Effect of Concurrent Src Kinase Inhibition with Short-Duration Hypothermia on Ca²⁺/Calmodulin Kinase IV Activity and Neuropathology after Hypoxia-Ischemia in the Newborn Swine Brain

Abstract

Background: Hypoxia-ischemia (HI) results in increased activation of Ca²⁺/calmodulin kinase IV (CaM kinase IV) mediated by Src kinase. Therapeutic hypothermia ameliorates neuronal injury in the newborn. Hypothesis: Inhibition of Src kinase concurrently with hypothermia further attenuates the hypoxia-induced increased activation of CaM kinase IV compared with hypothermia alone. Design/Methods: Ventilated piglets were exposed to HI, received saline or a selective Src kinase inhibitor (PP2), and were cooled to 33°C. Neuropathology, adenosine triphosphate (ATP) and phosphocreatine (PCr) concentrations, and CaM kinase IV activity were determined. Results: The neuropathology mean score (mean ± SD) was 0.4 ± 0.43 in normoxia-normothermia (p < 0.05 vs. hypoxia-normothermia), 3.5 ± 0.89 in hypoxia-normothermia (p < 0.05 vs. normoxia-normothermia), 0.7 ± 0.73 in hypoxia-hypothermia (p < 0.05 vs. normoxia-normothermia), and 0.5 ± 0.70 in normoxia-hypothermia (p < 0.05 vs. hypoxia-normothermia). The CaM kinase IV activity in cerebral tissue (pmol Pi/mg protein/min; mean ± SD) was 2,002 ± 729 in normoxia-normothermia, 1,704 ± 18 in normoxia-hypothermia, 6,017 ± 2,510 in hypoxia-normothermia, 4,104 ± 542 in hypoxia-hypothermia (p < 0.05 vs. normoxia-hypothermia), and 2,165 ± 415 in hypoxia-hypothermia with PP2 (p < 0.05 vs. hypoxia-hypothermia). The hypoxic groups with and without hypothermia or Src kinase inhibitor were comparable in the levels of ATP and PCr, indicating that they were similar in their degree of energy failure prior to treatments. Hypothermia or Src kinase inhibitor (PP2) did not restore the ATP and PCr levels. Conclusions: Hypothermia and Src kinase inhibition attenuated apoptotic cell death and improved neuropathology after hypoxia. The combination of short-duration hypothermia with Src kinase inhibition following hypoxia further attenuates the increased activation of CaM kinase IV compared to hypothermia alone in the newborn swine brain.

Transcriptomic profile of adverse neurodevelopmental outcomes after neonatal encephalopathy

“…This latter is responsible for the neuronal apoptosis following cerebral hypoxia and is a crucial point of action of therapeutic hypothermia neuroprotective effects 21 – 23 . A recent study has underlined the importance of this cascade, showing that its synergic downregulation together with therapeutic hypothermia, potentiates neuroprotection with reduced cell death and improved neuropathology in a piglet hypoxia model 24 . Of note, CaMKIV levels have been found to increase with the increase in the degree of cerebral tissue hypoxia assessed by cerebral tissue high energy phosphates 25 .…”

Epidermal Growth Factor Receptor Inhibition Reverses Cellular and Transcriptomic Alterations Induced by Hypoxia in the Neonatal Piglet Brain

“…Based on the biochemical results, we aimed to define the cellular effects of HX in the piglet cerebral cortex, and determined whether EGFRi pretreatment also affected HX-induced changes in cellular dynamics. First, we evaluated the overall cortical neuropathology following HX using H&E and a previously established and validated newborn piglet neuropathology score ( Hoque et al., 2014 ; Kratimenos et al., 2017a , 2017b ). The neuropathology score [median (interquartile range [IQR])] was 0 (0–1) in NX (n = 5) (p < 0.05 versus HX), 4 (3–4) in HX (n = 6) (p < 0.05 versus NX), and 2 (1–3) in EGFRi-HX (n = 7) (p < 0.05 versus HX) ( Figures 2 A–2C).…”

“…Cell death signaling in the neonatal brain involves the apoptotic and the rat sarcoma/mitogen-activated protein kinase (Ras/MAPK) pathways ( Chin et al., 1997 ; Hognason et al., 2001 ), and the initial HX insult linked to activation of these pathways causes oxygen free radical formation and lipid peroxidation of the neuronal membrane. We have previously shown that HX activates a set of apoptotic enzymes in the area of focal adhesions (FAs) and a variety of membrane receptors, including the epidermal growth factor receptor (EGFR) ( Kratimenos et al., 2017b ; Delivoria-Papadopoulou and Malaeb, 2014 ). Located at the cell membrane, the EGFR plays an essential role in cell growth and proliferation and has been shown to be neuroprotective following HX via nuclear factor (NF)-κB-dependent transcriptional upregulation of cyclin D1 ( Chen et al., 2016 ).…”

Effect of Src Kinase inhibition on Cytochrome c, Smac/DIABLO and Apoptosis Inducing Factor (AIF) Following Cerebral Hypoxia-Ischemia in Newborn Piglets

“…Our previous work has indicated that HI activates the enzyme Src kinase 24 – 26 . In the present study we investigated the regulatory mechanism by which the pre-apoptotic proteins translocate from mitochondria to the cytosol during HI through the Src kinase in the cerebral cortex of HI newborn piglets.…”