Hydrogen sulfide mitigates spatial learning deficits in angiotensin II-Induced hypertension via inhibition of endothelial NOX2 and neuroinflammation

Figures

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  Fig. 1. Pretreatment with NaHS in brain maintains H2S levels in Angiotensin II-Induced Hypertension. A Schematic diagram of the experimental design showing treatment groups in the current study. Animals from control, angiotensin II (600 ng/kg/min) plus vehicle, angiotensin II plus NaHS (4 mg/kg); saline plus NaHS (4 mg/kg) groups were subjected to the Morris water maze test. B line graph showing systolic blood pressure measured at day 0, 3, 7, and 14 in the control, Ang II, Ang II + NaHS NaHS, and groups. Day 0 value (2 day before osmotic pump implantation).Data are presented as mean ± SEM. C Scatter with Bar graph representing levels of H2S (in μM) in brain tissue extracts of animals in mentioned groups (saline), (n = 5). D Scatter with Bar graph representing concentration of total sulfide (in μM) in brain tissue extracts of animals groups as mentioned (n = 3) * indicates statistical significance of Ang II compared with control group, while # indicates statistical significance between the Ang II and Ang II + NaHS groups. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

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  Fig. 2. H2S protects spatial learning impairment and maintains Synaptic Protein Expression in Angiotensin II -Induced Hypertension. A Schematic diagram of the experimental design of the Morris water maze test. The animals were habituated to water for 2 days and, subsequently, trained to learn in the Morris water maze for 5 days. Line graphs representing day-wise changes in (B) latency (Second) and (C) path length (Meter) over the 5-day training period of animals in the Morris water maze. D Representative western blots of NMDAR2B, (E) PSD95 and synaptophysin in the Hippocampal samples obtained from the indicated groups. Scatter with Bar graph showing normalized protein expression of (F) NMDA R2B (G) Synaptophysin & (H) PSD95 Mean ± SEM is shown. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

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  Fig. 3. Administration of NaHS attenuates the increase in vessel-associated microglia and microglial activation in Angiotensin II infused hypertension. A Representative Co-immunofluorescence of CD31 (endothelial marker, red) and IBA1 (microglia, green) in mentioned groups magnified image shows microglia associated blood vessel region of images Scale bar: 20 μm. B Scatter with bar graph represent the percentage of microglia associated with blood vessel (C) Scatter with bar graph represents Pearson’s correlation coefficient between IBA1 and CD31 indicating microglia blood vessel overlapping. D Microglial morphology quantified by Skeleton analysis using Image J Skeletonize plugin microglia (E) Number of microglial branches and (F) length of total microglial branch in all groups. G Co-immunofluorescence images of IBA1 and CD68 showing CD68 foci in microglia in Ang II induced hypertension group along with other mentioned groups: group 1 is control very provided saline and vehicle, group 2, angiotensin II (600 ng/kg/min) plus vehicle; group 3, angiotensin II plus NaHS (4 mg/kg); group 4 (saline plus NaHS (4 mg/kg) Scale bar: 20 μm Mean ± SEM is shown. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 4. NaHS Pretreatment Exhibits Anti-inflammatory Effects by Modulating Cytokine and chemokine Profiles in Angiotensin II-Induced Neuroinflammation. A The layout of protein probed in cytokine and chemokine array along with positive control indicated as POS, each cytokine was detected in duplicate. B Representative images showing developed Cytokine and Chemokine arrays for control, Ang II induced hypertension with or without NaHS pre-treatment. Significantly modulated cytokines by NaHS pretreatment are denoted in the box. C Heat map illustrating the expression of cytokinesand chemokine in each group. Red and green colours represent relative high and low cytokine expression values, respectively. D The quantitative analysis of the cytokine was performed by densitometry using ImageJ. The data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 based on ANOVA; (n = 3) biological replicates.

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  Fig. 5. NaHS Pretreatment Reduces NOX2-Mediated Endothelial Dysfunction in the Hippocampus without modulating AT-1. A Representative immunofluorescence micrographs showing vWF in hippocampus section Scale bar: 20 μm (B) Scatter with Bar graph showing fluorescent signal estimated employing intensity thresholding method in Image J, NIH software. C Representative western blots of AT1 (angiotensin II type 1 receptor; AT1R), Nox2, Nox2 p67 in brain tissues of animals. The protein expression signals were normalized to actin (loading control) corresponding scatter with bar graphs showing change in mean intensity (expression) (D) AT1, Nox2 and Nox2 p67. (n = 3). E CO-Immunofluorescence on cortical sections Nox2 (red); cerebral vessels were labeled with CD31(Green) Scale bar: 20 μm. F Scatter with bar graph represents Pearson’s correlation coefficient between NOX2 and CD31 indicating microglia blood vessel overlapping (G) Corresponding bar graphs showing mean fluorescent intensity of NOX2 (Mean ± SEM) Scale bar: 20 μm. n = 3 individual brains and 3 sections/per brain. Statistical analysis was performed using 1-way ANOVA followed by Bonferroni’s post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 6. Proposed scheme of events in the present study. A summary figure depicting the neurovascular unit in physiological and Ang II-induced hypertension with NaHS supplementation, which includes astrocytes, pericytes, microglial cells, neurons, and the associated vasculature.In the physiological state, it shows normal microglial morphology, which enables them to survey the physiological status of blood vessels. Ang II-induced hypertension (600 ng/kg/day) causes activation of microglia, an increase in vessel-associated microglia, and endothelial Nox2 activation. Such activated microglia and endothelial cells are associated with upregulation of cytokines and chemokines, which could partially cause the neuroinflammatory milieu and increase endothelial-to-microglial interaction, eventually perturbing neuronal function. Restoration of H2S levels via exogenous supplementation NaHS restores endogenous H2S levels in the hypertensive brain and curtails microglial endothelial interaction, microglial and endothelial activation, and neuronal dysfunction.