Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Summary

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

Abstract

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain's response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

Introduction

Intracerebral hemorrhage (ICH) is a relatively common form of cerebrovascular disease with approximately 40–50% of afflicted patients dying within 30 days ¹. Unfortunately, little improvement has been made in the mortality rate over the last 20 years ². Reports from the National Institutes of Health ³ and guidelines from the American Heart Association ⁴ stressed the importance of developing clinically relevant models of ICH to extend the understanding of pathophysiology and develop targets for new therapeutic approaches.

Several models exist to mimic human ICH ⁵. As understanding of ICH pathophysiology matures, it has become evident that a variety of models may be used to examine different aspects of the disease. Previously used models include murine amyloid angiopathy ⁶, intraparenchymal microballoon insertion and inflation ⁷, and direct arterial blood infiltration ^8,9. Lobar hemorrhage from amyloid angiopathy has been modeled with the use of transgenic mice and represents a distinct ICH subtype. Microballoon models mimic acute mass effect from hematoma formation but fail to capture the brain’s cellular response to the presence of blood. Finally, direct arterial blood infiltration subjects the brain to arterial pressures from the femoral artery. Thus, this model mimics arterial pressures and the presence of blood but does not subject the brain to microvascular injury from small blood vessel rupture. Further, this model has inherently high variability. Interestingly, spontaneously hypertensive rats ¹⁰ develop spontaneous ICH as they age. Study of these animals after ICH development may mimic the disease in the presence of one of the major comorbidities predisposing humans to ICH. While these other models exist, intrastriatal injection of Clostridial collagenase ¹¹ or instrastiatal injection of autologous whole blood ¹² are, currently, the two most common models used in preclinical ICH research.

ICH model selection should be made based on the objective of the experimental question, including species selection and method of inducing hematoma formation. For instance, pigs are large animals with relatively large white matter brain volumes compared to mice. Thus, porcine models are suited to study white matter pathophysiology following ICH. In contrast, rodent brains are largely gray matter, but transgenic systems make rodents useful to assess molecular mechanisms of injury and recovery after ICH. Each model has its inherent strengths and weaknesses (Table 1), which should be carefully considered prior to experimentation.

The following protocols demonstrate the autologous blood and collagenase injection models in mice. These models have each been translated from models originally developed in rats ^13,14 and allow the use of widely available transgenic technology to explore molecular mechanisms associated with cell death after ICH. Both represent distinctly different injury mechanisms from human ICH, and both have distinctly different expected outcome in terms of behavioral and histological measures. Thus, certain hypotheses may lend themselves to one model over the other, but many ideas may require validation in both models.

Table 1. Comparison of characteristics of collagenase- and autologous blood injection intracerebral hemorrhage models.

	Collagenase Injection	Blood Injection
Ease of Use	+++	++
Reproducibility	++	++
Control of Hemorrhage Size	++	+++
Blood Reflux	+	++
Simulates Human Disease	+	-
Simplicity	++	++
Use in Multiple Species	++	++

Protocol

Ethics Statement: This protocol has been approved by the Duke University Institutional Animal Care and Use Committee and follows all the guidelines for the ethical use of animals.

1. Preparation of Equipment

1. Autoclave the surgical tools prior to surgery.
2. Disinfect the stereotactic apparatus with 70% ethanol.
3. Turn on water bath and keep water temperature at 42 °C.
4. Dissolve Type IV-S clostridial collagenase in normal saline at a concentration of 0.075 U per 0.4 µl.

2. Collagenase Injection Model

1. Weigh the mouse.
2. Anesthetize the mouse in an induction chamber with 5% isoflurane in 30% O₂/70% N₂. Adequate anesthesia is signaled after approximately 2 min when mouse respirations have slowed to 1 per second.
3. Intubate the trachea with a 30 mm 20 G intravenous catheter.
4. Connect the catheter to a rodent ventilator and mechanically ventilate the lungs with 1.6% isoflurane in 30% O₂/70% N₂ at a rate of 105 breaths per minute with a delivered tidal volume of 0.75 ml.^.
5. Shave the scalp with an electronic shaver. Once the mouse is anesthetized and intubated, move it to a different work station for shaving and then returned to the surgical bench.
6. Secure the head in a stereotactic frame, and level the head with both coronal and sagittal suture as reference points.
7. Apply ophthalmic ointment to eyes.
8. Insert a rectal temperature probe. Maintain rectal temperature at 37.0 ± 0.2 °C using an underbody circulating waterbed.
9. Wipe the surgical area with betadine followed with 70% ethanol and repeat 3 times.
10. Make a 1 cm midline scalp incision and wipe periosteum laterally with a sterile cotton-tipped applicator to expose bregma.
11. Drill 1 mm diameter burr hole 2.2 mm left lateral to bregma with a water-cooled drill.
12. Rotate collagenase vial 5 times, then wash a 0.5 μl syringe with 25 G needle (attached to stereotactic frame) with 0.5 µl collagenase solution 5 times (Leave 0.5 µl of collagenase solution in syringe after last wash).
13. Align needle tip with burr hole then expel 0.1 µl from syringe and wipe needle bevel with razor to discard.
14. Using a micromanipulator, advance the needle 3 mm deep to cortex and leave motionless for 30 sec.
15. Inject 0.4 µl over 90 sec.
16. Decrease isoflurane to 1% and leave needle motionless for 5 min.
17. Withdraw needle slowly.
18. Apply 1 - 2 drops of 0.25% bupivacaine subcutaneously and suture the skin.
19. Turn off isoflurane vaporizer and remove mouse from the stereotactic frame.
20. Allow mouse to recover spontaneous ventilation with subsequent tracheal extubation.
21. Return mouse to a clean cage and allow free access to food and water.

3. Autologous Blood Injection Model

1. Follow the steps 2.1 - 2.11 for the collagenase injection model.
2. Draw 50 µl of sterile normal saline into a 30 G 50 µl syringe.
3. Connect the microliter syringe with a 70 cm PE10 tube.
4. Expel all the normal saline from microliter syringe into PE10 tube to completely de-air tubing.
5. Pull the microliter syringe piston out 1 mm to make an air bubble at the distal opening of the PE10 tube-microliter syringe apparatus to avoid mixture of saline and blood during later procedures.
6. Wipe the distal central tail artery region of the mouse with 70% ethanol, and cut the artery with a razor at 0.5 to 1 cm to the tail tip.
7. Collect 40 µl of blood from the tail cut into the PE10 tube-microliter syringe apparatus. Note: that heparin is not used in the needle, tubing, or mouse.
8. Attach the microliter syringe onto the injection pump.
9. Connect the metal cannula portion of a 27 G needle to the end of the PE10 tube, and secure the needle to a micromanipulator on the stereotactic frame.
10. Expel 2 µl of blood out of 27 G needle and wipe needle bevel with razor to discard.
11. Align needle tip with burr hole and insert needle 3 mm deep to cortex.
12. Inject 35 µl of autologous blood at a rate of 2 µl per min.
13. Decrease isoflurane to 1% and leave needle motionless for 10 min.
14. Withdraw needle over 30 sec.
15. Apply 1 - 2 drops of 0.25% bupivacaine subcutaneously and suture the skin.
16. Turn off isoflurane vaporizer and remove mouse from the stereotactic frame.
17. Allow mouse to recover spontaneous ventilation with subsequent extubation.
18. Return mouse to a clean cage and allow free access to food and water.

4. Sham Operation

1. Follow the same procedures for collagenase injection model, except without injection after needle insertion.

5. Post-surgery Care

1. Inject 0.5 ml of normal saline subcutaneously in the evening of the surgical procedure at the back of the animal’s neck.
2. Provide softened food with water and gel food in small plastic cups placed on the floor of the cage. Replace the food daily for 7 days.
3. Check for weight loss, wound healing, and signs of discomfort daily for 7 days.
4. If recovery intervals of greater than 7 days are required, suture removal may be performed under light inhaled anesthesia (approximately 1% isoflurane in 30% O₂/70% N₂), if necessary.

Results

Due to differences in hematoma formation (Figure 1), ipsilateral turning is shown immediately after wake up for autologous blood injected mice and within 2 - 4 hr after collagenase injection, as hematoma expansion occurs (Figure 2). Absence of ipsilateral turning should raise concern for absence of significant injury. On the first post injury day, mice in both models should demonstrate significant neurological deficits (Figure 3). At 24 hr after injection, ipsilateral he...

Discussion

Despite emerging preclinical research and resultant large clinical trials for promising therapeutics ^15-18, there are no pharmacological interventions demonstrated to improve outcome in ICH, and care remains largely supportive. Lists of possible therapies may be generated by high throughput technologies, such as transcriptomic and proteomic work. While these technologies continue to advance our knowledge of potential therapeutic targets, forward and backward translation of promising targets may be best examine...

Materials

Name	Company	Catalog Number	Comments
Stereotactic frame	Stoelting Co.	51603
Probe holder with corner clamp	Stoelting Co.	51631
Mini grinder	Power Glide	Model 60100002
0.5 μl syringe	Microliter	86259	25 G needle
5 μl syringe	Microliter	7637-01
30 G microliter syringe	Microliter	7762-03
Syringe pump	KD Scientific	Model 100
Heat therapy water pump	Gaymar Industries, Inc.	Model# TP650
Circulating waterbed	CMS Tool & Die, Inc.
Rodent ventilator	Harvard Apparatus	Model 683
Isoflurane vaporizer	Drager	Vapor 19.1
Air flowmeter	Cole Parmer	Model PMR1-010295
Induction chamber			Self made
Otoscope	Welch Allyn	22820
Intravenous catheter	Becton-Dickinson	381534	20 G, 1.16 inch Insyte-W
Isoflurane	Baxter Healthcare Corporation	NDC10019-360-69
Collagenase Type IV-S	Sigma	C1889
Polyethylene tubing PE10	Becton-Dickinson	427401
27 G 1 1/4 inch needle	Becton-Dickinson	305136
Surgical scissors	Miltex	21-539
Forceps	Miltex	17-307
Needle holder	Boboz	RS-7840
Monofilament suture	Ethicon	8698	Size 5-0
Indicating controller	YSI	73ATD