Fukui On, Kinugasa Yukiko, Fukuda Aya, Fukuda Hirotsugu, Tskitishvili Ekaterine, Hayashi Shusaku, Song Mihyon, Kanagawa Takeshi, Hosono Takayoshi, Shimoya Koichiro, Murata Yuji
Department of Obstetrics and Gynecology, Osaka University School of Medicine, 2-2 Yamada-oka, Suita, 565-0871, Osaka, Japan.
Brain Res. 2006 Nov 22;1121(1):35-45. doi: 10.1016/j.brainres.2006.08.121. Epub 2006 Sep 29.
Inflammation is an important factor for hypoxia-ischemia (HI) brain injury. Interleukin (IL)-18 is a proinflammatory cytokine which may be a contributor to injury in the immature brain after HI. To investigate the effects of post-HI hypothermia on IL-18 in the developing brain, 7-day-old rats were subjected to left carotid artery ligation followed by 8% oxygen for 60 min and divided into a hypothermia group (rectal temperature 32 degrees C for 24 h) and a normothermia group (36 degrees C for 24 h). The IL-18 mRNA was analyzed with real-time RT-PCR, and the protein level was analyzed by Western blot, and the location and source of IL-18 were assessed by immunohistochemistry. The significant increase of the IL-18 mRNA was observed in the ipsilateral hemispheres of the normothermia group at 24 h and 72 h after HI compared with controls, but the level in the ipsilateral hemispheres of the hypothermia group was significantly reduced at both time points, compared with the normothermia group, respectively. The IL-18 protein level in the ipsilateral hemispheres of the normothermia group significantly increased at 72 h after HI compared with controls, however, the protein level of the hypothermia group was significantly decreased, compared with the normothermia group. IL-18-positive cells were observed throughout the entire cortex, corpus callosum (CC) and striatum in the ipsilateral hemispheres of normothermia group at 72 h after HI, however, little positive cells were observed in the hypothermia group. Double labeling immunostaining found that most of the IL-18-positive cells were colocalized with lectin, which is a marker of microglia. The number of ameboid microglia (AM) in the normothermia group was significantly increased in cortex and CC, compared with the number in controls, but there were very few ramified microglia (RM) in these areas. In contrast, the number of AM in the hypothermia group was significantly decreased in cortex and CC, compared with the number in the normothermia group, and there were no significant differences in the number of AM and RM between the hypothermia group and controls. In conclusion, we found that IL-18 mRNA and the protein level were attenuated by post-HI hypothermia and that post-HI hypothermia may decrease microglia activation in the developing brain.
炎症是缺氧缺血性（HI）脑损伤的一个重要因素。白细胞介素（IL）-18是一种促炎细胞因子，可能是HI后未成熟脑损伤的一个促成因素。为了研究HI后低温对发育中脑内IL-18的影响，将7日龄大鼠进行左颈动脉结扎，然后置于8%氧气环境中60分钟，并分为低温组（直肠温度32℃，持续24小时）和正常体温组（36℃，持续24小时）。用实时逆转录聚合酶链反应（RT-PCR）分析IL-18 mRNA，用蛋白质印迹法分析蛋白质水平，并用免疫组织化学法评估IL-18的定位和来源。与对照组相比，正常体温组同侧半球在HI后24小时和72小时时IL-18 mRNA显著增加，但与正常体温组相比，低温组同侧半球在这两个时间点的水平均显著降低。与对照组相比，正常体温组同侧半球在HI后72小时时IL-18蛋白水平显著增加，然而，与正常体温组相比，低温组的蛋白水平显著降低。HI后72小时，在正常体温组同侧半球的整个皮质、胼胝体（CC）和纹状体内均观察到IL-18阳性细胞，然而，在低温组中观察到的阳性细胞很少。双重免疫标记染色发现，大多数IL-18阳性细胞与小胶质细胞标志物凝集素共定位。与对照组相比，正常体温组皮质和CC内阿米巴样小胶质细胞（AM）数量显著增加，但这些区域的分枝状小胶质细胞（RM）很少。相比之下，与正常体温组相比，低温组皮质和CC内AM数量显著减少，低温组与对照组之间AM和RM数量无显著差异。总之，我们发现HI后低温可使IL-18 mRNA和蛋白水平降低，且HI后低温可能会减少发育中脑内小胶质细胞的激活。
