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Sildenafil treatment restored thickness of the CC and ECL and improved oligodendrocyte populations in a dose-dependent manner.

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Medium and high doses increased both total (Olig2 +) and mature (CC1 +) oligodendrocyte counts in the CC and ECL, but not immature ones (Olig2 + /CC1-).

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These findings indicate that sildenafil may promote oligodendrogenesis and support remyelination. Previous studies in adult models of stroke, multiple sclerosis, and diabetes support this hypothesis, demonstrating that sildenafil enhances oligodendrocyte regeneration, increases myelin thickness, and protects myelinated axons by modulating immune responses24,28,29,74.

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HI reduced thickness of the CC (median [interquartile ranges]: 0.52 µm [0.12–0.63], p < 0.01) and ECL (0.14 [0.02–0.33], p < 0.05) compared to sham (CC: 0.89 [0.75–0.98]; ECL: 0.40 [0.32–0.60]) (Fig. No significant differences in Olig2 + or CC1 + oligodendrocytes were observed at baseline between HI (CC: Olig2 + : 3820 cells/mm2 [2367–4387]; CC1 + : 2314 [1202–2613]; ECL: Olig2 + : 3833 [2516–4013]; CC1 + : 2000 [896–2505]) and sham (CC: Olig2 + : 3561 [3283–3996]; CC1 + : 2391 [2288–3061]; ECL: Olig2 + : 4273 [3714–4734]; CC1 + : 2837 [2581–3504]) (Fig. Medium- and high-dose sildenafil significantly increased thickness of the CC (medium-dose: 0.68 [0.47–0.78]; high-dose: 0.64 [0.52–0.87]) and ECL (medium-dose: 0.28 [0.00–0.47]; high-dose: 0.27 [0.16–0.46]), restoring it to sham levels. Low dose also improved thickness of the ECL (0.18 [0.00–0.36]) but was less effective in the CC (0.57 [0.49–0.80], p < 0.05 vs. HI) (Fig.

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Similar effects were seen in the ECL (Olig2 + : low-dose: 4087 [3846–4484]; medium-dose: 4923 oligodendrocytes/mm2 [4630–6250], p < 0.05 vs. HI; high-dose: 5640 oligodendrocytes/mm2 [4739–6351], p < 0.01 vs. HI); CC1 + : mature oligodendrocytes (low-dose; 2798 [2218–3155]; medium-dose: 3641 oligodendrocytes/mm2 [3077–4688], p < 0.01 vs. HI; high-dose: 4210 oligodendrocytes/mm2 [3543–5006], p < 0.001 vs. HI) (Fig.

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There was no difference between experimental groups in immature Olig2 + /CC1- oligodendrocytes. HI caused a significant increase in cleaved PARP) levels at P12 in the ipsilateral cortex and white matter compared to sham rats (p < 0.01) (Fig. This increase was accompanied by a significant reduction in the antioxidant protein SOD1 (p < 0.05) and a marked decrease in NeuN expression at P30 (p < 0.05) (Fig. Synaptophysin levels also were reduced significantly following HI (p < 0.001) (Fig. Sildenafil treatment restored cleaved PARP levels to values no longer significantly different from sham at P12 (Fig. Sildenafil’s effects appear context-dependent, promoting oligodendrocyte maturation primarily under hypoxic-ischemic conditions.

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Its anti-inflammatory and pro-survival actions may create a permissive environment for oligodendrocyte maturation and myelin repair during injury, whereas in the absence of HI, these pathways are likely insufficiently activated to elicit further enhancement.

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SOD1 levels also returned to baseline with sildenafil treatment, indicating a reversal of HI-induced oxidative stress (Fig. Similarly, NeuN and synaptophysin expression at P30 were normalized following sildenafil administration (Fig. pAKT levels were decreased significantly at P12 following HI (p < 0.05) compared to sham animals but returned to baseline levels after sildenafil treatment (Fig. In our rat model of term neonatal HIE, HI induced an increase in reactive astrocytes and microglia in the cortex adjacent to the infarct boundary, as well as in the white matter, which remained evident 20 days post-insult. The number of activated, phagocytic macrophages also increased significantly in CC and ECL.

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Interestingly, the anti-inflammatory cytokine IL-1ra was transiently upregulated at P12. Early microglial activation, often associated with an M2 immunomodulatory phenotype, is considered neuroprotective during early brain repair41,42,43,44,45,46. However, persistent microgliosis and sustained pro-inflammatory cytokine expression are linked to oligodendrocyte and neuronal injury47. In line with human and animal data, glial cells and neurons in our model appeared to have released inflammatory cytokines that contributed to ongoing neuroinflammation48,49,50,51,52,53. Reactive astrocytes, infiltrating the injured brain after HI, further amplified the inflammatory response54,55,56.

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This chronic inflammatory environment characterizes the tertiary phase of HIE injury, driven by both resident glia and infiltrating peripheral immune cells. Together, they create a toxic niche that hinders white matter maturation, neurogenesis, and synaptogenesis57,58,59. Sildenafil treatment significantly decreased the number of reactive astrocytes and activated microglia in the cortex and white matter. These effects were most prominent with medium and high doses, consistent with previous findings showing a dose-dependent reduction in brain injury and improved neurological outcomes19,20. These data suggest that sildenafil may target the tertiary phase of injury, promoting neurorestoration. In addition to its effects on glial cells, HI reduced the expression of key neuronal and synaptic proteins, including NeuN and synaptophysin.

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Sildenafil restored their levels to those comparable to sham controls, which suggests neuronal preservation and synaptic repair. These results align with our prior data19 and with studies in adult disease models75,76,77 that report enhanced neuronal survival and increased synaptic protein expression following sildenafil treatment.

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We used chemiluminescence detection and an imaging system (Amersham Imager 600, General Electric, Boston, Massachusetts, USA), and we quantified band intensities using Image Lab® software (Bio-Rad®, Hercules, California, USA), normalized to ß-actin (mouse anti-ß-actin; Millipore Sigma, Oakville, Ontario, Canada; dilution: 1:5000). We randomly assigned pups to treatment groups. We made group comparisons using Kruskal–Wallis non-parametric tests, followed by Dunn’s post hoc tests to adjust the α–level as necessary. We considered a p-value < 0.05 to be statistically significant. We conducted analyses using GraphPad Prism® (GraphPad Software Inc., San Diego, CA, USA).

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At P30, HI significantly increased the number of GFAP + reactive astrocytes in the ipsilateral cortex (median [interquartile ranges]: 533 cells/mm2 [529–601], p < 0.05) (Fig. 2), compared to sham (cortex: 239 [187–275]; CC: 425 [269–553]; ECL: 692 [576–850]). HI also elevated Iba1 + microglia in the cortex (413 [383–506], p < 0.0001] (Fig. Microglial process length was reduced after HI (0.033 mm [0.031–0.035] vs sham: 0.068 mm [0.066–0.071], p < 0.0001] (Fig. Galectin-3/MAC-2 + macrophages were significantly increased in the ECL (273 [96–706] vs.

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0 [0–27], p < 0.05) (Fig. IL-1ra was transiently increased at P12 (p < 0.05), and then normalized (Fig. Sildenafil (all doses) reduced GFAP + astrocytes in all regions to levels no longer significantly different from sham (cortex: low-dose: 358 [281–375], medium-dose: 232 [164–337], and high-dose: 173 [140–197]; CC: low-dose: 687 [527–811], medium-dose: 500 [77–614], and high-dose: 495 [402–558]; ECL: low-dose: 1292 [973–1747], medium-dose: 689 [401–768], and high-dose: 781 [627–863]). Medium and high dose sildenafil had the strongest effect (cortex: high-dose: p < 0.01 vs. HI; CC: medium-dose: p < 0.01 vs. Furthermore, sildenafil has exhibited notable anti-apoptotic effects. While HI induced a marked increase in apoptosis at P12, sildenafil treatment attenuated this response.

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Similar anti-inflammatory effects of sildenafil have been observed in adult models of multiple sclerosis, stroke, and hepatic encephalopathy, where it reduced microglial/macrophage activation and pfizer sildenafil 100 pro-inflammatory cytokine release23,25,29,60. Chronic neuroinflammation contributes to white matter injury by promoting axonal and myelin degeneration61,62,63,64. In our model, persistent neuroinflammation coincided with a reduction in thickness of the CC and ECL by P30. This finding is particularly relevant, since oligodendrocytes are highly susceptible to HI70 and are responsible for myelination during the tertiary phase. White matter injury occurs in nearly one-quarter of term neonates with HIE71 and is associated with adverse long-term neurodevelopmental outcomes72,73. Together, these results support both a neuroprotective and neurorestorative role for sildenafil, in agreement with prior studies demonstrating its ability to reduce apoptosis in neurons and oligodendrocytes22,78,79,80,81,82,83. Additional immunohistochemistry is required in our model to pinpoint the cell types affected by apoptosis and to determine whether sildenafil selectively mitigated apoptotic injury in oligodendrocytes or neurons. Mechanistically, sildenafil may exert these effects via modulation of the PI3K/AKT/mTOR signaling pathway84.

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HI; high-dose: p < 0.05 vs. HI; ECL: medium-dose: p < 0.05 vs. HI). Similarly, medium and high doses significantly reduced Iba1 + microglia in all regions to sham levels (cortex: low-dose: 176 [147–252], p < 0.001 vs. sham; medium-dose: 123 [90–225], p < 0.01 vs.

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HI; and high-dose: 114 [79–116], p < 0.0001 vs. HI; CC: low-dose: 246 [223–369], medium-dose: 267 [178–327], and high-dose: 257 [227–290]; ECL: low-dose: 451 [303–590], p < 0.05 vs. sham, medium-dose: 318 [227–470], and high-dose: 350 [281–436]) and restored microglial process length in the cortex (low-dose: 0.051 mm [0.048–0.055], p < 0.05 vs. sham; medium-dose: 0.058 mm [0.055–0.062]; 0.066 mm [0.063–0.069], p < 0.001 vs. HI) (Figs.

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Galectin-3 + macrophages in the ECL were reduced by all doses, although only the low-dose brought levels to those of sham (low-dose: 103 [7–314]; medium-dose: 150 [52–225], p < 0.05 vs. sham; and high-dose: 134 [81–378], p < 0.05 vs. sham) (Fig. Sildenafil normalized TNFα and IL-1ra levels, respectively at P12, P17 and P30. IL-1β remained slightly elevated with sildenafil sildenafil citrate 200mg price but without statistical significance.

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