Inducing Massive Pontine Hemorrhage in a Rat Model by Autologous Blood Injection

Abstract

Source: Tang, X. et. al., Massive Pontine Hemorrhage by Dual Injection of Autologous Blood. J. Vis. Exp. (2021)

This protocol describes the controlled infusion of autologous blood into the pontine region of a rat's brain to model massive pontine hemorrhage. The purpose is to simulate a localized brain bleed by increasing localized pressure.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Animal and instruments
2. Use 8-week-old male Sprague-Dawley rats weighing 250 ± 10 g.
3. House the rats for at least 7 days before surgery under controlled environmental conditions with an ambient temperature of 25 °C, relative humidity of 65%, and a 12/12-h light-dark cycle.
4. Provide food and water with no limit.
5. Prepare the instruments (Figure 1A-E).

2. Inject the blood in the pons

1. Weigh the rats again 3 days before surgery to select rats with suitable body weight for the experiments.
2. During the 3 days before modeling, train the rats to walk on balance beam 3 times per day to ensure normal rats could pass the balance beam without pause.
3. Preheat the heating pad to 37 °C before anesthesia.
4. Attach a microdrill to the holder on the stereotaxic frame.
5. Inject the rats with 50 mg/kg ketamine and 5 mg/kg xylazine intraperitoneally. Wait until there is no toe-pinch response.
6. Transport the rat into the stereotaxic frame in a prone position. Put the ear bars above the ear canal to secure the head. Fix the skull in a horizontal position to avoid skewing of the injection (Figure 1F).
7. Maintain anesthesia by isoflurane (97.5% oxygen and 2.5% isoflurane) through a stereotaxic nose cone with an inlet and outlet port.
8. Use eye ointment to keep the cornea moist.
9. Shave the hair above the skull with a micro shaver.
10. Draw a 3 cm mid-line with a marker pen in the skull from the line of the bilateral lateral canthus to 0.5 cm behind the posterior fontanelle, as shown in Figure 1F.
11. Apply Entoiodine surgical scrub in a circular fashion, starting at the middle of the marked mid-line and rotating outward.
12. Place the surgical drape.
13. Make an incision with a scalpel along the marked mid-line.
14. Use a cotton swab to remove any potential blood.
15. Place a piece of forceps on each side of the scalp flap to expose the skull (Figure 1G).
16. Dip a cotton swab in 0.9% saline and gently remove the connective tissues from the skull bone to avoid the connective tissues getting caught in the microdrill.
17. Mark the central point of the bregma as the origin point via a marker pen.
18. Perform a craniotomy (1 mm in diameter) using a microdrill at AP-9.0 mm, Lat 0 mm (Figure 2B and Table 1). Proceed carefully because this point is very close to the venous sinus (Figure 1G).
19. Remove the microdrill from the stereotaxic frame.
20. Put a 100 µL Hamilton syringe into the stereotaxic holder (Figure 1K). Turn on the injection pump switch, click the Rapid Inhalation button, aspirate heparin solution (12500 U diluted in 100 mL of saline) to 100 µL and then drain it completely to prevent blood clotting too fast.
21. Apply chlorhexidine and 75% alcohol surgical scrub to the whole tail from root to tip at least 3 times to disinfect the skin, soften the horniness, and dilate the tail vein to increase the success rate of injection.
22. Attach a scalp acupuncture to a 1 mL syringe.
23. Insert the scalp acupuncture into a lateral tail vein 3 cm from the tail tip and take 150 µL of blood (Figure 1H).
24. Remove the scalp acupuncture from the 1 mL syringe.
25. Transfer the blood into a tube (Figure 1I).
    NOTE: Transfer quickly to prevent blood clotting.
26. Change the surgical gloves.
27. Choose Withdraw mode, set the volume to 100 µL, set the speed at 200 µL/min, click the RUN button, and aspirate 100 µL of blood into the Hamilton syringe (Figure 1J).
28. Choose Infuse mode and set the speed at 1 µL/min.
29. Advance the syringe until the tip reaches 9.2 mm below the surface of the brain (Figure 2C and Figure 1L).
30. Click the Run button to inject the first 10 µL of blood at a speed of 1 µL/min (Table 1).
31. Stop the injection and leave the syringe in position for 20 min to prevent blood from flowing into the subarachnoid space.
32. Retract the syringe until the tip arrives at 9.0 mm below the surface of the brain.
33. Restart the injection at the same speed of 1 µL/min until the residual blood has been injected completely.
34. Leave the syringe in position for 10 min to avoid blood backflow.
35. Remove the syringe from the brain slowly.
36. Use bone cement to cover the craniotomy hole.
37. When the cement dries, suture the wound with a 4-0 polyamide suture filament. After sewing 3 or 4 stitches, tie 2-1-1 standard surgical knots.
38. To prevent infection, inject the rat with penicillin (0.25 mL, 80 IU diluted in 4 mL saline) intraperitoneally.
39. Inject the rats subcutaneously with Butorphanol tartrate (2.5 mg/kg) and repeat the injection every 2 hours to relieve postoperative pain until 24 hours after surgery.
40. Observe the rat every 15 min until it fully recovers from anesthesia. Return it to the original cage, with a heating pad underneath the cage. Provide the rat free access to food and water until sacrifice.

Table 1: Injection of autologous blood.

	Mild	Moderate	Massive
Total volume	30 μL	60 μL	100 μL
First injection
Stereotactic coordinates	AP -9.0 mm; Lat 0; Vert -9.2 mm
Volume	10 μL	10 μL	10 μL
Speed	1 μL/min	1 μL/min	1 μL/min
Interval time	20 min	20 min	20 min
Second injection
Stereotactic coordinates	AP -9.0 mm; Lat 0; Vert -9.2 mm		AP -9.0 mm; Lat 0; Vert -9.0 mm
Volume	20 μL	50 μL	90 μL
Speed	1 μL/min	1 μL/min	1 μL/min
Before withdrawal of the needle	10 min	10 min	10 min
AP: anteroposterior position
Lat: lateral
Vert: vertical

Results

Figure 1: Instruments and procedure of surgery. (A) Anesthesia machine. (B) Surgical instruments. (C) Microdrill. (D) Micro-injection pump. (E) Stereotaxic apparatus. (F) A line is marked i...

Materials

List of materials used in this article

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
Bone cement	Shanghai Xinshiji	20180306	Surgicl instruments
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
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
Micro-injection pump	Baoding Leifu	TFD03-01-C	Injection of autologous blood
Needle holder	Shanghai Jinzhong	J32020	Surgicl instruments
Penicilin	Guoyao of China		Infection Prevention
Q-tips	Jiangxi Songhe		Surgicl instruments
Scalp needle	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 forceps	Shanghai Jinzhong	J31080	Surgicl instruments