Reperfusion after hypoxia-ischemia exacerbates brain injury with compensatory activation of the anti- ferroptosis system: based on a novel rat model.

Hypoxic-ischemic encephalopathy, which predisposes to neonatal death and neurological sequelae, has a high morbidity, but there is still a lack of effective prevention and treatment in clinical practice. To better understand the pathophysiological mechanism underlying hypoxic-ischemic encephalopathy, in this study we compared hypoxic-ischemic reperfusion brain injury and simple hypoxic-ischemic brain injury in neonatal rats. First, based on the conventional Rice-Vannucci model of hypoxic-ischemic encephalopathy, we established a rat model of hypoxic-ischemic reperfusion brain injury by creating a common carotid artery muscle bridge. Then we performed tandem mass tag-based proteomic analysis to identify differentially expressed proteins between the hypoxic-ischemic reperfusion brain injury model and the conventional Rice-Vannucci model and found that the majority were mitochondrial proteins. We also performed transmission electron microscopy and found typical characteristics of ferroptosis, including mitochondrial shrinkage, ruptured mitochondrial membranes, and reduced or absent mitochondrial cristae. Further, both rat models showed high levels of glial fibrillary acidic protein and low levels of myelin basic protein, which are biological indicators of hypoxic-ischemic brain injury and indicate similar degrees of damage. Finally, we found that ferroptosis-related Ferritin (Fth1) and glutathione peroxidase 4 were expressed at higher levels in the brain tissue of rats with hypoxic-ischemic reperfusion brain injury than in rats with simple hypoxic-ischemic brain injury. Based on these results, it appears that the rat model of hypoxic-ischemic reperfusion brain injury is more closely related to the pathophysiology of clinical reperfusion. Reperfusion not only aggravates hypoxic-ischemic brain injury but also activates the anti-ferroptosis system.

Neferine Protects against Hypoxic-Ischemic Brain Damage in Neonatal Rats by Suppressing NLRP3-Mediated Inflammasome Activation.

Hypoxic-ischemic encephalopathy (HIE) is recognized as the main cause of neonatal death, and efficient treatment strategies remain limited. Given the prevalence of HIE and the associated fatality, further studies on its pathogenesis are warranted. Oxidative stress and neuroinflammatory injury are two important factors leading to brain tissue injury and nerve cell loss in HIE. Neferine, an alkaloid extracted from lotus seed embryo, exerts considerable effects against several diseases such as cancers and myocardial injury. In this study, we demonstrated the neuroprotective effect of neferine on HIE and hypothesized that it involves the inhibition of neuronal pyroptosis, thereby ameliorating neurological inflammation and oxidative stress. We demonstrated that the mRNA levels of proteins associated with pyroptosis including caspase-1, the caspase adaptor ASC, gasdermin D, interleukin- (IL-) 18, IL-1ß, and some inflammatory factors were significantly increased in neonatal HIBD model rats compared to those in the control group. The increase in these factors was significantly suppressed by treatment with neferine. We stimulated PC12 cells with CoCl2 to induce neuronal HIBD in vitro and investigated the relationship between neferine and pyroptosis by altering the expression of the NLRP3 inflammasome. The overexpression of NLRP3 partially reversed the neuroprotective effect of neferine on HIBD, whereas NLRP3 knockdown further inhibited caspase-1 activation and IL-1ß and IL18 expression. In addition, simultaneous alteration of NLRP3 expression induced changes in intracellular oxidative stress levels after HIBD. These findings indicate that neferine ameliorates neuroinflammation and oxidative stress injury by inhibiting pyroptosis after HIBD. Our study provides valuable information for future studies on neferine with respect to neuroinflammation and pyroptosis.

LXA4 protects against hypoxic-ischemic damage in neonatal rats by reducing the inflammatory response via the IκB/NF-κB pathway.

Hypoxia and the resultant decreases in cerebral blood flow in the perinatal period can lead to neonatal hypoxic-ischemic (HI) brain injury, which can, in turn, cause severe disability or even death. However, the efficacy of current treatment strategies remains limited. Several studies have demonstrated that lipoxin A4 (LXA4), as one of the earliest types of endogenous lipid mediators, can inhibit the accumulation of neutrophils, arrest inflammation, and promote the resolution of inflammation. However, research on LXA4 in the nervous system has rarely been carried out. In the present study, we sought to investigate the protective effect of LXA4 on HI brain damage in neonatal rats, as well as the underlying mechanisms. Through experiments conducted using an HI animal model, we found that the LXA4 intervention promoted the recovery of neuronal function and tissue structure following brain injury while maintaining the integrity of the blood-brain barrier in addition to reducing cerebral edema, infarct volume, and inflammatory responses. Our results suggest that LXA4 interfered with neuronal oxygen-glucose deprivation insults, reduced the expression of inflammatory factors, inhibited apoptosis, and promoted neuronal survival in vitro. Finally, the LXA4 intervention attenuated HI-induced activation of inhibitor kappa B (IκB) and degradation of nuclear factor-κB (NF-κB). In conclusion, our data suggest that LXA4 exerts a neuroprotective effect against neonatal HI brain damage through the IκB/NF-κB pathway. Our findings will help inform future studies regarding the effects of LXA4 on neuroinflammation, blood-brain barrier integrity, and neuronal apoptosis.

Glycine Protects against Hypoxic-Ischemic Brain Injury by Regulating Mitochondria-Mediated Autophagy via the AMPK Pathway.

Hypoxic-ischemic encephalopathy (HIE) is detrimental to newborns and is associated with high mortality and poor prognosis. Thus, the primary aim of the present study was to determine whether glycine could (1) attenuate HIE injury in rats and hypoxic stress in PC12 cells and (2) downregulate mitochondria-mediated autophagy dependent on the adenosine monophosphate- (AMP-) activated protein kinase (AMPK) pathway. Experiments conducted using an in vivo HIE animal model and in vitro hypoxic stress to PC12 cells revealed that intense autophagy associated with mitochondrial function occurred during in vivo HIE injury and in vitro hypoxic stress. However, glycine treatment effectively attenuated mitochondria-mediated autophagy. Additionally, after identifying alterations in proteins within the AMPK pathway in rats and PC12 cells following glycine treatment, cyclosporin A (CsA) and 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside (AICAR) were administered in these models and indicated that glycine protected against HIE and CoCl2 injury by downregulating mitochondria-mediated autophagy that was dependent on the AMPK pathway. Overall, glycine attenuated hypoxic-ischemic injury in neurons via reductions in mitochondria-mediated autophagy through the AMPK pathway both in vitro and in vivo.

Treatment With Liraglutide Exerts Neuroprotection After Hypoxic-Ischemic Brain Injury in Neonatal Rats via the PI3K/AKT/GSK3ß Pathway.

Neonatal hypoxic-ischemic (HI) brain injury is a detrimental disease, which results in high mortality and long-term neurological deficits. Nevertheless, the treatment options for this disease are limited. Thus, the aim of the present study was to assess the role of liraglutide in neonatal HI brain injury in rats and investigate the associated mechanisms. The results showed that treatment with liraglutide significantly reduced infarct volume and ameliorated cerebral edema, decreased inflammatory response, promoted the recovery of tissue structure, and improved prognosis following HI brain injury. Moreover, treatment with liraglutide inhibited apoptosis and promoted neuronal survival both in the rat model and following oxygen-glucose deprivation (OGD) insult. LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), partially reversed these therapeutic effects, suggesting that the PI3K/protein kinase B (Akt) pathway was involved. In conclusion, our data revealed that treatment with liraglutide exerts neuroprotection after neonatal HI brain injury via the PI3K/Akt/glycogen synthase kinase-3ß (GSK3ß) pathway and may be a promising therapy for this disease.

[Expression rhythm of autophagic gene in neurons of neonatal rats with hypoxia/ischemia and its regulatory mechanism].

OBJECTIVE: To investigate the expression of autophagic gene and circadian gene in the neurons of neonatal rats after hypoxic-ischemic brain damage and the mechanism of nerve injury induced by hypoxia/ischemia. METHODS: Twelve Sprague-Dawley (SD) rats were randomly divided into hypoxic-ischemic (HI) group and sham-operation group, with 6 rats in each group. Ligation of the right common carotid artery and hypoxic treatment were performed to establish a model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of the circadian protein Clock in the cortex and hippocampus. The neurons of the rats were cultured in vitro and randomly divided into oxygen glucose deprivation (OGD) group and control group. The neurons in the OGD group were treated with DMEM medium without glucose or serum to simulate ischemic state, and hypoxic treatment was performed to establish an in vitro model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of autophagy-related proteins Beclin1 and LC3 and Clock protein at different time points. The changes in the expression of Beclin1 and LC3 were measured after the expression of Clock protein in neurons was inhibited by small interfering RNA technique. RESULTS: The expression of autophagy-related proteins Beclin1 and LC3Ⅱ in neurons cultured in vitro displayed a rhythmic fluctuation; after OGD treatment, the expression of Beclin1 and LC3Ⅱ gradually increased over the time of treatment and no longer had a rhythmic fluctuation. Compared with the sham-operation group, the HI group had a significant reduction in the expression of Clock protein in the cortex and hippocampus (P<0.05). After OGD treatment, the neurons cultured in vitro had a significant reduction in the expression of Clock protein (P<0.05). Compared with the negative control group, the Clock gene inhibition group had significant reductions in the expression of Beclin1 and LC3Ⅱ (P<0.05). CONCLUSIONS: Hypoxia/ischemia induces the disorder in the expression rhythm of autophagy-related proteins Beclin1 and LC3, and the mechanism may be associated with the fact that the circadian protein Clock participates in the regulation of the expression of Beclin1 and LC3.

[Effects of PINK1 gene on cell apoptosis and cell autophagy in neonatal mice with hypoxic-ischemic brain damage].

OBJECTIVE: To study the effect of PINK1 (phosphatase and tensin homolog deleted on chromosome ten induced putative kinase 1) gene on cell apoptosis and cell autophagy in neonatal mice with hypoxic-ischemic brain damage (HIBD). METHODS: Seventy-two wild-type C57BL/6 mice and 72 PINK1 gene knockout neonatal C57BL/6 mice were randomly divided into four groups: sham-operated wild-type (SWT), HIBD model wild-type (MWT), sham-operated knockout (SKO) and HIBD model knockout (MKO). HIBD model was prepared by low oxygen exposure for 2.5 hours after right carotid artery ligation. After 24 hours of hypoxia-ischemia treatment, TTC (2,3,5-triphenyl four azole nitrogen chloride) staining was used to measure brain infarct volume. The immunohistochemical staining was used to measure the expression of cell apoptosis protein cleaved-caspase-3 (CC3) in brain tissues. The TUNEL method was used to measure cell apoptosis. The immunofluorescence staining and Western blot were used to measure the expression of cell autophagy protein LC3. RESULTS: Compared with the MWT group, the infarct volume of brain tissues was markedly reduced in the MKO group (P<0.05), the number of apoptotic cells and the cell apoptosis index were markedly decreased in the MKO group (P<0.05), the expression of apoptosis protein CC3 was significantly reduced in the MKO group (P<0.05), the expression of cell autophagy protein LC3 was significantly decreased in the MKO group, and the autophagy indicator LC3II/LC3I was also markedly reduced in the MKO group (P<0.05). CONCLUSIONS: PINK1 gene knockout can protect neonatal mice from HIBD.

[Clinical features and homological analysis of Pichia ohmeri-caused hospital-acquired fungemia in premature infants].

OBJECTIVE: To analyze the clinical features of fungemia caused by Pichia ohmeri (P. ohmeri) in neonate intensive care unit and explore its molecular biological characteristics so as to improve its diagnosis and treatment level. METHODS: The clinical data of 6 infected infants were retrospectively analyzed. The strains obtained from them were identified and homological analysis was performed through randomly amplified polymorphic assay to explore the epidemiological characteristics of this nosocomial infection. RESULTS: Before the isolation of P. ohmeri, they received intravenous antibacterial therapy for 13 - 45 days. Among them, 4 received mechanical ventilation and 5 had a peripheral insertion of central venous catheters. Five infants were healed after a therapy of caspofungin for 15 - 30 days. One neonate recurred after a 30-day administration of fluconazole. The strain was identified and confirmed as P. ohmeri. RAPD genotyping results showed that all 6 strains were from the same clone. No similar cases occurred after positive control measures despite a negative epidemiological sampling. CONCLUSIONS: P. ohmeri may cause premature infant fungemia and lead to its spread in hospital. Hospital infection control is a key point. And caspofungin is both safe and effective in the therapy of neonate fungemia.

[Clinical analysis of 31 cases of neonatal purulent meningitis caused by Escherichia coli].

OBJECTIVE: Neonatal purulent meningitis is a severe infection responsible for high mortality and disabling sequelae. Escherichia coli is the main pathogen of neonatal purulent meningitis. This study explored the clinical characteristics and antibiotic resistance of Escherichia coli-induced neonatal meningitis. METHODS: A retrospective chart review was performed. A total of 31 cases of neonatal purulent meningitis caused by Escherichia coli were identified in the neonatal intensive care unit between January 1, 2001 and December 31, 2011. The clinical characteristics and antibiotic sensitivity test results were analyzed. RESULTS: Fever, poor feeding, lethargy and seizure were common clinical signs of neonatal purulent meningitis caused by Escherichia coli. Acute complications mainly included hyponatremia (17 cases), hydrocephalus (8 cases), subdural collection (2 cases), ventriculitis (2 cases) and cerebral infarction (1 case). Thirty neonates (97%) had increased CRP levels. Of the 31 patients, 14 cases were cured and 12 had adverse outcomes (5 patients died during hospitalization). Escherichia coli strains were resistant (>50%) to commonly used penicillins and cephalosporins between 2007 and 2011, presenting significantly higher resistance rates than between 2001 and 2006. The detection rate of extended spectrum ß-lactamases (ESBLs)-producing strains between 2007 and 2011 increased significantly compared with between 2001 and 2006 (57% vs 0). CONCLUSIONS: The clinical manifestations of neonatal purulent meningitis caused by Escherichia coli are non specific. The outcome is poor. Monitoring of CRP levels is valuable for the early diagnosis of neonatal purulent meningitis. The antimicrobial resistance rates of Escherichia coli are increasing, especially to cephalosporins. The percentage of ESBLs-producing strains is increasing over the years.

[Comparative analysis of the pathogens responsible for hospital acquired and community acquired late onset neonatal septicemia].

OBJECTIVE: Late onset neonatal septicemia (systemic infection after 72 hours of life) remains a major cause of neonatal morbidity and mortality. Early treatment with appropriate antibiotics is critical since infected infants can deteriorate rapidly. The aim of this study was to review the pathogens responsible for late onset neonatal septicemia (LONS) and their antimicrobial susceptibilities in order to guide the initial selection of appropriate antibiotics for infants with suspected LONS. METHODS: A retrospective chart review of all cases with LONS seen in the neonatal intensive care unit (NICU) of Yuying Children's Hospital of Wenzhou Medical College from January 1, 2002 to December 31, 2005 was conducted. All cases were selected based on the clinical presentation and at least one positive result of blood culture. The basic clinical characteristics and the results of blood culture and antimicrobial susceptibilities were analyzed. RESULTS: A total of 102 cases with LONS were identified. Among those 102 cases, 80 were community acquired (infants admitted from home and the blood culture was done on admission) and 22 were hospital acquired (infants became sick while in the NICU and the blood culture was done prior to use of antibiotics). The clinical presentations were non-specific. Compared to the infants with community acquired LONS, infants with hospital acquired LONS were usually born more prematurely (mean gestational age 33 +/- 3 vs 39 +/- 2 wks, t = 2.255, P < 0.01), with lower weight (mean weight 1.79 +/- 0.70 vs 3.23 +/- 0.67 kg, t = 8.818, P < 0.01) and with younger age (mean age 12 +/- 6 vs 16 +/- 7 days, t = 7.581, P < 0.05). Of the 102 cases, a total of 103 strains of bacteria were isolated. Among the pathogenic bacteria isolated, the most common were coagulase-negative Staphylococcus (CoNS) (50/103, 48.5%), followed by Klebsiella pneumoniae (16/103, 15.5%). The main pathogens for community acquired LONS were Staphylococcus species and Escherichia coli. The most important pathogen responsible for hospital acquired LONS was Klebsiella pneumoniae. Most (> 80%) of the Staphylococcus especially CoNS were resistant to common antibiotics such as penicillin, erythromycin and cefazolin. Significant numbers (6/9) of Staphylococcus aureus isolated were methicillin-resistant Staphylococcus aureus (MRSA). However, all of the Staphyloccus isolates were sensitive to vancomycin. Almost all (15/16) of the Klebsiella pneumoniae isolated were multi-drug resistant due to production of extended-spectrum beta-lactamases (ESBLs). They were sensitive only to a few antibiotics such as carbapenems, aminoglycosides and quinolones. There was also one strain of vancomycin-resistant Enterococcus (VRE). Furthermore, there was no a single case of late onset neonatal sepsis due to infection with group B Streptococcus (GBS). CONCLUSIONS: The clinical manifestations of late onset neonatal sepsis are usually non-specific. GBS is not a significant pathogen responsible for community acquired LONS in the Wenzhou area. There are increasing numbers of multi-drug resistant bacterial species isolated from the newborn infants with late onset neonatal septicemia, which is most likely due the non-restricted use of antibiotics in the hospitals as well as in the communities. A routine blood culture should be taken from any newborn infant who is suspected of LONS and empirical use of appropriate antibiotics should be initiated as soon as the blood specimen for culture has been drawn. To reduce the occurrence of multi-drug resistant bacteria, the use of antibiotics especially the third generation cephalosporins in neonates should be restricted as much as possible.