Distinct Pharmacological Profiles of Monosulfide and Trisulfide in an Experimental Model of Intracerebral Hemorrhage in Mice

Abstract

Hydrogen sulfide and polysulfides are increasingly recognized as bioactive signaling molecules to produce various actions and regulate (patho)physiological processes. Here we examined the effects of sodium sulfide (Na₂S) and sodium trisulfide (Na₂S₃) on an experimental model of intracerebral hemorrhage (ICH) in mice. Na₂S or Na₂S₃ (25 µmol/kg, intraperitoneally (i.p.)) was administered 30 min before ICH induction by intrastriatal injection of collagenase. We found that Na₂S significantly ameliorated sensorimotor functions of mice after ICH. Histopathological examinations revealed that Na₂S inhibited neuron loss in the striatum, prevented axon degeneration in the internal capsule, and ameliorated axonal transport dysfunction in the striatum and the cerebral cortex where the edge of hematoma was located. Although Na₂S did not suppress accumulation of activated microglia/macrophages in the peri-hematoma region, it suppressed ICH-induced upregulation of inflammatory mediators such as C-X-C motif ligand 2. On the other hand, Na₂S₃ did not ameliorate ICH-induced sensorimotor dysfunction. Although the effect of Na₂S₃ on several parameters such as axon degeneration and axonal transport dysfunction was comparable to that of Na₂S, Na₂S₃ did not significantly inhibit neuron loss and upregulation of inflammatory mediators. These results suggest that the regulation of multiple pathological events is involved in the effect of Na₂S leading to amelioration of neurological symptoms associated with ICH.

INTRODUCTION

Gaseous molecules such as nitric oxide, carbon monoxide and hydrogen sulfide (H₂S) are now widely accepted as important signaling molecules that are produced endogenously via specific biosynthetic pathways. For example, several enzymes including cystathionine β-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulfur transferase are involved in the production of H₂S.^1,2) Accumulating lines of evidence indicate that H₂S produces various biological actions in the central nervous system as well as the other organ systems, which include positive modulation of N-methyl-D-aspartate subtype of glutamate receptors, activation of ATP-dependent K⁺ channels and enhancement of transport of cystine and cysteine into cells.^2,3) On the other hand, persulfide/polysulfide molecular species such as hydrogen polysulfides (H₂S_n), glutathione persulfide and cysteine persulfide are also considered to be produced endogenously by various biosynthetic pathways.^3–5) These persulfide/polysulfide species may exhibit distinct biological and pharmacological profiles from those of H₂S. For example, H₂S_n has been shown to activate TRPA1 channels much more potently than H₂S.³⁾ Concerning the difference between monosulfide and polysulfides, we have previously reported that sodium trisulfide (Na₂S₃) provides a more potent neuroprotective effect than sodium sulfide (Na₂S) on dopaminergic neurons in rat midbrain slice cultures.⁶⁾

Intracerebral hemorrhage (ICH) is a type of hemorrhagic strokes, which is caused due to bleeding into the brain parenchyma. Brain tissue damage associated with hemorrhage results in severe sensorimotor dysfunctions, for which effective drug therapies are yet to be established.⁷⁾ The disease processes in the brain include various events such as brain edema, neuronal cell death, axon tract degeneration and recruitment of activated inflammatory cells.^8,9) Therapeutic strategies are expected to combat with these multiple complications.¹⁰⁾

H₂S is known to produce neuroprotective effects in several neurological disorders,¹¹⁾ and the effect of H₂S on experimental models of ICH has been addressed by several studies using H₂S donors such as sodium hydrogen sulfide (NaHS) and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH). Zhao et al.¹²⁾ showed that intraperitoneal (i.p.) administration of NaHS at 5.6 mg/kg given at 0.5, 24 and 48 h after ICH induction inhibited cell death in the peri-hematoma region, improved sensorimotor functions, reduced the increase in brain water content, and suppressed neutrophil infiltration and microglial activation. A study by Shan et al.¹³⁾ provided similar results by employing i.p. injection of 25 µmol/kg NaHS at 30 min before ICH induction. Another H₂S donor ADT-OH used at 50 mg/kg/d for 3 d, starting from 3 h after ICH induction, was also shown to inhibit the increase in brain water content, suppress the production of inflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, and improved motor performance.¹⁴⁾ In contrast, the effect of polysulfides on pathogenic events in ICH has not been addressed so far. Accordingly, we performed head-to-head comparison of the effects of monosulfide Na₂S and polysulfide Na₂S₃ on an ICH model in mice.

MATERIALS AND METHODS

Preparation of ICH Model in Mice

Experimental procedures in the present study have been approved by the Animal Care and Use Committee of Kumamoto University, and animals were handled according to the United States National Institute of Health Guide for the Care and Use of Laboratory Animals. Male ICR mice (Nihon SLC, Shizuoka, Japan) at age of 8–10 weeks old were maintained with constant ambient temperature (22 ± 1 °C) and 12-h light–dark cycle. ICH was induced by intrastriatal injection of collagenase, as described previously.^15,16) Briefly, under anesthesia by i.p. injection of 0.3 mg/kg medetomidine, 4.0 mg/kg midazolam and 5.0 mg/kg butorphanol, type VII collagenase (Sigma-Aldrich, St. Louis, MO, U.S.A.) at 0.035 U in a volume of 0.5 µL was administered into the striatum (stereotaxic coordinates; 2.3 mm lateral to the midline, 0.2 mm anterior to the bregma and 3.5 mm deep below the skull) at a rate of 0.2 µL/min. Mice were returned to their home cage after surgery and maintained under the same conditions as those of pre-operation.

Drug Administration

The dose and the route of administration of Na₂S and Na₂S₃ essentially followed the methods of a previous report examining the effect of NaHS.¹³⁾ Na₂S and Na₂S₃ were purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Both agents were dissolved in 0.9% physiological saline immediately before use. Mice were divided randomly into four groups; 1) sham group that received i.p. injection of vehicle and intrastriatal injection of saline, 2) vehicle group that received i.p. injection of vehicle and was subjected to ICH, 3) Na₂S group that received i.p. injection of 25 µmol/kg Na₂S 30 min prior to ICH induction, 4) Na₂S₃ group that received i.p. injection of 25 µmol/kg Na₂S₃ 30 min prior to ICH induction.

Assessment of Sensorimotor Performance

Sensorimotor functions of mice were assessed by modified limb-placing test and beam-walking test. Both tests were carried out at 6, 24, 48, and 72 h intervals after induction of ICH. The modified limb-placing test evaluated the reflexes of forelimbs and hindlimbs by 7-point scale as described previously,¹⁵⁾ where 7 points meant the maximal deficits. In the beam-walking test, the rate of footstep faults and the walking distance during crossing a beam with 15 mm width and 110 mm length were recorded as an average value from three trials.¹⁶⁾

Immunohistochemistry

Immunohistochemical examinations essentially followed the procedures described in our previous reports.¹⁵⁾ At 3 d after ICH induction, mice were anesthetized and transcardially perfused with ice-cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde. Brains were isolated and post-fixed in 4% paraformaldehyde overnight, then soaked in 15% sucrose overnight at 4 °C. After storage at −80 °C, brains were cut into 16 µm frozen coronal sections. Then, specimens were processed for immunohistochemistry of cell type-specific marker proteins. For detection of neurons, microglia/macrophages and neutrophils, anti-NeuN (rabbit polyclonal) (1 : 500; cat# ABN78, Millipore Corporation, Billerica, MA, U.S.A.), rabbit anti-Iba1 antibody (1 : 500; cat# 019–19741, Wako Pure Chemical Corporation, Osaka, Japan) and rabbit anti-myeloperoxidase (MPO) (1 : 500, Dako, Glostrup, Denmark) were used as primary antibodies, respectively. Biotinylated goat anti-rabbit immunoglobulin G (IgG) (1 : 200 or 1 : 500; cat# BA-1000, Vector Laboratories, Burlingame, CA, U.S.A.) was used as a secondary antibody. Specimens were incubated with avidin-biotinylated horseradish peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories), and peroxidase was visualized by diaminobenzidine and hydrogen peroxide.

The number of NeuN-positive cells and MPO-positive cells in the central region of hematoma was counted by an investigator blinded to the treatment conditions. The center of hematoma was identified by placing the whole hematomal region in the central view of a bright-field microscope under the objective lens at low magnification (×2). Then, without moving position of the section, the objective lens was changed to higher magnification (×40) to examine an area of 270 × 360 µm². In the case of quantitation of Iba1-positive cells in the peri-hematoma region, the field of observation with an area of 270 × 360 µm² in each section was set along the edge of hematoma to contain the maximal number of immunopositive cells with round or oval shape.¹⁵⁾

For evaluation of morphological changes in axonal fibers and dysfunction of axonal transport, images of immunofluorescence staining of neurofilament-H and amyloid precursor protein (APP) were examined. Frozen coronal brain sections were obtained at 24 h after ICH induction. Visualization of axonal fibers in the internal capsule was carried out with mouse anti-neurofilament-H (1 : 500; Cell Signaling Technology Japan, Tokyo, Japan) and Alexa Fluor 488 donkey anti-mouse IgG (H + L) antibody (1 : 500; Invitrogen™, Life Technologies Japan, Tokyo, Japan) as the primary antibody and the secondary antibody, respectively. Axonal shape index^16,17) was calculated as a length: width ratio of singular neurofilament-H-immunopositive signals. In an image of 120 × 120 µm², fifteen fibers were randomly selected for measurement. APP was visualized with rabbit anti-Amyloid Precursor Protein (1 : 100; Invitrogen™) as a primary antibody and Alexa Fluor594 donkey anti-rabbit IgG (H + L) antibody (1 : 500; Invitrogen™) as a secondary antibody, respectively. Threshold-based quantification of APP-immunopositive area was carried out with NIH ImageJ software, in the total area of an image of 3.6 × 2.7 mm².¹⁷⁾

Measurement of Evans Blue Extravasation

Evans Blue extravasation was used for evaluation of the integrity of blood-brain barrier.¹⁸⁾ At 48 h after induction of ICH, physiological saline containing 2% Evans Blue was intraperitoneally administered to mice at 4 mL/kg body weight. After 24 h, mice were deeply anesthetized and perfused transcardially with ice-cold PBS. A brain slice with 4 mm thickness of the hemisphere ipsilateral to the hemorrhage was obtained 2 mm posterior from the frontal pole. The slice was incubated in 400 µL formamide at 55 °C overnight to extract Evans Blue. The content of Evans Blue in the extract was measured by the absorbance at 655 nm, and was expressed as the content per gram tissue.

Quantitative RT-PCR

At 6 h after ICH operation, mice were anesthetized deeply and transcardially perfused with 30 mL ice-cold PBS. A brain slice with 4 mm thickness was obtained from the hemisphere ipsilateral to the hemorrhage and at 2 mm posterior from the frontal pole where the whole hemorrhagic region was located.¹⁵⁾ Total RNA was extracted with the use of RNAiso Plus reagent (TaKaRa Bio Inc., Shiga, Japan). Reverse transcription of RNA into cDNA (1 cycle at 37 °C for 15 min, 85 °C for 5 s) was carried out by PrimeScript™ RT Master Mix (TaKaRa Bio Inc.) and used for quantification of mRNAs encoding IL-6 and chemokine C-X-C motif ligand 2 (CXCL2). Sequences of primers for PCR were IL-6 forward, 5′-TCCAGTTGCCTTCTTGGGAC-3′; IL-6 reverse, 5′-GTGTAATTAAGCCTCCGACTTG-3′; CXCL2 forward, 5′-CGCTGTCATGCCTGAAGAC-3′; CXCL2 reverse, 5′-CCTTGAGAGTGGCTATGACTTCTG-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH; as internal control) forward, 5′-ACCATCTTCCAGGAGCGAGA-3′; GAPDH reverse, 5′-CAGTCTTCTGGGTGGCAGTG-3′. The PCR program was run at 95 °C for 30 s, then 40 cycles at 95 °C for 15 s, 55 °C for 45 s and 72 °C for 30 s. Results were automatically analyzed with a comparative Ct method.

Statistics

All numerical data are presented as means ± standard error of the mean (S.E.M.) and analyzed statistically with the use of GraphPad Prism software (Graph Pad, San Diego, CA, U.S.A.). Data on motor functions were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test. The other sets of data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test, or non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Two-tailed probability values less than 0.05 were considered significant.

RESULTS

Na₂S but Not Na₂S₃ Alleviates Sensorimotor Dysfunction of Mice after ICH

A previous study reported that single intraperitoneal administration of 25 µmol/kg NaHS at 30 min before ICH induction alleviated ICH-induced motor deficits of mice.¹³⁾ Based on these observations, we examined the effects of Na₂S and Na₂S₃ at an intraperitoneal dose of 25 µmol/kg given at 30 min before induction of ICH. In these settings, Na₂S significantly improved the performance of mice in the modified limb-placing test at 48 and 72 h after ICH (Fig. 1A). Na₂S also tended to improve the performance of mice in the beam-walking test at the same time points, although the difference from vehicle-treated group did not reach statistical significance either in the foot fault rate (Fig. 1B) or in the walking distance (Fig. 1C), possibly due to the spontaneous recovery of performance. On the other hand, Na₂S₃ produced no significant effect on the sensorimotor performance of mice in the modified limb-placing test and the beam-walking test (Fig. 1).

Fig. 1. Na₂S but Not Na₂S₃ Improves Sensorimotor Performance after ICH

(A) Sensorimotor functions were evaluated by the modified limb-placing test. Higher scores reflect more severe deficits. (B, C) Results of the beam-walking test quantified as the foot fault rate (B) and the walking distance (C). The number in parentheses indicate the number of animals in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sham group; ^###  p < 0.001 vs. ICH + vehicle group.

Na₂S Attenuates Neuronal Injury by ICH

To evaluate the effects of Na₂S and Na₂S₃ on tissue damage induced by ICH, we conducted immunohistochemical examinations of a neuronal marker NeuN in brain tissues obtained at 3 d after ICH. As shown in Fig. 2A, ICH resulted in a drastic decrease in the number of remaining neurons in the central region of hematoma located in the striatum. Treatment with Na₂S partially but significantly promoted neuronal survival within the hematoma. Na₂S₃ also tended to increase the number of neurons, but the effect was weaker than that of Na₂S and did not reach statistical significance (Fig. 2B).

Fig. 2. Effects of Na₂S and Na₂S₃ on Neuronal Survival and Axonal Integrity

(A) Representative images of NeuN immunohistochemistry on brain sections obtained 3 d after induction of ICH. The rectangle in the low-magnification image (the scale bar represents 500 µm) indicates the region for cell counting. Scale bars in the other images at high magnification represent 50 µm. (B) Quantitative results on the number of NeuN-positive cells in each group. (C) Representative images of neurofilament-H immunofluorescence histochemistry for evaluation of axonal structures in the internal capsule at 24 h after induction of ICH. Scale bars = 20 µm. (D) Quantitative results on the axonal shape index of each group. (E) Representative images of APP immunofluorescence histochemistry for evaluation of axonal transport functions at 24 h after induction of ICH. Scale bars = 500 µm. (F) Quantitative results on APP-positive area. The number attached to each column of graphs indicates the number of animals examined. *** p < 0.001 vs. sham group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001; n.s., not significant.

Na₂S and Na₂S₃ Preserve Structure and Function of Axon Tracts after ICH

In our experimental model of ICH, hematoma consistently invaded into the internal capsule located adjacent to the striatum. The internal capsule contains descending and ascending axonal projections that connect the cerebral cortex and the spinal cord, and invasion of hematoma into the internal capsule is closely associated with the severity of neurological symptoms after ICH.^9,19) Neurofilament-H immunofluorescence visualized the structural damage of axonal fibers in the internal capsule. That is, coronal sections of the internal capsule in sham-treated mice contained lengthy fibrous immunopositive signals, while the shape of the immunopositive signals has changed drastically at 24 h after ICH to punctate pattern (Fig. 2C). Na₂S markedly and significantly prevented ICH-associated fragmentation of axonal structures in the internal capsule. Na₂S₃ also prevented fragmentation of axonal structures considerably, although the effect did not reach statistical significance (Fig. 2D).

ICH-induced neuropathological changes also involve impairment of axonal transport, as reflected by accumulation of APP, a substrate of fast axonal transport.^17,20) Indeed, intense APP-immunoreactive signals were observed in the peri-hematoma region distributed in the striatum and the cerebral cortex at 24 h after ICH (Fig. 2E). Na₂S and Na₂S₃ were equally effective in inhibiting accumulation of APP around the hematoma (Fig. 2F), suggesting preservation of axonal transport function.

Na₂S₃ Inhibits Recruitment of Inflammatory Cells after ICH

Inflammatory events in and around the hematoma are prominent features of the pathogenic events in ICH, which include accumulation of activated microglia/macrophages in the peri-hematoma region and infiltration of neutrophils into the hematoma. Results of Iba1 immunohistochemistry on brain tissues at 3 d after ICH revealed that accumulation of Iba1-positive microglia/macrophages with round or amoeboid shape in the peri-hematoma region was not affected by treatment with Na₂S. On the other hand, Na₂S₃ partially but significantly decreased the number of activated microglia/macrophages (Figs. 3A, B). Concerning neutrophil infiltration, MPO-positive cells were not detected in the striatum of sham-operated mice, whereas numerous counts of MPO-positive cells were detected in the center of the hematoma of ICH-induced mice at 3 d (Fig. 3C). Na₂S significantly decreased the number of MPO-positive cells in the central region of hematoma. The number of MPO-positive cells was significantly decreased also by Na₂S₃ as compared to vehicle-treated group, and the effect of Na₂S₃ was more prominent than that of Na₂S (Fig. 3D).

Fig. 3. Effects of Na₂S and Na₂S₃ on Recruitment of Inflammatory Cells

(A) Results of Iba1 immunohistochemistry on brain sections obtained at 3 d after induction of ICH. The rectangle in the low-magnification image (the scale bar represents 500 µm) indicates the region for cell counting. Scale bars in the other images at high magnification represent 50 µm. (B) The graph shows the number of Iba1-positive cells with activated appearance (round or amoeboid shape) in each group. (C) Results of MPO immunohistochemistry at 3 d after induction of ICH. Scale bars represent 500 µm in the low-magnification image and 50 µm in high-magnification images, respectively. (D) The graph summarizes the number of MPO-positive cells in the central region of hematoma. (E) Effects of Na₂S and Na₂S₃ on vascular permeability. Quantitative results of Evans Blue extravasation at 3 d after induction of ICH are shown. Number of animals examined is indicated in each column of graphs. *** p < 0.001 vs. sham group; ^# p < 0.05, ^### p < 0.001; n.s., not significant.

Because retention of the integrity of blood–brain barrier (BBB) may be responsible for the inhibitory effect of Na₂S and Na₂S₃ on infiltration of inflammatory cells, Evans Blue extravasation was used to evaluate the brain vascular permeability. Compared to sham-operated mice, mice at 72 h after ICH showed higher degree of vascular permeability in the brain as reflected by the increase in Evans Blue content. Pretreatment with either Na₂S or Na₂S₃ had no significant effect on ICH-induced increase in the brain vascular permeability (Fig. 3E).

Na₂S Suppresses Upregulation of Pro-inflammatory Factors after ICH

Inflammatory events triggered by ICH are associated with robust increases in the expression of pro-inflammatory cytokines and chemokines.²¹⁾ We examined the expression levels of mRNAs encoding IL-6 and CXCL2 in the ipsilateral hemisphere at 6 h after ICH induction. Treatment with Na₂S at 30 min before ICH induction resulted in considerable decreases in the expression of IL-6 and CXCL2 mRNAs as compared to the vehicle-treated group, and the inhibitory effect on CXCL2 mRNA expression was statistically significant. In contrast, the effect of Na₂S₃ on these parameters was weaker than Na₂S, which did not reach statistical significance (Figs. 4A, B).

Fig. 4. Effects of Na₂S and Na₂S₃ on the Expression of Inflammatory Mediators

Expression levels of mRNAs encoding IL-6 (A) and CXCL2 (B) at 6 h after induction of ICH were determined by quantitative RT-PCR. GAPDH mRNA was used as internal control. Number of animals examined is indicated in each column. * p < 0.05, **  p  <  0.01, ***  p  <  0.001 vs. sham group; ^##  p  <  0.01; n.s., not significant.

DISCUSSION

Previously we have addressed the neuroprotective effects of several sulfide species in rat midbrain slice cultures.⁶⁾ In that study, Na₂S₃ at concentrations of 10 µM or higher produced a significant protective effect on dopaminergic neurons against 1-methyl-4-phenylpyridinium cytotoxicity. When compared at the same molar concentration (10 µM), Na₂S₃ was most effective among other sulfide species including Na₂S, Na₂S₂ and Na₂S₄.⁶⁾ Based on these observations, the present study compared the effects of Na₂S (as monosulfide) and Na₂S₃ (as a representative polysulfide) on various pathological parameters of ICH in mice. The results show that these sulfide species exhibit different pharmacological profiles. That is, (1) Na₂S but not Na₂S₃ improved motor function of mice after ICH (Fig. 1), (2) Na₂S prevented neuron loss in the hematoma and fragmentation of axon structures in the internal capsule, and both Na₂S and Na₂S₃ reduced axonal transport dysfunction (Fig. 2), (3) Na₂S₃ inhibited accumulation of activated microglia/macrophages and infiltration of neutrophils more potently than Na₂S (Fig. 3), but (4) Na₂S suppressed ICH-induced expression of inflammatory cytokine/chemokine more potently than Na₂S₃ (Fig. 4).

Beneficial effect of monosulfide species on neurological functions after ICH incidents has been reported in several previous studies using NaHS or ADT-OH as H₂S donors.^12–14) Shan et al.¹³⁾ demonstrated that single intraperitoneal administration of 25 µmol/kg NaHS 30 min before ICH induction improved recovery of motor performance in a wire grip test. Consistent with these observations, we showed in the present study that single intraperitoneal administration of 25 µmol/kg Na₂S 30 min before ICH induction improved sensorimotor performance of mice at 48 and 72 h after ICH in the modified limb-placing test (Fig. 1).

Results of immunohistochemical examinations indicate that Na₂S showed protective effect on neuronal structures and functions. Previous studies reported that NaHS inhibited neuronal death in the striatum at 1 d after ICH,^12,13) and our results demonstrated a significant neuroprotective effect of Na₂S even at 3 d after induction of ICH (Figs. 2A, B). Moreover, we found that monosulfide could prevent fragmentation of axon structures in the internal capsule and dysfunction of fast axonal transport in the peri-hematoma region (Figs. 2C–F). Particularly, preservation of axonal structures in the internal capsule may be a prerequisite for enhanced recovery of sensorimotor functions, as the internal capsule contains axon bundles that connect the cerebral cortex with the spinal cord.^9,19)

In addition to these neuroprotective effects, Na₂S was able to attenuate inflammatory reactions triggered by ICH. In a study by Zhao et al.,¹²⁾ NaHS reduced the number of MPO-positive cells and Iba1-positive cells in the peri-hematoma region at 1 d after ICH. Our immunohistochemical results with brain sections obtained at 3 d after ICH indicated that Na₂S significantly decreased the number of MPO-positive neutrophils within the hematoma, although the number of activated Iba1-positive microglia/macrophages was unaffected (Figs. 3A–D). Results of quantitative RT-PCR at 6 h after ICH demonstrating significant suppression of CXCL2 mRNA expression as well as considerable reduction of IL-6 mRNA expression are consistent with anti-inflammatory actions of Na₂S (Fig. 4). In this context, other monosulfide donors such as NaHS and ADT-OH were also shown to suppress ICH-induced upregulation of several pro-inflammatory factors including IL-1β, IL-6 and TNF-α.^12,14)

In contrast to Na₂S, Na₂S₃ did not enhance the recovery of sensorimotor functions of mice after ICH (Fig. 1). In addition, Na₂S₃ produced only modest and insignificant protective effect on neurons in the hematoma (Figs. 2A, B). On the other hand, Na₂S₃ significantly prevented dysfunction of axonal transport and considerably inhibited fragmentation of axonal structures at a comparable extent to Na₂S (Figs. 2C–F). The latter observation raises an important possibility that the preservation of axonal structures in the internal capsule is required but not sufficient for improvement of sensorimotor functions after ICH insults.

Distinct pharmacological profiles of Na₂S₃ and Na₂S were also evident in their effects on inflammatory parameters. Indeed, inhibitory effect of Na₂S₃ on upregulation of IL-6 and CXCL2 mRNAs at 6 h after ICH was much less than that of Na₂S (Fig. 4). By contrast, inhibitory effect of Na₂S₃ on accumulation of activated microglia/macrophages and infiltration of neutrophils at 3 d after ICH was more potent than that of Na₂S (Figs. 3A–D). The inhibitory effect on infiltrating macrophages and neutrophils may not be attributable to the effect of sulfides on BBB, because no significant effect of either Na₂S or Na₂S₃ was observed on Evans Blue extravasation at 3 d after ICH (Fig. 3E). The underlying mechanisms that explain differential effects of Na₂S or Na₂S₃ on ICH pathologies are unclear at present, but two possibilities may deserve consideration. First, different sets of signaling molecules and pathways may be involved in the biological actions of monosulfide and polysulfide.³⁾ Previous studies addressing the effect of H₂S donors on ICH pathologies proposed several mechanisms of actions that include inhibition of P2X₇ receptor upregulation and NLRP3 inflammasome expression,¹²⁾ inhibition of Bcl-2 downregulation and inhibition of autophagy,¹³⁾ and sulfide-quinone oxidoreductase-mediated mitochondrial uncoupling and AMP kinase activation.¹⁴⁾ Whether or not polysulfides affect these signaling events have not been addressed so far, but the lack of effect of polysulfide on one or more of these signaling machineries might be responsible for the less potent therapeutic effect of Na₂S₃ on ICH than Na₂S. Second, BBB permeability of polysulfide may be lower than that of monosulfide, which may explain weaker effect of Na₂S₃ as compared to Na₂S. In this context, the significant effect of Na₂S₃ on the number of infiltrating cells might be mediated by its peripheral actions on monocytes/macrophages and neutrophils in the bone marrow or in the blood. Utilization of polysulfide donors with enhanced cell permeability²²⁾ might provide deeper insights into the biological actions of polysulfides in the brain.

In conclusion, we demonstrated that monosulfide applied as Na₂S ameliorated sensorimotor functions of mice after ICH, which was associated with beneficial influences on various histochemical and biochemical parameters including preservation of axon tract integrity and suppression of pro-inflammatory gene expression. Polysulfide applied as Na₂S₃ did not ameliorate ICH-induced sensorimotor dysfunctions, although it mimicked the effect of Na₂S on several pathological parameters. Regulation of multiple pathological events may be required for sulfide species to provide therapeutic effect on neurological symptoms in ICH.

Acknowledgments

This work was supported by The Smoking Research Foundation; JSPS KAKENHI, MEXT, Japan Grants 19K22813, 20H04126, 21J20376 and 22K19756.

Conflict of Interest

The authors declare no conflict of interest.

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