DOI: 10.5061/dryad.k3j9kd5ds
title: Electron microscopy images of thalamus from acutely hydrogen sulfide poisoned mice

description: 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.
description: 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 H<sub>2</sub>S at
 1000 ppm or room air by inhalation (whole body exposure) for 30 min. This
 exposure causes 50% mortality (LCt<sub>50</sub>). Male mice
 were used because previous work in the lab showed that they are more
 sensitive to acute H2S exposure than females<sup>50</sup>. 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 H<sub>2</sub>S gas (&gt; 99.9 %) or room air (&gt; 98 %) were supplied from gas cylinders (Airgas, IA). The concentration of H<sub>2</sub>S in the exposure chamber was monitored in real-time using a H<sub>2</sub>S 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 H<sub>2</sub>S on mitochondrial morphology. The dose of 1000 ppm was chosen to mimic acute H<sub>2</sub>S 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.  <em>Electron microscopy</em> 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<sup>®</sup> 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).
description: Not applicable
description: # Electron microscopy images of thalamus in brain from acutely poisoned
 mice with hydrogen sulfide gas # Dataset contents: This data set contains
 13 raw electron microscopy images that were analyzed to investigate the
 effects of acute hydrogen sulfide exposure on the brain mitochondria. #
 Experimental procedures: ## Exposure paradigm 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 (&gt; 99.9 %)
 or room air (&gt; 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¡ÆC 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 ¡ÆC. 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¢ªC.
 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 ¡É 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). # Results: High
 doses of hydrogen sulfide (H2S) are considered mitotoxic. As a result of
 Complex IV inhibition and through other mechanisms, mitochondrial
 dysfunction plays a critical role in H2S-induced toxicity with no detailed
 reports available on the morphology of brain mitochondria. To date, the
 mitochondrial morphological changes that take place after an acute H2S
 exposure are not well described. In the present date set, the images of
 transmission electron microscopy of thalami from mice subjected to a
 single acute (1000 ppm) H2S exposure at various time points were analyzed
 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. # Description of the data and
 file structure This data set contains the following images. * B
 CONTROL\_0003 2um.tif Thalamus of mice exposed to room air * B
 CONTROL\_0004 2um w Nu ER.tif Thalamus of mice exposed to room air * C
 CONTROL\_0005.tif Thalamus of mice exposed to room air * D 12
 hour\_0009.tif Thalamus of mice 12 h post exposure to 1000 ppm hydrogen
 sulfide * G 24 hour\_0001 2um.tif Thalamus of mice 24 h post exposure to
 1000 ppm hydrogen sulfide * G 24 hour\_0007 2um.tif Thalamus of mice 24 h
 post exposure to 1000 ppm hydrogen sulfide * J 48 hour\_0007.tif Thalamus
 of mice 48 h post exposure to 1000 ppm hydrogen sulfide * M
 72HOUR\_0004.tif Thalamus of mice 72 h post exposure to 1000 ppm hydrogen
 sulfide * N 72 hour\_0003 2um.tif Thalamus of mice 72 h post exposure to
 1000 ppm hydrogen sulfide * N 72 hour\_0009 2um.tif Thalamus of mice 72 h
 post exposure to 1000 ppm hydrogen sulfide * O 72 hour\_0001.tif Thalamus
 of mice 72 h post exposure to 1000 ppm hydrogen sulfide * O 72 hour\_0017
 2um.tif Thalamus of mice 72 h post exposure to 1000 ppm hydrogen sulfide *
 O 72 hour\_0024 500nm.tif Thalamus of mice 72 h post exposure to 1000 ppm
 hydrogen sulfide