Name: continuous iv infusion is the choice treatment route for arginine-vasopressin receptor blocker conivaptan in mice to study stroke-evoked brain edema
Uploaded: 2016-09-01T14:00:00.000+00:00
Duration: 8 min 44 s
Description: 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

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
<ol>
	<li>Pre-coat the Filament with Dental Resin Prior to the Surgery.
	<ol>
		<li>Cut a 12 mm long piece of filament from a 7-0 nylon suture. Mix 2 parts of the resin with 1 part of the hardener, and then immediately dip the tip of the filament into the mixture to cover about 1/3 of the entire length. Remove it quickly and verify that it is covered with a smooth-surfaced coat of dental resin.</li>
		<li>Air dry the filament for 2 hr before use.</li>
	</ol>
	</li>
	<li>Place the mouse into an anesthesia induction chamber (2 L volume), and set the flow of 1.5% isoflurane in 25% oxygen-enriched room air at 2 L/min going into the chamber.</li>
	<li>Allow the mouse to remain in the induction chamber until fully anesthetized,8 as determined by lack of response to tail pinch.</li>
	<li>Place the mouse onto the operating table with a heated pad, and set the isoflurane concentration on 1% for maintenance of anesthesia delivered spontaneously through the nose cone.</li>
	<li>Clip hair on the front and the back of the neck, and spray the neck with rubbing alcohol. Apply veterinary ophthalmic ointment on both eyes.</li>
	<li>To continuously monitor body temperature, use a rectal temperature probe connected to digital display during the surgery.</li>
	<li>Place the mouse in the supine position, apply povidone-iodine 10% scrub solution for disinfection, cover with a sterile drape, and then make a midline incision along the neck with a size 10 surgical scalpel blade.</li>
	<li>Temporarily ligate the left common carotid artery (CCA) with 6.0 silk suture.</li>
	<li>Place another tie on the left external carotid artery (ECA).</li>
	<li>Place a microvascular clip on the internal carotid artery (ICA).</li>
	<li>Cut a small hole on the external carotid artery with microvascular scissors. Insert a filament made from 7-0 nylon suture coated with dental resin into the opening in the ECA, and advance it cephalically, while removing the clip, until resistance is felt (about 5-7 mm from the bifurcation point of the CCA).</li>
	<li>Place a temporary tie onto the internal carotid artery to secure the filament in position. Refer to the diagram, Figure 1A. Close the skin with silk suture, and infiltrate the wound on the front of the neck with 0.2 ml of 0.5% bupivacaine, as in the step 2.6.</li>
	<li>Allow the mouse to awaken from anesthesia and regain consciousness in a recovery chamber for 60 min. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbence.</li>
	<li>Test the mouse for neurological deficit scoring (NDS) as follows: 0 = normal motor function, 1 = flexion of torso and of contralateral forelimb on tail lift, 2 = circling to the contralateral side but normal posture at rest, 3 = leaning to contralateral side at rest, 4 = no spontaneous motor activity.4,9
	<ol>
		<li>For the NDS testing, lift mouse by the tail and place on a flat surface for observation (2-3 min). Alternatively, observe mice in the recovery chamber. Exclude mice that exhibit NDS lower than 2 from the experiment due to insufficient occlusion of the MCA. 
		&#8203;Note: Laser Doppler flowmetry technique can be used to confirm MCA occlusion.9-11</li>
	</ol>
	</li>
	<li>Re-anesthetize the animal after 60 min of recovery in the induction chamber as described above, re-sterilize the previous skin incision with 10% povidone-iodine scrub solution, and reopen the surgical wound on the front of neck by cutting and removing the silk suture.</li>
	<li>To remove the filament from the ECA perform the following steps:
	<ol>
		<li>Place a microvascular clip on the ICA. Untie the suture on the ICA and pull the filament out while holding clip open.</li>
		<li>Close the clip and place a tie onto the ECA proximally to the cut.</li>
		<li>Remove the clip and remove the ligature from the CCA to restore blood flow to the brain. Refer to the diagram, Figure 1B.</li>
	</ol>
	</li>
</ol>
2. Installation of IV Catheter into the Jugular Vein for Continuous 48 hr Treatment
Note: Proceed to installation of the catheter immediately after the step 1.16.3 without awakening of the mouse.
<ol>
	<li>Use two different sizes of flexible tubing: insert the inner tubing (0.94 mm outside diameter; to contain the drug being infused) into the outer tubing (3.18 mm outside diameter; for protection of the inner tubing).</li>
	<li>Place the mouse in the prone position, apply 10% povidone-iodine scrub solution for disinfection, and make a 1 cm incision on the back of the neck with a size 10 surgical scalpel blade. Turn the mouse into the supine position, re-sterilize the front of the neck with 10% povidone-iodine scrub solution, and reopen the surgical wound on the front of the neck. Tunnel the tubing under the skin from the incision made on the back to the front of the neck, and exteriorize it from the open wound 1 cm.</li>
	<li>Remove subcutaneous adipose tissue from the left side of the front of the neck about 1 cm laterally from the midline. Locate the left jugular vein, place two ties along the vein 5 mm apart, and slightly stretch the vein. Make a small hole on the jugular vein between the two ties with microvascular scissors; insert the tip of the inner tubing 5 mm deep caudally (towards the heart).</li>
	<li>Secure the tubing with both ties to the jugular vein. Close the skin on the front of the neck with a 3-0 silk suture. Refer to the diagram, Figure 1C.</li>
	<li>Secure the tubing to the skin on the back of the neck with a suture, and connect the tubing to a microinfusion IV pump through a swivel. The swivel allows free movement of mice inside the cage with full access to the food and water.</li>
	<li>Infiltrate the skin incisions on the front and the back of the neck with 0.2 ml of 0.5% bupivacaine to prevent post-operative pain. 
	Note: Depending on institutional specific animal protocols, animals may be treated with opioids, NSAIDs, or antibiotics pre- and post-operatively. However, mentioned treatments may alter results of ischemic stroke outcome (see Discussion Section).11-14</li>
	<li>Allow the animal to awaken in the recovery chamber, and then house the animal in a separate cage until the end point of the experiment. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbence.</li>
	<li>Set the infusion rate for continuous 48 hr IV treatment of conivaptan (0.14 mg/ml in 5% dextrose) or vehicle at 1.5 ml/kg/hr,4 and start the infusion. 
	Note: The total amount of IV infused conivaptan should be approximately 0.2 mg/mouse/day in a total volume of 1.44 ml. 
	&#8203;Note: Perform Intraperitoneal (IP) injection of conivaptan twice daily with total amount of 0.2 mg in 1.44 ml of 5% dextrose. Implement IP injection was as previously described.15 Restrain mice by gripping the skin of the posterior surface of the body and the tail. Inject conivaptan or vehicle into the left lower quadrant of the abdomen using a 26 G/1/2 inch needle.15</li>
	<li>Observe the animals twice a day for the entire duration of the experiment (48 hr). Allow the animals to have full access to food and water. If the animal exhibits signs of distress or dyspnea consult a veterinarian. If euthanasia is necessary for the animal in severe distress refer to the step 3.1 below, and exclude the animal from the study.</li>
</ol>
3. Evaluation of Stroke-evoked Brain Edema at the end Point
<ol>
	<li>Sacrifice the mouse by over-anesthetizing with 5% isoflurane.</li>
	<li>Use a scalpel to make a skin incision over the skull bone along the midline, and retract the skin to expose the entire skull over the brain. Beginning at the foramen magnum, cut the skull bone laterally along the perimeter of the brain on each side, and lift it carefully without touching the brain beneath it. Sever the brain from the spine and the nerves attached on the base of the skull.</li>
	<li>Remove the brain, separate it from the olfactory bulb and the cerebellum, and dissect the cerebrum along the interhemispheric fissure into the ischemic and non-ischemic cerebral hemispheres, as previously described.9</li>
	<li>Assess brain edema by comparing wet-to-dry ratios (WDR) as described previously4,9. Weigh tissues before and after drying for 3 days at 100 &#176;C in an oven. Calculate brain water content (BWC) as % H2O = (1-dry wt. /wet wt.) x 100%.</li>
</ol>

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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.

<table><tbody><tr><td>Heated Pad</td><td>K&amp;amp;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><tr><td>Laboratory Tubing, 0.94 mm x 0.51 mm</td><td>Dow Corning</td><td>508-002</td><td></td></tr><tr><td>Laboratory Tubing, 3.18 mm x 1.98 mm</td><td>Dow Corning</td><td>508-009</td><td></td></tr></tbody></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 ...

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8.8K Views.  Swedish Medical Center. 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. 

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

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Neuroscience

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Intracranial Pressure Monitoring In Nontraumatic Intraventricular Hemorrhage Rodent Model

Survivors of intraventricular hemorrhage are often left with significant long-term memory impairment; thus, research utilizing intraventricular hemorrhage animal models is essential. In this study, we sought out ways to measure intracranial pressure, mean arterial pressure, and cerebral perfusion pressure during nontraumatic intraventricular hemorrhage in rats. The experimental design included three Sprague Dawley groups: sham, standard 200 &#181;l intraventricular hemorrhage, and vehicle control groups. By introducing an intraparenchymal fiberoptic pressure sensor, precise intracranial pressure measurements were obtained in all groups. Cerebral perfusion pressures were calculated with the knowledge of intracranial pressure and mean arterial pressure values. As expected, the intraventricular hemorrhage and vehicle control groups both experienced a rise in the intracranial pressure and subsequent decline in cerebral perfusion pressure during intraventricular injection of autologous blood and artificial cerebrospinal fluid, respectively. The addition of an intraparenchymal fiberoptic pressure sensor is beneficial in monitoring precise intracranial pressure changes.

Monitoring intracranial pressure in rodent&#160;models of nontraumatic intraventricular hemorrhage is not common in the current literature. Herein, we demonstrate a technique for measuring intracranial pressure, mean arterial pressure, and cerebral perfusion pressure during intraventricular hemorrhage in a rat&#160;animal model.

Survivors of intraventricular hemorrhage are often left with significant long-term memory impairment; thus, research utilizing intraventricular hemorrhage ...

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

Fluorescent Dye-Based Visualization of Blood-Brain Barrier Breakdown in Mice

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...

Continuous Drug Infusion System in Mouse Model: A Surgical Procedure to Implant Micro-osmotic Pump Infusion System in the Mouse Brain for Continuous Drug Delivery

One day before the surgery, prepare brain osmotic pumps by using a 1-milliliter syringe and blunt-head needle to fill the pumps with artificial cerebrospinal fluid, or aCSF. Then, immerse the pump in aCSF and place it on a shaker for gentle overnight shaking. On the following day, remove the aCSF from the pump, and fill it completely with previously prepared drug solution diluted in aCSF, until the excess leaks out.
Then, use surgical scissors to cut the catheter tubes to desired length. Using a blunt-end brain infusion needle, attach the tubes to the brain infusion kit. Then, fill the tubes in the infusion kit with the drug solution. After assembling the kit, attach it to the micro-osmotic pump. Finally, to prevent the pump from drying out, immerse the osmotic pump brain infusion setting in aCSF within a sterilized 50-milliliter tube.
To start the surgery, mount and fix the head of an anesthetized mouse onto a stereotactic apparatus. Using a pair of surgical scissors and pincers, cut open the outer skin covering the skull. Then, use iodine to clean the peripheral skull. Next, use a pair of blunt-head scissors near the neck region to separate the outermost layer of skin from the subcutaneous skin for the osmotic pump brain fusion set implantation.
Use the stereotactic apparatus to mark the infusion point with reference to the brain map. Use a nail drill to drill a hole around the area marked on the skull, being careful not to break the mouse meninges and blood vessels. Then, place the micro-osmotic pump brain fusion set containing the drug of interest, or aCSF, as a control under the skin behind the neck region.
To infuse the drug into the brain, insert the brain infusion needle into the drilled hole. Use surface desensitizing gel to fix the needle in place on the skull, and wait until the glue dries. Cut off the projecting part on top of the needle.