Simiao Wu was supported by the Science &#38; Technology Department of Sichuan Province (2024YFHZ0330) and the West China Hospital of Sichuan University. Weihong He was supported by the Science &#38; Technology Department of Sichuan Province (2023YFS0297) and Sichuan University. We thank Yi Zhang and Yue Li at the Research Core Facility, West China Hospital of Sichuan University, for their technical support on TUNEL imaging and analysis.

This protocol and the experiments reported here were approved by the Sichuan University Medical Ethics Committee. The care and use of animals were in compliance with local and international ethical criteria.
NOTE: Adult male Sprague-Dawley rats weighing 250-280 g were used in this study. The rats were housed in a standard environmental condition (20-22 &#176;C temperature, 12 h light/dark cycle), fed with a standard rat formula diet and high-purity water. The surgical procedure was performed under sterile conditions.
1. Rat model of MCAO
<ol>
	<li>Induce initial anesthesia using 4% isoflurane with oxygen at 1 L/min. Monitor depth of anesthesia by foot pinch. While anesthesia is achieved, administer an ocular ointment to prevent dryness. Inject rat with 1 mL of saline subcutaneously as volume replenishment and with 5 mg/kg carprofen and 0.1 mg/kg buprenorphine for preoperative analgesia. Reduce isoflurane to 3.5% initially and then to 2% gradually throughout the surgery.</li>
	<li>Place the rat in a supine position and maintain body temperature at 36.5 &#177; 0.1 &#176;C on a regulated heat mat with a rectal temperature monitor probe. Shave and disinfect the skin in the neck with an iodophor disinfecting solution.</li>
	<li>Make a 2 cm midline incision in the neck and retract the soft tissues to expose the carotid vessels. Use forceps to isolate the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA).</li>
	<li>Use sutures to ligate the proximal right CCA and clamp the right ICA and ECA with a micro-clip at their origin.</li>
	<li>Insert a nylon monofilament coated with a round silicone tip into the lumen of the CCA through a small incision and tie a knot in the CCA to prevent bleeding and dislodging of the nylon monofilament.</li>
	<li>Open the right ICA micro-clip and insert the nylon monofilament into the right middle cerebral artery (MCA) through the ICA stump until the silicone tip occludes the origin of MCA (~18-20 mm).</li>
	<li>Cut off the exposed part of the nylon monofilament and close the neck midline incision.</li>
	<li>After 2 h occlusion of MCA, open the neck incision and untie the knot in the CCA. Then withdraw the nylon monofilament to reperfuse the MCA. Ligate the distal right CCA, and close the neck incision with either continuous or interrupted sutures.</li>
	<li>Post-operatively, give buprenorphine by subcutaneous injection every 8 h, at a dose of 0.03 mg/kg. Observe rats throughout the recovery from the anesthesia in a heated cage with a temperature-regulated heating mat. To encourage eating, place a Petri dish with mashed chow in the cage. House one rat per cage after the surgery.</li>
	<li>Subject the sham rats to the same procedure without the monofilament insertion. 
	NOTE: A total of 9 animals are used in each group for measurement of infarct size (n = 3), immunofluorescence analysis (n = 3), and apoptosis detection(n = 3).</li>
	<li>Evaluate neurological function at 2 h after MCAO and 22 h after reperfusion using Zea-Longa behavioral rating scale with a 0-5 point scale14. Exclude rates with a score of 0 or 4.</li>
</ol>
2. Measurement of infarct size (Figure 1)
<ol>
	<li>Sacrifice rats under deep anesthesia (4% isoflurane) by guillotine and carefully isolate the whole brain from the skull.</li>
	<li>Freeze the brain at -20 &#176;C for 20 min and cut the frozen brain into six coronal slices with a 2 mm distance between slices. Stain the brain slices with 2% TTC for 15 min at 37 &#176;C in the dark.</li>
	<li>Fix brain slices with 4% paraformaldehyde for 24 h at room temperature (RT). Photograph the stained slices with a digital camera, with auto mode setting for close-up objects.</li>
	<li>Transfer the pictures to a computer and measure the infarct size using ImageJ. Present the infarct size as the ratio of the sum of the infarct area in 6 slices to the sum of the whole brain area in the same slices. Express data as mean &#177; standard error of the mean (SEM).</li>
</ol>
3. Immunofluorescence analysis on brain cryosection (Figure 2)
<ol>
	<li>Induce anesthesia with 4% isoflurane and 1 L/min oxygen. Incise the skin and subcutaneous tissue to expose the heart.</li>
	<li>Remove the pericardium and make a small cut in the right atrium. Perfuse 200 mL of saline through the left ventricle, followed by 200 mL of 4% paraformaldehyde.</li>
	<li>Open the skull and remove the intact brain.</li>
	<li>Fix the brain in 4% paraformaldehyde at RT for over 24 h. Then, dehydrate it in a series of sucrose solution gradients of 10%, 15%, and 30% concentrations until the tissue sinks, approximately 6-8 hours for each concentration.</li>
	<li>Embed the brain in optimal cutting temperature (OCT) compound and remove air bubbles if there are any. Then, snap-freeze the brain in liquid nitrogen and store it in the -80 &#176;C freezer.</li>
	<li>Section the brain using a cryostat at a thickness of 5 &#181;m. Throughout sectioning, ensure the cabinet temperature remains between -18 &#176;C and -20 &#176;C. Label the sectioning location based on the model site and experimental conditions. Store the sections in a freezer.</li>
	<li>Allow the frozen sections to equilibrate at RT for ~30 min to perform immunofluorescence staining. Then, soak the sections with sterile PBS 3x for 5 min each.</li>
	<li>Outline the tissue with an immunohistochemistry pen. Incubate with 20 &#181;g/mL Proteinase K (RNase-free) and 0.3% Triton X-100 at RT for 10 min.</li>
	<li>Soak the sections with sterile PBS 3x for 5 min each. Apply 3% BSA to the outlined tissue and incubate at RT for 1 h for blocking.</li>
	<li>After blocking, remove the solution, add the appropriate primary antibody (Rabbit recombinant monoclonal cathepsin-B antibody, 1:200), and incubate the sections overnight in the dark at 4 &#176;C.</li>
	<li>Remove the primary antibody and wash the sections with sterile PBS containing 0.1% Tween-20 3x for 5 min each.</li>
	<li>Add the appropriate secondary antibody (Goat Anti-Rat IgG [H+L] [FITC conjugated], 1:200) with the required fluorophore, and incubate the sections in a humidified chamber at RT for 1 h, protecting them from light.</li>
	<li>After secondary antibody incubation, wash the sections with sterile PBS containing 0.1% Tween-20 3x for 5 min each. Stain the sections with a mounting medium containing DAPI and cover them with a coverslip.</li>
	<li>Capture images using a fluorescence microscope with the following settings: excitation wavelength of 488 nm, emission wavelength of 520 nm, and exposure time of 500 ms. Store the sections in the dark at 4 &#176;C for long-term preservation.</li>
</ol>
4. Detection of apoptosis by TUNEL staining (Figure 3)
NOTE: A commercial TUNEL assay kit is used for TUNEL staining. This TUNEL kit includes a DNase I Buffer working solution, deoxynucleotidyl transferase (TdT) equilibration buffer, labeling working solution, and DAPI working solution.
<ol>
	<li>Perfuse and isolate the brain using the same procedure as above (steps 3.2-3.3). Immerse the brain in 4% paraformaldehyde for 3-4 h for fixation.</li>
	<li>Cut the brain into coronal slices approximately 2 mm thick, starting from the rostral end to the caudal end, and immerse in 4% paraformaldehyde for an additional 12 h. Following fixation, wash the brain slices in running water for 1 h.</li>
	<li>Immerse the brain slices in ethanol solutions of 50%, 70%, 80%, 90%, 95%, and 100% concentrations sequentially for 35-50 min each for dehydration.</li>
	<li>Treat the brain slices with TO-type transparent biological agent I for 35-50 min, followed by clearing agent II for 35-50 min, until the tissue is fully transparent.</li>
	<li>Immerse the brain slices sequentially in embedding medium I for 60 min, embedding medium II for 60 min, embedding medium III for 60 min, and embedding medium IV for 60 min.</li>
	<li>Place the brain slices into an embedding cassette. Seal the cassette, label it clearly, and place it in a cold storage room for solidification.</li>
	<li>Set the microtome to an initial thickness of 10 &#181;m to trim the tissue block. Then, adjust the thickness to 5 &#181;m for slicing.</li>
	<li>Heat the water bath to 45 &#176;C, float the tissue sections on the water surface, and pick them up onto slides. After approximately 30 s, remove the slides, dry them at RT, and store them in a drying box.</li>
	<li>Deparaffinize the stored brain sections by immersing them in a clearing agent for 2 cycles, 10 min each.</li>
	<li>To rehydrate the sections, soak them in anhydrous ethanol for 5 min, repeating 3 times. Transfer the sections to 90% ethanol for 3 min. Further, soak the sections in 80% ethanol for 3 min. Finally, immerse the sections in 70% ethanol for 3 min.</li>
	<li>Rinse the samples in PBS 3x for 5 min each. Gently remove excess moisture using filter paper.</li>
	<li>Administer 100 &#181;L of 1x proteinase K working solution to each sample and incubate at 37 &#176;C for 20 min. Following permeabilization, rinse the samples in PBS 3x for 5 min each.</li>
	<li>Prepare samples for positive control.
	<ol>
		<li>Add 100 &#181;L of 1x DNase I Buffer working solution (TUNEL assay kit) to the permeabilized sample and allow it to equilibrate at RT for 5 min. Carefully remove any excess liquid from the sample using absorbent paper.</li>
		<li>Add 100 &#181;L of diluted DNase I working solution (200 U/mL) to the sample and incubate at 37 &#176;C for 10-30 min. Rinse the samples in PBS 3x for 5 min each.</li>
	</ol>
	</li>
	<li>Prepare samples for negative control.
	<ol>
		<li>Apply 100 &#181;L of 1x DNase I Buffer working solution to the permeabilized sample and equilibrate at RT for 5 min. Incubate the negative control sample with DNase I Buffer at 37 &#176;C for 10-30 min, ensuring no DNase I enzyme is added. Rinse the samples in PBS 3x for 5 min each.</li>
	</ol>
	</li>
	<li>Apply 100 &#181;L of terminal deoxynucleotidyl transferase (TdT) equilibration buffer (TUNEL assay kit) to each sample and incubate in a humidified chamber at 37 &#176;C for 10-30 min. Then, remove the TdT Equilibration Buffer with absorbent paper.</li>
	<li>Add 50 &#181;L of labeling working solution (TUNEL assay kit) to each sample, then incubate them at 37 &#176;C in a humidified, dark chamber for 60 min. If the signal intensity is low, consider extending the DNA labeling reaction time. Then, rinse the samples in PBS 3x for 5 min each.</li>
	<li>After blotting excess moisture, apply the DAPI working solution (TUNEL assay kit) and incubate at RT in the dark for 5 min to counterstain the nuclei. Then, rinse the samples in PBS 4x for 5 min each. Remove excess liquid with absorbent paper, and seal the slides using an anti-fade mounting medium.</li>
	<li>Scan the stained slides using the Vectra Polaris multispectral imaging system and analyze data with the system software.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Present the experimental results as means &#177; SEM and analyze data with a Student&#39;s t-test for two group comparisons using GraphPad Prism 10 software (GraphPad Software, San Diego, CA).</li>
	<li>Set a p-value less than 0.05 as a statistical difference.</li>
</ol>

Immunofluorescence Staining to Study Cell Death in Rat MCAO Stroke Models

The authors have nothing to disclose.

Occlusion of a cerebral artery leads to deprivation of oxygen and nutrition, followed by the activation of reactive oxygen species, intracellular calcium overload, glutamate release, and induction of inflammatory responses15. This series of events results in cell death and irreversible tissue damage in the infarcted brain. The penumbra is potentially salvageable through medical intervention within an appropriate therapeutic window16. The only approved treatment for ischemic...

Cerebral stroke is a leading cause of death and disability worldwide1. The high mortality and morbidity of stroke result in immense public healthcare burden and serious socio-economic consequences. Although the rate of stroke incidence and mortality remain stable, the number of stroke patients and stroke-related deaths are increasing over decades2,3. Strokes can be categorized as ischemic or hemorrhagic. The majority of stroke cases are ischemic strokes caused by the occlusion of a main cerebral artery4. To date, tissue plasminogen activator (tPA) is the only FDA-approved drug for ischemic stroke, but its application is highly limited by the narrow therapeutic window, complications, and contraindications5,6,7. Thus, it is urgent to develop new treatment options with a wider therapeutic window to mitigate the effects of stroke.
Experimental animal models are useful tools to study the pathophysiology of ischemic stroke. In most human patients, ischemic stroke is caused by the blockage of the middle cerebral artery (MCA)8. Therefore, rodent models of middle cerebral artery occlusion (MCAO) have been developed to resemble human cerebral infarction. While there are several MCAO approaches that are used in small animal studies, the method most widely used is the intraluminal suture model, which involves inserting a nylon filament into the middle cerebral artery from either the external or internal carotid arteries, resulting in transient or permanent occlusion of the blood flow4,9. This model leads to a large volume of cerebral infarction and allows examination of cell death signaling in the infarcted brain tissue with immunofluorescent techniques.
The area surrounding the infarcted core, referred to as penumbra, is the target for potential stroke therapies10,11. Since blood flow in the penumbra is partially maintained, injured neurons and non-neuronal cells in this area may be salvaged by inhibiting the activation of cell death signaling. Targeting cell death pathways may be a promising strategy for neuroprotection after stroke12. Therefore, evaluating cell death signaling is crucial for experimental stroke research. Recently, cathepsin-B, a lysosomal protease, has been shown to play a role in mediating programmed cell death after stroke, and it has gained substantial attention in the neurological field13. Cathepsin-B represents a potential target for neuroprotection that merits further investigation.
This article demonstrates how to induce MCAO using the intraluminal suture approach in rats. We also show how to perform 2,3,5-triphenyltetrazalium chloride (TTC) staining to determine infarct size and detect apoptotic cells using Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Akoya Vectra Polaris</td>
			<td>Akoya Biosciences</td>
			<td>N/A</td>
			<td>Vectra Polaris multispectral imaging system&#160;</td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Chengdu Jinshan Chemical Reagent Co.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>West China Hospital</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>West China Hospital</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cathepsin-B antibody</td>
			<td>Abcam</td>
			<td>Ab214428</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>LEICA&#160;</td>
			<td>LEICA CM 1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>OLYMPUS</td>
			<td>TG-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rat IgG (H+L) (FITC conjugated)</td>
			<td>Elabscience</td>
			<td>&#160;E-AB-1021</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Solarbio</td>
			<td>SL038</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 10 software</td>
			<td>N/A</td>
			<td>https://www.graphpad-prism.cn/?c=i&#38;a=prismdownload_cn</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>N/A</td>
			<td>https://imagej.net/ij/</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunohistochemistry pen</td>
			<td>Biosharp</td>
			<td>http://www.biosharp.cn/index/product/details/language/en/product_id/2828.html</td>
			<td></td>
		</tr>
		<tr>
			<td>InForm software</td>
			<td>Akoya Biosciences</td>
			<td>Version 2.6.0</td>
			<td>system software for the multispectral imaging system&#160;</td>
		</tr>
		<tr>
			<td>Iodophor disinfecting solution</td>
			<td>Huatian Technology Industry Co.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co.</td>
			<td>https://www.rwdstco.com/inhalation-anesthesia-solutions/</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium containing DAPI</td>
			<td>Solarbio</td>
			<td>S2110</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon monofilament</td>
			<td>RWD Life Science Co.</td>
			<td>https://www.rwdstco.com/</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal cutting temperature (OCT) compound</td>
			<td>Epredia</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Biosharp</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer solution (PBS)</td>
			<td>Solarbio</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad&#160;</td>
			<td>Version 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Tiangen</td>
			<td>RT403-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Kelun Co.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-Dawley rats</td>
			<td>Huafukang Co.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BioFroxx</td>
			<td>1245GR500</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instruments</td>
			<td>RWD Life Science Co.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>TO-type transparent biological agent&#160;&#160;</td>
			<td>Beijing Solarbio Science &#38; Tecnology Co., Ltd.</td>
			<td>http://www.solarbio.net/</td>
			<td></td>
		</tr>
		<tr>
			<td>Triphenyltetrazolium chloride (TTC) solution</td>
			<td>Solarbio</td>
			<td>G3005</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BioFroxx</td>
			<td>1139ML100</td>
			<td></td>
		</tr>
		<tr>
			<td>TUNEL kit</td>
			<td>Elabscience</td>
			<td>&#160;E-CK-A321</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating cell death signaling by immunofluorescence in a rat model of ischemic stroke

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery (MCA). Current pharmacological approaches for the treatment of ischemic stroke are limited; therefore, novel therapies providing effective neuroprotection against ischemic injury following stroke are urgently needed. In the brain tissue following ischemic stroke, the area of the ischemic penumbra is salvageable but at risk of progressing to irreversible damage. The penumbral area surrounding the infarcted core is a conceptual target for neuroprotection. Since the blood flow is partially maintained in the penumbral area, neuronal and non-neuronal cells in this area transiently survive after stroke, and these cells that are still viable may be rescued by appropriate medical interventions. Understanding the pathophysiology of the penumbra is important in the development of neuroprotective therapies because the cell death pathways activated in the ischemic penumbra may indicate therapeutic targets, such as RUNX1 and cathepsins. These protein targets functioning as mediators of programmed cell death can be further exploited in translational research. With moderate size and similarity to the human brain, the in vivo rat model of MCA occlusion (MCAO) mimics the human ischemic stroke and offers an applicable tool to investigate the penumbral pathology, examine the cell death signaling, and evaluate the effects of potential targets in the context of MCAO. Here, we describe how to induce MCAO in rats and how to perform immunofluorescence staining for the detection of cell death signaling in the rat brain following MCAO.

Rats are subjected to 120 min ischemia during MCAO followed by 22 h reperfusion. The mortality rate was minimal during MCAO surgery and was around 25% within the period of reperfusion. Significant brain damage was observed in the MCAO group, whereas no infarct was observed in the cerebrum of the Sham group (24.87 &#177; 1.21% vs. 0% sum of infarct area to the whole brain area; MCAO [n = 3] vs. Sham [n = 3], P &#60; 0.001; Figure 1A,B). In the MCAO group, immunoreact...

Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke

Rat in vivo models are indispensable tools to investigate the pathological mechanisms and therapeutic targets for ischemic stroke. This work will describe performing immunofluorescence staining in infarcted brain slices following middle cerebral artery occlusion (MCAO) in rats.

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery ...

evaluating-cell-death-signaling-immunofluorescence-rat-model-ischemic

Research

JoVE Journal

Neuroscience

543 Views.  Sichuan University. In vivo modellen van ratten zijn onmisbare hulpmiddelen om de pathologische mechanismen en therapeutische doelen voor ischemische beroerte te onderzoeken. Dit werk beschrijft het uitvoeren van immunofluorescentiekleuring in hersenplakjes met een infarct na occlusie van de middelste hersenslagader (MCAO) bij ratten.

Video: Evaluatie van celdoodsignalering door immunofluorescentie in een rattenmodel van ischemische beroerte

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

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

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

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

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

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Optimized Management of Endovascular Treatment for Acute Ischemic Stroke

This manuscript describes a streamlined protocol for the management of patients with acute ischemic stroke, which aims at the minimization of time from hospital admission to reperfusion. Rapid restoration of cerebral blood flow is essential for the outcomes of patients with acute ischemic stroke. Endovascular treatment (EVT) has become the standard of care to accomplish this in patients with acute stroke due to large vessel occlusion (LVO). To achieve reperfusion of ischemic brain regions as fast as possible, all in-hospital time delays have to be carefully avoided. Therefore, management of patients with acute ischemic stroke was optimized with an interdisciplinary standard operating procedure (SOP). Stroke neurologists, diagnostic as well as interventional neuroradiologists, and anesthesiologists streamlined all necessary processes from patient admission and diagnosis to EVT of eligible patients. Target times for every step were established. Actually achieved times were prospectively recorded along with clinical data and imaging scores for all endovascularly treated stroke patients. These data were regularly analyzed and discussed in interdisciplinary team meetings. Potential issues were evaluated and all staff involved was trained to adhere to the SOP. This streamlined patient management approach and enhanced interdisciplinary collaboration reduced time from patient admission to reperfusion significantly and was accompanied by a beneficial effect on clinical outcomes.

The outcome of patients with acute ischemic stroke depends on swift restoration of cerebral blood flow. This protocol aims at optimizing the management of such patients by minimizing peri-procedural timings and rendering the time from hospital admission to reperfusion as short as possible.

This manuscript describes a streamlined protocol for the management of patients with acute ischemic stroke, which aims at the minimization of time from ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32&#8211;36 weeks&#39; gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants.

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke

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