Short-term effects of hypothermia on axonal injury, preoligodendrocyte accumulation and oligodendrocyte myelination after hypoxia-ischemia in the hippocampus of immature rat brain.

ABSTRACT

Hypothermia is known to improve neurological recovery of animals and humans exposed to hypoxic-ischemic (HI) injury. However, the underlying mechanisms of the neuroprotective effects of hypothermia are only partially understood, including decreased excitotoxicity and apoptosis, and suppressed inflammation. There are few studies about the hypothermic effects on axonal injury and oligodendrocyte (OL) lineage degeneration, which are important components of neonatal brain injuries that cause cognitive disability. We hypothesized that mild hypothermia would reduce axonal injury and increase myelination in the hippocampus after HI. We performed left carotid artery ligation followed by 8% oxygen for 2 h in 7-day-old rats. Animals were divided into a hypothermic group (rectal temperature 32-33°C for 24 h) and a normothermic group (36-37°C for 24 h) immediately after HI. Animals were sacrificed at 1, 3 and 7 days for immunohistochemistry or Western blot analysis. We detected neuron loss by microtubule-associated protein 2 labeling and axonal injury by non-phosphorylated neurofilament (SMI32) with neurofilament 200 (NF200) double staining. We examined early OL progenitors by A2B5 or NG2, preoligodendrocytes (preOLs) by O4, and mature OLs by 2,3-cyclic nucleotide 3-phosphodiesterase (CNPase) and glutathione S-transferase (GST)-pi staining. Apoptosis was studied by active caspase-3. Hypothermia was associated with a significant elevation of neurons and axons in the hippocampal CA1 region after HI. Early OL progenitors (A2B5(+)) were elevated, but preOLs (O4(+)) and active caspase-3 were dramatically reduced in the hypothermic rat brain. Further study showed that the apoptotic rate of preOLs (caspase-3(+)-O4(+)/O4(+)) was markedly attenuated by hypothermic treatment compared to normothermic animals. The immunoreactivity of CNPase and GST-pi and the protein level of the myelin basic protein significantly increased in the hippocampus of hypothermia-treated rat brain. Axonal myelination also increased in hypothermic animals, which were tested by myelin basic protein and NF200 double staining and electron microscopy. These results showed that hypothermia reduced HI damage to axons and OL myelination coincided with increased early OL progenitor proliferation and decreased preOL accumulation and apoptosis. This study suggested new aspects that may contribute to elucidate the mechanism of hypothermic neuroprotection in neonatal rat brain.