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This study investigated the effects of sildenafil on neuroinflammation and white matter injury in a rat model of term neonatal HIE.

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Daily monitoring was performed by facility technicians and researchers. To model brain injury in term human neonates with HIE31,32,33,34,35,36, we used the well-established Vannucci model31,32,33,34,37,38. 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).

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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). 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).

Induction of term neonatal HIE

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. 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. Hypoxia–ischemia (HI) was induced in postnatal day 10 (P10) male Long-Evans rats via a left carotid ligation followed by 2 h of hypoxia (8% oxygen). Pups were randomized to receive oral sildenafil or vehicle starting 12 h post-HI, twice daily for 7 days. White matter integrity (corpus callosum and external capsule), oligodendrocyte presence, and glial activation were assessed by histology and immunohistochemistry.

Inflammatory markers were measured by enzyme-linked immunosorbent assay (ELISA), and signaling pathways were examined by Western blot. Outcomes were compared to sham and untreated HI controls.

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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). Thickness of the corpus callosum and left external capsule was reduced. Sildenafil treatment — particularly at medium and high doses — attenuated astrocytes and microglia activation, restored microglial morphology, and normalized cytokine expression. White matter thickness was significantly improved, with increased numbers of total Olig2 + and mature CC1 + oligodendrocytes. Mechanistically, sildenafil restored p-AKT levels, which suggests involvement of the PI3K/AKT/mTOR pathway. Sildenafil significantly reduced neuroinflammation, improved white matter integrity, and supported oligodendrocyte recovery after neonatal HI. These findings highlight the potential of sildenafil as a neurorestorative therapy during the tertiary phase of injury in neonatal HIE. Neonatal hypoxic-ischemic encephalopathy (HIE) due to birth asphyxia remains a leading cause of neonatal death and long-term neurological disability affecting approximately 3 per 1000 live births worldwide1. Therapeutic hypothermia (TH), initiated within 6 h of life and for 72 h, is the only proven treatment for HIE and reduces mortality and neurodevelopmental impairments2,3,4. However, TH is not effective for all affected neonates and offers no neurorestorative properties3. A critical need exists for adjunct therapies that promote brain repair (“neurorestorative” therapies) after an initial injury. The initial phase (0–6 h) involves energy failure and necrosis5. The secondary phase (6–48 h) triggers apoptosis through excitotoxicity, oxidative stress, and mitochondrial damages6,7. The tertiary phase (beyond 48 h) is marked by chronic neuroinflammation and epigenetic changes that impair recovery and lead to further neuronal and oligodendrocyte loss through apoptosis and autophagy8. Persistent glial activation and inflammatory cytokine release disrupt essential neurodevelopmental processes such as neurogenesis, oligodendrogenesis, and synaptogenesis9,10,11,12,13,14. While TH targets the early phases of injury, therapies that address the tertiary phase may enhance long-term recovery. Sildenafil (Viagra®), a phosphodiesterase type 5 (PDE5) inhibitor that increases intracellular cGMP, shows promise as a neurorestorative therapy.

• Sildenafil white is a common name for the generic version of Viagra.
• It is used to treat erectile dysfunction in men.
• Sildenafil white is known for its rapid onset of action.
• The medication works by enhancing blood flow to the penis.
• It is typically taken about 30 to 60 minutes before sexual activity.
• Sildenafil white is available in various doses, usually 25mg, 50mg, and 100mg.
• The drug should not be used with nitrate medications to avoid dangerous interactions.
• Common side effects include headache, flushing, and nasal congestion.
• It is important to follow dosage instructions provided by a healthcare provider.
• Sildenafil white can cause low blood pressure in some users.
• Store sildenafil white in a cool, dry place, away from direct sunlight.

It has been found to enhance both functional and structural recovery in adult stroke models15,16 and is already used safely in neonates with persistent pulmonary hypertension17,18.

In a rat model of term neonatal HIE, sildenafil enhanced brain and retinal function and structure after hypoxia–ischemia19,20,21 and increased neuronal density near injury sites19. However, its effects on neuroinflammation and white matter injury in this model remain unclear. In adult models of neurodegenerative sildenafil gel disease, sildenafil has been shown to reduce neuroinflammation22,23,24,25,26,27 and promote white matter repair by supporting oligodendrocyte survival23,28,29. The present study aimed to evaluate the effects of sildenafil on neuroinflammation and white matter injury in a rat model of term HIE.

Chemical synthesis

This study investigated the effects of sildenafil on neuroinflammation and white matter injury in a rat model of term neonatal HIE. Hypoxia–ischemia (HI) was induced in postnatal day 10 (P10) male Long-Evans rats via a left carotid ligation followed by 2 h of hypoxia (8% oxygen). Pups were randomized to receive oral sildenafil or vehicle starting 12 h post-HI, twice daily for 7 days. White matter integrity (corpus callosum and external capsule), oligodendrocyte presence, and glial activation were assessed by histology and immunohistochemistry. Inflammatory markers were measured by enzyme-linked immunosorbent assay (ELISA), and signaling pathways were examined by Western blot.

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Outcomes were compared to sham and untreated HI controls. Thickness of the corpus callosum and left external capsule was reduced. Sildenafil treatment — particularly at medium and high doses — attenuated astrocytes and microglia activation, restored microglial morphology, and normalized cytokine expression. White matter thickness was significantly improved, with increased numbers of total Olig2 + and mature CC1 + oligodendrocytes. Mechanistically, sildenafil restored p-AKT levels, which suggests involvement of the PI3K/AKT/mTOR pathway.

How it works for PAH

Sildenafil significantly reduced neuroinflammation, improved white matter integrity, and supported oligodendrocyte recovery after neonatal HI. These findings highlight the potential of sildenafil as a neurorestorative therapy during the tertiary phase of injury in neonatal HIE. Neonatal hypoxic-ischemic encephalopathy (HIE) due to birth asphyxia remains a leading cause of neonatal death and long-term neurological disability affecting approximately 3 per 1000 live births worldwide1. Therapeutic hypothermia (TH), initiated within 6 h of life and for 72 h, is the only proven treatment for HIE and reduces mortality and neurodevelopmental impairments2,3,4. However, TH is not effective for all affected neonates and offers no neurorestorative properties3. All procedures followed institutional standard operating procedures (SOPs) and the Canadian Council on Animal Care’s (CCAC) guidelines in accordance with the Animals for Research Act, were approved by the local Animal Care Committee, and were reported in accordance with ARRIVE guidelines30. Adult female Long-Evans rats male-only litters (Harlan Laboratories) were housed under standard conditions with food and water ad libitum. Pups remained with their mother until weaning at postnatal day 21 (P21). Daily monitoring was performed by facility technicians and researchers. To model brain injury in term human neonates with HIE31,32,33,34,35,36, we used the well-established Vannucci model31,32,33,34,37,38. On P10, rat pups underwent left carotid artery ligation (ischemia) followed by 2 h of hypoxia (8% oxygen)19.

4.1 Nitrates

A critical need exists for adjunct therapies that promote brain repair (“neurorestorative” therapies) after an initial injury. The initial phase (0–6 h) involves energy failure and necrosis5. The secondary phase (6–48 h) triggers apoptosis through excitotoxicity, oxidative stress, and mitochondrial damages6,7. The tertiary phase (beyond 48 h) is marked by chronic neuroinflammation and epigenetic changes that impair recovery and lead to further neuronal and oligodendrocyte loss through apoptosis and autophagy8. Persistent glial activation and inflammatory cytokine release disrupt essential neurodevelopmental processes such as neurogenesis, oligodendrogenesis, and synaptogenesis9,10,11,12,13,14.

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While TH targets the early phases of injury, therapies that address the tertiary phase may enhance long-term recovery. Sildenafil (Viagra®), a phosphodiesterase type 5 (PDE5) inhibitor that increases intracellular cGMP, shows promise as a neurorestorative therapy. It has been found to enhance both functional and structural recovery in adult stroke models15,16 and is already used safely in neonates with persistent pulmonary hypertension17,18. In a rat model of term neonatal HIE, sildenafil enhanced brain and retinal function and structure after hypoxia–ischemia19,20,21 and increased neuronal density near injury sites19. However, its effects on neuroinflammation and white matter injury in this model remain unclear.

Dosage for erectile dysfunction (ED)

In adult models of neurodegenerative sildenafil gel disease, sildenafil has been shown to reduce neuroinflammation22,23,24,25,26,27 and promote white matter repair by supporting oligodendrocyte survival23,28,29. The present study aimed to evaluate the effects of sildenafil on neuroinflammation and white matter injury in a rat model of term HIE. All procedures followed institutional standard operating procedures (SOPs) and the Canadian Council on Animal Care’s (CCAC) guidelines in accordance with the Animals for Research Act, were approved by the local Animal Care Committee, and were reported in accordance with ARRIVE guidelines30. Adult female Long-Evans rats male-only litters (Harlan Laboratories) were housed under standard conditions with food and water ad libitum. Pups remained with their mother until weaning at postnatal day 21 (P21). 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). 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. 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). 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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The 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. 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. We also assessed the microglial activation by measuring the maximal process length from five randomly selected microglia cells per animal.

• The term 'sildenafil white' often refers to unbranded, generic pills.
• It may be available online through various pharmacies and vendors.
• Users should verify the legitimacy of sildenafil sources to avoid fake products.
• This medication is not suitable for individuals with certain heart conditions.
• Sildenafil white is sometimes used recreationally for its euphoric effects.
• It may interact with certain antibiotics and antifungals, altering effectiveness.
• Patients should inform their doctor of all medications before use.
• The medication is not an aphrodisiac; it only facilitates erectile response.
• Overuse or misuse of sildenafil white can lead to serious health risks.
• The recommended maximum frequency is once per day unless advised otherwise.
• Consultation with a healthcare professional is essential prior to use.

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 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. 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.

AN 351

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). 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.

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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. 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. 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).