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HI; high-dose: 5640 oligodendrocytes/mm2 [4739–6351], p < 0.01 vs.

Adverse effects

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 but without statistical significance. 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. HI); CC1 + : mature oligodendrocytes (low-dose; 2798

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[2218–3155]; medium-dose: 3641 oligodendrocytes/mm2

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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. Similar effects were seen in the ECL (Olig2 + : low-dose: 4087 [3846–4484]; medium-dose: 4923 oligodendrocytes/mm2 [4630–6250], p < 0.05 vs. [3077–4688], p < 0.01 vs. HI; high-dose: 4210 oligodendrocytes/mm2 [3543–5006], p < 0.001 vs.

Property	Description	Typical Dosage	Appearance	Packaging Type
Name	Sildenafil White	50 mg, 100 mg	White, Round Pills	Blister Pack
Chemical Formula	C22H30N6O4S	N/A	N/A	N/A
Manufacturer	Example Pharma Co.	N/A	N/A	N/A
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HI) (Fig.

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There was no difference between experimental

Other Requirements

On P10, rat pups underwent left carotid artery ligation (ischemia) followed by 2 h of hypoxia (8% oxygen)19. Sham-operated controls underwent identical procedures without hypoxia–ischemia (HI), and sham pups were kept normothermic using warming blankets (Cincinnati Sub-Zero, Cincinnati, USA) during separation from dams. Sample size (n = 6–12 per group) was based on feasibility, ethical constraints, and the 3Rs principle (replacement, reduction, refinement). Sildenafil (Viagra®, Pfizer; 100-mg tablets) was prepared as previously described19,20. From 12 h post-HI, we randomized pups to receive sildenafil or vehicle by oral gavage twice daily for 7 days (P10-17).

Sildenafil Citrate

Doses in HI pups included 2, 10, and 50 mg/kg for histology/immunohistochemistry, and 50 mg/kg for Western blot/ELISA, based on human-equivalent dosing39 and previous efficacy studies in cortex/retina19,20. We collected brains at P3019,20 and sectioned them at –2.16 mm from Bregma. We imaged hematoxylin and eosin-stained coronal sections using a Leica DM4000B microscope and a Leica DFC450C digital camera (Leica DFC450C, Leica Microsystems, Wetzlar, Hessen, Germany). We stitched overlapping 5 × images using a Microsoft Image Composite Editor. Using ImageJ (Image Processing and Analysis in Java)40, we blindly measured the thicknesses of the corpus callosum and ipsilateral external capsule on two sections per animal and averaged them.

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At P30, we initially incubated the sections in a universal antigen retrieval reagent at 95 °C for 20 min, and then blocked them in phosphate-buffered saline containing tween20 and gelatin (PBS-GT). We incubated the primary antibodies overnight at 4 °C in PBS-GT. We also used the appropriate AlexaFluor® or Cy™3 secondary antibodies. We counterstained the sections with DAPI and mounted them with Vectashield (H1200; Vector Laboratories, Burlingame, CA, USA). We performed the imaging using a fluorescent microscope (20x). groups in immature Olig2 + /CC1- oligodendrocytes.

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For quantitative measurements, coronal brain sections were selected at a consistent anatomical level characterized by visible hippocampi, reduced lateral ventricles, a visible third ventricle, and clear delineation of the thalamus (Figs. Stained brain sections were imaged using a fluorescent microscope (Leica DM4000B LED, Leica Microsystems, Wetzlar, Germany) with a 20 × objective. For each animal, 2–3 fields were captured in the left (ipsilateral) cortex of HI animals near the infarct boundary zone and extending toward the midline (Fig. 1G); corresponding cortical fields were captured in sham-vehicle animals at matched anatomical landmarks to ensure valid comparisons across groups. Additional 20 × micrographs were also obtained from the corpus callosum (CC) and the left (ipsilateral) external capsule (ECL) to evaluate white matter, using identical anatomical reference points across animals; the CC was measured at the midline between hemispheres, and the ECL on the left side at the same coronal level (Fig.

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We also assessed the microglial activation by measuring the maximal process length from five randomly selected microglia cells per animal. One investigator took pictures of the respective fields of view and another blinded investigator, performed the cell counts. For western blotting, we euthanized the rats at P12, P17, and P30. The sildenafil otc middle third of the ipsilateral cortex and white matter (excluding basal ganglia, hippocampus, and cerebellum) was flash frozen. We lysed the tissues by sonication in an ice-cold RIPA buffer (89,901, ThermoFisher Scientific, Waltham, Massachusetts, USA) with protease inhibitors (S8820, Sigma-Aldrich, St.

Patient resources

Louis, Missouri, USA), centrifuged (12,000 RCF, 10 min, 4 °C), and measured the protein concentration (BCA assay) (23,225; Thermo Fisher Scientific, PierceTM, Waltham, Massachusetts, USA). We loaded equal protein amounts on SDS–polyacrylamide CriterionTM TGXTM precast gels (567–1085; Bio-Rad®, Hercules, California, USA) and then transferred them to a Polyvinylidene difluoride (PVDF) membrane (10,600,023; AmershamTM HybondTM, Boston, Massachusetts, USA). We blocked the membranes with 5% dried non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (9005-64-5; Fisher BioReagentsTM, Hampton, New Hampshire, USA), and then incubated them overnight at 4 °C with the following primary antibodies: cleaved poly-ADP-ribose polymerase (PARP) (rabbit anti-PARP, 9542; Cell Signaling Technology, Danvers, Massachusetts, USA; dilution 1:1000), a marker of apoptosis; superoxide dismutase 1 (SOD1) (rabbit anti-SOD1, ab13498; Abcam, Cambridge, UK; dilution 1:1000), a marker of oxidative stress; synaptophysin (rabbit anti-synaptophysin, 5461; Cell Signaling Technology, Danvers, Massachusetts, USA; dilution 1:1000), a marker of synaptic density; oligodendrocyte lineage transcription factor 2 (Olig2) (mouse anti-Olig2, MABN50; Sigma-Aldrich, St. Louis, Missouri, USA; dilution 1:2000), a marker of total oligodendrocytes; neuronal nuclei antibody (NeuN) (mouse anti-NeuN, MAB377; Millipore, Burlington, Massachusetts, USA; dilution 1:500), a marker of mature neurons; and phosphorylated protein kinase B (pAKT) (rabbit anti-phospho-Akt Ser473 D9E, 4060; Cell Signaling Technology, Danvers, Massachusetts, USA; dilution 1:1000), an upstream regulator of the mechanistic target of rapamycin (mTOR) pathway. 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). HI caused a significant increase in cleaved PARP) levels at P12 in the

8.5 Geriatric Use

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 sildenafil 45 mg chewable tablets [147–252], p < 0.001 vs. sham; medium-dose: 123 [90–225], p < 0.01 vs. HI; and high-dose: 114 [79–116], p < 0.0001 vs.

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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. 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. ipsilateral cortex and white matter compared

4.3 Concomitant Guanylate Cyclase (GC) Stimulators

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). 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.

Sildenafil improved white matter thickness and promoted oligodendrogenesis

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. 0 [0–27], p < 0.05) (Fig.

Sildenafil may reduce neuroinflammation through mTOR pathway activation

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. HI; high-dose: p < 0.05 vs. to sham rats (p < 0.01) (Fig.

Step	Description	Equipment Needed
Raw Material Preparation	Sourcing and testing chemical reagents	Reaction vessels, pH meters
Chemical Synthesis	Formation of sildenafil via chemical reactions	Reactors, stirring devices
Purification	Removing impurities through filtration or chromatography	Filtration units, chromatography columns
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This increase was accompanied by a significant

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reduction in the antioxidant protein SOD1 (p

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< 0.05) and a marked decrease in NeuN expression at P30 (p < 0.05) (Fig.

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