This work was supported by research funds from the National Health and Medical Research Council of Australia, the Hunter Medical Research Institute, and the University of Newcastle.&#160;

All procedures are performed with approval from the University of Newcastle Animal Care and Ethics Committee, as well as in accordance with the relevant guidelines and regulations, including the NSW Animal Research Act, the NSW Animal Research Regulation, and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
1. Dissection of Brain Tissue
<ol>
	<li>Sacrifice twelve week old male rats by decapitation. Rats can either be sacrificed by stunning and decapitation, or can be first anesthetized with isoflurane (5% induction, 1.5-2% maintenance) in 70% N2 and 30% O2, and then decapitated.</li>
	<li>Remove the brain from the skull as rapidly as possible (ideally, this should be performed in less than 2 min).</li>
	<li>Remove the cortex by free-hand dissection. The dissection should be performed in the minimum time possible, with as little damage to the brain as possible so as to maintain the integrity of the slices.</li>
	<li>In order to remove any residual skin, fur, blood, etc., immerse the brain in a small amount (3-5 ml) of 37 &#176;C Krebs buffer (118 mM sodium chloride, 4.7 mM potassium chloride, 1.3 mM calcium chloride, 1.2 mM potassium dihydrogen phosphate, 1.2 mM magnesium sulfate, 25 mM sodium hydrogen carbonate, 11.7 mM glucose, 0.03 mM disodium ethylenediaminetetraacetic acid (EDTA), equilibrated with 95% oxygen/5% carbon dioxide (carbogen), pH 7.4).</li>
</ol>
2. Preparation of Microslices
<ol>
	<li>Place the brain on the stage of a McIlwain chopper on which two slices of filter paper have been placed and moistened with warm Krebs buffer. Keep the brain and filter paper moistened with warm Krebs buffer, but not so wet that there is free liquid on the surface of the paper that can cause the brain to slide around.</li>
	<li>Slice the brain into 250 &#181;m coronal sections. As nutrients, oxygen, and waste materials that are normally exchanged with the blood supply are exchanged with a fluid which surrounds the tissue in isolated neural tissue, the size of the slices should not be more than 400 &#181;m in thickness to allow adequate oxygenation and nutrient exchange to occur.</li>
	<li>Turn the McIlwain stage 90&#176;.</li>
	<li>Slice brains into 250 &#181;m sagittal slices. Herein, these brain sections (250 &#181;m&#160;x 250 &#181;m slices of variable length depending on the thickness of the tissue) are referred to as microslices.</li>
	<li>Place the microslices into a round bottom tube with 10-15 ml 37 &#176;C Krebs buffer (the amount of Krebs buffer used will depend on the amount of tissue).</li>
</ol>
3. Equilibration of Microslices
<ol>
	<li>Gently agitate until individual microslices are suspended, and then allow them to settle under gravity.</li>
	<li>Carefully remove the supernatant, which will be cloudy due to suspended cell debris, and add 10 ml fresh 37 &#176;C Krebs buffer to the tube.</li>
	<li>Repeat this washing procedure four times, which will result in the removal of the majority of debris.</li>
	<li>Following this, equilibrate the microslices at 37 &#176;C with gentle shaking/inversion, and change the Krebs buffer every 15 min for 1 hr in total.</li>
	<li>At this time, add 2 ml fresh 37 &#176;C Krebs buffer, and transfer 15-20 microslices (150 &#181;l of microslice suspension) to flat bottom polystyrene 5 ml tubes using an extra wide bore pipette tip with smoothed edges (which can be created by cutting ~2 mm from the end of a P1000 tip). Spread the microslices evenly over the base of the tube in a single layer, so that each microslice is not further than a few millimeters from the oxygenated atmosphere.</li>
</ol>
4. Excitotoxic Stimulation
<ol>
	<li>Incubate the microslices at 37 &#176;C, and continually pass carbogen over the surface of the 150 &#181;l microslice suspension, using a gas line that is positioned just above the surface of the microslice suspension (to avoid mechanical disruption).</li>
	<li>Treat microslices with either 150 &#181;l 5 mM glutamate + 100 &#181;M glycine in Krebs buffer (excitotoxic stimulus: final concentration 2.5 mM glutamate + 50 &#181;M glycine) or 150 &#181;l Krebs buffer alone (control nonstimulated). A variety of stimuli can be used at this step (including, but not limited to, AMPA, NMDA + glycine, KCl depolarization, and oxygen/glucose deprivation).</li>
	<li>Remove the incubation solution at various times post-stimulation (0, 30, 60, 90, 120, and 300 sec), and replace it with 300 &#181;l of ice-cold homogenization buffer (30 mM Tris, pH 7.4, 1 mM ethylene glycol tetraacetic acid, 4 mM EDTA, 100 &#181;M ammonium molybdate, 5 mM sodium pyrophosphate, 25 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonylfluoride, complete protease inhibitor cocktail).</li>
	<li>Homogenize microslices on ice, using a glass-Teflon Dounce Homogenizer (20 strokes; 700 rpm).</li>
	<li>Store homogenates at -80 &#176;C, and various signaling molecules, death molecules, etc. can be measured by western blot.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+cAMP/PKA+and+MAPK+signaling+in+neuronal+cells+is+dependent+on+developmental+stage.">Coupling of cAMP/PKA and MAPK signaling in neuronal cells is dependent on developmental stage.</a> Exp. Neurol. 169 (1), 44-55 (2001).</li><li>Kharlamov, E., Cagnoli, C. M., Atabay, C., Ikonomovic, S., Grayson, D. R., Manev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+effect+of+protein+synthesis+inhibitors+on+potassium+deficiency-induced+apoptotic+cell+death+in+immature+and+mature+neuronal+cultures.">Opposite effect of protein synthesis inhibitors on potassium deficiency-induced apoptotic cell death in immature and mature neuronal cultures.</a> J. Neurochem. 65 (3), 1395-1398 (1995).</li><li>Kristensen, B. 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The authors declare that they have no conflict of interest regarding any of the work conducted within this manuscript.

Herein, an in vitro technique for the generation of microslices that can be used to examine the molecular mechanisms involved in excitotoxicity and ischemia-mediated cell death in intact mature brain tissue is described. This technique produces viable tissue (Figures 1-3), that is metabolically similar to larger organotypic slices15. Furthermore, this microslice model closely corresponds to the response observed following excitotoxicity mediated brain injuries in vivo10<...

The most common form of stroke is ischemic stroke, which occurs when a cerebral blood vessel becomes occluded. The tissue ischemia which results from cessation of blood flow causes widespread depolarization of membranes, release of excitatory neurotransmitters, and sustained elevation of intracellular calcium, which leads to the activation of cell death pathways 1. This process has been termed &#39;excitotoxicity&#39;, and is a common pathway involved in neuronal death produced by a variety of pathologies, including stroke 2. Inhibition of the signaling pathways involved in excitotoxicity and other neuronal cell death cascades is an appealing approach to limit neuronal damage following stroke.
Identifying the precise molecular mechanisms involved in excitotoxicity and ischemic stroke can be difficult when using whole animal systems. As such, primary embryonic and &#39;neuronal-like&#39; (e.g. neuroblastoma and adenocarcinoma immortalized lines) cell culture systems are often used. The main advantages of these models are that they are easy to manipulate, relatively cost-effective, and cell death can be readily measured and quantitated. However, signaling pathways can be altered by the culturing conditions used 3,4, and immature neurons and immortalized lines can express different receptors and signaling molecules when compared to mature brain 5-8. Furthermore, cultured neurons only allow the examination of one cell type (or two, if a coculture system is used), whereas intact brain tissue is heterogeneous, containing a variety of cell types that interact with each other. Organotypic slice culture systems (thin explants of brain tissue) are also used, and these models allow the study of heterogeneous populations of cells as they are found in vivo. However, only a limited amount of tissue can be obtained from each animal when using this technique, slices cannot be cultured for as long as immortalized cell lines, and medium to long-term culture can result in alterations in signaling pathways and receptors in the slices. Whilst mature brain can be used to generate organotypic slices, slices from immature brain are more amenable to culture, and are more commonly utilized. There is therefore the need to develop models which mimic or represent intact mature brain, that are easy to use, in which neuronal signaling pathways can be examined.
Herein, an in vitro technique involving intact nervous tissue that can be used to elucidate molecular mechanisms involved in cell death following an excitotoxic or ischemic insult is described. This technique reduces the number of animals required to perform an experiment, is reproducible, and generates viable tissue that behaves in a metabolically similar fashion to larger organotypic slices. Additionally, the neuronal circuitry, cellular interactions, and postsynaptic compartment remains partly intact. The physiological buffer used allows the cell membranes to &#39;reseal&#39;, and enables cells to recover their original membrane resistance 9. This brain slice model is able to faithfully mimic responses observed following excitotoxicity mediated brain injuries 10, and can be used to examine the molecular mechanisms involved in stroke.

<table><tbody><tr><td>Guillotine</td><td></td><td></td><td>Used to decapitate animal</td></tr><tr><td>Surgical equipment</td><td></td><td></td><td>Forceps, scissors, tweezers, &lt;em&gt;etc.&lt;/em&gt;, for brain removal and dissection</td></tr><tr><td>McIlwain chopper</td><td>McIlwain choppers are manufactured/distributed by a range of companies including Mickle Engineering, Harvard Apparatus, Campden Instruments and Ted Pella.</td><td></td><td>Used to generate 100-400 &amp;#181;m brain sections.</td></tr><tr><td>Round bottom plastic tubes</td><td>Greiner</td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td>For keeping tissue at 37 &amp;deg;C</td></tr><tr><td>Humidifier/aerating apparatus</td><td></td><td></td><td>Used to keep microslices in a humidified, oxygenated environment</td></tr><tr><td>Flat bottomed polystyrene tubes</td><td>Nunc</td><td></td><td></td></tr><tr><td>Dounce Homogenizer</td><td></td><td></td><td></td></tr></tbody></table>

excitotoxic stimulation of brain microslices as an in vitro model of stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Microslices generated using this procedure are viable, and a variety of species (for example, rat, mouse, and chicken) can be used to produce microslices. Three independent measures of viability have been utilized: respiration rate (Figure 1), adenine nucleotide ratios (Figure 2), and tissue potassium content (Figure 3). Using these measures, it has been demonstrated that microslices remain viable for at least 2 hr post-generation.
Brain micro...

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Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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8.4K Views.  The University of Newcastle. We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury. This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Video: Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Immunology and Infection

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Neuroscience

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

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Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model