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. 2021 Sep 18;22(18):10099.
doi: 10.3390/ijms221810099.

Hydrogen Sulfide Reduces Ischemia and Reperfusion Injury in Neuronal Cells in a Dose- and Time-Dependent Manner

Stefanie Scheid  1 , Max Goeller  1 , Wolfgang Baar  1 , Jakob Wollborn  1   2 , Hartmut Buerkle  1 , Günther Schlunck  3 , Wolf Lagrèze  3 , Ulrich Goebel  4 , Felix Ulbrich  1
Affiliations

Hydrogen Sulfide Reduces Ischemia and Reperfusion Injury in Neuronal Cells in a Dose- and Time-Dependent Manner

Stefanie Scheid et al. Int J Mol Sci. .

Abstract

Background: The ischemia-reperfusion injury (IRI) of neuronal tissue, such as the brain and retina, leads to possible cell death and loss of function. Current treatment options are limited, but preliminary observations suggest a protective effect of hydrogen sulfide (H2S). However, the dosage, timing, and mechanism of inhaled H2S treatment after IRI requires further exploration.

Methods: We investigated possible neuroprotective effects of inhaled H2S by inducing retinal ischemia-reperfusion injury in rats for the duration of 1 h (120 mmHg), followed by the administration of hydrogen sulfide (H2S) for 1 h at different time points (0, 1.5, and 3 h after the initiation of reperfusion) and at different H2S concentrations (120, 80, and 40 ppm). We quantified the H2S effect by conducting retinal ganglion cell counts in fluorogold-labeled animals 7 days after IRI. The retinal tissue was harvested after 24 h for molecular analysis, including qPCR and Western blotting. Apoptotic and inflammatory mediators, transcription factors, and markers for oxidative stress were investigated. Histological analyses of the retina and the detection of inflammatory cytokines in serum assays were also performed.

Results: The effects of inhaled H2S were most evident at a concentration of 80 ppm administered 1.5 h after IRI. H2S treatment increased the expression of anti-apoptotic Bcl-2, decreased pro-apoptotic Bax expression, reduced the release of the inflammatory cytokines IL-1β and TNF-α, attenuated NF-κB p65, and enhanced Akt phosphorylation. H2S also downregulated NOX4 and cystathionine β-synthase. Histological analyses illustrated a reduction in TNF-α in retinal ganglion cells and lower serum levels of TNF-α in H2S-treated animals after IRI.

Conclusion: After neuronal IRI, H2S mediates neuroprotection in a time- and dose-dependent manner. The H2S treatment modulated transcription factor NF-κB activation and reduced retinal inflammation.

Keywords: H2S; hydrogen sulfide; ischemia–reperfusion injury; neuroprotection; retinal ganglion cells.

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Conflict of interest statement

There are no competing conflict of interest.

Figures

Figure 1
Figure 1
Time- and dose-dependent influence of H2S inhalation on retinal ganglion cell count after ischemia–reperfusion injury (IRI). Quantification of retinal ganglion cell density (cells/mm2, data are the mean ± SD, n = 8, *** = p < 0.001; IRI vs. IRI + 80 ppm H2S at 0 h and 1.5 h, vs. IRI + 120 ppm H2S at 1.5 h).
Figure 2
Figure 2
Time- and dose-dependent influence of H2S inhalation on vital retinal ganglion cells after ischemia–reperfusion injury (IRI). Representative flat mount images (n = 8) of fluorogold-labeled retinal ganglion cells 7 days after IRI and immediate (0 h) and delayed (i.e., 1.5 and 3 h) H2S inhalation (40, 80 and 120 ppm).
Figure 3
Figure 3
Effect of H2S inhalation on retinal expression of Bax and Bcl-2 mRNA. (a–c) Fold induction of Bax mRNA expression in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR after (a) 120 ppm H2S inhalation (data are mean ± SD, n = 8; ** = p < 0.01, IRI vs. 120 ppm H2S at 1.5 h), (b) 80 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. IRI + 80 ppm H2S at 1.5 h; ** = p < 0.01, IRI vs. IRI + 80 ppm H2S at 0 h), and (c) 40 ppm H2S inhalation. (d–f) Fold induction of Bcl-2 mRNA expression in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR after (d) 120 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. 120 ppm H2S at 1.5 h), (e) 80 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. IRI + 80 ppm H2S at 1.5 h; *** = p < 0.001, IRI vs. IRI + 80 ppm H2S at 0 h) and (f) 40 ppm H2S inhalation.
Figure 4
Figure 4
Effect of H2S inhalation on retinal expression of NOX-4 mRNA. Fold induction of NOX-4 mRNA expression in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR after (a) 120 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. 120 ppm H2S at 1.5 h), (b) 80 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. IRI + 80 ppm H2S at 0 h and vs. IRI + 80 ppm H2S at 1.5 h), and (c) 40 ppm H2S inhalation.
Figure 5
Figure 5
Effect of H2S inhalation on the retinal expression of IL-1ß and TNF-α mRNA. (a–c) Fold induction of IL-1ß mRNA expression in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR after (a) 120 ppm H2S inhalation (data are mean ± SD, n = 8; ** = p ≤ 0.01, IRI vs. 120 ppm H2S at 1.5 h), (b) 80 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001 IRI vs. IRI + 80 ppm H2S at 0 h and vs. IRI + 80 ppm H2S at 1.5 h), and (c) 40 ppm H2S inhalation. (d–f) Fold induction of TNF-α mRNA expression in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR after (d) 120 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. 120 ppm H2S at 1.5 h), (e) 80 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. IRI + 80 ppm H2S at 0 h and vs. IRI + 80 ppm H2S at 1.5 h), and (f) 40 ppm H2S inhalation.
Figure 5
Figure 5
Effect of H2S inhalation on the retinal expression of IL-1ß and TNF-α mRNA. (a–c) Fold induction of IL-1ß mRNA expression in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR after (a) 120 ppm H2S inhalation (data are mean ± SD, n = 8; ** = p ≤ 0.01, IRI vs. 120 ppm H2S at 1.5 h), (b) 80 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001 IRI vs. IRI + 80 ppm H2S at 0 h and vs. IRI + 80 ppm H2S at 1.5 h), and (c) 40 ppm H2S inhalation. (d–f) Fold induction of TNF-α mRNA expression in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR after (d) 120 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. 120 ppm H2S at 1.5 h), (e) 80 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. IRI + 80 ppm H2S at 0 h and vs. IRI + 80 ppm H2S at 1.5 h), and (f) 40 ppm H2S inhalation.
Figure 6
Figure 6
TNF-α expression after unilateral IRI and H2S inhalation. Double staining was performed to clarify in which areas of the retina the expression of TNF-α occurs. Cross-sections of the retinae 7 days after unilateral IRI showed that IRI led to an increased expression of TNF-α mainly in the retinal ganglion cell layer, as well as in the inner nuclear layer. After the application of H2S, the expression was attenuated. Abbreviations: NFL  = nerve fiber layer; GCL  = ganglion cell layer; INL  = inner nuclear layer; ONL  = outer nuclear layer.
Figure 7
Figure 7
Effect of H2S inhalation on IL-1β and TNF-α levels in peripheral blood samples. (a) Analysis of IL-1β levels after IRI and subsequent H2S inhalation (80 ppm) (pg/mL). (b) Analysis of TNF-α levels after IRI and subsequent H2S inhalation (80 ppm) (pg/mL; data are mean ± SD, n = 8; * = p < 0.05, IRI vs. IRI + 80 ppm H2S at 1.5 h).
Figure 8
Figure 8
Effect of H2S inhalation on the retinal expression of CBS mRNA. Fold induction of CBS mRNA expression in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR after (a) 120 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. 120 ppm H2S at 0 h and vs. 120 ppm H2S at 1.5 h), (b) 80 ppm H2S inhalation (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. IRI + 80 ppm H2S at 0 h and vs. IRI + 80 ppm H2S at 1.5 h), and (c) 40 ppm H2S inhalation.
Figure 9
Figure 9
(a,c) Effect of H2S inhalation on the retinal expression of NF-κB phosphorylation. Densitometric analysis of n = 8 Western blots for NF-κB phosphorylation after time-dependent inhalation of 80 ppm H2S (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. IRI + 80 ppm H2S at 1.5 h) and representative Western blot image (n = 8) showing the suppression of retinal phosphorylation of NF-κB compared to total NF-κB. (b,d) Effect of H2S inhalation on the retinal expression of Akt phosphorylation. Densitometric analysis of n = 8 Western blots for Akt phosphorylation after time-dependent inhalation of 80 ppm H2S (data are mean ± SD, n = 8; *** = p < 0.001, IRI vs. IRI + 80 ppm H2S at 1.5 h, and * = p < 0.05, IRI vs. IRI + 80 ppm H2S at 0 h) and representative Western blot image (n = 8) showing the increase of retinal phosphorylation of Akt compared to total Akt.
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