This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) (18K09424 to Toshihide Kurihara and 20K18393 to Yukihiro Miwa) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Keio University School of Medicine.
1. Preparation of surgical instruments and animals
<ol>
	<li>Autoclave surgical instruments and keep them in 70% ethyl alcohol. Prior to each new surgical procedure, clean surgical instruments carefully using 70% ethyl alcohol.</li>
	<li>Prepare male BALB/cAJc1 mice (6 weeks old, 26-28 kg) in a specific-pathogen-free (SPF) room to maintain sterile conditions before, during and after the surgery.</li>
</ol>
2. Transient bilateral common carotid artery occlusion (tBCCAO)
<ol>
	<li>Put a mouse under anesthesia via intraperitoneal injection with a combination of midazolam (40 &#956;g/100 &#956;L), medetomidine (7.5 &#956;g/100 &#956;L) and butorphanol tartrate (50 &#956;g/100 &#956;L), termed &#8220;MMB&#8221;, as previously described8,9. Hold the mouse&#8217;s back skins to keep the mouse away from bumping its eyes until the mouse is completely anesthetized.
	<ol>
		<li>Judge the depth of anesthesia by pinching the mouse toe until it has no response, of which method is commonly used for checking complete anesthesia10. 
		NOTE: Generally, less than 5 min are required for mice to fall asleep. Proper recipes for general anesthesia may be different by institutions.</li>
	</ol>
	</li>
	<li>Apply one drop of 0.1% purified sodium hyaluronate eye drop solution to the eyes to prevent dryness on the eyes under anesthesia.</li>
	<li>Place the mouse on its back and fix the mouse&#39;s paws using adhesive tapes.</li>
	<li>Disinfect the neck area of the mouse using 70% ethyl alcohol before the surgery. 
	NOTE: Additional clipping of the fur was not performed as this may cause subsequent skin inflammation11,12.</li>
	<li>Perform sagittal incision of the neck with a blade (Figure 1). 
	NOTE: Incision needs to be made on the midline between the neck, sternum and trachea.</li>
	<li>Separate both salivary glands carefully using two forceps and mobilize them to visualize the underlying CCAs.</li>
	<li>Isolate the right CCA carefully from the respective vagal nerves and accompanying veins without harming their structures, and place two 6-0 silk sutures under the CCA. Tie the two ties tightly to block the blood flow (Figure 1). 
	NOTE: During the procedure, small veins could be damaged. If bleeding is seen, wiping is required to visualize the CCAs clearly.</li>
	<li>Find the left CCA carefully from the respective vagal nerves and accompanying veins without harming their structures, and occlude the left CCA for 2 seconds by a clamp (Figure 1). 
	NOTE: A 6-0 silk suture needle is needed to be placed under the left CCA to mark a site for clamping.</li>
	<li>After reopening of the left CCA, suture wounds of the neck by a 6-0 silk suture and apply a dab of antibiotic (50 &#956;L) onto the neck to inhibit bacterial infection. 
	NOTE: Softly remove a clamp to avoid damaging the arterial wall when reopening of the left CCA.</li>
	<li>Inject 0.75 mg/kg of atipamezole hydrochloride intraperitoneally to the mouse to help the mouse recovered from deep anesthesia quickly. Return the mouse to a mouse cage with pre-heated pads. 
	NOTE: Do not let the mouse left unattended until the mouse regains sufficient consciousness to maintain sternal recumbency.</li>
	<li>Inject 0.4 mg/kg of butorphanol tartrate to the mouse for the management of pain when the mouse wakes up. 
	NOTE: The protocol can be paused here. As a first hint for successful tBCCAO, eyelid drooping of the mouse can be observed (Figure 2).</li>
	<li>For euthanasia, inject 3x of MMB mixture to the mice and sacrifice them for experiments.</li>
</ol>
3. General observations (survival rates and eyelid drooping)
<ol>
	<li>After the surgery, check survival rates for all causes of death at day 0 (after the surgery), 1, 3 and 7.</li>
	<li>Assess eyelid drooping by a 4-point rating scale: 1 = no drooping, 2 = mild drooping (~50%), 3 = severe drooping (over 50%), and 4 = severe drooping with eye discharge.</li>
</ol>
4. Retinal blood perfusion
<ol>
	<li>Inject 200 &#956;L of FITC-dextran (25 mg/mL) into the left ventricle of the mouse, which is commonly used for the observation of blood perfusion in mouse retinal vessels13,14.</li>
	<li>2 minutes after circulation, enucleate the eyes and fix in 4% paraformaldehyde for 1 hour. The retinas were carefully obtained and flat-mounted, as previously described15, and examined via a fluorescence microscope.</li>
	<li>Take photographs of the retinal whole mounts at 4x magnification and merge into a single using a merge analyzer, previously described16.</li>
	<li>Measure the perfused areas via a vessel analysis tool in NIH Fiji/ImageJ software.</li>
</ol>
5. Western blot
<ol>
	<li>3 and 6 hours after tBCCAO, obtain the eyes of mice and immediately transfer to a Petri dish containing cold PBS to isolate the retinas.</li>
	<li>After isolation of the retinas, perform western blotting, as previously described9.</li>
	<li>Incubate with antibodies for hypoxia-inducible factor-1&#945; (HIF-1&#945;; a general hypoxia marker) and for &#946;-Actin (an internal loading control) overnight followed by incubation of HRP-conjugated secondary antibodies. Visualize the signals via chemiluminescence.</li>
</ol>
6. Quantitative PCR (qPCR)
<ol>
	<li>6, 12 and 24 hours after tBCCAO, process the obtained retinas for qPCR, as previously described17.</li>
	<li>Perform qPCR via real-time PCR system. Primers used are listed in Table 1. Calculate fold changes between levels of different transcripts by the &#916;&#916;CT method.</li>
</ol>
7. Immunohistochemistry (IHC)
<ol>
	<li>3 days after tBCCAO, obtain the eyes of mice and embed in paraffin.</li>
	<li>Cut the paraffin-embedded eyes by a microtome to obtain the eye sections.</li>
	<li>De-paraffinize and stain the eye sections of 5 &#956;m thickness as previously described13.</li>
	<li>Incubate with an antibody for glial fibrillary acidic protein (GFAP; a reliable marker for astrocytes and M&#252;ller cells in the retina) overnight followed by incubation of Alexa Fluor 555-conjugated secondary antibody.</li>
	<li>Use DAPI (4&#8242;,6-diamidino-2-phenylindole) for staining the nucleus in the retina. Visualize signals via a fluorescence microscope.</li>
	<li>Assess morphology scoring by a 4-point rating scale, as previously described13,18: 0 = no signal, 1 = few positive glial end-feet in the ganglion cell layer (GCL), 2 = few labelled processes reaching from GCL to the outer nuclear layer (ONL), and 3 = most labelled processes reaching from GCL to ONL.</li>
</ol>
8. Electroretinography (ERG)
<ol>
	<li>3 and 7 days after tBCCAO, perform ERG using a Ganzfeld dome, acquisition system and LED stimulators, as previously described9.</li>
	<li>Following dark adaptation overnight, anesthetize mice with a combination of MMB under dim red light.</li>
	<li>Use a mixed solution of 0.5% tropicamide and 0.5% phenylephrine to dilate the pupils.</li>
	<li>Place the active electrodes on contact lens and place the reference electrode in the mouth.</li>
	<li>Obtain ERG responses from both eyes of each animal.</li>
	<li>Record scotopic responses under dark adaptation with various stimuli.</li>
	<li>Measure the amplitudes of a-wave from the baseline to the lowest point of a-wave.</li>
	<li>Measure the amplitudes of b-wave from the lowest point of a-wave to the peak of b-wave.</li>
	<li>Keep all mice warm during the procedure using heat pads.</li>
</ol>
9. Optical coherence tomography (OCT)
<ol>
	<li>2 weeks after tBCCAO, perform OCT using SD-OCT system, as previously reported8,9.</li>
	<li>For the measurement, subject mice to mydriasis by a mixed solution of 0.5% tropicamide and 0.5% phenylephrine, and to general anesthesia by a mixture of MMB.</li>
	<li>Obtain B scan images from equatorial slices of en-face scans.</li>
	<li>Examine the retinas at 0.2, 0.4 and 0.6 mm from the optic nerve head.</li>
	<li>Measure retinal thickness from the retinal nerve fiber layer (NFL) to the external limiting membrane (ELM), and consider the average of measured values as retinal thickness of an individual mouse.</li>
	<li>Plot the results as spider diagrams.</li>
</ol>

The authors have nothing to disclose.

In the study, we have shown that tBCCAO, using simple sutures and a clamp, could induce retinal ischemia and accompanying retinal dysfunction. Furthermore, we have demonstrated our current protocol for the development of a mouse model of retinal ischemia is easier and faster in comparison with other previous protocols for the development of retinal ischemic injury models2,3,7.
Anatomically, the left a...

The retina is a neurosensory tissue for visual function. Since a substantial amount of oxygen is needed for visual function, the retina is known as one of the highest oxygen demanding tissues in the body1. The retina is susceptible to vascular diseases as oxygen is delivered through blood vessels. Various types of vascular diseases, such as diabetic retinopathy and retinal blood vessel (veins or arteries) occlusion, can induce retinal ischemia. To investigate pathological mechanisms of retinal ischemia, reproducible and clinically relevant experimental models of retinal ischemia are considered necessary. Middle cerebral artery occlusion (MCAO) by insertion of an intraluminal filament is the most generally utilized method for the development of in vivo rodent models of experimental cerebral ischemia2,3. Due to the proximity of the ophthalmic artery (OpA) to MCA, MCAO models are also used simultaneously to understand the pathophysiology of retinal ischemia4,5,6. To induce cerebral ischemia along with retinal ischemia, long filaments are typically inserted through incision of the common carotid artery (CCA) or the external carotid artery (ECA). These methods are difficult to perform, require a long time to complete the surgery (over 60 minutes for one mouse), and lead to high variabilities in the outcomes after the surgery7. It remains important to develop a better model to improve these concerns.
In this study, we simply used short transient bilateral CCA occlusion (tBCCAO) with needles and a clamp to induce retinal ischemia in mice and analyzed typical results of ischemic injuries in the retina. In this video, we will give a demonstration of the tBCCAO procedure.

<table>
	<tbody>
		<tr>
			<td>Atipamezole hydrochloride</td>
			<td>Zenoaq</td>
			<td>Antisedan</td>
			<td>For anti-anesthesia</td>
		</tr>
		<tr>
			<td>Applied Biosystems 7500 Fast</td>
			<td>Applied Biosystems</td>
			<td>-</td>
			<td>For qPCR</td>
		</tr>
		<tr>
			<td>Butorphanol tartrate</td>
			<td>Meiji Seika Pharma</td>
			<td>Vetorphale</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>BZ-II Analyzer</td>
			<td>KEYENCE</td>
			<td>-</td>
			<td>For an image merge</td>
		</tr>
		<tr>
			<td>BALB/cAJc1</td>
			<td>CLEA</td>
			<td>-</td>
			<td>Mouse strain</td>
		</tr>
		<tr>
			<td>&#946;-Actin (8H10D10) Mouse mAb</td>
			<td>CST</td>
			<td>3700</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Clamp Forcep</td>
			<td>World Precision Instruments</td>
			<td>WPI 500451</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Dumont forceps #5</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>Dojindo</td>
			<td>340-07971</td>
			<td>For IHC</td>
		</tr>
		<tr>
			<td>Envisu SD-OCT system</td>
			<td>Leica</td>
			<td>R4310</td>
			<td>For OCT</td>
		</tr>
		<tr>
			<td>FITC-dextran</td>
			<td>Merk</td>
			<td>FD2000S</td>
			<td>For retinal blood perfusion</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>KEYENCE</td>
			<td>BZ-9000</td>
			<td>For fluorescence detection</td>
		</tr>
		<tr>
			<td>Gatifloxacin hydrate</td>
			<td>Senju Pharmaceutical</td>
			<td>Gachifuro</td>
			<td>For anti-bacterial infection</td>
		</tr>
		<tr>
			<td>GFAP Monoclonal Antibody (2.2B10)</td>
			<td>Thermo</td>
			<td>13-0300</td>
			<td>For IHC</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Marukan</td>
			<td>RH-200</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>HIF-1&#945; (D1S7W) XP Rabbit mAb</td>
			<td>CST</td>
			<td>36169</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000 mini</td>
			<td>GE Healthcare</td>
			<td>-</td>
			<td>For chemiluminescence</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Sandoz K.K</td>
			<td>SANDOZ</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Microtome Tissue-Tek TEC 6</td>
			<td>Sakura</td>
			<td>-</td>
			<td>For sectioning</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Orion Corporation</td>
			<td>Domitor</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Handaya</td>
			<td>HS-2307</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>PuREC</td>
			<td>MAYO Corporation</td>
			<td>-</td>
			<td>For ERG</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Sodium hyaluronate</td>
			<td>Santen Pharmaceutical</td>
			<td>Hyalein</td>
			<td>For eye lubrication</td>
		</tr>
		<tr>
			<td>Tropicamide/Penylephrine hydrochloride</td>
			<td>Santen Pharmaceutical</td>
			<td>Mydrin-P</td>
			<td>For mydriasis</td>
		</tr>
		<tr>
			<td>6-0 silk suture</td>
			<td>Natsume</td>
			<td>E12-60N2</td>
			<td>For surgery</td>
		</tr>
	</tbody>
</table>

a murine model of ischemic retinal injury induced by transient bilateral common carotid artery occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

After systemic circulation of FITC-dextran for 2 minutes, retinal vasculatures of the left and right retinas in the sham-operated mice and tBCCAO-operated mice were examined (Supplementary Figure 1). FITC-dextran was fully visible in the both retinas in the sham-operated mice and the left retina in the tBCCAO-operated mice, while it was partially detectible in the right retina in the tBCCAO-operated mice.
After tBCCAO, eyelid drooping was examined (Figure ...

Watch this Scientific Journal Video about A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion at JoVE.com

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

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7.3K Views.  Keio University School of Medicine. Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Video: A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...