Neuroprotective effect of peroxynitrite decomposition catalyst and poly(adenosine diphosphate—ribose) polymerase inhibitor alone and in combination in rats with focal ischemia

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Object. The authors evaluated the neuroprotective effect of 5,10,15,20-tetrakis(N-methyl-4′-pyridyl)porphyrinatoiron(III) (FeTMPyP), a peroxynitrite decomposition catalyst, and 1,5-isoquinolinediol (ISO), a poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitor, alone and in combination in rats with focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO).

Methods. Male Sprague—Dawley rats were subjected to 2 hours of MCAO followed by 22 hours of reperfusion. Cerebral infarction and neurological deficits were estimated after ischemia. Intraperitoneal injections of FeTMPyP (1 and 2 mg/kg) and ISO (0.05 and 0.1 mg/kg) were administered alone or in combination in ischemic animals. The PARP activity in vehicle- and drug-treated groups was estimated using anti—poly(ADP-ribose) antibody in immunofluorescence and immunoblotting studies.

Two hours of MCAO and 22 hours of reperfusion produced significant cerebral infarction and neurological deficits. Treatment with FeTMPyP (1 and 2 mg/kg) and ISO (0.05 and 0.1 mg/kg) produced a significant reduction in cerebral infarction and neurological deficits. Combination therapy (2 mg/kg FeTMPyP and 0.1 mg/kg ISO) enhanced the inhibition of ischemic volume (77.81 ± 0.86%) compared with monotherapies (FeTMPyP 54.07 ± 5.6% and ISO 53.06 ± 3.88%). Immunoblotting and immunofluorescence studies showed PARP activation after ischemia, which was reduced by drug treatment.

Conclusions. Neuroprotection observed with FeTMPyP and ISO alone and in combination may be attributed to inhibition of the peroxynitrite—PARP cascade of cerebral ischemia/reperfusion injury.

Article Information

Address reprint requests to: Shyam S. Sharma, Ph.D., Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar (Mohali) 160 062, Punjab, India. email: sssharma@niper.ac.in.

© AANS, except where prohibited by US copyright law.

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Figures

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    Photographs of representative coronal brain sections (2 mm thick) stained with 2% TTC after 2 hours of MCAO and 22 hours of reperfusion showing infarction in vehicle-treated, FeTMPyP-treated (2 mg/kg), ISO-treated (0.1 mg/kg), and combined therapy (2 mg/kg FeTMPyP and 0.1 mg/kg ISO) groups. Animals in the sham-operated group underwent surgery but not MCAO. Dark areas on the TTC-stained sections indicate the absence of ischemia, and the highlighted regions indicate the ischemic portions of brain tissue. A significant decrease in ischemia was demonstrated with the use of drug treatments.

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    Bar graph depicting the infarct volume in ischemic rats treated with vehicle (saline or 1% DMSO), FeTMPyP (1 or 2 mg/kg), ISO (0.05 or 0.1 mg/kg), and combination therapy (2 mg/kg FeTMPyP and 0.1 mg/kg ISO). Statistically significant neuroprotection was observed in FeTMPyP- and ISO-treated ischemic rats. Enhanced neuroprotection was observed in animals treated with the combination therapy. *p < 0.05, compared with the saline-treated group; #p < 0.05, compared with DMSO-treated group; and @p < 0.05, compared with FeTMPyP-treated (2 mg/kg) group. All pairwise comparisons were made using the Tukey post-hoc test after ANOVA.

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    Bar graph depicting neurological deficit scores in ischemic rats treated with vehicle, FeTMPyP, ISO, and combined therapy. Statistically significant neuroprotection was observed in ischemic rats treated with either dose of FeTMPyP and ISO. *p < 0.05, compared with the saline-treated group; and #p < 0.05, compared with DMSO-treated group. All pairwise comparisons were made using the Tukey post-hoc test after ANOVA.

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    Representative immunofluorescence photomicrographs showing PAR residues detected using anti—PAR antibody in brain sections from ischemic rats. Arrows and arrowheads point toward nuclei and cytosol, respectively. Upper Left: Negative control (primary antibody not added). Upper Center: Tissue section obtained from a DMSO-treated ischemic rat, showing a dense signal at the nucleus compared with that in the cytoplasm. Upper Right: Section obtained from an ISO-treated (0.1 mg/ kg) ischemic rat, demonstrating a low-intensity signal in the nucleus compared with that in the cytoplasm. Lower Left: An FeTMPyP-treated (2 mg/kg) section showing uniform signal in the nucleus and cytoplasm. Lower Right: Brain section obtained in a rat treated with the combination therapy (2 mg/kg FeTMPyP and 0.1 mg/kg ISO), showing reduced signal in both the nucleus and the cytoplasm.

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    Upper: Western immunoblot demonstrating PAR immunoreactivity in ischemic rats treated with vehicle (Lane 1), 1 mg/kg FeTMPyP (Lane 2), and 2 mg/kg FeTMPyP (Lane 3). Treatment with FeTMPyP produced a reduction in PAR immunoreactivity. Lower: Bar graph depicting immunoreactivity estimated by using the densitometry analysis method normalized to percentage. *p < 0.05, compared with the saline-treated group. All pairwise comparisons were made using the Tukey post-hoc test after ANOVA.

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