This work was supported by a grant from the Institute of Medical System Engineering (iMSE) &#38; GIST-Caltech Collaborative Fund (K04592) from GIST and by the Basic Science Research Program through NRF of Korea funded by the Ministry of Science, ICT and future Planning (NRF-2013R1A2A2A01067890).

All procedures were conducted according to the institutional guidelines of Gwangju Institute of Science and Technology (GIST), and all procedures were approved by the Institutional Animal Care and Use Committee at GIST.
1. Pre-lesioning Steps
<ol>
	<li>Identification of the Forelimb Area in the Internal Capsule using AAV-GFP
	<ol>
		<li>House and handle Sprague Dawley rats (~400 g, 11 - 13 weeks) in compliance with institutional and national guidelines.</li>
		<li>Sterilize all surgical tools and electrodes using an appropriate sterilizer (Steam or Plasma sterilizer). Use steam sterilizer at 121 &#176;C as setting of 30 min&#160;for sterilization and 30 min&#160;for dry.</li>
		<li>Anesthetize the animal with a mixture of ketamine hydrochloride (100 mg/kg) and xylazine (7 mg/kg) via an intramuscular injection. Check the depth of anesthesia by paw pinching. Maintain the body temperature at 37.5 &#177; 0.5 &#176;C via a heating pad under the body of the animal.</li>
		<li>Place the animal in a stereotactic frame using an ear bar and mouth holder.</li>
		<li>Clean and disinfect the surgical site with 70% alcohol and povidone iodine solution. Infiltrate 2% lidocaine hydrochloride under the scalp in the intended skull incision area to reduce the intraoperative pain.</li>
		<li>Apply vet ophthalmic ointment to prevent drying of the eyes. Place a sterile drape over the animal to the operative sites. Maintain all procedures in sterile conditions.</li>
		<li>Perform a midline skull incision of 2 cm using a scalpel and retract the skin bilaterally with wire retractors. Dry the skull with cotton swabs and 30% hydrogen peroxide.</li>
		<li>Make a hole using a hand piece drill over the forelimb area of motor cortex (AP: +2.5 from bregma, ML: &#177;2.5 from the midline) and clear the tract with micro-currette for virus-injection.</li>
		<li>Thaw the AAV-GFP (2 x 1012 virus molecules/ml) on ice and load 1 &#181;l of the virus in a 10 &#181;l syringe. Place the syringe on the stereotactic frame.</li>
		<li>Move the needle to the pre-made hole and lower the needle 1 mm deep into the dura.</li>
		<li>Inject the virus slowly (0.1 &#181;l/min) using a high precision micropump and leave the needle in place for an additional 10 min to allow the virus to diffuse out.</li>
		<li>After cleaning the operative site with saline irrigation, secure the wound with 3-0 nylon suture; release the rat from the stereotactic frame and transfer it to a recovery chamber. Administer &#160;ketoprofen (2 mg/kg) via an intramuscular injection&#160;&#160;for postoperative pain control.</li>
		<li>Maintain the body temperature (37 &#176;C) with heating pad and administer second generation cephems-class antibiotics (0.1%, 1 ml) via an intramuscular injection and 2% lidocaine hydrochloride via subcutaneous injection as necessary.&#160;&#160;Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Single house the animal until full recovery.</li>
		<li>Following 2 - 3 week recovery, deeply anesthetize the rat with an overdose of ketamine hydrochloride (300 mg/kg) via an intramuscular injection in the hood. Confirm the death of animal by lack of toe pinching response, pulse, and breathing. Place the rat supine in the hood.</li>
		<li>Open the abdominal cavity via a &#39;Y&#39;-shaped incision to open the chest cavity. Tightly clamp the descending aorta with a hemostat and rupture the right atrium of the rat heart for blood drainage. Initiate perfusion into the left ventricle of the heart with cold 1% paraformaldehyde for 5 min (10 ml/min) followed by 4% paraformaldehyde for 30 min (10 ml/min).</li>
		<li>Remove the rat head from carcass using a pair of scissors. Make a midline incision from the neck to the nose and remove the neck muscles using scissor or rongeur so that skull is exposed. Gently dissect the skull bones and duras out from the brain.</li>
		<li>Extract the brain and place the rat brain in a 50 ml conical tube filled with 4% paraformaldehyde overnight. The next day, wash the brain with 1x PBS 3 times and place it in a 30% sucrose solution.</li>
		<li>After the brain completely sinks to the bottom of the 30% sucrose solution, place the brain in the cryomold with OCT compound at -20 &#176;C in cryotome. Slice the brain in coronal plane at a thickness of 40 &#956;m and an interval of 200 &#956;m.</li>
		<li>Perform GFP immunohistochemistry staining using the slide method14. Apply primary antibody (1:200 of Anti-Green Fluorescent Protein, Rabbit IgG fraction) to brain slices overnight at 4 &#176;C. On 2nd day, wash with 1% Phosphate Buffered Saline with Tween-20 (PBST) solution 3 times and apply the secondary antibody (1:500 of Goat Anti-Rabbit IgG (H + L)) for 1 hr. Rinse the slide with 1% PBST 3 times. Place the cover glass on the brain slice.</li>
		<li>Using a fluorescent microscope (excitation wavelength 470 nm, emission wavelength 525 nm, magnification 5X), observe the AAV-GFP transduced axons in the internal capsule. Compare the locations of transduced axons with the Rat Brain Atlas15 to determine the stereotactic coordinates of the transduced axons</li>
	</ol>
	</li>
	<li>Pre-lesioning Adjustment of the Light Intensity Appropriate for Capsular Infarct Modeling
	<ol>
		<li>Construction of the Optical Neural Interface
		<ol>
			<li>Cut an appropriate length (4 cm) of a 27 gauge spinal needle with a stylet inside using a cutting drill. 
			NOTE: Cutting may compress and crush the spinal needle tip; remove the stylet and polish the spinal needle tip to remove the crushed portion of the spinal needle and maintain the inner caliber of the spinal needle.</li>
			<li>Strip an appropriate length (10 cm) of the jacket of the optical fiber (125 &#181;m with a 62.5 &#181;m core) of one side patch cord.</li>
			<li>Insert the unjacked optical fiber into the metal tube (external diameter: 3.8 mm, internal diameter: 3.3 mm and length: 17 mm), which is then clamped around the fiber. The metal tube is helpful to fill the space between the optical fiber and the hub of the spinal needle. Clamp the lower 1/2 of the metal tube with a presser twice.</li>
			<li>Apply the heat-curable epoxy on the optical fiber and insert the optical fiber into the spinal needle. Apply an additional epoxy to the empty space in the hub. Cure the epoxy for 20 min at 100 &#176;C for stable fixation.</li>
			<li>Cleave the optical fiber that protrudes out of the spinal needle and polish the optical fiber at the tip of the spinal needle using diamond lapping (polishing) sheets.</li>
			<li>Connect the FC/PC connector part of patch cord to the coupler of green laser system and measure the light intensity from the tip of the optical fiber using digital optical power and energy meter.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Photothrombotic Infarct Lesioning in the Internal Capsule
<ol>
	<li>Sterilize all surgical tools and electrodes using an appropriate sterilizer (Steam or Plasma sterilizer). Use steam sterilizer at 121 &#176;C as setting of 30 min&#160;for sterilization and 30 min&#160;for dry.</li>
	<li>Anesthetize the animal (~400 g, 11 - 13 weeks) with a mixture of ketamine hydrochloride (100 mg/kg) and xylazine (7 mg/kg) via an intramuscular injection. Check the depth of anesthesia by paw pinching. Maintain the body temperature at 37.5 &#177; 0.5 &#176;C via a heating pad under the body of the animal.</li>
	<li>Place the animal in a stereotactic frame using an ear bar and mouth holder.</li>
	<li>Clean and disinfect the surgical site with 70% alcohol and povidone iodine solution. Infiltrate 2% lidocaine hydrochloride under the scalp in the intended skull incision area to reduce the intraoperative pain. Apply vet ophthalmic ointment to prevent drying of the eyes.</li>
	<li>Apply a sterile drape over the animal and expose the operative sites. Maintain all procedures in sterile conditions.</li>
	<li>Perform a midline skull incision of 2 cm and retract the skin bilaterally with wire retractors. Dry the skull with cotton swaps and hydrogen peroxide.</li>
	<li>Adjust the height of the nose clamp until the bregma and lambda are aligned at the same level. CRITICAL STEP: This alignment is critical to correctly approach a deeper structure, such as in performing an infarct lesion in the internal capsule in the main experiment.</li>
	<li>Make a hole (diameter: 2 mm; AP: -2.04 from bregma; ML: &#177;3.0 from the midline) using a drill to induce photothrombosis.</li>
	<li>Polish and clean the optical fiber tip of the optical interface. Fix the ONI to the stereotaxic frame without bending. Check the tip of the ONI and wipe it out clearly before and after the insertion of the optical interface.</li>
	<li>Measure the laser intensity from the tip of the optical fiber prior to the insertion of the optical interface to the target site of the rat brain. Adjust the laser intensity to 3.5 mW, as confirmed by pre-surgery steps, at the tip of the optical fiber.</li>
	<li>Insert the ONI into the target area of the internal capsule (-7.8 mm confirmed from pre-surgery step) through the drill hole.</li>
	<li>Maintain the body temperature at 37.5 &#177; 0.5 &#176;C during photothrombosis. A lower body temperature may not produce the expected extent of infarction. Inject Rose Bengal (2 ml/kg) through the tail vein.</li>
	<li>Turn on the 532 nm green laser for 90 sec 1 min after the Rose Bengal injection. After irradiation, gently remove the ONI from the brain. After cleaning the operative site, secure the wound secure the wound with 3-0 nylon suture; release the rat from the stereotactic frame and transfer it to a recovery chamber.</li>
	<li>For sham-operated group (SOG), perform an identical lesion-making procedure, except for injection of saline (0.2 ml/100 g) instead of Rose-Bengal.</li>
	<li>Maintain the body temperature (37 &#176;C) with heating pad after surgery and administer antibiotics (second generation cephalosporin, 0.1%, 1 ml) via an intramuscular injection. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return post-surgical animals to the cage occupied by other animals until fully recovered. 
	NOTE: Preliminary experiment was performed in the same procedures to find the optimal light intensity from 1 mW to 5 mW and the procedure may be required to acquire the satisfactory extent of lesion in different condition.</li>
</ol>
3. Evaluation of Capsular Infarct Lesioning
<ol>
	<li>Behavioral Testing and Animal Grouping
	<ol>
		<li>Perform single pellet reaching tasks as described by Whishaw et al.14 to evaluate the motor deficit of the forelimb every day for 1 week after the stroke modeling. Perform a single pellet reaching task (SPRT) in &#160;food-restricted animals (90% of control body weight)&#160;using clear Plexiglas (30 x 15 x 35 cm height) with a 1 cm wide slit and a food shelf in the front of the middle of the front wall.</li>
		<li>Place a pellet on the food shelf obliquely contralateral to the preferred forelimb. Administer 20 pellets per session for 3 weeks. 
		NOTE: A successful number of SPRTs is defined as a reach in which the animal grasps a food pellet and puts it into the mouth without dropping it.</li>
		<li>Calculate the score as a percentage of successful reaches, which is defined by the following formula: 
		<img alt="Equation1" src="/files/ftp_upload/53281/53281equation1.jpg" /> 
		NOTE: We divide animals into 3 groups: the sham-operated group (SOG), moderate recovery group (MRG), and poor recovery group (PRG). If a post-stroke SPRT score &#62;50%, we classify the rats as the MRG, which indicates the presence of a substantial lesion, but not complete destruction of the target. If the post-stroke SPRT score is &#60;50% compared with the pre-stroke SPRT score, we classify the group as PRG, which indicates complete lesioning in the target.</li>
	</ol>
	</li>
	<li>Neurohistological Confirmation of Infarct Lesioning
	<ol>
		<li>Perform cardiac perfusion with 4% paraformaldehyde as previously described. After the brain completely sinks in the 30% sucrose solution, perform coronal sectioning at a thickness of 10 &#181;m and an interval of 200 &#181;m using a microtome or cryotome4.</li>
		<li>Stain with H&#38;E, Nissl, Luxol fast blue-PAS, Neurofilament protein-L or Glial fibrillary acid protein staining and observe the histological findings to determine the optimal light intensity that can cover the entire breadth of the internal capsule in the target area&#160;to observe staining4,17.</li>
		<li>Using ImageJ software, measure the volume of the photothrombotic infarct area of the internal capsule on the brain slides.
		<ol>
			<li>To measure the volume of infarct area, launch the &#39;ImageJ&#39; software. To open the files to be stacked, select &#39;Image to Stacks&#39; (&#39;Image&#39; &#8594; &#39;Stacks&#39; &#8594; &#39;Image to Stacks&#39;). Edit file name and select &#39;Set the Scale&#39; (&#39;Analyze&#39;&#160;&#8594; &#39;Set the Scale&#39;) to edit scale.</li>
			<li>In &#39;Plugins&#39;, select &#39;Measure Stacks&#39; to calculate volume or area of images. Insert the distance interval of 2 images into &#39;Slice Spacing&#39;. Make a drawing of the ROI (Region Of Interest) of all images and click &#39;Measure&#39;. 
			NOTE: The software &#39;ImageJ&#39; automatically calculates the area and volume of each image and total volume of them.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The capsular infarct model presented here demonstrates a targeted lesion with marked and persistent motor impairment in forelimb function. Previous models of subcortical capsular stroke have demonstrated an insufficient degree of motor impairment and a rapid recovery process6,8,9 . In this sense, this model resembles the clinical capsular infarct cases which exhibit long-term functional impairment.
The most critical steps in the development of a circumscribed capsular infarct model ...

Until recently, the &#34;gray matter stroke (GMS) models&#34; have been exclusively used to understand the pathophysiology of stroke and to guide the development of new treatments. However, there has been an increasing prevalence of stroke that affects subcortical white matter in elderly individuals, which constitutes 15 - 25% of all strokes1,2. Numerous studies have been conducted regarding stroke using GMS models, whereas there are few studies that have used white matter stroke (WMS) models. White matter in rodents is substantially less than the white matter in humans or primates. Consequently, it is more difficult to selectively access and destroy the target regions in the white matter3. Additionally, no efficient tools have been developed to date to selectively destroy the planned extent of targeted white matter. Therefore, there has been lack of appropriate models for study of white matter strokes.
Animal stroke models are often used to monitor the progress of motor recovery for development of new rehabilitative and therapeutic methods. It is ideal to utilize an animal model that exhibits a long-term neurological deficit concordant with the anatomical alterations demonstrated in human stroke4,5. In this regard, rapid recovery of the motor deficit and wide involvement of the brain following infarct lesioning may not be realistic in the pursuit of stroke research. Previous capsular infarct models have been made by the occlusion of the internal carotid or anterior choroidal arteries and diffusion of endothelin-1 (ET-1) into the internal capsule6-9. Nonetheless, artery occlusion requires careful dissection of arteries, but it produces a wide area of infarct lesion, including the internal capsule, without persistent behavioral deficits. Moreover, ET-1 was not diffused to completely destroy the posterior limb of the internal capsule, and hence less marked or persisting behavioral deficit.
A photothrombotic infarction model has been widely used to generate various types of infarct lesions in the cortex and subcortical structures10. The technique include intravenous administration followed by focal illumination, which leads to platelet aggregation in the small vessels and generation of infarct lesions10. Photothrombotic technique has been extensively used to create GMS lesions, whereas it has rarely been used to generate WMS lesions5,11. For this technique, a combination of Rose Bengal dye and light irradiation has been demonstrated to be useful in destruction of the target structure, causing corresponding functional deficits. The key element of the photothrombotic technique is light irradiation, because it determines the size of infarct lesions. Light irradiation results in different effects on gray matter and white matter, because the scattering of light is more than 4 times higher in white matter compared with gray matter12; Accordingly, if the light intensity has a sufficiently low irradiance (&#60;1,140 mW/mm2), one can limit the extension to which photothrombotic lesion affect the extent to the white matter (i.e., internal capsule). For example, light of higher energy can induce infarcts in both gray and white matter, yet lower energy light may induce photothrombosis only in white matter. Furthermore, the penetration of light energy was very limited. Approximately 99% of light energy was lost beyond 1 mm from the source of light13. Therefore, it is expected that accurately targeted, lower energy light induces photothrombosis only in the white matter with a minimal encroachment of the neighboring gray matter.
Here, we describe a novel method to create infarct lesions in the forelimb area of the internal capsule in rodents. We describe the method of identification of the forelimb area in the internal capsule, the technology of light irradiation, including the adjustment and delivery of light, and the generation of an infarct lesion. We also describe behavioral testing used to evaluate the completeness of the capsular modeling.

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circumscribed capsular infarct modeling using a photothrombotic technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

&#160; The method presented here is intended to create a circumscribed capsular infarct with a persistent motor deficit. Therefore, it is critical to correctly determine the target within the internal capsule in the pre-surgery step. The somatotopic mapping of pyramidal fibers in the internal capsule has not been settled to date. To correctly identify the target within the internal capsule, the forelimb area must be delineated. An injection of AAV-GFP into the forelimb area of the motor c...

Watch this Scientific Journal Video about Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique at JoVE.com

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

circumscribed-capsular-infarct-modeling-using-photothrombotic

Research

JoVE Journal

Medicine

7.8K Views.  Gwangju Institute of Science and Technology. This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Video: Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

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 ...

Neuroscience

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes and are defined as the sudden blockage of a vessel supplying blood to the brain. Within minutes of the blockage, cells begin to die and result in irreparable tissue damage. Current therapeutic treatments focus on clot removal or lysis to allow for the reperfusion and prevent more severe brain damage. Although transient brain plasticity may salvage some of the damaged tissue over time, significant fractions of patients are left with neurological deficits that will never resolve. There is a lack of therapeutic options to treat neurological deficits caused by stroke, emphasizing the need to develop new strategies to treat this growing patient population. Injectable biomaterials are currently being designed to enhance brain plasticity and improve endogenous repair through the delivery of active agents or stem cells. One method to test these approaches is to utilize a rodent stroke model, inject the biomaterial into the stroke core, and assess repair. Knowing the precise location of the stroke core is imperative for the accurate treatment after stroke, therefore, a stroke model that results in a predictable stroke location is preferable to avoid the need for imaging prior to injection. The following protocol will cover how to induce a photothrombotic stroke, how to inject a hydrogel in a controlled and precise manner, and how to extract and cryosection the brain while keeping the biomaterial intact. In addition, we will highlight how these same hydrogel materials can be used for the co-delivery of stem cells. This protocol can be generalized to the use of other injectable biomaterials into the stroke core.

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes ...

Biology

Modified MCAO/R Model with Preserved ECA for Enhanced Reperfusion and Stability

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.

Author Spotlight: A Novel Approach to Cerebral Ischemia Modeling &#8211; Enhancing Reperfusion and Simplifying Procedure

This protocol describes the process of preparing for middle cerebral artery occlusion reperfusion via the common carotid artery.

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Summary

Explore More Videos

Chapters in this video

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion