This study was supported by grant Scientific Research Foundation for the High-level Talents, Fujian University of Traditional Chinese Medicine (X2019002-talents).

Procedures involving animal subjects have been approved by the Medical Norms and Ethics Committee of Wenzhou Medical University and are in accordance with the China legislation on the use and care of laboratory animals.
1. Preparation of the thread plug, SD rats, and experimental equipment
<ol>
	<li>Use a nylon thread with a diameter of 0.28 mm as the main body of the thread plug. 
	NOTE: The softness and hardness of the nylon thread should be moderate.</li>
	<li>Cover one end of the nylon thread with silicone material. The length of the silicone part of the thread plug is approximately 1.2 cm, and the diameter is approximately 1.2 mm. The head end is tapered, and the silicone part is cylindrical. Reserve another 5-7 cm. The nylon thread is easy to clamp and can be cut according to specific needs after the operation.</li>
	<li>Use 75% ethanol to soak the thread plug for 3 min before the operation and rinse any residual ethanol with normal saline before plugging.</li>
	<li>Select 12 male SD rats weighing between 280 and 320 g, and randomly divide them into a sham group and experimental group (n = 6 per group). After a week of environmental adaptation, fast the rats for 12 h and water-deprive them for 4 h before the operation.</li>
	<li>Prepare the following experimental equipment needed for the experiment: a small animal anesthesia machine, a brain stereotaxic instrument, a dissecting microscope, a high-speed skull drill, scissors, tweezers, vascular forceps, a needle holder, a needle thread, a 2 mL syringe, a laser-speckle blood-flow imaging system, and a small-animal MRI scanner.</li>
</ol>
2. Construction of SD-Rat SSS-Embolization Model via Thread Embolization
<ol>
	<li>Place the SD rat into an anesthesia-induction box and use a small-animal anesthetic machine to deliver 4% isoflurane to induce anesthesia. Thereafter, use forceps to confirm that the SD rat&#39;s hind limbs and toes do not respond to moderate pinching.</li>
	<li>Quickly fix the SD ratwith shaved top hair in the prone position on a brain stereotaxic device. Maintain anesthesia with 1.5-2.0% isoflurane (at a speed of 0.5 L/min), and stabilize the respiratory rate at 40-60 breaths/min. Stabilize the rat&#39;s body temperature via a heating pad at 37&#177;0.2 &#176;C.
	<ol>
		<li>Apply sterile ophthalmic eye lubricant after the rat is placed on the stereotaxic frame to protect the corneas from drying during anesthesia.</li>
	</ol>
	</li>
	<li>Sterilize the surface on the top of the rat&#39;s head with 5% povidone iodine alternating three times with 75% ethanol. Make a skin incision (2.0-cm long) in the middle of the head, and then carefully peel off the top fascia and periosteum to fully expose the skull.
	<ol>
		<li>Confirm the positions of the anterior fontanelle, posterior fontanelle, coronal suture, sagittal suture, and herringbone suture.</li>
	</ol>
	</li>
	<li>Use the area between the coronal suture and the herringbone suture as the blood-flow observational area. To prevent the skull from affecting observation during laser-speckle blood-flow imaging, thin the skull in the observation area until the blood vessels are clearly visible. The size of the thinned skull should be approximately 1.0 cm &#215; 1.0 cm. This step and the following are all performed under a dissecting microscope.
	<ol>
		<li>During skull grinding, use normal-temperature saline to rinse the drill repeatedly to avoid high-temperature burns to the cerebral cortex.</li>
	</ol>
	</li>
	<li>Use a high-speed drill to grind the skull within a 6.0 mm x 4.0 mm bone window centered at bregma to expose the SSS of the bregma area.
	<ol>
		<li>Use normal saline to cool the skull during grinding. When the skull becomes thin, use tweezers to carefully remove the remaining bone pieces to avoid tearing the SSS.</li>
	</ol>
	</li>
	<li>Choose a suitable thread plug, use the SSS bregma point as the plug point, carefully puncture it with a 2 mL syringe needle, and quickly insert the thread plug head into the plug point.
	<ol>
		<li>At this time, the angle between the end of the thread-plug head and the SSS should be approximately 30-45&#176;; then adjust the angle between the end of the thread plug and the SSS to 0-10 degrees, and slowly insert the SSS into the center until the head reaches the posterior edge of the sinus confluence. Thereafter, cut off the excess part of the tail. 
		&#8203;NOTE: Rapid bleeding may occur when the SSS is punctured. If the end of the thread plug cannot be quickly inserted into the plug point at one time, use a small gauze or cotton ball to gently press the plug point while slowly sliding down to expose the plug point carefully, and then quickly insert the end of the wire bolt into the SSS. After the thread plug is inserted, if there is bleeding at the plug point, hemostatic materials such as a gelatin sponge can be used to stop the bleeding.</li>
	</ol>
	</li>
</ol>
3. Detection of Blood Flow on the Brain Surface of SD Rats
<ol>
	<li>Use a laser light source to uniformly illuminate the blood-flow observational area. The reflected light is collected by a camera and is transmitted to a computer for analysis. Use the following parameter settings for the laser-speckle blood-flow imaging system: wavelength: &#955; = 785 nm; and image exposure time: T = 10 ms.</li>
	<li>Center the SD-rat blood-flow observational area into the field of view of the laser-speckle blood-flow imaging system and conduct continuous monitoring of blood flow on the brain surface for 2 min. Collect and process the blood flow data before and after embolization for each SD rat, and obtain the laser-speckle blood-flow map of the observed area.</li>
	<li>Repeatedly rinse the operated area with normal saline to wash away bone debris and residue. Suture the skin (0# thread) and disinfect with iodophor.</li>
	<li>Maintain body temperature until the rat awakens after surgery and then house in a single cage with food and water provided ad libitum. The sham group cannot be plugged.</li>
	<li>After data collection is completed, perform post-processing.
	<ol>
		<li>Complete selection of the region of interest (ROI) via the tools provided by the laser-speckle blood-flow imaging system software. The values obtained are the average blood-flow value in the ROI, and the local cerebral-blood-flow values before and after embolization. Use the blood-flow value before embolization as the baseline value.</li>
		<li>Select four ROIs and measure the relative change in cerebral blood flow in each ROI, expressed as the percent change from the baseline value.</li>
	</ol>
	</li>
</ol>
4. Detection of thread position on small animals MRI
<ol>
	<li>Use the following T2-weighted imaging (T2WI) parameters for MRI imaging system: time of echo (TE) = 33 ms, time of repeat (TR) = 3000 ms, number of excitation (NEX) = 4, Slices = 28, slice thickness = 0.8 mm, matrix size = 256*256 mm2, flip angle = 80&#176;, field of view (FOV) = 30*30 mm2, scan time = 6 min 24 s; magnetic resonance angiography (MRA) parameters were set as follows: TE = 4.4 ms, TR = 12 ms, NEX = 4, slices = 80, slice thickness = 0.4 mm, matrix size = 256*256 mm2, flip angle = 80&#176;, FOV = 30*30 mm2, scan time = 16 min 23 sec 40 ms.</li>
	<li>Fix the animal on the MRI scanning table, calibrate the brain position by positioning scanning, and perform T2WI and MRA sequence scanning after confirming the position.</li>
	<li>Use continuous anesthesia via the animal anesthesia machine during the detection. Then, euthanize the SD rats by intraperitoneal injection of excessive pentobarbital.</li>
	<li>Image acquisition and post-processing: after collecting the image data, in order to observe the state of the thread plug in the SSS more clearly, use the pseudo color enhancement method to display the T2WI image of the rat brain.</li>
</ol>

<ol>
	<li>Bousser, M. G., Ferro, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+venous+thrombosis:+an+update.">Cerebral venous thrombosis: an update.</a> Lancet Neurology. 6 (2), 162-170 (2007).</li><li>Guenther, G., Arauz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+venous+thrombosis:+A+diagnostic+and+treatment+update.">Cerebral venous thrombosis: A diagnostic and treatment update.</a> Neurologia. 26 (8), 488-498 (2011).</li><li>Stam, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombosis+of+the+cerebral+veins+and+sinuses.">Thrombosis of the cerebral veins and sinuses.</a> New England Journal of Medicine. 352 (17), 1791-1798 (2005).</li><li>Einh&#228;upl, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EFNS+guideline+on+the+treatment+of+cerebral+venous+and+sinus+thrombosis+in+adult+patients.">EFNS guideline on the treatment of cerebral venous and sinus thrombosis in adult patients.</a> European Journal of Neurology. 17 (10), 1229-1235 (2010).</li><li>Coutinho, J. M., Zuurbier, S. M., Stam, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Declining+mortality+in+cerebral+venous+thrombosis:+a+systematic+review.">Declining mortality in cerebral venous thrombosis: a systematic review.</a> Stroke. 45 (5), 1338-1341 (2014).</li><li>Gotoh, M., Ohmoto, T., Kuyama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+study+of+venous+circulatory+disturbance+by+dural+sinus+occlusion.">Experimental study of venous circulatory disturbance by dural sinus occlusion.</a> Acta Neurochir (Wien). 124 (2-4), 120-126 (1993).</li><li>Miyamoto, K., Heimann, A., Kempski, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculatory+alterations+in+a+mongolian+gerbil+sinus-vein+thrombosis+model.">Microcirculatory alterations in a mongolian gerbil sinus-vein thrombosis model.</a> Journal of Clinical Neuroscience. 8 (4), (2001).</li><li>Ungersb&#246;ck, K., Heimann, A., Kempski, a. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+Blood+Flow+Alterations+in+a+Rat+Model+of+Cerebral+Sinus+Thrombosis.">Cerebral Blood Flow Alterations in a Rat Model of Cerebral Sinus Thrombosis.</a> Stroke. 24 (4), (1993).</li><li>R&#246;ttger, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+reversible+sinus+sagittalis+superior+thrombosis+in+the+rat:+magnetic+resonance+imaging+changes.">A new model of reversible sinus sagittalis superior thrombosis in the rat: magnetic resonance imaging changes.</a> Neurosurgery. 57 (3), 573-580 (2005).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothrombosis+combined+with+thrombin+injection+establishes+a+rat+model+of+cerebral+venous+sinus+thrombosis.">Photothrombosis combined with thrombin injection establishes a rat model of cerebral venous sinus thrombosis.</a> Neuroscience. 306, 39-49 (2015).</li><li>Yang, H., Meng, Z., Zhang, C., Zhang, P., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+a+new+rat+model+of+central+venous+sinus+thrombosis+and+analyzing+its+pathophysiological+and+apoptotic+changes.">Establishing a new rat model of central venous sinus thrombosis and analyzing its pathophysiological and apoptotic changes.</a> Journal of Neuroscience Methods. 203 (1), 130-135 (2012).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</li><li>Fluri, F., Schuhmann, M. K., Kleinschnitz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ischemic+stroke+and+their+application+in+clinical+research.">Animal models of ischemic stroke and their application in clinical research.</a> Drug Design, Development and Therapy. 9, 3445-3454 (2015).</li><li>Wang, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+tissue+pH+in+an+experimental+model+of+acute+stroke+-+Determination+of+graded+regional+tissue+pH+changes+with+non-invasive+quantitative+amide+proton+transfer+MRI.">Mapping tissue pH in an experimental model of acute stroke - Determination of graded regional tissue pH changes with non-invasive quantitative amide proton transfer MRI.</a> Neuroimage. 191, (2019).</li><li>Liu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Vigilin+as+a+Potential+Ischemia+Biomarker+by+Brain+Slice-Based+Systematic+Evolution+of+Ligands+by+Exponential+Enrichment.">Identification of Vigilin as a Potential Ischemia Biomarker by Brain Slice-Based Systematic Evolution of Ligands by Exponential Enrichment.</a> Analytical Chemistry. 91 (10), 6675-6681 (2019).</li></ol>

The authors declare that they have no competing financial interests.

In this study, a new type of CVST model was successfully established by inserting a self-made thread plug into the SSS of SD rats. Additionally, laser-speckle blood-flow imaging and small-animal MRI were combined to monitor changes in blood flow on the brain surface of SD rats before and after the embolization in order to standardize ischemic timing and location.
In 1989, Longa et al. made a reversible MCA occlusion model by retrogradely inserting a self-made nylon suture into the external car...

Cerebral venous sinus thrombosis (CVST) is a rare disease of the cerebral venous system that accounts for only 0.5-1.0% of all causes of stroke but has a relatively high occurrence rate in children and young adults1. During autopsy, CVST was found to be the cause of 10% of cerebrovascular disease deaths2. Thrombosis can occur in any part of the intracranial venous system. The superior sagittal sinus (SSS) is one of the most commonly affected areas in CVST and can involve multiple blood vessels. Owing to stenosis or occlusion of the venous sinuses, intracranial venous return is blocked, which is often accompanied by increased intracranial pressure3. The clinical manifestations of CVST are complex and vary over time; although there is a lack of specificity of symptoms, the most common symptoms include headache (77.2%), seizures (42.7%), and neurological deficits (39.9%). In severe cases, coma and even death may occur4,5. In recent years, due to the overall improvement of medical and health standards and public health awareness, the proportion of related risk factors has changed, the proportion of trauma and infection has decreased, and the proportion of CVST caused by pregnancy, puerperium, oral contraceptives, and other reasons has gradually increased5.
At present, the pathogenesis of CVST is still not well understood. To explore CVST in depth, further pathophysiological research is needed. However, most of these research methods are invasive and therefore difficult to implement clinically. Owing to many limitations of clinical research, animal models have irreplaceable advantages in terms of basic and translational research.
The cause of CVST is complex, as its initial onset is often unrecognized and the location of thrombus formation is highly variable. Fortunately, animal models can achieve better control of these factors. In the past few decades, a variety of CVST animal models have been established, and each model has its own disadvantages. According to different production methods, they can be roughly divided into the following categories: the simple SSS-ligation model6,7; the SSS internal-injection-accelerator model8; the ferric-chloride-induced SSS thrombosis model9; the photochemical-induced SSS thrombosis model10; and the self-made embolism-occlusion SSS model11. However, most of these models are unable to circumvent invasive damage to the animal's cerebral cortex and are not able to accurately control the ischemic time and location. In some models, the thrombus will recanalize spontaneously; in other models, the SSS becomes permanently occluded. In addition, complicated operations and/or serious injuries may affect subsequent pathophysiological findings in these models.
In the present study, a thread plug was inserted into the SSS of Sprague-Dawley (SD) rats to successfully establish a CVST model that minimized damage, enabled precise controllability, and yielded good stability. Additionally, small-animal magnetic resonance imaging (MRI) and laser-speckle blood-flow imaging were combined to verify the model's effectiveness. We evaluated changes in cerebral blood flow before and after establishment of our model, as well as evaluated the stability of our model, laying a foundation for further studies exploring the occurrence, development, and related pathophysiological mechanisms of CVST.

<table><tbody><tr><td>2 mL syringe</td><td>Becton,Dickinson and Company</td><td>301940</td><td></td></tr><tr><td>brain stereotaxic instrument</td><td>Shenzhen RWD Life Technology Co., Ltd</td><td>68025</td><td></td></tr><tr><td>dissecting microscope</td><td>Wuhan SIM Opto-technology Co.</td><td>SIM BFI-HR PRO</td><td></td></tr><tr><td>high-speed skull drill</td><td>Shenzhen RWD Life Technology Co., Ltd</td><td>78046</td><td></td></tr><tr><td>laser-speckle blood-flow imaging system</td><td>Wuhan SIM Opto-technology Co.</td><td>SIM BFI-HR PRO</td><td></td></tr><tr><td>needle holder</td><td>Shenzhen RWD Life Technology Co., Ltd</td><td>F31022-12</td><td></td></tr><tr><td>needle thread</td><td>Shenzhen RWD Life Technology Co., Ltd</td><td>F33303-08</td><td></td></tr><tr><td>scissors</td><td>Shenzhen RWD Life Technology Co., Ltd</td><td>S13029-14</td><td></td></tr><tr><td>silica gel</td><td>Heraeus Kulzer</td><td>302785</td><td></td></tr><tr><td>small animal anesthesia machine</td><td>Shenzhen RWD Life Technology Co., Ltd</td><td>R540</td><td></td></tr><tr><td>small-animal MRI</td><td>Bruker Medical GmbH</td><td>Biospec 94/30 USR</td><td></td></tr><tr><td>tweezers</td><td>Shenzhen RWD Life Technology Co., Ltd</td><td>F11029-11</td><td></td></tr><tr><td>vascular forceps</td><td>Shenzhen RWD Life Technology Co., Ltd</td><td>F22003-09</td><td></td></tr></tbody></table>

establishment of a rat model of superior sagittal-sinus occlusion via a thread-embolism method

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors are involved in the course of the disease, resulting in great limitations in clinical research. Therefore, the establishment of stable CVST animal models that can standardize a variety of uncontrollable confounding factors have helped to circumvent shortcomings in clinical research. In recent decades, a variety of CVST animal models have been constructed, but the results based on these models have been inconsistent and incomplete. Hence, in order to further explore the pathophysiological mechanisms of CVST, it is necessary to establish a novel and highly compatible animal model, which has important practical value and scientific significance for the diagnosis and treatment of CVST. In the present study, a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis was established via a thread-embolization method, and the stability and reliability of the model were verified. Additionally, we evaluated changes in cerebral venous blood flow in rats after the formation of CVST. Collectively, the SD-rat SSS-thrombosis model represents a novel CVST animal model that is easily established, minimizes trauma, yields good stability, and allows for accurately controlling ischemic timing and location.

To establish the SD-rat SSS-thrombosis model via the suture method, the suture should be prepared in advance (Figure 1A), and the equipment required for the experiment (Figure 1B) should be prepared. Due to the delicate nature of the operation, the preparation of the model needs to be completed under a dissecting microscope. The main steps are shown in Figure 2. To facilitate the description of the specific details of the blood-flow...

Watch this Scientific Journal Video about Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method at JoVE.com

Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors ...

establishment-rat-model-superior-sagittal-sinus-occlusion-via-thread

Research

JoVE Journal

Neuroscience

3.4K Views.  the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University. Door het ontbreken van een ideaal diermodel is de studie van CVST sterk beperkt. Nu werd met succes een nieuw type SD-Rat CVST-model vastgesteld door zelfgemaakte draadembolie centripetally in te voegen op de oorspronkelijke locatie van SSS, die verschillende of oncontroleerbare interferentiefactoren in klinische toestand standaardiseerde. Nadat de rat met succes is verdoofd door intra-peritoneale injectie, brengt u gel oftalmisch oogsmeermiddel aan nadat de rat op stereotaxic frame is geplaa...

Video: Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method

A Low Mortality Rat Model to Assess Delayed Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage

Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) in rats, in order to identify the initiating events, pathophysiological changes and potential targets for treatment.
Methods: Twenty-eight male Sprague-Dawley rats (250 - 300 g) were arbitrarily assigned to one of two groups - SAH or saline control. Rat subarachnoid hemorrhage in the SAH group (n=15) was induced by double injection of autologous blood, 48 hr apart, into the cisterna magna. Similarly, normal saline (n=13) was injected into the cisterna magna of the saline control group. Rats were sacrificed on day five after the second blood injection and the brains were preserved for histological analysis. The degree of vasospasm was measured using sections of the basilar artery, by measuring the internal luminal cross sectional area using NIH Image-J software. The significance was tested using Tukey/Kramer's statistical analysis.
Results: After analysis of histological sections, basilar artery luminal cross sectional area were smaller in the SAH than in the saline group, consistent with cerebral vasospasm in the former group. In the SAH group, basilar artery internal area (.056 &mu;m &plusmn; 3) were significantly smaller from vasospasm five days after the second blood injection (seven days after the initial blood injection), compared to the saline control group with internal area (.069 &plusmn; 3; p=0.004). There were no mortalities from cerebral vasospasm.
Conclusion: The rat double SAH model induces a mild, survivable, basilar artery vasospasm that can be used to study the pathophysiological mechanisms of cerebral vasospasm in a small animal model. A low and acceptable mortality rate is a significant criterion to be satisfied for an ideal SAH animal model so that the mechanisms of vasospasm can be elucidated 7, 8. Further modifications of the model can be made to adjust for increased severity of vasospasm and neurological exams.

Aneurysmal subarachnoid hemorrhage (SAH) is bleeding that occurs into the subarachnoid space when an aneurysm ruptures. While the morbidity and mortality from this event has been on a decline due to improved treatment approaches, the risk of vasospasm after subarachnoid hemorrhage continues to be the same as it was several years ago. The importance of establishing a comprehensive and reproducible animal model to identify initiating events of cerebral vasospasm has been the focus of research since the first use of rats in an experimental vasospasm model in 1979 by Barry et al. Early work in rats demonstrated that a single injection of autologous blood into the cisterna magna led to acute (within minutes) but not delayed cerebral vasospasm 3, 6, 14. Here we characterize a low mortality SAH rat model that results in reproducible delayed vasospasm.

Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) ...

Medicine

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

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

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

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

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

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

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

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

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 ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented.

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely ...

Middle Cerebral Artery Occlusion in Rats with Preserved Vascular Anatomy

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have significantly contributed to our understanding of its pathological mechanisms and the development of potential treatments. Presently, there are two common methods involving filament (endovascular suture) techniques to induce animal models of cerebral ischemia. However, these methods have inherent limitations, such as reduced blood perfusion to the brain, damage to the external carotid artery system, impaired food and/or water intake, and sensory dysfunction of the face. This article introduces a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. In this study, the common carotid artery (CCA) of Sprague-Dawley rats was exposed, and an incision was made. A filament was then inserted through the incision into the internal carotid artery to occlude the middle cerebral artery. After 1.5 h of induced ischemia, the occluding filament was fully removed from both the internal carotid artery and the CCA. The incision in the CCA was subsequently sutured using 11-0 microsurgical sutures under a microscope (magnification 4x). Through the utilization of microsurgical techniques to repair the CCA, this study successfully developed a unique method to induce an ischemic stroke model in rats while preserving the anatomical integrity of cerebral blood vessels.

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have ...

Standardizing Rat Calvarial Suture-Bony Defect Model for Regenerative Studies

The Establishment of Calvarial Suture-Bony Composite Defects in Rats: A Standardized Model for Suture-Regenerative Therapy Investigation

Large-scale calvarial defects often coincide with cranial suture disruption, leading to impairments in calvarial defect restoration and skull development (the latter occurs in the developing cranium). However, the lack of a standardized model hinders progress in investigating suture-regenerative therapies and poses challenges for conducting comparative analyses across distinct studies. To address this issue, the current protocol describes the detailed modeling process of calvarial suture-bony composite defects in rats.
The model was generated by drilling full-thickness rectangular holes measuring 4.5 mm &#215; 2 mm across the coronal sutures. The rats were euthanized, and the cranium samples were harvested postoperatively at day 0, week 2, week 6, and week 12. &#181;CT results from samples collected immediately post-surgery confirmed the successful establishment of the suture-bony composite defect, involving the removal of the coronal suture and the adjacent bone tissues.
Data from the 6th and 12th postoperative weeks demonstrated a natural healing tendency for the defect to close. Histological staining further validated this trend by showing increased mineralized fibers and new bone at the defect center. These findings indicate progressive suture fusion over time following calvarial defects, underscoring the significance of therapeutic interventions for suture regeneration. We anticipate that this protocol will facilitate the development of suture-regenerative therapies, offering fresh insights into the functional restoration of calvarial defects and reducing adverse outcomes associated with suture loss.

Author Spotlight: Development and Evaluation of a Standardized Rat Model for Calvarial Suture-Bony Composite Defects

We describe the detailed surgical procedures of calvarial suture-bony composite defects in rats, alongside the investigations into the short-term and long-term prognoses of the model. We aim to construct a standardized model for developing suture-regenerative therapies.

Large-scale calvarial defects often coincide with cranial suture disruption, leading to impairments in calvarial defect restoration and skull development ...

Establishment of a Minimally Invasive Rat Model of Pulmonary Embolism Using Autologous Blood Clots

Pulmonary embolism (PE) is one of the leading causes of cardiovascular death, resulting in a significant socioeconomic burden. Although current treatments primarily focus on anticoagulation and thrombolysis, there is an urgent need for a better understanding of its pathophysiology and the development of new treatment strategies. Animal models play a crucial role in understanding PE and developing new therapies for the disease, with rodents commonly used due to ethical and cost considerations. However, existing rodent models for PE are limited by a lack of standardized procedures, which hampers reproducibility and cross-study comparisons. This study aims to establish a minimally invasive rat model of PE using autologous blood clots. The model features a minimally invasive blood sampling technique, a standardized thrombus generation procedure, and minimally invasive vein access. Additionally, protocols for quantifying infarcted areas and visualizing the pulmonary arterial tree are provided. These procedures aim to improve the reliability of rodent models for studying PE progression and facilitate the development of novel treatments.

A detailed methodology for establishing a minimally invasive rat model of pulmonary embolism using autologous blood clots is described. Additional methods for quantifying the infarcted area and visualizing the pulmonary arterial tree are also provided.

Pulmonary embolism (PE) is one of the leading causes of cardiovascular death, resulting in a significant socioeconomic burden. Although current treatments ...

Establishing a Subarachnoid Hemorrhage Rat Model Induced by Autologous Blood Injection

Source: B&#246;mers, J. et al. <a href="https://app.jove.com/v/62567">Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure for establishing a subarachnoid hemorrhage rat model. An anesthetized rat is implanted with probes to measure intracranial pressure and cerebral blood flow. The target site is identified, and the needle is inserted at a pre-chiasmatic position. Autologous blood is then injected into the subarachnoid space, increasing intracranial pressure and decreasing cerebral blood flow, establishing the model.

Source: B&#246;mers, J. et al. Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model. J. Vis. ...

Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method

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