1Department of Neurology, University of Connecticut Health Center, 2Department of Neurology, School of Medicine, University of Pennsylvania, 3Department of Neurosurgery, Hartford Hospital, 4Department of Neurosurgery, School of Medicine, University of Pennsylvania
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Sansing, L. H., Kasner, S. E., McCullough, L., Agarwal, P., Welsh, F. A., Kariko, K. Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice. J. Vis. Exp. (54), e2618, doi:10.3791/2618 (2011).
Investigation of the pathophysiology of injury after intracerebral hemorrhage (ICH) requires a reproducible animal model. While ICH accounts for 10-15% of all strokes, there remains no specific effective therapy. The autologous blood injection model in mice involves the stereotaxic injection of arterial blood into the basal ganglia mimicking a spontaneous hypertensive hemorrhage in man. The response to hemorrhage can then be studied in vivo and the neurobehavioral deficits quantified, allowing for description of the ensuing pathology and the testing of potential therapeutic agents. The procedure described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.
1. Preparation of equipment
2. Preparation of mouse for injection
Note: Have mice delivered to your animal facility at least 7 days before the surgery to allow them to acclimate to the new environment and reduce stress.
3. Intracerebral hemorrhage surgery
Notes: During the entire surgery the mouse is anesthetized with 30% Oxygen, 70% Nitrous oxide, and 1-3% Isoflurane, continuously maintained at 37 ± 0.5°C using a thermistor-controlled heating pad and monitored by rectal thermometer.
4. Representative results:
Figure 1. Coronal section of mouse brain 15 minutes after ICH surgery. Immediately after sacrifice the brain was inspected for ICH success based on gross inspection of a coronal section at the needle insertion site. Hemorrhages that tracked down to the base of the brain, up the needle track past the corpus callosum, or into the ventricles were deemed unsuccessful and that mouse was eliminated from all analyses. Overall ICH success rates were 75-85% in 50 mice with 0% mortality.
Figure 2. Cylinder testing demonstrates left hemiparesis after right basal ganglia ICH. (A) Sample mouse rear after ICH surgery. Note the placement of only the right forelimb on the wall of the cylinder after left basal ganglia ICH. (B) Graph of cylinder testing 1 results from cohort of mice after ICH surgery (n=5) compared to sham (n=4). Sham mice had all procedures except blood injection (needle was inserted into brain). Each mouse was placed in a 12-cm diameter clear glass cylinder and observed for 20 rears. The initial placement of the forelimbs on the wall of the cylinder was scored per rear. Subsequent movements (such as lateral exploration) were not scored until the mouse returned to the ground and the next rear scored. The laterality index was calculated as (# right forelimb placements on the side of the cylinder - # left forelimb placements)/(# right + # left + # both), where 0 indicated no forelimb preference and 1 indicated only the right forelimb was used.
This surgical model of intracerebral hemorrhage in mice using autologous tail artery blood results in a reproducible model of spontaneous basal ganglia hemorrhage. An ICH model in mice offers the advantage of the availability of transgenic animals to investigate pathophysiology; however, their small size makes neurosurgical procedures more technically difficult than in larger animals.
The collagenase model and the autologous blood injection model are two well-established models of experimental ICH. While the collagenase model offers an easier procedure and a highly reproducible hemorrhage 2, the bacterial protein used to degrade the basement membrane could potentially effect any investigation of innate inflammatory responses. In addition, collagenase-disrupted BBB could unnaturally facilitate drug access to the brain during pharmacological (e.g., neuroprotection) experiments. A warfarin-associated ICH model has also recently been developed 3, which allows investigation of hemorrhage expansion for this subset of patients. The benefits of the autologous blood injection model include presence of mechanical damage associated with mass effect, a sterile system without exogenous proteins, the ability to eliminate anticoagulation in order to investigate the natural coagulation and inflammation pathways after spontaneous hemorrhage, and exquisite control over the size of the hemorrhage. Since all mice have the same hemorrhage size, the effects of therapeutic interventions on both tissue and functional outcome can be studied with precision with relatively small sample sizes.
The surgical procedure described here is similar to other published models using autologous blood injection(4-7), and several steps in our protocol were based on these published protocols. Significant improvements in this technique include the elimination of all expandable tubing and dead space in the system, which could potentially interfere with accurate measurement of the volume of blood injected, elimination of all anticoagulants, and a moderately large hemorrhage volume compared to other models of non-anticoagulated blood. A 15 uL ICH in an average 450 uL adult mouse brain accounts for 3% of brain volume. This is roughly comparable to a 40 mL ICH in man, assuming normal average adult brain volume is 1400 mL. This ICH volume results in measurable neurological deficits that persist over two weeks for the study of recovery while maintaining zero mortality rate, which is of practical importance when using expensive transgenic animals.
Direct visualization of this surgery should eliminate common mistakes and aid in ease of replication. Hopefully this will translate to further investigation into the mechanisms of injury and accelerate the development of potential therapeutics.
No conflicts of interest declared.
The work was funded by a fellowship from the Institute for Translational Medicine and Therapeutics, and a training grant from the Institute for Medicine and Engineering (T32HL007954) at the University of Pennsylvania and the Marlene L. Cohen and Jerome H. Fleisch Scholar Grant at the University of Connecticut Health Center (LHS) and NIH NS-029331(FAW).
|Stereotaxic frame for mouse neurosurgery (Stoelting, 51925)|
|Microinfusion pump and processor (UMP-3 and Micro4, World Precision Instruments, Sarasota, FL)|
|Mouse warmer (Stoelting, 50300)|
|Inhalational mouse anesthesia (Braintree Scientific, EZ-AF9000)|
|25 ÁL gastight borosilicate Hamilton syringe with coated plunger and no needle|
|(Hamilton company, Reno, NV, 1702RN syringe: 765401, ferrule: 30949, spacer: 30946)|
|fused silica needle cut to 2 cm length (Hamilton, 17739)||note Hamilton syringe and fused silica needle may be reused for multiple surgeries if sterilized prior to each surgery. These materials are crucial to avoid blood clotting.|
|Sterile surgical gloves|
|Surgical gown, bonnet and mask|
|sterile 27 g needle (single use)|
|sterile 1 cc syringe (single use)|
|sterile surgical blade|
|buprenorphine and isoflurane|
|paraffin wax paper squares|
|Veterinary surgical glue (Vetbond, 3M, St. Paul, MN)|