Name: a high-performance liquid chromatography measurement of kynurenine and kynurenic acid: relating biochemistry to cognition and sleep in rats
Uploaded: 2018-08-19T13:00:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats at JoVE.com

本研究部分由国立卫生研究院 (R01 NS102209) 资助, 并由克莱尔. 福布斯信托基金捐赠。

我们的实验性议定书得到了马里兰大学动物保育和使用委员会的批准, 并遵循了国家卫生研究院的护理和使用实验动物指南。
注: 所有实验均采用成年雄性大鼠 (250–350 g)。不同的动物群被用于生化分析、行为实验和睡眠唤醒记录。这些动物被安置在一个温度控制的设施在马里兰精神病学研究中心。他们被保持在一个12/12 小时的光黑暗的周期, 与灯在授时时间 (ZT) 0 和熄灯在 ZT 12。在实验中, 这些动物收到了随意食物和水的机会。该设施得到了美国实验室动物护理认证协会的充分认可。
1. 腹腔犬尿氨管理对大鼠的作用
注: 在本议定书中, 犬尿氨是在 zt 0 (光相开始), 并收集在 zt 2 和 zt 4, 以确定一个时间路线的犬尿氨新陈代谢。用盐水注射的动物作为控制。例如, 如果一只老鼠重500克, 所需剂量为100毫克/千克, 则大鼠应接受5毫升注射10毫克/毫升溶液的犬尿氨。
<ol><li>犬尿氨硫酸 ("犬尿氨"), 并将其放入一个20毫升玻璃瓶。一定要把犬尿氨在冰上。</li>
	<li>
添加生理盐水以达到所需浓度, 并将 pH 值调整为 7.6, 使用 1 N 氢氧化钠 (氢氧化钠)。涡旋和油脂实验溶液直到犬尿氨完全溶解。 注意事项: 在处理氢氧化钠时, 遵循所有建议预防措施。<ol>
<li>例如, 要达到10毫克/毫升溶液, 重出200毫克的犬尿氨, 并把它放在玻璃瓶。加入15毫升盐水和漩涡和油脂实验 (20 赫为 5–10 s, 1/8 英寸直径 microtip) 它溶化。使用氢氧化钠, 调整溶液的 pH 值。最后, 用生理盐水调节体积到20毫升。</li>
	</ol>
</li>
	<li>根据动物的体重和所需的治疗剂量, 对每只实验鼠进行称量, 计算每种注射量。小心地将动物从笼子里取出, 注入溶液腹腔, 将动物送回笼子。</li>
</ol>2. 用高效液相色谱法测量犬尿氨
<ol><li>组织收集 注: 在本议定书中, 犬尿氨是在 zt 0 (光相开始), 并收集在 zt 2 和 zt 4, 以确定一个时间路线的犬尿氨新陈代谢。用盐水注射的动物作为控制。<ol>
<li>用二氧化碳弄死动物。呼吸症状停止1分钟后, 用斩首法进行二次安乐死。</li>
		<li>收集全树干血, 放置一个一次性漏斗到一个15毫升管含有 20 ul 的 K3-EDTA (0.5 米)。让血液从体内排出到管子里。反复翻转管子, 确保 EDTA 混合在一起。</li>
		<li>使用剪刀, 把头上的皮肤切开中线, 露出头骨。去掉头骨后部多余的颈部肌肉, 在脊髓的开口处, 从头骨向后切开。<ol>
<li>轻轻拉两侧的头骨打开, 以揭露大脑, 然后小心地删除的大脑与镊子, 以免损害它。用剃刀刀片取出嗅球, 并将大脑从中线的一半切开。使用钳, 解剖 hemibrain 到感兴趣的区域 (即海马和皮质)。</li>
		</ol>
</li>
		<li>把所有解剖过的脑部放在铝箔上干冰上。组织完全冻结后, 将其转移到20毫升的塑料瓶, 并将其存储在-80 摄氏度。</li>
		<li>将整个树干的血液离心在 300 x g 10 分钟, 取出上清 (等离子) 并将其转移到新的离心管中, 然后将其储存在-80 摄氏度。</li>
	</ol></li>
	<li>样品准备
	<ol>
<li>血浆: 从被处理的动物收集<ol>
<li>用等离子解冻冷冻管 (见步骤2.1 为组织收集) 在湿冰。</li>
			<li>在一个新的离心管, 稀释样品与超纯水 (1:2 为犬尿氨, 1:10 为 KYNA)。到 100 ul 稀释样品, 添加 25 ul 6% 高氯酸。 注意事项: 在处理高氯酸时遵循所有建议预防措施。</li>
			<li>涡流所有样品, 然后离心他们在 1.2万 x g为10分钟。一旦完成, 删除 100 ul 的上清 (从每管), 并把它们放入一个小0.25 毫升离心管犬尿氨或 KYNA 的决心。</li>
		</ol>
</li>
		<li>大脑: 从被处理的动物收集<ol>
<li>放置管与冰冻脑标本 (见步骤2.1 为组织收集) 在干冰。在精确的分析天平上单独称量每个脑样本。把它还给管子, 放回干冰上。</li>
			<li>在所有的样品被权衡后, 把管子移到湿冰上。稀释每个样品1:10 瓦特/v 与超纯水和融汇他们使用一个 sonicator (20 赫 5–10 s, 1/8 英寸直径 microtip)。例如, 对于100毫克的脑组织, 添加 900 ul 的超纯水。</li>
			<li>整除 100 ul 每稀释的样品到一个新的管子和酸化它与 25 ul 25% 高氯酸。涡旋, 然后离心 1.2万 x g 10 分钟。</li>
			<li>离心后, 去除 100 ul 的上清, 并把它放到小0.25 毫升离心管 KYNA 的决心。</li>
		</ol>
</li>
	</ol></li>
	<li>高效液相色谱仪的建立与运行
	<ol>
<li>准备50毫米醋酸钠移动相。在1升的超纯水中溶解6.81 克醋酸钠三水。用冰乙酸将 pH 值调整为 6.2, 然后将真空过滤成清洁玻璃器皿。将醋酸钠移动相转移到干净的1升玻璃瓶中。</li>
		<li>准备500毫米醋酸锌。在500毫升的超纯水中溶解54.88 克醋酸锌, 然后将其真空过滤。把它转移到一个干净的500毫升玻璃瓶。用超纯水和500毫升瓶填充1升瓶, 高效液相色谱级乙腈。</li>
		<li>将进气管从 HPLC 机分别放入流动相、超纯水和100% 乙腈。将醋酸锌溶液分别通过与移动相结合的蠕动泵, 并在衍生之前进行泵浦。</li>
		<li>使用该机器上的控制面板, 程序它运行5% 乙腈和95% 超纯水0.5 毫升/分钟. 允许它运行20–30分钟, 以达到稳定的压力和基线。</li>
		<li>建立稳定基线后, 将溶液组成改为5% 乙腈和95% 醋酸钠流动相。平衡30分钟。</li>
		<li>打开荧光探测器中的灯, 让它预热。</li>
		<li>同时, 开机后柱泵的流量可达到0.1 毫升/分. 允许它在大约20分钟后到达稳定压力约 500 psi。</li>
		<li>制定标准。<ol>
<li>对于 KYNA, 从1毫米的库存溶液开始, 稀释它以准备5标准浓度 (即10 nm、5 nm、2.5 nm、1 nm 和 500 pM)。 注: 这将产生一个标准曲线范围从 200 fmoles 到 10 fmoles 每 20 ul 注射液。</li>
			<li>对于犬尿氨, 从1毫米的库存溶液开始, 稀释它以制备5标准浓度 (即10 微米、5微米、2.5 微米、1微米和 500 nM)。 注: 这将产生一个标准曲线范围从 200 pmoles 到 10 pmoles 每 20 ul 注射液。</li>
		</ol>
</li>
		<li>建立高效液相色谱系统软件, 控制样本序列参数, 允许多样本 autoinjections。<ol>
<li>当在 "运行示例" 屏幕上, 单击 "文件" 并向下拖动以 "创建新的示例集"。当新窗口打开时, 选择 "空"。单独列出所有标准 (请参阅步骤 2.3.8) 或按应运行的顺序排列示例。</li>
			<li>在 "函数" 列中, 指定标准和示例。标准应在序列的开始和结束时进行测定。在每5个样品之间注入水。</li>
			<li>将每个样本的运行时间设置为15分钟. 从标准和等离子制剂中注入 20 ul, 或从大脑匀浆制剂中注入 30 ul 样品。</li>
			<li>设置激发和发射波长 (取决于被评估的化合物) 为犬尿氨到励磁: 365 毫微米, 发射: 480 毫微米和保留时间: ~ 6 分钟, 并且为 KYNA 到励磁: 344 毫微米, 发射: 398 毫微米, 并且保留时间: ~ 11 分钟。</li>
			<li>设置好所有参数后, 机器平衡, 运行该序列。</li>
			<li>要量化数据, 请导航到 "浏览项目" 屏幕。在 "样本集" 选项卡下, 双击要量化的样本集。从所有注射的列表中, 突出显示所有标准并右键单击。在新窗口中, 选择 "进程", 然后使用下拉菜单选择 "如何" ("如何") 旁边的 "校准和定量":。</li>
			<li>再次突出显示所有标准, 右键单击并选择 "审阅"。当图谱打开时, 使用顶部的按钮 "集成", 然后 "校准" 每个标准;这将自动创建标准曲线。</li>
			<li>返回到 "示例集" 选项卡, 然后双击示例集。其次, 突出所有标准。重复上面提到的过程, 而是从下拉菜单中选择 "定量"。再次突出显示所有示例, 然后右键单击并选择 "审阅"。当图谱打开时, 使用顶部的按钮 "集成", 然后 "定量" 每个示例。 注意: 这将根据先前创建的标准曲线输出每个样本的浓度。</li>
		</ol>
</li>
	</ol></li>
</ol>3. 被动回避范式
注意: 这些行为实验是根据我们的生化发现和急性犬尿氨挑战而设计的。为了最大限度地增加脑 KYNA, 犬尿氨 (100 毫克/千克) 是在 zt 0, 2 h 的训练课程之前, 在被动回避范式测试海马介导的学习, 这发生在 zt 2。该仪器包括2个同等大小的隔间 (21.3 厘米高, 20.3 厘米宽, 15.9 厘米深), 由断头台门隔开, 并包含在隔音盒内。测试仪器的两个隔间被称为 "光边" 和 "暗面"。光明面的墙壁是清晰的, 在试验期间, 灯会打开, 以进一步照亮这个车厢。黑暗的车厢的墙壁被完全覆盖, 以保持一个黑色的条件。
<ol><li>
1天: 训练试验
	<ol>
<li>在测试之前, 用70% 乙醇清洗仪器。</li>
		<li>把动物带到测试室去。</li>
		<li>轻轻地将实验动物放到仪器的光边, 然后关闭并闩锁设备门和隔音盒。</li>
		<li>使用关联的软件, 开始试用。从主屏幕中, 从下拉菜单中选择 "文件", 然后从 "打开会话"。在新窗口中, 确保选中了正确的过程和设备, 然后单击 "关闭"。接下来, 选择 "配置" 然后 "信号"。在新窗口中, 选择正确的设备, 然后单击 "问题"。 注: 该软件将启动灯打开在设备的轻的一面, 并打开断头台门之间的两个车厢。将启动计时器。</li>
		<li>当动物完全进入设备的黑暗面时, 断头台门就会关闭, 而一个不可避免的脚震动 (0.56 mA 为1秒) 将被交付。</li>
		<li>记录在动物进入黑暗的车厢之前已经过去了的时间。</li>
		<li>在动物受到惊吓后, 允许三十年代的恢复时间, 然后从仪器中取出动物。</li>
		<li>把动物放回笼子里。</li>
		<li>在与下一只动物开始试验之前, 用湿毛巾擦拭器具并处理任何粪便颗粒。</li>
		<li>所有的动物都经过训练后, 把它们送回动物设施。</li>
	</ol>
</li>
	<li>
2天: 测试试验
	<ol>
<li>训练试验结束后, 将动物带回测试室24小时。开始对第一个动物的测试试验, 把它放到仪器的光边, 关闭并闩上门和隔音盒。</li>
		<li>使用与前一天相同的软件命令启动测试试验;灯将在仪器的光边打开, 两个车厢之间的断头台门将会打开。将启动计时器。</li>
		<li>当动物进入黑暗的车厢, 断头台门将关闭。记录所经过的时间。 注: 如果动物留在测试仪器的光边上, 试验将在最大300s 后通过软件终止。</li>
		<li>将动物放回自己的笼子里, 清洁仪器 (如步骤3.1.9 中所述), 然后再开始与下一只动物的试验。</li>
	</ol>
</li>
</ol>4. 睡眠分析
<ol><li>无线脑电图/肌电图发射机的外科植入 注: 遥测发射机应在手术前进行消毒和准备。<ol>
<li>使用剃刀刀片, 轻轻地除去小长度的塑料涂层, 露出导线引线。将发射机放在一个50毫升的锥形管中, 充满杀菌液 [例如, Wavecide (2.65% 戊二醛)]。<ol>
<li>将发射机放在消毒液中至少12小时。然后, 在手术之前, 把溶液转移到一个废物容器里。用无菌盐水冲洗发射机。</li>
		</ol>
</li>
		<li>在手术当天, 在将动物置于麻醉室之前称其为体重。诱导3–5% 异氟醚 100% O2。</li>
		<li>成功的麻醉后, 取出动物并小心剃须, 使用电动剃须刀, 头部和颈部的顶部, 以及在身体背部的一小块头发, 只是稍微左和下方的胸腔。</li>
		<li>管理卡洛芬 (5 毫克/千克) 皮下术后镇痛。</li>
		<li>将恒温控制的温水加热垫设置在37摄氏度的动物下, 以保持动物的体温在手术期间。</li>
		<li>在立体定向的框架内, 将动物放在鼻锥上以保持麻醉状态, 并放置耳棒以稳定头部。用 betadine 擦洗和70% 乙醇的交替拭子消毒剃毛皮肤。将 opthalamic 软膏涂抹在眼睛上以润滑它们。</li>
		<li>为确保在第一切口前有足够的麻醉, 请使用脚趾捏试验。</li>
		<li>用一把无菌手术刀, 从眼睛后部切开到颈部后方。颅骨和颈背颈部肌肉应暴露。</li>
		<li>如果需要暴露头骨, 使用棉尖喷头去除任何膜。微型牛头犬钳可以用来保持皮肤远离头骨。定位并标记 bregma 的位置。钻2毛刺孔和插入金属螺丝2.0 毫米前/+ 1.5 毫米侧向和7.0 毫米后/-1.5 毫米侧面相对于 bregma。完成2次革命拧紧螺钉。用一块纱布遮住裸露的头骨。</li>
		<li>使用无菌手术剪刀, 使切口进入体腔, 就在肋骨下面。</li>
		<li>在手术单上, 一定要把要植入的发射机的序列号记录下来。</li>
		<li>将无菌发射机插入体腔内。导线引线应该仍然是在身体外面。</li>
		<li>使用可吸收缝线缝合肌肉壁, 确保所有的线索聚集到一个切口的一端。</li>
		<li>从头上取出纱布, 把切口下面的皮肤抬起来。从头部切口到侧面切口, 在皮肤下运行一双长的不育止血剂。保持对皮肤的压力, 以减少任何伤害。<ol>
<li>一旦止血剂在身体切口附近可见, 夹住任何可能用手术剪刀覆盖的膜, 并抓住四发射器引线与止血剂。慢慢地抽出止血剂, 使电线躺在皮肤下, 跑到头骨上。</li>
		</ol>
</li>
		<li>指定哪些2引线将连接到头骨。使用镊子, 用一只手抓住电线的一端, 并在塑料护套的末端下方用另一只。拉上电线, 直到单独的循环可以做出来。</li>
		<li>将暴露的导线紧裹在金属螺钉3–4x 的周围。一旦安全, 拧紧螺丝, 以防止电线解开和剪辑任何额外的电线。用第二个引线和螺钉重复此操作。</li>
		<li>拉两个脑电图线索从体腔, 使他们坐在紧贴头骨。</li>
		<li>使用牙科水泥覆盖螺钉和皮质引线, 在暴露的头骨上创建一个上限。确保没有牙科水泥粘在皮肤或肌肉, 没有锋利的边缘被创造。允许牙科水泥干燥。 注意: 瓶盖应该足够小, 可以在上面缝合皮肤。</li>
		<li>对于肌电图的引线, 抓住的线和塑料鞘的末端, 如步骤4.1.15 所述。伸展电线, 直到各个回路清晰可见。</li>
		<li>在2–3毫米的右侧, 在颈部肌肉下运行22克针, 然后才允许它出现。使用美容整形缝合, 放置一个单一的, 松散缝合周围的嵌入针。</li>
		<li>将肌电信号线插入针中, 拔出针, 使肌电信号在肌肉下线。拧紧缝合线, 以确保导线保持到位, 并夹住可能从肌肉中出现的多余导线。</li>
		<li>重复步骤4.1.20 和4.1.21 的第二个肌电信号引线, 约1毫米向下从第一个铅。</li>
		<li>拉两个肌电信号的线索从体腔, 使他们坐在舒适的肌肉。</li>
		<li>使用美容整形针闭合头部和颈部切口。把所有多余的电线塞在身体的皮肤下, 用伤口夹来闭合身体切口。</li>
		<li>在两个切口部位应用利多卡因软膏, 协助动物术后疼痛。</li>
		<li>把动物送回笼子, 让笼子坐在暖气垫上, 直到动物从麻醉中恢复。 注意: 动物应在开始录音前7–10天恢复。</li>
	</ol></li>
	<li>脑电图/肌电信号记录 注: 在本议定书中, 我们在 ZT 0 中管理生理盐水或犬尿氨, 然后记录动物的睡眠-唤醒模式为24小时。<ol>
<li>在指定的房间里完成所有的睡眠记录, 在这段时间内动物不会被打断。</li>
		<li>将动物的家庭笼子 (轴承植入的脑电图/肌电信号发射机) 放在接收器单元上。用磁铁打开手术植入的发射机。 注: 当发射器被激活时, 接收装置上应出现一盏灯。</li>
		<li>使用相关软件设置脑电图/肌电信号数据的记录。<ol>
<li>选择 "实验", 然后从下拉菜单中 "创建"。命名实验, 然后保存它。接下来, 选择 "硬件", 然后 "编辑 MX2 配置"。</li>
			<li>在新窗口中, 选择实验中使用的所有发射器, 并指示每个接收板的配对。准备就绪后, 按 "开始所有连续" 启动录制。</li>
		</ol>
</li>
		<li>在实验结束时, 选择 "停止所有连续" 在相关的软件和关闭发射机使用磁铁。</li>
		<li>弄死动物由 CO2窒息然后心脏穿刺。通过用手术剪刀在头部顶部重新打开切口, 并从牙科水泥和颈部肌肉中切开无引线, 小心地取出发射机。</li>
		<li>接着, 打开体腔, 找到发射器, 慢慢地将其从体内移除。</li>
	</ol></li>
	<li>脑电图/肌电信号分析
	<ol>
<li>使用关联的软件分析睡眠唤醒数据。打开程序并选择 "添加新位置"。在新窗口中, 选择要导入到评分软件中的数据。 注意: 这将在软件内创建一个新文件, 其中包含实验期间收集的所有原始波形。</li>
		<li>手动为所需的实验评分警戒状态。评分后, 分析的参数, 如总持续时间, 回合次数, 和平均回合期间的各种警戒状态 [快速眼动 (rem), 非 REM (NREM), 和唤醒]。 注意: 分析可以在整个记录或缩短到特定的时间点进行。在此, 对 zt 0 至 zt 2 之间的记录期进行了分析。</li>
	</ol></li>
</ol>

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作者没有什么可透露的。

为了可靠地评估周围犬尿氨管理后的脑 KYNA, 必须结合和解释生化和功能性实验。在这里, 我们提出了一个详细的协议, 允许新用户建立有效的方法来测量血浆犬尿氨和脑 KYNA 的大鼠。血浆中犬尿氨的测定证实了准确的注射和代谢产物的测定 KYNA 证实了大脑中的从头合成。所描述的荧光液相色谱法有以下几个优点: 从柴田18到 derivatize KYNA, 在锌2 +的存在下, 包括在 nanom...

金伯利进程负责降解近95% 的必需氨基酸色氨酸1。在哺乳动物的大脑中, 犬尿氨被带入星形胶质细胞代谢成 neuroactive 小分子 KYNA 主要由酶犬尿氨转氨酶 (KAT) II2。KYNA 作为拮抗剂在 NMDA 和α7nACh 受体在大脑2,3,4, 也靶向信号受体包括芳烃受体 (AHR) 和 G 蛋白耦合受体 35 (GPR35)5 ,6。在实验动物中, 大脑 KYNA 的海拔在行为分析的2、7、8、9、10中被证明会损害他们的认知能力。.一个新的假说表明, KYNA 在调节认知功能方面起着不可或缺的作用, 它影响睡眠唤醒行为11, 从而进一步支持星形胶质细胞衍生分子在调节睡眠神经生物学方面的作用, 并认知12。
临床上, KYNA 的海拔在脑脊液和验尸后的脑组织中发现, 精神分裂症13,14,15,16, 一个衰弱的精神紊乱以认知障碍为特征的。精神分裂症患者也经常被睡眠紊乱所困扰, 这可能使病情恶化17。了解 KP 代谢和 KYNA 在调节睡眠与认知之间的关系, 特别是学习和记忆之间的作用, 可能会导致新的治疗精神分裂症和其他一些不良结局的治疗方法的发展。精神疾病。
一个可靠和一致的方法来测量金伯利的代谢物, 是重要的, 以确保从各个机构的研究可以融入到科学的理解, kp 生物学。目前, 我们描述了用高效液相色谱法测定大鼠脑血浆和 KYNA 中犬尿氨的方法。本议定书在锌2 +的存在下使用荧光检测, 最初是由柴田18和最近适应和优化到 derivatize 以500毫米醋酸锌作为后柱试剂, 从而使在脑内内源性、nanomolar 量 KYNA 的检测11。
为刺激本议定书所述的 KYNA 内源性生产, 直接 bioprecursor 犬尿氨在大鼠体内注入腹腔 (ip)。结合生化评估来确定 KYNA 生产的程度, 犬尿氨挑战对海马依赖性记忆 (被动回避范式) 和睡眠唤醒体系 (EEG 和肌电信号) 的影响也是调查11。这些技术的结合允许研究犬尿氨挑战在大鼠体内的生物化学和功能影响。

<table><tbody><tr><td>Wistar rats</td><td>Charles River Laboratories</td><td></td><td>adult male, 250-350 g</td></tr><tr><td>L-kynurenine sulfate</td><td>Sai Advantium</td><td></td><td></td></tr><tr><td>ReproSil-Pur C18 column (4 mm x 150 mm)</td><td>Dr. Maisch GmbH</td><td></td><td></td></tr><tr><td>EZ Clips</td><td>Stoelting Co.</td><td>59022</td><td></td></tr><tr><td>Mounting materials screws</td><td>PlasticsOne</td><td>00-96 X 1/16</td><td></td></tr><tr><td>Nonabsorbable Sutures</td><td>MedRep Express</td><td>699B</td><td>CP Medical Monomid Black Nylon Sutures, 4-0, P-3, 18&quot;, BOX of 12</td></tr><tr><td>Absorbable Sutures</td><td>Ethicon</td><td>J310H</td><td>4-0 Coated Vicryl Violet 1X27&#039;&#039; SH-1</td></tr><tr><td>Dental Cement</td><td>Stoelting Co.</td><td>51458</td><td></td></tr><tr><td>Drill Bit</td><td>Stoelting Co.</td><td>514551</td><td>0.45 mm</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Alliance HPLC system&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>E2695 separation module</td><td>Waters</td><td>176269503</td><td></td></tr><tr><td>2475 fluorescence detector</td><td>Waters</td><td>186247500</td><td></td></tr><tr><td>post-column reagent manager</td><td>Waters</td><td>725000556</td><td></td></tr><tr><td>Lenovo computer</td><td>Waters</td><td>668000249</td><td></td></tr><tr><td>Empower software</td><td>Waters</td><td>176706100</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Passive avoidance box for rat&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Extra tall MDF sound attenuating cubicle</td><td>MedAssociates</td><td>ENV-018MD</td><td>Interior: 22&quot; (W) x 22&quot; (H) x 16&quot; (D)</td></tr><tr><td>Center channel modulator shuttle box chamber</td><td>MedAssociates</td><td>ENV-010MC</td><td></td></tr><tr><td>Stainless steel grid floor for rat</td><td>MedAssociates</td><td>ENV-010MB-GF</td><td></td></tr><tr><td>Auto guillotine door</td><td>MedAssociates</td><td>ENV-010B-S</td><td></td></tr><tr><td>Quick disconnect shuttle grid floor harness for rat</td><td>MedAssociates</td><td>ENV-010MB-QD</td><td></td></tr><tr><td>Stimulus light, 1&quot; white lens, mounted on modular panel</td><td>MedAssociates</td><td>ENV-221M</td><td></td></tr><tr><td>Sonalert module with volume control for rat chamber</td><td>MedAssociates</td><td>ENV-223AM</td><td></td></tr><tr><td>SmartCtrl 8 input/16 output package</td><td>MedAssociates</td><td>DIG-716P2</td><td></td></tr><tr><td>8 Channel IR control for shuttle boxes</td><td>MedAssociates</td><td>ENV-253C</td><td></td></tr><tr><td>Infrared source and dectector array strips</td><td>MedAssociates</td><td>ENV-256</td><td></td></tr><tr><td>Tabletop interface cabinet, 120 V 60 Hz</td><td>MedAssociates</td><td>SG-6080C</td><td></td></tr><tr><td>Dual range constant current aversive stimulation module</td><td>MedAssociates</td><td>ENV-410B</td><td></td></tr><tr><td>Solid state grid floor scrambler module</td><td>MedAssociates</td><td>ENV-412</td><td></td></tr><tr><td>Dual A/B shock control module</td><td>MedAssociates</td><td>ENV-415</td><td></td></tr><tr><td>2&#039; 3-Pin mini-molex extension</td><td>MedAssociates</td><td>SG-216A-2</td><td></td></tr><tr><td>10&#039; Shock output cable, DB-9 M/F</td><td>MedAssociates</td><td>SG-219G-10</td><td></td></tr><tr><td>Shuttle shock control cable 15&#039;, 6</td><td>MedAssociates</td><td>SG-219SA</td><td></td></tr><tr><td>Small tabletop cabinet and power supply, 120 V 60 Hz</td><td>MedAssociates</td><td>SG-6080D</td><td></td></tr><tr><td>PCI interface package</td><td>MedAssociates</td><td>DIG-700P2-R2</td><td></td></tr><tr><td>Shuttle box avoidance utility package</td><td>MedAssociates</td><td>SOF-700RA-7</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Sleep-Wake Monitoring Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ponehmah software</td><td>Data Sciences International (DSI)</td><td>PNP-P3P-610</td><td></td></tr><tr><td>MX2 8 Source Acquisition interface</td><td>Data Sciences International (DSI)</td><td>PNM-P3P-MX204</td><td></td></tr><tr><td>Dell computer, Optiplex 7020, Windows 7, 64 bit</td><td>Data Sciences International (DSI)</td><td>271-0112-013</td><td></td></tr><tr><td>Dell 19&quot; computer monitor</td><td>Data Sciences International (DSI)</td><td>271-0113-001</td><td></td></tr><tr><td>Receivers for plastic cages, 8x</td><td>Data Sciences International (DSI)</td><td>272-6001-001</td><td></td></tr><tr><td>Cisco RV130 VPN router</td><td>Data Sciences International (DSI)</td><td>RV130</td><td></td></tr><tr><td>Matrix 2.0</td><td>Data Sciences International (DSI)</td><td>271-0119-001</td><td></td></tr><tr><td>Network switch</td><td>Data Sciences International (DSI)</td><td>SG200-08P</td><td></td></tr><tr><td>Neuroscore software</td><td>Data Sciences International (DSI)</td><td>271-0171-CFG</td><td></td></tr><tr><td>Two biopotential channels transmitter, model TL11M2-F40-EET</td><td>Data Sciences International (DSI)</td><td>270-0134-001</td><td></td></tr></tbody></table>

a high-performance liquid chromatography measurement of kynurenine and kynurenic acid: relating biochemistry to cognition and sleep in rats

色氨酸降解的犬尿氨通路 (KP) 与精神疾病有牵连。具体地说, 星形胶质细胞衍生代谢物 kynurenic 酸 (KYNA) 是n-甲基-d-天门冬氨酸 (NMDA) 和α7烟碱乙酰胆碱 (α7nACh) 受体的拮抗剂, 与健康和疾病的认知过程有牵连。随着精神分裂症患者大脑中 KYNA 水平的升高, 谷氨酸和胆碱能受体的故障被认为是与认知功能障碍相关的因果关系, 这是疾病精神病理学的核心领域。KYNA 可能在精神分裂症的个体中扮演 pathophysiologically 重要的角色。通过直接 bioprecursor 犬尿氨对动物进行治疗, 可以提高啮齿动物脑内内源性 KYNA, 大鼠的临床前研究表明, KYNA 的急性海拔可能会影响他们的学习和记忆过程。本协议详细描述了这一实验方法, 并结合 a) 对血液犬尿氨水平和脑 KYNA 形成 (使用高效液相色谱) 的生化分析, b) 行为测试, 以探讨与海马相关的上下文记忆 (被动回避范式) 和 c) 对睡眠唤醒行为的评估 [将脑电图 (EEG) 和肌电图 (肌电信号) 信号结合在一起的遥测记录] 在大鼠中。结合起来, 研究了高 KYNA、睡眠和认知之间的关系, 该协议详细描述了一种实验方法来了解大鼠体内犬尿氨海拔和 KYNA 形成的功能结果。通过这一协议的变化得到的结果将测试的假说, KP 和 KYNA 在调节睡眠和认知的健康和疾病状态的关键作用。

为了验证采用腹腔犬尿氨注射液作为提高脑 KYNA 的方法, 对组织进行了 HPLC 分析。标准曲线 (图 1) 是使用相关软件构造的, 并允许对组织样本进行量化。犬尿氨和 KYNA 的代表性图谱如图 2所示。犬尿氨在6分钟的保留时间内观察到, KYNA 的保留时间为11分钟。为了观察 KP 动力学, 在本协议中管理了100毫克/千克的犬尿氨, 并在注射后立...

Watch this Scientific Journal Video about A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats at JoVE.com

A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats

高效液相色谱法测定犬尿氨和 Kynurenic 酸: 对大鼠认知和睡眠的相关生物化学研究

犬尿氨通路 (KP) neuroactive 代谢物的改变与精神疾病有牵连。调查啮齿类动物体内改变的犬尿氨通路代谢的功能性结果可能有助于阐明新的治疗方法。目前的协议结合了生物化学和行为学的方法来研究急性犬尿氨挑战对大鼠的影响。

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite ...

a-high-performance-liquid-chromatography-measurement-kynurenine

Research

JoVE Journal

Behavior

10.4K 次观看.  University of Maryland School of Medicine. 犬尿氨通路 (KP) neuroactive 代谢物的改变与精神疾病有牵连。调查啮齿类动物体内改变的犬尿氨通路代谢的功能性结果可能有助于阐明新的治疗方法。目前的协议结合了生物化学和行为学的方法来研究急性犬尿氨挑战对大鼠的影响。

视频: A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

用高压液相色谱法提取和分析儿茶酚胺神经递质及其代谢物的简便方法

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

科研

化学

Noninvasive, High-throughput Determination of Sleep Duration in Rodents

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a long recovery period. To perform an EEG recording, the animal is connected to a receiver, creating an unnatural tether to the head-mount. EEG monitoring is time consuming, carries risk to the animal, and is not a completely natural setting for the measurement of sleep. Alternative methods to detect sleep, particularly in a high-throughput fashion, would greatly advance the field of sleep research. Here, we describe a validated method for detecting sleep via activity-based home-cage monitoring. Previous studies have shown that sleep assessed via this method has a high degree of agreement with sleep defined by traditional EEG-based measures. Whereas this method is validated for total sleep time, it is important to note that sleep bout duration should be assessed by an EEG which has better temporal resolution. The EEG can also differentiate rapid eye movement (REM) and non-REM sleep, giving more detail about the exact nature of sleep. Nevertheless, activity-based sleep determination can be used to analyze multiple days of undisturbed sleep and to assess sleep as a response to an acute event (like stress). Here, we show the power of this system to detect the response of mice to daily intraperitoneal injections.

We describe a high-throughput method of measuring sleep by means of activity-based home-cage monitoring. This method offers advantages over traditional EEG-based methods. It is well validated for the determination of total sleep duration and can be a powerful tool to monitor sleep in rodent models of human disease.

无创高通量测定啮齿类动物睡眠时间

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a ...

行为学

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the interpretation of the biological activity of any compound in drug development or in basic research projects. In epigenetics, target engagement is most often assessed by the analysis of proxy markers instead of measuring the union of the compound to the target. Downstream biological readouts that have been analyzed include the histone mark modulation or gene expression changes. KDM1A is a lysine demethylase that removes methyl groups from mono- and dimethylated H3K4, a modification associated with the silencing of gene expression. Modulation of the proxy markers is dependent on the cell type and function of the genetic make-up of the cells investigated, which can make interpretation and cross-case comparison quite difficult. To circumvent these problems, a versatile protocol is presented to assess the dose effects and dynamics of the direct KDM1A target engagement. The assay described makes use of a KDM1A chemoprobe to capture and quantify uninhibited enzyme, can be broadly applied to cells or tissue samples without the need for genetic modification, has an excellent window of detection, and can be used both for basic research and analysis of clinical samples.

Here, we present a protocol to measure KDM1A target engagement in a human or animal cell, tissue or blood samples treated with KDM1A inhibitors. The protocol employs chemoprobe tagging of the free KDM1A enzyme and direct quantification of the target occupation using chemoprobe-based immunoassays and can be used in preclinical and clinical studies.

使用基于化学探针的免疫测定直接测量KDM1A目标参与

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the ...

生物化学

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

神经科学

Collecting Sleep, Circadian, Fatigue, and Performance Data in Complex Operational Environments

Sleep loss and circadian misalignment contribute to a meaningful proportion of operational accidents and incidents. Countermeasures and work scheduling designs aimed at mitigating fatigue are typically evaluated in controlled laboratory environments, but the effectiveness of translating such strategies to operational environments can be challenging to assess. This manuscript summarizes an approach for collecting sleep, circadian, fatigue, and performance data in a complex operational environment. We studied 44 airline pilots over 34 days while they flew a fixed schedule, which included a baseline data collection with 5 days of mid-morning flights, four early flights, four high-workload mid-day flights, and four late flights that landed after midnight. Each work block was separated by 3&#8211;4 days of rest. To assess sleep, participants wore a wrist-worn research-validated activity monitor continuously and completed daily sleep diaries. To assess the circadian phase, pilots were asked to collect all urine produced in four or eight hourly bins during the 24 h after each duty block for the assessment of 6-sulfatoxymelatonin (aMT6s), which is a biomarker of the circadian rhythm. To assess subjective fatigue and objective performance, participants were provided with a touch-screen device used to complete the Samn-Perelli Fatigue Scale and Psychomotor Vigilance Task (PVT) during and after each flight, and at waketime, mid-day, and bedtime. Using these methods, it was found that sleep duration was reduced during early starts and late finishes relative to baseline. Circadian phase shifted according to duty schedule, but there was a wide range in the aMT6s peak between individuals on each schedule. PVT performance was worse on the early, high-workload, and late schedules relative to baseline. Overall, the combination of these methods was practical and effective for assessing the influence of sleep loss and circadian phase on fatigue and performance in a complex operational environment.

Sleep loss and circadian misalignment contribute to numerous operational accidents and incidents. The effectiveness of countermeasures and work scheduling designs aimed at mitigating fatigue can be challenging to evaluate in operational environments. This manuscript summarizes an approach for collecting sleep, circadian, fatigue, and performance data in complex operational environments.

在复杂操作环境中收集睡眠、循环、疲劳和性能数据

Sleep loss and circadian misalignment contribute to a meaningful proportion of operational accidents and incidents. Countermeasures and work scheduling ...

Human Circadian Phenotyping and Diurnal Performance Testing in the Real World

In our continuously developing &#39;around the clock&#39; society, there is a need to increase our understanding of how changes in biology, physiology and psychology influence our health and performance. Embedded within this challenge, is the increasing need to account for individual differences in sleep and circadian rhythms, as well as to explore the impact of time of day on performance in the real world. There are a number of ways to measure sleep and circadian rhythms from subjective questionnaire-based methods to objective sleep/wake monitoring, actigraphy and analysis of biological samples. This paper proposes a protocol that combines multiple techniques to categorize individuals into Early, Intermediate or Late circadian phenotype groups (ECPs/ICPs/LCPs) and recommends how to conduct diurnal performance testing in the field. Representative results show large differences in rest-activity patterns derived from actigraphy, circadian phase (dim light melatonin onset and peak time of cortisol awakening response) between circadian phenotypes. In addition, significant differences in diurnal performance rhythms between ECPs and LCPs emphasizes the need to account for circadian phenotype. In summary, despite the difficulties in controlling influencing factors, this protocol allows a real-world assessment of the impact of circadian phenotype on performance. This paper presents a simple method to assess circadian phenotype in the field and supports the need to consider time of day when designing performance studies.

Here, we present a method to investigate diurnal rhythms in performance following accurate categorization of participants into circadian phenotype groups based on the Munich ChronoType Questionnaire, gold standard circadian phase biomarkers and actigraphic measures.

现实世界中的人类昼夜Phenotyping和二年性能测试

In our continuously developing &#39;around the clock&#39; society, there is a need to increase our understanding of how changes in biology, physiology and ...

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

印刷业记录程序在小鼠体内测量睡眠

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

生物工程

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...