Given the tight reciprocal activation between inflammatory and coagulation cascades, it is quite possible that inflammation promotes thrombus formation within placental, umbilical cord or other vessels feeding the cerebral blood flow. According to a classic pathophysiological hypothesis of NAIS, such thrombus would then migrate and occlude cerebral arteries leading to embolic stroke (26). This embolic hypothesis is also supported by the preponderant distribution of NAIS in the middle cerebral arterial territories, and in few instances by the detection of thrombotic/embolic events proximal or distal to the NAIS (27).

However, this embolic hypothesis is challenged by: (i) the imbalanced distribution of NAIS between the anterior versus posterior intracranial arterial territories even when the asymmetry of anterior versus posterior blood flows is taken into account (13, 28, 29); (ii) the infrequent occurrence of extracerebral infarcts concomitant to NAIS (13, 28, 29); and (iii) angiographic findings from newborns with NAIS showing that 22–65% of them present focal disruptions of the anterior circulation which, in certain cases might correspond to arterial wall defects, or to thrombi generated in situ from an inflamed arterial wall (28, 29). Based on these elements, we hypothesized that maternofetal inflammation induces a focal arteritis specifically affecting NAIS susceptible cerebral arteries, namely the middle cerebral artery (MCA), anterior carotid artery and intracranial internal carotid artery (13, 28, 29).

Using a preclinical rat model of chorioamnionitis induced by pathogen components [lipopolysaccharide (LPS) from Escherichia coli], we showed that end-gestational inflammation combined with a classic prothrombotic stress (transcutaneous laser exposure of the artery of interest), but not sole prothrombotic stress, targeting the MCA triggers NAIS (30). On one hand, the walls from neonatal cerebral arteries susceptible to stroke displayed a constitutively higher expression of proinflammatory cytokines in NAIS-susceptible versus non-susceptible arteries. On the other hand, pups from LPS-exposed dams presented a cerebral arteritis characterized by an increased number of inflammatory cells and expression of proinflammatory cytokines [interleukin (IL)-1/IL-1 receptor antagonist (IL-1Ra) ratio] within NAIS-susceptible, but not non-susceptible, arteries (30).

These preclinical results support, beside the embolic hypothesis, the contribution of a focal arteritis and thrombosis in the pathophysiology of NAIS.

The neural cell stress associated with NAIS is mainly due to the combination of energy failure, excess of intracellular Ca²⁺, and glutamate release, as well as ionic imbalance and oxidative stress (64–66). All these pathways are key mechanisms driving neural cell death (64, 66–68). We learned from a preclinical model of NAIS, that excitotoxic cell death, necrosis and programmed necrosis (necroptosis) occur between 0 and 6 h after exposures to sole HI or pathogen components plus HI (Figure 2A) (66, 69–72).

Pattern recognition receptors (PRR) such as TLR recognize pathogens as well as DAMP (73, 74, 90–94). Inflammation sensitizes the neonatal brain to subsequent HI injury (54, 73, 74, 95). For instance, Stridh et al. showed in a newborn mouse model [at postnatal day (P) 9, i.e., a level of brain development equivalent to the term human newborn] of NAIS that TLR-2 deficiency protected the brain from infarcts (94). Energy failure due to hypoxia and inflammation combine their effects to increase the oxidative stress (96, 97). Besides, LPS and other pathogen components interact with various TLR to increase the synthesis of a wide set of proinflammatory cytokines and chemokines (Figure 2B) (54, 73, 74, 95). For instance, in a preclinical model of NAIS, exposure to LPS plus HI leads to an autocrine/paracrine loop of neuronal self-injury, mediated by inflammatory molecules: IL-1β, TNF-α, reactive oxygen species (ROS) production, and mitogen-activated protein kinases (MAPK)-induced apoptosis (Figure 2B) (73, 74). LPS combined with HI leads to glial activation and neurotoxic molecule release such as IL-1β-induced matrix metalloproteinase (MMP)-9, nitric oxide (NO), and inducible NO synthase (iNOS) (66, 73). In these LPS plus HI-exposed brains, NO and MMP-9 combine their effects to alter the blood brain barrier (BBB) by degrading the lamina of intracerebral blood vessels (67, 74). Such disruption enables proinflammatory and/or neurotoxic mediators to leak through the BBB, and thus increasing the extent of NAIS in preclinical models (74, 98) (Figure 2A).

Necroptosis is an early cell death pathway which is triggered by inflammatory mediators led by TNF-α and TNF family death receptor (TNFR): TNFR-1, FAS, and TLR, namely TLR-3 and TLR-4 (75, 79, 84, 99). The necrosome is a complex that requires the presence of activated receptor interacting protein kinase (RIP)-1, RIP-3 and the pseudo-kinase mixed lineage kinase domain-like (MLKL) to execute necroptosis (75, 99) (Figure 2B). All the mechanisms by which MLKL induces cell death are not totally elucidated (75, 81, 99). It was shown in a preclinical model that cerebral expression of TNF-α was triggered by LPS plus HI exposure (73). Accordingly, in this NAIS model RIP-3 expression was increased after LPS plus HI (74). IL-1β-induced MMP-9 can also activate necroptosis via Fas-ligand (Fas-L) interaction through Fas-associated death domain (FADD) (70, 96, 99). In line with these findings, it was shown that necrostatin-1—a RIP-1 inhibitor—administered after HI injury in P7 mice was neuroprotective: this inhibitor is able to prevent forebrain injury, as well as attenuates oxidative stress and mitochondrial dysfunction (69, 71). However, since RIP-1 is an important crosstalk molecule between apoptosis and necroptosis pathway, it has been observed in neonatal HI model that RIP-1 blockade increased apoptotic cell death (69).

The secondary phase of ischemic injury occurs between 24 and 72 h after NAIS and implicates apoptosis, anoikis and autophagy cell deaths. Intrinsic and extrinsic forms of apoptosis are known to be involved in NAIS due to pure HI or HI combined to pathogen exposure (67, 68, 72) (Figure 2B). Many preclinical studies characterized the involvement of apoptotic cell death in the genesis of NAIS and provided evidence in favor of neuroprotective strategies targeting apoptotic pathways (86–88, 100–102).

It is well known that apoptotic and autophagic cell death pathways crosstalk, and that autophagy can block apoptosis by sequestration of mitochondria (68) (Figure 2B). The induction of autophagy just after neonatal HI may be a neuroprotective mechanism by limiting apoptosis (68, 103). On the other hand, autophagy seems to be implicated in HI-induced cell death (104). Besides, another form of apoptosis triggered by cell detachment from the extracellular matrix—namely anoikis—could follow HI and/or inflammation exposures. Anoikis is induced by increased MMPs, including MMP-9 and activation of Fas receptor, which initiates the apoptosis cascade (105). This cell death pathway was assessed by our group: we showed an overexpression of MMP9 after HI plus inflammation injury (73, 74). Besides, the use of an MMP-9 competitive inhibitor shrank the size of LPS plus HI-induced brain infarcts (73, 74) (Figure 2A). To our knowledge, this is the first demonstration of anoikis-induced cell death in a model of combined inflammation and/or HI.

Our team and others uncovered that the upregulation of IL-1 plays a key role in chorioamnionitis, and in associated neonatal ischemic brain injuries (54, 79, 82, 84, 94–97). Our preclinical studies, and others, showed that prenatal IL-1 blockade using IL-1Ra is protective against chorioamnionitis, associated FIRS, and subsequent brain injuries (73, 74, 106–108). Postnatal administration of IL-1Ra is also effective in alleviating mortality (from 40 to 18%) as well as morbidities arising from postnatal inflammatory-sensitized NAIS (82, 92, 94): 66% decrease of the core (cavitary lesion), and 54% decrease of the penumbra (rim of mild to moderately ischemic tissue lying between the core and the unaffected tissue), and preventing the loss of motor skills (73, 74). IL-1Ra is an already approved drug to treat chronic inflammatory conditions, including those affecting pregnant mothers and newborns (96). IL-1Ra is, among the various molecules interfering with the IL-1 signaling, the one which dominates its pharmacological field due to its: (i) blocking effect on both IL-1α and IL-1β; (ii) short 4–6 h half-life (blood levels falling within a few hours of treatment stoppage); (iii) multiple routes of administration; (iv) approval for several pediatric inflammatory conditions (1–10 mg/kg/24 h), knowing that the repurposing of well-studied drugs used in the pediatric population is a cost-effective and efficient strategy to identify new therapies for pediatric diseases; and (v) excellent safety record (absence of opportunistic infection; reversible increase of liver enzyme, decrease of polymorphonuclear cells, slight increase of infection, that are mostly observed in patients on chronic treatment) after more than 10 years of use in more than 150,000 patients (80). Altogether, this provides encouraging preclinical evidence in favor of the efficacy and feasibility of end-gestational or neonatal interventions using IL-1Ra.

Hypothermia therapy is now a mandatory standard of care for term newborns suffering for diffuse HI encephalopathy (109–111). However, cooling treatment is modestly effective and leaves 50% of the treated patients with major sequelae (109, 112). Besides, it is uncertain why HT is effective for some, but not all, human newborns. Clinical studies reported that HT might have less beneficial effects on newborns exposed to infection-inflammation plus HI, than those exposed to HI alone (113–115). Furthermore, evidence in favor of an anti-inflammatory role of HT within the newborn brain is limited and conflicted. Only a few clinical or preclinical models address this question. However, a well-established anti-inflammatory effect of HT is the down regulation of oxidative stress within the brain (91, 97, 116–118). It has also been reported that HT is neuroprotective by limiting apoptotic cascades in human term newborns (110, 119–121). The potential effect of HT on neuroinflammatory cytokines expression has been poorly investigated up to now in preclinical models as well as in term newborns. It has been recently shown that HT did not modulate inflammatory molecules, including IL-1β, TNF-α, IL-1Ra and MMP-9, on LPS plus HI-exposed pups (118). Other downregulating effect of HT within preclinical NAIS brains remained unclear. In the clinical settings, HT has not been tested yet in NAIS patients, even though it is feasible and possibly effective.

Erythropoietin presents anti-inflammatory and neuroprotective properties mainly through dampening free radical release and neural cells apoptosis that have been well-established on preclinical models of neonatal brain infarcts (122, 123). Erythropoietin seems to be well-tolerated and neuroprotective against perinatal brain lesions of premature newborns: Benders et al. performed a study in 21 consecutive NAIS patients diagnosed by magnetic resonance imaging (MRI) using erythropoietin (1,000 IU/kg intravenously administered just after the diagnostic confirmation by MRI, and repeated at 24 and 48 h) (124).

There was no adverse effect on blood cells counts, or coagulation. The residual versus initial MRI injuries were quantitatively compared at 3 months of age between the erythropoietin-treated patients versus 10 untreated matched historical controls. The percentage of tissue loss within the ischemic area was not different between the treated versus untreated group. Hence, the effectiveness of erythropoietin in terms of neuroprotection in the NAIS context remains to be established.

Before considering human therapeutic trials aiming to prevent the occurrence of NAIS, it is mandatory to improve our ability to rapidly and efficiently detect prenatal, and immediately postnatal inflammation, and to correlate inflammatory profiles with the occurrence of NAIS. Hence, large scale case–control studies are a necessary prerequisite to further identify subpopulation(s) of newborns at risk of NAIS (28), by comparing pertinent profiles of expression of inflammatory and prothrombotic markers—reflecting acute activation instead of genetic predisposition—in the umbilical cord tissues and cord blood samples, between NAIS versus non NAIS patients. In addition, there is an urgent need to design efficient diagnostic tools adapted to the rapid and non-invasive diagnosis of chorioamnionitis and other maternofetal infectious/inflammatory diseases. In this line, MRI of the placenta, which has been shown to rapidly detect abnormal placental signals in a preclinical model of chorioamnionitis might be a promising diagnostic option (125). Based on these potential predictive biomarkers of NAIS, preventive anti-inflammatory strategies might then be tested.

A low threshold for early head MRI should be applied in newborns presenting acute neurological symptoms compatible with neonatal stroke. Adapted and thorough protocol of parenchymal and angiographic imaging should be continuously updated and more systematically applied (126), as recently recommended (127). The acute activation of inflammatory and prothrombotic factors at the time of the NAIS is an important research avenue to improved our physiopathological knowledge, and provide much needed biomarkers of NAIS. This might be performed first in retrospect using immunoassay on dried neonatal blood, as previously described (128). IL-1 blockers, erythropoietin, and/or hypothermia seem to be the most promising avenues to be tested in multicentric randomized control studies in the aim to limit the extent of NAIS and/or to promote recovery. Some teams already carried out retrospective and prospective phase I–II studies addressing such therapeutic approaches (124, 129, 130).