Effects of hypoxic preconditioning on the changes of expression of neuroglobin mRNA and labeled positive cells following cerebral ischemia in gerbils

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Abstract

BACKGROUND:

Neuroglobin (NGB), as newly discovered the third member of the globin family binding oxygen, mainly exists in brain of human beings and vertebrates, and it is closely correlated with the oxygen supply in brain.

OBJECTIVE:

To observe the changes of the expression of neuroglobin and number of positive cells labeled immunohistochemically following cerebral ischemia in gerbils after hypoxic preconditioning.

DESIGN:

A complete randomized grouping design and controlled experiment.

SETTING:

Department of Neurology, Department of Anesthesia, Shandong Provincial Hospital, Shandong University.

MATERIALS:

Sixty-six adult male Mongolian gerbils of clean degree, about 50–65 g, at an average of 57.5 g were provided by the Experimental Animal Center of Capital Medical University [certificate number of animal quality: SCXK(Beijing)2000-0012]. TRNzil (Tianwei Shidai Company, Beijing), polymerase chain reaction (PCR) primers (synthetized by Invitrogen Company, Shanghai); reverse transcription-PCR (RT-PCR) one-step kit (Toyobo Company); PCR instrument (GeneAmp PCR System 240); mice brain NGB monoclonal antibody (Academy of Military Medical Sciences); DAB (Zhongshan Company, Beijing).

METHEDS:

The study was completed from December 2004 to June 2005 in Shandong Provincial Hospital. ① The 66 gerbils were randomly divided into sham-operated group (n =6), cerebral ischemia group (n =30) and hypoxic preconditioning group (n =30). The gerbils in the hypoxic preconditioning group were put in the environment which contained O2 (0.08 in volume fraction) and N2 (0.92 in volume fraction) at temperature of 25 °C for 2 hours. After 5 hours, the gerbils in the hypoxic preconditioning group and cerebral ischemia group were anesthetized, then bilateral common carotid arteries were ligated. In the sham-operated group, bilateral common carotid arteries were only isolated without ligation and hypoxic preconditioning. ② At 1, 5, 10, 30 and 60 minutes after cerebral ischemia, the expression of NGB mRNA was detected with RT-PCR, and the number of NGF positive cells was counted with immunohistochemical staining. The NGB mRNA expression and number of NGB positive cells at different time points after ischemia were compared between the gerbils treated with and without hypoxic preconditioning.

MAIN OUTCOME MEASURES:

NGB mRNA expression and number of NGB positive cells at 1, 5, 10, 30 and 60 minutes after cerebral ischemia.

RESULTS:

All the 66 Mongolian gerbils were involved in the analysis of results. ① Results of NGB mRNA: In the cerebral ischemia group, the NGB mRNA expression began to increase at 1 minute after cerebral ischemia, but had no obvious difference as compared with that in the sham-operated group (P > 0.05), that at 5 minutes was obviously higher than that in the sham-operated group (0.951 ±0.034, 0.597±0.008, P < 0.05), it decreased gradually 10, 30 and 60 minutes after cerebral ischemia, but had no obvious difference as compared with that in the sham-operated group (P > 0.05). In the hypoxic preconditioning group, the NGB mRNA expression increased rapidly 1 minute after cerebral ischemia, which was obviously higher than that in the cerebral ischemia group (0.641±0.010, 0.618±0.015, P < 0.05), and the expressions at 5, 10 and 30 minutes were still obviously higher than those in the cerebral ischemia group (0.995±0.020 vs. 0.951±0.034; 0.941±0.010 vs. 0.615±0.018; 0.642±0.010 vs. 0.608±0.010, P< 0.05–0.01), whereas the expression at 60 minutes were not obviously different from that in the cerebral ischemia group (P > 0.05). ② Number of NGB positive cells: The numbers of NGB positive cells at 1, 10 and 30 minutes after cerebral ishcemia in the hypoxic preconditioning group [(50.2±3.3), (67.2±3.3), (35.0±4.3) cells] were obviously more than those in the cerebral ischemia group [(33.0±2.1), (60.5±1.9), (23.3±3.1) cells, P < 0.05–0.01], whereas those at 5 and 60 minutes had no obvious differences between the two groups (P > 0.05).

CONCLUSION:

Hypoxic preconditioning can rapidly accelerate the expression of NGB mRNA following cerebral ischemia, and it plays its neuroprotective role in hypoxic preconditioning.

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