Neuroprotective effect of the peptides ADNF-9 and NAP on hypoxic-ischemic brain injury in neonatal rats.

Perinatal asphyxia is an important cause of neonatal mortality and subsequent serious sequelae such as motor and cognitive deficits and seizures. Recent studies have demonstrated that short peptides derived from activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP) are neuroprotective at femtomolar concentrations. However, the effect of these peptides on the hypoxic-ischemic brain injury model is unknown. The aim of this study is to investigate the effects of the peptides ADNF-9 and NAP on neurodegeneration and cerebral nitric oxide (NO) production in a neonatal rat model of hypoxic-ischemic brain injury. Seven-day-old Wistar Albino rat pups have been used in the study (n=42). Experimental groups in the study were: sham-operated group, ADNF-9-treated hypoxia-ischemia group, NAP-treated hypoxia-ischemia group, ADNF-9+NAP-treated hypoxia-ischemia group, and vehicle-treated group. In hypoxia-ischemia groups, left common carotid artery was ligated permanently on the seventh postnatal day. Two hours after the procedure, hypoxia (92% nitrogen and 8% oxygen) was applied for 2.5 h. ADNF-9, NAP, and ADNF-9+NAP were injected (intraperitoneally; i.p.) as a single dose immediately after the hypoxia period. Brain nitrite levels, neuronal cell death, and apoptosis were evaluated in both hemispheres (carotid ligated or nonligated) 72 h after the hypoxic-ischemic insult. Histopathological evaluation demonstrated that ADNF-9 and NAP significantly diminished number of "apoptotic cells" in the hippocampal CA1, CA2, CA3, and gyrus dentatus regions in both hemispheres (ligated and nonligated). When compared with vehicle-treated group, combination treatment with ADNF-9+NAP did not significantly reduce "apoptotic cell death" in any of the hemispheres. ADNF-9 and NAP, when administered separately, significantly preserved the number of neurons CA1, CA2, CA3, and dentate gyrus regions of the hippocampus, when compared with vehicle-treated group. The density of the CA1, CA2, and dentate gyrus neurons was significantly higher when combination therapy with ADNF-9+NAP was used in the carotid ligated hemispheres. In the nonligated hemispheres, combination therapy preserved the number of neurons only in the CA1 and dentate gyrus regions. Brain nitrite levels were evaluated by Griess reagent and showed that hypoxic-ischemic injury caused a significant increase in NO production. Brain nitrite levels in ADNF-9+NAP-treated animals were not different in carotid ligated or nonligated hemispheres. The peptides ADNF-9 and NAP significantly decreased NO overproduction in the hypoxic-ischemic hemisphere, whereas no significant change appeared in hypoxia alone and also in the sham-operated group. These results suggest the beneficial neuroprotective effect of ADNF-9 and NAP in this model of neonatal hypoxic-ischemic brain injury. To our knowledge, this is the first study that demonstrates a protective effect of these peptides against hypoxia-ischemia in the developing brain.