Name: autoradiographic measurements of [14c]-iodoantipyrine in rat brain following central post-stroke pain
Uploaded: 2016-07-18T15:00:00.000+00:00
Duration: 7 min 27 s
Description: Watch this Scientific Journal Video about Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain at JoVE.com

本研究是由国家科学委员会授予柏庄徐中雄博士（NSC 99-2320-B-001-016-MY3，NSC 100-2311-B-001-003-MY3和NSC 102-2320-支持B-001-026-MY3）。这项工作是在生物医学科学，并获得资助中央研究院研究所进行。

在本研究中的协议接收来自中科院动物管理审批和利用委员会在台湾。 1.动物准备<ol><li>获取雄性SD大鼠（约300 - 400克）。保持在空调房间的老鼠 - 12小时/ 12小时光/暗周期下（21 22°C，湿度为50％）（在8:00 am开灯），自由获取食物和水。 </li></ol> 2.实验步骤<ol><li>允许所有的大鼠的实验前适应环境在他们的家笼1周。在适应，执行冯弗雷和足底测试，以建立基线。 </li><li> 冯弗雷测试 <ol><li>放置在丙烯酸类外壳（30厘米×30厘米×80厘米）的大鼠用于居住30分钟。 </li><li>得到的von Frey长丝（长丝没有11日 - 20）具有相同的长度，但变化的直径，以提供范围的2力量 - 百克。 </li><li>为了评估在大鼠后爪的爪缩回阈值，使用的von Frey长丝通过一个网状端口刺激后爪的中心上丙烯酸板在5分钟intertrial间隔。以记录最大施加压力，使用长丝以升序，从低到高，直到最大施加压力被记录。10 </li><li>当大鼠显示出爪缩回响应于刺激，记录长丝数目。按最低刺激确定本试验的戒断反应。 </li><li>立即重复相同的方法，对在连续共三次在同一老鼠。转换长丝数为相应的力（克）和平均值。 </li></ol></li><li> 足底测试 <ol><li>放置老鼠在一个透明的有机玻璃盒（分为四个帧80厘米* 30厘米* 15厘米）对于居住30分钟。 </li><li>用INFrared光束通过玻璃板刺激后爪的中心。调整的红外光的强度来获得大约10秒的平均缩爪反应潜伏期。通过按下，轮流红外光源，并启动数字固态计时器一键进行审判。操纵红外光束的持续时间。 </li><li>记录的红外光的持续时间时，大鼠表现出缩爪反应。最长持续时间不应该在每次试验中超过20秒，以避免组织损伤。使用至少5分钟的intertrial间隔，以避免相继的刺激。 </li><li>重复试验用三个试验用于左和右后爪，并计算平均每个后爪每只大鼠。 </li></ol></li><li> 胶原酶病变手术 <ol><li>麻醉用4％的异氟醚大鼠直到脚趾捏失去响应和躯体反应，手术刺激的发生。保持与1.5麻醉 - 2％isoflur烷手术的持续时间。 </li><li>放置在立体定位装置中的鼠以简单的加热垫维持体温在36.5 - 37.5℃。对眼睛涂抹眼霜，以防止干燥时的麻醉下。 </li><li>刮皮毛用无菌电推剪，并进行了切口光滑（约2-2.5厘米），沿头皮中线手术刀。清洁皮肤和颅骨交替普罗维登斯碘和消毒75％的酒精溶液。在手术阶段中，使用75％的酒精消毒的所有设备，工具和工作台保持无菌条件。在此之前，所有的手术材料必须在蒸汽高压灭菌器进行灭菌。 </li><li>使用无菌手套和工具，以及钻一小孔（直径3mm）的头骨用电钻在VB（包括腹后丘脑核[VPM]和腹后外侧丘脑核[VPL]）丘脑（3.0 -3.5毫米后，3.0-3.5毫米横向到前囟门，从颅骨表面5.5-5.8毫米深）。 </li><li> Microinject 0.5微升生理盐水或分别在对照组和实验组，4型胶原酶溶液0.125Ú。 </li><li>保持到位注射针对另外5分钟，以允许药物扩散。 </li><li>使用牙科水泥填补在颅骨孔，缝合切口。缝合切口后，应用局部止痛麻醉剂（利多卡因软膏）的伤口，他们回到自己的家乡笼。 </li><li>手术后，单独的房子在塑料保持架的老鼠，直到他们保持胸骨斜卧，直到从麻醉中恢复保持温暖。 </li></ol></li><li> 手术后的恢复行为测试 <ol><li>经过7天手术后恢复的，重复的部分2.2和2.3的测试阶段的过程。在4周内进行行为测试，同时监测健康和动物的发育状况。 </li><li&gt;监控对照组和实验组的手术后阶段之间的动物的健康状况（ 例如 ，体重，摄食量，和自由运动）。 </li></ol></li><li> 与PE-50管插管进行植入 <ol><li>麻醉，用4％的异氟醚的大鼠，和在36.5保持体温 - 37.5℃，用简单的加热垫。 </li><li>用手术刀，在前肢的背侧部和交汇分别左肩和胸腔，腹部分的中线切开两个孔（直径2厘米每个）。 </li><li>解离的皮肤和肌肉有一对的两个孔之间剪刀。 </li><li> PE-50管（20厘米长），以颈外静脉的一端连接通过腹孔。连接相同的PE-50管向背侧孔的终端，并加盖到皮肤上。 </li><li>用注射器注入盐水进入颈静脉，以验证该PE-50管不受阻碍。 </li><li>缝合切口，并与6毫克/公斤庆大霉素（腹膜内）注射大鼠。 </li><li>冲洗管手术后每隔一天用0.3毫升0.9％盐水，随后用0.1毫升20单位/毫升肝素的生理盐水。 </li></ol></li><li> 最终的行为测试 <ol><li>手术后恢复的步骤相比2.5 2.6步后，重复步骤2.2-2.3，以确认该行为一周是稳定的。 </li></ol></li><li> 注射放射性示踪剂 <ol><li>放置老鼠在静止笼5 - 10分钟的适应。 </li><li>使用分离器，PE-50管连接两个1毫升注射器。填充一个针筒生理盐水并填充其它与[14 C] -IAP溶液（125微居里/公斤的0.3体积- 0.5毫升）中。 </li><li>注入示踪剂进入外部颈静脉，用填充有氯化物的3M钾另一个注射器更换注射器。 </li><li>放射性示踪剂注射十秒钟后，挠度，按照标准的安乐死方法注入3氯化钾异氟醚过量之下，牺牲动物（ 即从50分钟蒸发器异氟醚）的基础上中央研究院机构动物护理在台湾的指导方针和利用委员会。 </li><li> 1分钟后，暴露颅骨，并剪掉剩余的肌肉。用咬骨钳，剥远离大脑头骨的背水面。剪掉用咬骨钳头骨的侧面。接着，用刮刀，切割沿脑腹面嗅球和神经的连接，并取出大脑。 </li><li>使用最佳切割温度（OCT）化合物冻结脑在干冰和甲基丁烷（约-55℃）。存储脑组织中的冷冻11,12 </li></ol></li><li> 脑切片 <ol><li>定向组织的切片，与后脑朝下。使用低温恒温器向大脑切成20＆＃181;微米厚的切片。 </li><li>放置在显微镜玻片上的脑切片中，在-20℃的低温恒温器，其中每个切片之间的240微米的间隔。 </li><li>放置在显微镜载玻片并用分级放射性成曝光盒在-20°C 3天五个标准滤纸。根据脑切片的顺序，安排在显微镜载玻片从上到下。最后，将滤纸在盒的底部。12 </li><li>从曝光盒除去荧光屏，并使用可变模式成像器读取荧光屏并生成图像显示[14 C] -IAP摄取对大脑切片。 </li></ol></li></ol> 3.数据分析<ol><li> 2.9.4步后，使用统计参数映射（SPM）和ImageJ的软件调整图像。重建全部采用串行冠状面图像。平滑和根据参考大鼠脑模型标准化图像。12,13 </li><li>为定量评估，使用ImageJ软件，以确定该像素的信号强度，并使用统计软件，用于分析测量该区域的感兴趣的脑图像（ROI）12，13 </li><li>为了研究不同的大脑核团之间的连接，使用MATLAB相关分析软件在区域间相关矩阵，显示放射性比值，和可视化矩阵彩色地图。最后，使用Pajek软件进行网络分析。12，13 </li><li>用2×5双向方差混合分析（ANOVA），用基团和周为因素，耐热性的持续时间在基线比较在假和CPSP组的von Frey试验的足底试验和机械力和周1 - 5，使用2×31双向ANOVA根据组和大脑区域测量放射性比。在适当的时候，进行Tukey的真实显着性差异（HSD） 事后检验。卡尔culate Pearson相关系数在所有的假和CPSP组选定的大脑区域，以评估相关性。 </li></ol>

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在行为测试中，CPSP集团展出在冯弗雷测试热痛试验和机械力在基线和缩足阈值的降低周1 - 5，发现与先前的研究相一致14 的[14 C] -IAP方法依赖于大脑图像的不同的脑切片的定量分析的像素强度。为了评估在大脑的图像数据，所述像素信号强度被定义。在本研究中，对环境的背景信号定义为25.811 - 46.979 CPM。在[14 C] -IAP 0.001微居里滤纸的信号被定义为42 CP...

中风出血已被证明是谁的神经性疼痛患者遭受超过8％的发生，4称为中央中风后疼痛（CPSP）1-3 CPSP可以从体感功能障碍，从而诱发过敏和异常性造成的。但是，体感功能障碍的CPSP病理生理机制仍然不明朗。例如，躯体感觉的损失从出血脑区的神经元传入神经阻滞造成的。痛觉过敏可由中央伤害感受神经元或中央去抑制，5，6，但所涉及的CPSP症状神经基板保持未知的过度兴奋引起的。一些研究表明，背外侧前额叶皮层（DPFC），喙前扣带皮层（ACC），杏仁核，海马，脑导水管周围灰质（PAG），喙腹内侧髓质，以及它们相互连接的调解处理痛觉7附加被证明LY，内侧前额叶皮质（内侧前额叶皮质）-amygdala电路参与疼痛相关的看法。在CPSP的病理生理机制8数据是多种多样的，神经基板的CPSP的激活需要进一步推敲。 [14 C] -Iodoantipyrine（IAP）的摄取来间接观察区域脑血流量（rCBF），假设大脑活动和CBF之间的关系。尽管[14 C] -IAP不能实时评估脑活动，如与功能性磁共振成像（fMRI），它具有若干优点。例如，[14 C] -IAP适合测量自发过程的病理状态中发生脑活动。9此外，[14 C] -IAP摄取没有麻醉测量。它还成本低于其它成像方法，包括磁共振成像和正电子发射断层扫描（PET）。的[14 C] -IAP方法已经建议适合measurin克自发性疼痛（ 例如 ，CPSP）由丘脑的腹侧基底核（VB）的病变引起的。9 本协议描述了如何执行[14 C] -IAP的方法来评估由丘脑VB的病变动物模型引起CPSP的神经基础的参与。该技术提供了确定所依据的行为和神经元水平CPSP症状的病理生理机制的一种方式。

<table><tbody><tr><td>&lt;strong&gt;Anesthetic:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Halocarbon Products Corporation&amp;nbsp;</td><td>NDC 12164-002-25</td><td>4%</td></tr><tr><td>&lt;strong&gt;Surgery&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>homeothermic blanket system</td><td>Harvard Apparatus</td><td>Model 50&amp;ndash;7079</td><td>body temperature were maintained at 36.5 - 37.5 &amp;deg;C.</td></tr><tr><td>10 &amp;micro;l micro syringe</td><td>Hamilton</td><td>80008, Model 1701SN</td><td>injected with collagenase</td></tr><tr><td>polyethylene-50 tubing</td><td>Becton, Dickinson and Company</td><td>427411</td><td>catheterized into external jugular vein</td></tr><tr><td>1 c.c syringe</td><td>Terumo Medical Products</td><td>SS-01T</td><td>injected with &lt;sup&gt;14&lt;/sup&gt;C-IAP and saline.</td></tr><tr><td>saline (Sodium Chloride 0.9 gm)</td><td>Taiwan Biotech Co., LTD.</td><td>100-130-0201</td><td>To flush the tube</td></tr><tr><td>&lt;strong&gt;Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>type 4 collagenase</td><td>Sigma</td><td>C5138-500MG</td><td>0.125 U</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G1264-250MG</td><td>6 mg/kg</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H9399</td><td>20 U/ml; 0.1 ml/day</td></tr><tr><td>&lt;sup&gt;14&lt;/sup&gt;C-iodoantipyrine (IAP)</td><td>PerkinElmer</td><td>NEC712</td><td>125 mCi/kg in 300 ml of 0.9% saline</td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>1.04936.1000</td><td>3 M</td></tr><tr><td>&lt;strong&gt;Behavior system:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>von Frey esthesiometer</td><td>Fabrication Enterprises, Inc.</td><td>Baseline Tactile Monofilaments 12-1666</td><td>mechanical hyperalgesia was assessed by measuring the withdrawal response to a mechanical stimulus</td></tr><tr><td>plantar test apparatus</td><td>IITC Life Science</td><td>IITC 390G Plantar Test</td><td>Thermal hyperalgesia was assessed by measuring the hind paw withdrawal latency in response to radiant heat.</td></tr><tr><td>&lt;strong&gt;Brain slice:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature compound</td><td>Sakura Fintek Inc</td><td>4583</td><td>embedded the brain</td></tr><tr><td>dry ice</td><td></td><td></td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>methylbutane</td><td>Sigma</td><td>M32631-1L</td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>Cryostat</td><td>&amp;nbsp;Leica Biosystems Nussloch GmbH, Germany</td><td>Leica CM1850</td><td>Coronal brain slice were sectioned on this machine.</td></tr><tr><td>&lt;strong&gt;Data analyze&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>exposure cassettes with a phosphor screen</td><td>Amersham Biosciences&amp;nbsp;</td><td>20 cm x 25 cm</td><td>The slices were dried on glass slides and placed alongside five standard filter papers with graded radioactivity. All of the slides were exposed to the cassettes at &amp;minus;20 &amp;deg;C.</td></tr><tr><td>&amp;gamma;-counter</td><td>Beckman Coulter</td><td>Beckman LS 6500 Liquid Scintillation Counter</td><td>To measure the radioactivity count of the filter papers.</td></tr><tr><td>Typhoon 9410 Variable Mode Imager</td><td>&amp;nbsp;GMI, Inc.</td><td>WS-S9410</td><td>To read&amp;nbsp; phosphor screen which was exposed by brain slice</td></tr><tr><td>Statistical Parametric Mapping (SPM)</td><td>Wellcome Centre for Neuroimaging</td><td>version 8</td><td>all of the brains were averaged to create the final brain template. To determine significant differences between the images in these two groups, the images were derived by subtracting the sham group from the CPSP group.</td></tr><tr><td>ImageJ</td><td>http://imagej.nih.gov/ij</td><td>version 1.46</td><td>Adjacent sections were aligned both manually and using Stack- Reg, an automated pixel-based registration algorithm in ImageJ software. All of the original three-dimensionally reconstructed brains were smoothed and normalized to the reference rat brain model.</td></tr><tr><td>Matlab</td><td>MathWorks</td><td>version 2009b</td><td>used Pearson correlation coefficients to examine the relationships between the CPSP and sham groups. An inter-regional correlation matrix was calculated across animals from each group.</td></tr><tr><td>Pajek</td><td>http://Pajek.imfm.si/</td><td>version 3.06</td><td>Graphical theoretical analysis was performed on networks defined by the above correlation matrices using Pajek software.</td></tr></tbody></table>

autoradiographic measurements of [14c]-iodoantipyrine in rat brain following central post-stroke pain

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to nociceptive stimuli. Although some studies have shown that neuropathic pain may involve the dorsolateral prefrontal cortex, rostral anterior cingulate cortex, amygdala, hippocampus, periaqueductal gray, rostral ventromedial medulla, and medial thalamus, the neural substrates and their connections that mediate CPSP remain unclear. [14C]-Iodoantipyrine (IAP) uptake can be measured to evaluate spontaneously active pain. It can be used to assess the activation of neural substrates that may be involved in CPSP in an animal model. The [14C]-IAP method in rats is less expensive to perform compared with other brain mapping techniques. The present [14C]-IAP protocol is used to measure the activation of neural substrates that are involved in CPSP that is induced by lesions of the ventral basal nucleus (VB) of the thalamus in a rodent model.

图1A描绘了实验时间表。大鼠被分配到假和CPSP群体的行为测试（ 即冯弗雷测试和足底试验）。在实验的第一天担任基线，并在周1重复测试 - 在周中的外颈静脉进行5 PE-50导管4.肝素（20单位/毫升，0.1毫升/天）期间，注射周4和5的肝素注射后五分钟，[14 C] -IAP注射，接着10秒后通过麻醉剂的牺牲过量。一分钟后，将大鼠断头，和OCT包埋脑切片进行?...

Watch this Scientific Journal Video about Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain at JoVE.com

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

的放射自显影测量[<sup&gt; 14</sup&gt; J] -Iodoantipyrine大鼠脑继中央脑卒中后疼痛

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to ...

autoradiographic-measurements-14c-iodoantipyrine-rat-brain-following

Research

JoVE Journal

Medicine

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

8.7K 次观看.  Fo Guang University. We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

视频: Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2血管闭塞/低血压：全脑缺血模型大鼠

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

科研

医学

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

一种中脑动脉闭塞技术在大鼠脑卒中后抑郁中的诱导

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

行为学

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

神经科学

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

生物化学

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

无创成像和脑缺血分析生活中的大鼠使用正电子发射断层扫描<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

颅内药物治疗和啮齿动物疼痛检测

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

定量自动放射法测定维沃脑蛋白合成区域速率

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

Neuroinflammation 的 PET 成像 [11C] DPA-713 在小鼠缺血性中风模型中的应用

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

测量一组啮齿动物脑样本中的中风后脑水肿、梗塞区和血脑屏障破裂

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

的放射自显影测量[

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