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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 preparation^2,3. 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 research^{5,6,7,8,9,10,11}. Another critical point for the survival rate of OHSC is the thickness of the slice itself as the diffusion and thus nutrient supply are limited^12,13,14.

Protokół

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

1. For preparation of OHSC use the following set of instruments: two small scissors, two curved tweezers, one tweezer with a fine tip, three blades (two of size 11, one of size 15), three scalpel holders, round filter papers (diameter: 35 mm), agar, one razor blade, one unmodified Pasteur pipette, and one modified Pasteur pipette without a tip. Sterilize all materials in an autoclave before usage (Figure 1).
2. Weigh 5 g agar and dissolve it in 100 mL distilled water. Sterilize the solution for 20 min at 121.7 °C at 210.8 kPa in an autoclave. Distribute the 3 mL liquid agar solution in 60-mm Petri dishes using a sterile glass pipette, allow it to solidify for 5 h, cover with plastic paraffin film to avoid contamination and store at 4 °C until further use. Agar blocks are needed to stabilize the brains during the slicing procedure.
3. Media
   1. Make 200 mL of the preparation medium (pH 7.35) consisting of 198 mL minimal essential medium (MEM) and 2 mL L-glutamine solution (final concentration 2 mM). Prepare the solution on the day of media preparation and store it at 4 °C.
   2. Prepare 100 mL of the culture medium consisting of 49 mL MEM, 25 mL Hanks' balanced salt solutions (HBSS), 25 mL (v/v) normal horse serum (NHS), 1 mL L-glutamine solution (final concentration 2 mM), 100 µg insulin, 120 mg glucose, 10 mg streptomycin, 10,000 U penicillin, and 800 µg vitamin C as reported previously. Warm the medium (37 °C), adjust the pH value to pH 7.4 and sterile filter (0.2 µm pore size). Repeat the procedure (warming up, pH adjustment, etc.) every second day before changing the medium. Use the medium at the most for one week when stored at 4 °C.
4. Fill one 35-mm Petri dish with preparation medium to store the brain. Place two empty Petri dishes for collecting the tissue on a cooling pack in the working area.

2. Preparation and Slicing with a Vibratome

1. Use brains from 7-9 day old rats or 4-5 day old mice for the OHSC preparation according to Stoppini et al.⁴ After decapitation of animals, remove the skin from the skull with scissors.
2. Introduce the blade of a fine scissor into the foramen magnum and open the skull by cutting along the caudal (back) rostral (front) axis. Make two cuts perpendicular to the first one, so that the scissors point towards the left and right ear, respectively ("T-incision").
3. Open the skull carefully with fine forceps, paying attention not to injure the brain. Use a scalpel (blade size 11) to cut the most rostral part of the frontal pole and the cerebellum.
4. By means of a spatula, remove the brain and place it carefully in the Petri dish filled with preparation medium (Figure 2). Position the brain on the specimen holder and fix it with medical cyanoacrylate glue. Use the pieces of agar to assure mechanical stabilization.
5. Dissect the tissue horizontally in 350 µm thick OHSC using a sliding vibratome.
6. Evaluate the slices optically using a binocular microscope. Discard OHSC of low quality immediately. It is important to take only those slices with intact cytoarchitecture isolated from the middle part of the hippocampus (see Figures 3 and 4) between the dorsal and the ventral hippocampus.
7. Separate the hippocampal region and the entorhinal cortex using a scalpel (round blade size 15; Figure 3). The perforant pathway and entorhinal cortex must be preserved.
8. Six to eight OHSC are obtained from each brain. Transfer 2-3 slices into one cell culture insert (pore size 0.4 µm) and place it in one well of a 6-well culture dish containing 1 mL culture medium per well.
9. Incubate the 6-well dishes at 35 °C in a fully humidified atmosphere with 5% (v/v) CO₂ and change the cell culture medium every second day.
   NOTE: Conduct your experiments at 6 day in vitro (div). Inflammatory reactions associated with the slicing procedure disappear by day 6. At this stage, microglial cells show a ramified morphology again and synaptic connections have matured.

3. Evaluation of Tissue Quality

1. Fixation, labeling and visualization of degenerating neurons in OHSC
   1. After performing the experiments, incubate the OHSC with 5 µg/mL propidium iodide (PI) for 2 h prior to fixation, in order to stain the nuclei of degenerating neurons.
      CAUTION: PI is a suspected carcinogen, always wear personal protective equipment (PPE) such as gloves.
   2. Wash the OHSC with 0.1 M phosphate buffer (pH 7.4) and fix them with a 4% (v/v) solution of paraformaldehyde (PFA) for 24 h.
      CAUTION: PFA is a toxic and suspected carcinogen. Work under fume hood and wear PPE.
   3. Wash the OHSC in inserts with 1 mL PBS and use a rigger brush to separate slices from the membrane.
   4. Label the OHSC with IB₄ dye in a 24-well plate (Figure 5).
2. Conduction of tumor invasion experiments using fluorescently labeled single cells
   1. 24 h prior to the start of an experiment, label the tumor cells by using the fluorescent dye Carboxyfluorescein diacetate succinimidyl ester (CFDA SE).
   2. Detach and count the tumor cells using a Neubauer chamber.
   3. Resuspend the cells in medium, such that 10 µL of suspension contains the desired cell number (normally 50,000 or 100,000 cells).
   4. Apply 10 µL of the cell suspension onto the slice culture and allow the cells to invade for 3 days.
   5. At the end of the experiment, fix the slice cultures using 4% (v/v) PFA for 24 h, and label the co-cultures with PI for another 24 h to visualize the cytoarchitecture.
      CAUTION: PFA is a toxic and suspected carcinogen. Work under fume hood and wear PPE.
   6. Mount the co-cultures onto a cover slip for further analysis using the mounting media.

4. Evaluation of OHSC Experiments

Analyze the OHSC with a confocal laser scanning microscope (CLSM). For detection of PI labeled, degenerating neurons or the PI labeled cytoarchitecture use monochromatic light of the wavelength λ = 543 nm and an emission band pass filter for wavelengths λ = 585-615 nm. For CFDA labeled tumor cells or IB₄ microglia, use an excitation wavelength of λ = 488 nm. For both experimental types, record a z-stack with 2 µm thick optical slices and used for evaluation.

Wyniki

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)...

Dyskusje

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.

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Materiały

Name	Company	Catalog Number	Comments
6-Well	Falcon	35-3046
Agar	Fluka	5040
Autoclav	Systec	DX-45
CFDA	Thermo Fisher	V12883
Confocal laser scanning microscope (CLSM) LSM700	Carl Zeiss
Eagle´s Minimal Essential Medium	Invitrogen	32360-034
Fluorescein labeled Griffonia (Bandeiraea) Simplicifolia Lectin I	Vector Labs	FL-1101
Glucose	Merk	1083371000
Glutamin	Invitrogen	25030-024
Hank´s Balanced Salt Solution (with Ca2+ and Mg2+)	Invitrogen	24020-133
Hank´s Balanced Salt Solution (without Ca2+  and Mg2+)	Invitrogen	14170-138
Insulin	Sigma Aldrich	I5500
L-ascorbic acid	Sigma Aldrich	A5960
L-Glutamin	Invitrogen	25030-024
LN229	Cell-Lines-Service	300363
Medical cyanoacrylate glue (Histoacryl glue)	B.Braun	1050052
Millicell Culture Inserts	Millipore	PICMORG50
NMDA N-methyl-D-aspartic acid	Sigma Aldrich	M3262
Normal Horse Seum	Invitrogen	26050-088
Penicillin Streptomycin	Invitrogen	15140-122
Petri dishes (all sizes)	Greiner	627160/664160/628160
PFA	Roth	0335.1	toxic
Propidium iodid (PI)	Sigma Aldrich	81845-25MG	toxic
U138	ATCC	HTB-14
Vibratome	Leica	Leica VT 1200