Characterization of Circulating Cold Shock Proteins FGF21 and RBM3 in a Multi-Center Study of Pediatric Cardiac Arrest.

Fibroblast Growth Factor 21 (FGF21) is a neuroprotective hormone induced by cold exposure that targets the ß-klotho co-receptor. ß-klotho is abundant in the newborn brain but decreases rapidly with age. RNA-Binding Motif 3 (RBM3) is a potent neuroprotectant upregulated by FGF21 in hypothermic conditions. We characterized serum FGF21 and RBM3 levels in patients enrolled in a prospective multi-center study of pediatric cardiac arrest (CA) via a secondary analysis of samples collected to evaluate brain injury biomarkers. Patients (n = 111) with remnant serum samples available from at least two of three available timepoints (0-24, 24-48 or 48-72 hours post-resuscitation) were included. Serum samples from 20 healthy controls were used for comparison. FGF21 was measured by Luminex and internally validated enzyme-linked immunoassay (ELISA). RBM3 was measured by internally validated ELISA. Of postarrest patients, 98 were managed with normothermia, while 13 were treated with therapeutic hypothermia (TH). FGF21 increased >20-fold in the first 24 hours postarrest versus controls (681 pg/mL [200-1864] vs. 29 pg/mL [15-51], n = 99 vs. 19, respectively, p < 0.0001, median [interquartile range]) with no difference in RBM3. FGF21 did not differ by sex, while RBM3 was increased in females versus males at 48-72 hours postarrest (1866 pg/mL [873-5176] vs. 1045 pg/mL [535-2728], n = 40 vs. 54, respectively, p < 0.05). Patients requiring extracorporeal membrane oxygenation (ECMO) postresuscitation had increased FGF21 versus those who did not at 48-72 hours (6550 pg/mL [1455-66,781] vs. 1213 pg/mL [480-3117], n = 7 vs 74, respectively, p < 0.05). FGF21 and RBM3 did not correlate (Spearman's rho = 0.004, p = 0.97). We conclude that in a multi-center study of pediatric CA patients where normothermic targeted temperature management was largely used, FGF21 was markedly increased postarrest versus control and highest in patients requiring ECMO postresuscitation. RBM3 was sex-dependent. We provide a framework for future studies examining the effect of TH on FGF21 or use of FGF21 therapy after pediatric CA.

FGF21 modulates hippocampal cold-shock proteins and CA2-subregion proteins in neonatal mice with hypoxia-ischemia.

BACKGROUND: Fibroblast growth factor 21 (FGF21) is a neuroprotectant with cognitive enhancing effects but with poorly characterized mechanism(s) of action, particularly in females. Prior studies suggest that FGF21 may regulate cold-shock proteins (CSPs) and CA2-marker proteins in the hippocampus but empirical evidence is lacking. METHODS: We assessed in normothermic postnatal day (PND) 10 female mice, if hypoxic-ischemic (HI) brain injury (25 min 8% O2/92% N2) altered endogenous levels of FGF21 in serum or in the hippocampus, or its receptor ß-klotho. We also tested if systemic administration of FGF21 (1.5 mg/kg) modulated hippocampal CSPs or CA2 proteins. Finally, we measured if FGF21 therapy altered markers of acute hippocampal injury. RESULTS: HI increased endogenous serum FGF21 (24 h), hippocampal tissue FGF21 (4d), and decreased hippocampal ß-klotho levels (4d). Exogenous FGF21 therapy modulated hippocampal CSP levels, and dynamically altered hippocampal CA2 marker expression (24 h and 4d). Finally, FGF21 ameliorated neuronal damage markers at 24 h but did not affect GFAP (astrogliosis) or Iba1 (microgliosis) levels at 4d. CONCLUSIONS: FGF21 therapy modulates CSP and CA2 protein levels in the injured hippocampus. These proteins serve different biological functions, but our findings suggest that FGF21 administration modulates them in a homeostatic manner after HI. IMPACT: Hypoxic-ischemic (HI) injury in female post-natal day (PND) 10 mice decreases hippocampal RNA binding motif 3 (RBM3) levels in the normothermic newborn brain. HI injury in normothermic newborn female mice alters serum and hippocampal fibroblast growth factor 21 (FGF21) levels 24 h post-injury. HI injury in normothermic newborn female mice alters hippocampal levels of N-terminal EF-hand calcium binding protein 2 (NECAB2) in a time-dependent manner. Exogenous FGF21 therapy ameliorates the HI-mediated loss of hippocampal cold-induced RNA-binding protein (CIRBP). Exogenous FGF21 therapy modulates hippocampal levels of CA2-marker proteins after HI.

Hypoxia-ischemia-mediated effects on neurodevelopmentally regulated cold-shock proteins in neonatal mice under strict temperature control.

BACKGROUND: Neonates have high levels of cold-shock proteins (CSPs) in the normothermic brain for a limited period following birth. Hypoxic-ischemic (HI) insults in term infants produce neonatal encephalopathy (NE), and it remains unclear whether HI-induced pathology alters baseline CSP expression in the normothermic brain. METHODS: Here we established a version of the Rice-Vannucci model in PND 10 mice that incorporates rigorous temperature control. RESULTS: Common carotid artery (CCA)-ligation plus 25 min hypoxia (8% O2) in pups with targeted normothermia resulted in classic histopathological changes including increased hippocampal degeneration, astrogliosis, microgliosis, white matter changes, and cell signaling perturbations. Serial assessment of cortical, thalamic, and hippocampal RNA-binding motif 3 (RBM3), cold-inducible RNA binding protein (CIRBP), and reticulon-3 (RTN3) revealed a rapid age-dependent decrease in levels in sham and injured pups. CSPs were minimally affected by HI and the age point of lowest expression (PND 18) coincided with the timing at which heat-generating mechanisms mature in mice. CONCLUSIONS: The findings suggest the need to determine whether optimized therapeutic hypothermia (depth and duration) can prevent the age-related decline in neuroprotective CSPs like RBM3 in the brain, and improve outcomes during critical phases of secondary injury and recovery after NE. IMPACT: The rapid decrease in endogenous neuroprotective cold-shock proteins (CSPs) in the normothermic cortex, thalamus, and hippocampus from postnatal day (PND) 11-18, coincides with the timing of thermogenesis maturation in neonatal mice. Hypoxia-ischemia (HI) has a minor impact on the normal age-dependent decline in brain CSP levels in neonates maintained normothermic post-injury. HI robustly disrupts the expected correlation in RNA-binding motif 3 (RBM3) and reticulon-3 (RTN3). The potent neuroprotectant RBM3 is not increased 1-4 days after HI in a mouse model of neonatal encephalopathy (NE) in the term newborn and in which rigorous temperature control prevents the manifestation of endogenous post-insult hypothermia.

Serum levels of the cold stress hormones FGF21 and GDF-15 after cardiac arrest in infants and children enrolled in single center therapeutic hypothermia clinical trials.

OBJECTIVE: Fibroblast Growth Factor 21 (FGF21) and Growth Differentiation Factor-15 (GDF-15) are putative neuroprotective cold stress hormones (CSHs) provoked by cold exposure that may be age-dependent. We sought to characterize serum FGF21 and GDF-15 levels in pediatric cardiac arrest (CA) patients and their association with use of therapeutic hypothermia (TH). METHODS: Secondary analysis of serum samples from clinical trials. We measured FGF21 and GDF-15 levels in pediatric patients post-CA and compared levels to both pediatric intensive care (PICU) and healthy controls. Post-CA, we compared normothermia (NT) vs TH (33 °C for 72 h) treated cohorts at < 24 h, 24 h, 48 h, 72 h, and examined the change in CSHs over 72 h. We also assessed association between hospital mortality and initial levels. RESULTS: We assessed 144 samples from 68 patients (27 CA [14 TH, 13 NT], 9 PICU and 32 healthy controls). Median initial FGF21 levels were higher post-CA vs. healthy controls (392 vs. 40 pg/mL, respectively, P < 0.001). Median GDF-15 levels were higher post-CA vs. healthy controls (7,089 vs. 396 pg/mL, respectively, P < 0.001). In the CA group, the median change in FGF21 from PICU day 1-3 (after 72 h of temperature control), was higher in TH vs. NT (231 vs. -20 pg/mL, respectively, P < 0.05), with no difference in GDF-15 over time. Serum GDF-15 levels were higher in CA patients that died vs. survived (19,450 vs. 5,337 pg/mL, respectively, P < 0.05), whereas serum FGF21 levels were not associated with mortality. CONCLUSION: Serum levels of FGF21 and GDF-15 increased after pediatric CA, and FGF21 appears to be augmented by TH.

Hippocampal and Prefrontal Cortical Brain Tissue Levels of Irisin and GDF15 Receptor Subunits in Children.

Cold-stress hormones (CSHs) stimulate thermogenesis and have direct neuroprotective effects on the brain. The obligatory receptor components of two new CSHs (irisin and growth differentiation factor-15 [GDF15]) were recently discovered. Irisin binds integrin-αV/ß5 heterodimers while GDF-15 binds to the orphan receptor glial cell-derived neurotrophic factor (GDNF) family receptor α-like (GFRAL). In addition, integrin-αV/ß5 was just identified as the key receptor mediating Zika virus infection in the CNS. We measured integrin-αV, integrin-ß5, and GFRAL protein levels across 78 high-quality human male/female brain tissues in infants, toddlers, preschoolers, adolescent, and adults-providing the most robust analysis to date on their levels in the human cortex and hippocampus. We report that integrin-αV was detected at all ages in the prefrontal cortex with levels greatest in adults. Integrin-αV was also detected in the hippocampus in all age groups. In contrast, integrin-ß5 was detected in cortex and hippocampus largely restricted to infants. Co-expression of integrin-αV/ß5 in the human infant hippocampus and cortex suggests the possibility that irisin has a more robust effect on the developing vs. the adult brain and may have implications for Zika virus infection in infants and young children.

Beyond Ussing's chambers: contemporary thoughts on integration of transepithelial transport.

In the mid-20th century, Hans Ussing developed a chamber that allowed for the simultaneous measurement of current and labeled probe flux across epithelia. Using frog skin as a model, Ussing used his results to propose mechanisms of transcellular Na(+) and K(+) transport across apical (exterior/luminal) and basolateral (interior) membranes that is essentially unchanged today. Others took advantage of Ussing's chambers to study mucosal tissues, including bladder and intestines. It quickly became clear that, in some tissues, passive paracellular flux, i.e., across the tight junction, was an important component of overall transepithelial transport. Subsequent work demonstrated that activation of the apical Na(+)-glucose cotransporter SGLT1 regulated paracellular permeability such that intestinal paracellular transport could coordinate with and amplify transcellular transport. Intermediates in this process include activation of p38 MAPK, the apical Na(+)/H(+) exchanger NHE3, and myosin light chain kinase (MLCK). Investigators then focused on these processes in disease. They found that TNF induces barrier dysfunction via MLCK activation and downstream caveolin-1-dependent endocytosis of the tight junction protein occludin. TNF also inhibited NHE3, and both barrier loss and PKCα-dependent NHE3 inhibition were required for TNF-induced acute diarrhea, emphasizing the interplay between transcellular and paracellular transport. Finally, studies using immune-mediated inflammatory bowel disease models showed that mice lacking epithelial MLCK were initially protected, but became ill as epithelial damage progressed and provided a tight junction-independent means of barrier loss. None of these advances would have been possible without the insights provided by Ussing and others using Ussing's ingenious, and still useful, chambers.