Hydrogen inhalation ameliorated mast cell-mediated brain injury after intracerebral hemorrhage in mice

Abstract

Objective: Hydrogen inhalation was neuroprotective in several brain injury models. Its mechanisms are believed to be related to antioxidative stress. We investigated the potential neurovascular protective effect of hydrogen inhalation especially effect on mast cell activation in a mouse model of intracerebral hemorrhage.

Design: Controlled in vivo laboratory study.

Setting: Animal research laboratory.

Subjects: One hundred seventy-one 8-week-old male CD-1 mice were used.

Interventions: Collagenase-induced intracerebral hemorrhage model in 8-week-old male CD-1 mice was used. Hydrogen was administrated via spontaneous inhalation. The blood-brain barrier permeability and neurologic deficits were investigated at 24 and 72 hours after intracerebral hemorrhage. Mast cell activation was evaluated by Western blot and immuno-staining. The effects of hydrogen inhalation on mast cell activation were confirmed in an autologous blood injection model intracerebral hemorrhage.

Measurement and main results: At 24 and 72 hours post intracerebral hemorrhage, animals showed blood-brain barrier disruption, brain edema, and neurologic deficits, accompanied with phosphorylation of Lyn kinase and release of tryptase, indicating mast cell activation. Hydrogen treatment diminished phosphorylation of Lyn kinase and release of tryptase, decreased accumulation and degranulation of mast cells, attenuated blood-brain barrier disruption, and improved neurobehavioral function.

Conclusion: Activation of mast cells following intracerebral hemorrhage contributed to increase of blood-brain barrier permeability and brain edema. Hydrogen inhalation preserved blood-brain barrier disruption by prevention of mast cell activation after intracerebral hemorrhage.

Conflict of interest statement

The authors have not disclosed any potential conflicts of interest

Figures

FIGURE 1

(A) Intracerebral hemorrhage induced a significant increase of brain water content in ipsilateral basal ganglia and cortex ( N=7) when compared to sham operated ( N=7) animals. One hour of hydrogen inhalation starting one hour after inducing of intracerebral hemorrhage ( N=7) significantly diminished brain edema. Two hours of hydrogen inhalation starting one hour after inducing of intracerebral hemorrhage ( N=7) showed a strong tendency to ameliorate intracerebral hemorrhage induced increase of brain water content. (B–C) All animals after intracerebral hemorrhage demonstrated significant neurological deficit according to performance on the modified Garcia (B), wire hanging and beam balance (C) tests. One hour of hydrogen treatment ameliorated neurological deficits in all three tests. Two hours of hydrogen treatment significantly decreased the neurological deficits evaluated by wire hanging and beam balance tests and showed a tendency to improvement in the Garcia test (B–C). * Significant vs. sham, # significant vs. vehicle, p<0.05 ANOVA, Tukey test. At 72 hours after inducing of intracerebral hemorrhage, brain water content in ipsilateral basal ganglia and cortex of ICH ( N=8) remained elevated compared to that of sham operated ( N=7) animals. Both a single one hour treatment with hydrogen starting one hour after inducing of intracerebral hemorrhage ( N=8) and multiple treatments (one hour daily for 3 days N=8) showed a tendency to decrease brain water content (D) and to ameliorate neurological deficits (E-F). * significant vs. sham, p<0.05 ANOVA, Tukey test.

FIGURE 2

(A) Intracerebral hemorrhage caused significant disruption of blood-brain barrier at 24 hours ( sham operated animals N=6, vehicle-treated animals N=8). One hour of hydrogen therapy ( N=8) significantly reduced intracerebral hemorrhage induced disruption of blood-brain barrier at 24 hrs. (B) One hour hydrogen therapy showed a tendency to preserve intracerebral hemorrhage induced disruption of blood-brain barrier at 72 hours ( Sham operated animals N=6, vehicle treated animals N=6, hydrogen treated animals N=6). * significant vs. sham, p<0.05 ANOVA, Tukey test.

FIGURE 3

(A) Neither intracerebral hemorrhage nor hydrogen treatment effected expression of Lyn kinase at 6 hours after inducing of intracerebral hemorrhage. (B) Intracerebral hemorrhage significantly increased phosphorylation of Lyn kinase at 6 hours after inducing of intracerebral hemorrhage ( Sham operated animals N=6, Vehicle N=6). One hour of hydrogen treatment ( N=6) significant diminished Lyn kinase phosphorylation when compared to intracerebral hemorrhage animals treated with vehicle. Pretreatment with catalase inhibitor at three hours before intracerebral hemorrhage ( N=6) prevented effect of hydrogen on Lyn kinase phosphorylation. * significant vs. sham, # significant vs. vehicle, p<0.05 ANOVA, Tukey test.

FIGURE 4

Intracerebral hemorrhage increased the release of tryptase at 6 hours after induction of intracerebral hemorrhage ( Sham operated animals N=6, (Vehicle N=6). One hour of hydrogen treatment ( N=6) significant diminished intracerebral hemorrhage induced release of tryptase. Pretreatment with catalase inhibitor at three hours before induction of intracerebral hemorrhage ( N=6) reversed effect of hydrogen on the release of tryptase. * significant vs. sham, # significant vs. vehicle, p<0.05 ANOVA, Tukey test.

FIGURE 5

6 hours after intracerebral hemorrhage induction, there were more tryptase positive cells in brain of vehicle (B) animals compared to sham-operated animals (A). One hour of hydrogen treatment (C) diminished number of tryptase positive cells.

FIGURE 6

Effect of intracerebral hemorrhage and hydrogen treatment on mast cells proliferation and activation was investigated by Toluidine Blue staining at 6 hours after inducing of intracerebral hemorrhage. More mast cells were observed in brain of vehicle animals (Panel I-B) compared to sham-operated animals (Panel I-A). One hour of hydrogen treatment reduced the number of mast cells (Panel I-C). Additionally if compared to granulated intensive stained cells in brain of sham operated animals (Panel II-A white arrow), most of the mast cells detected in brain of animals after intracerebral hemorrhage showed the clear signs of degranulation with less intensive Toluidine Blue staining and the appearance of `ghost' cells (Panel II-B white arrow, black outline). Hydrogen inhalation decreased the number of such cells and increased the amount of granulated mast cells (Fig 7C, white arrow).

FIGURE 7

Collagenase-induced intracerebral hemorrhage and intracerebral hemorrhage induced by direct injection of autologous blood demonstrated comparable release of mast cell proteins. No differences between these two models were detected. * significant vs. sham, p<0.05 ANOVA, Tukey test.

FIGURE 8

Intracerebral hemorrhage ( Vehicle N=6) significantly increased the production of hydrogen peroxide (H2O2) evaluated by Amplex Red peroxidase assay at 2 hours after ICH, when compared to sham operated animals ( Sham N=6). One hour of hydrogen treatment significantly diminished ICH-induced production of H2O2 ( N=6). Pretreatment with catalase inhibitor at three hours before inducing of intracerebral hemorrhage ( N=6) reversed effect of hydrogen on the H2O2 production. * significant vs. sham, # significant vs. vehicle, p<0.05 ANOVA, Tukey test.

FIGURE 9

At 24 hours after inducing of intracerebral hemorrhage, significant accumulation of nitrotyrosine was observed in brain of animals after intracerebral hemorrhage ( N=6) compared to sham operated animals ( N=6). Hydrogen inhalation decreased intracerebral hemorrhage induced accumulation of nitrotyrosine at 24 hours (). At 72 hour after inducing of intracerebral hemorrhage, hydrogen treatment showed a strong tendency to diminish nitrotyrosine accumulation in brain of hydrogen treated ( N=6) compared to untreated ( N=6) animals. * significant vs. sham, # significant vs. vehicle, p<0.05 ANOVA, Tukey test