Name: intrastriatal injection of autologous blood or clostridial collagenase as murine models of intracerebral hemorrhage
Uploaded: 2014-07-03T13:00:00
Description: Watch this Scientific Journal Video about Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage at JoVE.com

This work was supported by the American Heart Association Scientist Development Grant and the Foundation for Anesthesia Education and Research (MLJ). We would like to thank Talaignair N. Venkatraman PhD for his assistance with magnetic resonance imaging.

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
<ol>
	<li>Autoclave the surgical tools prior to surgery.</li>
	<li>Disinfect the stereotactic apparatus with 70% ethanol.</li>
	<li>Turn on water bath and keep water temperature at 42 &#176;C.</li>
	<li>Dissolve Type IV-S clostridial collagenase in normal saline at a concentration of 0.0...

<ol>
	<li>Asch, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence,+case+fatality,+and+functional+outcome+of+intracerebral+haemorrhage+over+time,+according+to+age,+sex,+and+ethnic+origin:+a+systematic+review+and+meta-analysis.">Incidence, case fatality, and functional outcome of intracerebral haemorrhage over time, according to age, sex, and ethnic origin: a systematic review and meta-analysis.</a> Lancet Neurology. 9, 167-176 (2010).</li><li>Qureshi, A. I., Mendelow, A. D., Hanley, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+haemorrhage.">Intracerebral haemorrhage.</a> Lancet. 373, 1632-1644 (2009).</li><li>Participants, N. I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priorities+for+clinical+research+in+intracerebral+hemorrhage:+report+from+a+National+Institute+of+Neurological+Disorders+and+Stroke+workshop.">Priorities for clinical research in intracerebral hemorrhage: report from a National Institute of Neurological Disorders and Stroke workshop.</a> Stroke. 36, (2005).</li><li>Morgenstern, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+management+of+spontaneous+intracerebral+hemorrhage:+a+guideline+for+healthcare+professionals+from+the+American+Heart+Association/American+Stroke+Association.">Guidelines for the management of spontaneous intracerebral hemorrhage: a guideline for healthcare professionals from the American Heart Association/American Stroke Association.</a> Stroke. 41, 2108-2129 (2010).</li><li>James, M. L., Warner, D. S., Laskowitz, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+intracerebral+hemorrhage:+a+translational+perspective.">Preclinical models of intracerebral hemorrhage: a translational perspective.</a> Neurocrit Care. 9, 139-152 (2008).</li><li>Winkler, D. 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Part 1: Description of technique, ICP changes and neuropathological findings.</a> Neurol Res. 6, 184-188 (1984).</li><li>Sang, Y. H., Su, H. X., Wu, W. T., So, K. F., Cheung, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+blood+pressure+aggravates+intracerebral+hemorrhage-induced+brain+injury.">Elevated blood pressure aggravates intracerebral hemorrhage-induced brain injury.</a> J Neurotrauma. 28, 2523-2534 (2011).</li><li>Krafft, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+intracerebral+hemorrhage+in+mice:+injection+of+autologous+blood+or+bacterial+collagenase.">Modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase.</a> J Vis Exp. , (2012).</li><li>Sansing, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+blood+injection+to+model+spontaneous+intracerebral+hemorrhage+in+mice.">Autologous blood injection to model spontaneous intracerebral hemorrhage in mice.</a> J Vis Exp. , (2011).</li><li>Rosenberg, G. A., Mun-Bryce, S., Wesley, M., Kornfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase-induced+intracerebral+hemorrhage+in+rats.">Collagenase-induced intracerebral hemorrhage in rats.</a> Stroke. 21, 801-807 (1990).</li><li>Nath, F. P., Jenkins, A., Mendelow, A. D., Graham, D. I., Teasdale, G. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+recombinant+activated+factor+VII+for+acute+intracerebral+hemorrhage.">Efficacy and safety of recombinant activated factor VII for acute intracerebral hemorrhage.</a> N Engl J Med. 358, 2127-2137 (2008).</li><li>Mendelow, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+surgery+versus+initial+conservative+treatment+in+patients+with+spontaneous+supratentorial+lobar+intracerebral+haematomas+(STICH+II):+a+randomised+trial.">Early surgery versus initial conservative treatment in patients with spontaneous supratentorial lobar intracerebral haematomas (STICH II): a randomised trial.</a> Lancet. , (2013).</li><li>James, M. L., Blessing, R., Bennett, E., Laskowitz, D. 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M., Lascola, C. D., Vitek, M. P., Laskowitz, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacogenomic+effects+of+apolipoprotein+e+on+intracerebral+hemorrhage.">Pharmacogenomic effects of apolipoprotein e on intracerebral hemorrhage.</a> Stroke. 40, 632-639 (2009).</li><li>James, M. 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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...

Intracerebral hemorrhage (ICH) is a relatively common form of cerebrovascular disease with approximately 40&#8211;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&#8217;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.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="width:197px;"></td>
			<td style="width:80px;">Collagenase Injection</td>
			<td style="width:66px;">Blood Injection</td>
		</tr>
		<tr>
			<td style="width:197px;">Ease of Use</td>
			<td style="width:80px;">+++</td>
			<td style="width:66px;">++</td>
		</tr>
		<tr>
			<td style="width:197px;">Reproducibility</td>
			<td style="width:80px;">++</td>
			<td style="width:66px;">++</td>
		</tr>
		<tr>
			<td style="width:197px;">Control of Hemorrhage Size</td>
			<td style="width:80px;">++</td>
			<td style="width:66px;">+++</td>
		</tr>
		<tr>
			<td style="width:197px;">Blood Reflux</td>
			<td style="width:80px;">+</td>
			<td style="width:66px;">++</td>
		</tr>
		<tr>
			<td style="width:197px;">Simulates Human Disease</td>
			<td style="width:80px;">+</td>
			<td style="width:66px;">-</td>
		</tr>
		<tr>
			<td style="width:197px;">Simplicity</td>
			<td style="width:80px;">++</td>
			<td style="width:66px;">++</td>
		</tr>
		<tr>
			<td style="width:197px;">Use in Multiple Species</td>
			<td style="width:80px;">++</td>
			<td style="width:66px;">++</td>
		</tr>
	</tbody>
</table>

<table border="0" cellpadding="0" cellspacing="0" width="416">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Name</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Stereotactic frame</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51603</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Probe holder with corner clamp</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51631</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Mini grinder</td>
			<td style="width:88px;">Power Glide</td>
			<td style="width:88px;">Model 60100002</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">0.5 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co.</td>
			<td style="width:88px;">86259</td>
			<td style="width:88px;">25 gauge needle</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">50 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7637-01</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">26G Hamilton needle</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7804-03</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Syringe pump</td>
			<td style="width:88px;">KD Scientific</td>
			<td style="width:88px;">Model 100</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Heat therapy water pump</td>
			<td style="width:88px;">Gaymar Industries, Inc.</td>
			<td style="width:88px;">Model# TP650</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Circulating waterbed</td>
			<td style="width:88px;">CMS Tool &#38; Die, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Rodent ventilator</td>
			<td style="width:88px;">Harvard Apparatus</td>
			<td style="width:88px;">Model 683</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Isoflurane vaporizer</td>
			<td style="width:88px;">Drager</td>
			<td style="width:88px;">Vapor 19.1</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Air flowmeter</td>
			<td style="width:88px;">Cole Parmer</td>
			<td style="width:88px;">Model PMR1-010295</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Induction chamber</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
			<td style="width:88px;">Self made</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Otoscope</td>
			<td style="width:88px;">Welch Allyn</td>
			<td style="width:88px;">22820</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">intravenous catheter</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">381534</td>
			<td style="width:88px;">20-gauge, 1.16 inch Insyte-W</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Isoflurane</td>
			<td style="width:88px;">Baxter Healthcare Corporation</td>
			<td style="width:88px;">NDC10019-360-69</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Collagenase Type IV-S</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:88px;">C1889</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE20</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427406</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE10</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427401</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">30G 1 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305128</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">27G 1 1/4 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305136</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Surgical scissors</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">21-539</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Forceps</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">17-307</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Needle holder</td>
			<td style="width:88px;">Boboz</td>
			<td style="width:88px;">RS-7840</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Monofilament suture</td>
			<td style="width:88px;">Ethicon</td>
			<td style="width:88px;">8698</td>
			<td style="width:88px;">Size 5-0</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Indicating controller</td>
			<td style="width:88px;">YSI</td>
			<td style="width:88px;">73ATD</td>
			<td style="width:88px;"></td>
		</tr>
	</tbody>
</table>

intrastriatal injection of autologous blood or clostridial collagenase as murine models of intracerebral hemorrhage

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&#39;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.

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...

Watch this Scientific Journal Video about Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage at JoVE.com

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

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.

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

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