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Isolation of Rodent Brain Vessels
啮齿动物脑血管分离   

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Abstract

The prevalence of neurodegenerative diseases is increasing worldwide. Cerebrovascular disorders and/or conditions known to affect brain vasculature, such as diabetes, are well-known risk factors for neurodegenerative diseases. Thus, the evaluation of the brain vasculature is of great importance to better understand the mechanisms underlying brain damage. We established a protocol for the isolation of brain vessels from rodents. This is a simple, non-enzymatic isolation protocol that allows us to perform comparative studies in different animal models of disease, helping understand the impact of several pathological conditions on brain vasculature and how those alterations predispose to neurodegenerative conditions.

Keywords: Blood-brain barrier(血脑屏障), Cerebrovascular and neurodegenerative disorders(脑血管和神经变性病), Isolated brain vessels(分离的脑血管), Non-enzymatic isolation protocol(非酶介导的分离方法), Rodents(啮齿动物)

Background

The brain is highly dependent on a constant supply of oxygen and nutrients that arrive through a vast network of blood vessels. The blood-brain barrier (BBB), mainly composed of microvascular endothelial cells that line cerebral microvessels along with periendothelial structures, which include pericytes, astrocytes and a basement membrane (Saraiva et al., 2016; Librizzi et al., 2017), guarantees the control of an homeostatic environment, necessary to maintain the health of brain cells. Thus, the study of how certain pathologies that can interfere with the integrity of cerebrovasculature is of great importance. Indeed, strong evidence from clinical, imaging, epidemiological and neuropathological studies confirmed over the past two decades that the presence of cerebrovascular disease has a pivotal role in Alzheimer disease (AD) and other dementias associated with aging (Chui et al., 2006; Schneider et al., 2007; Gorelick et al., 2011; Wharton et al., 2011; Yarchoan et al., 2012; Bennett et al., 2013; DeCarli, 2013; Toledo et al., 2013; Yates et al., 2014). Besides the low number of papers dedicated to the study of isolated brain vessels, the isolation protocols used in those studies present some inconsistencies rendering difficult the comparison and interpretation of the published observations. With this protocol, we intend to offer a standardized procedure to help researchers working in this field. This protocol was adapted from a previous protocol described by McNeill et al. (1999) and used in our laboratory to isolate total (arterial and venous) brain vessels from rodents (Figures 1 and 4) (Carvalho et al., 2010; Carvalho et al., 2013; Plácido et al., 2017).


Figure 1. Evaluation of the activity of the mitochondrial enzymatic complexes of mice brain vessels. Mitochondrial complexes I-III (A), II-III (B) and IV (C) were determined in vessels isolated from the brains of 11-month-old male wild type (WT; C57BL6/129S), type 2 diabetes-like mice (WT mice exposed to 20% sucrose solution during 7 months) and triple transgenic mice for Alzheimer disease (3xTg-AD, B6;129-Psen1 Tg(APPSwe,tauP301L)1Lfa/Mmjax). A significant decrease in the activity of mitochondrial complexes I-III was observed in brain vessels isolated from 3xTg-AD and type 2 diabetes-like mice. Also, a significant decrease in the activity of complex IV was observed in brain vessels isolated from 3xTg-AD mice. Data shown represent mean ± SEM from 6-8 pools of n = 3. Statistical significance: *P < 0.05; **P < 0.01 when compared with WT mice. Statistical significance was determined using the paired Student’s t-test and Kruskal-Wallis test for multiple comparisons, followed by the posthoc Dunn test (GraphPad Prism 5). These graphs have been previously published in Journal of Alzheimer’s Disease (DOI: 10.3233/JAD-130005) with permission from IOS Press.

Materials and Reagents

  1. 50 ml Oak Ridge polysulfone centrifuge tubes w/screw caps (Thermo Fisher Scientific, catalog number: 3115-0050 )
  2. Eppendorf microtubes 1.5 ml (VWR, catalog number: 700-5239 )
  3. PIPETMAN TIPS Diamond–ECOPACKTM D1000 (Gilson, catalog number: F161670 )
  4. PIPETMAN TIPS Diamond–ECOPACKTM D200 (Gilson, Catalog number: F161930 )
  5. PIPETMAN TIPS Diamond–ECOPACKTM D10 (Gilson, catalog number: F161630 )
  6. Stainless steel surgical blades (Swann Morton, catalog number: 0308 )
  7. Rodent brain
    Note: This protocol has only been tested with brains from male young and mature (3- and 12-month-old) Wistar rats and wild type, type 2 diabetes-like and triple transgenic for Alzheimer disease (3xTg-AD) mice (11-month-old). Nevertheless, we believe that this protocol can be applied to different strains, ages and sex, though the amount of obtained sample can be a limiting factor.
  8. Ice
  9. Distilled water
  10. Isoflurane (Lab. Vitória, Portugal)
  11. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  12. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
  13. Dextran from Leuconostoc mesenteroides (Sigma-Aldrich, catalog number: 31398 )
  14. Phosphate buffer (0.01 M) (see Recipes)
  15. Dextran (16%) (see Recipes)

Equipment

  1. 50 ml FisherbrandTM reusable glass low-form Griffin beakers (Fisher Scientific, catalog number: FB10050 )
  2. Bone cutting forceps (Aesculap, catalog number: FO611R )
  3. Centrifuge (Refrigerated Centrifuge) (Sigma Laborzentrifugen, model: SIGMA 3-16K )
  4. Curved fine tip forceps (Aesculap, catalog number: FB401R )
  5. Heidolph mechanical overhead stirrers, Brinkmann (Heidolph Instruments, model: RZR 1 )
  6. Laboratory bottles, narrow mouth, with screw cap (VWR, catalog number: 215-1594 )
  7. Micropipette PIPETMAN L (Light) type P200L (Gilson, catalog number: FA10005 )
  8. Micropipette PIPETMAN L (Light) type P1000L (Gilson, catalog number: FA10006 )
  9. Nickel/SS Lab spatulas with 1.63” Flat Rounded Ends (Cole-Parmer, catalog number: EW-06369-05 )
  10. Petri dish with cover 60 x 15 mm (Corning, catalog number: 70165-60 )
  11. Potter-Elvehjem with PTFE pestle and glass tube (DWK Life Sciences, Kimble®, catalog number: 886000-0023 )
  12. Precision scale (Mettler-Toledo International, model: AE240 )
  13. Precision balance PLE-N (KERN, model: PLE-N )
  14. Scalpel handle (Aesculap, catalog number: BB084R stainless)
  15. Soft hair brush, 3 mm dia. (CONTROLS, catalog number: 86-D1672 )
  16. Swing-out rotor (Sigma Laborzentrifugen, catalog number: 11133 )

Procedure

All the isolation protocol steps must be performed at 4 °C (always maintain solutions, homogenates and pellets on ice).

  1. Euthanize rodents by cervical displacement (mice) and decapitation (rats and mice). All procedures are approved by the Federation of Laboratory Animal Science Associations (FELASA).
  2. Perform a midline incision, posterior to anterior, along the scalp to reveal the skull (Video 1).

    Video 1. Brain removal, dissection and homogenization

  3. Cut the cranium carefully from the neck to the nose through the lateral cranial sutures. Two additional cuts can be performed, in the occipital hole to the ears direction, to facilitate the access to the brain (Figure 2A, Video 1).


    Figure 2. Illustrative images of brain dissection and homogenization. Cut the cranium carefully from the neck to the nose through the lateral cranial sutures (A). Remove the intact brain from the cranial box using a small spatula (B). Then, remove the cerebellum, olfactory bulbs and white matter using fine dissection forceps and keep the brain cortices (C). Finally, homogenize brain tissue, at 4 °C, in 10 ml PBS using a Potter-Elvehjem with PTFE pestle and glass tube, at 800 rpm/min (D).

  4. Remove the intact brain from the cranial box using a small spatula (Figure 2B, Video 1).
  5. Remove the cerebellum, olfactory bulbs and white matter using fine dissection forceps; keep the brain cortices (Figure 2C, Video 1, see Note 1).
  6. Perform a quick wash (approximately 30 sec) in non-sterile PBS (Video 1; see Recipes).
  7. Homogenize fresh brain tissue, at 4 °C, in 10 ml PBS using a Potter-Elvehjem with PTFE pestle and glass tube, at 800 rpm/min (Figure 3, Video 1).


    Figure 3. Schematic illustration of mice brain vessels isolation

  8. Centrifuge at 720 x g for 5 min, at 4 °C (Figure 3).
  9. Discard the supernatant by decantation (Figure 3).
  10. Resuspend the pellet in 10 ml PBS using a soft hair brush, 3 mm dia (Figure 3).
  11. Repeat the steps 8-10 two more times.
  12. Layer the PBS resuspended pellet over 20 ml 16% dextran (Video 2; see Recipes).
    Note: This step must be gently done to avoid mixing the two layers (Figure 3).

    Video 2. Brain vessels isolation

  13. Centrifuge at 4,500 x g for 20 min, at 4 °C (Figure 2).
  14. Collect the supernatant and the middle layer, and keep the pellet on ice (Figure 3).
  15. Repeat step 14.
  16. Discard the supernatant and resuspend the two resulting pellets in 100 μl PBS.
    Note: Resuspend the first pellet in 100 μl PBS and use the suspension to resuspend the second pellet. The vessels can be visualized at optic microscope (Figure 4) using the DiffQuick staining as described by Mota and Ramalho-Santos (2006).
  17. Store the vessels at -80 °C until use (used until 2 years upon isolation; Figure 3).


    Figure 4. Representative images of isolated mice brain vessels after DiffQuik staining. Unstained (A) and stained (B, C) brain vessels observed under the optic microscope. 100x amplification; scale bars = 50 μM.

Notes

  1. In order to minimize the day-to-day variability that can bias the results, in studies using animal models of disease, researchers must always isolate vessels from control and diseased animal models.
  2. Minimum number of animals required for the isolation of brain vessels: 1 rat brain or 3 mice brains. It is possible to use only 1 mice brain. However, the pellet size will be very small, which will difficult its visualization with the naked eye.

Recipes

  1. Phosphate buffer (0.01 M)
    8.5 g/L NaCl
    1.42 g/L Na2HPO4
    pH = 7.4
  2. Dextran (16%)
    160 g/L Dextran from Leuconostoc mesenteroides

Notes:

  1. Distilled water type 1 must be used in the preparation of the solutions.
  2. For short periods of time solutions must be conserved at 4 °C (e.g., one week); for longer periods solutions must be conserved at -20 °C (except Dextran solution).
  3. Dextran solution must not be frozen; it is stable for one week at 4 °C.

Acknowledgments

The authors’ work is supported by ‘FEDER funds through the Operational Programme Competitiveness Factors–COMPETE 2020 and national funds by FCT–Foundation for Science and Technology under the strategic project with COMPETE-attributed reference: POCI-01-0145-FEDER-007440’. Cristina Carvalho has a Postdoc fellowship from FCT (SFRH/BPD/107741/2015). The authors of the manuscript have no conflicts of interest to declare. This protocol was adapted from a previous protocol described by McNeill et al. (1999). Reprinted from Journal of Alzheimer’s Disease, vol. 35, Carvalho, Cristina; Machado, Nuno; Mota, Paula; Correia, Sónia C.; Cardoso, Susana; Santos, Renato X.; Santos, Maria S.; Oliveira, Catarina R.; Moreira, Paula I., Type 2 Diabetic and Alzheimer’s Disease Mice Present Similar Behavioral, Cognitive, and Vascular Anomalies, pp. 623-635, Copyright (2013), with permission from IOS Press.

References

  1. Bennett, D. A., Wilson, R. S., Arvanitakis, Z., Boyle, P. A., de Toledo-Morrell, L. and Schneider, J. A. (2013). Selected findings from the Religious Orders Study and Rush Memory and Aging Project. JAlzheimers Dis 33 Suppl 1: S397-403.
  2. Carvalho, C., Machado, N., Mota, P. C., Correia, S. C., Cardoso, S., Santos, R. X., Santos, M. S., Oliveira, C. R. and Moreira, P. I. (2013). Type 2 diabetic and Alzheimer's disease mice present similar behavioral, cognitive, and vascular anomalies. JAlzheimers Dis 35(3): 623-635.
  3. Carvalho, C., Santos, M. S., Baldeiras, I., Oliveira, C. R., Seica, R. and Moreira, P. I. (2010). Chronic hypoxia potentiates age-related oxidative imbalance in brain vessels and synaptosomes. Curr Neurovasc Res 7(4): 288-300.
  4. Chui, H. C., Zarow, C., Mack, W. J., Ellis, W. G., Zheng, L., Jagust, W. J., Mungas, D., Reed, B. R., Kramer, J. H., Decarli, C. C., Weiner, M. W. and Vinters, H. V. (2006). Cognitive impact of subcortical vascular and Alzheimer’s disease pathology. Ann Neurol 60(6): 677-687.
  5. DeCarli, C. (2013). Clinically asymptomatic vascular brain injury: a potent cause of cognitive impairment among older individuals. JAlzheimers Dis 33 Suppl 1: S417-426.
  6. Gorelick, P. B., Scuteri, A., Black, S. E., Decarli, C., Greenberg, S. M., Iadecola, C., Launer, L. J., Laurent, S., Lopez, O. L., Nyenhuis, D., Petersen, R. C., Schneider, J. A., Tzourio, C., Arnett, D. K., Bennett, D. A., Chui, H. C., Higashida, R. T., Lindquist, R., Nilsson, P. M., Roman, G. C., Sellke, F. W., Seshadri, S., American Heart Association Stroke Council, C. o. E., Prevention, C. o. C. N. C. o. C. R., Intervention, Council on Cardiovascular, S. and Anesthesia (2011). Vascular contributions to cognitive impairment and dementia: a statement for healthcare professionals from the american heart association/american stroke association. Stroke 42(9): 2672-2713.
  7. Librizzi, L., de Cutis, M., Janigro, D., Runtz, L., de Bock, F., Barbier, E. L. and Marchi, N. (2017). Cerebrovascular heterogeneity and neuronal excitability. Neurosci Lett.
  8. McNeill, A. M., Kim, N., Duckles, S. P., Krause, D. N. and Kontos, H. A. (1999). Chronic estrogen treatment increases levels of endothelial nitric oxide synthase protein in rat cerebral microvessels. Stroke 30(10): 2186-2190.
  9. Mota, P. C. and Ramalho-Santos, J. (2006). Comparison between different markers for sperm quality in the cat: Diff-Quik as a simple optical technique to assess changes in the DNA of feline epididymal sperm. Theriogenology 65(7): 1360-1375.
  10. Plácido, A. I., Pereira, C. M., Correira, S. C., Carvalho, C., Oliveira, C. R. and Moreira, P. I. (2017). Phosphatase 2A inhibition affects endoplasmic reticulum and mitochondria homeostasis via cytoskeletal alterations in brain endothelial cells. Mol Neurobiol 54(1): 154-168.
  11. Saraiva, C., Praca, C., Ferreira, R., Santos, T., Ferreira, L. and Bernardino, L. (2016). Nanoparticle-mediated brain drug delivery: Overcoming blood-brain barrier to treat neurodegenerative diseases. J Control Release 235: 34-47.
  12. Schneider, J. A., Arvanitakis, Z., Bang, W. and Bennett, D. A. (2007). Mixed brain pathologies account for most dementia cases in community-dwelling older persons. Neurology 69(24): 2197-2204.
  13. Toledo, J. B., Arnold, S. E., Raible, K., Brettschneider, J., Xie, S. X., Grossman, M., Monsell, S. E., Kukull, W. A. and Trojanowski, J. Q. (2013). Contribution of cerebrovascular disease in autopsy confirmed neurodegenerative disease cases in the National Alzheimer’s Coordinating Centre. Brain 136(Pt 9): 2697-2706.
  14. Wharton, S. B., Brayne, C., Savva, G. M., Matthews, F. E., Forster, G., Simpson, J., Lace, G., Ince, P. G., Medical Research Council Cognitive, F. and Aging, S. (2011). Epidemiological neuropathology: the MRC Cognitive Function and Aging Study experience. JAlzheimers Dis 25(2): 359-372.
  15. Yarchoan, M., Xie, S. X., Kling, M. A., Toledo, J. B., Wolk, D. A., Lee, E. B., Van Deerlin, V., Lee, V. M., Trojanowski, J. Q. and Arnold, S. E. (2012). Cerebrovascular atherosclerosis correlates with Alzheimer pathology in neurodegenerative dementias. Brain 135(Pt 12): 3749-3756.
  16. Yates, P. A., Desmond, P. M., Phal, P. M., Steward, C., Szoeke, C., Salvado, O., Ellis, K. A., Martins, R. N., Masters, C. L., Ames, D., Villemagne, V. L., Rowe, C. C. and Group, A. R. (2014). Incidence of cerebral microbleeds in preclinical Alzheimer disease. Neurology 82(14): 1266-1273.

简介

全世界神经退行性疾病的发病率正在增加。 已知会影响脑血管系统的脑血管疾病和/或病症,如糖尿病,是神经变性疾病的众所周知的危险因素。 因此,脑血管系统的评估对于更好地了解脑损伤的机制是非常重要的。 我们建立了从啮齿动物分离脑血管的方案。 这是一个简单的非酶分离方案,允许我们在不同的疾病动物模型中进行比较研究,帮助了解几种病理状况对脑血管系统的影响以及这些改变如何易于发生神经退行性疾病。
【背景】大脑高度依赖于通过庞大的血管网络到达的氧气和营养物质的不断供应。主要由微血管内皮细胞组成的血脑屏障(BBB),其包括脑内微血管以及周围结构,包括周细胞,星形胶质细胞和基底膜(Saraiva等,2016; Librizzi等,2017),保证控制稳态环境,保持脑细胞的健康。因此,研究如何干扰脑血管系统完整性的某些病理学是非常重要的。事实上,临床,成像,流行病学和神经病理学研究的有力证据证实了过去二十年来,脑血管疾病的存在在阿尔茨海默病(AD)和其他与衰老有关的痴呆中具有关键作用(Chui et al。,2006; Schneider et al。,2007; Gorelick et al。,2011; Wharton et al。,2011; Yarchoan et al。,2012; Bennett et al。,2013; DeCarli,2013; Toledo et al。,2013; Yates et al。 2014)。除了专门研究孤立的脑血管的文章数量少之外,这些研究中使用的隔离方案存在一些不一致,使得对发表的观察结果的比较和解释变得困难。有了这个协议,我们打算提供一个标准化的程序来帮助在这个领域工作的研究人员。该协议根据McNeill等人描述的先前协议进行了改进。 (1999),并用于我们的实验室以分离来自啮齿动物的总(动脉和静脉)脑血管(图1和图4)(Carvalho等人,2010; Carvalho等人,2013;Plácido等,2017)。

关键字:血脑屏障, 脑血管和神经变性病, 分离的脑血管, 非酶介导的分离方法, 啮齿动物

材料和试剂

  1. 50毫升橡胶岭聚硫离心管,带螺帽(Thermo Fisher Scientific,目录号:3115-0050)
  2. Eppendorf微管1.5 ml(VWR,目录号:700-5239)
  3. PIPETMAN TIPS Diamond-ECOPACK TM D1000(Gilson,目录号:F161670)
  4. PIPETMAN TIPS Diamond-ECOPACK TM D200(Gilson,目录号:F161930)
  5. PIPETMAN TIPS Diamond-ECOPACK TM D10(Gilson,目录号:F161630)
  6. 不锈钢手术刀片(Swann Morton,目录号:0308)
  7. 啮齿动物大脑
    注意:该方案仅用来自雄性年轻和成熟(3和12个月大的)Wistar大鼠和野生型,2型糖尿病和三重转基因阿尔茨海默病(3xTg-AD)的大脑测试,小鼠(11个月大)。尽管如此,我们认为,该方案可以应用于不同的品系,年龄和性别,尽管获得的样品量可能是限制因素。

  8. 蒸馏水
  9. 异氟烷(Lab。Vitória,葡萄牙)
  10. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  11. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S7907)
  12. 来自肠膜明串珠菌的右旋糖酐(Sigma-Aldrich,目录号:31398)
  13. 磷酸盐缓冲液(0.01 M)(见配方)
  14. 葡聚糖(16%)(见食谱)

设备

  1. 50ml Fisherbrand TM 可重复使用的玻璃低温格里芬烧杯(Fisher Scientific,目录号:FB10050)
  2. 骨切割钳(Aesculap,目录号:FO611R)
  3. 离心机(冷冻离心机)(Sigma Laborzentrifugen,型号:SIGMA 3-16K)
  4. 弯曲的精细镊子(Aesculap,目录号:FB401R)
  5. Heidolph机械式塔顶搅拌器,Brinkmann(Heidolph Instruments,型号:RZR 1)
  6. 实验室瓶,窄口,带螺帽(VWR,目录号:215-1594)
  7. 微型PIPETMAN L(轻型)P200L(Gilson,目录号:FA10005)
  8. Micropipette PIPETMAN L(Light)型P1000L(Gilson,目录号:FA10006)
  9. 镍/ SS实验室刮刀与1.63"扁圆头(Cole-Parmer,目录号:EW-06369-05)
  10. 培养皿盖60 x 15毫米(康宁,目录号:70165-60)
  11. Potter-Elvehjem用PTFE杵和玻璃管(DWK Life Sciences,Kimble ®,目录号:886000-0023)
  12. 精密秤(Mettler-Toledo International,型号:AE240)
  13. 精密平衡PLE-N(KERN,型号:PLE-N)
  14. 手术刀(Aesculap,目录号:BB084R不锈钢)
  15. 软毛刷,3毫米直径。 (CONTROLS,目录号:86-D1672)
  16. 摆动转子(Sigma Laborzentrifugen,目录号:11133)

程序

所有隔离方案步骤必须在4°C(始终保持冰上的溶液,匀浆和颗粒)下进行。

  1. 通过颈部置换(小鼠)和斩首(大鼠和小鼠)安乐死啮齿动物。 所有程序均经实验动物科学协会联合会(FELASA)批准。
  2. 在头皮后方进行中线切口,沿头皮露出头骨(视频1)。

    Video 1. Brain removal, dissection and homogenization

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  3. 通过侧颅缝线将颅骨从颈部小心地切割到鼻子。可以在枕骨方向的枕孔中进行两次额外的切口,以方便进入大脑(图2A,视频1)。


    图2.脑解剖和匀浆的说明图。 通过侧面颅缝线(A)将颅骨从颈部小心地切割到鼻子。使用小铲(B)从头盖箱中取出完整的大脑。然后用精细夹层钳去除小脑,嗅球和白质,保持脑皮层(C)。最后,使用具有PTFE杵和玻璃管的Potter-Elvehjem,以800rpm / min(D),在4℃下,在4℃,在10ml PBS中均匀化脑组织。

  4. 使用小铲子从颅骨盒中取出完整的大脑(图2B,视频1)。
  5. 使用精细夹层钳去除小脑,嗅球和白质;保持脑皮层(图2C,视频1,见注1)
  6. 在非无菌PBS中进行快速洗涤(约30秒)(视频1;请参阅食谱)。
  7. 使用具有PTFE杵和玻璃管的Potter-Elvehjem,以800rpm / min(图3,视频1),在4℃,在4℃下,在4℃下均匀化新鲜脑组织。

    图3.小鼠脑血管分离示意图

  8. 在720℃离心5分钟,4℃离心(图3)。
  9. 通过倾析弃去上清液(图3)
  10. 使用柔软的毛刷,3mm直径将沉淀重悬于10ml PBS中(图3)
  11. 再次重复步骤8-10两次。
  12. 将PBS重悬于沉淀超过20ml 16%葡聚糖的层(视频2;参见食谱)。
    注意:必须轻轻地完成此步骤,以避免混合两层(图3)。

    Video 2. Brain vessels isolation

    To play the video, you need to install a newer version of Adobe Flash Player.

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  13. 以4,500 x g离心20分钟,4℃离心(图2)。
  14. 收集上清液和中间层,并将沉淀物保持在冰上(图3)
  15. 重复步骤14.
  16. 弃去上清液,将得到的两个沉淀重悬于100μlPBS中 注意:将第一个沉淀重悬于100μlPBS中,并使用悬浮液重新悬浮第二颗粒。可以使用Mota和Ramalho-Santos(2006)所述的DiffQuick染色,在光学显微镜下观察血管(图4)。
  17. 将容器储存在-80°C直到使用(隔离后使用2年;图3)

    图4. DiffQuik染色后分离的小鼠脑血管的代表性图像。 在光学显微镜下观察到的未染色(A)和染色(B,C)脑血管。 100倍放大;比例尺= 50μM。

笔记

  1. 为了尽量减少可能偏向结果的日常变异性,在使用动物疾病模型的研究中,研究人员必须始终将血管与控制和患病动物模型隔离开。
  2. 分离脑血管所需动物的最小数量:1只大鼠脑或3只小鼠脑。然而,可以仅使用1只小鼠脑,颗粒尺寸将非常小,这将难以肉眼可视化。

食谱

  1. 磷酸盐缓冲液(0.01M)
    8.5克/升NaCl
    1.42g / L Na 2 HPO 4
    pH = 7.4
  2. 葡聚糖(16%)
    160克/升来自肠膜明串珠菌的葡聚糖

注意:

  1. 在制备溶液时必须使用1号蒸馏水。
  2. 在短时间内,解决方案必须在4°C(例如一周)内保存;对于更长的时间,溶液必须在-20°C(除葡聚糖溶液之外)保存。
  3. 不要冻结葡聚糖溶液;在4℃下稳定一周。

致谢

作者的工作得益于"FEDER基金通过运营计划竞争力因素 - COMPETE 2020"和FCT基金会在战略项目下的COMPETE归因参考:POCI-01-0145-FEDER-007440' 。克里斯蒂娜·卡瓦略(Cristina Carvalho)拥有FCT博士后研究奖学金(SFRH / BPD / 107741/2015)。手稿的作者没有声明利益冲突。该协议根据由McNeill等人(1999)描述的先前协议进行了修改。转载自"阿尔茨海默病杂志",vol。 35,Carvalho,Cristina;马多多,努诺莫塔,保拉Correia,SóniaC.卡多索,苏珊娜; Santos,Renato X。桑托斯,玛丽亚。 Oliveira,Catarina R。 Moreira,Paula I.,Type 2 Diabetic and Alzheimer's Disease Mice Present Similar Behavioral,Cognitive and Vascular Anomalies,pp。623-635,版权所有(2013),经IOS出版社许可。

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引用:Carvalho, C. I. and I. Moreira, P. (2017). Isolation of Rodent Brain Vessels. Bio-protocol 7(17): e2535. DOI: 10.21769/BioProtoc.2535.
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