Abstract OBJECTIVE: To study the protective effects of multiple course hyperbaric oxygen (HBO) treatment against hypoxic-ischemic brain damage (HIBD) in neonatal rats when HBO treatment is delayed (96 hrs after the HIBD event). METHODS: Eighty-eight 7-day-old Sprague-Dawley rat pups were randomly assigned to control, HIBD and HBO groups. The HBO group was subdivided into cohorts receiving treatment 2 h, 48 h and 96 h, respectively, after HIBD was induced. The three subgroups comprising different therapeutic windows were further randomly assigned to receive 1, 2 or 3 courses of HBO treatment ("HBO-1, -2 and -3 sub-groups"). HBO was administered once daily (2 ATA), a course lasting for seven days. There was an interval of three days between the courses. All pups were sacrificed at the end of HBO treatment (31 days after HIBD). TUNEL staining was used for testing neuronal apoptosis in the cortex and the CA1 of the hippocampus, and NSE staining was used to ascertain cortical neuronal population. RESULTS:① There were significantly more TUNEL positive cells in the HIBD group than in the control group; NSE positive cells were significantly lower than in controls (P<0.01). ② With the more delayed therapeutic window, the effects of apoptosis inhibition and neuronal protection of a single course of HBO were gradually reduced. ③ With increasing courses of HBO treatment, the effects of apoptosis inhibition and neuronal protection of HBO increased gradually in rats receiving treatment 48 and 96 hrs after HIBD. In the HBO group receiving treatment 2 hrs after HIBD, the number of apoptotic cells and NSE positive cells were close to that of the control group after one course of HBO treatment. CONCLUSIONS: One course of HBO administered within 2 hrs after HIBD can effectively inhibit neuron apoptosis and protect neurons. The effects of apoptosis inhibition and neuron protection of HBO can be increased through increasing the number of HBO treatment courses in neonatal rats with HIBD even if initiation of treatment is delayed after HIBD.［Chin J Contemp Pediatr, 2009, 11 (6):464-470］
［3］Raghupathi R, Graham DI, Mclntosh TK. Apoptosis after traumatic brain injury［J］. J Neuroltrauma, 2000, 17(10):927-938.
［4］Nakajima W, Ishida A, Lange MS, Gabrielson KL, Wilson MA, Martin LJ, et al . Apoptosis has a prolonged role in the neurodegeneration after hypoxic ischemia in the newborn rat［J］. J Neurosci, 2000, 20(21):7994-8004.
［5］Springer JE, Nottingham SA, Mcewen ML. Caspase-3 apoptotic signaling following injury to the central nervous system［J］. Clin Chem Lab Med, 2001, 39(4):299-307.
［6］Hayashi T, Iwai M, Ikeda T, Jin G, Deguchi K, Nagotani S, et al. Neural precursor cells division and migration in neonatal rat brain after ischemic/hypoxic injury［J］. Brain Res, 2005, 1038(1):41-49.
［7］Yun L, Changman Z, John WC, Austin RTC, John HZ. Multiple effects of hyperbaric oxygen on the express of HIF-1a and apoptotic gene in a global ischemia-hypotension rat model ［J］.Exp Neurol, 2005, 191(2005):198-210.
［8］Lou M, Chen Y, Ding M, Eschenfelder CC, Deuschl G. Involvement of the mito-chondrial ATP-sensitive potassium channel in the neuroprotective effect of hyperbaric oxygenation after cerebral ischemia［J］. Brain Res Bull, 2006, 69(2):109-116.
［9］Calvert JW, Zhou C, Nanda A, Zhang JH. Effect of hyperbaric oxygen on apoptosis in neonatal hypoxia-ischemia rat model ［J］. J Appl Physiol, 2003, 95(5):2072-2080.
［13］Rice JE, Vannucci RC, Brierley JB. The influence of immaturity on hypoxic-ischemic brain damage in the rat ［J］. Ann Neurol, 1981, 9(2):131-141.
［14］Yin D, Zhou C, Kusaka I, Calvert JW, Parent AD, Nanda A, et al. Inhibition of apoptosis by hyperbaric oxygen in a rat focal cerebral ischemic model［J］. J Cereb Blood Flow Metab, 2003, 23(7):855-864.
［15］Zhao H, Yenari MA, Cheng D, Sapolsky RM, Steinberg GK. Bcl-2 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome c translocation and caspase-3 activity ［J］. J Neurochem, 2003, 85(4):1026-1036.
［16］Ferrer I, Friguls B, Dalfo E, Justicia C, Planas AM. Caspase-dependent and caspase-independent signalling of apoptosis in the penumbra following middle cerebral artery occlusion in the adult rat ［J］. Neuropathol Appl Neurobiol, 2003, 29(5):472-481.
［17］Lossi L, Tamagno I, Merighi A. Molecular morphology of neuronal apoptosis: analysis of caspase-3 activation during postnatal development of mouse cerebellar cortex［J］. J Mol Hostol, 2004, 35(6):621-629.
［19］Li Y, Zhou C, Calvert JW, Colohan AR, Zhang JH. Multiple effects of hyperbaric oxygen on the expression of HIF-1 alpha and apoptotic genes in a global ischemia-hypotension rat model［J］. Exp Neurol, 2005, 191(1):198-210.
