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

Summary

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.

Abstract

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 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.

Introduction

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 injuries^3-5.

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 vivo^{6, 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 days^{8, 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.

Protocol

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

1. Prepare entorhino-hippocampal organotypic slice cultures (EHOSCs) from postnatal day 4 (P4) mouse pups using the static incubation method^4,11. Here, use C57Bl6 and CB6F1 mice.
   1. 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.
   2. 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.
2. Cut off the head of a P4 mouse pup with surgical scissors. No anesthesia is required for this step.
   1. 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.
3. 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.
   1. Transfer the tissue to a tissue chopper and cut 400 µm thick slices under aseptic conditions transversal to the septo-temporal axis of the hippocampus.
4. 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 °C. Change the medium the next day and then every other day up to 1 week.

2. In Vitro Hypoxia and Oxygen-glucose Deprivation with Reperfusion

1. 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.
2. 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.
   1. 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 °C.
   2. Use anaerobic strips as hypoxic indicators. Consider the medium to be oxygen free when the color of the strips changed from pink to white.
   3. 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.
3. Before the slices are exposed to OGD, perfuse the OGD medium again with N₂ for 30 min. Add 2 µl of a 1 mg/ml propidium iodide (PI) solution per well (for a final concentration of 2 µg/ml) to the slices for 30 min and select only healthy slices for experimentation as indicated by low numbers of PI positive cells.
4. 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.
   1. 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.
   2. 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 °C with 5% CO₂. Add PI again prior to fixation for the determination of cell death.

3. Pharmacological Treatments of Entorhino-hippocampal Organotypic Slice Cultures

1. 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.
2. Treatments for protection from OGD.
   1. Add 100 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 1 µM 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.
3. Treatments for induction of excitotoxic death.
   1. After 5 days in incubation medium switch cultures to serum free medium containing 100 µM (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) for 30 min.
   2. 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.

4. Fixation and Immunostaining of Entorhino-hippocampal Organotypic Slice Cultures

1. Propidium iodide (PI) staining.
   1. Add PI for 30 min to the culture medium of living slices at a concentration of 2 µg/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.
2. Fixation of the slices.
   1. 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 °C.
   2. Remove the PFA solution the following day and wash the slices 3-4 times with phosphate buffered saline (PBS).
3. Removal of the slices from the cell culture insert and blocking procedure for immunohistochemistry of free-floating sections.
   1. 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 under constant agitation by an orbital shaker.
4. Immunostaining of the slice cultures
   1. For labeling blood vessels use polyclonal anti-laminin at a dilution of 1:200 in PBS containing 1% NGS and 0.5% Triton-X100.
   2. 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.
   3. Incubate the slices with primary antibodies for 48 hr at 4 °C with constant agitation. After the incubation period, wash them in PBS with 0.5% Triton-X100 for 3-4 times.
   4. 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.
   5. 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.
5. Microscopy of the stained slices
6. 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).

5. Determination of Regional Vessel Density in the Cultures

1. Quantify the vessel density based on vessel crossings as described in Reference 8.
2. 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 µm per square, total field of 0.25 mm².
3. Overlay three such grids in the CA1, CA3, DG, and EC region (Figure 2). Count the vessels crossing the lines of the grid.
4. 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.
5. 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<0.05 defined as significant) using appropriate statistics software.

Results

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 con...

Discussion

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 ischemia^{6, 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...

Materials

List of materials used in this article

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