<meta charset="utf8"></head><body>쥐 MCAO 뇌졸중 모델의 세포 사멸을 연구하기 위한 면역형광 염색

<table><tbody><tr><td>Akoya Vectra Polaris</td><td>Akoya Biosciences</td><td>N/A</td><td>Vectra Polaris multispectral imaging system&amp;nbsp;</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&amp;nbsp;</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>&amp;nbsp;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&amp;amp;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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;&amp;nbsp;</td><td>Beijing Solarbio Science &amp;amp; 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>&amp;nbsp;E-CK-A321</td><td></td></tr></tbody></table>

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

<meta charset="utf8"></head><body>허혈성 뇌졸중의 쥐 모델에서 면역형광에 의한 세포 사멸 신호 평가

허혈성 뇌졸중의 쥐 모델에서 면역형광에 의한 세포 사멸 신호 평가

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

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

558 조회수.  Sichuan University. 쥐 생체 내 모델은 허혈성 뇌졸중의 병리학적 메커니즘과 치료 표적을 조사하는 데 필수적인 도구입니다. 이 연구는 쥐의 중대뇌동맥 폐색(MCAO) 후 경색된 뇌 절편에서 면역형광 염색을 수행하는 방법을 설명합니다.

비디오: 허혈성 뇌졸중의 쥐 모델에서 면역형광에 의한 세포 사멸 신호 평가

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.

쥐에서 자당의 갈망의 보육을 평가하기위한 일반적인 방법

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 ...

연구

신경과학

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.

전자관 X-선 마이크로 분석에 의한 뉴런의 총 칼슘의 측정

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.

신경 발사 환율의 조작에 의해 성상 세포 수용체의 가소성을 유도

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.

기존의 UV 레이저 공 초점 현미경을 사용하여 트리거 세포 스트레스와 죽음

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.

신생아 Hypoxic-허 혈 성 뇌 손상의 마우스 모델에서 Neurobehavioral 평가

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.

급성 허 혈 성 뇌졸중 혈관 내 수술 치료의 최적화 관리

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.

허 혈 성 뇌졸중의 쥐 모델에서 뇌혈관 장애의 비보에 평가

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.

염증 과민성 후기 조저산소-허혈성 뇌 손상의 페렛 모델

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.

쥐에서 급성 뇌졸중 후 뇌 회복을 평가하는 전임상 모델

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 ...