Neuroprotective efficacy and therapeutic time window of peroxynitrite decomposition catalysts in focal cerebral ischemia in rats

Abstract

Free radicals have been implicated in cerebral ischemia reperfusion (IR) injury. Massive production of nitric oxide and superoxide results in continuous formation of peroxynitrite even several hours after IR insult. This can produce DNA strand nicks, hydroxylation and/or nitration of cytosolic components of neuron, leading to neuronal death. Peroxynitrite decomposition catalysts 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron (III) (FeTMPyP) and 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) (FeTPPS) have been demonstrated to protect neurons in in vitro cultures; however, their neuroprotective efficacy in cerebral IR injury has not been explored. In the present study, we investigated the efficacy and the therapeutic time window of FeTMPyP and FeTPPS in focal cerebral ischemia (FCI). FCI was induced according to the middle cerebral artery occlusion (MCAO) method. After 2 h of MCAO and 70 h of reperfusion, the extent of neurological deficits, infarct and edema volume were measured in Sprague-Dawley rats. FeTMPyP and FeTPPS were administered at different time points 2, 6, 9 and 12 h post MCAO. FeTMPyP and FeTPPS (3 mg kg(-1), i.v.) treatment at 2 and 6 h post MCAO produced significant reduction in infarct volume, edema volume and neurological deficits. However, treatment at latter time points did not produce significant neuroprotection. Significant reduction of peroxynitrite in blood and nitrotyrosine in brain sections was observed on FeTMPyP and FeTPPS treatment. As delayed treatment of FeTMPyP and FeTPPS produced neuroprotection, we tested whether treatment had any influence over the apoptotic neuronal death. DNA fragmentation and in situ nick end-labeling assays showed that FeTMPyP and FeTPPS treatment reduced IR injury-induced DNA fragmentation. In conclusion, peroxynitrite decomposition catalysts (FeTMPyP and FeTPPS) produced prominent neuroprotection even if administered 6 h post MCAO and the neuroprotective effect is at least in part due to the reduction of peroxynitrite and apoptosis.

Figure 1

Chemical structure of PDCs 5,10,15,20-tetrakis(N-methyl-4′-pyridyl)porphyrinato iron (III) (FeTMPyP) and 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) (FeTPPS).

Figure 2

Study design for neuroprotective efficacy, therapeutic time window and mechanism of PDCs action: For neuroprotective efficacy and therapeutic time window, FeTMPyP and FeTPPS were tested at two dose levels (either 0.3 and 1 or 1 and 3 mg kg⁻¹) at 2, 6, 9, 12 h post MCAO along with a vehicle-treated control, which accounted for a total of 17 different groups of rats. The numbers mentioned in parenthesis are the doses (mg kg⁻¹). The times scheme, at which FeTMPyP and FeTPPS treatments were done after MCAO for different groups of animals, is indicated by their doses in parenthesis. Rats were killed 70 h post MCAO and extent of neurological damage was measured. For mechanistic studies PDCs were administered 30 min post MCAO. Peroxynitrite levels were estimated (dihydrorhodamine123 method) 3 h post MCAO and nitrotyrosine immunofluorescence analysis was performed 6 h post MCAO. DNA ladder, TUNEL and electron microscopy were conducted 72 h post MCAO.

Figure 3

Representative coronal brain sections (2-mm thick) from FeTMPyP (a) (0.3 and 1 or 1 and 3 mg kg⁻¹) and FeTPPS (b) (0.3 and 1 or 1 and 3 mg kg⁻¹)-treated rats stained with 2% triphenyl tetrazolium chloride (TTC) after 2 h of MCAO and 70 h of reperfusion showing infarction. FeTMPyP was administered at 2, 6, 9, 12 h post MCAO. Dark colored regions in the TTC-stained sections indicate nonischemic and pale colored regions indicate ischemic portion of brain.

Figure 4

Dihydrorhodamine123 was injected to rats 1 h post MCAO and rhodamine123 fluorescence estimated in plasma 1 h post reperfusion in vehicle and FeTMPyP and FeTPPS (3 mg kg⁻¹)-treated rat. Vehicle or FeTMPyP/FeTPPS (3 mg kg⁻¹) treatment was done 30 min post MCAO. The extent of rhodamine123 fluorescence reflects the extent of peroxynitrite formed during IR insult. FeTMPyP and FeTPPS treatment (3 mg kg⁻¹, i.v.) reduced the rhodamine123 fluorescence by decomposing peroxynitrite in a significant manner (P<0.05, F(2,9)=6.59, ANOVA followed by Tukey Test).

Figure 5

Immunofluorescence photomicrographs show nitrated tyrosine residues of cytosolic proteins. (a) Sham-operated rat brain section, (b) Vehicle-treated rat brain section (n=3). (c) FeTMPyP (3 mg kg⁻¹, i.v.)-treated animal (n=3). (d) FeTPPS (3 mg kg⁻¹, i.v.)-treated rat brain sections (n=3). Vehicle or FeTMPyP/FeTPPS (3 mg kg⁻¹) treatment was done 30 min post MCAO. All the FITC fluorescent images were acquired using the CCD camera (DC300F–Leica) with a constant exposure time of 0.805 s. FeTMPyP and FeTPPS treatment reduced the nitrotyrosine immunoreactivity.

Figure 6

Electron photomicrographs of sham-operated and MCAO rat brain after 2 h of ischemia and 70 h MCAO. (a) Normal brain cells from sham-operated animal. N indicates nucleus and C indicates cytoplasm of the cell (b) Apoptotic cell in its early stage of apoptosis from the penumbral area of MCAO rat brain. The chromatin gets compacted and condensed on the inner surface of the nuclear membrane. (c) Vacuoles formed in the cytoplasm. V indicates vacuole. (d) At an advanced stage, more chromatin condensation occurs. This stage is followed by the formation of membrane-packed apoptotic bodies which are phagocytosed by the nearby glial cells. (e) Early necrotic cell: the cell membrane is ruptured and the cellular contents coming out of the cell. (f) Completely necrotic cell observed at the ischemic zone after 2 h of ischemia and 70 h MCAO.

Figure 7

DNA ladder pattern observed in vehicle-treated and FeTMPyP-treated MCAO rats. Lane (1) Hind III digest (2) vehicle-treated ipsilateral penumbral cortex (3) FeTMPyP-treated ipsilateral penumbral cortex (4) vehicle-treated, ipsilateral ischemic cortex (5) FeTMPyP-treated ipsilateral ischemic cortex. The arrows indicate the fragmented DNA.

Figure 8

(a) Representative photomicrographs of saline (vehicle), FeTMPyP or FeTPPS (3 mg kg⁻¹, i.v.)-treated (n=3 in each group) MCAO rat brain sections at the level of anterior commissure, temporal limb (AP=−0.26 to −0.51 mm to bregma) labeled with DNA nick end-labeling (TUNEL) method. (I) Sham-operated rat brain sections after TUNEL. (II) Vehicle-treated rat brain sections labeled according to TUNEL method. The TUNEL-positive cells were observed in the ischemic and penumbral area of cerebral cortex and striatum. (III) FeTMPyP (3 mg kg⁻¹ i.v)-treated rat brain sections labeled according to TUNEL method. (IV) FeTPPS (3 mg kg⁻¹ i.v)-treated rat brain sections labeled according to TUNEL method. (b) TUNEL-positive cells were counted in each field (dimensions 0.42 × 0.35 mm) using Leica Qwin image analysis software and the cell count obtained from the same group of rats were averaged and translated to the number of positive cells per mm³. FeTMPyP and FeTPPS (3 mg kg⁻¹, i.v.) treatment significantly reduced the number of TUNEL-positive cells in the penumbral area (P<0.05, F(2,9)=9.25, ANOVA followed by Tukey test). Statistical significance was evaluated using one-way analysis of variance. ^*P<0.05 as compared to the vehicle-treated group.