Figure 1 was created with BioRender software (https://www.biorender.com/). This study was supported by grants from the guiding project of the Natural Science Foundation of Hubei Province (No. 2022CFC057).

The experimental protocol was approved by the Institutional Animal Care and Use Committee of Jianghan University (JHDXLL2024-080) and conducted in accordance with the Experimental Animal Ethical Guidelines issued by the Center for Disease Control of China. Adult male C57BL/6J mice weighing 21&#8211;26 g were used in this study. The details of the reagents and equipment used are listed in the Table of Materials.
1. Animal grouping and acute hyperglycemia inducing
<ol>
	<li>House the mice in the laboratory animal center of Jianghan University in a 12-h light/dark cycle controlled environment (20&#8211;22 &#176;C, 50%&#8211;55% humidity) with food and water ad libitum.</li>
	<li>Randomly divide the mice into four groups: Sham + saline (n = 12), Sham + glucose (n = 12), MCAO + saline (n = 12), and MCAO + glucose (n = 20).</li>
</ol>
2. Preoperative preparation
NOTE: All experimental mice fasted for 12 h before surgery.
<ol>
	<li>Administer systemic analgesia (meloxicam, 5 mg/kg subcutaneous) and local anesthesia to the incision site (bupivacaine, 2 mg/kg subcutaneous) peri-operatively.</li>
	<li>Sterilize all surgical instruments, cotton swabs, and surgical sutures by autoclaving at the conditions of 121 &#176;C and 15 psi for 30 min.</li>
	<li>Wipe the surgical platform and surrounding work area with 75% ethanol, and turn on the heating switch with the temperature set to 37 &#176;C.</li>
	<li>Anesthetize the mouse with a 3% isoflurane-20% oxygen gas mixture in the anesthesia induction chamber. Then, evaluate&#160;the&#160;toe pinch reflex to test&#160;the depth of anesthesia.</li>
	<li>Move the mouse on the surgical platform in the prone position, and quickly insert the mouse&#39;s nose in the nose cone.&#160;Open the gas flow to the nose cone, closing off flow to the anesthesia induction chamber. Adjust the proportion of isoflurane to 2% and set the flow of the gas mixture to 0.4 mL/min for anesthesia maintenance.</li>
	<li>Apply eye gel to the mouse&#39;s eyes to maintain moisture during surgery.</li>
	<li>Shave the hair of the neck region by using the animal clipper. Clean and disinfect the skin by alternating applying with iodine and 70% ethanol using a disinfectant cotton stick.</li>
	<li>Cover a sterile gauze on the neck and cut an opening to expose the operative region.</li>
</ol>
3. Baseline cerebral blood flow measurement
<ol>
	<li>Shave the hair of scalp region by using the animal clipper. Clean and disinfect the scalp by alternating applying with iodine and 70% ethanol using a disinfectant cotton stick.</li>
	<li>Make a 1.0 cm long midline incision in the skin over the frontal region to expose the cranial fontanelle. Keep the skull moist with normal saline.</li>
	<li>Prepare the Laser Doppler Flowmetry (LDF) instrument and hold the LDF probe tip perpendicular to surface of the left parietal skull (1 mm posterior and 5 mm lateral to the bregma); once the FLUX is stable and record the baseline value. 
	NOTE: The unit of measurement for FLUX is Perfusion Units (PU), with a measurement range of 0-1000 PU. Ideal baseline CBF on a Laser Doppler Flowmetry are &#62;600 PU. A continuous period of 5 seconds with a fluctuation range of less than 10% is considered as valid FLUX reading.</li>
	<li>Suture the muscle and the skin separately using 5.0 PGA absorbable suture. Apply diclofenac sodium gel and mupirocin ointment to the wound.</li>
</ol>
4. MCAO surgical procedure
NOTE: MCAO is performed using a modified thread-occlusion method, as previously described by Chiang et al.13.
<ol>
	<li>Gently turn the mouse over to a supine position and administer intraperitoneally 30% glucose (7.2 mL/kg) or normal saline to the mice 15 min before the surgical procedure. 
	NOTE: Mice from Sham + glucose and MCAO + glucose groups received intraperitoneal injections of glucose. Mice from Sham + saline and MCAO + saline groups received intraperitoneal injections of normal saline.</li>
	<li>Shave the hair of neck region by using the animal clipper. Clean and disinfect the skin by alternating applying with iodine and 70% ethanol using a disinfectant cotton stick. Cover a sterile gauze on the neck and cut an opening to expose the operative region. 
	NOTE: Ensure the incision is kept straight to achieve optimal visualization of the carotid artery.</li>
	<li>Make a 1.5 cm midline incision on the ventral aspect of the neck using a scalpel. Pull apart the subcutaneous tissue and superficial fascia by using surgical tweezers. 
	NOTE: Make sure that the incision is kept straight to achieve optimal visualization of the carotid artery.</li>
	<li>Beneath the superficial fascia, locate the submandibular gland and an inverted triangle formed by three muscles: the sternohyoid, positioned at the midline over the trachea; the posterior belly of the digastric muscle, recognizable by its shiny white tendinous part; and finally, the sternomastoid muscle.</li>
	<li>Perform blunt dissection within the inverted triangle to identify and separate the left common carotid artery (CCA).</li>
	<li>Dissect the vagus nerve adjacent to the CCA and mucosal tissue surrounding the blood vessels using ophthalmic forceps. Make a ringer on CCA using a 5.0 polyglycolic acid (PGA) absorbable suture, but keep the suture untensioned.</li>
	<li>Separate upward along the CCA, a &#34;Y&#34; bifurcation can be observed to divide the CCA into the external carotid artery (ECA) and internal carotid artery (ICA).</li>
	<li>Separate the bifurcation to fully expose the ECA and ICA. Loop and tightly tie a 5.0 PGA absorbable suture around the ECA distally from the bifurcation. 
	NOTE: Ensure a sufficient distance between the permanent ligation site and vascular bifurcation to prevent the thread from dislodging during the rotation of the ECA.</li>
	<li>Temporarily clamp both the CCA and ICA using two 8 mm x 2 mm light micro serrafine arterial artery clamps to block the blood flow from the CCA to the ICA.</li>
	<li>Stretch the suture tie on the ECA distal and CCA to straighten the ECA segment.</li>
	<li>Use microscissors to make a small incision between the two suture ties on the ECA.</li>
	<li>Insert a silicon-coated monofilament suture (30 mm long, 3&#8211;4 mm coated silicon) into the ECA. Loop and slightly tie a second 5.0 PGA absorbable suture on the ECA near the bifurcation to prevent the monofilament suture from backing out.</li>
	<li>Completely cut the ECA distal to the permanent ligation and remove the artery forceps clamp from the ICA.</li>
	<li>Withdraw the suture to the bifurcation of the CCA, then carefully retract and rotate the ECA stump. Adjust the insertion direction of the suture and slowly insert it, approximately 9.0&#8211;10.0 mm from the CCA bifurcation into the ICA, to occlude the MCA.</li>
	<li>Tighten the second PGA absorbable suture around the ECA and remove the artery clamp from the CCA.</li>
	<li>Suture the muscle and the skin separately using 5.0 PGA absorbable suture. Apply diclofenac&#160;sodium&#160;gel and mupirocin ointment to the wound.</li>
	<li>Gently turn the mouse over to the prone position and repeat step 3.4. to record the blood flow following MCA occlusion. 
	NOTE: Mice exhibiting a blood flow reduction of less than 40% of the baseline value following MCAO were excluded from the study14.</li>
	<li>Turn on the heating switch of recovery cage with the temperature set to 37&#176;C. Place the mouse in the recovery cage during the post-occlusion period (60 min).</li>
</ol>
5. Monofilament removal and reperfusion
<ol>
	<li>Re-anesthetize the mouse as previously described just before the occlusion period should end.</li>
	<li>Clamp the CCA using a microclip artery clamp.</li>
	<li>Partially retract the monofilament from the ICA until the silicon-coated tip becomes visible through the ICA.</li>
	<li>Place another microclip artery clamp on the ICA above the silicon-coated tip.</li>
	<li>Completely withdraw the monofilament and tightly ligate the ECA stump.</li>
	<li>Remove the microclip artery clamp from ICA and CCA, respectively.</li>
	<li>Suture the muscle and the skin layer by layer using 5.0 PGA absorbable suture.</li>
	<li>Place the mouse in the recovery chamber and monitor all mice until they are fully awake. Administer meloxicam (5 mg/kg, subcutaneously) for pain relief every 12 h for up to 24 h.</li>
</ol>
6. Blood glucose measurement
NOTE: Blood glucose levels were measured at the following time points: (1) just before MCAO surgery (baseline), (2) immediately after the insertion of the monofilament (15 min after glucose injection), (3) immediately after the withdrawal of the monofilament immediately after the insertion of the monofilament (75 min after glucose injection).
<ol>
	<li>Wipe the mouse&#39; tail with an alcoholic cotton ball to make the tail vein fully hyperaemic.</li>
	<li>Cut off the tail tip by 1&#8211;2 mm using surgical scissors.</li>
	<li>Gently squeeze along the root of the tail to the tip of the tail, facilitating the blood to flow out of the incision.</li>
	<li>Position the sample absorption tank of the test paper at the edge of the blood droplet. 
	NOTE: The blood will be drawn into the test paper due to the siphoning effect.</li>
	<li>Read the blood glucose meter reading and record the result. 
	NOTE: Exclude the mice whose blood glucose levels did not rise above 10 mmol/L after 15 min of glucose injection.</li>
	<li>Remove any surplus blood using a cotton ball and apply pressure to halt the bleeding.</li>
</ol>
7. 2,3,5-Triphenyltetrazolium Chloride (TTC) staining
<ol>
	<li>Euthanize the mouse by intraperitoneal injection of an overdose of pentobarbital (300 mg/kg).</li>
	<li>Once the mouse stops breathing, promptly immobilize it with a supine position.</li>
	<li>Open the thorax using scissors, and cut out the diaphragm to expose the heart.</li>
	<li>Insert a 23 G injection needle into the left ventricle of the mouse and incise the auricular appendix with scissors.</li>
	<li>Perfuse 15 mL of normal saline until the fluid draining from the auricular appendix appears clear and transparent.</li>
	<li>Decapitate the mouse and cut open the scalp to fully expose the skull using scissors. Insert the scissors tip 2 mm slightly in front of the coronal suture of the skull to pry it open.</li>
	<li>Use tweezers to remove the mouse&#39;s brain tissue intact.</li>
	<li>Place the brain tissue in a rodent brain matrice and cut it into 2 mm coronal slices.</li>
	<li>Transfer the slices to a 24-well plate and incubate them with 2% TTC solution at 37 &#176;C for 15 min.</li>
	<li>Start the scanning software and set the parameter settings to a resolution of 1200 dpi and a JPG format for image analysis.</li>
	<li>Clip the tissue sections out from the 24-well plate with curved forceps and arrange the sections on the glass scanning plate.</li>
	<li>Scan the tissue sections and then export the images for measurement analysis.</li>
	<li>Measure the infarct area by correcting for tissue swelling. Subtract the non-infarct area of the ipsilateral side from the area of the contralateral side using ImageJ software15. Assess the infarct size as a percentage of the contralateral hemisphere. For additional details on infarct area analysis, refer to Friedl&#228;nder et al.16.</li>
</ol>
8. Gross observation
<ol>
	<li>Repeat steps 7.1&#8211;7.8.</li>
	<li>Clip the tissue sections out from the brain matrice with curved forceps and arrange the sections on the glass scanning plate.</li>
	<li>Scan the brain coronal sections as described in steps 7.10 and 7.12.</li>
</ol>
9. Hematoxylin and eosin (H&#38;E) staining
<ol>
	<li>Repeat steps 7.1&#8211;7.4. Slowly perfuse 15 mL of normal saline, followed by perfusion of approximately 15 mL of paraformaldehyde (PFA).</li>
	<li>Repeat steps 7.6&#8211;7.7. Fix the brain tissue with 4% PFA for 24 h at room temperature.</li>
	<li>Place the fixed brain tissue into the automated tissue dehydrator for dehydration, vitrification, and wax immersion.</li>
	<li>Embed the tissue in paraffin wax and cut the brain tissue into 5 &#956;m thick coronal slices by microtome.</li>
	<li>Dewax the slice by immersing it in xylene three times, with each immersion lasting 8 min. Then, gradually immerse the slice in ethanol solutions of decreasing concentrations (100%, 95%, 90%, 80%, 70%), followed by distilled water, with each step lasting 5 min.</li>
	<li>Stain the slice with hematoxylin solution for 3 min. Then, differentiate it by immersing it in 5% hydrochloric acid alcohol for 5 s.</li>
	<li>Incubate the slice with eosin solution for 1 min, then dehydrate and hyalinize it with gradient ethanol solution (90%, 95%, 100%) and xylene.</li>
	<li>Mount the section with neutral resins.</li>
	<li>Capture the images of H&#38;E stained brain slices under a bright field using fluorescence microscopy.</li>
</ol>
10. Determination of Evans Blue (EB) lekage
NOTE: For details on this procedure, please refer to Wang et al.17.
<ol>
	<li>Inject 2% (w/v) Evans Blue (EB) solution (2 mL/kg) via the tail vein 23 h post-MCAO.</li>
	<li>Repeat steps 7.1&#8211;7.7 one hour after EB injection, and capture a photo of the entire brain.</li>
	<li>Place the brain in a rodent brain matrice and cut it coronally into 2 mm slices. Scan the brain coronal sections as described in steps 7.10&#8211;7.12 to observe EB leakage.</li>
	<li>Divide the slices into right and left portions, and put each one into a centrifuge tube.</li>
	<li>Homogenize every slice of brain tissue in 50% trichloroacetic acid, with a ratio of 100 mg of brain tissue to 0.4 mL of trichloroacetic acid.</li>
	<li>After being incubated at 4 &#176;C overnight, centrifuge the homogenate for 30 min at 10,000 x g (at 4 &#176;C).</li>
	<li>Transfer the supernatant liquid into another centrifuge tube and dilute it four-fold with ethanol.</li>
	<li>Measure the absorbance of the supernatant liquid at 620 nm using a spectrophotometer.</li>
	<li>Convert absorbance values to the concentration of EB using a standard curve of EB in ethanol (e.g., at 31.25, 62.5, 125, and 250 ug/mL). 
	NOTE: The result is presented as a microgram of EB per gram of brain tissue.</li>
</ol>
11. Determination of FITC-Dextran leakage
<ol>
	<li>Inject&#160; FITC-Dextran&#160; (10 KDa, 6 mg /mL, diluted in 0.01M PBS; 4 mL/kg) into the tail vein 24 h post-MCAO, allowing it to circulate in the blood for 10 min.</li>
	<li>Repeat steps 7.1&#8211;7.7 to perform perfusion and remove the intact brain tissue.</li>
	<li>Fix the brain tissue in 4% PFA for 24 h at room temperature in darkness.</li>
	<li>Transfer the fixed brain tissue into the sucrose solution with gradient concentrations of 10%, 20%, and 30% (diluted in 0.01M PBS) for dehydration.</li>
	<li>Embed the brain tissue using optimal cutting temperature compound (OCT) and cut into 30 &#956;m thick brain tissue sections. Transfer the sections onto a microscope slide using an inoculating loop and ensure they adhere to the surface of the slide.</li>
	<li>Mount the brain tissue sections using a mounting medium containing 4&#39;,6-diamidino-2-phenylindole (DAPI).</li>
	<li>Start the Laser confocal microscopy and its control software.</li>
	<li>Fix the microscope slide on the object stage and locate the infarct area under 200x eyepiece.</li>
	<li>Set the resolution to 1024 x 1024 and adjust the gain value and exposure time to obtain the clearest image possible.</li>
	<li>Acquire the images of the FITC-Dextran stained region of cerebral infarction under an excitation wavelength of 488 nm.</li>
</ol>

Hyperglycemia-Enhanced Hemorrhagic Transformation in tMCAO-Induced Stroke

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E., Dor&#233;, S., Candelario-Jalil, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammatory+mechanisms+of+blood-brain+barrier+damage+in+ischemic+stroke.">Neuroinflammatory mechanisms of blood-brain barrier damage in ischemic stroke.</a> Am J Physiol Cell Physiol. 316 (2), C135-C153 (2019).</li><li>Natarajan, R., Northrop, N., Yamamoto, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+isothiocyanate+(FITC)-dextran+extravasation+as+a+measure+of+blood-brain+barrier+permeability.">Fluorescein isothiocyanate (FITC)-dextran extravasation as a measure of blood-brain barrier permeability.</a> Curr Protoc Neurosci. 79, 9.58.1-9.58.15 (2017).</li><li>Ahishali, B., Kaya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+blood-brain+barrier+integrity+using+vascular+permeability+markers:+Evans+Blue,+Sodium+Fluorescein,+Albumin-Alexa+Fluor+conjugates,+and+Horseradish+Peroxidase.">Evaluation of blood-brain barrier integrity using vascular permeability markers: Evans Blue, Sodium Fluorescein, Albumin-Alexa Fluor conjugates, and Horseradish Peroxidase.</a> Methods Mol Biol. 2367, 87-103 (2021).</li><li>Kov&#225;cs, K. B., Bencs, V., Hud&#225;k, L., Ol&#225;h, L., Csiba, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemorrhagic+transformation+of+ischemic+strokes.">Hemorrhagic transformation of ischemic strokes.</a> Int J Mol Sci. 24 (18), 14067 (2023).</li></ol>

The authors have no conflicting interests to disclose.

The current protocol is designed to create a reliable animal model of hemorrhagic transformation following ischemic stroke, which can replicate the harmful effects of vessel revascularization under hyperglycemic conditions. Among the various risk factors for ischemic stroke, the blood glucose level within 24 h after the onset of stroke is positively correlated with the exacerbation of cerebral injury and increased mortality3,18. Numerous clinical investigations have also demonstrated that poststroke hyperglycemia is one of the most critical risk factors for HT and poorer neurological outcomes19. In the present protocol, 19 out of 20 mice were successfully induced to HT under acute hyperglycemia, resulting in a successful modeling ratio of approximately 95%. Unfortunately, four mice died from severe cerebral hematoma within 24 h after the MCAO surgery and were therefore excluded from further analysis.
Hyperglycemia-induced HT animal models have been utilized in recent preclinical research. Won et al. demonstrated that hyperglycemia promoted HT in a rat model of tPA stroke treatment20. However, the pathogenic factors in Won&#39;s HT model were more complex, as tPA itself may increase the risk of HT. In contrast, the current protocol utilized acute hyperglycemia combined with MCAO and achieved a significant success rate of HT in mice. Notably, variability exists among the hyperglycemia-induced HT models regarding glucose concentration, duration of ischemia, model reliability, and occurrence of HT21,22,23,24. Some studies utilized 50% glucose injection to induce significantly higher blood glucose levels (20-28 mmol/L), surpassing the typical range observed in stroke patients. The rate of HT in these experiments ranges from 70% to 73.3%, with a mortality rate recorded at 37.5% in one experimental model. In contrast, the current experimental procedures involved using a 30% glucose injection to induce acute hyperglycemia maintained between 14.3 mmol/L and 20.3 mmol/L, better aligning with the range of blood glucose values found in patients. Additionally, these procedures required a fasting period for experimental mice prior to surgery to regulate their preoperative baseline blood glucose levels to approximately around 5 mmol/L; failure to do so may result in an elevated baseline blood level of around 10 mmol/L, leading subsequently to excessively high blood glucose levels, and ultimately, higher mortality rates among the mice.
In a recent study, MacDougall et al. discovered that hyperglycemia led to an increase in the size of the infarct, particularly in a type 1 diabetes model25. The current findings indicate that the group administered with glucose exhibited a larger infarct size after 60 min of ischemia compared to the group under saline conditions. This result is consistent with a previous experiment that demonstrated the detrimental impact of high glucose on infarct progression in the penumbra area18. In this protocol, preoperative 30% glucose injection resulted in cerebral punctate hemorrhage after 24 h post-MCAO. Consistent with macroscopic gradation findings, histological analysis using H&#38;E staining revealed significant blood cell infiltration into the ischemic striatum and cortex in acute hyperglycemia mice. Naturally, there are other methods available for evaluating HT, such as hemoglobin measurement through ELISA or magnetic resonance imaging (MRI). Although the pathophysiological mechanism underlying HT remains elusive, BBB disruption is considered to be its leading cause after cerebral ischemia26. Therefore, BBB disruption can indirectly reflect the severity of HT. This protocol used EB staining to test BBB permeability. EB-stained brain slices visually demonstrate changes in BBB permeability and allow for quantitative measurement of EB dye content that has permeated into brain tissue. In recent years, other tracers such as Fluorescein-sodium and FITC-Dextran have been extensively utilized to assess BBB permeability in animal models of neurological diseases27,28. So, we also performed a Dextran staining experiment to delineate the location and extent of vascular endothelial injury in more detail by observing the leakage degree of FITC-Dextran.
The current protocol has several limitations that cannot be overlooked. Firstly, the MCAO surgery requires a high level of proficiency from the operator. It is crucial to ensure timely delivery of the embolic thread into the MCA and accurate placement and depth of the occlusion. Meeting these requirements necessitates extensive training for the experiment operator. Secondly, due to the potential for HT to worsen cerebral injury, postoperative care is essential in improving the survival rates of experimental animals. Lastly, it is important to acknowledge that clinical stroke patients often have multiple risk factors for HT, such as diabetes, high blood pressure, atrial fibrillation, and age29. Therefore, the present animal model cannot fully replicate the complex mechanisms involved in these risk factors.
In conclusion, this protocol effectively replicated the HT triggered by acute hyperglycemia in a cerebral ischemia-reperfusion mouse model. This model demonstrates good repeatability, high stability, and a high animal survival rate, making it suitable for widespread use in preclinical research of HT.

<a href="https://app-jove-com.remotexs.ntu.edu.sg/t/6615">This corrects</a> the article <a href="https://dx-doi-org.remotexs.ntu.edu.sg/10.3791/67371">10.3791/67371</a>

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/67371">A Mouse Model of Hemorrhagic Transformation Induced by Acute Hyperglycemia Combined with Transient Focal Ischemia</a>. The Protocol section was updated.

Erratum: A Mouse Model of Hemorrhagic Transformation Induced by Acute Hyperglycemia Combined with Transient Focal Ischemia

Cerebral infarction is the primary cause of disability and the second leading cause of death in adults worldwide1. The acute phase plays a crucial role in the progression of cerebral infarction, serving as a pivotal time point for disease treatment. Early and timely restoration of blood flow in the penumbra area is essential to prevent further brain cell death, with thrombolysis and interventional therapy representing the mainstays for acute cerebral infarction (ACI) treatment. However, hemorrhagic transformation (HT) poses a significant complication following thrombolysis and interventional therapy, occurring in 15%-30% of patients with ischemic stroke, thereby limiting their application to some extent2,3. The occurrence of HT significantly increases the risk of mortality and disability, affecting the prognosis of ACI. Therefore, it is of great clinical significance to investigate the pathological mechanisms of HT and to identify effective therapeutic targets.
Currently, thread embolism-induced middle cerebral artery occlusion (MCAO) is frequently utilized as a model of HT in rodents4. Prolonged obstruction can result in massive cerebral infarction involving the cortex and striatum, potentially leading to secondary HT. Thread MCAO does not require craniotomy, is highly reproducible, and produces focal brain damage and HT similar to a human stroke. However, this mechanical model has some distinct disadvantages, including high early mortality rates and low long-term survival rates5. Another frequently used HT model is the thrombolysis model, in which blood clot formation is induced firstly in the target vessel, followed by the use of thrombolytic drugs (e.g., rt-PA, warfarin) to dissolve the clot, mimicking the clinical process of HT in ischemic stroke6,7. Despite replicating the pathological process of clinical thrombolytic therapy to a large extent, HT animal models induced by rt-PA or warfarin are intricate to implement and associated with high animal mortality as well as variable incidence and location of bleeding. In order to advance basic and clinical translational research on HT after cerebral infarction, it is essential to establish a reproducible animal model of HT that is easy to operate and offers high stability.
Hyperglycemia is a significant contributor to HT following cerebral ischemia/reperfusion (I/R)8. Several retrospective studies have analyzed the clinical data of patients undergoing mechanical thrombectomy, revealing that elevated blood glucose levels upon admission are linked to a higher incidence of spontaneous HT3. In diabetic stroke patients, hyperglycemia significantly increases the risk of HT and leads to more severe neurological deficits9,10. Researchers have developed HT models by inducing cerebral I/R in diabetic animal models through MCAO. However, the diabetes-MCAO model has a long experimental duration, complex procedure, and high costs11,12. A reliable model of HT can be established by inducing acute hyperglycemia through intraperitoneal injection of glucose and integrating it with a cerebral I/R model generated by the suture technique. This method is easily performed with consistent bleeding position and effectively imitates the clinical features of post-stroke hyperglycemia. However, there are significant differences in crucial conditions such as ischemic time and glucose concentration; additionally, the stability of the model and the incidence of HT are inconsistent in different literature.
Our research group extensively utilized the acute hyperglycemia-MCAO method to establish an HT model. Furthermore, we conducted a comprehensive series of experiments to explore the relationship between ischemic time, blood glucose concentration, HT incidence rate, and animal mortality. These experiments ultimately led to identifying optimal conditions for creating a post-cerebral infarction HT model. This study presents a detailed protocol for establishing an acute hyperglycemia-induced HT model using intraperitoneal injection of 30% glucose combined with embolic MCAO.

<table><tbody><tr><td>2,3,5-Triphenyltetrazolium Chloride (TTC)</td><td>Sigma-Aldrich</td><td>108380</td><td>The dye for TTC staining</td></tr><tr><td>24-well culture plate</td><td>Corning Incorporated</td><td>CLS3527</td><td>The vessel for TTC staining</td></tr><tr><td>30% glucose injection</td><td>Kelun Pharmaceutical</td><td>H42021188</td><td>Acute hyperglycemia induction</td></tr><tr><td>4% paraformaldehyde</td><td>Wuhan Servicebio Technology&lt;br/&gt; Co., Ltd.</td><td>G1101</td><td>Tissue fixation</td></tr><tr><td>5.0 Polyglycolic acid absorbable suture</td><td>Jinhuan Medical Co., Ltd</td><td>KCR531</td><td>Equipment for surgery</td></tr><tr><td>96-well culture plate</td><td>Corning Incorporated</td><td>CLS3596</td><td>EB content measuring</td></tr><tr><td>Anesthesia machine</td><td>Midmark Corporation</td><td>VMR</td><td>Anesthesia for animal</td></tr><tr><td>Antifade Mounting Medium with DAPI</td><td>Beyotime Biotech</td><td>P0131</td><td>Mount for tissue sections</td></tr><tr><td>Automation-tissue-dehydrating&amp;nbsp;&lt;br/&gt; machine</td><td>Leica Biosystems</td><td>TP1020</td><td>Dehydrate tissue</td></tr><tr><td>Confocal microscopy</td><td>Leica Biosystems</td><td>STELLARIS 5</td><td>Image acquisition</td></tr><tr><td>Diclofenac sodium gel</td><td>MaYinglong Pharmaceutical</td><td>H10950214</td><td>Analgesia for animal</td></tr><tr><td>Eosin staining solution</td><td>Servicebio Technology</td><td>G1001</td><td>The dye for H&amp;amp;E staining</td></tr><tr><td>Evans Blue</td><td>Aladdin</td><td>E104208</td><td>EB staining</td></tr><tr><td>Eye gel</td><td>Guangzhou Pharmaceutical</td><td>H44023098</td><td>Material for surgery</td></tr><tr><td>Fitc-dextran</td><td>Sigma-Aldrich</td><td>60842-46-8</td><td>BBB permeability assessing</td></tr><tr><td>Fluorescence microscope</td><td>Olympus</td><td>BX51</td><td>Image acquisition</td></tr><tr><td>Frozen microtome</td><td>Leica Biosystems</td><td>CM1900</td><td>Use for frozen sections</td></tr><tr><td>Glucometer</td><td>YuWell</td><td>580</td><td>Blood glucose measurement</td></tr><tr><td>Hematoxylin staining Solution</td><td>Servicebio</td><td>G1004</td><td>The dye for H&amp;amp;E staining</td></tr><tr><td>Iodine</td><td>Lircon</td><td>20020059</td><td>Material for surgery</td></tr><tr><td>Isoflurane</td><td>Rwd Life Science</td><td>R510-22-10</td><td>Anesthesia for animal</td></tr><tr><td>Laser doppler blood flow meter</td><td>Moor Instruments</td><td>moorVMS</td><td>Blood flow monitoring</td></tr><tr><td>MCAO Sutures</td><td>Rwd Life Science</td><td>907-00023-01</td><td>Material for surgery</td></tr><tr><td>Meloxicam</td><td>Boehringer-Ingelheim</td><td>J20160020</td><td>Analgesia for animal</td></tr><tr><td>Microsurgical instrument kit</td><td>Rwd Life Science</td><td>SP0003-M</td><td>Equipment for surgery</td></tr><tr><td>Microtome</td><td>Thermo Fisher Scientific</td><td>HM325</td><td>Tissue section production</td></tr><tr><td>Microtome blade</td><td>Leica Biosystems</td><td>819</td><td>Tissue section production</td></tr><tr><td>Mupirocin ointment</td><td>GlaxoSmithKline</td><td>H10930064</td><td>Anti-infection for animal</td></tr><tr><td>Neutral balsam</td><td>Absin Bioscience</td><td>abs9177</td><td>Seal for H&amp;amp;E staining</td></tr><tr><td>Paraffin embedding center</td><td>Thermo Fisher Scientific</td><td>EC 350</td><td>Produce paraffin blocks</td></tr><tr><td>Pentobarbital sodium</td><td>Sigma-Aldrich</td><td>P3761</td><td>Euthanasia for animal</td></tr><tr><td>Phosphate buffered saline</td><td>Beyotime Biotech</td><td>C0221A</td><td>Rinse for tissue section</td></tr><tr><td>Scanner</td><td>EPSON</td><td>V330</td><td>Tissue scanning</td></tr><tr><td>Shaver</td><td>Shenzhen Codos Electrical Appliances Co.,Ltd.</td><td>CP-9200</td><td>Equipment for surgery</td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>1510-02362</td><td>EB content measuring</td></tr><tr><td>Sucrose solution</td><td>Shanghai Macklin Biochemical</td><td>57-50-1</td><td>Dehydration for tissue</td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura</td><td>4583</td><td>Tissue embedding medium</td></tr><tr><td>Trichloroacetic acid</td><td>Sigma-Aldrich</td><td>T6399</td><td>EB content measuring</td></tr></tbody></table>

a mouse model of hemorrhagic transformation induced by acute hyperglycemia combined with transient focal ischemia

Hemorrhagic transformation (HT) is a serious complication that can occur as a result of thrombolytic therapy following ischemic stroke (IS), and it poses significant limitations on the clinical application of recombinant tissue plasminogen activator (rt-PA). Unfortunately, there are currently no effective interventions available for HT in clinical practice. Therefore, there is an urgent need for stable and reliable experimental animal models to elucidate the pathogenesis of HT and develop effective intervention strategies. This study presented a protocol for establishing a mouse model of HT induced by acute hyperglycemia combined with transient focal ischemia (tMCAO). Male C57BL/6J mice were injected with 30% glucose to induce hyperglycemia and then subjected to 60 min of tMCAO with reperfusion. The infarct volume, integrity of the blood-brain barrier (BBB), and degree of intracranial hemorrhage were assessed at 24 h after MCAO. The results showed that glucose injection led to transient hyperglycemia (14.3-20.3 mmol/L), which significantly increased both the infarct volume and the incidence of HT. Hematoxylin-eosin (H&#38;E) staining indicated significant hemorrhagic lesions within the infarction zone in hyperglycemic mice. Additionally, hyperglycemic mice exhibited aggravated BBB disruption, as shown by more severe leakage of Evans blue (EB) and FITC-Dextran. In conclusion, acute hyperglycemia reliably and consistently resulted in macroscopic HT in a mouse model of tMCAO. This reproducible model offers a valuable tool for investigating the pathological mechanisms of HT and developing corresponding therapeutic interventions.

The experimental procedure of this study is illustrated in Figure 1. Briefly, the mice underwent thread occlusion-induced MCAO for 60 min, followed by reperfusion. Glucose (30% in normal saline, 7.2 mL/kg body weight) was intraperitoneally administered 15 min before MCAO. Blood glucose levels were measured at baseline (before glucose injection), immediately after MCAO, and at the time of reperfusion. After 24 h of reperfusion, the mice were euthanized, and brain tissues were collected for analysis of infarct volume, blood-brain barrier leakage, and histopathology. As depicted in Figure 2, all four groups exhibited similar baseline blood glucose levels. However, the mice that received glucose injections showed significantly elevated blood glucose levels compared to those in the saline groups. 
As depicted in Figure 3A, no visible infarction was observed on brain slices in the Sham + saline or Sham + glucose group, while marked cerebral infarction was clearly revealed by TTC staining on brain slices from both MCAO groups. Furthermore, mice injected with glucose exhibited a greater infarct volume compared to those injected with normal saline in the different MCAO groups. According to the statistical analysis, the infarct volume was 20.8% &#177; 0.8% and 45.1% &#177; 1.6% in the MCAO + saline group and MCAO + glucose group, respectively (Figure 3B).
Macroscopic observations revealed no hemorrhages in the left brain hemisphere of either the MCAO + saline group or the Sham + saline/glucose group. In contrast, multiple punctate hemorrhages were observed in the ischemic brain hemisphere of the MCAO + glucose group (Figure 4A). Scanning images of consecutive coronal brain sections showed that the hemorrhages in the MCAO + glucose group were distributed within the infarction regions of the cortex and striatum (Figure 4A). Additionally, histological analysis using H&#38;E staining demonstrated significant blood cell infiltration into the infarction regions of the cortex and striatum in the MCAO+glucose group (Figure 4B). Conversely, no blood cell infiltration was observed in either the MCAO+saline group or the Sham + saline/glucose group.
As illustrated in Figure 5A, no leakage of EB was observed in the brain slices of the Sham + glucose group. However, both MCAO groups showed distinct blue staining due to EB leakage. Additionally, compared to mice injected with saline, those injected with glucose exhibited increased EB permeability at 24 h post-MCAO. In comparison to the Sham + saline group, the MCAO + saline group demonstrated a significant increase in EB content (4.2 &#181;g/g &#177; 1.8 &#181;g/g vs. 28 &#181;g/g &#177; 0.7 &#181;g/g, p &#60; 0.01), which was further elevated in the MCAO + glucose group (28 &#181;g/g &#177; 0.7 &#181;g/g vs. 65.5 &#181;g/g &#177; 6.0 &#181;g/g, p &#60; 0.01) (Figure 5B).As illustrated in Figure 5C, the leakage of FITC-Dextran was more intense in the ischemic cerebral cortex and striatum of the MCAO + glucose group compared with MCAO + saline group.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67371/67371fig01.jpg" /> 
Figure 1: Schematic diagram of the experimental procedure. Mice were intraperitoneally injected with 30% glucose or saline before MCAO surgery, with blood glucose monitored during the procedure. 24 h after reperfusion, mice were euthanized for pathological and histological analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67371/67371fig01large.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/67371/67371fig02.jpg" /> 
Figure 2: Changes in blood glucose levels at various time points following the injection of 30% glucose. Data are presented as mean &#177; SD. N = 10-20. ***p &#60; 0.001 compared to the Sham + saline group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67371/67371fig02large.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/67371/67371fig03.jpg" /> 
Figure 3: Hyperglycemia worsened ischemic brain damage 24 h after reperfusion. (A) Representative images of TTC-stained coronal slices, with normal brain tissue appearing pinkish-red and ischemic infarct areas appearing grayish-white. Scale bar = 5 mm. (B) Statistical analysis of infarct volume. Data are presented as mean &#177; SD. N = 3. ***p &#60; 0.001 compared to the Sham + saline group, ##p &#60; 0.01 compared to the MCAO + saline group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67371/67371fig03large.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/67371/67371fig04.jpg" /> 
Figure 4: Hyperglycemia increased the risk of HT at 24 h after reperfusion. (A) Representative images of gross observation and coronal slices. The asterisk indicated the visible hemorrhagic foci. Scale bar = 5 mm. (B) Representative H&#38;E-stained half-brain images (top) and enlarged images (bottom). Scale bar = 200 &#181;m for half-brain images; Scale bar = 20 &#181;m for enlarged images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67371/67371fig04large.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/67371/67371fig05.jpg" /> 
Figure 5: Hyperglycemia aggravated BBB disruption at 24 h after reperfusion. (A) Representative images of Evans blue leakage. Scale bar = 5 mm. (B) Statistical analysis of Evans blue leakage in the ischemic hemispheres. Data are presented as mean &#177; SD, N = 3, **p &#60; 0.01, ***p &#60; 0.001 compared with Sham+saline group, ##p &#60; 0.01 compared with MCAO + saline group. (C) Representative images of FITC-Dextran stained ischemic cerebral cortex and striatum. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67371/67371fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

A Mouse Model of Hemorrhagic Transformation Induced by Acute Hyperglycemia Combined with Transient Focal Ischemia

This study presents a protocol for establishing a highly reproducible animal model of hemorrhagic transformation (HT) using middle cerebral artery occlusion/reperfusion (MCAO/R) in C57BL/6 mice with acute hyperglycemia.

Hemorrhagic transformation (HT) is a serious complication that can occur as a result of thrombolytic therapy following ischemic stroke (IS), and it poses ...

a-mouse-model-hemorrhagic-transformation-induced-acute-hyperglycemia

Nanyang Technological University

Research

JoVE Journal

Medicine

308 Views.  Jianghan University. Deze studie presenteert een protocol voor het opstellen van een zeer reproduceerbaar diermodel van hemorragische transformatie (HT) met behulp van occlusie/reperfusie van de middelste hersenslagader (MCAO/R) bij C57BL/6-muizen met acute hyperglykemie.

Video: Een muismodel van hemorragische transformatie geïnduceerd door acute hyperglykemie in combinatie met voorbijgaande focale ischemie

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e., medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Purpose. Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroCardiographic evaluatiOn of ischeMia comParing invAsive to phaRmacological trEatment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Method. Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

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

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

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

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

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

A Mouse Model of Hemorrhagic Transformation Induced by Acute Hyperglycemia Combined with Transient Focal Ischemia

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