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Evaluation of Caspase-1 Activation and IL-1β Production in A Kainic Acid Microdyalisis Brain Injury Model
在红藻氨酸损伤透析模型中评估半胱天冬酶1的活化和IL-1β的产生   

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Abstract

Intracerebral infusion of kainic acid (KA) by a microdialysis probe induces a focal swelling in the brain-perfused area which promotes inflammation (Compan et al., 2012; Oprica et al., 2003). The microdialysis technique allows the local in vivo perfusion of KA and the simultaneous collection of inflammatory mediators, and other neuroactive substances, released in the injured brain. This protocol also allows the perfusion of different solutions in each cerebral hemisphere at the same time. By perfusing KA in isotonic solution of Krebs-Ringer Bicarbonate (KRB) (280-290 mOsm) in one hippocampus and KA in hypertonic KRB solution (1,400-1,500 mOsm) in the contralateral side, we can evaluate in vivo the efficiency of hypertonic solutions in preventing inflammation induced by swelling after KA infusion. Once the inflammatory response has been induced, it is possible to infuse through the microdialysis probe a biotinylated specific inhibitor of caspase-1 allowing the detection of the brain regions and cells involved in IL-1 production in response to the injury (Oprica et al., 2003).

Keywords: ELISA(ELISA), In situ caspase-1 detection(原位检测caspase - 1), Brain injury(脑损伤), Inflammasome(炎性体), Microdyalisis(microdyalisis)

Materials and Reagents

  1. Female Sprague-Dawley rats* of 200-220 g body weight
  2. Isoflurane (Baxter S. L, catalog number: 691477H )
  3. Local analgesic Bupivacaine 0.25% (Braun Medical S. A, catalog number: 671065 )
  4. Betadine (Meda Farma Sau, catalog number: 644625.6 )
  5. Ocular lubricant (Lab Pérez Giménez, catalog number: 973370 )
  6. Sorbitol (Sigma-Aldrich, catalog number: S6021 )
  7. Albumin from rat serum (Sigma-Aldrich, catalog number: A6414 )
  8. Kainic Acid 100 mM (Sigma-Aldrich, catalog number: K0250 )
  9. Biotinylated Caspase Inhibitor 100 mM biotinyl-YVAD-CMK (AnaSpec, USA)
  10. Capillary Gap microscope slides (Dako REAL, catalog number: K8020 )
  11. Fluorescein-streptavidin conjugate (BD Pharmingen, PharmingenTM, catalog number: 554060 A-2001 )
  12. NeuN (Chemicon International, catalog number: MAB377 )
  13. Goat anti-mouse IgG secondary antibody Alexa Fluor-568 (Life Technologies, Molecular Probes®, catalog number: A11031 )
  14. Hoechst 33342 (Sigma-Aldrich, catalog number: B2261 )
  15. Paraformaldehyde
  16. KH2PO4
  17. MgSO4
  18. NaHCO3
  19. CaCl2
  20. KCl
  21. Isotonic Krebs Ringer Bicarbonate (see Recipes)
  22. Hypertonic KRB (see Recipes)
  23. Isotonic Krebs Ringer Bicarbonate (KRB) (see Recipes)
  24. Hypertonic KRB (see Recipes)
  25. Sacrifice anesthesia solution (see Recipes)

Equipment

  1. Small-animal sterotaxic apparatus (David Kopf, catalog number: 962 )
  2. Perfusion pumps (Bas, catalog number: MD-1001 )
  3. 1 ml syringes (Bas, catalog number: MD-000 )
  4. Dental drill and # 1 burrs
  5. Scalpel
  6. Anesthesia Isoflurane vaporizer (Parkland Scientific, catalog number: V3000PK )
  7. Rat ear bars (David Kopf, catalog number: 957 )
  8. Rat anesthesia mask (David Kopf, catalog number: 1929-B )
  9. Hypodermic needle (25 G)
  10. Microdialysis pumps  (BAS, catalog number: MD-1001 Babe Bee)
  11. Microdialysis probes, Cut-off 100.000 Daltons CMA-12 14/02 PES, (CMA, USA)
  12. Liquid Switch Valve (Univentor, Zejtun ZTN08, catalog number: 8401405 )
  13. Tygon tubing R3603; 0.38 mm ID (Cole Parmer Instruments Co. USA)

Experimental set-up

Procedure

  1. Animal Preparation
    1. The rat must be anesthetized prior to be placed in the stereotaxic equipment instrument. Place the animal into a vaporizer isoflurane box (breathing chamber), and keep it until the rat it is completely anesthetized. The anesthetic system automatically mixes pure isoflurane solution with oxygen in a desired proportion. During the performance of the experiment, a 2% mix of isoflurane is used.
    2. Shave the hair from the head.
    3. Stabilize the head of the animal by using rat ear bars and the rat anesthesia mask, adjusted dorso-ventrally at -3.0. To properly mark the coordinates for the microdialysis probes implant, the incisor bar was adjusted until the heights of lambda and bregma were equal. This flat skull position was achieved when the incisor bar was lowered 3.3 ± 0.4 mm below horizontal zero, according to Paxinos atlas (Paxinos and Watson, 1997).
    4. Apply ocular lubricant on eyes to avoid drying up.
    5. Decontaminate the skin with Betadine applying it on the head of the rat with the aid of a tip of cotton.
    6. To avoid extra bleeding from the skull, we administrate a vasoconstrictor local analgesic subcutaneously in the middle line of the head (0.1-0.2 ml).
    7. Make a 1-1.5 cm incision in the middle line of the skin head.
    8. Identify bregma and lambda after gently scraping away the periosteal connective tissue.
  2. Microdialysis probes implant
    1. According to the Paxinos atlas (Paxinos and Watson, 1997) the coordinates used for the implant of microdialysis probes are: - 4.6 mm posterior to bregma, + 2.5 mm lateral to midline (right hemisphere), - 2.5 mm lateral to midline (left hemisphere) and + 3.0 mm ventral from dura mater.
    2. Mark stereotaxically these points in order to drill the bone for the insertion of the microdialysis probes. The holes must penetrate the full skull but not the dura mater. The dura mater can be cut with a hypodermic needle (25 G). Apply lint if a small bleeding is produced.
    3. Mount the microdialysis probe in the probe/guide holder and attach it to a sterotaxic manipulator.
    4. Connect syringes to the liquid switch valve and then to the inlet canula of the probe through a 500 mm length Tygon tubing.
    5. Connect 25 mm of Tygon tubing to the outlet canula of the probe to collect the perfusate.
    6. Implant one probe in each hemisphere.
    7. In the left hemisphere we have two syringes connected to the probe: Syringe A filled with isotonic KRB and syringe B with isotonic KRB + Kainic Acid. Both connected to a microdialysis pump. Run the pump at flow rate of 1 μl/min.
    8. In the right hemisphere, we also have two syringes: Syringe C is filled with hypertonic KRB and syringe B with hypertonic KRB + Kainic Acid. Both connected to the microdyalysis pump.
    9. Start KRB perfusion in the left hemisphere immediately after the probe implant. The first 100 min of perfusion are discarded (stabilization period). After this period, collect the basal sample for 1 h. Then, perfuse kainic acid for 60 min, followed by a washout period of 6 h. At the end of the experiment, perfuse the biotinylated caspase-1 inhibitor for 1 h. The same protocol is used for the right hemisphere perfusion.
    10. Collect samples in an Eppendorf tube every hour and immediately transfer them to the freezer at -20 °C for its posterior IL-1 analysis.
    11. Stitch up the skin head and transfer the animal to a cage overnight.
  3. Tissue processing and histological detection of caspase-1 inhibitor
    1. Sacrifice the animal the following day under deep sacrifice anesthesia solution through intracardiac perfusion fixation with 250 ml of 4% paraformaldehyde, after a previous perfusion wash with 50 ml isotonic saline.
    2. Postfix brain in the same fixative solution for 24 h at 4 °C, wash and cryoprotect the tissue with 30% sucrose in phosphate-buffered saline (PBS). Freeze down the tissue and cut 20-μm-thick coronal sections of the hyppocampus in a cryostat.
    3. Mount tissue sections on positive-charged slides (Capillary Gap microscope slides), and treat them with 10 mM sodium acetate (pH 6.0) at 95 °C for 4 min for antigen retrieval.
    4. Incubate sections in fluorescein-streptavidin conjugate (1:200) for fluorochrome detection of caspase-1 inhibitor.
    5. For double-staining with a neuronal marker, incubate sections with the anti-neuronal nuclei primary antibody NeuN (1:500), after a previous treatment with 5% normal goat serum in 0.1% Triton X-100 in PBS.
    6. Incubate sections with Alexa Fluor-568 goat anti-mouse IgG secondary antibody (1:400).
    7. Coverslip slides in a medium containing p-phenylenediamine and bisbenzimide (Hoechst 33342) for nuclei detection.
      Note: All procedures used in this work were in accordance with the European Union Council Directive (86/609/EEC). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Hospital “Ramón y Cajal” (animal facilities ES280790002001).

Recipes

  1. Isotonic Krebs Ringer Bicarbonate (KRB)
    0.4 mM KH2PO4
    1.2 mM MgSO4
    25 mM NaHCO3
    1.2 mM CaCl2
    3 mM KCl
    0.5 mg/ml Rat Albumin
  2. Hypertonic KRB
    0.4 mM KH2PO4
    1.2 mM MgSO4
    25 mM NaHCO3
    1.2 mM CaCl2
    3 mM KCl
    20% Sorbitol
    0.5 mg/ml Rat Albumin
  3. Sacrifice anesthesia solution
    0.4-0.5 ml/rat of a mixture prepared as follows:
    0.5 ml of Ketamine (50 mg/ml)
    0.4 ml of Valium (4 mg/ml)
    0.1 ml Atropine (1 mg/ml)

Acknowledgments

This protocol has been adapted from the previously published paper: Compan et al. (2012). This work was supported by grants from PN I+D+I 2008-2011-Instituto Salud Carlos III-FEDER (EMER07/049 and PI09/0120) and Fundación Séneca (11922/PI/09). The authors declare no conflict of interests.

References

  1. Compan, V., Baroja-Mazo, A., López-Castejón, G., Gomez, A. I., Martínez, C. M., Angosto, D., Montero, M. T., Herranz, A. S., Bazán, E. and Reimers, D. (2012). Cell volume regulation modulates NLRP3 inflammasome activation. Immunity 2012;37:487-500.
  2. Oprica, M., Eriksson, C. and Schultzberg, M. (2003). Inflammatory mechanisms associated with brain damage induced by kainic acid with special reference to the interleukin‐1 system. J Cell Mol Med 7(2): 127-140.   
  3. Paxinos G and Watson C. The rat brain in sterotaxic coordinates, 3ed Edition.(1997). Academic Press, Inc. (Ca, USA)

简介

通过微透析探针脑内输注红藻氨酸(KA)在脑灌注区域诱导局灶性肿胀,这促进炎症(Compan等人,2012; Oprica等人, 。,2003)。微透析技术允许局部的体内灌注KA和同时收集炎性介质和其他神经活性物质,释放在受伤的脑中。该协议还允许在每个大脑半球同时灌注不同的溶液。通过在对侧中在一个海马中的Krebs-Ringer碳酸氢盐(KRB)(280-290mOsm)的等渗溶液中灌注KA和在高渗KRB溶液(1,400-1,500mOsm)中的KA,我们可以在体内评价高渗溶液在KA输注后预防由肿胀引起的炎症的效率。一旦已经诱导炎症反应,就有可能通过微透析探针输入胱天蛋白酶-1的生物素化特异性抑制剂,从而允许检测响应于损伤的IL-1产生的脑区域和细胞(Oprica等人, et al。,2003)。

关键字:ELISA, 原位检测caspase - 1, 脑损伤, 炎性体, microdyalisis

材料和试剂

  1. 体重为200-220g的雌性Sprague-Dawley大鼠*
  2. 异氟烷(Baxter S.L,目录号:691477H)
  3. 局部镇痛剂布比卡因0.25%(Braun Medical S.A,目录号:671065)
  4. Betadine(Meda Farma Sau,目录号:644625.6)
  5. 眼用润滑剂(LabPérezGiménez,目录号:973370)
  6. 山梨醇(Sigma-Aldrich,目录号:S6021)
  7. 来自大鼠血清的白蛋白(Sigma-Aldrich,目录号:A6414)
  8. 红藻氨酸100mM(Sigma-Aldrich,目录号:K0250)
  9. 生物素化半胱天冬酶抑制剂100mM生物素-YVAD-CMK(AnaSpec,USA)
  10. 毛细管间隙显微镜载玻片(Dako REAL,目录号:K8020)
  11. 荧光素 - 链霉抗生物素蛋白缀合物(BD Pharmingen,Pharmingen</sup>,目录号:554060A-2001)
  12. NeuN(Chemicon International,目录号:MAB377)
  13. 山羊抗小鼠IgG二抗Alexa Fluor-568(Life Technologies,Molecular Probes ,目录号:A11031)
  14. Hoechst 33342(Sigma-Aldrich,目录号:B2261)
  15. 多聚甲醛
  16. KH 2 PO 4
  17. MgSO 4 4 /
  18. NaHCO 3
  19. CaCl <2>
  20. KCl
  21. 等容克雷布斯林格碳酸氢盐(见配方)
  22. 高渗KRB(参见配方)
  23. 等容克雷布斯林格氏碳酸氢盐(KRB)(见配方)
  24. 高渗KRB(参见配方)
  25. 牺牲麻醉溶液(见配方)

设备

  1. 小动物sterotaxic装置(David Kopf,目录号:962)
  2. 灌注泵(Bas,目录号:MD-1001)
  3. 1ml注射器(Bas,目录号:MD-000)
  4. 牙钻和#1毛刺
  5. Scalpel
  6. 麻醉异氟烷蒸发器(Parkland Scientific,目录号:V3000PK)
  7. 大鼠耳棒(David Kopf,目录号:957)
  8. 大鼠麻醉面罩(David Kopf,目录号:1929-B)
  9. 皮下注射针(25 G)
  10. 微透析泵 (BAS,目录号:MD-1001 Babe Bee)
  11. 微透析探头,截止值100.000道尔顿CMA-12 14/02 PES,(CMA,USA)
  12. 液体开关阀(Univentor,Zejtun ZTN08,目录号:8401405)
  13. 聚乙烯管R3603; 0.38mm ID(Cole Parmer Instruments Co.USA)

实验设置

程序

  1. 动物准备
    1. 在放入立体定位仪器仪器前,必须麻醉大鼠。将动物放入蒸发器异氟醚盒(呼吸室),并保持直到大鼠完全麻醉。麻醉系统自动地将纯异氟醚溶液与所需比例的氧混合。在实验过程中,使用2%的异氟烷​​混合物
    2. 从头部剃掉头发。
    3. 使用大鼠耳杆和大鼠麻醉面罩稳定动物的头部,调整背腹侧-3.0。为了适当地标记微透析探针植入物的坐标,调节门牙棒直到λ和前囱的高度相等。根据Paxinos atlas(Paxinos和Watson,1997),当门牙杆降低到水平零点以下3.3±0.4mm时,实现了这种平坦的颅骨位置。
    4. 在眼睛上涂抹眼润滑剂以避免干涸。
    5. 借助于棉花尖端,将贝他丁应用于大鼠头部,对皮肤进行净化。
    6. 为了避免头颅额外出血,我们在头部中线(0.1-0.2ml)皮下施用血管收缩剂局部止痛剂。
    7. 做一个1-1.5厘米切口在皮肤头的中间行。
    8. 轻轻刮去骨膜结缔组织后,确定前囟和λ
  2. 微透析探针植入
    1. 根据Paxinos atlas(Paxinos and Watson,1997),用于植入微透析探头的坐标是: - 前囟后4.6 mm,中线外侧2.5 mm(右半球), - 中线外侧2.5 mm(左半球 )和来自硬脑膜的+ 3.0mm腹侧。
    2. 立体地标记这些点,以便钻出骨头用于插入微透析探针。孔必须穿透整个颅骨,而不是硬脑膜。硬脑膜可以用皮下注射针(25G)切割。如果出现小出血,则应用棉绒
    3. 将微透析探头安装在探头/导板固定器中,并将其连接到一个sterotaxic操纵器。
    4. 将注射器连接到液体开关阀,然后通过500 mm长的聚乙烯管连接到探头的入口导管。
    5. 将25 mm的Tygon管连接到探针的出口套管以收集灌注液
    6. 在每个半球植入一个探头。
    7. 在左半球,我们有两个注射器连接到探针:注射器A充满等张KRB和注射器B与等渗KRB +红藻酸。两者都连接到微透析泵。以1μl/min的流速运行泵。
    8. 在右半球,我们还有两个注射器:注射器C充满高渗KRB和注射器B与高渗KRB +红藻氨酸。两者都连接到微透析泵。
    9. 在探头植入后立即在左半球开始KRB灌注。丢弃第一个100分钟的灌注(稳定期)。此后,收集基础样品1小时。然后,灌注红藻氨酸60分钟,然后6小时的清洗期。在实验结束时,灌注生物素化的caspase-1抑制剂1小时。相同的协议用于右半球灌注。
    10. 每小时收集样品在Eppendorf管中,立即将其转移到-20°C的冰箱进行后期IL-1分析。
    11. 缝上皮肤头,将动物转移到笼子过夜。
  3. 组织处理和组织学检测caspase-1抑制剂
    1. 在先前用50ml等渗盐水灌注洗涤后,通过用250ml的4%多聚甲醛进行心脏内灌注固定,在第二天在深度牺牲麻醉溶液下牺牲动物。
    2. 在相同的固定溶液中在4℃下后脑修复24小时,用磷酸盐缓冲盐水(PBS)中的30%蔗糖洗涤和冷冻保护组织。冻结组织,切下低温恒温器中海马的厚度为20μm的冠状切片。
    3. 在带正电的载玻片(毛细管间隙显微镜载玻片)上装载组织切片,并在95℃下用10mM乙酸钠(pH 6.0)处理4分钟以进行抗原修复。
    4. 在荧光素 - 链霉亲和素结合物(1:200)中孵育切片用于胱天蛋白酶-1抑制剂的荧光染料检测。
    5. 对于使用神经元标记物的双染色,在用PBS中的0.1%Triton X-100中的5%正常山羊血清预先处理后,孵育具有抗神经元核初级抗体NeuN(1:500)的切片。
    6. 用Alexa Fluor-568山羊抗小鼠IgG二抗(1:400)孵育切片
    7. 盖玻片在含有对苯二胺和双苯酰亚胺的培养基(Hoechst 33342)中进行细胞核检测。
      注意:本工作中使用的所有程序均符合欧盟理事会指令(86/609/EEC)。 该方案由医院"Ramóny Cajal"动物实验伦理委员会(动物设施ES280790002001)批准。

食谱

  1. 等张克雷布林格氏碳酸氢盐(KRB)
    0.4mM KH 2 PO 4 sub/
    1.2mM MgSO 4 25mM NaHCO 3/v/v 1.2mM CaCl 2 3 mM KCl
    0.5 mg/ml大鼠白蛋白
  2. 高渗KRB
    0.4mM KH 2 PO 4 sub/
    1.2mM MgSO 4 25mM NaHCO 3/v/v 1.2mM CaCl 2 3 mM KCl
    20%山梨醇 0.5 mg/ml大鼠白蛋白
  3. 牺牲麻醉溶液
    0.4-0.5ml /大鼠制备的混合物 如下:
    0.5毫升氯胺酮(50毫克/毫升) 0.4ml缬氨酸(4mg/ml) 0.1ml阿托品(1mg/ml)

致谢

该协议已经从先前发表的文章改编而来:Compan em et al。(2012)。 这项工作得到了来自PN I + D + I 2008-2011 - Salud Carlos III-FEDER(EMER07/049和PI09/0120)和FundaciónSéneca(11922/PI/09)的资助。 作者声明没有利益冲突。

参考文献

  1. Compan,V.,Baroja-Mazo,A.,López-Castejón,G.,Gomez,AI,Martínez,CM,Angosto,D.,Montero,MT,Herranz,AS,Bazán,E.and Reimers, 2012)。 细胞体积调节调节NLRP3炎症小体 激活。 免疫力 2012; 37:487-500
  2. Oprica,M.,Eriksson,C.and Schultzberg,M。(2003)。 与红藻氨酸诱导的脑损伤相关的炎症机制,特别参考白细胞介素-1系统。 J Cell Mol Med 7(2):127-140。   
  3. Paxinos G和Watson C.The ster brain in sterotaxic coordinates,3ed Edition。(1997)。 Academic Press,Inc.(Ca,USA)
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Herranz, A. S., Bazán, E., Reimers, D., Montero-Vega, M. T., Jménez-Escrig, A. and Pelegrín, P. (2013). Evaluation of Caspase-1 Activation and IL-1β Production in A Kainic Acid Microdyalisis Brain Injury Model. Bio-protocol 3(8): e433. DOI: 10.21769/BioProtoc.433.
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