Pentoxifylline Alleviates Perinatal Hypoxic-Ischemia-Induced Short-term Memory Impairment by Suppressing Apoptosis in the Hippocampus of Rat Pups Je Hoon.

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Pentoxifylline Alleviates Perinatal Hypoxic-Ischemia-Induced Short-term Memory Impairment by Suppressing Apoptosis in the Hippocampus of Rat Pups Je Hoon Park1, Sung Eun Kim2, Jun Jang Jin2, Han Sung Choi3, Chang Ju Kim2, Il Gyu Ko2 1Department of Surgery, International St. Mary’s Hospital, College of Medicine, Catholic Kwandong University, Incheon, Korea 2Department of Physiology, College of Medicine, Kyung Hee University, Seoul, Korea 3Department of Emergency Medicine, College of Medicine, Kyung Hee University, Seoul, Korea

PURPOSE Perinatal hypoxic-ischemic brain damage is a major cause of acute mortality and chronic neurologic morbidity in infants and children. We investigated the effects of pentoxifylline, a methylxanthine derivative and type-4 phosphodiesterase inhibitor, on short-term memory and apoptotic neuronal cell death in the hippocampus following perinatal hypoxic-ischemia in newborn rats.

MATERIALS AND METHODS We used a step-down avoidance task to evaluate short-term memory and 3ʹ-5ʹ-cyclic adenosine monophosphate (cAMP) assay to detect cAMP levels. We evaluated apoptosis using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for evidence of DNA fragmentation, immunohistochemistry for caspase-3 levels, and western blot for Bcl-2 and Bax.

RESULTS Perinatal hypoxic-ischemic injury increased apoptotic cell death in the hippocampus, resulting in impaired short-term memory with decreased cAMP levels. Pentoxifylline treatment improved short-term memory by suppressing apoptotic cell death in the hippocampus with elevated cAMP levels. CONCLUSIONS Pentoxifylline ameliorated perinatal hypoxic-ischemia in rat pups. This alleviating effect could be ascribed to the inhibition apoptosis due to increased cAMP production by pentoxifylline.

Fig. 1. Effect of pentoxifylline on latency in the step-down avoidance task. A, Sham-operation group; B, perinatal hypoxic-ischemia-induced group; C, perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated group; D, perinatal hypoxic-ischemia-induced and 100-mg/kg pentoxifylline-treated group. *P<0.05 compared to the sham-operation group. #P<0.05 compared to the perinatal hypoxic-ischemia-induced group. §P<0.05 compared to the perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated group.

Fig. 2. Effect of pentoxifylline on 3ʹ-5ʹ-cyclic adenosine monophosphate (cAMP) levels in the hippocampus. A, Sham-operation group; B, perinatal hypoxic-ischemia-induced group; C, perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated group; D, perinatal hypoxic-ischemia-induced and 100-mg/kg pentoxifylline-treated group. *P<0.05 compared to the sham-operation group. #P<0.05 compared to the perinatal hypoxic-ischemia-induced group. §P<0.05 compared to the perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated group.

Fig. 3. Effect of pentoxifylline on DNA fragmentation in the hippocampal CA1 region. Upper panel: Photomicrographs of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells in the hippocampal CA1 region. A, Sham-operation group; B, perinatal hypoxic-ischemia-induced group; C, perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated group; D, perinatal hypoxic-ischemia-induced and 100-mg/kg pentoxifylline-treated group. Scale bar represents 400 μm. Lower panel: Number of TUNEL-positive cells in each group. *P<0.05 compared to the sham-operation group. #P<0.05 compared to the perinatal hypoxic-ischemia-induced group. §P<0.05 compared to the perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated group.

Fig. 4. Effect of pentoxifylline on caspase-3 expression in the hippocampal CA1 region. Upper panel: Photomicrographs of caspase-3-positive cells in the hippocampal CA1 region. A, Sham-operation group; B, perinatal hypoxic-ischemia-induced group; C, perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated group; D, perinatal hypoxic-ischemia-induced and 100-mg/kg pentoxifylline-treated group. Scale bar represents 400 μm. Lower panel: Number of caspase-3-positive cells in each group. *P<0.05 compared to the sham-operation group. #P<0.05 compared to the perinatal hypoxic-ischemia-induced group. §P<0.05 compared to the perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated group.

Fig. 5. Effect of pentoxifylline on Bax and Bcl-2 expression in the hippocampus. A, Sham-operation group; B, perinatal hypoxic-ischemia-induced group; C, perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated group; D, perinatal hypoxic-ischemia-induced and 100-mg/kg pentoxifylline-treated group. Upper panel: Results of band detection using an enhanced chemiluminescence (ECL) detection kit. Lower panel: Relative expression of Bax and Bcl-2 in the hippocampus. (1) Relative expression of Bax. (2) Relative expression of Bcl-2. (3) Ratio of Bax to Bcl-2. *P<0.05 compared to the sham-operation group. #P<0.05 compared to the perinatal hypoxic-ischemia-induced group. §P<0.05 compared to the perinatal hypoxic-ischemia-induced and 50-mg/kg pentoxifylline-treated group.