Sildenafil has been shown to have a beneficial effect on cognitive function in several animal models of adult diseases. For example, in a physiological mouse model of aging, sildenafil exerted neuroprotection against age-related cognitive dysfunction (Palmeri et al., 2013). Also, in a mouse model of Alzheimer’s disease, sildenafil administrated for 5 weeks improved spatial learning and memory retention (Cuadrado-Tejedor et al., 2011). Similarity, in a transgenic mouse model of amyloid deposition (Alzheimer’s disease), sildenafil acted as a memory enhancer via modulating memory storage, improving object memory and counteracting spatial learning impairment, both immediately during treatment and for a prolonged period beyond the drug administration (Puzzo et al., 2009). In a transgenic mouse model of Huntington’s disease, sildenafil treatment improved deficits in object recognition memory and passive avoidance learning (Saavedra et al., 2013). Furthermore, in stroke rat models, sildenafil administered for 7 consecutive days—starting either 2 h, 24 h, or 7 days after the stroke—promoted functional recovery measured by a foot fault test and an adhesive removal test 7 or 28 days after hypoxia-ischemia (Zhang et al., 2002, 2003). Sildenafil also restored motor coordination in beam walking, which implied a promotion of cerebellar function (Agusti et al., 2017) in rats with portacaval shunt (PCS). In both stressed and non-stressed animals, sildenafil markedly reduced serum cortisol levels (Socała et al., 2016), which could suggest protection against stress. Similarly, sildenafil was shown to improve cognitive function (measured by an object recognition test) and reduce anxiety-like behavior in sedentary rats exposed to acute stress exposure (Ozbeyli et al., 2015).

In addition, in a mouse model of neuropathic pain, sildenafil has been proposed as an analgesic, secondary to its antinociceptive effect by the induction of both GABAA and GABAB receptors and by increasing the threshold to pain (Huang L. J. et al., 2009). Sildenafil also has been shown to induce peripheral analgesia in a dose-dependent way (Jain et al., 2001). Finally, the anticonvulsant properties of sildenafil are still being debated, since sildenafil may have both pro- and anticonvulsant activity, depending on the experimental model of epilepsy, the animal species and the sildenafil dosage (Nieoczym et al., 2010; Tawfik et al., 2018). With respect to human patients, sildenafil has enhanced their ability to focus attention on streams of auditory stimuli (Schultheiss et al., 2001).

Thus, treatment with sildenafil may have an impact on cognitive function (spatial learning, memory retention, and object recognition), motor function (motor coordination), emotional function (anxiety-like behavior), analgesia, and seizures.

Sildenafil’s potential effects on neonates have been tested using animal models after hypoxia-ischemia. Sildenafil decreased neurological deficits 20 days post-injury as measured by a gait analysis (Yazdani et al., 2016) and promoted motor functional recovery 7 days post-injury as measured by an open-field test (Charriaut-Marlangue et al., 2014) and 30 days post-injury as measured by an elevated plus maze (EPM) and wire hang test (Engels et al., 2017).

Clinical trials (NCT02812433 and NCT04169191) are ongoing to determine whether similar beneficial functional effects can be observed in human neonates after birth asphyxia.

In rat models of adult stroke, the effects of sildenafil on the extent of ischemic brain injury are still being debated. One study has found a clear reduction of the cerebral infarct volume 24 h post-injury, when a single intravenous or intraperitoneal dose of sildenafil (8–32 mg/kg) was given 2 h post-hypoxia-ischemia (Chen et al., 2014). However, in a rat model of embolic stroke, sildenafil treatment (10 mg/kg) started 24 h after injury and continued daily for 6 days after embolus placement did not significantly reduce the lesion size 6 weeks after injury (Li et al., 2007), even though it improved function and enhanced angiogenesis. In a rat model of middle cerebral artery (MCA) stroke, the administration of sildenafil for 1 week in escalating doses from 25 to 150 mg did not affect brain infarct size, even though it preserved synapse function and increased angiogenesis (Bednar, 2008). The individual variations in the severity of injury associated with the different animal models of adult stroke (Patel et al., 2014) and the different regimens (doses and duration) of the sildenafil treatments may explain the differences in outcomes.

Several studies on the neonatal brain have now demonstrated that sildenafil treatment may reduce the extent of brain injury after hypoxia-ischemia. For example, Yazdani et al. (2016) found that per os treatment with sildenafil—started 24 h after hypoxia-ischemia at P10 and continued for 7 consecutive days (14 doses) —reduced the extent of injury 20 days post-injury and that higher doses (10 and 50 mg/kg) appeared to be more efficient than smaller doses (2 mg/kg). Charriaut-Marlangue et al. (2014) found that at postnatal day 7 (P7), a single dose of sildenafil that had been given intraperitoneally immediately after hypoxia-ischemia reduced brain injury, and the reduction was again dose-dependent—the 10 mg/kg was effective, but not the 5 mg/kg dose. Their follow-up study showed that this effect was not evident 72 h after hypoxia-ischemia, but was evident 8 days post-injury (Moretti et al., 2016). However, Engels et al. (2017) at P9, did not observe any improvement of brain injury with a single intraperitoneal dose of sildenafil 10 mg/kg given 2 h post-hypoxia-ischemia at P9, despite an observed functional improvement. Similar to adults, the effects of sildenafil may be dose- and duration-dependent.

Sildenafil treatment appears to be neuroprotective. It reduces neuronal death by inhibiting apoptosis (Díaz-Lucena et al., 2018) and balances the degree of autophagy (Venkat et al., 2019). In a rat model of chronic cerebral hypoperfusion, sildenafil given for 10 consecutive days post-injury reduced neurodegeneration in both the CA1-CA4 striatum of the hippocampus and cerebral cortex, and preserved granular cells in the dentate gyrus 3 months post-injury (Ferreira et al., 2013). In a rat model of adult stroke, sildenafil administered for 1 day alleviated neuronal damage and synaptic structure degeneration (Chen et al., 2014).

Simultaneously, sildenafil appears to be neurorestorative and encourages recovery after injury by promoting neurogenesis, synaptogenesis, and synaptic plasticity, and by preserving axon ultrastructure. Sildenafil stimulates neurogenesis by promoting the proliferation of neural progenitor cells and neural stem cells and by inducing the maturation of immature neurons in the neural circuit (Zhang et al., 2012; Santos et al., 2014). Sildenafil also has been shown to enhance synaptogenesis (Zhang X. et al., 2005), especially around the ischemic boundary regions, and thus regulates synaptic plasticity (Uthayathas et al., 2013). This effect has been found not only in animal models of adult stroke, but also in animal models of adult neurodegenerative diseases. In a transgenic mouse model of Alzheimer’s disease, a 5-day treatment with sildenafil in 14-month-old mice increased synaptic function through the regulation of Arc proteins, which resulted in the restoration of spatial learning and memory retention (Cuadrado-Tejedor et al., 2011). A 14-day administration of sildenafil in a rat model of Huntington’s disease attenuated cell damage (Thakur et al., 2013). In a seizure model, sildenafil restored the normal hippocampal neuronal architecture and preserved the hippocampal neuronal cell count (Tawfik et al., 2018). In a vascular dementia model in aged rats, a 28-day treatment of sildenafil improved synaptic plasticity through upregulating synaptophysin (Venkat et al., 2019). Finally, in a cuprizone-induced rat model of multiple sclerosis, sildenafil treatment preserved axon ultrastructure (Nunes et al., 2012; Araújo et al., 2020).

Interestingly, the effects of sildenafil on neurons appear somewhat age-dependent. In Ding et al.’s (2008) study, the therapeutic response to sildenafil treatment was weaker and delayed after hypoxia-ischemia in aged rats compared to younger adult rats, which reflected poorer brain plasticity in aged rats This difference in brain plasticity could be explained, at least partly at the molecular level, by the fact that the function of the NO/cGMP signaling pathway—that regulates angiogenesis, neurogenesis, axonal outgrowth, and synaptic plasticity during development and in adulthood—is age-dependent, with observed impairment in this pathway with advanced age (Chalimoniuk and Strosznajder, 1998).

In a safety study of human adult patients with stroke, sildenafil was found to be safe (Silver et al., 2009).

After birth, the neonatal brain still undergoes a significant degree of neuronal, synaptic, and angiogenic growth (Jensen, 2006). Axonal growth, neuronal dendritic arborization, and the establishment of synaptic contacts, myelination, and glial differentiation are among the many processes still occurring during the first postnatal weeks of rodents (Andersen, 2003) and during the first years of life in humans (Giedd et al., 1999; Andersen, 2003). Considering that the effects of sildenafil on neurons appear somewhat age-dependent, its beneficial effects may be even more marked in the injured maturing neonatal brain. Interestingly, Gómez-Pinedo et al. (2010) have demonstrated that rats whose cGMP was blocked during gestation presented with a reduced differentiation of stem cells in neurons and an increased differentiation in non-neuronal cells, and this effect normalized when sildenafil was given at the same time.

Far fewer research studies have investigated the effects of sildenafil on the neurons in the neonatal brain after hypoxia and ischemia. In an animal model of fetal ischemia, sildenafil treatment of mothers proved to be anti-oxidative, which could prevent neuronal death (Ozdegirmenci et al., 2010). Yazdani et al. (2016) demonstrated that per os treatment with sildenafil—started 24 h after hypoxia-ischemia at P10 and continued twice a day for 7 consecutive days (14 doses)—could cause a significant increase in the number of neurons near the infarct boundary 20 days post-injury (Vannuci rat model at P10), with the higher doses being the most efficient (10 and 50 mg/kg). Their follow-up study demonstrated that sildenafil may be neuroprotective in the neonatal brain by preventing apoptosis, but also neurorestorative by promoting neurogenesis (Yazdani et al., 2021). Engels et al. (2017) found an increased number of immature neurons in the ipsilateral subventricular zone and striatum after a single intraperitoneal dose of sildenafil (10 mg/kg) given 2 h post-hypoxia-ischemia at P9.

In an effort to cope with pathogens, toxins, insult, trauma, and degeneration, the central nervous system can mount an elaborate response called neuroinflammation, which is characterized by the leakage of the blood-brain barrier (BBB) (Becher et al., 2017) and the orchestrated actions of microglia, astrocytes, cytokines release, recruited blood leukocytes, and infiltrated macrophages from the periphery (Xiong et al., 2016). Russo and McGavern (2016) have suggested that some degree of inflammation may be necessary in the acute stage of brain injury to clear damage and set the stage for remodeling efforts. However, excessive or persistent neuroinflammation may be harmful and may further contribute to the progression of brain injury in both adults (Agusti et al., 2017) and neonates (Järlestedt et al., 2010). Drug modulating neuroinflammation may exert different/opposite effects on neuroinflammation, depending on the maturity of the brain and the timing of when the drug is administrated after injury.

Sildenafil has been demonstrated to have potent anti-inflammatory properties throughout the body: i.e., in animals with severe acute pancreatitis (Fang et al., 2020), bronchial asthma (Laxmi et al., 2019), intestinal ischemia, and reperfusion injury (Moore et al., 2019); and in humans with type 2 diabetes (Schlindwein et al., 2010), diabetic cardiomyopathy (Nurnberg et al., 2002), and heart failure. In the brain, sildenafil appears to play a role in modifying the inflammatory balance. In microglia cell culture, sildenafil inhibits microglia activation. In astrocyte cells culture, sildenafil downregulated several inflammatory receptors, including the Toll-like receptor 4 (TLR4), the substance P receptor (NK-1), and the protease-activated receptor 2 (PAR-2) (Hansson et al., 2018). In a rat model of hepatic encephalopathy, sildenafil mitigated inflammation via inhibiting the activation of microglia and astrocytes, and by preventing the release of cytokines (Agusti et al., 2017). In a model of traumatic brain injury, sildenafil inhibited the activation of microglia (Prado et al., 2013). In a rat model of irradiation-induced brain injury, sildenafil showed anti-inflammatory and antioxidant properties through the modulation of the NO/tetrahydrobiopterin (BH4) pathway (Thabet et al., 2021). In addition, in a model of experimental autoimmune encephalomyelitis (EAE), sildenafil appeared neuroprotective by promoting the switch of microglia from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype (Pifarré et al., 2014), which reduced the infiltration of CD4+ T lymphocytes and the production of IL-17 and TNFα (Araújo et al., 2020), and activated autophagy (Duarte-Silva et al., 2021).

In the neonatal brain, the immaturity of the immune system, the ongoing developmental neuronal apoptosis, and the different balance between pro- and anti-oxidant enzymes appear to create a window of increased susceptibility to neuroinflammation secondary to hypoxic-ischemia injury (Vexler and Yenari, 2009). Shrivastava et al. (2012) observed a glial/inflammatory response with an activation of microglia/macrophages and astrocytes and a recruitment of neutrophils from 3 h after hypoxia-ischemia at P7 up to 100 days after HI in the ipsilateral hemisphere, and from 3 to 72 h in contralateral hemisphere. Järlestedt et al. (2010) found that the attenuation of reactive gliosis, which appeared protective in the adult model of focal brain ischemia, did not reduce the volume of infarct after neonatal hypoxia-ischemia, although it increased the number of surviving neonatal neurons.

Only two studies so far have demonstrated the potential anti-inflammatory role of sildenafil in the injured neonatal brain. Sildenafil (10 mg/kg) administered intraperitoneally has been proved to reduce reactive astrogliosis and macrophage/microglial activation at 72 h and 7 days after hypoxia-ischemia at P7 in Sprague-Dawley rats (Charriaut-Marlangue et al., 2014). Also, Moretti et al. (2016) demonstrated that a single dose of 10 mg/kg of sildenafil (10 mg/kg) administered intraperitoneally 5 min after hypoxia-ischemia at P9 reduced the recruitment and the activation of microglia in the penumbral tissue at 72 h and 8 days after injury. Interestingly, sildenafil appeared to have a distinct effect on the polarization of microglia—depending on the timing of administration compared to the timing of disease—since it functioned as an anti-inflammatory at 72 h post-HI by increasing M2-like genes expression and decreasing M1-like genes expression; and then functioned as a pro-inflammatory at 8 days post-HI by increasing M1-like genes expression and decreasing M2-like genes expression (Moretti et al., 2016).

Myelin, the multilaminar sheath around axons produced in the central nervous system by oligodendrocytes (OLs), is important not only for the rapid conduction of action potential, but also for providing trophic support for axons (Nave, 2010a,b). White matter injuries (WMI) are characterized by the loss of mature OLs, the maturation arrest of pre-OLs, the degeneration of myelin, the failure of OLs migration, and the failure of myelination regeneration (Segovia et al., 2008; Huang Z. et al., 2009). The reaction of OLs varies according to the severity of the injury and its nature (acute vs. acute), and the maturity of brain development at time of injury (Emery, 2010).

It has been reported that neural stem cells (NSC) generation in rodents declines with aging due to the decrease in the basal levels of cGMP and the increase in PDE5 activity over time (Ahlenius et al., 2009). Also, in oligodendroglial precursor cells, sildenafil appeared to inhibit myelination via exerting a negative impact on the intrinsic oligodendroglial differentiation processes (Muñoz-Esquivel et al., 2019). However, based on other studies, sildenafil appears to be mostly protective for white matter injury. Benjamins and Nedelkoska (2007) and Yoshioka et al. (2000) found that sildenafil treatment of OLs culture promoted the survival of differentiated OLs. In a mouse model of multiple sclerosis, Pifarre et al. (2011) reported that sildenafil administered orally at peak disease could prevent axonal loss and promote remyelination with an increased number of axons with a remyelinating appearance. The administration of sildenafil from disease onset prevented OLs death at different stages of differentiation (including immature and mature myelinating OLs), preserved axons and myelin, and thus prevented disease progression (Pifarré et al., 2014). Sildenafil not only directly promoted pre-OLs maturation, but also indirectly cleared demyelinated myelin debris via the regulation of the microglia/macrophage inflammatory phenotype (Díaz-Lucena et al., 2018). Nunes et al. (2012) also found that sildenafil inhibited the apoptosis of OLs and maintained the normal structure of myelin and axons, which is characterized as the typical myelin lamellar arrangement with only rare fibers with reduced myelin thickness and an absence of collapsed myelin. This effect was more prominent in the group of mice receiving sildenafil from day 0–30 after injury compared to the group receiving it from day 15 to 30 (Nunes et al., 2016a), which suggests that an earlier administration after initial injury was more efficient. Sildenafil also has been proved to alleviate WMI induced by ischemia. In a mouse model of adult stroke, sildenafil promoted oligodendrogenesis by amplifying nestin-expressing NSC and their differentiation into neuronal and OLs progenitor cells that could migrate to the ischemic boundary and become mature oligodendrocytes (Zhang et al., 2012). After treatment with sildenafil, the myelin damage in iNOS-/- mice was reversed, the number of oligodendrocytes’ increased and myelin integrity and ultrastructure improved (Rapôso et al., 2014). The role of sildenafil on oligodendrocytes by balancing demyelination and remyelination appears to be independent of its anti-inflammatory effects. Any similar effects of sildenafil treatment in human adults with demyelinating diseases remains to be explored.

The neonatal immature brain differs from the adult mature brain due to the rapid differentiation of immature oligodendrocytes into mature oligodendrocytes and the ongoing active myelination during the first postnatal weeks (Andersen, 2003). Shrivastava et al. (2012) have reported subcortical white matter damage and long-term atrophy related to the loss of the immature oligodendrocytes in the tracts and the loss of the subventricular zone (SVZ) OLs progenitors persisting from 3 h to 100 days after hypoxia-ischemia at P7. The vulnerability of oligodendrocytes to neonatal hypoxia-ischemia appears to be dependent on the timing of the injury related to the stage of differentiation of the oligodendrocytes (Alix et al., 2012), with the pre-OLs being markedly more susceptible to hypoxia-ischemia than the mature OLs (Back and Rosenberg, 2014).

So far, published research is lacking on the effects of sildenafil on myelination in neonates. However, the upregulation of cGMP has been shown to increase oligodendrogenesis in the developing white matter and cortex of uninjured P7 rats (Duy et al., 2015). Thus, the potential oligodendrogenesis effects of sildenafil should be further investigated in neonates.

PDE5—the target of sildenafil—is widely distributed in cerebral vasculature, including in the smooth muscle and endothelium of the major cerebral arteries, in the brain capillary endothelial cells, and in the cerebral microvessel pericytes, a key component in the control of microcirculation and the blood-brain barrier (Kruuse et al., 2005), which explains the multiple described effects of sildenafil on cerebral vasculature, including increasing cerebral blood flow (Zhang et al., 2006; Kruuse et al., 2012), promoting microvascular reactivity (Han et al., 2012), promoting angiogenesis, etc. Increasing cerebral blood flow, resulting from either a recanalization of occluded arteries or angiogenesis, could improve the regional cerebral tissue microenvironment, which may lead to improving neurologic function recovery (Slevin et al., 2000). In a rat model of embolic stroke, a 7-day administration of sildenafil enhanced angiogenesis and increased local cerebral blood flow at 4–5 weeks after hypoxia-ischemia (Ding et al., 2008, 2011). In a rat model of epilepsy, sildenafil enhanced expression of the endothelial cell marker CD34 and the vascular endothelial growth factor (VEGF) 21 days after injury (Tawfik et al., 2018). In a rat model of Alzheimer disease, sildenafil decreased histopathological changes by modulating the vascular endothelial growth factor and the vascular cell adhesion molecule-1 (Ibrahim et al., 2021).

This vascular effect of sildenafil also has been well reported in human patients. In patients with pulmonary hypertension, sildenafil has reduced pulmonary arterial pressure and vascular resistance (Rosengarten et al., 2006). In neurologically healthy patients, sildenafil improved cerebral microvascular reactivity (Diomedi et al., 2005). In addition, a single dose of sildenafil improved the cerebral hemodynamic and increased the cerebral oxygen metabolism of patients with Alzheimer’s disease (Sheng et al., 2017). Sildenafil also attenuated injury by improving the cerebrovascular reactivity of patients with Becker muscular dystrophy (Lindberg et al., 2017). In patients with subarachnoid hemorrhage, sildenafil promoted neurofunction recovery by reducing cerebral vasospasm (Washington et al., 2016). In a randomized, double-blind, crossover, placebo-controlled trial with patients with small vessel disease (SVD), sildenafil led to a reduction in cerebral pulsatility and increased cerebrovascular reactivity, which suggests the potential benefit of sildenafil to prevent the progression of SVD (Webb et al., 2021).

Again, current research on effects of sildenafil on the vessels of the neonatal brain is very limited. Notwithstanding, Charriaut-Marlangue et al. (2014) have demonstrated that sildenafil treatment can significantly increase cerebral blood flow after hypoxia-ischemia at P7. Moretti et al. (2016) have found that the NO/cGMP pathway plays a critical role in neovascularization. In addition, in growth-restricted fetal sheep, maternal treatment with sildenafil improved vascular function (Inocencio et al., 2019). The only cerebral example related to human neonates was a study that showed that sildenafil promoted cerebral oxygenation in infants after they had cardiac surgery by increasing cerebral blood flow (Nagdyman et al., 2006).

Sildenafil is metabolized mostly through hepatic metabolism by the CYP3A4 enzymes, and to a lesser extent by the CYP2C enzyme (Jackson et al., 1999). Sildenafil has a half-life of 0.4 h in adult rodents and approximately 4 h in human adults (Daugan et al., 2003). One mg/kg orally leads to a C_max (after 1.5 h) of 516 nM in human plasma.¹ Sildenafil crosses the blood brain barrier (BBB) (Gómez-Vallejo et al., 2016) to reach the central nervous system disease. Gómez-Vallejo et al. (2016) have shown sildenafil to reach a concentration in cerebrospinal fluid (CSF) high enough (6–8 nM) to achieve PDE5 inhibition (the concentration required to inhibit 50% of PDE5 activity is 3.5 nM).

Similar to adults, the metabolic clearance of sildenafil in neonates is catalyzed predominantly by two cytochrome P450 (CYP) isozymes: CYP3A4 (major route) and CYP2C9 (minor route) (Hyland et al., 2001); in neonates, CYP3A7 also can contribute to N-demethylation (Takahiro et al., 2015). In vitro studies have demonstrated a rapid maturation and increase in the expression of these enzymes immediately after birth from very low levels in the fetal liver to values similar to those in adults by the end of the first week of life, which explains why the clearance of sildenafil increased threefold from the first day after birth to the end of the first week of life (Mukherjee et al., 2009; Steinhorn et al., 2009). This suggests that the sildenafil pharmacokinetic will vary during the first week of life (Koukouritaki et al., 2004). In addition, the volume distribution of sildenafil was fourfold higher in neonates compared to adults, which resulted in a longer terminal half-life in neonates (48–56 h) compared to adults (Mukherjee et al., 2009). Recently, a population pharmacokinetics of sildenafil in extremely premature infants (≤28 weeks gestation and <32 weeks of gestation) demonstrated that oral bioavailability following enteral administration was lower in premature infants (29%) than in adults (41%) (Nichols et al., 2002). Thus, additional pharmacokinetic studies would be needed in human neonates with neonatal encephalopathy to understand optimal treatment doses, especially considering that these neonates may have liver failure and multiorgan dysfunction in addition to their neonatal encephalopathy as a result of their birth asphyxia.