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 0.075 U per 0.4 &#181;l.</li>
</ol>
2. Collagenase Injection Model
<ol>
	<li>Weigh the mouse.</li>
	<li>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.</li>
	<li>Intubate the trachea with a 30 mm 20 G intravenous catheter.</li>
	<li>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..</li>
	<li>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.</li>
	<li>Secure the head in a stereotactic frame, and level the head with both coronal and sagittal suture as reference points.</li>
	<li>Apply ophthalmic ointment to eyes.</li>
	<li>Insert a rectal temperature probe. Maintain rectal temperature at 37.0 &#177; 0.2 &#176;C using an underbody circulating waterbed.</li>
	<li>Wipe the surgical area with betadine followed with 70% ethanol and repeat 3 times.</li>
	<li>Make a 1 cm midline scalp incision and wipe periosteum laterally with a sterile cotton-tipped applicator to expose bregma.</li>
	<li>Drill 1 mm diameter burr hole 2.2 mm left lateral to bregma with a water-cooled drill.</li>
	<li>Rotate collagenase vial 5 times, then wash a 0.5 &#956;l&#160;syringe with 25 G needle (attached to stereotactic frame) with 0.5 &#181;l collagenase solution 5 times (Leave 0.5 &#181;l of collagenase solution in syringe after last wash).</li>
	<li>Align needle tip with burr hole then expel 0.1 &#181;l from syringe and wipe needle bevel with razor to discard.</li>
	<li>Using a micromanipulator, advance the needle 3 mm deep to cortex and leave motionless for 30 sec.</li>
	<li>Inject 0.4 &#181;l over 90 sec.</li>
	<li>Decrease isoflurane to 1% and leave needle motionless for 5 min.</li>
	<li>Withdraw needle slowly.</li>
	<li>Apply 1 - 2 drops of 0.25% bupivacaine subcutaneously and suture the skin.</li>
	<li>Turn off isoflurane vaporizer and remove mouse from the stereotactic frame.</li>
	<li>Allow mouse to recover spontaneous ventilation with subsequent tracheal extubation.</li>
	<li>Return mouse to a clean cage and allow free access to food and water.</li>
</ol>
3. Autologous Blood Injection Model
<ol>
	<li>Follow the steps 2.1 - 2.11 for the collagenase injection model.</li>
	<li>Draw 50 &#181;l of sterile normal saline into a 30 G 50 &#181;l&#160;syringe.</li>
	<li>Connect the microliter syringe with a 70 cm PE10 tube.</li>
	<li>Expel all the normal saline from microliter syringe into PE10 tube to completely de-air tubing.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Collect 40 &#181;l 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.</li>
	<li>Attach the microliter syringe onto the injection pump.</li>
	<li>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.</li>
	<li>Expel 2 &#181;l of blood out of 27 G needle and wipe needle bevel with razor to discard.</li>
	<li>Align needle tip with burr hole and insert needle 3 mm deep to cortex.</li>
	<li>Inject 35 &#181;l of autologous blood at a rate of 2 &#181;l per min.</li>
	<li>Decrease isoflurane to 1% and leave needle motionless for 10 min.</li>
	<li>Withdraw needle over 30 sec.</li>
	<li>Apply 1 - 2 drops of 0.25% bupivacaine subcutaneously and suture the skin.</li>
	<li>Turn off isoflurane vaporizer and remove mouse from the stereotactic frame.</li>
	<li>Allow mouse to recover spontaneous ventilation with subsequent extubation.</li>
	<li>Return mouse to a clean cage and allow free access to food and water.</li>
</ol>
4. Sham Operation
<ol>
	<li>Follow the same procedures for collagenase injection model, except without injection after needle insertion.</li>
</ol>
5. Post-surgery Care
<ol>
	<li>Inject 0.5 ml of normal saline subcutaneously in the evening of the surgical procedure at the back of the animal&#8217;s neck.</li>
	<li>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.</li>
	<li>Check for weight loss, wound healing, and signs of discomfort daily for 7 days.</li>
	<li>If recovery intervals of greater than 7 days are required, suture&#160;removal may be performed under light inhaled anesthesia (approximately 1% isoflurane in 30% O2/70% N2), if necessary.</li>
</ol>

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Lei, Sheng, Wang, Lascola, Warner, and Laskowitz have no conflicts of interest to declare. James received grant funding by American Heart Association, National Institutes of Health, and Cephalogics.

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 ...

intrastriatal-injection-autologous-blood-or-clostridial-collagenase

Research

JoVE Journal

Medicine

14.7K Views.  Duke University. 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. 

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

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

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.

The autologous blood injection model of intracerebral hemorrhage in mice 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.

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

Neuroscience

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Massive Pontine Hemorrhage by 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 &#181;L 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.

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 ...

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented.

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely ...

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

<a href='https://app.jove.com/v/62089/massive-pontine-hemorrhage-by-dual-injection-of-autologous-blood'>Source: Tang, X. et. al., Massive Pontine Hemorrhage by Dual Injection of Autologous Blood. J. Vis. Exp. (2021)</a>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.

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

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

Establishing a Subarachnoid Hemorrhage Rat Model Induced by Autologous Blood Injection

To place the laser Doppler probe, make a 15-millimeter incision caudally in the midline, starting just anterior to the eyes. After removing the connective tissue and the muscles with forceps, use the end of a sterile cotton swab as a regime to identify the bregma and the coronal sutures.
After placing the armed retractor, place a 25-gauge spinal needle in the stereotaxic frame exactly on the bregma and note the position. Next, remove the needle from the bregma. Move the frame anteriorly by 6.5 millimeters, then replace the needle in the midline to mark the side of drilling.
Drill until the dura mater is identified below the bone. Using straight forceps, gently remove the bone fragments, then fill the cavity with bone wax. Next, 3 to 4 millimeters lateral to the right of the bregma and just anterior to the coronal suture for the laser Doppler, drill another hole taking care not to penetrate the dura mater.
Look for the vessels where the laser Doppler can measure the blood flow. Place the laser Doppler and check the values. A minimum value of 100 flux units is required. If the values remain acceptable after removing the microscope, add 1 drop of glue to fix the probe. Recheck to confirm whether the value is above 80 flux units.
To induce subarachnoid hemorrhage, insert the needle gently through the skull in the midline between the hemispheres until resistance is felt due to the base of the skull. Retract the needle by 1 millimeter to ensure correct placement just anteriorly to the optic chiasm. To ensure the most homogeneous result when injecting the blood, turn the needle clockwise by 90 degrees such that the needle tip points to the right. Then, remove the stiletto.
After a 15-minute equilibration, adjust the level of anesthesia to obtain a mean arterial blood pressure in the range 80 to 100 millimeters of mercury. Then, perform a blood gas analysis to confirm that the pH, partial pressure of carbon dioxide and partial pressure of oxygen are within physiological parameters.
Next, using a 1-milliliter syringe with a blunt 23-gauge needle, withdraw 500 microliters of blood from the tail catheter. To avoid injection of air, fill the dead space of the spinal needle chamber with blood. After adjusting the volume of the blood in the syringe to 300 microliters, connect the syringe to the spinal needle. Then, grasping firmly around the syringe, inject the blood manually to surpass mean arterial pressure. Observe a steep rise in the intracerebral pressure and a concurrent steep fall in the cerebral blood flow.