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Introduction:Clinical studies have demonstrated that the initial rapid decrement in regional cerebral oxygen saturation (rSO2) level by near-infrared spectroscopy shortly after ROSC was associated with good neurological outcomes.Aim:To evaluate whether the timing of rSO2decline shortly after ROSC reflects the severity of brain injury in a well-established rat model of CA/CPR.Methods:We used a total of 85 Wister SD rats (male, 435.2±3.1 g).To produce variable severities of brain injury, CA was induced by different asphyxial times and resuscitated by finger chest compressions and mechanical ventilation. Survival time was recorded for 72 hours after ROSC. Daily neurofunctional scores (NFS) were evaluated by an investigator blinded to the experiments. Electroencephalography (EEG), immunohistochemistry, plasma IL-6, and the gene expressions of cytokines (IL-1, IL-6, TNF-α, HMGB-1) and mitochondrial fission-related proteins (Dnm1L, Fis1, Mief1) in the brain were also evaluated.Result:Animals in the 12-min CA group exhibited a longer time to EEG recovery after CA/CPR compared with the 6-min group (P<.0001), suggesting the longer CA duration induces a delayed brain electrical recovery. Time from ROSC to achieving the initial minimum rSO2value (defined as Tnadir) was significantly prolonged as CA time increased; 15.3 ± 2.3, 32.1 ± 22.1, and 44.0 ± 2.8 min in 6, 9, and 12min CA, respectively (ANOVA: P<.0001). Tnadircut-off of 24-min provided the optimal sensitivity and specificity for predicting good neurologic outcomes (NFS≥60%) at 72 hours after ROSC (AUC, 0.88; sensitivity, 89%; specificity, 86%; P<.01). Tnadirshowed a better predictive power for the good neurological outcome compared with plasma IL-6. Immunohistochemistry at 24 hours post-CA revealed that FJB-positive degenerating neurons in cortex, caudoputamen, and hippocampus were conspicuous in animals with Tnadir>24-min, but not in animals with Tnadir≤24-min. Animals with Tnadir>24 tended to have a higher gene expression level of IL-6 in the brain at 2 hours post-ROSC compared to those with Tnadir≤24. Tnadirdid not show associations with other inflammatory or mitochondrial fission markers.Conclusion:Tnadircan be a novel predictor for a good neurological outcome after CA/CPR.

Objective:We recently reported that post-resuscitation normoxic therapy attenuates oxidative stress in multiple organs and improves post-cardiac arrest (CA) organ injury, oxygen metabolism, and survival. Yet, detailed mechanisms of gene expression patterns and signaling pathways mitigated by normoxic therapy have not been elucidated. Therefore, we assessed post-resuscitation normoxic therapy-modified gene expression of oxidative stress-related signaling molecules.Methods:Rats were resuscitated from 10 minutes of asphyxial CA and divided into 2 groups: those that inhaled 100% supplemental O2(CA-FIO2 1.0) and those that inhaled 30% supplemental O2(CA-FIO2 0.3). Control groups were also prepared for comparison (control-FIO2 1.0, control-FIO2 0.3). At 2 hours after resuscitation, brain and heart tissues were collected, and mRNA purifications followed by real-time PCR measurements were performed to compare gene expression of hyperoxia-induced inflammatory and apoptosis-related signaling pathways amongst these groups.Results:In the brain, relative IL-1 beta mRNA gene expression levels, which represent inflammatory signaling pathways, increased post-CA (8.1±2.3 in CA-FIO2 1.0 and 1.0±0.4 in control-FIO2 0.3, p<0.05), but were significantly attenuated by normoxic therapy (2.3±0.2 in CA-FIO2 0.3, p<0.05). Likewise, normoxic therapy significantly reduced oxidative stress-induced inflammatory (NFKB1, TGFB1, MAPK14, TRAF6) and apoptosis-related (BAX, EGF) mRNA gene expression levels in the brain, whereas no statistical differences were detected in the heart.Conclusions:Post-CA normoxic therapy significantly attenuated the gene expression of oxidative stress-induced inflammation and apoptosis in the brain, while there were no remarkable changes in the heart. Therefore, it is inferred that the heart is more tolerant to hyperoxic injury compared to the brain.

Background:Oxygen & Glucose Deprivation (OGD) and Reperfusion (OGD-R) is considered one of the best models for studying ischemia and reperfusion injury (IRI) in vitro. Cell viability (CV) experimentation can aid in estimating cell survival after IRI insults and potential benefits from therapeutic attempts. However, the different metabolites used in various CV assays may compromise the usefulness and validity of various CV tools. We conducted two independent cell viability assays (WST-8 & ATP) after OGD and OGD-R on neurons (NE) and astrocytes (AS).Methods:NE (HT-22) and AS (C8-D1A) were subjected to 6h OGD only and 6h OGD followed by 20h Reperfusion (OGD-R). OGD is based on the combination of chemical ischemia (standard culture media depleted of glucose and other energy sources) and severe hypoxia (O2 ~1.4 to 1.8%) for 6h, while 20h reperfusion consists of readdition of standard culture media and conditions (21% O2, 5% CO2, 37°C). CV was determined by WST-8 and ATP “CellTiter-Glo 2.0” assays.Results:By WST-8 assay, OGD decreased CV in NE (Fig. 1A, P value = 0.0121), but increased CV in AS (Fig. 1A, P value = 0.0079). OGD-R decreased CV in NE and AS (Fig. 2A, P value = 0.0010, 0.0146, respectively). By ATP assay, OGD decreased CV in NE (Fig. 1B, P value <0.0001) and AS (Fig. 1C, P value <0.0001). OGD-R also decreased CV in NE (Fig. 2B, P value <0.0001) and AS (Fig. 2C, P value = 0.0001).Conclusions:The current data reveals opposite CV results after OGD in AS when comparing WST-8 and ATP assays; OGD caused CV decrease in NE but increased in AS, which constitutes a paradoxical response based on previous literature. However, OGD-R caused similar injury patterns in NE and AS from WST-8 and ATP assays. It is unknown why OGD increased CV in AS through WST-8 after OGD, however, it can potentially occur due to AS hypermetabolism leading to increased NADPH+ production, which is the main substrate in this CV estimation. We conclude that CV should be carefully assessed with multiple, independent CV assays.

Introduction:Lysophosphatidylcholine (LPC) was found to be decreased in plasma in the early phase of resuscitation after cardiac arrest (CA), including a species containing docosahexaenoic acid (LPC-DHA). Supplementing this deficiency of plasma LPC-DHA post-CA significantly attenuated brain dysfunction implicating a causative role of plasma decreased LPC-DHA for brain injury. Previous studies showed the importance of LPC-DHA as a carrier of DHA to maintain proper brain function. However, the role of LPC-DHA for brain function has not been fully understood.Objective:This study is aimed at determining the importance of maintaining proper brain LPC-DHA level via plasma supplementation to prevent brain damage after CA using human patients, animal model, and in-vitrocell studies.Methods and Results:We first evaluated associations between the plasma LPC-DHA levels and neurological outcomes using 45 post-CA patients. We then measured LPC-DHA levels and histological, biochemical, metabolic alterations in the plasma and brain after 10 min CA rat model and examined how these alterations were attenuated by supplementing LPC-DHA. Finally, we further investigated the beneficial effect of LPC-DHA using cell cultures. We found that the decreased plasma LPC-DHA was strongly associated with neurological outcomes and disappearance of difference between gray and white matter in the brain after CA in human patients. In rats, the decreased plasma LPC-DHA was associated with decreased level of brain LPC-DHA after CA, and supplementing plasma LPC-DHA normalized the brain levels of LPC-DHA and alleviated neuronal cell death, activation of astrocyte, and expression of various inflammatory and mitochondrial dysfunction genes. We also found normalized overall metabolic alterations from the untargeted metabolomics analysis. Furthermore, LPC treatment showed a similar protective effect for neurons and astrocytes in mixed primary brain cell cultures.Conclusion:The attenuation of biochemical and physiologic alterations, and the normalization of decreased brain LPC-DHA post-CA with LPC-DHA supplementation demonstrate plasma LPC-DHA is important for the maintenance of proper brain LPC-DHA levels, which is essential for preventing brain damage post-CA.