We thank Swedish Medical Center for providing the funding and facilities. We also thank Craig Hospital for the generous use of laboratory space.

Experiments were carried out in accordance with the guidelines of the National Institutes of Health for the care and use of animals in research and were approved by the Swedish Medical Center Animal Care and Use Committee. All procedures were performed with appropriate aseptic techniques. Experimental animals utilized for the study were male, 3 months old, wild type C57 mice with body weight from 25 to 27 g.
1. In Vivo Stroke Induction
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	<li>Pre-coat the Filament with Dental Res...

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	<li>Gueniau, C., Oberlander, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kappa+opioid+agonist+niravoline+decreases+brain+edema+in+the+mouse+middle+cerebral+artery+occlusion+model+of+stroke.">The kappa opioid agonist niravoline decreases brain edema in the mouse middle cerebral artery occlusion model of stroke.</a> J Pharmacol Exp Ther. 282, 1-6 (1997).</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=Etiology+of+stroke+and+choice+of+models.">Etiology of stroke and choice of models.</a> Int J Stroke. 7, 398-406 (2012).</li><li>Vakili, A., Kataoka, H., Plesnila, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+arginine+vasopressin+V1+and+V2+receptors+for+brain+damage+after+transient+focal+cerebral+ischemia.">Role of arginine vasopressin V1 and V2 receptors for brain damage after transient focal cerebral ischemia.</a> J Cereb Blood Flow Metab. 25, 1012-1019 (2005).</li><li>Zeynalov, E., Jones, S. M., Seo, J. W., Snell, L. D., Elliott, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine-Vasopressin+Receptor+Blocker+Conivaptan+Reduces+Brain+Edema+and+Blood-Brain+Barrier+Disruption+after+Experimental+Stroke+in+Mice.">Arginine-Vasopressin Receptor Blocker Conivaptan Reduces Brain Edema and Blood-Brain Barrier Disruption after Experimental Stroke in Mice.</a> PloS one. 10, e0136121 (2015).</li><li>Med Lett Drugs Ther.. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conivaptan+(Vaprisol)+for+hyponatremia.">Conivaptan (Vaprisol) for hyponatremia.</a> The Medical letter on drugs and therapeutics. 48, 51-52 (2006).</li><li>Zhao, X. Y., 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=Effect+of+arginine+vasopressin+on+the+cortex+edema+in+the+ischemic+stroke+of+Mongolian+gerbils.">Effect of arginine vasopressin on the cortex edema in the ischemic stroke of Mongolian gerbils.</a> Neuropeptides. 51, 55-62 (2015).</li><li>Manaenko, A., Chen, H., Kammer, J., Zhang, J. H., Tang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+Evans+Blue+injection+routes:+Intravenous+versus+intraperitoneal,+for+measurement+of+blood-brain+barrier+in+a+mice+hemorrhage+model.">Comparison Evans Blue injection routes: Intravenous versus intraperitoneal, for measurement of blood-brain barrier in a mice hemorrhage model.</a> J Neurosci Methods. 195, 206-210 (2011).</li><li>Adams, S., Pacharinsak, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+anesthesia+and+analgesia.">Mouse anesthesia and analgesia.</a> Curr Protoc Mouse Biol. 5, 51-63 (2015).</li><li>Zeynalov, 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=The+perivascular+pool+of+aquaporin-4+mediates+the+effect+of+osmotherapy+in+postischemic+cerebral+edema.">The perivascular pool of aquaporin-4 mediates the effect of osmotherapy in postischemic cerebral edema.</a> Crit Care Med. 36, 2634-2640 (2008).</li><li>Zeynalov, E., 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=Low+doses+of+carbon+monoxide+protect+against+experimental+focal+brain+ischemia.">Low doses of carbon monoxide protect against experimental focal brain ischemia.</a> Neurotox Res. 15, 133-137 (2009).</li><li>Zeynalov, E., Nemoto, M., Hurn, P. D., Koehler, R. C., Bhardwaj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+selective+kappa+opioid+receptor+agonist+is+gender+specific+and+linked+to+reduced+neuronal+nitric+oxide.">Neuroprotective effect of selective kappa opioid receptor agonist is gender specific and linked to reduced neuronal nitric oxide.</a> J Cereb Blood Flow Metab. 26, 414-420 (2006).</li><li>Ma, M. C., Qian, H., Ghassemi, F., Zhao, P., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-sensitive+{delta}-opioid+receptor-regulated+survival+and+death+signals:+novel+insights+into+neuronal+preconditioning+and+protection.">Oxygen-sensitive {delta}-opioid receptor-regulated survival and death signals: novel insights into neuronal preconditioning and protection.</a> J Biol Chem. 280, 16208-16218 (2005).</li><li>Ahmad, M., Zhang, Y., Liu, H., Rose, M. E., Graham, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+opportunity+for+neuroprotection+in+experimental+stroke+with+selective+blockade+of+cyclooxygenase-2+activity.">Prolonged opportunity for neuroprotection in experimental stroke with selective blockade of cyclooxygenase-2 activity.</a> Brain Res. 1279, 168-173 (2009).</li><li>Meisel, 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=Preventive+antibacterial+treatment+improves+the+general+medical+and+neurological+outcome+in+a+mouse+model+of+stroke.">Preventive antibacterial treatment improves the general medical and neurological outcome in a mouse model of stroke.</a> Stroke. 35, 2-6 (2004).</li><li>Miner, N. A., Koehler, J., Greenaway, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+injection+of+mice.">Intraperitoneal injection of mice.</a> Appl Microbiol. 17, 250-251 (1969).</li><li>Adis International Limited. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conivaptan:+YM+087.">Conivaptan: YM 087.</a> Drugs in R&amp;D. 5, 94-97 (2004).</li><li>Murphy, T., Dhar, R., Diringer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conivaptan+bolus+dosing+for+the+correction+of+hyponatremia+in+the+neurointensive+care+unit.">Conivaptan bolus dosing for the correction of hyponatremia in the neurointensive care unit.</a> Neurocrit Care. 11, 14-19 (2009).</li><li>Liu, X., Nakayama, S., Amiry-Moghaddam, M., Ottersen, O. P., Bhardwaj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine-vasopressin+V1+but+not+V2+receptor+antagonism+modulates+infarct+volume,+brain+water+content,+and+aquaporin-4+expression+following+experimental+stroke.">Arginine-vasopressin V1 but not V2 receptor antagonism modulates infarct volume, brain water content, and aquaporin-4 expression following experimental stroke.</a> Neurocrit Care. 12, 124-131 (2010).</li><li>Wallace, B. K., Jelks, K. A., O'Donnell, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-induced+stimulation+of+cerebral+microvascular+endothelial+cell+Na-K-Cl+cotransport+involves+p38+and+JNK+MAP+kinases.">Ischemia-induced stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransport involves p38 and JNK MAP kinases.</a> Am J Physiol Cell Physiol. 302, C505-C517 (2012).</li><li>O'Donnell, M. 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=Intravenous+HOE-642+reduces+brain+edema+and+Na+uptake+in+the+rat+permanent+middle+cerebral+artery+occlusion+model+of+stroke:+evidence+for+participation+of+the+blood-brain+barrier+Na/H+exchanger.">Intravenous HOE-642 reduces brain edema and Na uptake in the rat permanent middle cerebral artery occlusion model of stroke: evidence for participation of the blood-brain barrier Na/H exchanger.</a> J Cereb Blood Flow Metab. 33, 225-234 (2013).</li><li>Walcott, B. P., Kahle, K. T., Simard, J. 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The authors have nothing to disclose.

This study has important value for preclinical stroke research. This study reveals that continuous IV infusion of conivaptan (0.2 mg/day) after experimental stroke in mice efficiently reduces brain edema after 48 hr of treatment. The effect of IP injection of the same dose of conivaptan on brain edema was also investigated. Conivaptan treatment by both IV and IP routes produces aquaresis in mice as indicated by: 1) increase in plasma osmolality slightly above physiological levels; and 2) decrease in urine osmolality due ...

Stroke continues to be an enormous burden for patients and clinicians. Animal stroke models have been used in the laboratory setting for nearly two decades.1 Nevertheless, experimental treatments that work in animals often fail in humans.2 This discrepancy in treatment outcomes may be due to various factors, such as administration routes for drugs used in animal research, drug metabolism and elimination rate, and many other aspects. One of the major complications of stroke, brain edema, is a focus of current research in neuroscience. Mechanisms of brain edema formation involve disturbances in water and electrolyte balance triggered by the arginine-vasopressin (AVP) response to ischemic brain injury.3 Therefore, AVP and its receptors (V1a and V2) are a major focus of research studies of post-ischemic brain edema formation.3
We have developed a methodology to study the effects of mixed arginine-vasopressin (V1a and V2) receptor blocker conivaptan on post-ischemic brain edema in mice.4 Molecular targets of conivaptan5 make the drug a suitable candidate for exploration of its properties in alleviation of brain edema. Furthermore, conivaptan needs to be studied in the context of pathophysiological events of stroke.6 In designing this study, we considered comparing treatment outcomes using two different routes of administration for conivaptan: intravenous (IV)4 and intraperitoneal (IP).7 Effects of the treatments on stroke-induced brain edema were evaluated. Here detailed protocols are described for surgical induction of experimental stroke by middle cerebral artery occlusion (MCAO), and followed by continuous conivaptan treatment using the installation of a jugular IV catheter. After induction of stroke, animals were randomly allocated into the following groups: vehicle or conivaptan (0.2 mg/day) treated IV or IP.

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			<td>Heated Pad</td>
			<td>K&#38;H Manufacturing Inc</td>
			<td>1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Monitor with Rectal Probe</td>
			<td>Physitemp</td>
			<td>7029</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk Suture Spool, 6-0</td>
			<td>Surgical Specialties Corporation</td>
			<td>SP114</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk Suture on a Needle, 3-0</td>
			<td>Ethicon</td>
			<td>1684G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Suture, 7-0</td>
			<td>Ethicon</td>
			<td>1696G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental Resin Polysiloxane with Hardener</td>
			<td>Heraeus Kulzer</td>
			<td>65817930</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinfusion IV Pump</td>
			<td>Kent Scietific</td>
			<td>GT0897</td>
			<td></td>
		</tr>
		<tr>
			<td>Swivel 22GA</td>
			<td>Instech</td>
			<td>375/22PS</td>
			<td></td>
		</tr>
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			<td>Laboratory Tubing, 0.94 x 0.51 mm</td>
			<td>Dow Corning</td>
			<td>508-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Tubing, 3.18 x 1.98 mm</td>
			<td>Dow Corning</td>
			<td>508-009</td>
			<td></td>
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</table>

continuous iv infusion is the choice treatment route for arginine-vasopressin receptor blocker conivaptan in mice to study stroke-evoked brain edema

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among the most important players in the development and evolution of stroke-evoked brain edema is the hormone arginine-vasopressin and its receptors, V1a and V2. Recently, the V1a and V2 receptor blocker conivaptan has been attracting attention as a potential drug to reduce brain edema after stroke. However, animal models which involve conivaptan applications in stroke research need to be modified based on feasible routes of administration. Here the outcomes of 48 hr continuous intravenous (IV) are compared with intraperitoneal (IP) conivaptan treatments after experimental stroke in mice. We developed a protocol in which middle cerebral artery occlusion was combined with catheter installation into the jugular vein for IV treatment of conivaptan (0.2 mg) or vehicle. Different cohorts of animals were treated with 0.2 mg bolus of conivaptan or vehicle IP daily. Experimental stroke-evoked brain edema was evaluated in mice after continuous IV and IP treatments. Comparison of the results revealed that the continuous IV administration of conivaptan alleviates post-ischemic brain edema in mice, unlike the IP administration of conivaptan. We conclude that our model can be used for future studies of conivaptan applications in the context of stroke and brain edema.

Body temperature of the animals was within the physiological range and stable throughout the surgical procedure of stroke induction. Two mice that exhibited NDS lower than 2 immediately after MCAO were excluded from the study.
MCAO in mice produces infarct volume in the ipsilateral hemisphere at 48 hr. Evaluation of the TTC-stained slices shows that about 50% of the hemisphere is affected by infarct after MCAO (Figure 1D...

Watch this Scientific Journal Video about Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema at JoVE.c

Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema

Our studies have revealed that the beneficial effects of conivaptan are dependent on the method of delivery after experimental stroke in mice. We have developed a research protocol for delivery of the receptor blocker via IV catheter on stroke-evoked brain edema formation in mice.

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among ...

The overall goal of this pre-clinical study, is to create a successful mouse model of Conivaptan administration after stroke, to help improve the design of future research studies in the stroke field. This method can help answer key questions in the stroke research field, such as, what is the importance of the delivery route to the beneficial effects of Conivaptan on stroke recovery in mice. The main advantage of this technique, is that it will assist researchers in the modelling studies of Conivaptan and it's effects on stroke-evoked brain edema.At least two hours before the surgery, cut a 12 millimeter piece of filament from a 7-0 nylon suture, then mix two parts dental resin with one part hardener, and quickly dip the filament into the mixture, until approximately four millimeters of the filament have been coated. Immediately verify that the filament tip is covered with a smooth coat of dental resin. Then, air-dry the filament for two hours.When the resin is ready, place an anesthetized mouse onto a heating pad on the operating table, and confirm a lack of response to toe-pinch. Remove the hair on the front and back of the neck with clippers, spray the neck with rubbing alcohol, and apply ointment to both eyes. To continuously monitor body temperature during the surgery, use a rectal temperature probe connected to a digital display.Place the mouse in the supine position under a dissecting microscope, and if necessary, position a piece of rolled gauze underneath the neck to arch it. Then disinfect the neck with a Povidone Iodine 10%scrub solution. Then, cover the animal with a sterile drape, and use a size 10 surgical blade to make a mid-line incision along the neck.Using a 6-0 silk suture, temporarily ligate the left common carotid artery, and follow this with a tie on the left external carotid artery. Place a microvascular clip on the internal carotid artery, and use microvascular scissors to cut a small hole on the external carotid artery. After inserting the dental resin-coated filament into the opening, advance the filament cephalically while removing the clip until resistance is felt.Place a temporary tie onto the internal carotid artery to secure the filament in position, and close the skin with a silk suture. Then infiltrate the wound on the front of the neck with 0.2 millimeters of 0.5%Bupivacaine and transfer the mouse to a recovery chamber with monitoring until sternal recumbance is maintained. For Neurological Deficit Scoring, lift the mouse by the tail, and place the animal on a flat surface for two to three minutes.Score the animal's behavior according to the text, excluding mice that exhibit a Neurological Deficit Score lower than two, due to an insufficient occlusion of the middle cerebral artery. To remove the filament, re-sterilize the incision with a 10%Povidone Iodine scrub solution, and cut and remove the suture to re-open the surgical wound. Then, under a dissecting microscope, place a microvascular clip on the internal carotid artery, and untie the internal carotid arterial suture.Hold the clip open to slide out the filament. Then immediately close the clip again, and suture the external carotid artery proximal to the cut. When the external carotid artery ligature is secure, remove the clip and untie the suture around the common carotid artery to restore the blood flow to the brain.Immediately after removing the common carotid artery suture, disinfect the back of the neck with a 10%Povidone Iodine scrub solution, and use a size 10 surgical scalpel blade to make a one centimeter incision. Then re-sterilize the front of the neck, and re-open the surgical wound. Now tunnel a piece of 0.94 millimeter outer-diameter tubing, nested within a piece of 3.18 millimeter outer-diameter tubing under the skin of the animal from the back to the front of the neck, and exteriorize the tubing when it reaches the front incision.Remove the subcutaneous adipose tissue from the left side of the front of the neck about one centimeter laterally from the mid-line. Then locate the left jugular vein under the dissecting microscope, and place two ties along the vein 5 millimeters apart, slightly stretching the vessel. Next, use microvascular scissors to make a small hole on the jugular vein between the two ties, and insert the tip of the inner tubing five millimeters deep caudally toward the heart.Secure the tubing with both ties to the jugular vein, and use a 3-0 silk suture to close the skin on the front of the neck. Then secure the tubing to the skin on the back of the neck with a suture and connect the tubing to a micro-infusion intravenous pump through a swivel. Infiltrate the skin incisions on the front and the back of the neck with 0.2 millimeters of 0.5%Bupivacaine to prevent post-operative pain, and allow the animal to awaken in the recovery chamber with monitoring.When the animal is recumbent, set the infusion rate for a continuous 48 hour intravenous treatment of Conivaptan or vehicle, at 1.5 millimeters per kilogram per hour, and start the infusion. Alternatively, administer 0.2 milligrams of Conivaptan and 1.44 milliliters of 5%Dextrose twice intraperitoneally, daily for two days, followed by a brief observation of the animals after each injection. Middle cerebral arterial occlusion in mice, produces an infarct volume in the ipsilateral hemisphere at 48 hours with approximately 50%of the hemisphere affected as visualized by tetrazolium chloride staining.The evaluation of the brain water content at 48 hours after a 60 minute middle cerebral arterial occlusion induction, reveals that intravenous Conivaptan administration significantly reduces the brain edema, in both the ipsilateral and contralateral hemispheres. In contrast, daily intraperitoneal Conivaptan treatment by a 0.2 millimeters bolus fails to produce similar positive effects on the stroke-evoked brain edema. Once mastered, this technique can be completed in two hours, if it is performed properly.While attempting this procedure, it's important to remember to avoid excessive blood loss in the experimental animal because it can affect your study result and lead to increased mortality. Following this procedure, other studies like evaluating the blood brain barrier disruption can be performed to answer additional questions about the involvement of inflammation in post-ischemic brain edema. After the development, this technique paved the way for researchers in the field of neuroscience, to explore the importance of route of drug administration for clinical studies.After watching this video, you should have a good understanding of how to surgically create ischemic conditions in mice and stow an intravenous catheter into the jugular vein and administer Conivaptan by continuous IV infusion in mice. Don't forget that working with live animals can be extremely hazardous and that precautions such as wearing surgical gloves and masks should always be taken while performing this procedure.

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8.7K vistas.  Swedish Medical Center. The overall goal of this pre-clinical study, is to create a successful mouse model of Conivaptan administration after stroke, to help improve the design of future research studies in the stroke field. This method can help answer key questions in the stroke research field, such as, what is the importance of the delivery route to the beneficial effects of Conivaptan on stroke recovery in mice. The main advantage of this technique, is that it will assist researchers in the modelling studies of C...

Video: Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema