This work was supported by the University of Basel, Department of Biomedicine, and the Swiss National Science Foundation (31003A_141007). Markus Saxer provided technical support.

Animal experiments were carried out in accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU) and were reviewed and permitted by Swiss authorities.
1. Setting up Organotypic Entorhino-hippocampal Slice Cultures
<ol>
	<li>Prepare entorhino-hippocampal organotypic slice cultures (EHOSCs) from postnatal day 4 (P4) mouse pups using the static incubation method4,11. Here, use C57Bl6 and CB6F1 mice.
	<ol>
		<li>Take approximately 30 min per mouse pup for the preparation of slice cultures, i.e., 3 hr for a litter of 6 mouse pups. Carry out all steps under sterile conditions in a laminar flow workbench with sterilized surgical instruments.</li>
		<li>In order to avoid a possible interference from the position of the slice along the septo-temporal axis, use only hippocampal slices of the septal half. If not stated otherwise, carry out all steps at RT.</li>
	</ol>
	</li>
	<li>Cut off the head of a P4 mouse pup with surgical scissors. No anesthesia is required for this step.
	<ol>
		<li>Remove the skull around the brain and identify the fissure between the caudal end of the cerebral hemispheres and the midbrain. Carefully remove the part of the brain rostral to the midbrain from the skull and place it in ice-cold preparation medium consisting of MEM supplemented with a stabilized form of L-glutamine.</li>
	</ol>
	</li>
	<li>Continue the dissection under a stereomicroscope with the brain being immersed in the ice cold preparation medium. Remove remaining meninges from the cortical hemispheres. Identify the hippocampus at the medial caudal surface of the hemisphere and separate it together with the adjacent entorhinal cortex from the remainder of the brain.
	<ol>
		<li>Transfer the tissue to a tissue chopper and cut 400 &#181;m thick slices under aseptic conditions transversal to the septo-temporal axis of the hippocampus.</li>
	</ol>
	</li>
	<li>Place the slices on cell culture inserts (approximately 6 slices per insert) and incubate them in 6-well plates with 1 ml per well of incubation medium (47 ml MEM, 25 ml Eagle Medium, 25 ml horse serum, 1 ml glutamax I, 1 ml of a sterile 10% glucose solution, pH 7.3) in a humidified atmosphere with 5% CO2 at 37 &#176;C. Change the medium the next day and then every other day up to 1 week.</li>
</ol>
2. In Vitro Hypoxia and Oxygen-glucose Deprivation with Reperfusion
<ol>
	<li>See Table 1 for an exact time schedule of the procedure. Culture EHOSCs for 5 days (DIV5) before exposure to hypoxia or OGD. At this time point switch the medium from incubation medium containing 25% horse serum to serum free medium with the following composition: Neurobasal medium with 2% B27 supplement, 1% glutamax and additional 0.1% glucose.</li>
	<li>Prepare the OGD medium as detailed in Reference 7 .The final concentrations of the components will be the following: 0.3 mM CaCl2, 70 mM NaCl, 5.25 mM NaHCO3, 70 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, and 10 mM sucrose at pH 6.8. Add 1 ml of OGD medium to each well of two 6-well tissue culture plates.
	<ol>
		<li>Perfuse the OGD medium with N2 for 1 hr in a hypoxia chamber and then seal and keep O/N in an incubator at 37 &#176;C.</li>
		<li>Use anaerobic strips as hypoxic indicators. Consider the medium to be oxygen free when the color of the strips changed from pink to white.</li>
		<li>For the induction of hypoxia, use serum free medium with glucose instead of OGD medium. Before use, perfuse the medium with N2 in a hypoxia chamber as detailed above for OGD medium.</li>
	</ol>
	</li>
	<li>Before the slices are exposed to OGD, perfuse the OGD medium again with N2 for 30 min. Add 2 &#181;l of a 1 mg/ml propidium iodide (PI) solution per well (for a final concentration of 2 &#181;g/ml) to the slices for 30 min and select only healthy slices for experimentation as indicated by low numbers of PI positive cells.</li>
	<li>Use the schedule of OGD exposure and reperfusion indicated in Figure 1. Deprive slices of either oxygen only (hypoxia) or oxygen and glucose (OGD) for 15 min.
	<ol>
		<li>For induction of hypoxia or OGD, transfer slices into OGD medium and keep for 15 min in the hypoxia chamber. Ensure that all fluids that remain on the sides of the insert are aspirated away when the cultures are switched from regular medium to OGD medium and back.</li>
		<li>After hypoxia or OGD, replace the OGD medium with oxygenated serum free medium and cultures are allowed to recover for 3, 24 or 48 hr in serum free medium in the regular incubator at 37 &#176;C with 5% CO2. Add PI again prior to fixation for the determination of cell death.</li>
	</ol>
	</li>
</ol>
3. Pharmacological Treatments of Entorhino-hippocampal Organotypic Slice Cultures
<ol>
	<li>Culture EHOSCs for 5 days (DIV5) in incubation medium before exposure to pharmacological treatments. Start pharmacological treatments either during OGD or immediately after OGD when slices are transferred to serum free medium.</li>
	<li>Treatments for protection from OGD.
	<ol>
		<li>Add 100 &#181;M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 1 &#181;M tetrodotoxin (TTX) to the culture medium during OGD induction (for 15 min) or immediately after OGD induction for 30 min. Then, incubate in normoxic serum free medium for 48 hr before fixation.</li>
	</ol>
	</li>
	<li>Treatments for induction of excitotoxic death.
	<ol>
		<li>After 5 days in incubation medium switch cultures to serum free medium containing 100 &#181;M (RS)-&#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) for 30 min.</li>
		<li>Remove the culture medium containing AMPA by aspiration (take care to remove any residual medium on inserts), then transfer the slices to fresh plates with normal serum free medium and keep them for 48 hr until fixation.</li>
	</ol>
	</li>
</ol>
4. Fixation and Immunostaining of Entorhino-hippocampal Organotypic Slice Cultures
<ol>
	<li>Propidium iodide (PI) staining.
	<ol>
		<li>Add PI for 30 min to the culture medium of living slices at a concentration of 2 &#181;g/ml. View live cultures with an inverted microscope equipped with fluorescence optics and select slices for further treatments. For PI staining in combination with immunohistochemistry, add PI solution 30 min prior to fixation of the cultures.</li>
	</ol>
	</li>
	<li>Fixation of the slices.
	<ol>
		<li>Remove the culture medium from the 6-well plate and add 3 ml of freshly prepared 4% paraformaldehyde (PFA) to each well. Then place the 6-well plate in a refrigerator and fix O/N at 4 &#176;C.</li>
		<li>Remove the PFA solution the following day and wash the slices 3-4 times with phosphate buffered saline (PBS).</li>
	</ol>
	</li>
	<li>Removal of the slices from the cell culture insert and blocking procedure for immunohistochemistry of free-floating sections.
	<ol>
		<li>Carefully detach and remove the slices from the culture inserts using an art paint brush of the size 0 or smaller. Transfer each individual slice into a well of a 96 well plate filled with blocking buffer (PBS with 3% normal goat serum (NGS) and 2% Triton-X100). Keep the slices in the blocking buffer for 2 hr at RT&#160;under constant agitation by an orbital shaker.</li>
	</ol>
	</li>
	<li>Immunostaining of the slice cultures
	<ol>
		<li>For labeling blood vessels use polyclonal anti-laminin at a dilution of 1:200 in PBS containing 1% NGS and 0.5% Triton-X100.</li>
		<li>For labeling neurons use anti-microtubule associated protein 2 (MAP2) at a dilution of 1:500 in PBS containing 1% NGS and 0.5% Triton-X100.</li>
		<li>Incubate the slices with primary antibodies for 48 hr at 4 &#176;C with constant agitation. After the incubation period, wash them in PBS with 0.5% Triton-X100 for 3-4 times.</li>
		<li>Dilute secondary antibodies 1:500 in PBS containing 1% NGS and 0.5% Triton-X100. Incubate the slices for 2 hr at RT in the dark with the Alexa fluorophore conjugated secondary antibodies. Use the appropriate goat anti-rabbit or goat anti-mouse secondary antibody conjugated to Alexa 350 or 488.</li>
		<li>Remove the secondary antibody solution and wash the slices three times with Tris-buffered saline (TBS); mount them on glass slides and coverslip the slides with Mowiol.</li>
	</ol>
	</li>
	<li>Microscopy of the stained slices</li>
	<li>View the stained slices either on a standard microscope with epifluorescence equipment or with a confocal microscope. Take images with an appropriate camera. For some images, invert the fluorescence contrast in order to yield darkly stained vessels on a bright background (Figures 5 and 6).</li>
</ol>
5. Determination of Regional Vessel Density in the Cultures
<ol>
	<li>Quantify the vessel density based on vessel crossings as described in Reference 8.</li>
	<li>Use slices stained for laminin for these measurements. Superimpose recorded images of 10X magnification with a 6 x 6 grid, where each grid is equal to 100 x 100 &#181;m per square, total field of 0.25 mm2.</li>
	<li>Overlay three such grids in the CA1, CA3, DG, and EC region (Figure 2). Count the vessels crossing the lines of the grid.</li>
	<li>For obtaining results with a good statistical relevance, make measurements in at least 3 independent experiments, each including data from 3 mouse pups with 3 slices per mouse pup.</li>
	<li>Determine the average value of vessel crossings from the grids for each region and perform a statistical analysis by ANOVA with Bonferroni correction as post hoc test (p&#60;0.05 defined as significant) using appropriate statistics software.</li>
</ol>

<ol>
	<li>Turner, R. C., Dodson, S. C., Rosen, C. L., Huber, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+science+of+cerebral+ischemia+and+the+quest+for+neuroprotection:+navigating+past+failure+to+future+success.">The science of cerebral ischemia and the quest for neuroprotection: navigating past failure to future success.</a> J Neurosurg. 118, 1072-1085 (2013).</li><li>Bacigaluppi, M., Comi, G., Hermann, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ischemic+stroke.+Part+two:+modeling+cerebral+ischemia.">Animal models of ischemic stroke. Part two: modeling cerebral ischemia.</a> Open Neurol J. 4, 34-38 (2010).</li><li>Woodhams, P. L., Atkinson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+entorhino-dentate+projections+in+organotypic+slice+cultures:+mode+of+axonal+regrowth+and+effects+of+growth+factors.">Regeneration of entorhino-dentate projections in organotypic slice cultures: mode of axonal regrowth and effects of growth factors.</a> Exp Neurol. 140, 68-78 (1996).</li><li>Prang, P., Del Turco, D., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+entorhinal+fibers+in+mouse+slice+cultures+is+age+dependent+and+can+be+stimulated+by+NT-4,+GDNF,+and+modulators+of+G-proteins+and+protein+kinase.">Regeneration of entorhinal fibers in mouse slice cultures is age dependent and can be stimulated by NT-4, GDNF, and modulators of G-proteins and protein kinase.</a> C. Exp Neurol. 169, 135-147 (2001).</li><li>Bonnici, B., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+regeneration+of+intrinsic+spinal+cord+axons+in+a+novel+spinal+cord+slice+culture+model.">Spontaneous regeneration of intrinsic spinal cord axons in a novel spinal cord slice culture model.</a> Eur J Neurosci. 27, 2483-2492 (2008).</li><li>Laake, J. H., Haug, F. M., Wieloch, T., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+in+vitro+model+of+ischemia+based+on+hippocampal+slice+cultures+and+propidium+iodide+fluorescence.">A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence.</a> Brain Res Brain Res Protoc. 4, 173-184 (1999).</li><li>Rytter, A., Cronberg, T., Aszt&#233;ly, F., Nemali, S., Wieloch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+hippocampal+organotypic+tissue+cultures+exposed+to+in+vitro+&#8216;ischemia&#8217;+show+selective+and+delayed+CA1+damage+that+is+aggravated+by+glucose.">Mouse hippocampal organotypic tissue cultures exposed to in vitro &#8216;ischemia&#8217; show selective and delayed CA1 damage that is aggravated by glucose.</a> J Cereb Blood Flow Metab. 23, 23-33 (2003).</li><li>Bendfeldt, K., Radojevic, V., Kapfhammer, J., Nitsch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+modulates+density+of+blood+vessels+and+preserves+tight+junctions+in+organotypic+cortical+cultures+of+mice:+a+new+in+vitro+model+of+the+blood-brain+barrier.">Basic fibroblast growth factor modulates density of blood vessels and preserves tight junctions in organotypic cortical cultures of mice: a new in vitro model of the blood-brain barrier.</a> J Neurosci. 27, 3260-3267 (2007).</li><li>Camenzind, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+transendothelial+glucose+transporter+1+and+P-glycoprotein+transporters+in+a+cortical+slice+culture+model+of+the+blood-brain+barrier.">Preservation of transendothelial glucose transporter 1 and P-glycoprotein transporters in a cortical slice culture model of the blood-brain barrier.</a> Neuroscience. 170, 361-371 (2010).</li><li>De Jong, G. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+hypoperfusion+yields+capillary+damage+in+the+hippocampal+CA1+area+that+correlates+with+spatial+memory+impairment.">Cerebral hypoperfusion yields capillary damage in the hippocampal CA1 area that correlates with spatial memory impairment.</a> Neuroscience. 91, 203-210 (1999).</li><li>Cavaglia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+variation+in+brain+capillary+density+and+vascular+response+to+ischemia.">Regional variation in brain capillary density and vascular response to ischemia.</a> Brain Res. 910, 81-93 (2001).</li><li>Chip, S., Nitsch, C., Wellmann, S., Kapfhammer, J. 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Animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were reviewed and permitted by Swiss authorities. The authors have nothing to disclose.

With the methods presented here, hippocampal organotypic slice cultures can be used as a versatile tool to study plastic changes in neural tissue after injury. While organotypic slice cultures have been used in the past for studying neuronal reactions after ischemia6, 7 the new aspect of the simultaneous study of vascular changes significantly enhances the potential applications of this method. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads t...

The central nervous system is particularly sensitive to a loss or reduction of oxygen and glucose supply by the vasculature. Even a rather short interruption of blood supply to the brain can induce a permanent loss of function of the relevant brain areas leading to the typical stroke syndromes. In addition to the neuronal loss in the primary affected areas, there typically is additional delayed neuronal loss through secondary damage. Unfortunately until now, no neuroprotective treatment for the reduction of secondary neuronal death was available1. Research efforts for studying the mechanisms of secondary damage rely on the use of animal models of cerebral ischemia like middle cerebral artery occlusion and various thrombotic occlusion techniques (for a recent review see2). In parallel, also due to limitations and ethical concerns with the use of animal models, organotypic slice culture of various CNS tissues have been used that allow the study of neuronal reactions to various type of injuries3-5.
For studying the neuronal reaction under conditions that mimic ischemic brain injury, the model system of oxygen glucose deprivation (OGD) has been developed. In this model, slice cultures are temporarily exposed to a medium that lacks glucose and has been equilibrated with nitrogen gas in the absence of oxygen. With such a treatment, it is possible to induce neuronal injury and loss which is rather similar to the one observed after ischemic injury in vivo6, 7. In the hippocampus, such a treatment induces neuronal loss specifically in the CA1, but not in the CA3 area or the dentate gyrus of the hippocampus. In contrast, the study of vascular reactions in slice cultures so far has not been widely undertaken. An obvious reason is the lack of circulation and blood vessel perfusion in the slice culture model. However, we have shown previously that it is possible to maintain blood vessels in CNS slice cultures for several days8, 9.
The overall goal of this method is to not only monitor the fate of neurons after OGD but extend the study to the fate and remodeling of the vasculature which is an important part of the injury response. Until now such studies have required the use of animal experiments (De Jong et al., 1999; Cavaglia et al., 2001). In the protocol presented here, we will detail how such studies can be done in entorhino-hippocampal organotypic slice cultures challenged either with hypoxia or by excitotoxic lesions followed by analysis of both neuronal survival and blood vessel reactions. This protocol is based on a previously published study on this subject10 and can be useful for any laboratory interested in neurovascular interactions in the CNS.

<table><tbody><tr><td>Minimum Essential Medium MEM</td><td>Gibco</td><td>11012-044</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td>stabilized form of L-glutamine</td></tr><tr><td>Millicell cell culture inserts</td><td>Millipore</td><td>PICM03050</td><td></td></tr><tr><td>Basal medium Eagle&amp;nbsp;</td><td>Gibco</td><td>41010-026</td><td></td></tr><tr><td>Horse serum</td><td>Gibco</td><td>26050-088</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>Anaerobic strips</td><td>Sigma-Aldrich</td><td>59886</td><td></td></tr><tr><td>Propidium iodide solution</td><td>Sigma-Aldrich</td><td>P4864</td><td></td></tr><tr><td>AMPA</td><td>R&amp;amp;D systems</td><td>0169-10</td><td></td></tr><tr><td>CNQX</td><td>R&amp;amp;D systems</td><td>0190/10</td><td></td></tr><tr><td>TTX</td><td>R&amp;amp;D systems</td><td>1078/1</td><td></td></tr><tr><td>polyclonal anti-laminin</td><td>Sigma-Aldrich</td><td>L9393&amp;nbsp;</td><td></td></tr><tr><td>anti-MAP2</td><td>Abcam</td><td>ab11267</td><td></td></tr><tr><td>Alexa anti-mouse 350</td><td>Molecular Probes</td><td>A11045</td><td></td></tr><tr><td>Alexa anti-mouse 488</td><td>Molecular Probes</td><td>A11001</td><td></td></tr><tr><td>Alexa anti-rabbit 350</td><td>Molecular Probes</td><td>A11046</td><td></td></tr><tr><td>Alexa anti-rabbit 488</td><td>Molecular Probes</td><td>A11008</td><td></td></tr><tr><td>Statistics software</td><td>GraphPad Software</td><td>GraphPad Prism</td><td></td></tr><tr><td>McIlwain tissue chopper</td><td>Ted Pella</td><td>10180</td><td></td></tr><tr><td>Hypoxia chamber</td><td>Billups-Rothenberg</td><td>MIC-101</td><td></td></tr></tbody></table>

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.

Oxygen glucose deprivation and hypoxia induce neuronal death and blood vessel reduction specifically in the hippocampal CA1 region. 
OGD or oxygen-deprivation alone for 15 min induced a strong induction of cell death as seen by propidium iodide staining specifically in the CA1 area of the hippocampus (Figure 3) similar as described previously7. With markers to visualize the network of blood vessels, it was found that blood vessel density and organization appeared similar to the c...

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

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 ...

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Neuroscience

9.7K Views.  University of Basel. 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.

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

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Developmental Biology

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

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. ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential 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 ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...