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Perinatal hypoxia ischemia (HI) is a significant cause of brain injury in surviving infants. Although hypothermia improves outcomes in some infants, additional therapies are needed since about 40% of infants still have a poor outcome. Acetyl-L-carnitine (ALCAR), an acetylated derivative of L-carnitine, protected against early changes in brain metabolites and mitochondrial function after HI on postnatal day (PND) 7 in a rat pup model of near-term HI injury. However, its efficacy in long-term structural and functional outcomes remains unexplored. We determined the efficacy of ALCAR therapy administered to rat pups after HI at PND 7, using both longitudinal in vivo magnetic resonance imaging and behavioral tests, in male and female rats. HI led to sex-specific behavioral impairment, with males exhibiting more global functional deficits than females. Interestingly, HI reduced the volume of the contralateral hemisphere in males only, suggesting that the brain injury is more diffuse in males than in females. Treatment with ALCAR improved both morphological and functional outcomes in both male and female rats. These results suggest that ALCAR may be a potential therapy for clinical use since the treatment attenuated the moderate injury produced under the experimental conditions used and improved the functional outcome in preclinical studies.

Background: Neonatal encephalopathy induced by perinatal asphyxia is a serious condition associated with high mortality and morbidity. Inflammation after the insult is thought to contribute to brain injury. This inflammatory response to hypoxia-ischemia (HI) may not only occur in the brain but also in peripheral organs. The aim of the present study was to investigate the effect of neonatal HI on the inflammatory response in the liver in comparison to inflammation in the brain. Methods: HI was induced in P7 Wistar rats by unilateral carotid artery occlusion and hypoxia. Cytokine and chemokine mRNA levels were determined in the brain and liver by quantitative PCR. Polarization of brain macrophages to the M1/M2-like phenotype and infiltration of neutrophils were characterized by immunohistochemistry. Results: 3 h after HI, an upregulation of the proinflammatory cytokines TNF-α and IL-1β and anti-inflammatory IL-10 was observed in the ipsilateral hemisphere of the brain compared to mRNA levels in sham-operated animals. Additionally, cerebral CINC-1 and MCP-1 mRNA expressions were increased. We also observed increased numbers of macrophages/microglia of the M1-like phenotype as well as a small increase in granulocyte influx in the ipsilateral hemisphere. Conversely, in the liver 3 h after HI, a downregulation of TNF-α, IL-1β, and MCP-1 and a trend towards an upregulation of IL-10 were observed compared to mRNA levels of sham-operated animals. However, hepatic CINC-1 expression was increased compared to levels in sham-operated animals. Following systemic hypoxia only, no significant changes in the expression of TNF-α, CINC-1 or MCP-1 were observed in the liver compared to sham-operated littermates, except for an upregulation in hepatic IL-1β expression 3 h after hypoxia. Twenty-four hours after insult, cerebral ipsilateral TNF-α, MCP-1 and CINC-1 mRNA expression was still increased, together with an increase in TGF-β expression. Moreover, an increase in macrophages/microglia of the M1-like phenotype was observed together with the appearance of macrophages/microglia of the M2-like phenotype around the cerebral lesion as well as an increase in granulocyte influx in comparison to 3 h after HI. In the liver, 24 h after HI, cytokine and chemokine responses were similar to mRNA levels in sham-operated animals except for a decrease in IL-10 and MCP-1. Conclusion: We describe for the first time that brain damage following neonatal HI induces an early downregulation of the proinflammatory response in the liver. HI induces an early proinflammatory response in the brain with a concomitant increase in influx of neutrophils and polarization of macrophages/microglia to the M1-like phenotype starting at 3 h and increasing up to 24 h after HI. The inflammatory state of the brain changes after 24 h, with an increase in the anti-inflammatory cytokine TGF-β together with the appearance of macrophages/microglia of the M2-like phenotype. The downregulation of proinflammatory cytokines in the liver is not due to systemic hypoxia only, but is induced by the cerebral damage.

Hypoxia is the most common cause of neonatal seizures and can lead to epilepsy, but the epileptogenic mechanisms are not yet understood. We have previously shown that hypoxia-induced seizures in the neonatal rat result in acutely decreased amplitudes and frequency of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) in hippocampal CA1 pyramidal neurons. In the current study, we asked whether such changes persist for several days following hypoxia-induced seizures. Similar to the acute findings, we observed decreased frequency and amplitudes of sIPSCs and decreased mIPSC amplitudes in CA1 pyramidal neurons at 3–5 days after hypoxia. However, in contrast to the acute findings, we observed no differences between hypoxia-treated and control groups in mIPSC frequency. Additionally, by 7 days after hypoxia, sIPSC amplitudes in the hypoxia group had recovered to control levels, but sIPSC frequency remained decreased. These data indicate that the persistently decreased sIPSC frequency result from decreased firing of presynaptic inhibitory interneurons, with only transient possible changes in postsynaptic responses to GABA release.

Hypoxia-ischemia (HI; concurrent oxygen/blood deficiency) and associated encephalopathy represent a common cause of neurological injury in premature/low-birth-weight infants and term infants with birth complications. Resulting behavioral impairments include cognitive and/or sensory processing deficits, as well as language disabilities, and clinical evidence shows that male infants with HI exhibit more severe cognitive deficits compared to females with equivalent injury. Evidence also demonstrates activation of sex-dependent apoptotic pathways following HI events, with males preferentially activating a caspase-independent cascade of cell death and females preferentially activating a caspase-dependent cascade following neonatal hypoxic and/or ischemic insults. Based on these combined data, the ‘female protection’ following HI injury may reflect the endogenous X-linked inhibitor of apoptosis (XIAP), which effectively binds effector caspases and halts downstream cleavage of effector caspases (thus reducing cell death). To test this theory, the current study utilized neonatal injections of vehicle or embelin (a small molecule inhibitor of XIAP) in male and female rats with or without induced HI injury on postnatal day 7 (P7). Subsequent behavioral testing using a clinically relevant task revealed that the inhibition of XIAP exacerbated HI-induced persistent behavioral deficits in females, with no effect on HI males. These results support sex differences in mechanisms of cell death following early HI injuries, and suggest a potential clinical benefit from the development of sex-specific neuroprotectants for the treatment of HI.

Hypoxic-ischemic brain injury is often delayed and involves both apoptotic and immunoregulatory mechanisms. In this study, we used a neonatal model of hypoxia-ischemia to examine the effect of the mixed lineage kinase (MLK) inhibitor CEP-1347 on brain damage, apoptosis and inflammation. The tissue volume loss was reduced by 28% (p = 0.019) in CEP-1347-treated versus vehicle-treated rats and CEP-1347 significantly attenuated microgliosis at 7 days (p = 0.038). CEP-1347 decreased TUNEL-positive staining as well as cleaved caspase 3 immunoreactivity. CEP-1347 did not affect the expression of pro-inflammatory cytokines IL-1β, IL-6 and MCP-1, nor did it affect the expression of OX-42 (CR3) and OX-18 (MHC I) 24 h after the insult. In conclusion, the MLK inhibitor CEP-1347 has protective effects in a neonatal rat model of hypoxia-ischemia, which is mainly related to reduced apoptosis.