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Neuroscience

Massive Pontine Hemorrhage by Dual Injection of Autologous Blood

Published: May 29th, 2021

DOI:

10.3791/62089

1The Second Affiliated Hospital of Guangzhou Medical University
* These authors contributed equally

We present a protocol to establish a massive pontine hemorrhage model in a rat via dual injection of autologous blood.

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred microliters of autologous blood was taken from the tail vein and stereotaxically injected into the pons. The injection process was divided into 2 steps: First, 10 µL of blood was injected into a specific location, anteroposterior position (AP) -9.0 mm; lateral (Lat) 0 mm; vertical (Vert) -9.2 mm, followed by a second injection of the residual blood located at AP -9.0 mm; Lat 0 mm; Vert -9.0 mm with a 20-minute interval. The balance beam test, limb placement test, and the modified Voestch neuroscore were used to evaluate neurological function. Magnetic Resonance Imaging (MRI) was used to assess the volume of hemorrhage in vivo. The symptoms of this model were in line with patients with massive pontine hemorrhage.

Intracerebral hemorrhage accounts for one-fifth of stroke patients. The prognosis of intracerebral hemorrhage depends on the speed, volume, and location of bleeding1,2. Compared to the forebrain hemorrhage, the brainstem hemorrhage has higher mortality and morbidity3. About 40% of brainstem hemorrhage occurs in the pons4. The etiology and pathophysiology of pontine hemorrhage are quite different and less studied than forebrain hemorrhage5.

There are two kinds of pontine hemorrhage animal models. One is spontaneo....

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The protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Guangzhou Medical University. Rats were provided by the Animal Center of Southern Medical University. The experimental design is shown in Figure 1.

1. Animal and instruments

  1. Use 8-week-old male Sprague-Dawley rats weighing 250 ± 10 g.
  2. House the rats for at least 7 days before surgery under controlled environmenta.......

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A total of 25 animals were used, 3 for control, 6 for 30 µL, 6 for 60 µL, and 10 for 100 µL blood injections. One rat that received a 100 µL injection of autologous blood (1/10) died within 24 hours after surgery.

Behavioral tests were conducted on Day 1, Day 3, Day 7 and Day 14 after surgery. The scores for the control group and blood-injection groups on different timepoints after surgery are presented in

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In the present study, we provided a protocol to generate a massive pontine hemorrhage rat model. This model can be used for the research on the pathophysiological mechanism and prognosis of massive pontine hemorrhage.

Throughout the experiment, 25 rats were used, of which only one died. The verification of MRI, gross anatomy, and the HE staining indicated that this method had a very low mortality rate and a high success rate. To establish massive pontine hemorrhage model, two problems must be .......

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This study was financially supported by the National Science Foundation of China (81471181 and 81870933) and the Opening Lab Program of Guangzhou Medical University (0506308) to Y Jiang, and by the National Science Foundation of China (81701471) and the Scientific Program of Guangzhou Municipal Health Commission (20191A011083) to Z Qiu, and by the National Science Foundation of China (81501009) to L Wu.

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Name Company Catalog Number Comments
100ml Saline solution Guangdong yixiang 191222201 C1 Preparing heparin diluent
100μl Microinjector Shanghai Gaoge Injection of autologous blood
1ml Syringe Jiangsu Zhiyu 20191014 Withdraw autologous blood from the tail vein
75% Alcohol Shandong Lierkang Disinfection of rat tail
Adhesive tape Shanghai Jinzhong Surgicl instruments
Animal anesthesia system RWD R510-31S-6 Inducing and maintaining anesthesia
Balance beam Jiangsu Saiangsi For neurological deficit scores
Blades Shanghai Feiying 74-C For gross anatomy
Bone cement Shanghai Xinshiji 20180306 Surgicl instruments
Brain tank Shenzhen LEIYEA For gross anatomy
Butorphanol tartrate Jiangsu Hengrui For pain management
Electric cranial drill Nanjing  Darwin biotechnology 20180090018 Making a burr hole on the skull
EP tube Nantong Surui Transfer autologous blood
Erythromycin eye cream Yunnan pharmacy Eyes protection
HE dye liquor Solarbio G1120 For HE staining
Heating pad Dangerous Jungle JR01 Keeping warm
Heparin sodium injection Chengdu Haitong Pharmacal Company 190701 Preparing heparin diluent
IndoPhors Guoyao of China Sterilization
Isoflurane RWD 20080701 Inducing and maintaining anesthesia
Light dark box Jiangsu Saiangsi For neurological deficit scores
Micro-injection pump Baoding Leifu TFD03-01-C Injection of autologous blood
MRI system Philips Confirmation of infarction in vivo
Needle holder Shanghai Jinzhong J32020 Surgicl instruments
Penicilin Guoyao of China Infection Prevention
Q-tips Jiangxi Songhe Surgicl instruments
Scalp heedle Jiangxi Hongda 20200313 Withdraw autologous blood from the tail vein
Scalpel Shanghai Kaiyuan 170902 Surgicl instruments
Shearing scissors Shanghai Jinzhong Y00040 Surgicl instruments
Stereotaxic apparatus RWD 900-00001-00 for surgical positioning
Surgical towel Xinxiang Huakangweicai 20070601 Surgicl instruments
Suture needle Shanghai Jinzhong Surgicl instruments
Suture scissors Shanghai Jinzhong J25041 Surgicl instruments
Tissue holding forcepts Shanghai Jinzhong J31080 Surgicl instruments

  1. Charidimou, A., et al. Brain hemorrhage recurrence, small vessel disease type, and cerebral microbleeds: A meta-analysis. Neurology. 89 (8), 820-829 (2017).
  2. Tao, C., et al. A novel brainstem hemorrhage model by autologous blood infusion in Rat: White Matter Injury, Magnetic Resonance Imaging, and Neurobehavioral features. Journal of Stroke and Cerebrovascular Diseases. 25 (5), 1102-1109 (2016).
  3. Ichimura, S., et al. Surgical treatment for primary brainstem hemorrhage to improve postoperative functional outcomes. World Neurosurgery. 120, 1289-1294 (2018).
  4. Behrouz, R. Prognostic factors in pontine haemorrhage: A systematic review. European Stroke Journal. 3 (2), 101-109 (2018).
  5. Guo, X., et al. Brainstem iron overload and injury in a rat model of brainstem hemorrhage. Journal of Stroke and Cerebrovascular Diseases. 29 (8), 104956 (2020).
  6. Chung, Y., Haines, S. J. Experimental brain stem surgery. Neurosurgery Clinics of North America. 4 (3), 405-414 (1993).
  7. Lekic, T., Tang, J., Zhang, J. H. A rat model of pontine hemorrhage. Acta Neurochirurgica Supplement. 105, 135-137 (2008).
  8. Lekic, T., et al. Evaluation of the hematoma consequences, neurobehavioral profiles, and histopathology in a rat model of pontine hemorrhage. Journal of Neurosurgery. 118 (2), 465-477 (2013).
  9. Shrestha, B. K., et al. Rat brainstem hemorrhage model: Key points to success in modeling. World Neurosurgery. 117, 106-116 (2018).
  10. Luo, M., Tang, X., Zhu, J., Qiu, Z., Jiang, Y. Establishment of acute pontine infarction in rats by electrical stimulation. Journal of Visualized Experiments. (162), (2020).
  11. Wu, L., et al. Keep warm and get success: The role of postischemic temperature in the mouse middle cerebral artery occlusion model. Brain Research Bulletin. 101, 12-17 (2014).

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