Effect of hypothermia on interleukin-1 receptor antagonist pharmacodynamics in inflammatory-sensitized hypoxic-ischemic encephalopathy of term newborns.

BACKGROUND: Hypothermia is increasingly tested in several neurological conditions, such as neonatal encephalopathy, stroke, traumatic brain injury, subarachnoid hemorrhage, spinal cord injury, and neurological outcomes of cardiac arrest. Current studies aim to increase benefits of hypothermia with new add-on therapies including immunomodulatory agents. Hypothermia has been shown to affect the metabolism of commonly used drugs, including those acting on neuroimmune pathways. OBJECTIVE: This study focuses on the effect of hypothermia on interleukin-1 receptor antagonist pharmacodynamics in a model of neonatal encephalopathy. METHODS: The effect of hypothermia on (i) the tissue concentration of the interleukin-1 receptor antagonist, (ii) the interleukin-1 inflammatory cascade, and (iii) the neuroprotective potential of interleukin-1 receptor antagonist has been assessed on our rat model of neonatal encephalopathy resulting from inflammation induced by bacterial compound plus hypoxia-ischemia. RESULTS: Hypothermia reduced the surface of core and penumbra lesions, as well as alleviated the brain weight loss induced by LPS+HI exposure. Hypothermia compared to normothermia significantly increased (range 50-65%) the concentration of the interleukin-1 receptor antagonist within the central nervous system. Despite this increase of intracerebral interleukin-1 receptor antagonist concentration, the intracerebral interleukin-1-induced tumor necrosis factor-alpha cascade was upregulated. In hypothermic condition, the known neuroprotective effect of interleukin-1 receptor antagonist was neutralized (50 mg/kg/12 h for 72 h) or even reversed (200 mg/kg/12 h for 72 h) as compared to normothermic condition. CONCLUSION: Hypothermia interferes with the pharmacodynamic parameters of the interleukin-1 receptor antagonist, through a bioaccumulation of the drug within the central nervous system and a paradoxical upregulation of the interleukin-1 pathway. These effects seem to be at the origin of the loss of efficiency or even toxicity of the interleukin-1 receptor antagonist when combined with hypothermia. Such bioaccumulation could happen similarly with the use of other drugs combined to hypothermia in a clinical context.

Perinatal Arterial Ischemic Stroke Is Associated to Materno-Fetal Immune Activation and Intracranial Arteritis.

The medium-size intra-cranial arteries arising from the carotid bifurcation are prone to perinatal arterial ischemic strokes (PAIS). PAIS' physiopathology needs to be better understood to develop preventive and therapeutic interventions that are currently missing. We hypothesized that materno-fetal inflammation leads to a vasculitis affecting selectively the carotidian tree and promoting a focal thrombosis and subsequent stroke. Dams were injected with saline or lipopolysaccharide (LPS) from Escherichia coli. A prothrombotic stress was applied on LPS-exposed vs. saline (S)-exposed middle cerebral arteries (MCA). Immunolabeling detected the inflammatory markers of interest. In S-exposed newborn pups, a constitutive higher density of macrophages combined to higher expressions of tumor necrosis factor-α (TNF-α), and interleukin 1ß (IL-1ß) was observed within the wall of intra- vs. extra-cranial cervicocephalic arteries. LPS-induced maternal and placental inflammatory responses mediated by IL-1ß, TNF-α and monocyte chemotactic protein 1 (MCP-1) were associated with: (i) increased density of pro-inflammatory macrophages (M1 phenotype); and (ii) pro-inflammatory orientation of the IL-1 system (IL-1ß/IL-1 receptor antagonist (IL-1Ra) ratio) within the wall of LPS-, vs. S-exposed, intra-cranial arteries susceptible to PAIS. LPS plus photothrombosis, but not sole photothrombosis, triggered ischemic strokes and subsequent motor impairments. Based on these preclinical results, the combination of pro-thrombotic stress and selective intra-cranial arteritis arising from end gestational maternal immune activation seem to play a role in the pathophysiology of human PAIS.

Neuronal self-injury mediated by IL-1ß and MMP-9 in a cerebral palsy model of severe neonatal encephalopathy induced by immune activation plus hypoxia-ischemia.

BACKGROUND: Inflammation due to remote pathogen exposure combined to hypoxia/ischemia (HI) is one of the most common causes of neonatal encephalopathy affecting at-term or near-term human newborn, which will consequently develop cerebral palsy. Within term-equivalent rat brains exposed to systemic lipopolysaccharide (LPS) plus HI, it was previously showed that neurons produce IL-1ß earlier than do glial cells, and that blocking IL-1 was neuroprotective. To further define the mechanisms whereby IL-1 exerts its neurotoxic effect, we hypothesize that IL-1ß plays a pivotal role in a direct and/or indirect mechanistic loop of neuronal self-injury through matrix metalloproteinase (MMP)-9. METHODS: An established preclinical rat model of LPS+HI-induced neonatal encephalopathy was used. In situ hybridization, ELISA, and immunolabeling techniques were employed. Selective blocking compounds allowed addressing the respective roles of IL-1 and MMP-9. RESULTS: In LPS+HI-exposed forebrains, neuronal IL-1ß was first detected in infarcted neocortical and striatal areas and later in glial cells of the adjacent white matter. Neuronal IL-1ß played a key role: (i) in the early post-HI exacerbation of neuroinflammation and (ii) in generating both core and penumbral infarcted cerebral areas. Systemically administered IL-1 receptor antagonist (IL-1Ra) reached the brain and bound to the neocortical and deep gray neuronal membranes. Then, IL-1Ra down-regulated IL-1ß mRNA and MMP-9 neuronal synthesis. Immediately post-HI, neuronal IL-1ß up-regulated cytokine-induced neutrophil chemoattractant (CINC-1), monocyte chemoattractant protein-1 (MCP-1), and inducible nitric oxide synthase. MMP-9 would disrupt the blood-brain barrier, which, combined to CINC-1 up-regulation, would play a role in polymorphonuclear cell (PMN) infiltration into the LPS+HI-exposed brain. IL-1ß blockade prevented PMN infiltration and oriented the phenotype of macrophagic/microglial cells towards anti-inflammatory and neurotrophic M2 profile. IL-1ß increased the expression of activated caspase-3 and of receptor-interacting-protein (RIP)-3 within infarcted forebrain area. Such apoptotic and necroptotic pathway activations were prevented by IL-1Ra, as well as ensuing cerebral palsy-like brain damage and motor impairment. CONCLUSIONS: This work uncovered a new paradigm of neuronal self-injury orchestrated by neuronal synthesis of IL-1ß and MMP-9. In addition, it reinforced the translational neuroprotective potential of IL-1 blockers to alleviate human perinatal brain injuries.

Stability of glutathione added as a supplement to parenteral nutrition.

BACKGROUND: Most very premature newborns (<32 weeks of gestation) receive parenteral nutrition (PN) that is inherently contaminated with peroxides. Oxidative stress induced by PN is associated with bronchopulmonary dysplasia, a main pathological complication in these infants who have weak antioxidant capacity to detoxify peroxides because of their glutathione deficiency. In animals, glutathione supplementation of PN prevented oxidative stress and alveolar loss (the main characteristic of bronchopulmonary dysplasia). Of its two forms-oxidized glutathione (GSSG) and reduced glutathione (GSH)-GSSG was used because of its better stability. However, a 30% loss of GSSG in PN is observed. The potentially high therapeutic benefits of GSSG supplementation on the health of very premature infants make the study of its stability highly important. METHODS: GSSG was incubated in combination with the following components of PN: dextrose, multivitamins, Primene, and Travasol, and with cysteine, cystine, and peroxides, for 24 h. Total glutathione in these solutions was measured 0-24 h after the addition of GSSG. RESULTS: The combination of cysteine and multivitamins caused the maximum loss of glutathione. The stability of GSSG was not affected by multivitamins. The cysteine was responsible for ∼20% of the loss of GSSG; in the presence of multivitamins, the loss reached >70%. Removing the cysteine prevented the degradation of glutathione. CONCLUSION: GSSG reacts with cysteine to form cysteine-glutathione mixed disulfide, another suitable glutathione substrate for preterm neonates. The study confirms that GSSG added to PN can potentially provide a precursor to de novo synthesis of glutathione in vivo.

Neonatal parenteral nutrition affects the metabolic flow of glucose in newborn and adult male Hartley guinea pigs' liver.

Extremely premature birth is associated with a permanent disruption of energy metabolism. The underlying mechanisms are poorly understood. The oxidative stress induced by parenteral nutrition (PN) during the first week of life is suspected to reprogram energy metabolism in the liver. Full-term male Hartley guinea pigs (to isolate PN from prematurity) receiving PN enriched or not with glutathione (to isolate PN effects from PN-induced oxidative stress effects) or an Oral Nutrition (ON) during the first week of life were used. At 1 week (neonatal) and 16 weeks (adult), measurements of liver glutathione (GSH and GSSG) and activities of three key enzymes of energy metabolism (glucokinase (GCK), phosphofructokinase (PFK), and acetyl-CoA carboxylase (ACC)) were performed. Differences between groups were reported if p ≤ 0.05 (Analysis of Variance). At 1 week, compared to ON, PN induced higher GSSG (oxidative stress), higher GCK activity, and lower PFK and ACC activity, the glutathione supplement prevented all PN effects. At 16 weeks, early PN induced lower GSSG (reductive stress) and lower GCK activity, which was prevented by added glutathione, and higher ACC activity independent of glutathione supplement. ACC was negatively associated (r2 = 0.33) with GSSG. Increased nicotinamide adenine dinucleotide phosphate levels confirmed the glucose-6-phosphate accumulation at 1 week, whereas our protocol failed to document lipid accumulation at 16 weeks. In adult male guinea pigs, neonatal exposure to PN affected glutathione metabolism leading to reductive stress (lower GSSG) and an altered metabolic flow of glucose. Partial prevention with glutathione supplementation suggests that, in addition to peroxides, other factors of PN are involved.

Relationship between redox potential of glutathione and DNA methylation level in liver of newborn guinea pigs.

The metabolism of DNA methylation is reported to be sensitive to oxidant molecules or oxidative stress. Hypothesis: early-life oxidative stress characterized by the redox potential of glutathione influences the DNA methylation level. The in vivo study aimed at the impact of modulating redox potential of glutathione on DNA methylation. Newborn guinea pigs received different nutritive modalities for 4 days: oral nutrition, parenteral nutrition including lipid emulsion Intralipid (PN-IL) or SMOFLipid (PN-SF), protected or not from ambient light. Livers were collected for biochemical determinations. Redox potential (p < 0.001) and DNA methylation (p < 0.01) were higher in PN-infused animals and even higher in PN-SF. Their positive correlation was significant (r2 = 0.51; p < 0.001). Methylation activity was higher in PN groups (p < 0.01). Protein levels of DNA methyltransferase (DNMT)-1 were lower in PN groups (p < 0.01) while those of both DNMT3a isoforms were increased (p < 0.01) and significantly correlated with redox potential (r2 > 0.42; p < 0.001). The ratio of SAM (substrate) to SAH (inhibitor) was positively correlated with the redox potential (r2 = 0.36; p < 0.001). In conclusion, early in life, the redox potential value strongly influences the DNA methylation metabolism, resulting in an increase of DNA methylation as a function of increased oxidative stress. These results support the notion that early-life oxidative stress can reprogram the metabolism epigenetically. This study emphasizes once again the importance of improving the quality of parenteral nutrition solutions administered early in life, especially to newborn infants. Abbreviation of Title: Parenteral nutrition and DNA methylation.

Glutathione Supplementation of Parenteral Nutrition Prevents Oxidative Stress and Sustains Protein Synthesis in Guinea Pig Model.

Peroxides contaminating parenteral nutrition (PN) limit the use of methionine as a precursor of cysteine. Thus, PN causes a cysteine deficiency, characterized by low levels of glutathione, the main molecule used in peroxide detoxification, and limited growth in individuals receiving long-term PN compared to the average population. We hypothesize that glutathione supplementation in PN can be used as a pro-cysteine that improves glutathione levels and protein synthesis and reduces oxidative stress caused by PN. One-month-old guinea pigs (7-8 per group) were used to compare glutathione-enriched to a non-enriched PN, animals on enteral nutrition were used as a reference. PN: Dextrose, amino acids (Primene), lipid emulsion (Intralipid), multivitamins, electrolytes; five-day infusion. Glutathione (GSH, GSSG, redox potential) and the incorporation of radioactive leucine into the protein fraction (protein synthesis index) were measured in the blood, lungs, liver, and gastrocnemius muscle. Data were analysed by ANOVA; p < 0.05 was considered significant. The addition of glutathione to PN prevented the PN-induced oxidative stress in the lungs and muscles and supported protein synthesis in liver and muscles. The results potentially support the recommendation to add glutathione to the PN and demonstrate that glutathione could act as a biologically available cysteine precursor.

Role of Perinatal Inflammation in Neonatal Arterial Ischemic Stroke.

Based on the review of the literature, perinatal inflammation often induced by infection is the only consistent independent risk factor of neonatal arterial ischemic stroke (NAIS). Preclinical studies show that acute inflammatory processes take place in placenta, cerebral arterial wall of NAIS-susceptible arteries and neonatal brain. A top research priority in NAIS is to further characterize the nature and spatiotemporal features of the inflammatory processes involved in multiple levels of the pathophysiology of NAIS, to adequately design randomized control trials using targeted anti-inflammatory vasculo- and neuroprotective agents.

Neuroprotective effects of hypothermia in inflammatory-sensitized hypoxic-ischemic encephalopathy.

BACKGROUND: Despite the recent introduction of hypothermia as a mandatory standard of care, the incidence of neonatal encephalopathy in full-term newborns and its devastating neuro-behavioral outcomes continues to be a major individual, familial and social issue. Neonatal encephalopathy is mainly due to the compounding and interacting effects of hypoxia-ischemia and inflammation resulting from placental and other perinatal infections. It is unclear why hypothermia is effective in alleviating neonatal encephalopathy in some, but not all, full-term newborns. However, newborns exposed to inflammatory-sensitized hypoxia-ischemia seem to have less therapeutic benefit from hypothermia than those exposed to hypoxia-ischemia alone. OBJECTIVES: To clarify this uncertainty, we tested the efficacy of hypothermia in a double-hit model of neonatal encephalopathy induced by inflammatory-sensitized hypoxia-ischemia. METHODS: Using a rat preclinical model of endotoxin plus hypoxia-ischemia-induced neonatal encephalopathy of term newborns, we assessed the following in pups exposed (or not) to hypothermia: the extent of brain injuries and the expressions of molecules implicated in neural cell death, namely: pro-inflammatory cytokines, matrix metalloproteinase-9, antioxidant enzymes, as well as receptor-interacting protein-3. RESULTS: Hypothermia was neuroprotective on inflammatory-sensitized hypoxia-ischemia-induced penumbra, but not core, brain injuries. This beneficial effect was associated with a hypothermia-induced increase of antioxidant enzymes (superoxide dismutase-1, glutathione peroxidase-1), but was not associated with any variations of the other inflammatory mediators tested, namely: interleukin-1ß, interleukin-1 receptor antagonist, tumor necrosis factor-α and matrix metalloproteinase-9. CONCLUSION: Hypothermia is neuroprotective against inflammatory-sensitized hypoxia-ischemia possibly through a hypothermia-induced increase of antioxidant enzymes. This neuroprotective effect seems to be independent of the interleukin-1 system.

Activation of the IL-1ß/CXCL1/MMP-10 axis in chorioamnionitis induced by inactivated Group B Streptococcus.

Infection or inflammation during pregnancy is known to lead to maternal immune activation triggering a fetal inflammatory response syndrome associated with deleterious effects, such as brain injury and neurodevelopmental disabilities. Group B Streptococcus (GBS) - one of the most common bacterium colonizing pregnant women - can be responsible for chorioamnionitis. Given that interleukin (IL)-1ß has a major role in anti-GBS host defense, we hypothesized that IL-1ß-driven innate immune response is implicated in GBS-induced chorioamnionitis. Using a rat model of GBS-induced chorioamnionitis, this study showed that inflammatory response to this pathogen was associated with maternal and placental IL-1ß hyper expression. Following placental chemokine (C-X-C motif) ligand 1 (CXCL1) production, polymorphonuclear leukocytes (PMN) placental infiltration started at 24 h post-GBS exposure, and MMP-10 was released within these placentas. At 72 h, PMN infiltration extended to membranes and to membranes' arteries. This was associated with IL-1ß release within the fetus blood at 72 h. Such a GBS-associated inflammatory cascade might be deleterious for fetal organs. These results pave the way toward targeted placento-protective anti-inflammatory strategies against GBS-induced chorioamnionitis.