Neuronal Death Assay to Evaluate Cell Death in Ex Vivo Epilepsy Model

Overview

This video demonstrates the technique of assessing neuronal death in rhinal cortex-hippocampus organotypic slices. Neuronal death occurs via evolving epileptic seizures — resembling in vivo epilepsy — and is detected through dual-staining of the dead cells using propidium iodide and immunofluorescence.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of rhinal cortex-hippocampus slices

NOTE: The preparation of rhinal cortex-hippocampus slices uses P6-7 Sprague-Dawley rats.

1. Culture setup and medium preparation
   1. On the day before the culture, prepare the required media and place them at 4 °C.
   2. Prepare dissection medium: 25 mM glucose in Gey's Balanced Salt Solution (GBSS).
   3. Prepare culture medium: 50% Opti-MEM, 25% HBSS, 25% Horse Serum (HS), 25 mM glucose, 30 µg/mL Gentamycin.
   4. Prepare maintenance medium: Neurobasal-A (NBA), 2% B27, 1 mM L-glutamine, 30 µg/mL Gentamicin, HS (15%, 10%, 5%, and 0%).
2. Brain harvesting
   1. Just before starting the culture, add 1.1 mL of culture medium to each well of the 6-well plate with a P1000 pipette and place it at 37 °C.
   2. Place all the equipment (dissection microscope, tissue chopper, dissecting lamp, dissecting tools, electrodes, plates, inserts, and filter papers) inside the biological safety cabinet and sterilize under UV light for 15 minutes.
   3. Adjust slice thickness to 350 µm.
   4. Withdraw the GBSS from the fridge. Add 5 mL of GBSS to six Petri dishes. Six Petri dishes will be required per animal.
   5. Euthanize the rat pup. Perform decapitation by using a sharp scissor at the base of the brain stem of the animal.
   6. Wash the animal head three times in cold GBSS and take it inside the safety cabinet.
3. Tissue isolation and preparation of slices
   1. Firmly insert sharp forceps into the eye sockets to hold the head.
   2. Using a thin scissor cut the skin/scalp along the midline starting from the vertebral foramen towards the frontal lobes and put it aside.
   3. Cut in the same way the skull and along the cerebral transverse fissure (space between brain and cerebellum). With curved long forceps, move it apart.
   4. Discard the olfactory bulbs with a spatula. Remove the brain from the head and place it in ice-cold GBSS with the dorsal surface faced up (Figure 1A).
   5. Insert the fine forceps into the cerebellum and go along the midline with the spatula opening each hemisphere very carefully (Figure 1B).
   6. With short curve forceps, carefully remove the excess tissue that covers the hippocampi, without touching the hippocampal structure. Then with a spatula, cut below each hippocampus (Figure 1C).
   7. Pick up one hemisphere and place it, with hippocampus facing up, onto a filter paper. Repeat the procedure with the other hemisphere and place it parallel to the first one, in the filter paper. Put the filter paper on the tissue chopper, with the hemispheres perpendicular to the blade, and cut the hemispheres in 350 μm slices (Figure 1D).
   8. Place the sliced tissue into a Petri dish with cold GBSS (Figure 1E).
   9. Carefully separate the slices using the round tip electrodes (Figure 1F). Keep only the slices with a structurally intact rhinal cortex and hippocampus. DG and CA areas should be perfectly defined, as well as the entorhinal and perirhinal cortex (Figure 1G).
   10. Place each slice onto the insert (Figure 1H-I), with a spatula and a round-tip electrode. Remove excess dissection medium around each slice with a P20 pipette (Figure 1J). Four rhinal cortex-hippocampus slices can be cultured in a single insert (Figure 1K).
4. Culture maintenance
   1. Change the medium every other day.
   2. Warm up the medium at 37 °C.
   3. Take the plates from the incubator. Pick up each insert by holding the plastic edge with forceps (Figure 1L).
   4. Use a free hand to aspirate the medium from the well. Place the insert back into the well and add 1 mL, with a P1000 pipette, of fresh warmed medium (Figure 1M). Repeat for all the inserts. Make sure no air bubbles are trapped between the membrane and the medium.
      NOTE: Epileptic-like slices undergo a gradual and controlled deprivation of serum in the medium. From 9 Days In Vitro (DIV) on, slices are maintained in NBA without HS.

2. PI uptake assay

NOTE: Cell death was assessed by monitoring the cellular uptake of the fluorescent dye propidium iodide (PI). PI is a polar compound, which enters cells with damaged cell membranes and interacts with DNA emitting red fluorescence (absorbance 493 nm, emission 630 nm). Since PI is not permeant to live cells, it is used to detect dead cells in a population.

1. PI incubation
   1. Prepare, in culture medium, a fresh 1:10 dilution of PI stock.
   2. For PI uptake assay remove the plate from the incubator and carefully raise the insert. Add 13 µL of PI to the medium, with a P20 pipette, obtaining a final concentration of 2 µM.  Agitate slowly the plate before putting the insert back in place. Make sure there are no bubbles beneath the slices.
   3. Put the slices back in the 37 °C incubator for 2 h.
   4. Proceed with the immunohistochemistry protocol, as described in the next section. Cover the plates with aluminium, since PI is light sensitive.

3. Immunohistochemistry

NOTE: In immunohistochemistry, a neuron-specific antibody, as well as antibodies able to discriminate resting and reactive phenotypes of microglia and astrocytes, were used to evaluate the extent of neuronal death and gliosis in rhinal cortex-hippocampus epileptic-like organotypic slices.

1. Tissue fixation
   1. Remove the plate from the incubator and aspirate the medium. Fix the slices with 4% paraformaldehyde (PFA) for 1 h at RT, by adding 1 mL of PFA beneath and above the slices, with a P1000 pipette.
   2. Remove the PFA and add 1 mL of PBS. Also, add PBS beneath and above the slices.
   3. Keep the slices at 4 °C, in PBS, until further use. Always put parafilm around the plates to avoid drying.
2. Immunostaining steps
   1. Wash twice, 10 min each time, with 1 mL of PBS.
   2. Prepare permeabilization/blocking solution containing 1% Triton-X100, 10% HS, and 10% BSA in PBS. Prepare 5% BSA solution.
   3. Draw two rectangles with the hydrophobic pen (Figure 2A). Cut the slices from the insert (Figure 2B) with a highly sharp blade. Put two slices per slide (Figure 2C) and add 140 µL of permeabilization/blocking solution on the top of each slice, using a P200 pipette. Incubate for 3 h at RT.
   4. Dilute the primary antibodies to the working dilution in 5% BSA in PBS. Incubate with the primary antibodies overnight at 4 °C.
   5. Incubate with the secondary antibodies for 4 h at RT. From this step on, protect the plate from light since fluorophores are being worked with.
   6. Place a 50 μL drop of Hoechst solution on the top of each slice and incubate for 20 min at RT.
   7. Wash between incubations. Always wash three times, for 10 min each time, with PBS-T.
   8. Remove Hoechst and wash as recommended.
   9. Add 50 μL of mounting medium on the top of each slice. Cover with a glass coverslip and surround with nail polish (Figure 2D).
   10. Let it dry at RT for 24 h.
   11. Visualize the immunostaining under a confocal microscope. Keep the stained slices at -20 °C.

Representative Results

Figure 1: Detailed procedure for the preparation of rhinal cortex-hippocampus organotypic slices. (A) Remove the brain from the head and place it in ice-cold GBSS with the dorsal surface faced up. (B) Insert the forceps into the cerebellum. Open the brain through the midline and remove the excess tissue over the hippocampus. (C) With a spatula cut below the hippocampus, as indicated by the arrows. (D) Place both hippocampi facing up and parallel to each other onto the filter paper and cut 350 µm slices on the tissue chopper. (E) Place the sliced hippocampus in ice-cold GBSS. (F) Separate the slices with the help of round-tipped glass electrodes. (G) Choose only the slices that depict an intact rhinal cortex and hippocampus. (H, I) With the help of a round-tipped glass electrode push each slice to the spatula and place it on the insert. (J) Remove the GBSS surrounding the slice. (K) Place four slices per insert. (L) To change the medium, lift the insert and aspirate the medium with a glass pipette. (M) Add fresh medium by placing the pipette between the insert and the walls of the 6-wells plate. Make sure there are no air bubbles beneath the slices.

Figure 2: Specific procedure for the immunohistochemistry assay. (A) With the hydrophobic pen draw two squares in the slide. (B) Cut the piece of insert that contains the slice. (C) Place each slice in the squares drawn with the hydrophobic pen and start the permeabilization/blocking step. (D) After concluding the protocol, finish by mounting the slices in mounting medium, covering with a glass coverslip, and surrounding it with nail polish.

Materials

Name	Company	Catalog Number	Comments
50 mL Centrifuge Tube, Conical Bottom	Corning	430829
70% Ethanol	Manuel Vieira, Lda	UN1170
Anti-NeuN (rabbit)	Werfen	16712943S	Dilute at a ratio 1:500
B-27™ Supplement (50X), serum free	Thermo Fisher Scientific	17504-044
Blades for scalpel handle	Fine Science Tools	10011-00
Bovine Serum Albumin (BSA)	NZYTech	MB04602	5% BSA is used to dilute the primary antibodies. Add 0.5g BSA in 10 mL PBS.
Brain/Tissue Slice Chamber System	Warner Instruments
Cell culture inserts, 30 mm, hydrophilic PTFE	Millipore SAS	PICM03050
Cold light source	SCHOTT	KL 300 LED
Confocal laser microscope	Zeiss	LSM 710
Conventional incubator	Thermo Scientific Heraeus	BB15, Function Line	Set to 37 °C and 5% CO2
D(+)-Glucose monohydrate	Merck Millipore	1.08342.1000
D-(+)-Glucose solution, 45% in water	Sigma	G8769
di-Sodium hydrogen phosphate dihydrate	Merck Milipore	1.06580.1000
Dissecting microscope/magnifier	MEIJI TECHNO CO. LTD	122285
Donkey anti-rabbit IgG (H+L) coupled to Alexa Fluor 488	Invitrogen	A21206	Dilute at a ratio 1:500
Dumont #5 Fine Forceps Biologie Inox	Fine Science Tools	11254-20
Dumont #5 Forceps Standard Inox	Fine Science Tools	11251-20
Dumont #7 Forceps Standard Dumoxel	Fine Science Tools	11271-30
Dumont Medical #7S Forceps Short Curve Inox	Fine Science Tools	11273-22
Gentamycin stock solution, 50 mg/mL	Thermo Fisher Scientific	15750-037
Gey’s Balanced Salt Solution (GBSS)	Biological Industries	01-919-1A
Glass Electrodes	Science Products	GB150F-10	Round tips homemade
Glass Pasteur pipettes, 230 mm	VWR International	612-1702
Hank’s Balanced Salt Solution (HBSS)	Thermo Fisher Scientific	24020-091
Horse Serum, Heat Inactivated (HS)	Thermo Fisher Scientific	26050-088
Hydrochloric acid	Merck Milipore	1.09057.1000
Hydrophobic Pen	Dako	S200230-2
INCU-Line IL10	VWR	390-0384
Iris Spatula Curved	Fine Science Tools	10092-12
Labculture Class II Biological Safety Cabinet	HERASafe	HS 12
Lens Cleaning Paper	TIFFEN
L-Glutamine solution 200 mM (Q)	Thermo Fisher Scientific	25030-024
Micro tube 0.5 mL, PP	SARSTEDT	72,699
Micro tube 1.5 mL, PP	SARSTEDT	72.690.001
Micro tube 2.0 mL, PP	SARSTEDT	72.691
Miroscope Cover Glasses, 24 mm x 60 mm	Marienfeld	102242
Nail polish	Cliché
Neurobasal-A Medium (NBA)	Thermo Fisher Scientific	10888-022
Opti-MEM® I Reduced-Serum Medium	Thermo Fisher Scientific	31985-047
Paraformaldehyde, powder	VWR Chemicals	2,87,94,295
Peristaltic pump	Gilson	M312
Phosphate saline buffer (PBS)			Homemade. PBS with 0.5% Tween-20 (PBS-T) is used to wash slices during the immunohistochemistry assay.
Phosphate standard solutions, PO43- in water	BDH ARISTAR	452232C
Pipette set	Gilson	P2, P10, P20, P100, P200, P1000
Platinum 5 blades	Gillette
Propidium iodide (PI)	Sigma-Aldrich	P4170-25MG
Qualitative Filter Paper, Cellulose, Grade 1, 55 mm	Whatman	1001-055	Medium retention 11µm
Qualitative Filter Paper, Cellulose, Grade 1, 90 mm	Whatman	1001-090	Medium retention 11µm
Scalpel handle	Fine Science Tools	91003-12
Slip Tip Insulin Syringe without Needle 1 mL	SOL-M	161000
Student Scissors Straight SharpSharp 12cm	Fine Science Tools	91402-12
SuperFrost Plus™ Adhesion slides	Thermo Fisher Scientific	J1800AMNZ
TC-Treated Sterile 60 x 15mm Tissue Culture Dish	Corning	CORN430166
TC-Treated Sterile 6-Wells Plates	Corning	CORN3516
Tissue Chopper	The Mickle Laboratory Engineering CO. LTD.	MTC/2	Set to 350 μm
Triton X-100	BDH	14630
Tween-20	Sigma	P2287