Fluoxetine-enhanced autophagy ameliorates early brain injury via inhibition of NLRP3 inflammasome activation following subarachnoid hemorrhage in rats

Fig. 1. Caspase-1 activation and necrotic cell death were induced in the early stages after SAH. a Representative western blot and the relative band densities of caspase-1 p20 in the ipsilateral cortex at different time points (6, 12, 24, 48, and 72 h) after SAH (n = 4/group). The bar represents the mean ± SD. *P < 0.05 vs sham, **P < 0.01 vs sham. b Representative photomicrographs and the quantification of the PI-positive neural cells in the ipsilateral cortex at 24 h after SAH (n = 3/group). Fluorescence colors: DAPI: blue and PI: red. Scale bar = 100 μm, the quantification of the PI-positive neural cells expressed as positive cells per square millimeter. The bar represents the mean ± SD. **P < 0.01 vs sham

Fig. 2. Double staining for PI with NeuN (neuron marker), Iba-1(microglia marker), GFAP(astrocyte marker), and caspase-1, and ultrastructural changes of necrotic cells. a Double staining for PI with NeuN (neuron marker), Iba-1(microglia marker), and GFAP(astrocyte marker). Scale bar = 50 μm. b Double staining for PI with caspase-1 in the ipsilateral cortex at 24 h after SAH. Scale bar = 50 μm. c Ultrastructural changes of necrotic cells at 24 h after SAH. Neurons in the left panel appeared normal with nuclei, mitochondria, and cytomembranes. Neurons in the right panel showed fragmented nuclei (black asterisk), swollen mitochondria (red asterisk), disrupted cytomembranes (white arrow), damaged organelles (white arrow), and double-membrane autophagosomes (red arrow). Scale bar = 2 μm

Fig. 3. Caspase-1 activation contributes to early brain injury after SAH. a Representative western blots and densitometric analyses showing levels of IL-1ß. The bar represents the mean ± SD. **P < 0.01 vs sham. ## P < 0.01 vs vehicle control. b Representative western blots and densitometric analyses showing levels of IL-18 (n = 6/group). The bar represents the mean ± SD. **P < 0.01 vs sham. ## P < 0.01 vs vehicle control. c Representative photomicrographs of PI staining of neural cells in different groups at 24 h after SAH (n = 3/group). Fluorescence colors: DAPI: blue and PI: red. Scale bar = 100 μm. d The quantification of the PI-positive neural cells. The quantification of the PI-positive neural cells expressed as positive cells per square millimeter. The bar represents the mean±SD. **P < 0.01 vs sham, ## P < 0.01 vs vehicle control. e Quantification of neurological scores (n = 9). The bars represent the mean with interquartile range. *P < 0.05 vs sham. # P < 0.05 vs vehicle control. f Quantification of SAH severity (n = 9). The bars represent the mean ± SD. No significant difference was observed between vehicle and SAH+AC-YVAD-CMK group

Fig. 4. Fluoxetine reduced expression of NLRP3, caspase-1 p20, IL-1ß, and IL-18, decreased the numbers of necrotic cells and brain edema, and improved the neurological scores. a Representative Western blots and densitometric analyses of levels of NLRP3, caspase-1 p20, IL-1ß, and IL-18. The bar represents the mean ± SD. **P < 0.01 vs sham. # P < 0.05 vs vehicle control, ## P < 0.01 vs vehicle control. b Representative photomicrographs of PI-positive neural cells in different groups at 24 h after SAH (n = 3/group). Fluorescence colors: DAPI: blue and PI: red. Scale bar = 100 μm. c The quantification of the PI-positive neural cells. The quantification of the PI-positive neural cells expressed as positive cells per square millimeter. The bar represents the mean ± SD. **P < 0.01 vs sham, ## P < 0.01 vs vehicle control. d Quantification of brain water content (n = 6). The bars represent the mean ± SD. **P < 0.01 vs sham control, ## P < 0.01 vs vehicle control. e Quantification of neurological scores (n=18). The bars represent the mean with interquartile range. *P < 0.05 vs sham control, # P < 0.05 vs vehicle control

Fig. 5. Autophagy activation was involved in the protective effect of fluoxetine on NLRP3 inflammasome activation. a Double fluorescence of PI staining (red) and cells with autophagy marker LC3 (green). b Representative micrographs showing double immunofluorescence with caspase-1 (red) and LC3 (green). Scale bar = 50 μm. c Representative Western blots and d densitometric analyses of levels of beclin-1 (n = 6). The bars represent the mean ± SD. *P < 0.05 vs sham control, # P < 0.05 vs vehicle control

Fig. 6. Autophagy inhibitor 3-MA blocks the effects of fluoxetine on expression of NLRP3, caspase-1 p20, IL-1β, and IL-18 after SAH induction. a Representative Western blots of NLRP3, caspase-1 p20, IL-1β, and IL-18. b Densitometric analyses of levels of NLRP3, caspase-1 p20, IL-1β, and IL-18. N = 6. The bar represents the mean ± SD. **P < 0.01 vs the sham group. ## P < 0.01 vs SAH + vehicle group. & P < 0.05 vs SAH + fluo group. Representative photomicrographs and the quantification of the PI-positive neural cells in different groups at 24 h after SAH (n = 3/group). Fluorescence colors: DAPI: blue and PI: red. Scale bar = 100 μm, the quantification of the PI-positive neural cells expressed as a percent of the total DAPI+ cells. The bar represents the mean ± SD. **P < 0.01 vs SAH + vehicle group. ## P < 0.01 vs SAH + fluoxetine group

Fig. 7. Autophagy inhibitor 3-MA abolishes the neuroprotective effects of fluoxetine on necrotic cell death, brain edema, and neurological function after SAH. a Representative photomicrographs of the PI-positive neural cells in different groups at 24 h after SAH (n = 3/group). Fluorescence colors: DAPI: blue and PI: red. Scale bar = 100 μm. b The quantification of the PI-positive neural cells expressed as positive cells per square millimeter. **P < 0.01 vs the sham group. ## P < 0.01 vs SAH + vehicle group. & P < 0.05 vs SAH + fluo group. c Quantification of brain water content (n = 6). The bars represent the mean ± SD. **P < 0.01 vs the sham group. ## P < 0.01 vs SAH + vehicle group. & P < 0.05 vs SAH + fluo group. d Quantification of neurological scores (n = 15/group). The bars represent the mean with interquartile range. *P < 0.05 vs the sham group. # P < 0.05 vs SAH + vehicle group. & P < 0.05 vs SAH + fluo group