Example of the histopathology of the lung in a control rat and a rat exposed to H₂S.

<p>Hematoxylin and eosin stained sections of perfused lungs from control and H₂S-exposed rats, 100X magnification. Lungs of both rats are normal (top panels) with no evidence of lesions that would be expected to occur during inhaled sulfide exposure. As illustrated on the lower panels, both unexposed controls and H₂S exposed rats displayed mild expansion of the peribronchial, peribronchiolar and perivascular interstitium by low to moderate numbers of lymphocytes and macrophages with fewer plasma cells consistent with chronic bronchus associated lymphoid tissue hyperplasia (BALT, see text for additional comments).</p

GFAP staining by DAB chromagen with hematoxylin counterstain in one rat exposed to saline (control) and one rat intoxicated with H₂S with normal recovery.

<p>There was no difference in astrocyte density or morphology between the 2 animals.</p

Summary of location of the various brain lesions.

<p>Summary of location of the various brain lesions.</p

Immediate outcome in keeping with the number of intra-peritoneal NaHS injections required to produce a coma.

<p>There was no difference in the mortality whether 1, 2 or 3 injections were administered. Of note is that the 2 rats that were unable to swim in the H₂S groups belonged to the rats that received 3 IP injections.</p

Brain histopathology.

<p>Sections of frontal cortex (panels a and b) and thalamus (c) from one rat that presented a coma but with no neurological deficit (a1, b1, c1) and from rat (#16, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131340#pone.0131340.t001" target="_blank">Table 1</a>) that was unable to swim after H₂S exposure (a2, b2, c2). In contrast to rat with no symptom, the brain of rat #16 showed diffuse and extended neuronal necrosis and neuropil edema affecting the outer frontal cortex (motor agranular cortex) and the cingulate gyrus (anterior limbic area). Neurons are hypereosinophilic with karyolytic or pyknotic nuclei and peri-nuclear edema, Bregma 0.0. 400x. Panel c1 shows normal thalamus at same level and magnification in the intoxicated rat with no deficit. Panel c2: Extensive neuronal necrosis in the lateral posterior nucleus of the thalamus. Bregma -4.8. 400x.</p

Histopathology of the hippocampus and piriform cortex in the rat # 38 that displayed severe neuronal necrosis in the frontal, temporal, parietal and occipital cerebral cortices.

<p>The same pattern was found in all the rats presenting cortical lesions. a) Note the diffuse acute neuronal necrosis and neuropil edema affecting the outer retrosplenial and occipital cortex, while the hippocampus was completely unaffected. Bregma -4.5. 40x; b) Note the sharp demarcation at the rhinal fissure between the necrotic temporal cortex (top) and the unaffected piriform cortex (bottom). Bregma -4.5. 100x.</p

Examples of swimming strategies in 2 intoxicated rats treat with MB.

<p>The recordings in the upper panels where obtained from a rat with no brain lesions while those in the lower panels were obtained in the rat #92, (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131340#pone.0131340.t001" target="_blank">Table 1</a>) that was later found to have neuronal cortical necrosis. The latter rat was unable to find the platform even though its behavior was normal during open field test.</p

Experimental design and outcomes.

<p>Twenty-one control rats received saline IP injection and were assigned to behavior test (control rats). A first series of 32 rats received H₂S injection, 8 rats did not present a coma. The 24 rats that presented a coma, were separated into a non-treatment (H₂S) group (12 rats) and methylene blue treated (H₂S-MB) group (12 rats). In the H₂S group, 8 died immediately (*1 out of 8 rats died within 12 h) and 4 survived from a coma (one these 4 rats was however unable to swim). Meanwhile 9 survived in the H₂S-MB group, they were all able to swim. Therefore, to match the number of surviving rats between the 2 groups, a second series of 10 rats received NaHS: 5 died and 5 survived. The 5 surviving rats were added to the H₂S group, one of them could not swim. Out of 9 surviving rats in the H₂S group, 7 could therefore complete the behavior test, while the 2 rats unable to swim were euthanized within 48 h. Out of 9 surviving rats in the H₂S-MB group, 9 completed the behavior test.</p

Body weight following H₂S induced coma.

<p>Body weight decreased gradually over the 4 days of training in the control group. In the H₂S group, body weight significantly dropped at D1 day (significantly different from control group, P<0.05), and then did not change thereafter. Note that the 2 rats unable to eat that were euthanized are not included in this computation. In the H₂S-MB group, weight also significantly dropped at D1 (significantly different from control group, P<0.05) then remained below baseline until D4. Values are shown as mean ± SD. *significantly different from control at P<0.05. # Significantly different from baseline (day 0) at P<0.05.</p

Probe trials.

<p>Average latency, distance, time spent in the platform quadrant, and number platform crossing are displayed during the probe trial. No significant difference was observed between the 3 groups. Values are shown as mean ± SD.</p