Excessive activation of the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) plays a prominent role in various of models of cellular injury. Here, we identify poly(ADP-ribose) (PAR) polymer, a product of PARP-1 activity, as a previously uncharacterized cell death signal. PAR polymer is directly toxic to neurons, and degradation of PAR polymer by poly(ADP-ribose) glycohydrolase (PARG) or phosphodiesterase 1 prevents PAR polymer-induced cell death. PARP-1-dependent, NMDA excitotoxicity of cortical neurons is reduced by neutralizing antibodies to PAR and by overexpression of PARG. Neuronal cultures with reduced levels of PARG are more sensitive to NMDA excitotoxicity than WT cultures. Transgenic mice overexpressing PARG have significantly reduced infarct volumes after focal ischemia. Conversely, mice with reduced levels of PARG have significantly increased infarct volumes after focal ischemia compared with WT littermate controls. These results reveal PAR polymer as a signaling molecule that induces cell death and suggests that interference with PAR polymer signaling may offer innovative therapeutic approaches for the treatment of cellular injury.excitotoxicity ͉ poly(ADP-ribose) glycohydrolase ͉ poly(ADP-ribose) polymerase ͉ stroke P oly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear protein that is involved in the DNA base excision repair system, where it is potently activated by DNA strand nicks and breaks (1, 2). Using NAD ϩ as a substrate, PARP-1 builds up homopolymers of ADP ribose units on various nuclear proteins including histones, DNA polymerases, topoisomerases, DNA ligase-2, transcription factors (3, 4), and PARP-1 itself (5, 6). Although the exact physiologic function of PARP-1 is not completely understood, in some tissues it plays an important role in DNA repair and genomic stability (5,7,8). Poly(ADP-ribose) (PAR) catabolism and metabolism is a dynamic process, with PAR glycohydrolase (PARG) playing the major role in the degradation of the polymer (9).Recent studies using pharmacologic inhibition of PARP or genetic KO of PARP-1 indicate that PARP-1 plays a dramatic and significant role in cellular injury after stroke, trauma, ischemiareperfusion of the heart, spleen, skeletal muscle, and retina, arthritis, ␤-islet cytotoxicity͞diabetes mellitus, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease, experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, endotoxic shock, multiple-system organ failure, and liver damage (for review, see refs. 1 and 10). PARP-1 activation also plays a prominent role in NMDA excitotoxicity, because PARP-1 KO mice are remarkably resistant both in vitro and in vivo to the excitotoxic effects of glutamate and NMDA (11,12). A cell-suicide hypothesis has been proposed (1,2,13,14) to explain the actions of PARP-1 in mediating cell death. However, studies in mice lacking PARG suggest that PAR polymer formed during the activation of PARP-1 might play a role in PARP-1-dependent cell death. PARG KO mice die at embryoni...

The proton exchange processes between water and solutes containing exchangeable protons have recently become of interest for monitoring pH effects, detecting cellular mobile proteins and peptides, and enhancing the detection sensitivity of various low-concentration endogenous and exogenous species. In this work, the analytic expressions for water exchange (WEX) filter spectroscopy, chemical exchange-dependent saturation transfer (CEST), and amide proton transfer (APT) experiments are derived by the use of Bloch equations with exchange terms. The effects of the initial states for the system, the difference between a steady state and a saturation state, and the relative contributions of the forward and backward exchange processes are discussed. The theory, in combination with numerical calculations, provides a useful tool for designing experimental schemes and assessing magnetization transfer (MT) processes between water protons and solvent-exchangeable protons. As an example, the case of endogenous amide proton exchange in the rat brain at 4.7 T is analyzed in detail. The proton exchange process between water and endogenous and exogenous dilute mobile solutes containing exchangeable protons has recently become of interest for several types of experiments. In so-called water-exchange (WEX) filter spectroscopy (1,2), the proton exchange (water to solute) is used to assess the exchange properties of amide protons between 6.8 and 8.3 ppm, and to selectively excite spectra of endogenous mobile proteins and peptides in the brain (3,4). This is in accordance with knowledge gained from in vitro protein studies using high-resolution magnetic resonance spectroscopy (MRS). For instance, the exchange rates of several types of amide protons in proteins are known to change by a factor of 10 per pH unit in the base-catalyzed pH range (2,5,6). The acquired WEX spectra are very similar to those from macromolecules (e.g., mobile proteins, lipids, and peptides) previously detected in the brain by alternative methods (7-11). Although it is possible to detect these solvent-exchangeable protons in vivo by WEX spectroscopy (3,4), it would be useful to have solute-sensitive image contrasts in the water signal to enable the spatial distribution of the solute content and related properties, such as pH and temperature, to be imaged. The imaging of pH (intra-and extracellular) by in vivo proton MR methods is an area of research that has recently become popular (12,13). One possible approach for this purpose is to perform continuous low-power radiofrequency (RF) irradiation of the exchangeable solute protons and follow the subsequent transfer of saturation to water. In such a chemical exchange-dependent saturation transfer (CEST) approach (14 -16), the reverse proton exchange process (solute to water) is used as a means of enhancing the detection sensitivity of low-concentration exogenous and endogenous agents (14 -23), including small molecules (such as urea (15,18)), polymers (dendrimers and poly-lysine (19,20), and polyuridilic acid (23))...

Background and Purpose-The importance of postmenopausal estrogen replacement therapy for stroke in females remains controversial. We previously showed that female rats sustain less infarction in reversible middle cerebral artery occlusion (MCAO) than their ovariectomized counterparts and that vascular mechanisms are partly responsible for improved tissue outcomes. Furthermore, exogenous estrogen strongly protects the male brain, even when administered in a single injection before MCAO injection. The present study examined the hypothesis that replacement of 17␤-estradiol to physiological levels improves stroke outcome in ovariectomized, estrogen-deficient female rats, acting through blood flow-mediated mechanisms. Methods-Age-matched, adult female Wistar rats were ovariectomized and treated with 0, 25, or 100 g of 17␤-estradiol administered through a subcutaneous implant or with a single Premarin (USP) injection (1 mg/kg) given immediately before ischemia was induced (nϭ10 per group). Each animal subsequently underwent 2 hours of MCAO by the intraluminal filament technique, followed by 22 hours of reperfusion. Ipsilateral parietal cortex perfusion was monitored by laser-Doppler flowmetry throughout ischemia. Cortical and caudate-putamen infarction volumes were determined by 2,3,5-triphenyltetrazolium chloride staining and digital image analysis. End-ischemic regional cerebral blood flow was measured in ovariectomized females with 0-or 25-g implants (nϭ4 per group) by 14 C-iodoantipyrine quantitative autoradiography. Results-Plasma estradiol levels were 3.0Ϯ0.6, 20Ϯ8, and 46Ϯ10 pg/mL in the 0-, 25-, and 100-g groups, respectively.Caudate-putamen infarction (% of ipsilateral caudate-putamen) was reduced by long-term, 25-g estrogen treatment (13Ϯ4% versus 31Ϯ6% in the 0-g group, PϽ0.05, and 22Ϯ3% in the 100-g group). Similarly, cortical infarction (% of ipsilateral cortex) was reduced only in the 25-g group (3Ϯ2% versus 12Ϯ3% in the 0-g group, PϽ0.05, and 6Ϯ3% in the 100-g group. End-ischemic striatal or cortical blood flow was not altered by estrogen treatment at the neuroprotective dose. Infarction volume was unchanged by acute treatment before MCAO when estrogen-treated animals were compared with saline vehicle-treated animals. Conclusions-Long-term estradiol replacement within a low physiological range ameliorates ischemic brain injury in previously ovariectomized female rats. The neuroprotective mechanism is flow-independent, not through preservation of residual ischemic regional cerebral blood flow. Furthermore, the therapeutic range is narrow, because the benefit of estrogen in transient vascular occlusion is diminished at larger doses, which yield high, but still physiologically relevant, plasma 17␤-estradiol levels. Lastly, unlike in the male brain, single-injection estrogen exposure does not salvage ischemic tissue in the female brain. Therefore, although exogenous steroid therapy protects both male and female estrogen-deficient brain, the mechanism may not be identical and depends on long-term hormone aug...