Hypothermia treatment in Hypoxia-Inducible Factor-1α Knockout Mice with Hypoxia-Ischemia

Hypoxia-inducible factor-1α (HIF-1α) has a wide-ranging role in the cellular responses to hypoxia. We previously found that neuron-specific HIF-1α-deficient mice (HIF-KO) that underwent neonatal hypoxia-ischemia (HI) had increased brain injury suggesting its neuroprotective function. To investigate whether HIF-1α is also involved in the mechanisms of protection by hypothermia (HT), the standard of care for hypoxic-ischemic encephalopathy, we tested the effect of HT on HIF-KO and wild-type (WT) littermates after HI in postnatal day 9 mice.

Cooling at 32°C began 1 h following HI and lasted for 3.5 h. Mice were perfused 5-7 days later for histological determination of injury severity. For Western blots, mice were killed 4 h or 24 h after HI with HT or HI with normothermia (NT) and ipsilateral cortices and hippocampi were evaluated for expression of HIF-1α, spectrin, ERK1/2, phosphorylated-ERK1/2 (p-ERK), and RNA-binding motif protein 3 (RBM3), one of the main cold-inducible mRNA-binding proteins.

Histological evaluation showed WT mice with HT had less injury than WT with NT, but HIF-KO mice showed no reduction of injury with HT. Regionally, the reduction of injury in WT with HT is greater in the hippocampus than in the cortex. Protein expression of HIF-1α was lower in HIF-KO cortex at 4 h with NT or HT and at 24 h with NT, but HIF-1α was higher in WT with NT at 24 h. Expression of spectrin 145/150 in WT cortex with HT was not different than sham at 4 h, indicating limitation of necrosis with HT. In the HIF-KO cortex at 4 h spectrin 145/150 was higher in both NT and HT, indicating no protection with HT. In the hippocampus at 4 h and 24 h, spectrin 145/150 was elevated in all groups compared to sham. ERK activity, as represented by the ratio of p-ERK/ERK, was upregulated at 24 h in the cortex in WT with NT or HT compared to sham and in HIF-KO mice with NT or HT treatment compared to sham. RBM3 was elevated at 4 h in both WT and HIF-KO cortex with HT, but there was no change in the hippocampus.

These results support a critical role for HIF-1α in the mechanisms of protection with HT.