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

Abstract

Source: Carvalho, M., et al., A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures. J. Vis. Exp. (2021)

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. Culture setup and medium preparation On the day before the culture, prepare the required media and place them at 4 °C. Prepare d…

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 indic…

Materials

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)
Hydrochloric acid	Thermo Fisher Scientific	26050-088
Hydrophobic Pen	Merck Milipore	1.09057.1000
INCU-Line IL10	Dako	S200230-2
Iris Spatula Curved	Warner Instruments	BSC-HT Haas Top
Labculture Class II Biological Safety Cabinet	Fine Science Tools	10092-12
Lens Cleaning Paper	HERASafe	HS 12
L-Glutamine solution 200 mM (Q)	TIFFEN
Micro tube 0.5 mL, PP	Merck Millipore	1.05886.0500
Micro tube 1.5 mL, PP	SARSTEDT	72,699
Micro tube 2.0 mL, PP	SARSTEDT	72.690.001
Miroscope Cover Glasses, 24 mm x 60 mm	Sutter Instrument	MP-285
Nail polish	Marienfeld	102242
Neurobasal-A Medium (NBA)	Cliché
Opti-MEM® I Reduced-Serum Medium	Thermo Fisher Scientific	10888-022
Paraformaldehyde, powder	Thermo Fisher Scientific	31985-047
Peristaltic pump	VWR Chemicals	2,87,94,295
Phosphate saline buffer (PBS)	Gilson	M312
Phosphate standard solutions, PO43- in water			Homemade. PBS with 0.5% Tween-20 (PBS-T) is used to wash slices during the immunohistochemistry assay.
Pipette set	BDH ARISTAR	452232C
Platinum 5 blades	Gilson	P2, P10, P20, P100, P200, P1000
Propidium iodide (PI)	Sigma-Aldrich	P5405-250g
Qualitative Filter Paper, Cellulose, Grade 1, 55 mm	Sigma-Aldrich	P4170-25MG	Stock solution at 1 mg/mL in water.
Qualitative Filter Paper, Cellulose, Grade 1, 90 mm	Whatman	1001-055	Medium retention 11µm
Scalpel handle	Whatman	1001-090	Medium retention 11µm
Slip Tip Insulin Syringe without Needle 1 mL	Fine Science Tools	91003-12
Student Scissors Straight SharpSharp 12cm	Astro Med Inc GRASS Product Group	S48 Stimulator
SuperFrost Plus™ Adhesion slides	Fine Science Tools	91402-12
TC-Treated Sterile 60 x 15mm Tissue Culture Dish	Thermo Fisher Scientific	J1800AMNZ
TC-Treated Sterile 6-Wells Plates	Corning	CORN430166
Tissue Chopper	MEDICAL SYSTEMS CORP.	TC-102
Triton X-100	The Mickle Laboratory Engineering CO. LTD.	MTC/2	Set to 350 μm
Tween-20	BDH	14630
	Sigma	P2287