Animals

A complementary DNA (cDNA) encoded with human wildtype Ngb was synthesized using a modification of recursive PCR strategy. Neuroglobin cDNA was then sub-cloned into an expression vector pcDNA3.1 (pcDNA3.1-Ngb). A BamH1- Xho1 fragment from the pcDNA3.1-Ngb was further sub-cloned into a pUB6 plasmid with the human ubiquitin C promoter to achieve ubiquitous expression of the transgene. The Ngb transgenic mice were then generated to incorporate and overexpress the human wild-type Ngb at the transgenic core facility at the University of Louisville (9). The first generation of Ngb overexpressing transgenic mice was then backcrossed with the C57/B6 strain for at least 10 generations and all the mice were genotyped to confirm continued expression of the transgene. Neuroglobin mRNA and protein expression in the brain were assessed at the age of 2 months, i.e., the age at which the mice were used for the study, and showed increased expression as previously reported (9). Furthermore, we have previously shown that sustained hypoxia, but not IH, elicit increased expression of Ngb (7). Male C57BL/6J (WT) mice were purchased from Jackson Laboratories (Bar Harbor, Maine) at 5 weeks of age, and housed at the animal facility at the University of Chicago until they attained 8 weeks of age at which time they were randomly paired to serve as controls for the Ngb transgenic mice. Animals were housed in a 12-h light/dark cycle-controlled room (lights on at 7:00 a.m. till 7:00 p.m.) with ambient temperature being kept at 26 ± 1°C. Male mice were placed in groups of four mice in standard clear mouse polycarbonate cages and were allowed unrestricted access to standard chow food and water. All behavioral experiments were conducted during the light period (between 9:00 a.m. and 12:30 p.m.). Mice were randomly allocated to either IH or room air (RA) exposures. The experimental protocols were approved by the Institutional Animal Use and Care Committee at the University of Chicago (protocol # 72043) and are in close agreement with the National Institutes of Health Guide in the Care and Use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.