Name: using a cell-tracer injection to investigate the origin of neointima-forming cells in a rat saccular side wall model
Uploaded: 2022-03-16T14:45:00
Description: Watch this Scientific Journal Video about Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model at JoVE.com

The authors thank Alessandra Bergadano, DVM, PhD, for the dedicated supervision of long-term animal health. This work was supported by the research funds of the Research Council, Kantonsspital Aarau, Aarau, Switzerland, and the Swiss national science foundation SNF (310030_182450).

Veterinary support was performed according to institutional guidelines. Experiments were approved by the Local Ethics Committee, Switzerland (BE 60/19). The ARRIVE guidelines and 3R principles have been strictly followed6,7. Thirty-one male Lewis rats, 12 weeks old and weighing 492&#160;&#177; 8 g, were included. House all rats at a room temperature of 23 &#176;C and a 12 h light/dark cycle. Provide free access to water and pellets. Statistical analyses have been...

<ol>
	<li>Vergouwen, M. 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=Definition+of+delayed+cerebral+ischemia+after+aneurysmal+subarachnoid+hemorrhage+as+an+outcome+event+in+clinical+trials+and+observational+studies:+proposal+of+a+multidisciplinary+research+group.">Definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: proposal of a multidisciplinary research group.</a> Stroke. 41 (10), 2391-2395 (2010).</li><li>Macdonald, R. 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=Preventing+vasospasm+improves+outcome+after+aneurysmal+subarachnoid+hemorrhage:+rationale+and+design+of+CONSCIOUS-2+and+CONSCIOUS-3+trials.">Preventing vasospasm improves outcome after aneurysmal subarachnoid hemorrhage: rationale and design of CONSCIOUS-2 and CONSCIOUS-3 trials.</a> Neurocritical Care. 13 (3), 416-424 (2010).</li><li>Wanderer, 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=Levosimendan+as+a+therapeutic+strategy+to+prevent+neuroinflammation+after+aneurysmal+subarachnoid+hemorrhage.">Levosimendan as a therapeutic strategy to prevent neuroinflammation after aneurysmal subarachnoid hemorrhage.</a> Journal of Neurointerventional Surgery. , (2021).</li><li>Wanderer, 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=Aspirin+treatment+prevents+inflammation+in+experimental+bifurcation+aneurysms+in+New+Zealand+White+rabbits.">Aspirin treatment prevents inflammation in experimental bifurcation aneurysms in New Zealand White rabbits.</a> Journal of Neurointerventional Surgery. 14 (2), 189-195 (2021).</li><li>Gruter, B. E., 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=Patterns+of+neointima+formation+after+coil+or+stent+treatment+in+a+rat+saccular+sidewall+aneurysm+model.">Patterns of neointima formation after coil or stent treatment in a rat saccular sidewall aneurysm model.</a> Stroke. 52 (3), 1043-1052 (2021).</li><li>Kilkenny, C., 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=Animal+research:+reporting+in+vivo+experiments:+the+ARRIVE+guidelines.">Animal research: reporting in vivo experiments: the ARRIVE guidelines.</a> British Journal of Pharmacology. 160 (7), 1577-1579 (2010).</li><li>Tornqvist, E., 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=Strategic+focus+on+3R+principles+reveals+major+reductions+in+the+use+of+animals+in+pharmaceutical+toxicity+testing.">Strategic focus on 3R principles reveals major reductions in the use of animals in pharmaceutical toxicity testing.</a> PLoS One. 9 (7), 101638 (2014).</li><li>Nevzati, E., 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=Aneurysm+wall+cellularity+affects+healing+after+coil+embolization:+assessment+in+a+rat+saccular+aneurysm+model.">Aneurysm wall cellularity affects healing after coil embolization: assessment in a rat saccular aneurysm model.</a> Journal of Neurointerventional Surgery. 12 (6), 621-625 (2020).</li><li>Marbacher, 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=The+Helsinki+rat+microsurgical+sidewall+aneurysm+model.">The Helsinki rat microsurgical sidewall aneurysm model.</a> Journal of Visualized Experiments: JoVE. (92), e51071 (2014).</li><li>Nevzati, E., 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=Biodegradable+magnesium+stent+treatment+of+saccular+aneurysms+in+a+rt+model+-+introduction+of+the+surgical+technique.">Biodegradable magnesium stent treatment of saccular aneurysms in a rt model - introduction of the surgical technique.</a> Journal of Visualized Experiments: JoVE. (128), e56359 (2017).</li><li>Gruter, B. E., 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=Testing+bioresorbable+stent+feasibility+in+a+rat+aneurysm+model.">Testing bioresorbable stent feasibility in a rat aneurysm model.</a> Journal of Neurointerventional Surgery. 11 (10), 1050-1054 (2019).</li><li>Kadirvel, 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=Cellular+mechanisms+of+aneurysm+occlusion+after+treatment+with+a+flow+diverter.">Cellular mechanisms of aneurysm occlusion after treatment with a flow diverter.</a> Radiology. 270 (2), 394-399 (2014).</li><li>Li, Z. 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=Endothelial+progenitor+cells+contribute+to+neointima+formation+in+rabbit+elastase-induced+aneurysm+after+flow+diverter+treatment.">Endothelial progenitor cells contribute to neointima formation in rabbit elastase-induced aneurysm after flow diverter treatment.</a> CNS Neuroscience &amp; Therapeutics. 19 (5), 352-357 (2013).</li><li>Marbacher, 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=Intraluminal+cell+transplantation+prevents+growth+and+rupture+in+a+model+of+rupture-prone+saccular+aneurysms.">Intraluminal cell transplantation prevents growth and rupture in a model of rupture-prone saccular aneurysms.</a> Stroke. 45 (12), 3684-3690 (2014).</li><li>Frosen, 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=Contribution+of+mural+and+bone+marrow-derived+neointimal+cells+to+thrombus+organization+and+wall+remodeling+in+a+microsurgical+murine+saccular+aneurysm+model.">Contribution of mural and bone marrow-derived neointimal cells to thrombus organization and wall remodeling in a microsurgical murine saccular aneurysm model.</a> Neurosurgery. 58 (5), 936-944 (2006).</li><li>Marbacher, S., Niemela, M., Hernesniemi, J., Frosen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrence+of+endovascularly+and+microsurgically+treated+intracranial+aneurysms-review+of+the+putative+role+of+aneurysm+wall+biology.">Recurrence of endovascularly and microsurgically treated intracranial aneurysms-review of the putative role of aneurysm wall biology.</a> Neurosurgical Review. 42 (1), 49-58 (2019).</li><li>Frosen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smooth+muscle+cells+and+the+formation,+degeneration,+and+rupture+of+saccular+intracranial+aneurysm+wall--a+review+of+current+pathophysiological+knowledge.">Smooth muscle cells and the formation, degeneration, and rupture of saccular intracranial aneurysm wall--a review of current pathophysiological knowledge.</a> Translational Stroke Research. 5 (3), 347-356 (2014).</li><li>Fang, X., 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=Bone+marrow-derived+endothelial+progenitor+cells+are+involved+in+aneurysm+repair+in+rabbits.">Bone marrow-derived endothelial progenitor cells are involved in aneurysm repair in rabbits.</a> Journal of Clinical Neuroscience. 19 (9), 1283-1286 (2012).</li><li>Morel, 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=Sex-related+differences+in+wall+remodeling+and+intraluminal+thrombus+resolution+in+a+rat+saccular+aneurysm+model.">Sex-related differences in wall remodeling and intraluminal thrombus resolution in a rat saccular aneurysm model.</a> Journal of Neurosurgery. , 1-14 (2019).</li><li>Gruter, B. E., 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=Fluorescence+video+angiography+for+evaluation+of+dynamic+perfusion+status+in+an+aneurysm+preclinical+experimental+setting.">Fluorescence video angiography for evaluation of dynamic perfusion status in an aneurysm preclinical experimental setting.</a> Operative Neurosurgery. 17 (4), 432-438 (2019).</li><li>Marbacher, S., Strange, F., Frosen, J., Fandino, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+extracranial+aneurysm+models+for+the+study+and+treatment+of+brain+aneurysms:+A+systematic+review.">Preclinical extracranial aneurysm models for the study and treatment of brain aneurysms: A systematic review.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 40 (5), 922-938 (2020).</li><li>Ravindran, K., 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=Mechanism+of+action+and+biology+of+flow+diverters+in+the+treatment+of+intracranial+aneurysms.">Mechanism of action and biology of flow diverters in the treatment of intracranial aneurysms.</a> Neurosurgery. 86, 13-19 (2020).</li><li>Marbacher, 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=Loss+of+mural+cells+leads+to+wall+degeneration,+aneurysm+growth,+and+eventual+rupture+in+a+rat+aneurysm+model.">Loss of mural cells leads to wall degeneration, aneurysm growth, and eventual rupture in a rat aneurysm model.</a> Stroke. 45 (1), 248-254 (2014).</li><li>Morosanu, C. O., 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=Neurosurgical+cadaveric+and+in+vivo+large+animal+training+models+for+cranial+and+spinal+approaches+and+techniques+-+systematic+review+of+current+literature.">Neurosurgical cadaveric and in vivo large animal training models for cranial and spinal approaches and techniques - systematic review of current literature.</a> Neurologia i Neurochirurgia Polska. 53 (1), 8-17 (2019).</li><li>Wanderer, 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=Arterial+pouch+microsurgical+bifurcation+aneurysm+model+in+the+rabbit.">Arterial pouch microsurgical bifurcation aneurysm model in the rabbit.</a> Journal of Visualized Experiments: JoVE. (159), e61157 (2020).</li></ol>

This study demonstrates that neointima formation is mediated via endothelial cells originating in the parent artery of the aneurysm complex but is supported by the recruitment of cells derived from the aneurysm wall in vital aneurysms. Nevertheless, the role of circulating progenitor cells in aneurysm healing remains controversial12,13. Overall, 31 male Lewis rats were included in this investigation; only 4 died prematurely (12.9% mortality).
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Subarachnoid hemorrhage caused by the rupture of an intracranial aneurysm (IA) is a devastating neurosurgical condition associated with high morbidity and mortality1,2,3,4. In addition to microsurgical clipping, which provides direct endothelium-to-endothelium contact, endovascular devices have gained increasing importance over the past decades for treating ruptured and incidentally discovered IAs. The healing response in endovascularly treated IAs mainly depends on neointima formation and thrombus organization. Both are synergic processes, depending on cell migration from the adjacent vessel and the aneurysm wall.5 To date, the origin of endothelial cells in neointima formation of endovascular treated aneurysms remains unclear. There is an ongoing debate in the literature about the source from which neointima-forming cells are recruited.
By using a cell-tracer injection of CM-Dil dye (see the Table of Materials) in the abdominal aorta of rats, we aimed to analyze the role of endothelial cells, originating in the parent artery, in neointima formation at two different FU time points (day 7 and day 21) (Figure 1). An advantage of the model is the direct local cell-tracer incubation in vivo in a parent artery prior to aneurysm suture, allowing FU at later time points. In vivo injection techniques, such as cell-tracer incubation, have not been described in the literature. An advantage of this technique is the direct, one-point, intraoperative, in vivo injection, which makes the model robust and reproducible.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-0 resorbable suture</td>
			<td>Ethicon Inc., USA</td>
			<td>VCP428G</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 non-absorbable suture</td>
			<td>B. Braun, Germany</td>
			<td>G0762563</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 non-absorbable suture</td>
			<td>B. Braun, Germany</td>
			<td>C0766070</td>
			<td></td>
		</tr>
		<tr>
			<td>9-0 non-absorbable suture</td>
			<td>B. Braun, Germany</td>
			<td>G1111140</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezol</td>
			<td>Arovet AG, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bandpass filter blue</td>
			<td>Thorlabs</td>
			<td>FD1B</td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Bandpass filter green</td>
			<td>Thorlabs</td>
			<td>FGV9</td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Bipolar forceps</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Bicycle spotlight</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Board (20 x 10 cm)</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Indivior, Switzerland</td>
			<td>1014197</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Sony NEX-5R, Sony, Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula (27-1/2 G)</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Cell count software</td>
			<td>Image-J version 1.52n, U.S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker CM-Dil dye</td>
			<td>ThermoFisher SCIENTIFIC, USA</td>
			<td>C7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Coil-Device</td>
			<td>Styker, Kalamazoo, MI, USA</td>
			<td></td>
			<td>2 cm of Target 360 TM Ultra, 2-mm diameter</td>
		</tr>
		<tr>
			<td>Desinfection</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Eye-lubricant</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Sintetica, S.A., Switzerland</td>
			<td>98683</td>
			<td>any generic</td>
		</tr>
		<tr>
			<td>Flumazenil</td>
			<td>Labatec-Pharma, Switerzland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresceine</td>
			<td>Curatis AG</td>
			<td>5030376</td>
			<td>any generic</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foil mask</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Glucose (5%)</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Homeothermic Control Unit, Harvard, Edenbridge, England</td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Isotonic sodium chloride solution (0.9%)</td>
			<td>Fresenius KABI</td>
			<td>336769</td>
			<td>any generic</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td></td>
			<td></td>
			<td>any generic</td>
		</tr>
		<tr>
			<td>Longuettes</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Boehringer Ingelheim</td>
			<td>P7626406</td>
			<td>any generic</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Virbac, Switzerland</td>
			<td>QN05CM91</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Roche, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monitoring-system</td>
			<td>Starr Life Sciences Corp., 333 Allegheny Ave, Oakmont, PA 15139, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>O2-Face mask</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Operation microscope</td>
			<td>OPMI, Carl Zeiss AG, Oberkochen, Germany</td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Rectal temperature probe</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Scalpell</td>
			<td>Swann-Morton</td>
			<td>210</td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Small animal shaver</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Smartphone</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (0.1%)</td>
			<td>Sigma-Aldrich</td>
			<td>11667289001</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft feed</td>
			<td>Emeraid Omnivore</td>
			<td></td>
			<td>any generic</td>
		</tr>
		<tr>
			<td>Soft tissue forceps</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Soft tissue spreader</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Stainless steel sponge bowls</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Stent-Device</td>
			<td>Biotroni, B&#252;lach, Switzerland</td>
			<td></td>
			<td>modified magmaris device, AMS with polymer coating, 6-mm length, 2-mm diameter</td>
		</tr>
		<tr>
			<td>Sterile micro swabs</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Straight and curved microforceps</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Straight and curved microscissors</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Straight and curved forceps</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Surgery drape</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Syringes 1 mL, 2 mL, and 5 mL</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Tape</td>
			<td></td>
			<td></td>
			<td>any other</td>
		</tr>
		<tr>
			<td>Vascular clip applicator</td>
			<td>B. Braun, Germany</td>
			<td>FT495T</td>
			<td></td>
		</tr>
		<tr>
			<td>Yasargil titan standard clip (2x)</td>
			<td>B. Braun Medical AG, Aesculap, Switzerland</td>
			<td>FT242T</td>
			<td>temporary</td>
		</tr>
	</tbody>
</table>

using a cell-tracer injection to investigate the origin of neointima-forming cells in a rat saccular side wall model

Microsurgical clipping creates a subsequent barrier of blood flow into intracranial aneurysms, whereas endovascular treatment relies on neointima and thrombus formation. The source of endothelial cells covering the endoluminal layer of the neointima remains unclear. Therefore, the aim of the present study was to investigate the origin of neointima-forming cells after cell-tracer injection in the already well-established Helsinki rat microsurgical sidewall aneurysm model.
Sidewall aneurysms were created by suturing decellularized or vital arterial pouches end-to-side to the aorta in male Lewis rats. Before arteriotomy with aneurysm suture, a cell-tracer injection containing CM-Dil dye was performed into the clamped aorta to label endothelial cells in the adjacent vessel and track their proliferation during follow-up (FU). Treatment followed by coiling (n = 16) or stenting (n = 15). At FU (7 days or 21 days), all rats underwent fluorescence angiography, followed by aneurysm harvesting and macroscopic and histological evaluation with immunohistological cell counts for specific regions of interest. 
None of the 31 aneurysms had ruptured upon follow-up. Four animals died prematurely. Macroscopically residual perfusion was observed in 75.0% coiled and 7.0% of stented rats. The amount of cell-tracer-positive cells was significantly elevated in decellularized stented compared to coiled aneurysms with respect to thrombus on day 7 (p = 0.01) and neointima on day 21 (p = 0.04). No significant differences were found in thrombus or neointima in vital aneurysms. 
These findings confirm worse healing patterns in coiled compared to stented aneurysms. Neointima formation seems particularly dependent on the parent artery in decellularized aneurysms, whereas it is supported by the recruitment from aneurysm wall cells in vital cell-rich walls. In terms of translation, stent treatment might be more appropriate for highly degenerated aneurysms, whereas coiling alone might be adequate for aneurysms with mostly healthy vessel walls.

A total of 31 animals were included in the laboratory setting: 27 rats were included in the final statistical analysis; 4 rats died prematurely (12.9% mortality rate). Intraoperatively, breath distension was significantly (p = 0.03) reduced in stent- (12.9 &#956;m &#177; 0.7) compared to coil-treated (13.5 &#956;m &#177; 0.6) rats. Fluorescence angiography was performed for every rat at the end of the final FU. Reperfusion was indicated in all 6 coil-treated animals, whereas reperfusion was observed in only 12.5...

Watch this Scientific Journal Video about Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model at JoVE.com

Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model

We performed a one-point, lipophilic cell-tracer injection to track endothelial cells, followed by an arteriotomy and suturing of sidewall aneurysms on the abdominal rat aorta. Neointima formation seemed dependent on the parent artery in decellularized aneurysms and was promoted by the recruitment from aneurysm wall cells in vital cell-rich walls.

Microsurgical clipping creates a subsequent barrier of blood flow into intracranial aneurysms, whereas endovascular treatment relies on neointima and ...

By using a direct intra-aortal cell-tracer application, we can analyze the role of endothelial cells originating from the parent artery in neointima formation at two different time points. A big advantage of this technique is a direct, one-point, interpretive in-vivo injection which makes this model a robust and reproducible technique. Before the surgery, incubate the arterial pouches from donor rats in 0.1%sodium dodecyl sulfate for 10 hours at 37 degrees Celsius to obtain decellularized aneurysms.Collect these pouches from donor animals a few days before the surgery. To begin, analyze the animal's behavior and inspect the mucus membranes and turgor as part of the preoperative clinical examination. Record the weight of each animal.For anesthesia induction, place all rats in a clean box provided with oxygen until loss of consciousness after five to 10 minutes. Check the depth of anesthesia by the absence of the pedal withdrawal reflex. Place the rats in a supine position and shave the thoracoabdominal area with an electric shaver.Using tape, fixate the rat's paws on a board covered by a heating pad connected to an auto-regulating rectal probe. Insert the rectal probe into the rat's anus to maintain the desired temperature of 37 degrees Celsius with the help of a heating pad. Then install a sensor on the right-hind leg connected to a computerized system for checking vital signs intraoperatively.For perianesthetic care, apply a sterile ophthalmic lubricant to the eyes, and cover them with an opaque foil mask to prevent drying and damage from the surgical lamp. Disinfect the surgical field with povidone iodine, and drape the surgical field in a sterile fashion. Put a micro swab with purple padding under the proximal and distal parts of the aorta to better visualize the artery as soon as it is separated from the caval vein.Then protect the abdomen with white gauze. On the day of operation, dissolve two microliters of the cell-tracer by pipetting in one milliliter of PBS. Transfer the mixture to a one-milliliter syringe fitted with a cannula.Turn off the light in the operating room. Using a microscope, perform the one-point injection in the middle ventral part of the aorta with micro forceps, and carefully inject one milliliter of heparinized 0.9%saline solution. Inject the cell-tracer carefully, and immediately turn off the operating microscope as well.Protect the abdomen with wet gauze again. Let the dye incubate for at least 15 minutes. Then turn on the microscope and operating room lights.Use micro forceps and micro scissors to perform the longitudinal arteriotomy so that its length averages the diameter of the harvested aneurysm. Place the aneurysm beside the aorta before performing arteriotomy to ensure the correct length. Suture the aneurysm with eight to 10 single stitches using a non-absorbable 10-0 suture.Before the final stitch, deliver the coil with a packing density of one centimeter. Either coil or stent treatment can be performed in this model. Then carefully remove the temporary clamps, starting distally, under continuous irrigation with heparinized saline.Close the wound in a layered fashion. At the end of the surgery, reverse the anesthesia with a subcutaneous injection. Let each operated animal recover in a clean cage until fully awake and warm as needed with a heating lamp.Pulled baseline aneurysm volumes for day seven and day 21 did not differ significantly for decellularized or vital aneurysms between the coil or stent treatment groups. Pulled FU volumes for decellularized aneurysms showed a non-significant aneurysm growth in coiled compared to the stented aneurysms. Pulled FU volume was significantly greater in the vital coiled than in the stented group.Amounts of cell-tracer positive cells in the neointima of decellularized aneurysms did not significantly differ between the stent or coiled-treated groups at day seven FU but were significantly higher in stented rats at day 21 FU.No significant differences were noted in vital-aneurysm sutured rats at either seven days or 21 days FU.In decellularized aneurysms at seven days FU, significantly more cell-tracer positive cells remained in the thrombus of the stent-treated compared to the coil-treated group. This difference was not observed in vital aneurysms at seven days FU.Remember to always turn off the lights in the operating room prior to injecting the cell-tracer to prevent fading. Also, let incubate the dye for at least 15 minutes.

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Research

JoVE Journal

Neuroscience

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

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% ...

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors. Using animal models to accomplish this goal, many methodological strategies require invasive techniques to manipulate the intensity of the behavior of interest (e.g., lesion methods, pharmacological manipulations, microdialysis techniques, genetically-engineered animal models). The utilization of a comparative species approach allows researchers to take advantage of naturally occurring differences in response strategies existing in closely related species. In our lab, we use two species of the Peromyscus genus that differ in paternal responses. The male California deer mouse (Peromyscus californicus) exhibits the same parental responses as the female whereas its cousin, the common deer mouse (Peromyscus maniculatus) exhibits virtually no nurturing/parental responses in the presence of pups. Of specific interest in this article is an exploration of the neurobiological factors associated with the affiliative social responses exhibited by the paternal California deer mouse. Because the behavioral neuroscience approach is multifaceted, the following key components of the study will be briefly addressed: the identification of appropriate species for this type of research; data collection for behavioral analysis; preparation and sectioning of the brains; basic steps involved in immunocytochemistry for the quantification of vasopressin-immunoreactivity; the use of neuroimaging software to quantify the brain tissue; the use of a microsequencing video analysis to score behavior and, finally, the appropriate statistical analyses to provide the most informed interpretations of the research findings.

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn&#x2019;t require invasive techniques to manipulate the expression of the behavior.

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Biodegradable Magnesium Stent Treatment of Saccular Aneurysms in a Rat Model - Introduction of the Surgical Technique

The steady progess in the armamentarium of techniques available for endovascular treatment of intracranial aneurysms requires affordable and reproducable experimental animal models to test novel embolization materials such as stents and flow diverters. The aim of the present project was to design a safe, fast, and standardized surgical technique for stent assisted embolization of saccular aneurysms in a rat animal model.
Saccular aneurysms were created from an arterial graft from the descending aorta.The aneurysms were microsurgically transplanted through end-to-side anastomosis to the infrarenal abdominal aorta of a syngenic male Wistar rat weighing &#62;500 g. Following aneurysm anastomosis, aneurysm embolization was performed using balloon expandable magnesium stents (2.5 mm x 6 mm). The stent system was retrograde introduced from the lower abdominal aorta using a modified Seldinger technique.
Following a pilot series of 6 animals, a total of 67 rats were operated according to established standard operating procedures. Mean surgery time, mean anastomosis time, and mean suturing time of the artery puncture site were 167 &#177; 22 min, 26 &#177; 6 min and 11 &#177; 5 min, respectively. The mortality rate was 6% (n=4). The morbidity rate was 7.5% (n=5), and in-stent thrombosis was found in 4 cases (n=2 early, n=2 late in stent thrombosis).
The results demonstrate the feasibility of standardized stent occlusion of saccular sidewall aneurysms in rats - with low rates of morbidity and mortality. This stent embolization procedure combines the opportunity to study novel concepts of stent or flow diverter based devices as well as the molecular aspects of healing.

Reproducable experimental animal models are needed for the testing of novel embolization materials, which have been designed to treat endovascular occlusion of intracranial aneurysms (IA). The present study aims to develop a safe and standardized surgical technique for stent assisted embolization of saccular aneurysms in a rat animal model.

The steady progess in the armamentarium of techniques available for endovascular treatment of intracranial aneurysms requires affordable and reproducable ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...