Fig 1.
Effects of hyperbaric oxygen therapy on neurological function of light-dark box (LDB) and Morris water maze (MWM) test.
(A) Number of transitions in the LDB test. (B) Latency time in the LDB test. (C) Percentage of time spent in the target quadrant during MWM test. (D) Latency to find the platform in the MWM test. NH, ischemia-reperfusion model and normobaric hyperoxia group; CT, control group; HO, ischemia-reperfusion model and hyperbaric oxygen therapy group; GM, ischemia-reperfusion model group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. CT group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. GM group; Δp < 0.05, ΔΔp < 0.01, ΔΔΔp < 0.001 vs. NH group.
Fig 2.
Histopathological, ultrastructural, nissl, and TUNEL changes in the hippocampal CA1 region of rats after global cerebral ischemia-reperfusion injury.
Representative images showing alterations in the hippocampal CA1 region of rats from different experimental groups: Control (CT, panels A, E, I, M), Ischemia-Reperfusion (GM, panels B, F, J, N), Ischemia-Reperfusion + Normobaric Hyperoxia (NH, panels C, G, K, O), and Ischemia-Reperfusion + Hyperbaric Oxygen Therapy (HO, panels D, H, L, P). A-D: H&E staining showing histopathological changes (Magnification: 400 × ; Scale bar: 400 μm). (A) Control group displays normal cellular architecture with intact pyramidal neurons. (B) Ischemia-reperfusion group exhibits marked neuronal loss and pyknotic nuclei. (C) Normobaric Hyperoxia shows moderate improvement compared to the ischemia group. (D) HBOT treatment demonstrates notable neuroprotection with preserved cellular morphology. E-H: Transmission electron microscopy of CA1 neurons (Magnification: 10,000 × ; Scale bar: 500 nm). (E) Control neurons show normal ultrastructure with intact organelles. (F) Ischemia-reperfusion causes severe mitochondrial swelling, endoplasmic reticulum dilation, and nuclear condensation. (G) Normobaric Hyperoxia shows intermediate ultrastructural preservation. (H) HBOT-treated samples display reduced ultrastructural damage with relatively preserved mitochondria. I-L: Nissl staining for neuronal survival assessment (Magnification: 400 × ; Scale bar: 100 μm). (I) Control section shows abundant Nissl bodies in neuronal cytoplasm. (J) Ischemia-reperfusion leads to significant loss of Nissl substance. (K) Normobaric Hyperoxia shows partial preservation of Nissl substance. (L) HBOT treatment preserves Nissl bodies, indicating improved neuronal viability. M-P: TUNEL assay for apoptosis detection (Magnification: 400 × ; Scale bar: 50 μm). (M) Control tissue shows minimal TUNEL-positive neurons. (N) Ischemia-reperfusion results in extensive TUNEL-positive neurons, indicating widespread apoptosis. (O) Normobaric Hyperoxia moderately decreases apoptotic neuron death compared to the ischemia group. (P) HBOT treatment significantly reduces TUNEL-positive neurons.
Fig 3.
Effects of hyperbaric oxygen therapy on biochemical parameters in global cerebral ischemia-reperfusion injury.
(A and B) Serum hepcidin protein and mRNA levels measured by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR), respectively. (C) Malondialdehyde (MDA) concentrations. (D) Glutathione peroxidase (GSH-Px) activity. (E) Superoxide dismutase (SOD) activity. *p < 0.05, **p < 0.01, ***p < 0.001 vs. CT group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. GM group; Δp < 0.05, ΔΔp < 0.01, ΔΔΔp < 0.001 vs. CA group.
Fig 4.
Western blot analysis of protein expression levels in hippocampal tissues across different experimental groups (n = 4 for each group).
(A) Western blot images showing protein bands for Smad1 (52 kDa), Smad2 (52 kDa), phosphorylated Smad1 (P-Smad1, 60 kDa), phosphorylated Smad2 (P-Smad2, 60 kDa), Ferroportin/SLC40A1 (62 kDa), and β-actin (42 kDa, loading control). B-F: Quantification of protein expression of (B) Smad1, (C) Smad2, (D) Ferroprotein (SLC40A1), (E) P-Smad1, and (F) P-Smad2. The protein expression levels are normalized to β-actin and presented as folds of control. *p < 0.05, **p < 0.01, ***p < 0.001 vs. CT group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. GM group; Δp < 0.05, ΔΔp < 0.01, ΔΔΔp < 0.001 vs. CA group.
Fig 5.
Western blot analysis of protein expression levels in hippocampal tissues of rats across different experimental groups (n = 4 for each group).
(A) Western blot images showing protein bands for Hepcidin (16 kDa), Glutathione Peroxidase 4 (GPX4, 22 kDa), Bone Morphogenetic Protein 6 (BMP6, 42 kDa), and GAPDH (36 kDa, loading control). B-D: Densitometric quantification of protein expression of (B) Hepcidin, (C) GPX4, and (D) BMP6. The protein expression levels are normalized to GAPDH and presented as folds of control. *p < 0.05, **p < 0.01, ***p < 0.001 vs. CT group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. GM group; Δp < 0.05, ΔΔp < 0.01, ΔΔΔp < 0.001 vs. CA group.