04陆姮磊--大鼠脑组织规范性取材_培训课件

1、大鼠脑组织规范性取材专业技术岗位汇报专业技术岗位汇报大鼠脑组织规范性取材专业技术岗位汇报专业技术岗位汇报秦秋平秦秋平陆姮磊秦秋平秦秋平2013-09-25上海药物所药物安全评价研究中心2016 10 12 中国科学院上海药物研究所药物安全评价研究中心中国科学院上海药物研究所药物安全评价研究中心2016-10-12主要内容主要内容大鼠脑的解剖结构大鼠脑的解剖结构不同的取材方式不同的取材方式常见人工假象常见人工假象不同取材方式之间的比较不同取材方式之间的比较脑的解剖结构脑的解剖结构脑的最前端（嗅球） 位于眶间最狭窄处横嵴-嗅球与大脑分界处大脑Bregma 前囟点大脑小脑分界处（前缘骨缝）松果体解剖时最容易损伤三处松果体解剖时，最容易损伤三处： 嗅球、垂体和小脑绒球脑的解剖结构脑的解剖结构嗅球 前额皮质前额皮质 尾状核 穹隆-胼胝体后端向下弯纤维束 胼胝体-两大脑半球之间弧形白 色神经纤维带 海马海马 丘脑上部 丘脑下部 横窦 小脑嗅球脚嗅球脚 前联合（连接同一半球不同部位） 梨状叶 视神经交叉 乳状体 脚间窝脚间窝 副海马皮质区 锥体重要的神经系统功能结构重要的神系统功能结构Regions

2、 that perform critical neurological functions should be sampled routinely.Motor sensoryCognitionHomeostasiscommon neurotoxicity target sitesCertain structures should be sampled regularly: Caudate putamen纹状体 Cerebellum小脑Cerebellum小脑 Cerebral cortex（frontal额区, parietal顶区, cingulate-扣带回, temporal颞区, occipital枕区, retrospenial压后皮质, piriform梨状叶, entorhinal内皮层） Choroid plexus脉络丛 Eye (with optic nerve)眼含视神经 Hi海马Hippocampus海马(Cornu Ammonis海马角and Dentate gyrus齿状回） Hypothalamus下丘脑 medulla oblongata延髓(Trige

3、minal三叉神经, Vagal level F) 中脑rostral to the midpoint of the cerebellum (level G), 小脑绒球through the caudal portion of the cerebellum (level H).For each level, anatomical structures of interest are shown: aca =anterior commissure; Acb =accumbens nucleus; Amy =amygdala; Arc =arcuate nuclei; Au =auditory cortex; B =basal nucleus; CA1 CA2 CA3 =cornu ammonis 1 2 and 3; cc =corpusAmy =amygdala; Arc =arcuate nuclei; Au =auditory cortex; B =basal nucleus; CA1, CA2, CA3 =cornu ammonis 1, 2 and 3; cc =corpus

4、 callosum; Cer =cerebellum; Cg =cingulate cortex; cg =cingulum; cic =inferior colliculi commissure; cp =cerebral peduncles; Cpu =caudate putamen; csc =superior colliculi commissure; DG=dentate gyrus; DR =dorsal raphe nucleus; Ect =ectorhinal cortex; Ent =entorhinal cortex; Fl =flocculus; fmi =forceps minor of the corpus callosum; f =fornix; fi =fimbria; GP =globus pallidus; Hb =habenular nuclei; Hyp =hypothalamus; ic =internal capsule; IC =inferior colliculi; ICj =Calleja islands; icp =inferior

5、cerebral peduncles; Lc =limbic cortex;7段法取材出处7段法取材出处3段取材遗漏 大量解剖功能 神经疾病发生率日益增加无法明确原因 的神经退行性 疾病临床试验中多 种药物引起神 经毒性改变潜在神经毒性 的化合物结构，漏诊漏诊。65段取材虽不 漏，耗时耗能，成立成立小组确小组确 个标个标漏，耗时耗能， 不适用STP成立成立Nervous System Sampling小组小组明明确确4个个目目标标 在GLP的毒性试验中每一动物种属（啮齿类、犬或猴）神经结构必须被取到 取材的标准要有广泛的实用性取材的标准要有广泛的实用性 界定传统的染色和神经组织学的流程并能适用于常规的毒理学实验 提倡被官方审计的报告中要最大程度和最大限度记录显微镜下神经病理评价的结果在原有3段取材基础上增加4段，比如增加嗅球、丘脑上部、丘脑下部、内 侧膝状体等，7段式取材可以包含大约50个左右的脑部功能结Rat: Seven transverse (coronal) hematoxylin and eosinstained sections corresponding to level

6、s based on anatomic targetbased on anatomic target landmarks. OB = olfactory bulb, OC = optic chiasm IF = infundibulum (and/or median eminence), CN V = cranial nerve V (trigeminal)(trigeminal), CP = cerebral peduncle.Bolon et al, 2013STP position paper: Recommended practices for sampling and processing the nervous system (brain, spinal cord, nerve, and eye) during nonclinical general toxicity studies. SectionAnatomical RegionFunctional Significance LevelggLevel 1Olfactory bulbHigh lifetime cell

7、turnover rate; vulnerability to inhaled toxicantsLevel 2Frontoparietal cortex, Learning and behavior; involuntary motor activity basal gangliaLevel 3Parietal cortex, thalamus, hypothalamus,hippocampusMotor; integration of neuroendocrine, sensory, and behavioral systemsldbbk dhldLevel 4MidbrainSubstantia nigra, Parkinsons disease;raphe nuclei and reward/addictive responsesLevel 5Posterior colliculusAuditory reflexes; high neuronal,metabolic rateLl 6Cb llPtb ll tditiflttLevel 6Cerebellum, ponsPost

8、ure, balance, muscle tone, coordination of voluntary motor activityLevel 7MedullaCircumventricular organ vulnerability;hypoglossal nucleus, amyotropic lateral sclerosisSection 1 Olfactory BulbsSection 1 Olfactory BulbsONL Olfactory Nerve Fiber Layer 嗅球神经纤维层嗅球神经纤维层 GL Glomerular Layer 血管层 EPL External Plexiform Layer 外网层外网层 MCL Mitral Cell Layer （僧帽细胞） GCL Granule Cell Layer （颗粒细胞）（颗粒细胞）Section 2: Forebrain (bregma 0.48 mm)Fr/Par - Frontoparietal Cortex (neocortex)额顶皮质区（新皮质）()额顶皮质新皮质 CC- Corpus c

9、allosum胼胝体 Cing Cingulate Cortex扣带回 ON Optic nerves视神经 Pir Piriform Cortex梨状皮质Pir Piriform Cortex梨状皮质 SN Septal Nuclei隔核（边缘系统） (limbic system) CP Caudate Putamen纹状体 (basal ganglia)（基底神经节）CC(basal ganglia)（基底神经节） NuA Nucleus accumbens（伏核） ICj Islands of Calleja（嗅结节岛） LO lateral olfactory tract嗅神经束NuATu olfactory tubercle嗅结节NuAICjLO ONICjLO TuSection 3 ThalamusSection 3 ThalamusFr/Par Frontoparietal Cortex额顶皮质区） RSR tli l Ct压后皮质RS Retrosplenial Cortex压后皮质 H Hippocampus海马 ChP Choroid Plexus脉络丛 EC External Capsule外囊ECTh Thalamus丘脑 HT Hypothalamus下丘脑 IC Internal Capsule内囊 A Amygdala杏仁核ChPECAAmygdala杏仁核 Au Auditory cortex听觉皮层 Pir Piriform Cortex梨状皮层AuICdb(b)Section 4 Midbrain (bregma 6.48 mm) Occ Occipital Cortex枕叶皮质区 RS Retrosplenial Cortex压后皮质 E tRE thil Ct内嗅皮层EntR Entorhinal Cortex内嗅皮层 Te Temporal Cortex颞叶皮质 H Hippocampus海马 SubN Substantia Nigra黑质 RN Red

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