Electron microscopy images of thalamus from acutely hydrogen sulfide poisoned mice

To date, the mitochondrial morphological changes that take place after an acute H2S exposure are not well described. In the present study, we analyzed images of transmission electron microscopy of thalami from mice subjected to a single acute (1000 ppm) H2S exposure and euthanized at various time points to assess the mitochondrial structure and morphometric parameters over time. From our observations, changes induced by H2S up to 48 h were mostly limited to increased cristae and matrix compartments' disorganization in mainly accumulated round-shaped mitochondria. At 72 h, H2S induced a spectrum of morphological cellular changes. Impaired H2S-mediated energy-producing capacity of mitochondria relative to their normal-sized counterparts was inferred from our observations of disorganization and paucity of the inner mitochondrial membrane, the infoldings of which—into cristae—are well-documented to increase the surface area for ATP production, the evaluation of a disorganized network, and likely a disrupted mitophagy because of changes in mitochondrial shape.

Methods
C57BL/6J male mice 7-8 week old were randomly assigned to each group (n=8 for H2S group and n=4 to room air group). Seven- to eight-week-old mice are considered as young adult mice and used in this study. Mice were exposed to a single dose of H2S at 1000 ppm or room air by inhalation (whole body exposure) for 30 min. This exposure causes 50% mortality (LCt50). Male mice were used because previous work in the lab showed that they are more sensitive to acute H2S exposure than females50. Small sample size of surviving mice (n=3 for each group) were used because this study is a pilot study where electron microscopic investigation was performed for the first time while providing minimal but significant statistical power. Mice in the control group were exposed to room air. High purity of H2S gas (> 99.9 %) or room air (> 98 %) were supplied from gas cylinders (Airgas, IA). The concentration of H2S in the exposure chamber was monitored in real-time using a H2S sensor (RKI Instruments, Union City, CA). Mice were euthanized at 12, 24, 48, and 72 h after the single acute exposure to assess time course effects of H2S on mitochondrial morphology. The dose of 1000 ppm was chosen to mimic acute H2S exposure following industrial accidents or nefarious acts. Only mice surviving the acute exposure were used in this study. Mice were euthanized at each time point by decapitation using a guillotine. Mouse brains were immediately removed. The thalamus was micro-dissected on ice and blocked in 1 % paraformaldehyde, 3 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 at 4°C and subsequently processed for electron microscopy. 

Electron microscopy

Micro-dissected thalamus brain tissues were placed into 1 % paraformaldehyde, 3 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and fixed for 48 h at 4 °C. The thalamus tissues were washed in the cacodylate buffer for 10 min 3 times and post-fixed with 1 % osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at 20-22˚C. Subsequently, the thalami were washed three times with deionized water for 15 min each. The thalamus tissues were then washed in distilled water for 10 min and dehydrated through a graded ethanol series (25, 50, 70, 85, 95, 100%) for 1 h each step. This was followed by dehydration, with 3 changes of pure acetone, 15 min each, and infiltrated with EmBed 812 formula (hard) for EPON epoxy resin (Electron Microscopy Sciences, Hatfield PA) with graded ratios of resin to acetone until fully infiltrated with pure epoxy resin (3:1, 1:1, 1:3, pure) for 6-12 h per step. Thalami were placed into BEEM® embedding capsules and polymerized at 70 ℃ for 48 h. Thick sections (1.5 mm) were made using a Leica UC6 ultramicrotome (Leica Microsystems, Buffalo Grove, IL) and stained with EMS Epoxy stain (a blend of toluidine blue-O and basic fuchsin). Thin sections were made at 50 nm and collected onto single slot carbon film grids. TEM images were collected using a 200kV JEOL JSM 2100 scanning transmission electron microscope (Japan Electron Optics Laboratories, USA, Peabody, MA) with a GATAN One View 4K camera (Gatan inc., Pleasanton, CA).