这项工作的一部分是由乌得勒支大学生命科学补助金资助的。

所有动物实验都是按照国际准则进行的, 并事先得到当地实验伦理委员会的批准。全血是从小型捐助者服务 (迷你捐助者 Dienst, MDD) 在大学医学中心乌得勒支 (UMCU) 在荷兰。MDD 得到了 UMCU (Medisch Ethische Toetscommissie) 的医学伦理学委员会的积极批准, 其编号为 07-125/C 号议定书。
1. 蛋白质的生产和表征
<ol><li>细胞培养 注意: 请参阅所用培养基的材料表。<ol>
<li>将 HEK293-F 细胞解冻37摄氏度, 直到有一小块冰块。将解冻后的细胞立即转移至10毫升的冰冷介质 (4 °c), 并将细胞悬浮在室温下 (RT), 在 500 x g 处进行4分钟 (分钟)。</li>
		<li>去除培养基和并用重悬在10毫升的新鲜培养基在 RT, 其次是离心 500 x 克4分钟。</li>
		<li>取出上清液, 并用重悬5毫升培养基中的细胞颗粒. 将细胞悬浮液直接添加到含有25毫升预热培养基 (37 °c) 的125毫升烧瓶中。将单元格在37°c、8% CO2和95% 湿度的轨道振动筛上以 125 rpm 的速度旋转。</li>
		<li>每周两次亚文化细胞。使用自动单元格计数器 (请参阅材料表) 来评估细胞数量和细胞活力。种子存活细胞在浓度为 3 x 105细胞/毫升成锥形烧瓶。保持培养基总容积在锥形瓶总容积的 1/5, 保持曝气最佳。</li>
	</ol></li>
	<li>转 染 注: 在细胞活力为 > 90% 时, 在转染和染细胞的三到四倍前亚文化。基于等离子膜完整性的自动 (电场多通道) cellcounting 系统对细胞生存能力进行评估。请参阅转染试剂的材料表。<ol>
<li>在 500 x g 上离心细胞在 RT 4 分钟;并用重悬细胞颗粒在10毫升预热培养基 (37 °c) 和稀释细胞悬浮到 ~ 1.0 x10 6 细胞/毫升在总容量 1 l 整除细胞悬浮在10毫升整除数 100 ml 锥形烧瓶。</li>
		<li>稀释1毫克的质粒 DNA 在33毫升减少血清最低基本培养基 (记忆, 参见材料表) 通过温和的混合。在一个单独的管, 稀释1330µL 的转染试剂在内存中通过温和的混合。孵育两个解决方案在 rt 5 分钟添加和混合的 DNA 稀释在内存中的转染试剂, 并孵化的混合30分钟的 rt。</li>
		<li>将 DNA 转染试剂组合添加到细胞中 (在1节中培养);添加6.6 毫升的混合到每500毫升锥形瓶含有100毫升细胞在培养基中。维护先前提到的文化条件中的细胞。在转染后三天收获含有分泌 IL4-10 融合蛋白的培养基上清液。</li>
		<li>根据制造商的协议执行 IL10 ELISA, 确定 IL4-10 融合蛋白在培养中清液中的浓度 (请参阅材料表)。 注: 在最佳培养条件下, 融合蛋白的浓度可预计为2.5 至5µg/毫升。</li>
	</ol></li>
	<li>亲和层析法纯化蛋白质 注意: 亲和层析是在室温下进行的。然而, 所有的分数 (文化上清, 负荷, 流动, 和洗脱分数) 是保存在冰过程中。<ol>
<li>根据制造商的协议 (参见材料表), 10 毫克的内部制作的单克隆抗体抗 IL4 到1克 CNBr 激活琼脂糖4B。将2.5 毫升的耦合琼脂糖珠慢慢倒入玻璃色谱柱 (30 厘米长和1.5 厘米内径, 请参阅材料表)。</li>
		<li>通过将含有1% 牛血清白蛋白的磷酸盐缓冲盐水 (PBS) 通过柱状体, 阻断珠的剩余结合部位。用25毫升的 pbs (ph = 7.4) 冲洗柱子, 后跟12.5 毫升的0.1 米甘氨酸缓冲液 (ph = 2.5) 和25毫升的 pbs (ph = 7.4)。</li>
		<li>将100毫升的10倍浓缩 HEK293 细胞培养液通过柱, 流速为1毫升/分钟. 用50毫升 PBS 冲洗柱。</li>
		<li>洗脱 IL4-10 融合蛋白与12.5 毫升的0.1 米甘氨酸缓冲 (pH = 2.5) 的流速为1毫升/分, 并收集2.5 毫升的分数。添加350µL 1 米三缓冲 (pH = 9) 的每一个分数, 使 pH 值为7。透析中和的分数过夜对2升的 PBS (pH = 7.4)。<ol>
<li>使用16毫米干径和 3.5 K 分子量切断的透析油管。无菌过滤器透析馏分使用0.45 µM 孔径的一次性过滤器。贮存无菌 IL4-10 融合蛋白溶液在整除数-80 摄氏度。</li>
		</ol>
</li>
		<li>用考马斯染色的12% 页凝胶和 HP 秒 (3 µm SEC-2000 柱) 评价洗脱 IL4-10 融合蛋白的纯度。对于 HP SEC 分析, 在柱上的浓度为1毫克/毫升的蛋白质中加载40µL。使用100毫米磷酸钠缓冲液, 150 毫米氯化钠 (pH = 6.8) 作为流动相。将流速设置为1毫升/分钟。</li>
		<li>根据制造商的协议, 确定每批纯化 IL4-10 融合蛋白的浓度, IL10 ELISA 法和二辛可宁酸蛋白检测试剂盒 (蛋白检测套件, 参见材料表)。</li>
		<li>使用重组人 IL10 在 ELISA 检测中创建标准曲线。为了纠正重组 IL10 (18 kDA) 和 IL4-10 融合蛋白 (34 kDA) 之间的大小差异, 将获得的浓度乘以1.8 的因子。</li>
	</ol></li>
	<li>IL4-10 融合蛋白的生物活性研究 注: 对每批 IL4-10 融合蛋白进行全血检测, 并将不同批次的活性作为质量控制的一部分进行比较。全血化验是在新的人类血液中收集的一天的检测肝素管。血液是在我们的内部捐助者服务的健康志愿者获得的。样品应作为潜在的生物危害处理。<ol>
<li>在浓度范围 5-1215 ng/毫升的 RPMI 培养基上制备3倍稀释的纯化 IL4-10 融合蛋白 (参见材料表)。</li>
		<li>制备脂多糖 (100 稀释/毫升) 和人血液稀释4次 RPMI 培养基。</li>
		<li>将 IL4-10 融合蛋白、LPS 和人血的预稀释为48井板。对每个井, 添加50µL IL4-10 融合蛋白 (最终浓度 1-243 ng/毫升), 75 µL RPMI 培养基, 25 µL LPS (最终浓度 10 ng/毫升), 100 µL 人类血液 (最后稀释)。 注: 每井总容积为250µL。</li>
		<li>在37°c、8% CO2和95% 湿度为 18 h 孵化板材。</li>
		<li>根据制造商的协议, 用 TNFα ELISA 法评价 TNFα在培养上清液中的浓度。</li>
		<li>根据公式计算 IL4-10 融合蛋白对炎症反应的抑制作用:% 抑制 = (1-(A-b)/(C b)) x 100 注: A = TNFα水平在 lps 被刺激的文化用 IL4-10 融合蛋白质处理, B = TNFα水平在静态文化和 C = TNFα水平在 lps 刺激的文化。</li>
	</ol></li>
</ol>2. 小鼠持续炎症性疼痛模型
注意: 使用雄性和雌性小鼠 (C57Bl/6) 是很重要的, 因为这将有助于识别性依赖的效果。一般来说, 使用的老鼠是在 8-16 周的年龄之间。要确定所需的动物数量, 应执行功率计算。在因特网上可以很容易地找到用于执行此类计算的基于 Web 的工具 (例如、http://www.powerandsamplesize.com; http://www.sample-size.net)。
<ol><li>
慢性炎症性疼痛的诱导 注: intraplantar 注射卡拉胶或完整的弗洛伊德佐剂 (CFA), 诱导成年小鼠持续炎症性疼痛。不同的测试不会相互干扰。<ol>
<li>在0.9% 氯化钠中通过溶液通过23口径的针, 以确保卡拉胶被正确溶解, 通过溶解λ-卡拉胶 (参见材料表) 来制备卡拉胶 (2% 瓦特/v)。在执行注入之前, 直接准备一个新的解决方案。</li>
		<li>或者, 如果使用 cfa, 在注入 intraplantarly 之前适当地混合 cfa 解决方案, 因为 cfa 是含1毫克结核分枝杆菌 (H37Ra) 热杀死和干、0.85 毫升石蜡油和0.15 毫升 mannide 的含油乳液。油酸酯 (请参阅材料表)。 注: 建议使用金属活塞的玻璃哈密尔顿注射器注射此化合物, 因为橡胶活塞可能与佐剂中的油反应。</li>
		<li>
Intraplantar 注射液
		<ol>
<li>在注射前至少15分钟维持2.1.1 或2.1.2 的混合物, 以允许对 rt 进行调整. 注射20µL 的化合物 (或溶剂作为一个控制) 皮下到后爪的非麻醉鼠标使用30口径针。</li>
			<li>插入针在拇指的底部指导到脚跟和注射的化合物时, 针插入约0.5 厘米。注射后, 慢慢地收回针头, 同时略微旋转注射器, 避免从注射部位漏出溶液。</li>
		</ol>
</li>
	</ol>
</li>
</ol>3. 疼痛测量
注意: 确保从事行为实验的研究人员对老鼠的治疗视而不见。在进行任何测量之前, 将动物与测试环境进行适应是很重要的。最好是在开始实验前的一周, 在每个用于执行测试的不同设备中, 动物被放置 1-2 倍于 15-30 分钟。在同一个实验中处理男性和女性时, 要独立地评价雄性和雌性。在不同的群体之间广泛清洁笼子和表面, 以避免对不同性别的行为产生潜在的有害影响。测量基线撤退潜伏期或机械阈值三倍, 以准确确定基线阈值, 并确定这些是否在 intraplantar 注入任何化合物之前是稳定的。
<ol><li>
冯弗雷试验: 对无害刺激的机械敏感性
	<ol>
<li>将动物单独放置在网架上的亚克力笼中, 并允许老鼠在测量之前至少15分钟的适应。</li>
		<li>通过测量从0.02 到4克的一组校准的冯弗雷毛发, 来评估机械灵敏度. 考虑到爪子的任何运动, 从应用的头发作为积极的撤退反应。将花丝垂直于足爪表面, 施加足够的力, 对皮肤产生轻微的弯曲, 并保持 3 s. 确保头发在应用到爪子时不会扭曲。</li>
		<li>使用上下方法15、21计算50% 爪提取阈值。 注: 第一个应用的灯丝是0.4 克 (其他起动灯丝可能会根据这些小鼠在基线显示的阈值来使用)。缺乏对灯丝的反应表明, 下一个较厚的灯丝用于以下刺激, 而积极的反应表明使用下一个较薄的灯丝。头发呈现的数量是由第一个差异反应决定的;此后, 还将执行5个应用程序。根据前面15、21中描述的方法计算50% 阈值。</li>
	</ol>
</li>
	<li>
热敏性: 哈格里夫斯测试
	<ol>
<li>将动物放在预热 (30 摄氏度) 的玻璃板上, 让老鼠在测量之前至少15分钟适应, 等待动物们静静地坐着。</li>
		<li>通过使用哈格里夫斯设备的聚焦可见光束加热动物的爪子来确定热提取延迟时间 (请参阅材料表)。 注: 光束的强度设置为 12%, 因为通过实验确定的强度设置, 光束唤起了在 C57Bl/6 小鼠基线上平均时间为8秒的撤退响应。强度设置应根据个别机器和鼠标应变确定。最大的截止时间为二十年代或更多的是用来避免伤害动物。</li>
		<li>测量每只动物4次的撤退延迟时间。测量所有响应的平均值。独立测量每只爪子, 并在同一爪的后续测量之间至少有1分钟的间隔。</li>
	</ol>
</li>
	<li>
体位缺陷: 动态负重试验
	<ol>
<li>测试前测量每种动物的体重, 因为负重分析要求每个动物的体重作为输入参数。</li>
		<li>将动物单独放置在地板上的动态重量轴承系统中, 组装到一个覆盖物上, 以支持将记录鼠标移动的摄像机。在录制5分钟之前, 让动物适应0.5 到1分钟。 注意: 在这个测试中, 一次只能评估一个自由运动的动物。</li>
		<li>调整设置以执行负重和分析数据。 注: 在这些实验中, 我们使用以下参数: (i) 低权重阈值为0.6 克: 这是激活传感器的一个细胞所必需的最小重量。(ii) 高权重阈值1克: 这个数字表示完全激活一个细胞所需的重量。(三) 2 个细胞的表面阈值: 这个数字表示需要被激活才能考虑的相邻单元格的数量。(iv) 图像的最小数目为 5: 捕获每张图像需要0.1 秒。该数字指示鼠标不移动的最小帧数。因此, 值为5意味着鼠标姿态需要保持稳定, > 0.5 秒才能获得用于分析的测量。</li>
		<li>通过重播视频, 对每段视频进行分析, 确保软件在显示的时间框架内正确识别每一个肢体。 注: 至少1分钟的记录时间需要验证。动态重量轴承软件计算并提供以下参数: (一) 每只爪在克中的重量, 以及整个身体重量的百分比。(ii) 由组合前爪或组合后爪在克内的重量和整个身体重量的百分比承担。(iii) 左/右爪子负重或前/后负重的比例。(iv) 激活压力敏感垫的每只爪子或组合前/后爪的表面积。(v) 每个参数的平均值和标准偏差。(六) 整个实验期间不同体位 (饲养、3爪或4爪) 的持续时间。(七) 每只爪子在整个实验过程中的重量。</li>
	</ol>
</li>
</ol>4. 鞘内注射 IL4-10 融合蛋白镇痛
注: 确认房间内的通风是开放的, 以确保适当的空气流通。检测 IL4-10 融合蛋白 (1 µg/小鼠的功效; 5 µL 总注射量) 应对照不同细胞因子 (IL4 + IL10、0.5 µg + 0.5 µg/小鼠、5µL 总注射量) 组合的车辆注射和注射进行试验。
<ol><li>麻醉 3-4% 异氟醚, 在感应腔内氧流速为2升/分的小鼠。通过捏爪子来检查鼠标是否被适当的麻醉, 以确保动物没有反应。</li>
	<li>把鼠标放在桌子上, 头放在仪器的鼻锥上, 用 1.5-2% 异氟醚保持鼠标镇静, 在进行鞘内注射时, 氧流速为2升/分。</li>
	<li>背部填充注射化合物成一个干净的25µL 玻璃汉密尔顿注射器连接到27口径针。</li>
	<li>按住鼠标的脊柱后, 肋骨和轻微地举起鼠标。将针垂直放置在腰椎 L5 和 L6 之间, 并小心地将其插入腰部中部的皮肤。把针放到椎间隙。 注意: 发现椎间隙可能需要一些 ' 搜索 ' 通过移动针沿延髓腹轴, 轻轻按压针, 直到失去了骨组织的抵抗力。<ol>
<li>通过验证是否发生尾弹来确认正确的目标定位。慢慢地注入5µL 的化合物, 等待几秒钟, 然后慢慢地收回针。如果只观察到单爪弹, 针可能无法正确放置。</li>
	</ol>
</li>
	<li>注射后, 检查鼠标密切的第一30分钟后, 从麻醉恢复, 以确保没有任何运动损伤/瘫痪。 注意: 疼痛行为可以测量 (如第3节所述) 恢复后15分钟。</li>
</ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+assessment+of+neuropathic+pain,+fatigue,+and+anxiety+in+experimental+autoimmune+encephalomyelitis+(EAE)+and+attenuation+by+interleukin-10+gene+therapy.">Behavioral assessment of neuropathic pain, fatigue, and anxiety in experimental autoimmune encephalomyelitis (EAE) and attenuation by interleukin-10 gene therapy.</a> Brain Behav Immun. 59, 49-54 (2017).</li><li>Kiguchi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+administration+of+interleukin-13+reverses+inflammatory+macrophage+and+tactile+allodynia+in+mice+with+partial+sciatic+nerve+ligation.">Peripheral administration of interleukin-13 reverses inflammatory macrophage and tactile allodynia in mice with partial sciatic nerve ligation.</a> J Pharmacol Sci. 133 (1), 53-56 (2017).</li><li>Eijkelkamp, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL4-10+Fusion+Protein+Is+a+Novel+Drug+to+Treat+Persistent+Inflammatory+Pain.">IL4-10 Fusion Protein Is a Novel Drug to Treat Persistent Inflammatory Pain.</a> J Neurosci. 36 (28), 7353-7363 (2016).</li><li>Schott, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins. , (1984).</li><li>Scopes, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+protein+purification.">Strategies for protein purification.</a> Curr Protoc Protein Sci. , (2001).</li><li>Gregory, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+animal+models+of+pain:+disease+models+and+outcome+measures.">An overview of animal models of pain: disease models and outcome measures.</a> J Pain. 14 (11), 1255-1269 (2013).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balancing+GRK2+and+EPAC1+levels+prevents+and+relieves+chronic+pain.">Balancing GRK2 and EPAC1 levels prevents and relieves chronic pain.</a> J Clin Invest. 123 (12), 5023-5034 (2013).</li><li>Willemen, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial/macrophage+GRK2+determines+duration+of+peripheral+IL-1beta-induced+hyperalgesia:+contribution+of+spinal+cord+CX3CR1,+p38+and+IL-1+signaling.">Microglial/macrophage GRK2 determines duration of peripheral IL-1beta-induced hyperalgesia: contribution of spinal cord CX3CR1, p38 and IL-1 signaling.</a> Pain. 150 (3), 550-560 (2010).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+dynamic+weight+bearing+as+a+novel+end-point+for+the+assessment+of+Freund's+Complete+Adjuvant+induced+hypersensitivity+in+mice.">Use of dynamic weight bearing as a novel end-point for the assessment of Freund's Complete Adjuvant induced hypersensitivity in mice.</a> Neurosci Lett. 524 (2), 107-110 (2012).</li><li>He, Y., Tian, X., Hu, X., Porreca, F., Wang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+reinforcement+reveals+non-evoked+ongoing+pain+in+mice+with+tissue+or+nerve+injury.">Negative reinforcement reveals non-evoked ongoing pain in mice with tissue or nerve injury.</a> J Pain. 13 (6), 598-607 (2012).</li><li>Bottros, M. M., Christo, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+perspectives+on+intrathecal+drug+delivery.">Current perspectives on intrathecal drug delivery.</a> J Pain Res. 7, 615-626 (2014).</li><li>Eijkelkamp, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Piezo2+in+EPAC1-dependent+mechanical+allodynia.">A role for Piezo2 in EPAC1-dependent mechanical allodynia.</a> Nat Commun. 4, 1682 (2013).</li><li>Bradman, M. 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作者没有什么可透露的。

本手稿描述了一种重组 IL4-10 融合蛋白的生产和表征方法, 以及检测其在持续炎症性疼痛小鼠模型中抑制炎症痛觉的有效性的方法。IL4-10 融合蛋白的生产和纯化是在小尺度上进行的。HEK293 细胞被选作蛋白质生产的表达系统, 因为它们能够在原核表达系统中无法实现转化后的修饰。翻译后的修改与蛋白质功能有关, 缺乏这种修饰可能会影响 IL4-10 融合蛋白的治疗效果。用亲和层析法从培养中清液中纯?...

慢性疼痛仍然是第二十一世纪最虚弱和未受治疗的医疗问题之一, 影响 > 20% 的成人人口1,2。然而, 提供缓解慢性疼痛的治疗往往无效或必须停止, 因为严重的副作用3。重要的是, 目前可用的药物只提供症状缓解, 但不显着改变或治疗慢性疼痛。虽然慢性疼痛似乎是神经紊乱, 证据表明免疫系统参与慢性疼痛发展4,5。此外, 以免疫为基础的治疗疼痛的方法正在出现。例如, 抗炎细胞因子能抑制几种慢性疼痛模型中的疼痛6,7,8。然而, 抗炎细胞因子有短半衰期, 减少其潜在的止痛效果。此外, 抗炎细胞因子最优化的工作, 以配合。为了克服这些局限性, 我们最近将抗炎细胞因子 interleukin-4 (IL4) 和 interleukin-10 (IL10) 融合成一个分子。与单个细胞因子9相比, IL4-10 融合蛋白在抑制慢性炎症和神经病理性疼痛方面具有优异的疗效。这里我们描述了这种融合蛋白是如何产生、纯化以及如何控制其质量的。
IL4-10 融合蛋白是通过对 HEK293-F 细胞进行瞬态转染, pUPE 表达载体进行 IL4-10 融合蛋白的 cDNA 序列编码, 在人体细胞中产生的。HEK293-F 细胞被选择允许对蛋白质的后转化修饰, 在细菌表达系统中不会发生的东西。为了优化糖盖与唾液酸, cDNA 编码 beta-galactoside-2, 3-唾液酸化酶转移酶被纳入在载体作为第二种转基因。融合蛋白的纯化使用亲和蛋白纯化的文化上清, 因为它比纯化更强大的其他方法例如大小排除或离子交换层析10,11。为了纯化 IL4-10 融合蛋白, 我们用自制的单克隆抗体对 IL4。对纯化 IL4-10 融合蛋白的纯度和生物活性进行了评价, 作为质量控制的一部分。用月桂酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 和高压尺寸排除色谱 (HP SEC) 对生产批次纯度进行了评价。通过测量其在全血培养中抑制脂多糖 (LPS) 诱导的肿瘤坏死因子α (TNFα) 生产的能力, 对 IL4-10 融合蛋白的生物活性进行评价, 并将其与个体的组合进行比较。细胞 因子。
最后, 为了测试 IL4-10 融合蛋白抑制慢性疼痛的能力, 我们描述了如何在广泛使用的持久性炎症疼痛的小鼠模型中测试融合蛋白的镇痛作用12,13,14.在这里, 我们描述了炎症疼痛模型的方法。然而, 重要的是要注意, 其他疼痛模型可以使用 (例如, 神经病理性疼痛模型), 取决于需要回答的研究问题。为了评估这些模型的疼痛, 重要的是要使用各种行为措施, 包括诱发和非诱发疼痛的措施。在这里, 我们描述了对机械和热诱发行为反应变化的评估方法。对无害刺激的机械敏感性是用弗雷测试来评估的, 而热敏度则使用哈格里夫斯测试进行评估。重要的是, 用动态负重试验测量非诱发痛觉/痛觉。这些措施被广泛接受作为疼痛措施和产生的重要信息的疼痛阈值和潜在的疼痛经历的动物15,16,17。评估非诱发疼痛的其他措施 (例如, 刺激独立), 如条件位置首选项测试, 可能是有价值的18。为了评估药物抑制疼痛的潜能, 我们对融合蛋白进行鞘内管理, 因为这一管理途径需要较少的蛋白质剂量来达到与疼痛相关的区域, 避免系统性 (副作用)19, 20。

<table border="0" cellpadding="0" cellspacing="0" style="width:869px;" width="868">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">FreeStyle 293-F cells</td>
			<td style="width:143px;">Invitrogen</td>
			<td style="width:161px;">R790-07</td>
			<td style="width:269px;">Human embryonic kidney cells&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO FreeStyle 293 Expression Medium</td>
			<td>Life technologies</td>
			<td>12338018</td>
			<td>Culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">293fectin Reagent</td>
			<td style="width:143px;">Invitrogen</td>
			<td>12347019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO Opti-MEM + GlutaMAX</td>
			<td>Life technologies</td>
			<td>51985026</td>
			<td>Reduced Serum Medium for use during cationic lipid transfections&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">GIBCO RPMI Medium 1640 (1x)</td>
			<td>Life technologies</td>
			<td style="width:161px;">52400-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CNBr-Activated Sepharose 4B</td>
			<td style="width:143px;">GE Healthcare</td>
			<td>17-0430-01</td>
			<td>pre-activated media for coupling antibodies or other large proteins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Hydrochloric acid fuming 37%&#160;</td>
			<td style="width:143px;">Merck</td>
			<td>1003171000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td style="width:143px;">Sigma</td>
			<td>S7653-1kg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium bicarbonate&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:161px;">31437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Trizma hydrochloride</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:161px;">T3253-500G</td>
			<td>TRIS hydrochloride&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic Acid 100%&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Merck</td>
			<td style="width:161px;">1.00063.1000&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glycin-HCl&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:161px;">G2879&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:143px;">Pharmacie, UMCU</td>
			<td style="width:161px;"></td>
			<td>Phosphate-Buffered Saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">10X TGS</td>
			<td style="width:143px;">BIO-RAD</td>
			<td style="width:161px;">161-0772</td>
			<td>Tris/Glycine/SDS Buffer for SDS electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Mini-PROTEAN TGX Gels</td>
			<td style="width:143px;">BIO-RAD</td>
			<td style="width:161px;">456-1046</td>
			<td>12% SDS precast gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Trans-Blot Turbo Transfer Pack</td>
			<td style="width:143px;">BIO-RAD</td>
			<td style="width:161px;">170-4157</td>
			<td>Western blot transfer packs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yarra 3u SEC-2000 column</td>
			<td style="width:143px;">Phenomenex</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InstantBlue Protein Stain</td>
			<td style="width:143px;">Expedeon</td>
			<td>ISB1L</td>
			<td>ready to use Coomassie protein stain for polyacrylamide gels</td>
		</tr>
		<tr height="21">
			<td>Human IL-10 DuoSet ELISA</td>
			<td>R&#38;D</td>
			<td>DY217B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Human TNF&#945; ELISA Set</td>
			<td>Diaclone</td>
			<td>851570020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>BCA Pierce Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carrageenan</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:161px;">22049</td>
			<td>plant mucopolysaccharide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">CFA</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:161px;">F5881</td>
			<td>vaccine adjuvant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Hamilton syringe</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:161px;">20779</td>
			<td>glass syringe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Animal Enclosure</td>
			<td>IITC Life Science</td>
			<td style="width:161px;">433</td>
			<td style="width:269px;">Animal Enclosure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Von Frey mesh stand</td>
			<td>IITC Life Science</td>
			<td style="width:161px;">410</td>
			<td>Mesh Stand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">von Frey hairs&#160;</td>
			<td style="width:143px;">Stoelting</td>
			<td>58011</td>
			<td>touch test sensory probes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Plantar Test (Hargreaves Method)</td>
			<td>IITC Life Science</td>
			<td style="width:161px;">390G</td>
			<td>plantar test with heated glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dynamic Weight Bearing test</td>
			<td style="width:143px;">Bioseb</td>
			<td style="width:161px;">BIO-DWB-AUTO-M</td>
			<td>postural deficit test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Econo-Column Columns, 1.5 &#215; 30 cm</td>
			<td style="width:143px;">BIO-RAD</td>
			<td>7371532</td>
			<td>glass chromatography column&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">SnakeSkin Dialysis Tubing&#160;</td>
			<td style="width:143px;">Thermo Scientific</td>
			<td style="width:161px;">88242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Minisart NML Syringe Filter&#160;</td>
			<td style="width:143px;">Sartorius</td>
			<td style="width:161px;">16555-K</td>
			<td>single use filter unit, 0.45 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CASY Cell Counter and Analyzer</td>
			<td style="width:143px;">Roche</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of recombinant proteins to treat chronic pain

慢性疼痛难以治疗, 迫切需要新的方法来解决持久性疼痛。抗炎性细胞因子是有希望的候选者, 以治疗虚弱的疼痛状况, 因为他们的能力, 调节异常的神经免疫相互作用。然而, 生理上他们在一个不同的细胞因子的网络工作, 因此他们的治疗作用可能不是最佳的当被使用作为独立药物。为了克服这一局限性, 我们开发了抗炎细胞因子 IL4 和 IL10 的融合蛋白。本文介绍了 IL4-10 重组蛋白的生产和质量控制方法, 并对 IL4-10 融合蛋白在小鼠持续炎症性疼痛模型中的镇痛效果进行了试验研究。

在图 1a中显示了含有不同分数的 SDS 页凝胶在亲和层析纯化过程中的代表性图片。在负载 (L) 和流经 (FT) 馏分中, 所有在 HEK293 上清液中存在的蛋白质都被观察到。在洗涤 (W) 分数中没有发现蛋白质。在洗脱 (E) 分数, 观察两个带35和 37 kDa 的两个不同 glycoforms 的 IL4-10 融合蛋白 (箭)。在图 1B中, 显示了两种?...

Watch this Scientific Journal Video about Development of Recombinant Proteins to Treat Chronic Pain at JoVE.com

Development of Recombinant Proteins to Treat Chronic Pain

本文为 IL4-10 重组融合蛋白的生产方法和质量控制提供了详细的资料。我们还展示了如何测试这种蛋白质的有效性, 以解决疼痛的老鼠模型的炎症疼痛。

重组蛋白治疗慢性疼痛的研究进展

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

development-of-recombinant-proteins-to-treat-chronic-pain

Research

JoVE Journal

Medicine

9.4K 次观看.  University Medical Center Utrecht, Utrecht University. 本文为 IL4-10 重组融合蛋白的生产方法和质量控制提供了详细的资料。我们还展示了如何测试这种蛋白质的有效性, 以解决疼痛的老鼠模型的炎症疼痛。

视频: Development of Recombinant Proteins to Treat Chronic Pain

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

慢性缩窄大鼠坐骨神经疼痛过敏测试

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

膀胱胀诱发内脏运动反应为膀胱疼痛小鼠模型

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral procedures to study trigeminal neuropathic pain in rats. To meet the specificity of trigeminal neuropathic pain syndromes, the infraorbital nerve (IoN) is subjected to a chronic constriction injury (CCI) by loosely ligating the nerve. An intra-orbital approach is presented here to expose and ligate the IoN in the orbital cavity. After IoN ligation, rats exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are indicative of persistent pain and mechanical allodynia. Two phases can be defined in the development of the behavioral changes. During the first week following IoN-CCI (phase 1), rats show an increased and asymmetric face grooming activity, i.e., with face wash strokes primarily directed to the nerve-injured IoN territory. A distinction is made between face grooming behavior that is part of a more general body grooming behavior, which remains largely unaffected by IoN-CCI, and face grooming that is neither preceded nor followed by body grooming, which is significantly increased after IoN-CCI. During this period, responsiveness to mechanical stimulation of the IoN territory is reduced. This hyporesponsiveness is abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior (phase 2). The phenomenological similarities between these behavioral alterations and reported signs of facial pain (i.e., responses to noxious stimulation of the face) suggest the presence of dysesthesia/paresthesia and mechanical allodynia in the ligated IoN territory.

Chronic Constriction Injury of the Rat's Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain

This manuscript describes a rodent model to study trigeminal neuropathic pain. The methods described include surgical procedures to perform a chronic constriction injury of the rat&#8217;s infraorbital nerve and the post-surgical behavioral tests to measure the changes in spontaneous and evoked behavior that are indicative of persistent pain and allodynia.

老鼠的眶下神经（ION-CCI）研究三叉神经性疼痛性损伤

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral ...

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.

We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

单关节膝关节疼痛小鼠模型疼痛的自发性和诱发性测量

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part of many experimental procedures where reagents need to be introduced directly into the bloodstream. Unwittingly, the injection can be subcutaneous, possibly altering the scientific outcomes. Utilizing a nanoemulsion-based biological probe with an incorporated near-infrared fluorescent (NIRF) dye, this method offers the capability of imaging a successful tail vein injection in vivo. With the use of a NIRF imager, images are taken before and after the injection of the agent. An acceptable IV injection is then qualitatively or quantitatively determined based on the intensity of the NIRF signal at the site of injection.

Here we present a method to validate tail vein injections in rats by utilizing near-infrared fluorescence imaging data from dyes incorporated into agents or biological probes. The tail is imaged before and after the injection, the fluorescent signal is quantified, and an assessment of the injection quality is made.

近红外标记剂对大鼠尾静脉注射的新的最佳验证

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part ...

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

分离肺内动脉和平滑肌细胞以研究血管反应

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of ...

Vessel-Sparing Microsurgical Longitudinal Intussusception Vasoepididymostomy to Treat Epididymal Obstructive Azoospermia

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of epididymal OA since 2000. There are two challenges in classic microscopic vasoepididymostomy. First, anastomosis of the vas deferens and epididymis is performed with double-needle sutures. However, there is a lack of good-quality and cost-effective double-needle sutures in China, which leads to increased difficulty and poor success rates of anastomosis. Second, the separation of the vas deferens does not retain vasculature, although the vas deferens vasculature plays an important role in the blood supply to the vas deferens, epididymis, and testis. This affects the blood supply to the anastomotic area and epididymis.
Therefore, this team has made innovative improvements to address these problems. Good-quality, cost-effective, single-needle sutures, which are easy to purchase in China and other countries, were used in microsurgical longitudinal intussusception vasoepididymostomy. This can optimize the operation procedure and shorten the operation time while ensuring the success rate of the anastomosis. The surgical method of preserving the vas deferens vessels was innovatively proposed because the etiology of epididymal OA is mostly inflammatory in China. The protection of the blood supply to the vas deferens and epididymis is maximized using microsurgical forceps to separate and protect the vasculature. Patency reached 81.7% in the postoperative follow-up, indicating a better surgical treatment effect.

The present protocol describes a vessel-sparing, longitudinal intussusception vasoepididymostomy using readily available single-needle sutures in China, a safe and effective procedure, which may improve patients' patency and natural pregnancy rates.

血管保留性显微外科纵向套叠血管表皮造口术治疗附睾梗阻性无精子症

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of ...

Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in the efficacy and mechanism between the preparation combination and the component combination in order to explore the two herb combination strategy to treat injured PC12 cells. Cobalt chloride (CoCl2) combined with a glucose-free medium was employed to induce oxidative damage of PC12 cells. Then, the optimal combination of Astragalus mongholicus (Ast) and Erigeron breviscapus (Eri) injection was selected and combined following uniform design methods after screening their safe and effective concentration on PC12 cells. Further, the component combination screened comprises 10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid in two herbs. Then, MTT, Annexin V-FITC/PI, immunofluorescence, and Western blot analysis were used to evaluate the efficacy and mechanism of the preparation combination and the component combination on injured PC12 cells. The results showed that the optimal preparation combination for cell pro-survival was Ast injection and Eri capsule with a concentration of 6:1.8 (&#181;M). The component combination (10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid) was more effective than the preparation combination. Both combinations remarkably reduced apoptotic rate, the fluorescence intensity of caspase-3, and intracellular reactive oxygen species (ROS) level; meanwhile, they upregulated the expression levels of p-Akt/Akt, Bcl-2/Bax, and Nrf2. These effects were more evident in the component combination. In conclusion, both combinations can inhibit the injury induced by CoCl2 combined with a glucose-free medium on PC12 cells, thus promoting cell survival. However, the efficiency of the component combination over the preparation combination may be due to its stronger regulation of the PI3K/Akt/Nrf2 signaling pathway related to oxidative stress and apoptosis.

This protocol provides a combination strategy of two herbs to treat injured PC12 cells. The protocol provides a reference for optimizing the best application mode of traditional Chinese medicine (TCM).

探索两种草药组合策略治疗受损PC12细胞

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

部分坐骨神经结扎术:慢性神经性疼痛的小鼠模型，用于研究新疗法的抗伤害感受作用

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...