Changes in waiting time, need for mechanical circulatory support and outcomes in paediatric heart transplant recipients.

AIMS: Heart transplantation is a well-established treatment for end-stage heart failure in paediatric patients, demonstrating excellent long-term outcomes. METHODS: This retrospective study analyses 35 years of data on 244 paediatric recipients (<18 years) at a single institution from 1986 to 2022. The analysis explores changes in diagnoses, survival, waiting times and mechanical circulatory support (MCS) over three decades (1991-2000, 2001-2010 and 2011-2020). RESULTS: Survival outcomes significantly improved over the study period, with 1-year survival rates increasing from 79.3% (1991-2000) to 92.3% (2011-2020, P = 0.041). The median overall survival was 18.0 years, and median conditional survival to 1 year post-transplant was 20.9 years. Survival differences were noted among age groups, with infants under 1 year of age experiencing higher early mortality, and adolescents (aged 11-17 years) facing increased long-term risks, possibly linked to adult donor characteristics. Waiting times for heart transplantation increased, especially for younger age groups (0-5 and 6-10 years), reflecting the growing demand for donor organs. The use of MCS, including ventricular assist devices (VAD), surged, with 67% of recipients in the most recent decade receiving pre-transplant VAD support. Importantly, despite prolonged waiting times and increased VAD use, overall survival continued to be favourable. CONCLUSIONS: Early and long-term results after paediatric heart transplantation have been continuously improving over the past decades. Despite an increased demand for donor organs and the growing reliance on VAD as bridge to transplantation, post-transplantation survival is not compromised.

Long-term experience using CNI-free immunosuppression in selected paediatric heart transplant recipients.

BACKGROUND: CNI-free immunosuppression with conversion to mTORi-based immunosuppression has been demonstrated to reduce CNI-toxicity and to exhibit anti-proliferative properties. However, the experience of CNI-free immunosuppression in paediatric heart transplantation is limited. METHODS: A retrospective analysis was conducted of 129 paediatric heart transplants performed between 1997 and 2015. Fifteen patients with clinically indicated conversion from CNI-based to CNI-free immunosuppression were identified. Survival data, rejection episodes, renal function, post-transplantation lymphoproliferative disorder and CAV, including examination with OCT were analysed. RESULTS: Immunosuppression conversion was successful in all patients. Fourteen of 15 patients (93%) are currently living with good graft function. Median post-transplant survival was 15 years (range, 5-23 years), and median follow-up since conversion was 6 years (range, 1-11 years). Mild (grade 1R) ACR was present in three patients after discontinuation of CNIs. The recovery of renal function with a significant increase in eGFR was observed at 1 and 3 years after conversion. No patient had angiographic signs of macroscopic CAV according to the current ISHLT classification; however, OCT showed the signs of angiographically silent CAV in all patients. CAV did not progress in any patient, implying CAV was stabilised by mTORi-based CNI-free immunosuppression. CONCLUSIONS: CNI-free immunosuppression based on mTORis is a safe and appropriate strategy for maintenance therapy in selected paediatric patients, significantly improves renal function and stabilises CAV. OCT revealed early development of angiographically silent CAV.

Education of Children With Cyanotic Congenital Heart Disease After Neonatal Cardiac Surgery.

BACKGROUND: The aim of the study was to evaluate the educational achievement of patients diagnosed with univentricular heart physiology (UVHP) or transposition of the great arteries (TGA) after neonatal cardiac surgery. METHODS: An exploratory online survey was performed with patients registered with the National Register for Congenital Heart Defects in Germany. For this publication, a subgroup analysis was conducted among patients diagnosed with TGA (n = 173; 36.3%) and UVHP (n = 304; 63.7%). RESULTS: Median age of the sample at school enrollment was 6 years (range, 5-8 years). The majority were enrolled at a standard elementary school (n = 368 of 477; 77.1%), although patients with UVHP were enrolled significantly more often at a special needs school (n = 52 of 304; 17.1%, TGA patients n = 11/ of 173; 6.4%, P < .001). A total of 45.8% (n = 66 of 144) of the patients graduated with a high school diploma. A substantial number of patients had been diagnosed with behavioral or learning disorders (TGA patients n = 63 of 173 [36.4%], UVHP patients n = 148 of 304 [48.7%]) and received early supportive therapy or remedial teaching before (TGA patients n = 89 of 173 [51.4%], UVHP patients n = 209 of 304 [68.8%]) and/or during their school careers (TGA patients n = 54 of 173 [31.2%], UVHP patients n = 120 of 304 [39.5%]). CONCLUSIONS: A large proportion of patients who underwent neonatal cardiac surgery graduated with a high school diploma. These results are of great importance to congenital heart defect patients, affected families, and treating physicians. Nevertheless, study participants, especially patients with UVHP, face some academic challenges. We conclude that long-term follow-up examinations and regular developmental assessments may be beneficial.

A Prospective Clinical Trial Measuring the Effects of Cardiopulmonary Bypass Under Mild Hypothermia on the Inflammatory Response and Regulation of Cold-Shock Protein RNA-Binding Motif 3.

Therapeutic hypothermia during cardiac surgery has been widely used for neuroprotection and to attenuate the systemic inflammatory response due to cardiopulmonary bypass (CPB). Experimental data suggest that cold-shock protein RNA-binding motif 3 (RBM3), which is induced in response to hypothermia, plays a key role in hypothermia-induced organ protection. To date, investigation on RBM3 has been performed exclusively in vitro or in animal models, and the detection and regulation of RBM3 in human blood has not been investigated until now. The aim of this study was to investigate the level of RBM3 protein and cytokine expression profile involved in the inflammatory response in patients with congenital heart disease undergoing cardiac surgery involving CPB and therapeutic hypothermia. A single-center prospective trial with 23 patients undergoing cardiac surgery with CPB was performed. RBM3 protein was quantified in blood serum samples collected from patients and healthy individuals employing a new developed enzyme-linked immunosorbent assay. Cytokine levels were analyzed from dry blood spot samples using a Quanterix Simoa Immunoassay. For the first time, RBM3 protein was detected in blood samples of patients with congenital heart disease undergoing cardiac surgery. Hereby, RBM3 protein concentrations were significantly elevated in patients after cardiac surgery with CPB and mild hypothermia as compared with pre-surgery levels. Moreover, a complex immune reaction with significant induction of pro-inflammatory cytokines (interleukin [IL]-1 beta, IL-6, IL-8, IL-16, IL-18, monocyte chemotactic protein 1, CC-chemokine ligand [CCL]3, CCL4, intercellular adhesion molecule-1) in response to CPB was detected. Significantly elevated vascular endothelial growth factor and matrix metallopeptidase 3 concentrations reflecting ischemia/reperfusion-induced injury were observed 24 hours after weaning from CPB. The use of CPB is still associated with a complex inflammatory response. RBM3 protein is measurable in blood samples of patients with significantly higher concentrations after cardiac surgery with CPB and mild-to-moderate hypothermia. RBM3 is a new candidate as a biomarker for therapeutic hypothermia and a possible new therapeutic target for organ protection.

RBM3 and CIRP expressions in targeted temperature management treated cardiac arrest patients-A prospective single center study.

BACKGROUND: Management of cardiac arrest patients includes active body temperature control and strict prevention of fever to avoid further neurological damage. Cold-shock proteins RNA-binding motif 3 (RBM3) and cold inducible RNA-binding protein (CIRP) expressions are induced in vitro in response to hypothermia and play a key role in hypothermia-induced neuroprotection. OBJECTIVE: To measure gene expressions of RBM3, CIRP, and inflammatory biomarkers in whole blood samples from targeted temperature management (TTM)-treated post-cardiac arrest patients for the potential application as clinical biomarkers for the efficacy of TTM treatment. METHODS: A prospective single center trial with the inclusion of 22 cardiac arrest patients who were treated with TTM (33°C for 24 hours) after ROSC was performed. RBM3, CIRP, interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and inducible nitric oxide synthase (iNOS) mRNA expressions were quantified by RT-qPCR. Serum RBM3 protein concentration was quantified using an enzyme-linked immunosorbent assay (ELISA). RESULTS: RBM3 mRNA expression was significantly induced in post-cardiac arrest patients in response to TTM. RBM3 mRNA was increased 2.2-fold compared to before TTM. A similar expression kinetic of 1.4-fold increase was observed for CIRP mRNA, but did not reached significancy. Serum RBM3 protein was not increased in response to TTM. IL-6 and MCP-1 expression peaked after ROSC and then significantly decreased. iNOS expression was significantly increased 24h after return of spontaneous circulation (ROSC) and TTM. CONCLUSIONS: RBM3 is temperature regulated in patients treated with TTM after CA and ROSC. RBM3 is a possible biomarker candidate to ensure the efficacy of TTM treatment in post-cardiac arrest patients and its pharmacological induction could be a potential future intervention strategy that warrants further research.

First steps to a clinical research unit for developmental research in paediatric cardiology: conception and progress of the LEADER project (Long Term Early Development Research) in CHD.

OBJECTIVE: We developed the Long-term Early Development Research (LEADER) project to investigate the development of children with CHD and/or after cardiopulmonary resuscitation. Both populations are at risk for delays in motor, cognitive, and language development. However, few studies to date have investigated the longitudinal development in these children. METHODS: To establish a clinical research unit, we planned three studies: a cross-sectional study in children after cardiopulmonary resuscitation (LEADER-REA Pilot Study), a longitudinal study in children after cardiopulmonary resuscitation, with a focus on evaluating various biomarkers as predictors for developmental outcome (LEADER-CPR study), and a longitudinal study in children with ventricular septal defect, tetralogy of Fallot, or transposition of the great arteries after cardiac surgery (LEADER-CHD study). RESULTS: Implementation of all three LEADER studies was successful and study protocols were conducted as planned. Findings from the LEADER-REA Pilot study have been recently published and data collection for both prospective trials is ongoing. Descriptive analysis of the first 20 assessments of the LEADER-CHD study showed no severe deficits in overall cognitive, motor, and language developments in the children. CONCLUSIONS: Children with CHD and/or after cardiopulmonary resuscitation are at risk for developmental delay. Therefore, a detailed developmental assessment is necessary as a pre-requisite for individual developmental support. Our LEADER project has been shown to be feasible in a clinical setting and is the first step towards the establishment of a clinical research unit in our clinic with a focus on longitudinal research.

Developmental Outcome in Infants with Cardiovascular Disease After Cardiopulmonary Resuscitation: A Pilot Study.

Unfavorable neurological outcome in children after cardiopulmonary resuscitation in infancy is frequent. However, few studies have investigated the development of these patients using comprehensive developmental tests and the feasibility of the Bayley Scales of Infant Development, 3rd Edition (BSID-III) has not been reported for this population. In this cross-sectional pilot study, we assessed the cognitive, language, and motor development in infants after cardiopulmonary resuscitation of ≥ 5 min with the BSID-III at the age of 12 or 24 months, depending on recruitment age. For analysis, 11 patients with in-hospital (n = 8) and out-of-hospital (n = 3) cardiac arrest were included. BSID-III results could not be quantified in three patients because of visual/hearing and/or motor impairment. In patients with quantifiable scores, 50.0% scored average in composite BSID-III scores, while the other 50.0% showed developmental delays, scoring distinctly below average. We conclude that the BSID-III is feasible for developmental assessment in the majority of the study population, but the use of instruments suitable for hearing/visually impaired and/or severely disabled infants is crucial to avoid biased results. Accurate characterization of developmental deficits is important to facilitate early identification and therapy of deficits.

Educational achievement of children with congenital heart disease: Promising results from a survey by the German National Register of Congenital Heart Defects.

BACKGROUND: Survival rates of children with congenital heart disease (CHD) have increased significantly in the decade. There is now increased interest in the long-term outcome and quality of life of these children. AIMS: To assess the educational achievement of patients with CHD in Germany. STUDY DESIGN: Cross-sectional study using an online survey. The recruitment of study participants was carried out via the database of the German National Register for Congenital Heart Defects (NRCHD). SUBJECTS: Patients born between 1992 and 2011 were enrolled in the study. For 2609 study participants (female = 1870 (71.7%); 1072 (41.1%) patients; 1537 (58.9%) parents), who participated in the survey, detailed information regarding the underlying CHD diagnosis and clinical data was available. OUTCOME MEASURES: Age at enrollment, secondary school form, school year repetition, school degree. RESULTS: The large majority of study participants were enrolled at a conventional elementary school (83.4%) and started school at the age of 6 years or below (73.3%). In total 45.7% of graduated study participants graduated with the qualification necessary to study at any university. In terms of analysis of the different CHD severity subgroups 57.3% of patients with a mild CHD, 47.5% with a moderate CHD and only 35.1% suffering from a severe CHD attained a high school diploma. CONCLUSIONS: In our study, the majority of participating CHD patients had a standard school career. These initial results are of great importance to affected families and treating physicians as they show that, in general, a normal school career is possible for all CHD patients.

Immunodepression after CPB: Cytokine dynamics and clinics after pediatric cardiac surgery - A prospective trial.

BACKGROUND: Corrective surgery for congenital heart defects is known to trigger a severe immune reaction. There has been extensive research on the effects of inflammation after cardiopulmonary bypass (CPB). Interestingly, monocytes are observed to be non-responsive to stimulation with lipopolysaccharide (LPS) under these conditions, indicating a state of immunodepression, which lays the ground for second hit infections after cardiosurgery with CPB. OBJECTIVES: The aim of this prospective study was to analyze immunodepression after pediatric cardiopulmonary bypass and to differentiate the effects of monocytic anergy on postoperative outcome. METHODS: In a prospective trial, we quantified the immune responses in 20 pediatric patients (median age 4.9months, range 2.3-38.2months; median weight 7.2kg, range 5.2-11.7kg) with congenital ventricular septal defect undergoing heart surgery with CPB. Ex vivo LPS-induced protein expression of IFN-γ, IL-1ß, IL-1Ra, IL-6, IL-8, IL-10, IL-12, IL-17, TNF-α, and MCP-1 was measured before (T1), immediately after (T2) and 4h after (T3) cardiopulmonary bypass surgery using Luminex technology. RESULTS: The innate immune system responds to CPB with an almost complete depression of monocytic function. Inflammatory IL-12, TNF-α, IL-1ß, IL-6, IL-8 and IFN-y are completely suppressed. IL-10, IL-1Ra and MCP-1 are still produced during suppression with IL-1Ra being overly secreted during reversion. Suppression of TNF-α expression after LPS-stimulation correlates closely with longer mechanical ventilation time (r=-0.619, p=0.004). CONCLUSION: Cardiosurgery with CPB causes a state of immunodepression making pediatric patients more vulnerable to second hit infections. MCP-1, IL-10, and IL-1Ra play an important role in monocyte recovery, eventually permitting new therapeutic options for controlling immunodepression and inflammation. Standardized glucocorticoid therapy should be evaluated carefully for each individual patient.

Educational level and employment status in adults with congenital heart disease.

Purpose Through this study we aimed to assess the educational level and employment status of adults with CHD in Germany. METHODS: Data were acquired from an online survey carried out in 2015 by the German National Register for Congenital Heart Defects. A total of 1458 adults with CHD participated in the survey (response rate: 37.6%). For 1198 participants, detailed medical information, such as main cardiac diagnosis and information from medical reports, was available. RESULTS: Of the participants surveyed (n=1198), 54.5% (n=653) were female, and the mean age was 30 years. The majority of respondents (59.4%) stated that they had high education levels and that they were currently employed (51.1%). Patients with simple CHD had significantly higher levels of education (p<0.001) and were more likely to be employed (p=0.01) than were patients with complex CHD. CONCLUSIONS: More than half of the participants had high education levels and the majority were employed. The association between CHD and its severity and individuals' educational attainment should be investigated more closely in future studies.

Dynamics in prevalence of Down syndrome in children with congenital heart disease.

We assessed the dynamics in the prevalence of children with congenital heart disease (CHD) and Down syndrome in Germany with regard to phenotype, severity, and gender. Data from patients with CHD and Down syndrome born between 1980 and 2014 were analyzed, who are registered with the German National Register for Congenital Heart Defects. One thousand six hundred eighteen CHD patients with Down syndrome were identified. The prevalence of children born with both Down syndrome and CHD was constant from 2005 to 2009 but increased from 2010 to 2014. Regarding CHD groups, complex and simple lesions have become more equal since 2005. The number of simple lesions with shunt has a peak prevalence in the period of 2010-2014. Atrioventricular septal defect was the most common CHD phenotype, but temporal changes were found within the group of CHD phenotypes over the observation period. CONCLUSION: Our findings suggest a growing number of CHD and Down syndrome, which may be the result of improved medical management and progress in educational, social, and financial support. This development is noteworthy as it adds new aspects to present discussions in the media and political settings. What is known: • Congenital heart disease is regarded to be the most important clinical phenomenon in children with Down syndrome, due to its significant impact on morbidity and mortality. • New developments in prenatal diagnostic and therapy management of congenital heart disease continue to influence the number of patients diagnosed with congenital heart disease and Down syndrome. What is New: • This study provides essential data giving the first overview of the dynamics in the prevalence of congenital heart disease and Down syndrome over an extended length of time up to 2015 in a large patient cohort, taking recent developments into account. • Our data suggest a growing prevalence of congenital heart disease and Down syndrome, which may be the result of improved medical management for Down syndrome patients and progress in educational, social, and financial support for their families; this development is noteworthy as it adds new aspects to the present discussion in the media and political settings.

Long-term early development research in congenital heart disease (LEADER-CHD): a study protocol for a prospective cohort observational study investigating the development of children after surgical correction for congenital heart defects during the first 3 years of life.

INTRODUCTION: Congenital heart disease (CHD) is the most common birth defect. Studies on the development of children with CHD point towards deficits in motoric, cognitive and language development. However, most studies are cross-sectional and there is a gap in the knowledge concerning developmental trajectories, risk and protective factors and a lack of research concerning environmental predictors. Specifically, no studies have so far considered the importance of early caregiving experiences and child temperament for the development of children with CHD. METHODS: In a single-centre prospective cohort study, cognitive, motoric and language development of 180 children after corrective surgery for a simple transposition of the great arteries (TGA), tetralogy of Fallot (TOF) or ventricular septal defect (VSD) will be assessed at ages 12, 24 and 36 months with the Bayley Scales of Infant Development 3rd Edition (BSID-III). At age 12 months, a free-play video observation will be conducted to investigate the relationship between primary caregiver and child, and child temperament will be assessed with the Infant Behavior Questionnaire-Revised Short Version. Medical information will be obtained from patient records and demographic information via questionnaires. ANALYSIS: Frequency and severity of developmental delays will be reported descriptively. Differences between groups (TGA, TOF, VSD) will be subjected to repeated-measures analysis across time points. Multiple regressions will be applied for the analysis of predictors at each time point. For the analysis of differential developmental trajectories, mixed-model analysis will be applied. ETHICS AND DISSEMINATION: The study has been approved by the local medical ethics committee. Written informed consent will be obtained from all participants. Parents have the option to be debriefed about BSID-III results after each assessment and about the study results after project completion. Results will be disseminated in peer-reviewed journals and presented at conferences. TRIAL REGISTRATION NUMBER: DRKS00011006; Pre-results.

Changing prevalence of severe congenital heart disease: Results from the National Register for Congenital Heart Defects in Germany.

OBJECTIVE: To assess the prevalence of congenital heart disease (CHD) in Germany in relation to phenotypes, severity and gender. DESIGN: Cross-sectional registry study. SETTING: We analyzed data from patients with CHD born between 1996 and 2015. PATIENTS: A total of 26 630 patients, registered with the NRCHD, were born between 1996 and 2015. 10 927 patients were excluded from the current analysis due to prior registration with the NRCHD under the German PAN Prevalence Study, which showed a potential bias in the inclusion of this patient population (proportion of mild cardiac lesions was comparatively high due to improved diagnostic capability for earlier identifying minor lesions). At least 15 703 patients with demographic data and detailed medical information were included in the current study. INTERVENTIONS: None. OUTCOME MEASURES: Prevalence of CHD in Germany differentiated into gender, severity, and phenotype. RESULTS: In total, 15 703 patients with CHD (47.1% female) were included in this study. The five most common phenotypes were found to be ventricular septal defect (19.2%), atrial septal defect (13.0%), Tetralogy of Fallot (9.3%), univentricular heart (9.4%), and coractation of the aortae (7.0%). The prevalence of CHD in regard to severity changed over the duration of the observation period. From 1996 to 2007, the number of simple CHD rose steadily (P < .001), whereas the number of severe CHD has grown significantly since 2008/2009 (P < .001). In regard to gender, the prevalence of simple CHD was higher in females, whereas complex lesions were more common in males (P < .001). CONCLUSIONS: Our study shows a growing number of registered severe CHD in the recent decade in Germany. This development is noteworthy as it implicates a growing demand for first intensive hospital care, expert pediatric cardiologic aftercare, and consequently higher economic impact for this patient population.

Neuroprotection via RNA-binding protein RBM3 expression is regulated by hypothermia but not by hypoxia in human SK-N-SH neurons.

OBJECTIVE: Therapeutic hypothermia is an established treatment for perinatal asphyxia. Yet, many term infants continue to die or suffer from neurodevelopmental disability. Several experimental studies have demonstrated a beneficial effect of mild-to-moderate hypothermia after hypoxic injury, but the understanding of hypothermia-induced neuroprotection remains incomplete. In general, global protein synthesis is attenuated by hypothermia, but a small group of RNA-binding proteins including the RNA-binding motif 3 (RBM3) is upregulated in response to cooling. The aim of this study was to establish an in vitro model to investigate the effects of hypoxia and hypothermia on neuronal cell survival, as well as to examine the kinetics of concurrent cold-shock protein RBM3 gene expression. METHODS: Experiments were performed by using human SK-N-SH neurons exposed to different oxygen concentrations (21%, 8%, or 0.2% O2) for 24 hours followed by moderate hypothermia (33.5°C) or normothermia for 24, 48, or 72 hours. Cell death was determined by quantification of lactate dehydrogenase and neuron-specific enolase releases into the cell cultured medium, and cell morphology was assessed by using immunofluorescence staining. The regulation of RBM3 gene expression was assessed by reverse transcriptase-quantitative polymerase chain reaction and Western blot analysis. RESULTS: Exposure to hypoxia (0.2% O2) for 24 hours resulted in significantly increased cell death in SK-N-SH neurons, whereas exposure to 8% O2 had no significant impact on cell viability. Post-hypoxia treatment with moderate hypothermia for 48 or 72 hours rescued the neurons from hypoxia-induced cell death. Moreover, exposure to severe hypoxia led to observable cell swelling, which was also attenuated by moderate hypothermia. Finally, moderate hypothermia but not hypoxia led to the induction of RBM3 expression on both transcriptional and translational levels. CONCLUSION: Moderate hypothermia protects neurons from hypoxia-induced cell death. The expression of the cold-shock protein RBM3 is induced by moderate hypothermia and could be one possible mediator of hypothermia-induced neuroprotection.

Moderate therapeutic hypothermia induces multimodal protective effects in oxygen-glucose deprivation/reperfusion injured cardiomyocytes.

OBJECTIVE: Therapeutic hypothermia has been shown to attenuate myocardial cell death due to ischemia/reperfusion injury. However, cellular mechanisms of cooling remain to be elucidated. Especially during reperfusion, mitochondrial dysfunction contributes to cell death by releasing apoptosis inductors. The aim of the present study was to investigate the effects of moderate therapeutic hypothermia (33.5°C) on mitochondrial mediated apoptosis in ischemia/reperfusion-injured cardiomyocytes. METHODS: Ischemic injury was simulated by oxygen-glucose deprivation for 6h in glucose/serum-free medium at 0.2% O2 in mouse atrial HL-1 cardiomyocytes. Simulation of reperfusion was achieved by restoration of nutrients in complete supplemented medium and incubation at 21% O2. Early application of therapeutic hypothermia, cooling during the oxygen-glucose deprivation phase, was initiated after 3h of oxygen-glucose deprivation and maintained for 24h. Mitochondrial membrane integrity was assessed by cytochrome c and AIF protein releases. Furthermore, mitochondria were stained with MitoTracker Red and intra-cellular cytochrome c localization was visualized by immunofluorescence staining. Moreover, anti-apoptotic Bcl-2 and Hsp70 as well as phagophore promoting LC3-II protein expressions were analyzed by Western-blot analysis. RESULTS: Therapeutic hypothermia initiated during oxygen-glucose deprivation significantly reduced mitochondrial release of cytochrome c and AIF in cardiomyocytes during reperfusion. Secondly, anti-apoptotic Bcl-2/Bax ratio and Hsp70 protein expressions were significantly upregulated due to hypothermia, indicating an inhibition of both caspase-dependent and -independent apoptosis. Furthermore, cardiomyocytes treated with therapeutic hypothermia showed increased LC3-II protein levels associated with the mitochondria during the first 3h of reperfusion, indicating the initiation of phagophores formation and sequestration of presumably damaged mitochondrion. CONCLUSION: Early application of therapeutic hypothermia effectively inhibited cardiomyocyte cell death due to oxygen-glucose deprivation/reperfusion-induced injury via multiple pathways. As hypothermia preserved mitochondrial membrane integrity, which resulted in reduced cytochrome c and AIF releases, induction of both caspase-dependent and -independent apoptosis was minimized. Secondly, cooling attenuated intrinsic apoptosis via Hsp70 upregulation and increasing anti-apoptotic Bcl-2/Bax ratio. Moreover, therapeutic hypothermia promoted mitochondrial associated LC3-II during the early phase of reperfusion, possibly leading to the sequestration and degradation of damaged mitochondrion to attenuate the activation of cell death.

Effects of moderate and deep hypothermia on RNA-binding proteins RBM3 and CIRP expressions in murine hippocampal brain slices.

Therapeutic hypothermia has emerged as an effective neuroprotective therapy for cardiac arrest survivors. There are a number of purported mechanisms for therapeutic hypothermia, but the exact mechanism still remains to be elucidated. Although hypothermia generally down-regulates protein synthesis and metabolism in mammalian cells, a small subset of homologous (>70%) cold-shock proteins (RNA-binding motif protein 3, RBM3 and cold-inducible RNA-binding protein, CIRP) are induced under these conditions. In addition, RBM3 up-regulation in neuronal cells has recently been implicated in hypothermia-induced neuroprotection. Therefore, we compared the effects of moderate (33.5°C) and deep (17°C) hypothermia with normothermia (37°C) on the regulation of RBM3 and CIRP expressions in murine organotypic hippocampal slice cultures (OHSC), hippocampal neuronal cells (HT-22), and microglia cells (BV-2). Moderate hypothermia resulted in significant up-regulation of both RBM3 and CIRP mRNA in murine OHSC, but deep hyporthermia did not. RBM3 protein regulation was also significantly up-regulated by 33.5°C, but no significant up-regulation of CIRP protein was observed in the OHSC. Additionally, OHSC exposed to 17°C for 24h were positive for Propidium Iodide (PI) immunostaining, indicating the onset of cell death. Similarly, RBM3 gene expression in a HT-22 neuronal cells mono-culture and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were also up-regulated at 33.5°C but only in the co-culture at 17°C. No significant up-regulation of RBM3 nor CIRP gene expression were observed in a BV-2 mono-culture at either temperature. We observed that RBM3 mRNA and protein expressions in murine OHSC, as well as in mono-culture of HT-22 neuronal cells and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were significantly up-regulated by exposure to moderate hypothermia. These findings further support the implication of RBM3 as a potential effector for hypothermia-induced neuroprotection.