Name: organotypic hippocampal slice cultures as a model to study neuroprotection and invasiveness of tumor cells
Uploaded: 2017-08-27T13:00:00
Description: Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells at JoVE.com

The authors would like to thank Christine Auste for her support with the video recording and Chalid Ghadban for his excellent technical assistance. Urszula Grabiec was supported by the Roux Programm FKZ 29/18.

Animal experiments were performed in accordance with the Policy of Ethics and the Policy on the Use of Animals in Neuroscience Research as approved by the European Communities Council Directive 2010/63/EU of the European Parliament and of the Council of the European Union on the protection of animals used for scientific purposes.
1. Preparation of Instruments and Culture Media
<ol>
	<li>For preparation of OHSC use the following set of instruments: two small scissors, two curved tweezers, one...

The present protocol describes the preparation of OHSC. This model allows testing of intrinsic capabilities and reactions of brain tissue after the application of physiological and pathological stimuli. Besides analyses of electrophysiological parameters, OHSC can be lesioned and the effects of damage on all cell types can be determined. Treatment with different substances and the detailed description of lesioning processes or healing in the absence of macrophages and lymphocytes is possible.
...

OHSC are a well-characterized in vitro model to study both physiological and pathological properties of neurons, astrocytes and microglia1. It is easy to control the extracellular environment and monitor the cellular and morphological changes after various stimuli. The organization of hippocampal neurons and their connections are well preserved after preparation2,3. Out of several advantages, OHSC allow monitoring of brain injury and tumor invasion without animal surgery. Six to eight OHSC can be obtained from a single rodent brain. OHSC therefore help to significantly reduce the number of animals and allow testing multiple drug concentrations, genetic manipulations or different lesion models in the same animal. In slice-based assays, experimental conditions can be precisely controlled. Additionally, time dependent development of pathological conditions like secondary damages can easily be monitored by time-lapse imaging.
In the given protocol, originally established by Stoppini et al.4, the preparation steps are described and important morphological landmarks for the selection of appropriate slices are highlighted. We recommend the preparation of postnatal day 7-9 rats or postnatal day 4-5 mice. In these periods, OHSC show a robust resistance to mechanical traumas and a high potential for reorganization of neuronal circuits. In contrast, preparations from embryonic or adult rats rapidly change their structure and lose their organotypic morphology during cultivation and are therefore less suitable for studying long-term processes in basic research5,6,7,8,9,10,11. Another critical point for the survival rate of OHSC is the thickness of the slice itself as the diffusion and thus nutrient supply are limited12,13,14.

<table border="0" cellpadding="0" cellspacing="0" width="874">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:412px;">6-Well</td>
			<td style="width:204px;">Falcon</td>
			<td style="width:179px;">35-3046</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>Fluka</td>
			<td>5040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Autoclav</td>
			<td>Systec</td>
			<td>DX-45</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFDA</td>
			<td>&#160;Thermo Fisher</td>
			<td>V12883</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal laser scanning microscope (CLSM) LSM700</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eagle&#180;s Minimal Essential Medium&#160;</td>
			<td>Invitrogen</td>
			<td>32360-034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein labeled Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td>Vector Labs</td>
			<td>FL-1101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein labeled GSL I &#8211; isolectin B4</td>
			<td>Vector Labs</td>
			<td>FL-1201&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Merk</td>
			<td>1083371000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutamin</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Hank&#180;s Balanced Salt Solution (with Ca2+ and Mg2+)</td>
			<td>Invitrogen</td>
			<td>24020-133</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Hank&#180;s Balanced Salt Solution (without Ca2+&#160; and Mg2+)</td>
			<td>Invitrogen</td>
			<td>14170-138</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin</td>
			<td>Sigma Aldrich</td>
			<td>I5500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-ascorbic acid</td>
			<td>Sigma Aldrich</td>
			<td>A5960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamin</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LN229</td>
			<td>Cell-Lines-Service</td>
			<td>300363</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medical cyanoacrylate glue (Histoacryl glue)</td>
			<td>B.Braun</td>
			<td>1050052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millicell Culture Inserts</td>
			<td>Millipore</td>
			<td>PICMORG50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NMDA N-methyl-D-aspartic acid</td>
			<td>Sigma Aldrich</td>
			<td>M3262</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal Horse Seum</td>
			<td>Invitrogen</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes (all sizes)</td>
			<td>Greiner</td>
			<td>627160/664160/628160</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PFA</td>
			<td>Roth</td>
			<td>0335.1</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium iodid (PI)</td>
			<td>Sigma Aldrich</td>
			<td>81845-25MG</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U138</td>
			<td>ATCC</td>
			<td>HTB-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vibratome</td>
			<td>Leica</td>
			<td>Leica VT 1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Neuroprotection studies: To determine neuronal damage, the number of PI positive nuclei and IB4 positive microglia in every third optical section of the granule cell layer (GCL) of the dentate gyrus (DG) was counted. For tumor invasion experiments, the maximal intensity z-projection of the stack was used for calculating the area covered by tumor cells, as a measure of invasion and to visualize different invasion patterns (Figure 6)...

Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells at JoVE.com

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

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. ...

Research

JoVE Journal

Neuroscience

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 ...

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 ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such 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 ...

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 ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...