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>

<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. T., 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=Spontaneous+hemorrhagic+stroke+in+a+mouse+model+of+cerebral+amyloid+angiopathy.">Spontaneous hemorrhagic stroke in a mouse model of cerebral amyloid angiopathy.</a> J Neurosci. 21, 1619-1627 (2001).</li><li>Sinar, E. J., Mendelow, A. D., Graham, D. I., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+intracerebral+hemorrhage:+effects+of+a+temporary+mass+lesion.">Experimental intracerebral hemorrhage: effects of a temporary mass lesion.</a> J Neurosurg. 66, 568-576 (1987).</li><li>Mendelow, A. D., Bullock, R., Teasdale, G. M., Graham, D. I., McCulloch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+haemorrhage+induced+at+arterial+pressure+in+the+rat.+Part+2:+Short+term+changes+in+local+cerebral+blood+flow+measured+by+autoradiography.">Intracranial haemorrhage induced at arterial pressure in the rat. Part 2: Short term changes in local cerebral blood flow measured by autoradiography.</a> Neurol Res. 6, 189-193 (1984).</li><li>Bullock, R., Mendelow, A. D., Teasdale, G. M., Graham, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+haemorrhage+induced+at+arterial+pressure+in+the+rat.+Part+1:+Description+of+technique,+ICP+changes+and+neuropathological+findings.">Intracranial haemorrhage induced at arterial pressure in the rat. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+hemodynamic+changes+in+experimental+intracerebral+hemorrhage.">Early hemodynamic changes in experimental intracerebral hemorrhage.</a> J Neurosurg. 65, 697-703 (1986).</li><li>Anderson, C. S., 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=Rapid+blood-pressure+lowering+in+patients+with+acute+intracerebral+hemorrhage.">Rapid blood-pressure lowering in patients with acute intracerebral hemorrhage.</a> N Engl J Med. 368, 2355-2365 (2013).</li><li>Clark, W., Gunion-Rinker, L., Lessov, N., Hazel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citicoline+treatment+for+experimental+intracerebral+hemorrhage+in+mice.">Citicoline treatment for experimental intracerebral hemorrhage in mice.</a> Stroke. 29, 2136-2140 (1998).</li><li>Mayer, S. 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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+E+modifies+neurological+outcome+by+affecting+cerebral+edema+but+not+hematoma+size+after+intracerebral+hemorrhage+in+humans.">Apolipoprotein E modifies neurological outcome by affecting cerebral edema but not hematoma size after intracerebral hemorrhage in humans.</a> J Stroke Cerebrovasc Dis. 18, 144-149 (2009).</li><li>James, M. L., Blessing, R., Phillips-Bute, B. G., Bennett, E., 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=S100B+and+brain+natriuretic+peptide+predict+functional+neurological+outcome+after+intracerebral+haemorrhage.">S100B and brain natriuretic peptide predict functional neurological outcome after intracerebral haemorrhage.</a> Biomarkers. 14, 388-394 (2009).</li><li>James, M. L., Sullivan, P. 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. L., 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=Brain+natriuretic+peptide+improves+long-term+functional+recovery+after+acute+CNS+injury+in+mice.">Brain natriuretic peptide improves long-term functional recovery after acute CNS injury in mice.</a> J Neurotrauma. 27, 217-228 (2010).</li><li>Indraswari, F., 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=Statins+improve+outcome+in+murine+models+of+intracranial+hemorrhage+and+traumatic+brain+injury:+a+translational+approach.">Statins improve outcome in murine models of intracranial hemorrhage and traumatic brain injury: a translational approach.</a> J Neurotrauma. 29, 1388-1400 (2012).</li><li>Laskowitz, D. T., 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=The+apoE-mimetic+peptide,+COG1410,+improves+functional+recovery+in+a+murine+model+of+intracerebral+hemorrhage.">The apoE-mimetic peptide, COG1410, improves functional recovery in a murine model of intracerebral hemorrhage.</a> Neurocrit Care. 16, 316-326 (2012).</li><li>Lei, 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=Interaction+between+sex+and+apolipoprotein+E+genetic+background+in+a+murine+model+of+intracerebral+hemorrhage.">Interaction between sex and apolipoprotein E genetic background in a murine model of intracerebral hemorrhage.</a> Translational Stroke Research. 3, (2012).</li><li>Lekic, T., 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=Evaluation+of+the+hematoma+consequences,+neurobehavioral+profiles,+and+histopathology+in+a+rat+model+of+pontine+hemorrhage.">Evaluation of the hematoma consequences, neurobehavioral profiles, and histopathology in a rat model of pontine hemorrhage.</a> J Neurosurg. 118, 465-477 (2013).</li><li>Nakamura, T., 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=Intracerebral+hemorrhage+in+mice:+model+characterization+and+application+for+genetically+modified+mice.">Intracerebral hemorrhage in mice: model characterization and application for genetically modified mice.</a> J Cereb Blood Flow Metab. 24, 487-494 (2004).</li><li>Yang, 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=Statins+Protect+the+Blood+Brain+Barrier+Acutely+after+Experimental+Intracerebral+Hemorrhage.">Statins Protect the Blood Brain Barrier Acutely after Experimental Intracerebral Hemorrhage.</a> J Behav Brain Sci. 3, 100-106 (2013).</li><li>Rynkowski, M. 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=A+mouse+model+of+intracerebral+hemorrhage+using+autologous+blood+infusion.">A mouse model of intracerebral hemorrhage using autologous blood infusion.</a> Nature Protocols. 3, 122-128 (2008).</li><li>Wang, J., Fields, J., Dore, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+an+improved+preclinical+mouse+model+of+intracerebral+hemorrhage+using+double+infusion+of+autologous+whole+blood.">The development of an improved preclinical mouse model of intracerebral hemorrhage using double infusion of autologous whole blood.</a> Brain Research. 1222, 214-221 (2008).</li><li>MacLellan, C. L., 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=Intracerebral+hemorrhage+models+in+rat:+comparing+collagenase+to+blood+infusion.">Intracerebral hemorrhage models in rat: comparing collagenase to blood infusion.</a> J Cereb Blood Flow Metab. 28, 516-525 (2008).</li></ol>

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 examined through use of clinically relevant preclinical models 19-22. Such models are useful because they allow rapid throughput of selected candidates, examination of mechanisms in vivo, inexpensive investigation of dosing, therapeutic window, and other parameters germane to developing clinical trials 23-25. While obvious advantages exist in using preclinical models, modeling should occur in the most clinically relevant but logistically feasible system available. While models exist for &#39;higher&#39; order animals such as primates, use of mice to model human disease provides an inexpensive, high-throughput, and powerful technology to examining pathologic mechanisms and therapeutic effects. Incorporating transgenic systems allows for an even more robust evaluation of mechanistic pathways and cell populations involved.
Currently two murine models are in common use: intrastriatal autologous blood or collagenase injection. Both models are versatile and easy to use, relative to other stroke models. Both models can induce ICH in different brain areas 26, allowing evaluation of regional responses; hematoma volume can be controlled and changed, allowing for evaluation of mild, moderate, and severe injury; and clinically relevant physiology (e.g., blood pressure, temperature, etc.) can be controlled. Finally, while each model was originally developed in the rat, both have since been translated into mice to allow the use of transgenic systems 21,24,25,27. However, each model lends itself to the study of different aspects of ICH, as each represents distinctly different components of ICH. Autologous blood-injection may recreate the brain&#39;s response to intraparenchymal blood exposure. Thus, initial mass affect and shear forces, mild inflammatory changes, apoptosis, and blood resorption may all be studied 10,28. Further, recent modifications to this model have resulted in the ability to mimic hematoma expansion 29,30. However, this model does not invoke the component of vascular injury and/or hematoma expansion found in the human disease. In contrast, collagenase-injection adds the elements of vascular rupture, early hematoma expansion, and enhanced neuroinflammatory effect. While obvious concerns exist about artifactual contribution of collagenase to this inflammatory effect, there is a lack of hard evidence for this 31, and our own (unpublished) data suggest that collagenase in isolation does not induce a marked inflammatory response in cell culture.
From a procedural standpoint, both models require limited skill with microsurgery and, thus, are easily learned so as to obtain reproducible effects. Pitfalls to be avoided include: 1) invasion of the dura or creating thermal brain injury when drilling, 2) or penetration of the ventricular system with needle insertion. Dural injury allows for reflux of injectant, and intraventricular injection results in little to no intraparenchymal hematoma formation. Further, care must be taken upon needle withdrawal to not disrupt newly formed/forming hematoma. Mortality is to be expected in a certain percentage of mice but is directly related to hematoma size and degree of injury desired; thus, this outcome may be titrated by injectant volume/concentration.
As with all models, protocols will be optimized for use by specific operators. Due to inherent variability in all in vivo systems, experience with a particular model as a key factor for success cannot be overstated. A model&#39;s distinctive characteristics, operator experience with a given model, outcome metrics of interest, and logistical factors must all be taken into account when selecting the best possible experimental model.

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 hemispheres show stable hematoma volumes (Figure 4); further, at 24 hours after injection, brain water content should be expected to be 79.8 + 0.34% in collagenase-injected mice and 79.3 + 0.23% in autologous blood-injected mice. Mortality should be expected to occur between 10 - 25% of collagenase-injected mice and less than 10% of autologous blood-injected mice. Unavoidable death due to hematoma volume, cerebral edema, and increased intracranial pressure usually occurs within the first 24 - 48 hr after intrastriatal injection. Death occurring after 72 hr may often be avoided with proper post-injury care (e.g., ready access to softened food and water). Functional recovery generally begins by post injury day 2 with autologous blood-injected mice recovering significantly faster than collagenase-injected mice.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51439/51439fig1highres.jpg" width="500" /> 
Figure 1. Serial magnetic resonance imaging of mouse brains comparing autologous blood and collagenase injection models of intracerebral hemorrhage. After intracerebral hemorrhage induction via left intrastriatal injection of 35 &#181;l autologous blood (A) or 0.075 U Type IV-S clostridial collagenase (B) in 10 - 12 week old C57/Bl6 male mice, serial magnetic resonance imaging demonstrates hematoma expansion in the collagenase-injected mice compared to stable hematoma formation in the autologous blood-injected mice. Hematoma volumes are 10.1, 23.1, 29.9 mm3 at 1, 6, &#38; 12 hr after collagenase injection, respectively, and 7.0, 5.8, 3.2 mm3 at 1, 6, and 24 hr after whole blood injection, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51439/51439fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51439/51439fig2highres.jpg" width="500" /> 
Figure 2. Corner turn test in mice 24 hr after intracerebral hemorrhage. Taken immediately after intrastriatal collagenase injection into the left basal ganglia, presence of expected ipsilateral turning response in 10 - 12 week old C57/Bl6 male mice denotes adequate injury. This turning should occur immediately after significant injury in mice injected with autologous blood and within 2 - 4 hr in collagenase-injected mice. Mice in both models showed more left turns after injury compared to uninjured mice (**p &#60;0.01; one-way ANOVA with post-hoc Scheffe&#8217;s test; n = 10/group).
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51439/51439fig3highres.jpg" width="500" /> 
Figure 3. Rotarod performance after intracerebral hemorrhage in mice. Baseline and post injury rotorod latencies of 10 - 12 week old C57/Bl6 male mice for one week after left intrastriatal 35 &#181;l autologous blood-, 0.075 U Type IV-S clostridial collagenase-injection, or sham operation (*p = 0.022; repeated measures ANOVA with post-hoc Scheffe&#8217;s test; F-value = 12.726; n = 10/group). Mice are assessed via rotorod testing every other day after injury to avoid significant training bias.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51439/51439fig4highres.jpg" width="500" /> 
Figure 4. Hematoxylin and eosin stains of mouse brain after intracerebral hemorrhage. Photomicrographs of 10 - 12 week old C57/Bl6 male mouse brains at 24 hr after left intrastriatal injection of 35 &#181;l autologous blood (right) or 0.075 U Type IV-S Clostridial collagenase (left). Hematoma volumes are 20.2 mm3 after collagenase injection and 6.4 mm3 after whole blood injection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51439/51439fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Medicine

14.6K 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

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, 2, 3, 4, 5 These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). 6, 7, 8, 9 It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. 10 This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. 11, 12, 13 Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. 14 Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. 15, 16, 17, 18 This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. 19, 20, 21 Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. 22, 23, 24, 25 The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma 26, 27, 28, 29, 30. 
There are numerous resuscitation methods (R) in association with HS and trauma. 31, 32, 33, 34 A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. 35, 36, 38 This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma 38, 39.

The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, ...

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

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 ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...