We thank all our collaboration partners of the Immunostroke Consortia (FOR 2879, From immune cells to stroke recovery) for suggestions and discussions. This work was funded by the Deutsche Forschungsgemeinschaft (DFG,German Research Foundation) under Germany&#39;s Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy - ID 390857198) and under the grants LI-2534/6-1, LI-2534/7-1 and LL-112/1-1.

The experiments reported in this video were conducted according to the national guidelines for the use of experimental animals, and the protocols were approved by the German governmental committees (Regierung von Oberbayern, Munich, Germany). The mice used in this study were male C57Bl/6J mice, 10-12 weeks old, and dispatched by Charles River Germany. The animals were housed under controlled temperatures (22 &#176;C &#177; 2 &#176;C), with a 12 h light-dark cycle period and access to pelleted food and water ad libitum.
1. Preparation of the material and instruments
<ol>
	<li>Dissolve Rose Bengal in 0.9% saline solution to reach a final concentration of 10 mg/mL. Connect the heat blanket to keep the operation area warm and maintain the mouse body temperature during anesthesia at 37 &#176;C.</li>
	<li>Prepare scissors, forceps, pieces of cotton, dexpanthenol eye ointment, and suture material. Prepare a syringe with saline solution (without needle) to maintain the operation area hydrated. Prepare the anesthesia gas (100% O2 + isoflurane).</li>
</ol>
2. Preparation of the animal
<ol>
	<li>Inject analgesia 30 min before surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).</li>
	<li>Record the mouse body weight to adjust the dose of Rose Bengal to be injected (10 &#181;L/g i.e., 100 &#181;g/g).</li>
	<li>Place the mouse in the induction chamber with an isoflurane flow rate of 4% to anesthetize it until the spontaneous movement of the body and vibrissae stops.</li>
	<li>Transfer the mouse into the stereotactic frame and place it in a prone position with its nose into the anesthesia mask. Fix the animal and maintain the isoflurane concentration at 4% for 1 min. Then reduce and maintain the isoflurane concentration at 2%.</li>
	<li>Gently insert the rectal probe to monitor the temperature throughout the surgical procedures. Set the associated feedback-controlled heating pad to maintain the mouse body temperature at 37 &#176;C.</li>
	<li>Apply dexpanthenol eye ointment to both eyes and clean the skin and surrounding fur with a disinfectant agent.</li>
</ol>
3. Photothrombosis model
<ol>
	<li>Make a 2.0-2.5 cm longitudinal incision and retract to expose the skull. Perform the skull exposure with a single cut to avoid wound complications.</li>
	<li>Remove the periosteum gently with cotton and identify the coronal sutures.</li>
	<li>Put on the protective glasses, switch on the 561 nm laser and mark the bregma +3 mm left.</li>
	<li>Switch off the laser, attach a sticker with a 4 mm diameter hole placed at the marked coordinates mentioned above.</li>
	<li>Inject the mouse with Bengal Rose (10 &#181;L/g), intraperitoneally. Place the laser beam at 4-5 cm from the skull, switch on the 561 nm laser and illuminate the skull for 20 min.</li>
	<li>Apply two drops of 0.9% saline on&#160;the skull to rehydrate, suture the wound, and place the animal in a recovery chamber at 37 &#176;C to recover from anesthesia. After 1 h, return the mice to their cages in a temperature-controlled room.</li>
	<li>Inject analgesia every 12 h for 3 days after surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).</li>
</ol>
4. Sham operation
<ol>
	<li>Carry out two different procedures of Sham operations as described in steps 4.1.1 and 4.1.2.
	<ol>
		<li>Perform all the procedures identically to the operation described above. Inject Rose Bengal without switching on the laser. After 20 min under anesthesia, allow the animals to stay in the recovery chamber for 1 h to recover, before being returned to their cages.</li>
		<li>Perform all the procedures identically to the operation described above, switching on the laser. Do not inject Rose Bengal. After 20 min of laser illumination, allow the animals to stay in the recovery chamber for 1 h to recover from anesthesia, before being returned to their cages.</li>
	</ol>
	</li>
</ol>
5. Laser speckle
<ol>
	<li>Connect the heated blanket to keep the operation area warm and maintain the mouse body temperature during anesthesia at 37 &#176;C.</li>
	<li>Place the mouse into the induction chamber with an isoflurane flow rate of 4% to anesthetize it until the spontaneous movement of the body and vibrissae stops and then transfer the mouse into the stereotactic frame.</li>
	<li>Place the mouse in a prone position with its nose into the anesthesia mask. Fix the animal and maintain the isoflurane concentration at 4% for 1 min. T hen reduce and maintain it at 2%.</li>
	<li>Gently insert the rectal probe to monitor the temperature throughout the surgical procedures. Set the associated feedback-controlled heating pad to maintain the mouse body temperature at 37 &#176;C and apply dexpanthenol eye ointment to both eyes. Clean the skin and the surrounding fur with a disinfectant agent.</li>
	<li>Make a 2.0-2.5 cm longitudinal incision and retract to expose the skull. Perform the skull exposure with a single cut to avoid wound complications.</li>
	<li>Place the sterotactic frame under the laser speckle and adjust the height to obtain a sharp image. Focus the laser speckle perfusion imaging (LSI) camera on the cranial window. Configure the high resolution laser speckle imaging (LSI) camera system as previously described15.</li>
	<li>Acquire data from a 1 cm x 1 cm field of view using a 785 nm wavelength and 80 mW lasers with a frame rate of 21 images/s at a working distance of 1 cm for 1 min.</li>
	<li>After imaging, apply two drops of 0.9% saline to the skull to rehydrate, suture the wound and place the animal in a recovery chamber at 37 &#176;C to recover from anesthesia for 1 h. After 1 h, return the mice to their cages in a temperature-controlled room.</li>
</ol>
6. Neuroscore
NOTE: For the neurological deficit analysis, a modified neurological scale published by Eckenstein et al. in 1997 is used15.
<ol>
	<li>Score the animals for general (Table 1) and focal deficits (Table 2). This composite scale ranges from 0 (no deficits) to 46 (severe impairments).</li>
	<li>Perform the neuroscore at the same time each day and use surgical clothes to keep a neutral smell.</li>
	<li>Habituate the mice for 30 min in the room with an open cage prior to the testing and allow them to observe each item for 30 s.</li>
</ol>
7. Perfusion
<ol>
	<li>Prepare a 20 mL syringe containing PBS-heparin (2 U/mL) and place it 1 m above the bench to facilitate/ensure gravity-driven perfusion.</li>
	<li>Inject intraperitoneally 100 &#181;L of ketamine and xylazine (120/16 mg/kg body weight, respectively). Wait for 5 min and corroborate the cessation of spontaneous body movement and vibrissae.</li>
	<li>Fix the animal in a supine position and disinfect the abdominal body surface with 100% ethanol. Make a 3 cm long incision in the abdomen; cut the diaphragm and the ribs to completely visualize the heart.</li>
	<li>Make a small incision in the right atrium and insert the perfusion cannula into the left ventricle and perfuse with 20 mL PBS-heparin.</li>
	<li>After perfusion, decapitate the animal and remove the brain, freeze it using dry ice and store them at -80 &#176;C until further use.</li>
</ol>
8. Infarct volumetry
<ol>
	<li>Cryosectioning: Cut the brain serially on a cryostat to 20 &#181;m thick sections every 120 &#181;m and mount on slides. Store the slides at -80 &#176;C until further processing.</li>
	<li>Cresyl violet (CV) staining
	<ol>
		<li>To prepare the staining solution, mix 0.5 g of CV acetate in 500 mL of H2O. Stir and heat (60 &#176;C) until the crystals are dissolved. Allow the solution to cool and store it in a dark bottle. Reheat to 60 &#176;C and filter (paper filter) before every use.</li>
		<li>Dry the slides at room temperature for 30 min. Place them in 95% ethanol for 15 min, followed by 70% ethanol for 1 min, and afterwards in 50% ethanol for 1 min.</li>
		<li>Place the slides in distilled water for 2 min, refresh the distilled water, and place the slides in water again for 1 min. Then, place the slides in the pre-heated staining solution for 10 min at 60 &#176;C. Wash the slides twice in distilled water for 1 min.</li>
		<li>Place the slides in 95% ethanol for 2 min. Then place them into 100% ethanol for 5 min, refresh the 100% ethanol and place the slides in 100% ethanol again for 2 min. Afterwards, cover the slides with a mounting medium.</li>
		<li>Analysis: Scan the slides and analyze the indirect infarct volume by the Swanson method16 to correct for edema: Ischemic area = (ischemic region)-((ipsilateral hemisphere) - (contralateral hemisphere)).</li>
	</ol>
	</li>
</ol>
9. Tunel staining (in situ apoptosis detection kit)
<ol>
	<li>Dry the slides, post-fix in 4% paraformaldehyde in PBS (ph 7.4) for 10-20 min at RT. Wash in PBS, post-fix in precooled ethanol: acetic acid 2:1 for 5 min at -20 &#176;C.</li>
	<li>Wash in PBS and apply equilibration buffer (10 s to a maximum of 60 min at RT) and apply working strength TdT enzyme (1 h at 37 &#176;C in humidified chamber)</li>
	<li>Apply working strength stop/wash enzyme (10 min at RT), wash in PBS and apply warmed (RT) working strength anti-digoxigenin conjugate (30 min at RT in dark)</li>
	<li>Wash in PBS, incubate with DAPI for 5 min at RT and mount the slides with fluoromount media.</li>
</ol>

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The authors have no competing interests to disclose.

The presented protocol describes the experimental stroke model of photothrombosis by illuminating the intact skull with a 561 nm laser, with a previous intraperitoneal injection of Rose Bengal. Until recently, the use of this model has been low but is steadily increasing.
Mortality during stroke induction in this model is absent. The overall mortality of less than 5% arises during operation due to anesthesiological complications or sacrifice after meeting the exclusion criteria. To warrant the low variability of this model and its reproducibility, the following exclusion criteria are suggested: 1) operation time longer than 30 min; 2) infection of the suture; 3) bite wound; and 4) no infarct or no fore asymmetry at 24 h after PT.
A widely used experimental stroke models is the transient occlusion of the MCA, by using a suture filament, which is introduced in the internal carotid artery until the silicon-coated tip occludes the origin of the MCA. This model allows the reperfusion by removing the filament and mimics the human clinical scenario, in which there is a restoration of the cerebral blood flow after spontaneous or therapeutic (rtPA) lysis of an embolic clot18,19. However, it involves a complex surgery with high variability of the final infarct and high mortality rate10. In contrast, the permanent occlusion of the MCA distal of the lenticulostriatal arteries can be achieved by coagulation of the artery20,21, which induces locally defined lesions in the neocortex22. Although this model has a lower mortality rate, it requires invasive surgery to the animal by trepanation of the skull over the MCA to later coagulate it23. Consequently, high surgical skills are required for a successful and unbiased in vivo stroke study.
Compared to other brain ischemia models, the photothrombotic model as carried out in this video has the advantage of no craniotomy or major surgery on the animal, unlike other models that involve complex surgeries or brain craniotomy. Moreover, the simple execution of the model makes the surgery accessible to many with low time-consuming training. Low mortality, moderate infarct volume, and flexibility to induce the lesion to a specific brain region, emphasize the advantage of this experimental paradigm for brain regeneration and stroke studies24,25,26,27.
Despite the obvious advantages, a few limitations of this stroke model should be taken into consideration. The long exposure of anesthetics to the animal might be a critical factor to take into account, as the impact of anesthetics on neuroprotection and stroke outcome is already well-known28. Although the duration of this surgical procedure takes approximately 30 min, the animal can be under low anesthetic concentrations due to the minimal manipulation of the animal during the 20 min of laser illumination. Because this model induces moderate brain injuries, only minor behavioral deficits are detectable. Thus, more advanced test systems with higher sensitivity and qualitative test parameters, such as the skilled reaching test29 and Neuroscore17, as described in here, maybe more suitable for detecting long-term functional outcomes in this model. Finally, due to the permanent aggregation of the platelets into the illuminated blood vessels, no reperfusion can be obtained, which is a feature observed in a substantial percentage of stroke patients due to spontaneous clot lysis or therapy30.
A similar phototrombotic stroke model was published in 2013 by Labat-gest and Tomasi, describing a PT protocol using a cold light lamp instead of a 561 nm green laser8. Both laser and cold light sources can be used to induce Rose Bengal excitation. An advantage of laser-based light sources over cold light lamps is that lasers can be used to target individual surface arterioles for in vivo vessel-specific clotting31. Although we were not targeting specific arterioles, we used a 561 nm green laser for brain illuminationn and phototrombosis induction, because of the Rose Bengal absortion peak at 562 nm. To ensure a proper laser intensity during the illumination, the Cobolt Monitor Software-6.1.0.0 was used to calibrate the laser. Moreover, in the present study a Rose Bengal dosage of 10 &#181;L/g (100 &#181;g/g) was sufficient to induce phototrombosis, while the previous protocol reported a higher dose (150 &#181;g/g)8. In addition, the protocol provides a behavioral method to analyze the stroke outcome (Neuroscore) and an additional sham-control group (laser illumination) in order to prove that the laser itself does not produce any tissue damage, so only the combination of Rose Bengal + laser illumination induce a brain lesion.
Overall, this non-invasive straightforward surgical procedure enables high reproducibility and directionality of the stroke lesion to the brain alongside the possibility of long-term observation due to minimal mortality. This photothrombotic stroke model is distinguished as a valuable experimental paradigm for basic and translational stroke research.

Ischemic stroke remains a principal cause of death and acquired adult disability in developed countries in the 21st century accounting for approximately 2.7 million deaths in 2017 worldwide1. Even with the immense efforts of the scientific community, few treatments are available. Furthermore, with such high exclusion criteria, these already limited options are not accessible to many patients, resulting in an urgent need for novel treatments to improve functional recovery after stroke.
Considering the incapability of in vitro models to replicate the complex interactions of the brain, animal models are essential for preclinical stroke research. Mice are the most frequently used animal model in the stroke research field. The majority of these mouse models aim to induce infarctions by blocking the blood flow within the middle cerebral artery (MCA) since the majority of human stroke lesions are located in the MCA territory2. Although these models better recapitulate human stroke lesions, they involve convulated surgeries with high infarct volume variability.
Since Rosenblum and El-Sabban&#39;s proposal of the photothrombotic model in 19773, and later the application of this model to rats Watson et al.4, it has become widely used in ischemic stroke research5,6. The photothrombotic stroke model induces a local and defined cortical infarct as a result of the photoactivation of a light-sensitive dye previously injected into the blood flow. This causes local thrombosis of the vessels in the areas exposed to light. Briefly, upon exposure to light from the injected photosensitive dye, localized oxidative injury of the endothelial cell membrane is induced, leading to platelet aggregation and thrombus formation, followed by local disruption of cerebral blood flow7.
The principal advantage of this technique resides in its simplicity of execution and the possibility to direct the lesion to the desired region. Unlike other experimental stroke models, minor surgical expertise is needed to perform the photothrombotic stroke model as the lesion is induced through illumination of the intact skull. Moreover, the well-delimited borders (Figure 2A and Figure 5B) and the flexibility to induce the lesion to a specific brain region can facilitate the study of cellular responses within the ischemic or intact cortical area8. For these reasons, this approach is suitable for the study of cellular and molecular mechanisms of cortical plasticity.
Over the past few decades, the growing concern regarding the lack of reproducibility between research groups has been coined the so-called replication crisis9. After the coordination of the first preclinical randomized controlled multicenter trial study in 201510, a proposed tool to improve preclinical research11,12,13, it was confirmed that one cause for failing reproducibility between preclinical studies from independent laboratories was the lack of sufficient standardization of experimental stroke models and outcome parameters14. Accordingly, when the ImmunoStroke consortium was established (https://immunostroke.de/), a collaboration which aims to understand brain-immune interactions underlying the mechanistic principles of stroke recovery, the standardization of all the experimental stroke models among each research group was essential.
Described here is the standardized procedure for the induction of the photothrombotic model as used in the above-mentioned research consortium. Briefly, an animal underwent anesthetics, received a Rose Bengal injection (10 &#181;L/g) intraperitonally, and the intact skull, 3 mm left from bregma, was immediately illuminated by a 561 nm laser for 20 min (Figure 1). Additionally, a related histological and behavioral method to analyze the stroke outcome in this model is reported. All methods are based on standard operating procedures developed and used in the laboratory.

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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.

The model described here is a photothrombotic stroke model by Rose Bengal injection and intact skull illumination for 20 min, at a constant 561 nm wavelength and 25 mW output power at the fiber. Although the complete photothrombotic surgery lasts 30 min, the animal is kept under low anesthesia and the brain damage is moderate. Approximately 10 min after transfer to their cages, all the animals were awake, freely moving in the cage, and interacting with littermates.
Infarct volumetry was performed using cresyl violet stained serial coronal brain sections 24 h after stroke induction (Figure 2A). The mean infarct volume was 29.3 mm3, representing 23% of one brain hemisphere. Moreover, the variability of this stroke model is exceptionally low with a standard deviation of approximately 3.5% (Figure 2B). The lesion area encompasses the motor cortex without the affection of subcortical structures.
Photothrombosis caused a moderate, long-term sensorimotor impairment, indicated by the composite Neuroscore17 (Figure 3); general and focal deficits were measured 24 h, 3 days and 7 days after surgery. The general Neuroscore has five items, including the evaluation of the fur, ears, eyes, posture, and spontaneous activity, with a maximum score of 18 (Table 1). The focal Neuroscore comprises seven items, including the evaluation of body symmetry, gait, climbing, circling behavior, forelimb symmetry, compulsory cycling, and whiskers response, with a maximum score of 28 (Table 2). Stroke animals had a significant change in the composite neuroscore 24 h after surgery compared to Sham-operated animals. These differences persisted, although stroke mice improved over time (Figure 3).
Mortality during the observation time rarely occurs in 1-2% of the animals. In this report, none of the 10 animals studied had to be excluded and all of them survived the 7-day observation period. The body weight and temperature changes in the mice were monitored at 24 h, 3 days, and 7 days after surgery (Figure 4A,B). Data showed that body weight and temperature were decreased 24 h after surgery only in the Rose Bengal + illumination group, but recovered to the level of the Sham-operated animals in 3 days after surgery.
To confirm an induction of ischemic changes, 24 h after surgery, the animals underwent a laser imaging test. A laser speckle contrast imaging measured blood perfusion of the cortex for a duration of 1 min and an averaged color-coded picture was obtained for each animal. This demonstrates&#160;that Rose Bengal or laser illumination alone does not produce a lesion, while simultaneous application of Rose Bengal and laser illumination generates a round hypoperfused area of 4 mm diameter surrounded by a narrow oligemic zone (Figure 5A). In addition, a cresyl violet and Tunel staining for assessment of the infarct volume 24 h after surgery revealed no tissue damage either in Rose Bengal or laser illumination surgeries. On the other hand, Rose Bengal + laser illumination generated a well-demarcated lesion (Figure 5B).
Table 1: General Neuroscore. For each of the five general deficits measured, animals can receive between 0 and 4 points depending on the severity. The scores on the five areas are then summed to provide a total general score ranging from 0-18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/Table 1-62536R1.pdf" target="_blank">Please click here to download this Table.</a>
Table 2: Focal Neuroscore. For each of the seven general deficits measured, animals can receive between 0 and 4 points depending on the severity. The scores on the five areas are then summed to provide a total general score ranging from 0-28. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/Table 2- 62536R1.pdf" target="_blank">Please click here to download this Table.</a>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62536/62536fig01.jpg" /> 
Figure 1: Photothrombosis (PT). Diagram depicting the photothrombotic area, 3 mm from Bregma. The green dot indicates the position of the laser. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/62536fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62536/62536fig02.jpg" /> 
Figure 2: Volumetric infarct analysis and infarct outcome 24 h after PT. (A) Representative cresyl violet stained coronal brain, sections every 120 &#181;m at 24 h after PT. Dashed lines demarcate the lesion area. (B) Infarct volume analysis of 10 brains (each dot representing one individual brain) 24 h after PT. The horizontal red line represents the mean (29.32 mm3), error bars indicate standard deviation (3.45 mm3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/62536fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62536/62536fig03.jpg" /> 
Figure 3: Neuroscore for functional deficits after PT. Composite Neuroscore before, 24 h, 3 days, and 7 days after PT. BL = before PT, RB = Rose Bengal. n = 5 per group. *p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/62536fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62536/62536fig04.jpg" /> 
Figure 4: Body weight and temperature analysis after PT. (A) Body weight and (B) temperature was slightly reduced in PT animals compared to Sham-operated groups at 24 h and recovered 3 days after PT. BL = before PT, RB = Rose Bengal. n = 5 per group.*p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/62536fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62536/62536fig05.jpg" /> 
Figure 5: Lesion confirmation after PT. (A) Laser Speckle imaging (B) Cresyl violet (upper panels) and Tunel staining (lower panels) confirmed the lesion only after administration of Rose Bengal and subsequent laser illumination. RB = Rose Bengal. Scale bar = 1,000 &#181;m in upper panel B, scale bar = 20 &#181;m in lower panel B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/62536fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis at JoVE.com

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

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, ...

modeling-stroke-in-mice-focal-cortical-lesions-by-photothrombosis

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.0K Views.  LMU Munich. 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.

Video: Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

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.

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.

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 ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed.
Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...

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

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

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

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