犬の鼻腔内癌における放射線治療予後因子と増感効果に関する研究

酪農学園大

Obstructive Jaundice Influences Expression of nNOS and NADPH-d in the Hepatic Afferent and Efferent Neurons of the Rabbit

肝臟疾病會增加肝臟代謝負荷而造成血液循環改變，並可能造成神經病變。先前研究指出，自主（傳出）神經在調控肝內血液動態、膽汁分泌、碳水化合物及脂質的代謝，還有肝實質組織的再生上，扮演著重要角色。肝臟感覺（傳入）神經能偵測肝醣合成速率、脂肪酸與胺基酸的代謝率、血液中血糖濃度或是能量改變、肝門靜脈及肝內微循環的血壓、滲透壓、溫度，而回饋給自主神經系統，參與反射性活動，進而影響及調控肝臟功能，進行局部性或一般性的恆定控制。一氧化氮是重要的調控因子影響肝臟功能甚鉅，例如：血管擴張、免疫與炎症反應的控制以及細胞增生。因此本研究意欲探討在阻塞性黃膽的慢性傷害下，觀察神經性一氧化氮合成酶（nNOS）與菸鹼醯胺腺嘌呤-黃素蛋白輔酶（NADPH-d）在支配肝臟之傳入與傳出神經元的表現情形，藉以評估一氧化氮的製造量變化。實驗結果顯示，成年紐西蘭大白兔經過膽管結紮後，兩三天內即出現阻塞性黃膽症狀，二至三星期內肝臟組織切片的H&E染色可見嚴重的肝硬化病變，小結神經節、迷走神經背側運動核、T7-T10背根神經節、T7-T10脊髓側角之中外側細胞柱、腹腔神經節的神經元更是出現nNOS與NADPH-d反應活性顯著增強，代表支配肝臟並以一氧化氮為神經遞質的傳入與傳出神經元有全面被激活現象。此現象一來可推測，肝臟在遭受膽管阻塞性黃膽的傷害後，其代謝、血液動態與溫度調節作用勢必改變、紊亂、甚至失功能，而感覺神經也勢必會接受到大量的機械性、化學性、滲透性等感知訊息，所以感覺神經元將一氧化氮製造量提昇，有助於感覺訊息的快速傳報，以發揮回饋調控的效果；二來可表示，自主神經系統不論是交感或副交感神經，為了因應膽管阻塞所造成肝臟的病變，以增加一氧化氮的製造，意欲刺激肝外膽道系統，排空滯留在肝臟內或膽囊內的膽汁，而達到恢復肝臟內代謝、血液循環、膽汁流動等功能控制的恆定狀態。Liver diseases would cause neuropathy and that has been influenced the pathogenesis of circulation by increasing the hepatic metabolic load. Earlier studies have indicated that the autonomic efferents play important roles in intrahepatic haemodynamic and bile flow regulation, control of carbohydrate and lipid metabolism, and parenchymal cell regeneration. The hepatic sensory innervations (afferents) can detect the rate of glycogen synthesis, metabolism of lipid acid and amino acid, changes of blood glucose concentration and energy, blood pressure and temperature of portal vein, and intrahepatic microcirculation that may be able to elicit reflex activities and contribute to liver functional regulation and local or general homeostatic control. Nitric oxide (NO) is an important regulator of some liver functions, such as enhancement of vasodilation, control of immune and inflammatory reactions, and affection of cell proliferation. To investigate whether obstructive jaundice would mediate an influence on the nerve innervations in the rabbit liver, we studied the effect of bile duct ligation (BDL) on the expression of nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d) and neuronal NO-synthase (nNOS) in the hepatic afferent and efferent neurons. Jaundice was present in the eyelids, ear skins and mucosa at 3 days postoperation (dpo). This was followed by the prominent bile duct proliferation and cirrhosis in the liver at 14-21 dpo. NADPH-d and nNOS reactivities were markedly elevated in the nodosal ganglion, dorsal motor vagal nucleus, T7-T10 dorsal root ganglia, T7-T10 intermediolateral cell column and celiac ganglion, suggesting an upregulation of NO production in these areas. Quantitatively, the optical density of NADPH-d positive neurons in all of the related nuclei was significantly increased in the BDL animals compared with controls. Since NO is thought to be able to increase the motilities of the gallbladder and the sphincter of Oddi in anesthelized rabbits, the elevation of NADPH-d and NOS reactivities is likely to magnify the excitatory effect on the extrahepatic biliary system for the purpose of depletion of the loitered bile juice from the jaundiced liver. These data may also have important implications for the altered state of circulatory, metabolic or regenerative responsiveness under pathological condition.目 次 第一章 文獻探討………………………………………………… 1 第一節 肝臟的解剖生理學……………………………………… 1 一、 肝臟的解剖構造……………………………………….…. 1 二、 肝臟的神經支配……………………………….……….… 2 1. 肝臟之感覺神經系統…………………………….…………. 2 2. 肝臟之運動神經系統..……………………………….……..3 （1）肝臟之交感神經系統……………………….…………... 3 （2）肝臟之副交感神經系統…………………….…………... 5 3. 肝臟實質的神經分布…………………………….………... 6 第二節 一氧化氮…………………………………………. .... 8 一、 一氧化氮……………………………………………...…. 8 二、 一氧化氮合成酶…………………………………………… 8 三、 ㄧ氧化氮合成酶在肝臟的分布…………………………… 9 四、 神經性一氧化氮合成酶活性之調節機制……………….. 12 第三節 阻塞性黃膽……………………………………………… 13 第二章 實驗目的………………………………………………… 15 第三章 材料與方法…………………………………………….. 16 【實驗動物】……………………………………………………..16 【誘導阻塞性黃膽】……………………………………………. 16 【組織固定】……………………………………………………. 17 【組織檢體取樣與切片流程】…………………………………. 18 【NADPH-d組織化學反應】……………………………………… 19 【神經性一氧化氮合成酶免疫組織化學反應】………………. 20 【定量分析與統計】……………………………………………. 21 第四章 結果………………………………………..…………… 23 第一節 阻塞性黃膽之血清生化學檢查………………………… 23 第二節 H&E組織染色法………………………………......... 24 第三節 NADPH-d與nNOS染色法之神經反應表現……………... 25 一、 第七至第十胸段脊髓背根神經節………………………… 25 二、 第七至第十胸段脊髓的中間外側細胞柱……………….. 26 三、 腹腔神經節……………………………………………….. 26 四、 小結神經節…………………………………………………. 27 五、 孤立徑核……………………………………………………. 28 六、 迷走神經背側運動神經核…………………………………. 28 第五章 討論………………………………………………………. 52 第一節 阻塞性黃膽………………………………………………. 52 第二節 NADPH-d與nNOS在神經反應表現之意義…………….... 53 一、 交感神經支配肝臟的神經迴路…………………………... 53 二、 副交感神經支配肝臟的神經迴路………………………... 55 第三節 結論……………………………………………………... 59 第六章 參考文獻…………………………………………………. 60 圖 次 圖一 總膽管結紮手術圖……………………………………………30 圖二 實驗兔黃膽症狀……………………………………………. 31 圖三 實驗兔總膽管結紮後肝臟外觀……………………………. 32 圖四 正常與膽管結紮兔肝H&E染色鏡下情形…………………… 33 圖五 正常與膽管結紮兔肝T7-T10背根神經節NADPH-d組織化學染色法之神經反應表現…………………………………………...... 34 圖六 正常與膽管結紮兔T7-T10背根神法經節nNOS免疫組織化學染色之神經反應表現………………………………………........… 35 圖七 正常與膽管結紮兔T7-T10中間外側細胞柱NADPH-d組織化學染色法之神經反應表現………………………………………...... 36 圖八 正常與膽管結紮兔T7-T10中間外側細胞柱nNOS免疫組織化學染色之神經反應表現……………………………………........… 37 圖九 正常與膽管結紮兔腹腔神經節NADPH-d組織化學染色法之神經反應表現……………………………………………………...... 38 圖十 正常與膽管結紮兔小結神經節NADPH-d組織化學染色法之神經反應表現……………………………………………………...... 39 圖十一 正常與膽管結紮兔小結神經節nNOS免疫組織化學染色之神經反應表現…………………………………………………........ 40 圖十二 正常與膽管結紮兔孤立徑核與迷走神經背側運動神經核NADPH-d組織化學染色法之神經反應表現……………......... 41 圖十三 正常與膽管結紮兔孤立徑核與迷走神經背側運動神經核nNOS免疫組織化學染色之神經反應表現………………............ 42 表 次 表一 正常與膽管結紮兔血清生化值變化………………………… 43 表二 正常與膽管結紮兔T7-T10背根神經節NADPH-d組織化學染色法之神經反應表現…………………………………………......... 44 表三 比較正常兔與膽管結紮兔的T7-T10背根神經節內受NADPH-d標誌神經元之視密度值………………………………………....... 45 表四 正常與膽管結紮兔T7-T10中間外側細胞柱NADPH-d組織化學染色法之神經反應表現……………………………………......... 46 表五 正常與膽管結紮兔腹腔神經節NADPH-d組織化學染色法之神經反應表現…………………………………………………......... 47 表六 正常與膽管結紮兔小節神經節NADPH-d組織化學染色法之神經反應表現……………………………………………………....... 48 表七 比較正常兔與膽管結紮兔的小結神經節內受NADPH-d標誌神經元之視密度值………………………………………………....... 49 表八正常與膽管結紮兔孤立徑核NADPH-d組織化學染色法之神經反應表現………………………………………………………......... 50 表九 正常與膽管結紮兔迷走神經背側運動神經核NADPH-d組織化學染色法之神經反應表現………………………………….......… 5

Apoptosis and Ki-67 as predictive factors for response to radiation therapy in feline nasal lymphomas

Nasal lymphoma is the most common nasal tumor in cats and is generally a solitary and radiosensitive tumor. We retrospectively evaluated the response to radiation and survival time in relation to apoptosis and Ki-67 indices in feline nasal lymphomas treated with radiation therapy. The apoptotic and Ki-67 indices were evaluated with TUNEL and immunohistochemical staining in 30 biopsy tissues that were taken before any treatment. These two indices were compared, and differences between different treatment response groups were analyzed. The correlation between the median survival times (MST) and the indices was estimated using the Kaplan Meier method, and statistical differences between survival curves were analyzed using a log-rank method. With regard to apoptotic index, a statistical difference was observed between the samples taken from cats with complete response and stable disease (1.22% vs. 0.45%; P=0.045). The Ki-67 index in cats with both complete response and partial response was significantly higher than in cats with stable disease (44.4% and 39.6% vs. 16.3%; P0.9%) nasal lymphoma were not significantly prolonged MSTs (P=0.202), however, high Ki-67-positive (>40%) cats experienced a statistically significant relationship with longer survival time (P=0.015). Our results indicate that spontaneous apoptotic and Ki-67 indices are strong predictors for response to radiation therapy in feline nasal lymphomas

Prognostic Utility of Apoptosis Index, Ki-67 and Survivin Expression in Dogs with Nasal Carcinoma Treated with Orthovoltage Radiation Therapy

Apoptosis, Ki-67 and survivin expression have been reported as prognostic values in human cancer treated with radiation therapy. The aim of this study was to evaluate the correlation between the outcome of canine nasal carcinomas treated with radiation therapy and these cancer markers. The apoptotic index (AI) was evaluated with TUNEL assays, and an immunohistochemical evaluation was performed on Ki-67 and survivin in 33 biopsy samples taken before treatment. Median survival times were estimated using Kaplan-Meier curves and the log-rank method. The AI ranged from 0 to 0.7%, and the percentage of Ki-67-positive cells defined as the proliferative index (PI) ranged from 0.8 to 77% in all samples. Neither the AI nor the PI had a significant relationship with survival time (P=0.056 and 0.211). Survivin expression was detected in 84.9% of samples of canine nasal carcinoma. Dogs with high survivin expression were associated with poorer response to treatment and had shorter survival times (P=0.017 and 0.031). Advanced-stage tumors were also significantly associated with a high level of survivin (P=0.026). Overexpression of survivin was shown to be an unfavorable prognostic factor in dogs with nasal carcinomas treated with radiation therapy

Peripheral Lymphocyte Subsets as a Prognostic Indicator of Mortality and Morbidity in Healthy Dogs

To evaluate the relationship among immune status and increased morbidity and mortality, peripheral blood lymphocytes (CD3+, CD4+, CD8+ and CD21+ cells) from 32 healthy dogs over 8 years of age were analyzed. Twenty-five of the 32 dogs were followed-up for 3 years after the analysis; and 14 dogs were found to be diseased, and nine dogs died. There was no notable difference between the ages of the dogs that died compared with the ones that survived. The relative percentage of CD4+ and the CD4+:CD8+ ratio decreased notably in dogs falling ill compared with healthy dogs. The relative percentage of CD3+ lymphocytes showed a notable decrease in dogs that died within 3 years in comparison with dogs that survived. In a discriminant analysis of morbidity and mortality, most patients were correctly classified as diseased or not and surviving or dead, respectively. These results indicate that the immunophenotypes of peripheral blood lymphocytes in older dogs offer promise as parameters for evaluating mortality and morbidity