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行政院國家科學委員會補助專題研究計畫成果報告
神經保護藥物對粒線體功能異常時鼠腦病變的影響
計畫類別：█個別型計畫 □整合型計畫
計畫編號：NSC89－2314－B－002－058－
執行期間： 88 年 8 月 1 日至 89 年 7 月 31 日
計畫主持人：李旺祚 台大醫學院小兒科
中文摘要 3-Nitropropionic acid (3-NP)為粒線體 呼吸鏈複合體 II 的抑制劑，它可在鼠導致 選擇性紋狀體病變，如同人類的 Huntington 病。過去的許多研究包括我們的研究顯 示 ， 抑 制 興 奮 性 氨 基 酸 的 釋 放 和 施 予 NMDA 受體的拮抗物，會減輕粒線體毒物 引 起 的 腦 病 變 。 最 近 的 研 究 也 顯 示 ， Dichloroacetate (DCA)對心臟衰竭及腦缺 血的鼠有保護作用。DCA 也被用於治療粒 線體腦病變。為了更進一步了解 DCA 是否 也可用於減輕 3-NP 所致的腦病變，及這些 藥物對腦代謝物的影響，在本計畫中，我 們利用磁共振影像(MRI)(T2 圖譜)和生物 活體磁共振質譜(in vivo 1 H MRS)的變化來 評估 DCA 對粒線體功能異常所引發腦病 變的治療效果。我們以 3-NP 為實驗藥物， 以迷你注射器包埋於二個月大的 Sprague-Dawley 株 鼠 腹 部 皮 下 ， 並 以 MK-801(2mg/kg)和 DCA (100mg/kg)為治療藥 物，比較治療的效果。我們先觀察慢性 3-NP 注射對鼠行為和腦病變的影響。然後觀 察 DCA 對亞急性 3-NP 腦病變的影響。結 果顯示慢性 3-NP 注射可使鼠產生肢體障 礙類似 Huntington 病，如同亞急性注射。 同時鼠腦紋狀體也會產生病變。生物活體 磁共振質譜也顯示 NAA 下降，表示神經元 的死亡。於亞急性的動物模式中，DCA 的 治療並不減輕 3-NP 對鼠腦紋狀體的傷 害。DCA 的臨床運用需更進一步的深入探 討其可行性。關鍵詞：3-Nitropropionic acid, Huntington
病, dichloroacetate.
Abstract
Systemic injection of 3-nitropropionic acid (3-NP), an irreversible inhibitor of complex II in mitochondrial respiratory chain, induces selective striatal lesions in rats and non-human primates mimicking those in Huntington’s disease. In recent studies, dichloroacetate (DCA) was shown to have protective effects in rat models of cerebral ischemia and myocardial dysfunction. However, its therapeutic effect on brain lesions induced by mitochondrial dysfunction is rarely investigated. In the past year, we established an in vivo animal model to evaluate the rat brain lesions in mitochondrial dysfuction. In the present study, we compared the therapeutic effect of DCA and MK-801 by in vivo animal model.
Two-month-old Sprague-Dawley rats were treated with 3-NP by continuous drug release from mini-pump, implanted subcutaneously. MK-801 (2mg/kg) and DCA (100mg/kg) were given in the same way. The rats were then evaluated by MRI (T2 maps) and in vivo 1H-MRS at indicated time points. We first evaluated the effect of chronic 3-NP injection on rats, and then evaluated the effect of DCA on the striatal lesions induced by 5-day 3-NP injection. The results showed that chronic 3-NP injection produced behavioral change and selective striatal lesions on rats. MRS also showed the decline of NAA/Cr ratio, indicating the neuronal loss or dysfunction. The simultaneous application of DCA showed no attenuation of the striatal lesions. The present results suggest that the
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application of DCA in brain lesions induced by mitochondrial inhibitors need further investigation.
Keywor ds: 3-Nitropropionic acid,
Huntington disease, dichloroacetate.
緣由與目的
Neurodegenerative diseases have been found to be associated with mitochondrial dysfunction as in mitochondrial diseases in recent years. Systemic administration of 3-nitropropionic acid (3-NP), an irreversible inhibitor of complex II in mitochondrial respiratory chain, induces selective striatal lesions in rats and non-human primates mimicking those in Huntington’s disease (Lee et al, 2000a and b). Several drugs have been shown to have neuroprotective effect in 3NP-treated rats, including glutamate release inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists. In recent years, dichloroacetate (DCA) was shown to have protective effect for cerebral ischemia in rats (Peeling et al., 1996), and was also used as metabolic therapy for myocardial ischemia and failure. DCA can stimulate the pyruvate dehydrogenase complex activity, and, therefore, can improve the recovery of cerebral lactate, acidosis and ATP following reperfusion in forebrain ischemia of rats. Because persistent lactic acidosis was supposed to be a factor contributing to the neuronal injury, decreasing cerebral lactic acidosis and enhancing cerebral metabolism may be one of the strategies in ameliorating brain injury related to elevated lactate, such as cerebral ischemia. DCA has also been implicated in the therapy of patients with MELAS and Leigh disease in recent years (DeStefano et al., 1995; Pavlakis et al., 1998; Saitoh et al., 1998), especially in Japan. However, there are limited in vivo researches about the application of DCA in neurodegenerative diseaseas. Therefore, further investigation of the therapeutic efficacy is highly needed.
方法
Two-month-old Sprague-Dawley rats were used. 3-NP was injected by mini-pump, implanted subcutaneously. MK-801 (2mg/kg) and DCA (100mg/kg) were used as therapeutic drugs. 3-NP-induced behavioral changes in the animals were recorded and graded according to the neurological scale described by Guyot et al. (1997). The 3-NP-induced brain lesions were evaluated by MRI (T2 maps) and in vivo proton MRS.
We first evaluated the effect of chronic 3-NP injection on the behavior and brain lesions, and then evaluated the effect of DCA and Mk-801 on the brain lesions induced by 5-day 3-NP injection.
Magnetic resonance measurements were performed on a 4.7 Tesla spectrometer (Bruker Biospec) with an active shielding gradient at 6.9 G/cm in 500 µsec. The rats were placed in a prone position with a custom-designed head-holder. A 20cm birdcage coil was used for RF excitation, and a 2cm-diameter surface coil placing directly over the head was used for signal receiving. After magnetic field optimization, a multi-slice multi-echo imaging was obtained with the following parameters: field of view = 5cm, 4 slices (2 mm thick with 1 mm gap), matrix = 256 x 128, TR = 4000msec, and initial TE = 20msec with an echo spacing of 20msec for 6 echoes. Data processing for T2 maps was performed with commercial image analysis software (MRVision Co. CA, U.S.A.).
The point-resolved spectroscopy (PRESS) sequence preceded by three consecutive CHESS pulses for water suppression was used to acquire the localized proton spectra over the striatum. Each CHESS pulse for water suppression was 15 msec in duration, and was followed by a spoiled gradient. CHESS pulses of 2msec duration for the 90 and 180 pulses were used. After manual adjustment of the transmitter and receiver, shimming of the region of interest and maximizing the suppression of water signal, localized spectral data was obtained with the following parameters: TR = 2000msec, TE = 136msec, scan no. = 256,
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spectral width = 4000 Hz. The peaks for N-acetylaspartate (NAA), choline (Cho), creatine (Cr), succinate (Suc) and lactate (Lac) were recognized. The ratios of NAA/Cr, Suc/Cr, Cho/Cr and Lac/Cr were used for data analysis.
結果與討論
The present results showed that chronic application of 3-NP produced behavioral changes mimicking those in Huntington disease. There were also selective striatal lesions on rat brain with gradual decrease of NAA/Cr ratios over the striatal areas, indicating the neuronal loss or dysfunction. Elevated Suc/Cr and Lac/Cr ratios were also noted in chronic 3-NP injection.
Subacute 3-NP injection by mini-pump also produced behavioral changes and selective striatal lesions as shown in our previous studies (Lee et al, 2000a). Simultaneous application of DCA, however, did not abolish the behavioral changed and striatal lesions induced by subacute 3-NP injection. Compared with DCA, MK-801 showed prominent protective effect with attenuation of the striatal lesions induced by 3-NP as shown before (Lee et al, 2000a).
Although DCA had been shown to be effective in attenuation of ischemic cerebral lesions in rats (Peeling et al, 1996), it is not effective in alleviating the striatal lesions induced by 3-NP. Hypoxia and ischemia may suppress the activity of pyruvate dehydrogenase complex with accumulation of lactate. DCA can stimulate the pyruvate dehydrogenase complex activity, and, therefore, can improve the recovery of cerebral lactate, acidosis and ATP following reperfusion in forebrain ischemia of rats. In contrast, the accumulation of lactate in 3-NP-induced brain lesions results from the inhibition of respiratory chain complex II. Therefore, the stimulation of pyruvate dehydrogenase complex activity may not be effective in improving the mitochondrial function. Another possibility is that 3-NP also inhibit the Kreb’s cycle and, therefore, affect the cell metabolism and survival.
DCA has been tried in several kinds of
mitochondrial encephalopathy. Although DCA was shown to be effective in treatment of patients with Leigh disease and MELAS (DeStefano et al., 1995; Pavlakis et al., 1998; Saitoh et al., 1998), its effect on other mitochondrial encephalopathies needs further investigation to see whether different
mechanisms of mitochondrial
encephalopathies will lead to different response of the patients to the treatment with DCA.
參考文獻
DeStefano N, Matthews PM, Ford B, et al. Short-term dichloroacetate treatment improves indices of cerebral metabolism in patients with mitochondrial disorders. Neurology 1995;45:1193-8.
Guyot M.C., Hantraye P., Dolan R., Palfi S., Maziere N. and Brouillet E. (1997) Quantifiable bradykinesia, gait abnormalities and Huntington's disease-like striatal lesions in rats chronically treated with 3-nitropropionic acid. Neuroscience 79,45-56.
WT Lee, Shen YZ and Change C. Neuroprotective Effect of lamotrigine and MK-801 on rat brain lesions induced by 3-nitropropionic acid: evaluation by magnetic resonance imaging and in vivo proton magnetic resonance spectroscopy. Neuroscience 2000a, 95: 89-95.
Lee WT, Lee CS, Pan YL, et al. Temporal Changes of cerebral metabolites and striatal lesions in Acute 3-Nitropropionic Acid Intoxication in the Rat. Magn Reson Med 2000b, 44, 29-34.
Pavlakis SG, Kingsley PB, Kaplan GP, Stacpoole PW, O'Shea M, Lustbader D. Magnetic resonance spectroscopy: use in monitoring MELAS treatment. Arch Neurol 1998;55:849-52.
Peeling J, Sutherland G, Brown RA, Curry S. Protective effect of dichloroacetate in a rat model of forebrain ischemia.
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Saitoh S, Momoi MY, Yamagata T, Mori Y, Imai M. Effects of dichloroacetate in three patients with MELAS. Neurology 1998; 50:531-4.
