織化學染色之間的關聯性並建立適用之表格，期望應用於實際推估法醫案例的死亡時間（time of death, TOD）。將大鼠人道犧牲後個別置放於保鮮盒內，放入恆溫（25℃及35℃）且氣流固定的培養箱內進行0-7 天的死後變化觀察。主要臟器包含腦、心臟、肝臟、脾臟、肺臟、腎臟、皮膚及背最長肌，於肉眼觀察後，皆會進行採樣並固定於10％之中性福馬林，後進行組織學及免疫化學染色的觀察，免疫組織化學染色；使用本研究室常規染色之抗體，包含CD3、CD79a、Desmin、Factor VIII、GFAP、Myoglobin、NF、SMA、Vimentin。 本研究結果證實於肉眼及組織學觀察中，大部分的變化於高溫下死後變化速率較快並具有規律性，如肉眼觀察中，皮膚變色、腹部鼓脹及臟器的軟爛程度；而屍溫、屍僵、屍水的產出及背最長肌的軟爛程度為非特異性；在脫毛，死後變化速率於低溫組快於高溫組。組織學中，僅有脾臟變化不具有特異性，其他臟器於皆為高溫組快於低溫組。此外，許多特殊變化也在本研究中發掘，例如死後變化造成的誤診，於1 dpm（day of postmortem），在小腦白質部呈現海綿樣變化，擬似病毒性疾病造成之海綿樣腦病；肝細胞內出現數量不等的囊泡，擬似脂肪肝造成之液狀小滴；肺泡腔出現嗜伊紅性且均質樣的液體，擬似肺水腫導致之液體蓄積。在免疫組織化學染色中，大部分抗體表現為陽性轉為陰性，陽性訊號的消失發生於2-3 dpm，然而在CK、desmin、GFAP、myoglobin及SMA的染色結果呈現於不同臟器、不同時段及不同溫度下的表現不一致，有些染色甚至出現交叉反應影響判讀，因此於辨識上有其困難性。 本研究將於2溫度下肉眼觀察、組織學及免疫組織化學染色中，具有顯著差異的變化統整並製作成與PMI相關之表格，期望藉由法醫解剖案例驗證本表格對於PMI估算的實用性。Animal welfare and medical disputes are getting public attention to raise development of veterinary forensic medicine. Estimation of postmortem interval (PMI) is always correlated with observation in postmortem changes (PMCs). However, PMCs are deeply influenced by extrinsic and intrinsic factors, leading to misestimate PMI frequently. Up to date, studies explored PMCs are almost at macroscopic level, but less investigation by light microscopy. The study aims to correlate PMCs in gross examination and light microscopy, following with immunohistochemistry to estimate PMI. The rats were sacrificed by humane method, whole bodies were stored under 25℃ and 35℃ individually, and then sampled from day 0 to 7. The major organs, including the brain, heart, liver, spleen, lung, kidney, skin, and m. longissimus dorsi were observed grossly, then fixed in 10% neutral formalin for microscopy and immunohistochemistry. As for the antibodies of immunohistochemistry, including CD3, CD79a, Desmin, Factor VIII, GFAP, Myoglobin, NF, SMA, and Vimentin were used. The results showed that postmortem rate in 35℃ was faster than 25℃, and the changes presented regularly. In gross, such as discoloration, abdominal swelling, and texture of the organs; however, the body temperature, rigor mortis, decomposition fluid, and the texture of m. longissimus dorsi were nonspecific; alopecia was faster in 25℃. In light microscopical examination, only spleen change was nonspecific, others were faster in 35℃. Some changes found in this study are worth our attention, such as in 1 dpm (day of postmortem), the spongy like change noted in the white matter might be comfused with spongiform change in the central nervous system; the intracytoplasmic vacuoles of hepatocytes should differentiate with lipid droplets of fatty liver; initiation of small amount of edematous exudates within the alveoli might be misdiagnosed in lung edema. In immunohistochemistry, most of the antibodies revealed positive to negative, and signals disappeared in 2-3 dpm; however, in CK, desmin, GFAP, myoglobin, and SMA showed variable results under different organs, dpm, and temperatures, some of them even displayed cross reaction, this leads difficulties in interpretation. The results from gross, microscopy, and immunohistochemistry study are summarized in flow charts for the purpose of PMI estimation, and it further needs to put it into practice of forensic cases to confirm the feasibility