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A Rat Model of Intracerebral Hemorrhage Induced by Collagenase IV

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Journal of Neuroinflammation
Mar 2014


Stroke, the second leading cause of death worldwide (Ingall, 2004), is one of the major causes of morbidity and mortality. Intracerebral hemorrhage (ICH), a lethal type of stroke, accounts for 20% of all strokes (Qureshi et al., 2001), and occurs in about 50-60% of Asians (Inagawa, 2002). In order to understand the disease process, three animal model of ICH have been used to study the pathophysiology and treatment of ICH, including the microballoon model, the bacterial collagenase injection model (Rosenberg et al., 1990) and the autologous blood injection model (Andaluz et al., 2002; Belayev et al., 2003). In the collagenase injection model, the hemorrhage size is controllable which was induced by small vessel breakdown. This model also can mimic the onset of spontaneous intraparenchymal bleeding and the expansion of continuous bleeding in ICH patients (Kazui et al., 1996; MacLellan et al., 2008; James et al., 2008). In the past several years, our previous studies have proven that our modified collagenase IV injection model is a reliable and reproducible model of ICH in rat (Lu et al., 2014; Gao et al., 2014; Wang et al., 2011). We hereby introduce our model of ICH as following.

Keywords: Intracerebral hemorrhage (脑出血), Model (模型), Collagenase IV (IV型胶原酶)

Materials and Reagents

  1. About 2 months old male Sprague-Dawley rats(280~320 g)
  2. Isoflurane
  3. Collagenase IV (Sigma-Aldrich, catalog number: C5138 )
  4. 4-0 monocryl


  1. Sterile iodophor wipes
  2. Sterile bone wax
  3. Induction chamber
  4. Stereotactic apparatus (KOPF®, model: 900 Small Animal Stereotaxic Instrument)
  5. Stereotaxic drill (KOPF®, model: 1474 High Speed Stereotaxic Drill)
  6. The animal gas anesthesia system (RDW Life Science)
  7. Temperature controller and temperature sensor system (RDW Life Science, model: 69001)
  8. Microinjection pump (KD Scientific, model: KDS310 )
  9. Hamilton syringe 26 G (5 μl)
  10. Magnetic Resonance Imaging (MRI)
  11. Brain matrix (RDW Life Science, catalog number: 68710 )


  1. The stereotactic apparatus are set up and the weight of rats are recorded before operation (Figure 1).
  2. The rats are deeply anesthetized with 3% isoflurane in an induction chamber.
  3. To ensure the depth of anesthesia, the rats are tested by toe pinch.
  4. After loss of consciousness, the rats are fixed on the stereotactic frame using a nose clamp and two ear bars (Figure 1).
  5. A temperature sensor is inserted into the rats’ anus, and the temperature of rats is maintained at 37 °C by a temperature controller.
  6. The rats are kept anesthetized with 2% isoflurane by gas mask.
  7. After shaving the fur, the superior surface of rat head is prepared by sterile iodophor wipes as surgical field.
  8. A longitudinal incision is made on the surface of head to expose the bregma.
  9. Using the stereotactic apparatus, a left point 3 mm lateral to the bregma is marked.
  10. A burr hole through the skull is drilled at the marked point by a high speed stereotaxic drill.
    Note: The Depth of the drilling depended on the experience of the operator. We can assess the damage by Immunohistochemistry.
  11. 1 μl collagenase type IV (0.25 IU/μl) is injected into corpus striatum (5 mm below the skull) by a 5 μl Hamilton syringe 26 G at a slow rate of 0.2 μl/min.
  12. The syringe is remained at the place about 7 min after the injection is completed.
  13. For about 7 min later, the syringe is removed slowly.
  14. Once the syringe is removed, sterile bone wax is used to plug the hole quickly.
  15. The skin on the surface of head is closed by using 4-0 monocryl.
  16. The rats are removed from the stereotactic apparatus and are allowed to recover in a warmed cage with free access to food and water.
    Note: The recovery period is about 20-30 min.
  17. To evaluate the rat Model of ICH induced by collagenase IV, the rats are sacrificed and the brain is cut into slices after operation to assess the volume of hematoma (see Note 3) (Figure 2A and 2B). Furthermore, the neurobehavior (Bederson test) of ICH rats is assessed (Figure 2C-2F). At last, the volume of hematoma also is analysed by MRI (Figure 2G-2H).

    Figure 1. A picture representing a set of the stereotactic apparatus system and a rat mounted in the stereotactic frame. A. Stereotactic frame; B. Infusion pump; C. Stereotaxic drill; D. Nose clamp; E. Ear bar; F. Hamilton syringe 26 G.

    Figure 2. Evaluation of ICH rat induced by collagenase IV. A. The hematoma at 3 h after operation. B. The hematoma at 3 days after operation. C-F. Neurobehavioral test at 3 days after operation, such as, failure to extend contralateral forelimb fully (C), failure to flex the hind legs (E), circling-walking (D), and failure to keep balance on a beam. G-J. The volume of hematoma tested by MRI at 3 days after operation.


  1. The concentration of collagenase IV should be exactly 0.25 IU/μl, because a small change in concentration of collagenase IV may cause a different in the volume of the hematoma.
  2. The stereospecific place collagenase IV injected is 3 mm lateral to the bregma and 5 mm below to skull.
  3. The brains are sliced by the brain matrix, which can slice the brains about 1 mm thickness. And before sliced, the brains should be freezed at -40 °C about 20 min.
  4. Bederson test (Bederson et al., 1986) is an evaluation for neurologic status of stroke rats. Rats will be suspended about 1m high by holding the tail, the status of the forelimb flexion will be observed and recorded. Rats that extend both of their forelimbs will be considered as normal or having no observable deficit, standing for grade 0. If any consistent flexion of forelimb occurs, such as only wrist flexion, or shoulder adduction with elbow extension, or even full flexion of wrist and elbow with adduction and internal rotation of the shoulder contralateral to the infarct side, rats will be assigned at least grade 1. If further, rats could not resist the lateral push toward the paretic side after gently providing a lateral pressure behind the shoulder, will be grade 2. Then rats are allowed to move freely. Whether the rats will circle toward the paretic side is the indicator for grade 3. Generally, rats that display circling behavior, being grade 3, have greater neurologic deficit.


The study was supported by the grant from the National Natural Science foundation of China (No. 81171088).


  1. Andaluz, N., Zuccarello, M. and Wagner, K. R. (2002). Experimental animal models of intracerebral hemorrhage. Neurosurg Clin N Am 13(3): 385-393.
  2. Belayev, L., Saul, I., Curbelo, K., Busto, R., Belayev, A., Zhang, Y., Riyamongkol, P., Zhao, W. and Ginsberg, M. D. (2003). Experimental intracerebral hemorrhage in the mouse: histological, behavioral, and hemodynamic characterization of a double-injection model. Stroke 34(9): 2221-2227.
  3. Bederson, J. B., Pitts, L. H., Tsuji, M., Nishimura, M. C., Davis, R. L. and Bartkowski, H. (1986). Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17(3): 472-476.
  4. Gao, L., Lu, Q., Huang, L. J., Ruan, L. H., Yang, J. J., Huang, W. L., ZhuGe, W. S., Zhang, Y. L., Fu, B., Jin, K. L. and ZhuGe, Q. C. (2014). Transplanted neural stem cells modulate regulatory T, gammadelta T cells and corresponding cytokines after intracerebral hemorrhage in rats. Int J Mol Sci 15(3): 4431-4441.
  5. Inagawa, T. (2002). What are the actual incidence and mortality rates of intracerebral hemorrhage? Neurosurg Rev 25(4): 237-246.
  6. Ingall, T. (2004). Stroke--incidence, mortality, morbidity and risk. J Insur Med 36(2): 143-152.
  7. James, M. L., Warner, D. S. and Laskowitz, D. T. (2008). Preclinical models of intracerebral hemorrhage: a translational perspective. Neurocrit Care 9(1): 139-152.
  8. Kazui, S., Naritomi, H., Yamamoto, H., Sawada, T. and Yamaguchi, T. (1996). Enlargement of spontaneous intracerebral hemorrhage. Incidence and time course. Stroke 27(10): 1783-1787.
  9. Lu, Q., Gao, L., Huang, L., Ruan, L., Yang, J., Huang, W., Li, Z., Zhang, Y., Jin, K. and Zhuge, Q. (2014). Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat. J Neuroinflammation 11: 44.
  10. MacLellan, C. L., Silasi, G., Poon, C. C., Edmundson, C. L., Buist, R., Peeling, J. and Colbourne, F. (2008). Intracerebral hemorrhage models in rat: comparing collagenase to blood infusion. J Cereb Blood Flow Metab 28(3): 516-525.
  11. Qureshi, A. I., Tuhrim, S., Broderick, J. P., Batjer, H. H., Hondo, H. and Hanley, D. F. (2001). Spontaneous intracerebral hemorrhage. N Engl J Med 344(19): 1450-1460.
  12. Rosenberg, G. A., Mun-Bryce, S., Wesley, M. and Kornfeld, M. (1990). Collagenase-induced intracerebral hemorrhage in rats. Stroke 21(5): 801-807.
  13. Wang, Z., Cui, C., Li, Q., Zhou, S., Fu, J., Wang, X. and Zhuge, Q. (2011). Intracerebral transplantation of foetal neural stem cells improves brain dysfunction induced by intracerebral haemorrhage stroke in mice. J Cell Mol Med 15(12): 2624-2633.


中风是世界第二大死亡原因(Ingall,2004),是发病和死亡的主要原因之一。脑内出血(ICH)是致命型中风,占所有中风的20%(Qureshi等人,2001),并发生在约50-60%的亚洲人中(Inagawa,2002) 。为了理解疾病过程,已经使用ICH的三种动物模型来研究ICH的病理生理学和治疗,包括微球模型,细菌胶原酶注射模型(Rosenberg等人,1990)和自体血液注射模型(Andaluz等人,2002; Belayev等人,2003)。在胶原酶注射模型中,出血尺寸是可控的,其由小血管分解诱导。该模型还可以模拟自发性实质内出血的发作和ICH患者中持续出血的扩张(Kazui等人,1996; MacLellan等人,2008; James et al。,2008)。在过去几年中,我们以前的研究已经证明我们的修饰的胶原酶IV注射模型是大鼠中ICH的可靠且可重复的模型(Lu等人,2014; Gao等人, ,2014; Wang 等人,2011)。我们在此引入我们的ICH模型如下。

关键字:脑出血, 模型, IV型胶原酶

  • Bencke-Malato,M.,Cabreira,C.,Wiebke-Strohm,B.,Bucker-Neto,L.,Mancini,E.,Osorio,MB,Homrich,MS,Turchetto-Zolet,A.,De Carvalho,M 。,Stolf,R.,Weber,R.,Westergaard,G.,Castagnaro,AP,Abdelnoor,RV,Marcelino-Guimaraes,FC,Margis-Pinheiro,M.and Bodanese-Zanettini, 大豆WRKY家族的全基因组注释和参与响应的基因的功能表征 Phakopsora pachyrhiziz 感染。 BMC Plant Biol 14(1):236。
  • Scherb,C.T。和Mehl,A。(2006)。 SBI杀真菌剂,也适用于其他杀真菌剂类 - 离体叶试验。 FRAC (杀菌剂抗性行动委员会)
  • Twizeyimana,M.,Ojiambo,P.,Ikotun,T.,Paul,C.,Hartman,G.and Bandyopadhyay,R。(2007)。 大豆种质田间,温室和独立叶评估的比较 Plant Dis 91(9):1161-1169。
  • Wiebke-Strohm,B.,Pasquali,G.,Margis-Pinheiro,M.,Bencke,M.,Bucker-Neto,L.,Becker-Ritt,AB,Martinelli,AH,Rechenmacher,C.,Polacco, Stolf,R.,Marcelino,FC,Abdelnoor,RV,Homrich,MS,Del Ponte,EM,Carlini,CR,De Carvalho,MC和Bodanese-Zanettini,MH(2012)。 无处不在的尿素酶影响大豆对真菌的易感性。 植物分子生物学> 79(1-2):75-87。
  • ...

    1. 无菌碘伏湿巾
    2. 无菌骨蜡
    3. 感应室
    4. 立体定位装置(KOPF ,型号:900小动物立体定位仪)
    5. 立体定位钻(KOPF ,型号:1474高速立体定位钻)
    6. 动物气体麻醉系统(RDW Life Science)
    7. 温度控制器和温度传感器系统(RDW Life Science,型号:69001)
    8. 显微注射泵(KD Scientific,型号:KDS310)
    9. Hamilton注射器26G(5μl)
    10. 磁共振成像(MRI)
    11. 脑基质(RDW Life Science,目录号:68710)


    1. 立体定向装置设置,并在手术前记录老鼠的体重(图1)
    2. 在诱导室中用3%异氟烷将大鼠深度麻醉
    3. 为了确保麻醉深度,通过脚趾测试大鼠。
    4. 失去知觉后,使用鼻夹和两个耳杆将大鼠固定在立体定位框架上(图1)。
    5. 将温度传感器插入大鼠肛门,并且通过温度控制器将大鼠的温度维持在37℃。
    6. 用2%异氟烷通过防毒面罩将大鼠保持麻醉
    7. 在剃毛后,通过无菌碘浮床擦拭器作为手术区域制备大鼠头部的上表面
    8. 在头部表面上形成纵向切口以暴露前囟
    9. 使用立体定向装置,标记前囟点侧边3mm处的左点
    10. 通过高速立体定位钻在标记点钻穿过颅骨的钻孔。
      注意:钻孔的深度取决于操作者的经验。 我们可以通过免疫组织化学评估损伤。
    11. 通过5μlHamilton注射器26G以0.2μl/min的缓慢速率将1μlIV型胶原酶(0.25IU /μl)注射入纹状体(在颅骨下方5mm)。
    12. 注射器在注射完成后约7分钟保持在该位置
    13. 约7分钟后,缓慢取出注射器。
    14. 一旦注射器被取出,无菌骨蜡被用来快速堵塞孔
    15. 头部表面的皮肤用4-0 monocryl封闭
    16. 将大鼠从立体定向装置中取出,并允许在温热的笼子中恢复,自由进食和水。
    17. 为了评估由胶原酶IV诱导的ICH的大鼠模型,处死大鼠,并且在手术后将脑切成切片以评估血肿的体积(参见注释3)(图2A和2B)。 此外,评估ICH大鼠的神经行为(Bederson检验)(图2C-2F)。 最后,还通过MRI分析血肿的体积(图2G-2H)

      图1.表示一组立体定位装置系统和安装在立体定位框架中的老鼠的图片。 A.立体定位框架; B.输液泵; C.立体定位钻; D.鼻夹; E.耳杆; F. Hamilton注射器26 G.

      图2.胶原酶IV诱导的ICH大鼠的评价 A.术后3小时的血肿。 B.手术后3天血肿。 C-F。手术后3天的神经行为测试,例如未充分伸展对侧前肢(C),未能屈曲后肢(E),环绕行走(D)以及未能在梁上保持平衡。 G-J。手术后3天MRI检查的血肿体积。


    1. 胶原酶IV的浓度应该精确地为0.25IU /μl,因为胶原酶IV的浓度的小变化可能导致血肿的体积不同。
    2. 注射的立体特异性位置胶原酶IV在前囱的侧面3mm和颅骨下面5mm
    3. 大脑由脑基质切片,其可切割约1mm厚度的脑。在切片前,大脑应在-40℃下冷冻约20分钟
    4. Bederson试验(Bederson等人,1986)是对中风大鼠的神经学状态的评价。大鼠将通过握住尾巴悬挂约1米高,将观察和记录前肢屈曲的状态。延伸其两个前肢的大鼠将被认为是正常的或没有可观察到的缺陷,代表0级。如果发生前肢的任何一致的弯曲,例如只有手腕弯曲或具有肘伸展的肩部内收,或甚至手腕的完全屈曲和肘部,其具有对应于梗塞侧的肩部的内收和内旋,大鼠将被分配至少1级。如果进一步,在轻轻地在肩部后面提供侧向压力之后,大鼠不能抵抗侧向推向偏瘫侧然后使大鼠自由移动。是否大鼠将朝向瘫痪侧圈是等级3的指标。通常,显示盘旋行为,即3级的大鼠具有更大的神经缺陷。




    1. Andaluz,N.,Zuccarello,M。和Wagner,K.R。(2002)。 脑内出血的实验动物模型。 Neurosurg Clin N Am 13(3):385-393。
    2. Belayev,L.,Saul,I.,Curbelo,K.,Busto,R.,Belayev,A.,Zhang,Y.,Riyamongkol,P.,Zhao,W.and Ginsberg, 小鼠实验性脑内出血:双注射模型的组织学,行为和血液动力学表征。 笔划 34(9):2221-2227。
    3. Bederson,J.B.,Pitts,L.H.,Tsuji,M.,Nishimura,M.C.,Davis,R.L。和Bartkowski,H。(1986)。 大鼠大脑中动脉闭塞:模型评估和神经系统检查的发展。 笔划 17(3):472-476。
    4. 高等学校学报(社会科学版)。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。 移植的神经干细胞在大鼠脑内出血后调节调节性T细胞,gammadelta T细胞和相应的细胞因子。/a> Int J Mol Sci 15(3):4431-4441
    5. Inagawa,T。(2002)。 脑内出血的实际发生率和死亡率是多少? Neurosurg Rev 25(4):237-246。
    6. Ingall,T。(2004)。 中风 - 发病率,死亡率,发病率和风险。 J Insur Med 36(2):143-152。
    7. James,M.L.,Warner,D.S。和Laskowitz,D.T。(2008)。 脑内出血的临床前模型:平移视角。 Neurocrit Care < em> 9(1):139-152。
    8. Kazui,S.,Naritomi,H.,Yamamoto,H.,Sawada,T.and Yamaguchi,T。(1996)。 扩大自发性脑内出血。发病和时间过程。 中风 27(10):1783-1787。
    9. Lu,Q.,Gao,L.,Huang,L.,Ruan,L.,Yang,J.,Huang,W.,Li,Z.,Zhang,Y.,Jin,K.and Zhuge, 2014)。 抑制雷帕霉素的哺乳动物靶标改善了大鼠脑内出血后的神经行为缺陷并调节免疫应答。 a> J Neuroinflammation 11:44
    10. MacLellan,C.L.,Silasi,G.,Poon,C.C.,Edmundson,C.L.,Buist,R.,Peeling,J.and Colbourne,F。(2008)。 大鼠脑内出血模型:比较胶原酶与血液输注。 Blood Flow Metab 28(3):516-525。
    11. Qureshi,A.I.,Tuhrim,S.,Broderick,J.P.,Batjer,H.H.,Hondo,H.and Hanley,D.F。(2001)。 自发性脑内出血 N Engl J Med 344( 19):1450-1460。
    12. Rosenberg,G.A.,Mun-Bryce,S.,Wesley,M。和Kornfeld,M。(1990)。 大鼠中胶原酶诱导的脑内出血 中风 21 (5):801-807。
    13. Wang,Z.,Cui,C.,Li,Q.,Zhou,S.,Fu,J.,Wang,X.and Zhuge,Q。(2011)。 胎儿神经干细胞的脑内移植改善了小鼠脑内出血中风引起的脑功能障碍。 J Cell Mol Med 15(12):2624-2633。
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    引用:Lu, Q., Huang, L. and ZhuGe, Q. (2015). A Rat Model of Intracerebral Hemorrhage Induced by Collagenase IV. Bio-protocol 5(14): e1541. DOI: 10.21769/BioProtoc.1541.