Transcriptomics of Rat Striata 16 h after Gamma Knife Surgery: Distinct Bilateral Effects in the Un-irradiated Striatum. Rattus norvegicus

Gamma knife surgery (GKS) is used for treatment of various brain disorders. The effects of gamma irradiation to targeted and un-targeted regions were evaluated by monitoring gene expression changes in the unilateral irradiated (60 Gy) and contralateral un-irradiated striata in the rat. Striata of irradiated and control brains were dissected 16 h post-irradiation for analysis by rat whole genome 44K DNA oligo microarray. Results revealed 230 induced and 144 repressed genes in the irradiated striatum and 432 induced and 239 repressed genes in the un-irradiated striatum. The number of altered genes in un-irradiated striatum was more than that in irradiated striatum. Results of RT-PCR and western analyses suggested that gamma-irradiation caused cellular damage, including oxidative stress, in both striata of both hemispheres. Our present results indicate that unilateral irradiation during GKS produce bilateral effects as early as 16 h, the time-period analyzed, and these molecular changes in the un-irradiated striatum are ample proof. Overall design: For the experiment, a total of four adult male Wistar rats were housed in acrylic cage at 24 ºC with tap water and laboratory chow ad libitum. The rats were anesthetized with pentobarbital (40 mg/kg intraperitoneally) and fixed in the Regis-Valliccioni frame (Neurospace, Neuilly, France). A central maximum dose of 60 Gy was administered (for 30 min) to the unilateral striatum with the Leksell gamma knife model C (Elekta Instrument AB) unit by means of 4-mm collimator. At 16 h post-irradiation, whole brains were rapidly removed and left and right striata were separated on ice according to the method of Glowinski and Iversen with some modifications. The irradiated and un-irradiated striata from irradiated rats and the ipsilateral and contralateral striata from control rats, respectively, were dissected from individual male rats and used for extraction of total RNA. Dye-swap or reverse labeling with Cy3 and Cy5 dyes procedure was applied followed by hybridization and wash processes, and the hybridized microarrays (G4131A) were scanned using a Standard protocol of Agilent DNA Microarray Scanner and data processing was done by Feature Extraction software Version 8.1 from Agilent Technologies.

伽玛刀手术（Gamma knife surgery, GKS）可用于治疗多种脑部疾病。本研究通过监测单侧照射（剂量60 Gy）及对侧未照射纹状体的基因表达变化，评估了伽玛射线对靶区与非靶区组织的生物学效应。
于照射后16小时，解剖照射组与对照组大鼠的纹状体，采用大鼠全基因组44K DNA寡核苷酸微阵列开展基因表达分析。结果显示，照射侧纹状体中有230个基因上调、144个基因下调，未照射侧纹状体则有432个基因上调、239个基因下调，未照射侧纹状体的差异表达基因数量多于照射侧。
逆转录聚合酶链式反应（Reverse Transcription-Polymerase Chain Reaction, RT-PCR）与蛋白质印迹（Western blot）分析结果表明，伽玛射线照射可导致双侧纹状体出现细胞损伤，包括氧化应激反应。本研究结果证实，伽玛刀手术中的单侧照射可在照射后16小时（本研究的检测时间节点）即产生双侧效应，未照射侧纹状体的分子变化即为该现象的充分证据。
实验整体设计：本实验共纳入4只成年雄性Wistar大鼠，饲养于亚克力笼中，环境温度维持24℃，自由饮用自来水、摄取标准实验室饲料。采用戊巴比妥钠（40 mg/kg，腹腔注射）对大鼠进行麻醉，将其固定于Regis-Valliccioni立体定向头架（Neurospace, Neuilly, France）。使用Leksell C型伽玛刀设备（Elekta Instrument AB），搭配4 mm准直器，对单侧纹状体给予中央最大剂量60 Gy的照射，照射时长30分钟。
照射后16小时，快速取出全脑，在冰上参照Glowinski与Iversen的方法并稍作修改，分离左右两侧纹状体。分别从照射组大鼠中解剖照射侧与未照射侧纹状体，从对照组大鼠中解剖同侧与对侧纹状体，提取总RNA用于后续实验。采用Cy3与Cy5染料进行反转标记（dye-swap）实验，随后完成杂交与洗涤步骤；将杂交后的微阵列（G4131A）使用安捷伦DNA微阵列扫描仪按照标准操作流程进行扫描，数据处理采用安捷伦科技公司的Feature Extraction软件Version 8.1完成。