Name: lateral chronic cranial window preparation enables in vivo observation following distal middle cerebral artery occlusion in mice
Uploaded: 2016-12-29T13:00:00.000+00:00
Duration: 8 min 41 s
Description: Watch this Scientific Journal Video about Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice at JoVE.com

VP is a participant in the Charit&#233; Clinical Scientist Program, funded by the Charit&#233; - Universit&#228;tsmedizin Berlin and the Berlin Institute of Health. TB is an SNSF PostDoc Mobility fellow. The authors receive grant support from EinsteinStiftung/A-2012-153 to PV.

ETHICS STATEMENT: Experiments involving animal subjects were performed in accordance with the guidelines and regulations set forth by Landesamt fuer Gesundheit und Soziales, Berlin, Germany (G0298/13) and the ARRIVE criteria, as applicable. For this study, 10- to 12-week-old male C57Bl/6J mice were used.
1. Lateral Chronic Cranial Window Preparation
<ol>
	<li>Perform anesthesia with a subcutaneous injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). Test for adequate sedation with a pain stimulus.</li>
	<li>Sterilize the surgical instruments and surgical field with 70% ethanol.</li>
	<li>Remove the scalp hair from the neck to the eyes using a rodent shaver.</li>
	<li>Fix the head in a stereotactic frame.</li>
	<li>Use dexpanthenol eye ointment on both eyes to avoid dehydration.</li>
	<li>Clean the surgical area to remove all hairs and sterilize it with 3 layers of 74.1% ethanol and 10% 2-propanolol.</li>
	<li>Perform a midline incision from the neck to the eyes using a scalpel.</li>
	<li>Span the skin flap with 4 tenting sutures.</li>
	<li>Remove the periosteum carefully on the left hemisphere by scraping it off with a scalpel to the point where the temporal muscle begins. 
	NOTE: This preparation also serves to create a good adhesion area for the dental glue, which fixes the cover glass.</li>
	<li>Perform a fronto-parietal craniotomy with a diameter of 4 mm by thinning the skull at the rim of the bone flap using a microdrill. Apply saline solution while drilling to avoid heat injury.</li>
	<li>Elevate the bone flap with cannulas and remove it using microforceps.</li>
	<li>Irrigate carefully and extensively with saline solution.</li>
	<li>Mix the dental glue until it has the proper consistency and is not fluid (i.e., the glue should not produce threads anymore). Place it on the bone around the craniotomy.</li>
	<li>Place a cover glass with a 6-mm diameter on the prepared glue and fix it with the rest of the dental glue. Be sure that it is watertight. Wait until the glue is dried and hard by testing it with forceps. 
	NOTE: An additional irrigation accelerates the curing process.</li>
	<li>Remove the tenting sutures and close the wound with skin sutures.</li>
	<li>Pull up the skin of the flank of the mouse. Insert a needle subcutaneously and gently substitute 0.5 ml of sterile saline subcutaneously for fluid balance.</li>
	<li>After surgery, keep the animals in the heated recovery cage for 90 min. Wait until the animals are fully awake before leaving them unattended. Wait until the animals are completely recovered before returning them to a cage with other animals.</li>
	<li>Repeat the subcutaneous saline volume substitution after about 12 hr, as described in step 1.16.</li>
	<li>Always apply analgesia via gavage or directly into the oral cavity&#160;after surgery (e.g., paracetamol (10 mg/ml) or other non-steroidal anti-inflammatory drugs).</li>
	<li>Check the condition of the animals every day after surgery, and always provide mashed animal food on the floor in a petri-dish to make eating simple and to avoid critical weight loss after surgery. 
	NOTE: Intravital microscopy can be performed on the first day after cranial window preparation.</li>
	<li>&#160;Apply isoflurane anesthesia and fix the animal in a head holder. Open the skin suture and clean the window with cotton buds and sterile saline.&#160;After 24 hr, the cranial window should be filled with cerebrospinal fluid by that time point, which enables imaging. Perform imaging by using established microscopy protocols18.</li>
</ol>
2. Distal MCAo
NOTE: The MCAo procedure should be performed about 5 d after CW preparation. This minimizes interference from the immune reaction caused by the CW preparation with the stroke-induced immune reaction.
<img alt="Figure 1" src="/files/ftp_upload/54701/54701fig1.jpg" /> 
Figure 1. Overview of Distal MCAo. A. This is a nice overview about the vessels before the op. B. The vessels after first bipolar contact. C. The vessels after second bipolar contact. D. The overview about the vessels, which are completely closed now. E. Final overview with lower magnification. <a href="https://cloudfront-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54701/54701fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Anaesthetize the mice using an anesthesia mask and an appropriate anesthetic regime, in consultation with veterinary staff (e.g., induction with 1.5 - 2% isoflurane and maintenance with 1.0 - 1.5% isoflurane in 2/3 N2O and 1/3 O2 via a vaporizer).</li>
	<li>Shave and remove the hair and disinfect the skin and surrounding fur with an appropriate agent (e.g., 70% ethyl alcohol), and dry it afterwards.</li>
	<li>Maintain the body temperature of the mice at 36.5 &#176;C &#177; 0.5 &#176;C during surgery. A feedback controlled heating pad, heated according to the rectal temperature of the mouse, is highly recommended.</li>
	<li>Place the animal in the lateral position. Fix the nose in the anesthesia mask and adjust the isoflurane concentration to 1.0 - 1.5%.</li>
	<li>Apply wet ointment (dexpanthenol) to both eyes.</li>
	<li>Use the skin incision made for the CW preparation.</li>
	<li>Gently separate the skin and identify the temporal muscle underneath.</li>
	<li>Adjust the energy of the high-frequency generator (5 - 7 W) and use the bipolar mode.</li>
	<li>Use the electrocoagulation forceps and carefully remove the temporal muscle from the skull, creating a muscle flap, without totally removing the muscle.</li>
	<li>Identify the MCA below the transparent skull in the rostral part of the temporal area, dorsal to the retro-orbital sinus. If the MCA bifurcation cannot be identified, try to identify the vessel most rostral.</li>
	<li>Thin the skull above the MCA branch with a microdrill while continuously irrigating to avoid heat damage.</li>
	<li>Lift the bone with cannulas and remove it with microforceps.</li>
	<li>Decrease the energy of the high-frequency generator to 3 - 5 W.</li>
	<li>Approach the artery from above and gently touch it with the bipolar forceps on both sides without lifting the vessel.</li>
	<li>Coagulate the artery proximally and distally to the vessel bifurcation.</li>
	<li>Wait for 30 sec, and then touch the artery gently to ensure that the blood flow is permanently interrupted. Repeat the electrocoagulation if a recanalization is observed.</li>
	<li>Fix the temporal muscle with 1 or 2 stitches at the muscle insertion to cover the bone defect, if possible.</li>
	<li>Suture the wound and place the animal into the heated recovery box. In general, animals recovery quickly after volatile anesthesia.</li>
	<li>For volume substitution, apply 0.5 ml of sterile saline subcutaneously, as described in step 1.16.</li>
	<li>After surgery, allow the animals to stay in the heated recovery cage for 90 min. Wait until the animals are conscious before leaving them unattended. Only return them to a cage with other animals when they are fully recovered.</li>
	<li>Repeat the volume substitution, as explained in step 1.16, after 12 hr.</li>
	<li>Apply postoperative analgesia via drinking water (e.g., paracetamol (10 mg/ml) or other non-steroidal anti-inflammatory drugs).</li>
	<li>Check the medical condition of the animals every day after surgery. Provide mashed animal food in a petri-dish on the floor to simplify eating and to minimize postoperative weight loss.</li>
</ol>
3. Sham Treatment
<ol>
	<li>Perform all procedures identically to steps 1 and 2, described above-including CW preparation-except do not coagulate the exposed middle cerebral artery.</li>
</ol>

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The authors have nothing to disclose.

Stroke is among the principal causes of long-term disability and death worldwide1. Beyond acute treatment, investigating new approaches and mechanisms to accelerate and enhance recovery after stroke remains a prime medical goal7. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. In fact, models inducing transient or permanent MCAo mimic one of the most common types of focal cerebral ischemia in patients4. Besides proximal occlusion of the MCA, the filament model for surgical occlusion of the distal MCAo is probably the most frequently used model in experimental stroke research5,19. Here, we describe the basic technique of permanent distal MCAo combined with a lateral CW, offering the opportunity for longitudinal intravital microscopy in mice. High reproducibility of lesion volume, as well as very low mortality rates, in particular with respect to studying long-term outcomes, are the main advantages of this murine model. In this cortical stroke model, blood vessels in the stroke area and peri-infarct region can be visualized via the chronic CW. Using a multi-fluorescence epifluorescence videomicroscopic system, blood flow dynamics and the dynamic recruitment of circulating cells can be visualized. Blood vessels are visualized through the use of fluorescently labeled macromolecules, like dextrans or albumin. Cells can be labeled either by fluorescent dyes or by genetic models, such as bone marrow chimeras with GFP-positive animals. Further, to study cell-to-cell interactions and the dynamics of extravascular cells, a two-photon laser scanning confocal microscope can be applied. Imaging up to 250 &#956;m below the cortical surface can be performed. Again, blood vessels are stained using fluorescently labeled macromolecules, and cells are labeled genetically (e.g., by using the transgenic GFP-Nestin mouse).
The cranial window surgery is performed via a craniotomy without opening the dura. One major pitfall is to accidently injure the dural layer and cortex underneath when opening the skull with the microdrill. Therefore, this technique requires some technical skill in order to avoid damage to the dura and cortex, which induces an immune reaction and influences microscopy results. Alternatively, a potential thinned-skull model is limited by less reliable microscopy quality because of the remaining skull, especially in the long term. Frequently, repetitive skull thinning is necessary, whereas in the CW model, the window quality lasts for several months, until skull regrowth or dural thickening influences imaging quality14,20. A modulation of this model with a thinned-skull preparation would be possible. The dural layer should be left on the brain to avoid any injury or excitation of the cortex. Only if a direct application to the ischemic area is desired in an experimental model can the dura be removed, carefully and without injuring any bridging veins.
In contrast to models combining a CW and inducing targeted vessel occlusion via irradiation of circulating photosensitizers, leading to only very small ischemic lesions, the distal MCAo model mimics the majority of human strokes that are located in the cortical MCA territory13. To avoid interference with a transient inflammatory reaction due to CW preparation, the window should be prepared several days before distal MCAo surgery.
A number of tests to assess functional as well as behavioral aspects in rodents are available (e.g., gait analysis, rotarod test, pole test, adhesive removal test, staircase test, open field test, and Morris water maze)21. In all of these tests, mice subjected to MCAo perform less successfully than control animals with respect to short-term and intermediate-term outcomes. However, regarding the assessment of long-term outcomes, it must be acknowledged that the sensitivity of functional testing is very limited in terms of distal MCAo, as well as mild proximal MCAo19,21-23.
Distal MCAo, performed by well-trained surgeons, can be induced within less than 20 min and can produce highly reproducible ischemic lesions. However, reproducibility requires a thorough control of confounders. Differences in surgical technique may lead to differences in infarct size24. Different mouse strains might show a different stroke outcome due to variances in cerebral vascular anatomy between strains. Furthermore, as body temperature affects neuronal damage and susceptibility to ischemia, with hypothermia leading to smaller lesions and hyperthermia to more severe deficits25,26, temperature control and maintenance are highly relevant in this model, as well as in other ischemia models27. In general, physiological parameters, such as blood pressure and blood gases, are important confounders of outcome and must be controlled for28. In addition, the choice of the anesthetic is highly important, as some agents may exert direct neuroprotective effects or act indirectly via vasoactive properties29. Therefore, exposure to anesthesia should be standardized and kept as short as possible. Finally, housing conditions, like the use of enrichment, may affect stroke outcome as well, and thus should be standardized and described in research reports30. To produce relevant preclinical results for the development of new therapeutic approaches, standardization, quality control, and reporting are of the utmost importance31.

Stroke is among the principal causes of long-term disability and death worldwide1, coming second after coronary heart disease. In addition, stroke is the primary cause of long-term disability, underscoring its tremendous socioeconomic impact6-8. Beyond acute treatment, investigating new approaches and mechanisms to accelerate and enhance recovery after stroke remains a prime medical goal7. 
In the last few decades, data from experimental stroke research has contributed substantially to understanding the complex pathophysiological cascades triggered by ischemia9,10. Excitotoxicity, apoptosis, peri-infarct depolarization, and inflammation have been identified as the most relevant mediators of cell death following focal cerebral ischemia. Moreover, using animal models of cerebral ischemia, important concepts, diagnostic modalities, and therapeutic approaches have been developed and validated (e.g., "penumbra" and thrombolysis)11.
The availability of experimental stroke models, combined with non-invasive imaging modalities (e.g., magnetic resonance imaging (MRI), computed tomography, or laser speckle contrast analysis), enables the researcher to investigate hyperacute and chronic pathophysiological changes induced by the ischemic insult in a longitudinal manner12. Along with studying the spatiotemporal profile of the evolving lesion, changes resembling neuronal plasticity can be investigated and correlated to functional outcomes and histological findings. Within the last few years, further methodological advances have been made using the combination of cerebral ischemia models and in vivo microscopy via cranial windows13. These new techniques allow investigators to analyze the neurovascular unit at the cellular and molecular level, with great analytic power in the acute, subacute, and chronic phases following focal cerebral ischemia14. Moreover, in vivo microscopy imaging of microcirculatory dynamics has revealed novel aspects of cerebral microvasculature function and angioarchitecture, with significant pathophysiological relevance15-17. 
In this protocol, we present how to perform a chronic CW preparation lateral to the sagittal sinus and how to surgically induce a distal stroke underneath the window. This mouse model can be applied to sequential imaging of acute, subacute, and chronic changes following focal cerebral ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

<table><tbody><tr><td>Binocular surgical microscope</td><td>Zeiss</td><td>Stemi 2000 C</td><td></td></tr><tr><td>Light source for microscope</td><td>Zeiss</td><td>CL 6000 LED</td><td></td></tr><tr><td>Heating pad with rectal probe</td><td>FST</td><td>21061-10</td><td></td></tr><tr><td>Stereotactic frame</td><td>Kopf</td><td>Model 930</td><td></td></tr><tr><td>Anaethesia system for isoflurane</td><td>Draeger</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Abott</td><td></td><td></td></tr><tr><td>Dumont forceps #5</td><td>FST</td><td>11251-10</td><td></td></tr><tr><td>Dumont forceps #7</td><td>FST</td><td>11271-30</td><td></td></tr><tr><td>Bipolar Forceps</td><td>Erbe</td><td>20195-501</td><td></td></tr><tr><td>Bipolar Forceps&amp;nbsp;</td><td>Erbe&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 20195-022</td><td></td><td></td></tr><tr><td>Microdrill</td><td>FST&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 18000-17&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Needle holder</td><td>FST</td><td>12010-14</td><td></td></tr><tr><td>5-0 silk suture</td><td>Feuerstein, Suprama</td><td></td><td></td></tr><tr><td>7-0 silk suture</td><td>Feuerstein,Suprama</td><td></td><td></td></tr><tr><td>8-0 silk suture</td><td>Feuerstein, Suprama</td><td></td><td></td></tr><tr><td>Veterinary Recovery Chamber</td><td>Peco Services</td><td>V1200</td><td></td></tr></tbody></table>

lateral chronic cranial window preparation enables in vivo observation following distal middle cerebral artery occlusion in mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

The timeline and representative results are shown in Figures 2 and 3. The cranial window preparation, with a small cranial window lateral to the superior sagittal sinus (Figure 2 B, C, D) results in a very low mortality and morbidity rate when performed by an experienced surgeon. All of the 10 animals survived, and all chronic CW could be used for high-quality imaging, even 28 days after surgery. There was no problem with wound infections or other complications.
Due to the short exposure to volatile anesthesia and only minor brain damage, approximately 10 - 15 min after distal MCAo and transfer to the heated recovery cage, all animals were awake, freely moving in the recovery cage, and interacting with littermates. In general, mortality of the distal MCAo model is less than 5% and mainly occurs as a result of vascular injury and subsequent hemorrhage during MCAo surgery. Mortality during the 28-day observation period following distal MCAo occurs only very rarely. Morphologically, the lesion can be assessed using histology or MRI (Figure 3 A, B). In addition, MRI measurements offer the opportunity to assess lesion volume and progression in a longitudinal manner. An MRI performed 24 hr after ischemia clearly depicts the ischemic lesion located underneath the chronic CW, while following sham surgery, no lesioned cortical tissue was found (Figure 3A). The MRI results clearly show the strict restriction of the lesion to the cortex, thus sparing the striatum, in contrast to the filamentous MCAo model (Figure 3B). The lateral chronic CW preparation enables long-term visualization of cortical vasculature and microcirculation by epi-fluorescence microscopy (Figure 3C, upper part) and of subcortical areas by two-photon microscopy (Figure 3C, lower part). Further, it is possible to image molecular pathways and cell-to-cell interactions with fluorescence-labeled cells or autofluorescence measurements, such as fluorescence lifetime imaging. As shown in Figure 3D, the distal MCAo model causes highly reproducible ischemic lesions. Regarding the infarct volume, we expect a standard deviation below 15% in a single set of surgeries. As mentioned above, in contrast to models of proximal MCAo, mortality rates are rather low for the distal model5. Using sequential MRI, lesion, volume, and edema progression following focal cerebral ischemia can be assessed. The MRI at 24 hr and 96 hr after permanent distal MCAo showed no significant progression of T2 hyperintensities.
<img alt="Figure 2" src="/files/ftp_upload/54701/54701fig2.jpg" /> 
Figure 2: Chronic Cranial Window Preparation. (A) Representative timeline. (B) Marking the area where the craniotomy takes place, lateral to the temporal muscle, medial to the superior sagittal sinus, and dorsal to bregma. (C) Brain surface after a craniotomy, with an intact dura layer and the cover glass in place; the circle shows the location of the ischemic area after distal MCAo. (D) Finished chronic cranial window with fixed cover glass, ready for repetitive imaging for several weeks (b = bregma, tm = temporal muscle, SSS = superior sagittal sinus, ai = area of ischemia). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54701/54701fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54701/54701fig3.jpg" /> 
Figure 3: Combination of the Lateral Chronic CW and Distal MCAo or Sham Surgery. (A) The MRI performed 24 hr after sham surgery does not show any lesioned cortical tissue. (B) The MRI performed 24 hr after ischemia clearly depicts the ischemic lesion (*) located underneath the chronic CW. (C) Intravital epi-fluorescence imaging (upper part) and two-photon imaging (lower part) of the cortical vasculature. (D) Infarct volume assessed via MRI at 24 hr and 96 hr after ischemia shows a mean lesion volume of 13.16 2.3 mm3 at 24 hr and 12.2 1.9 mm3 at 96 hr. Each dot represents an individual animal (n = 10 animals per group, mean &#177; SEM). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54701/54701fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice at JoVE.com

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

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11.6K Views.  Charité-Universitätsmedizin. Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Video: Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Medicine

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

A Model of Transient Cerebral Ischemia by Occlusion of Middle Cerebral Artery

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Author Spotlight: Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Summary

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Chapters in this video

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

Mouse Model of Middle Cerebral Artery Occlusion

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

Transient Middle Cerebral Artery Occlusion Model of Stroke

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique