phenanthrenes and Infarction--Middle-Cerebral-Artery

Stroke could cause long-term disability, even mortality around the world. Recently, Sodium tanshinone IIA sulfonate (STS), identified from Salvia miltiorrhiza Bunge and was found to have unique efficiency in clinical practice as a potential therapeutic agent for ischemic cerebral infarction. However, systematic investigation about the biological mechanism is still lacking. Herein, we utilized high-throughput proteomics approach to identify the underlying targets for the treatment of STS in stroke.. We investigated the effect of STS on stroke outcomes on rat model of the Middle Cerebral Artery Occlusion and Reperfusion (MCAO/R), assessing by Z-Longa score, infarct volume and HE staining. Pharmacoproteomic profiling of ischemic penumbra in cortical (IPC) was performed using DIA-based label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. Bioinformatics analysis was processed for further investigation. The expression of core proteins was semi-quantified by DIA, and the major protein correlating with stroke was examined using parallel reaction monitoring (PRM).. Rats in the MCAO/R group showed neurological function deterioration, which was improved by STS. There were 423 differentially expressed proteins (DEPs) in IPC being detected and quantified in both the sham group and the MCAO/R group. Meanwhile, 285 proteins were significantly changed in the STS treated group, compared to the MCAO/R model. Protein-protein interaction (PPI) network, pathway and biological function enrichment were processed for the DEPs across each two groups, the results of which were integrated for analysis. Alb, mTOR, Dync1h1, Stxbp1, Cltc, and Sptan1 were contained as the core proteins. Altered molecules were discovered to be enriched in 18 signal pathways such as phosphatidylinositol signaling system, PI3K/AKT signal pathway and HIF-1 signal pathway. The results also showed the correlation with sleep disturbances and depression post-stroke.. We concluded that STS could prevent penumbra from progressively ongoing damage and improve neurological deficits in MCAO/R model rats. The intersected pathways and protein networks predicted by proteomics might provide much more detailed information for the therapeutic mechanisms of STS in the treatment of CIS.

Topics: Animals; Brain Ischemia; Chromatography, Liquid; Infarction, Middle Cerebral Artery; Ischemic Stroke; Phenanthrenes; Phosphatidylinositol 3-Kinases; Proteomics; Rats; Rats, Sprague-Dawley; Stroke; Tandem Mass Spectrometry

The diterpenoid cryptotanshinone (CTS) has wide biological functions, including inhibition of tumor growth, inflammation and apoptosis. The present study aimed to explore the possible effect of CTS on cerebral ischemia/reperfusion (I/R) injury and the underlying mechanisms.. Male C57BL/6J mice underwent transient middle cerebral artery occlusion (tMCAO) and murine microglia BV2 cells were challenged by Oxygen/glucose deprivation, to mimic I/R and ischemic/hypoxic and reperfusion (H/R) injury, respectively. CTS was administered 0.5 h (10 mg/kg) after the onset of MCAO or 2 h (20μM) post OGD. Infarct volume and neurological deficit were measured. Immunofluorescence, qPCR, and western blot, were performed to detect the expression of cytokines, apoptotic marker, and M1/M2 phenotype-specific genes. Flow cytometry was applied for M1/M2 subpopulation or Annexin V/PI apoptosis assessment.. CTS significantly reduced cerebral infarct volume, neurologic deficit scores, pro-inflammatory cytokine production (IL-6, TNF-α, and IL-1β), apoptotic protein expression (cleaved caspase-3) of mice after tMCAO challenge. Furthermore, CTS attenuated CD16+ M1-type and elevated CD206+ M2-type microglia in vivo or in vitro.. We propose that the neuroprotective effect of CTS in the I/R or H/R context are explained modulation of microglial polarization, suggesting therapeutic potential for cerebral ischemic stroke.

Topics: Animals; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred C57BL; Microglia; Phenanthrenes; Reperfusion Injury

Sodium tanshinone IIA sulfonate (STS) can protect against brain damage induced by stroke. However, the neural protection mechanism of STS remains unclear. We investigated whether STS performs its protective function by suppressing autophagy and inflammatory activity during brain injury. We established a transient middle cerebral artery occlusion and reperfusion (MCAO/R) model by blocking the left middle cerebral artery with a thread inserted through the internal carotid artery for 1 h, followed by reperfusion for 48 h either with or without STS and the autophagy inhibitor 3-methyladenine (3-MA). Neuroprotective effects were determined by evaluating infarction, brain edema, and neurological deficits. The numbers of microglia-derived macrophages, monocyte-derived microglia, T cells, and B cells in the brains were measured, based on the surface marker analyses of CD45, CD11b, B220, CD3, and CD4 using fluorescence-assisted cell sorting. STS (10, 20, 40 mg/kg) was able to significantly reduce infarct volumes, improve neurological deficits, and reduce brain water contents. STS treatment reduced neuroinflammation, as assessed by the infiltration of macrophages and neutrophils, corresponding with reduced numbers of macrophages, T cells, and B cells in ischemia/reperfusion (I/R) brains. In addition, STS treatment also attenuated the upregulation of autophagy associated proteins, such as LC3-II, Beclin-1 and Sirt 6, which was induced by MCAO. These results demonstrated that STS can provide remarkable protection against ischemic stroke, possibly via the inhibition of autophagy and inflammatory activity.

Topics: Animals; Autophagy; Brain Ischemia; Disease Models, Animal; Infarction, Middle Cerebral Artery; Inflammation; Neuroprotective Agents; Phenanthrenes; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Benefits from thrombolysis with recombinant tissue plasminogen activator (rt-PA) after ischemic stroke remain limited due to a narrow therapeutic window, low reperfusion rates, and increased risk of hemorrhagic transformations (HT). Experimental data showed that rt-PA enhances the post-ischemic activation of poly(ADP-ribose)polymerase (PARP) which in turn contributes to blood-brain barrier injury. The aim of the present study was to evaluate whether PJ34, a potent PARP inhibitor, improves poor reperfusion induced by delayed rt-PA administration, exerts vasculoprotective effects, and finally increases the therapeutic window of rt-PA. Stroke was induced by thrombin injection (0.75 UI in 1 μl) in the left middle cerebral artery (MCA) of male Swiss mice. Administration of rt-PA (0.9 mg kg

Topics: Animals; Edema; Endothelial Cells; Hemorrhage; Infarction, Middle Cerebral Artery; Inflammation; Male; Mice; Neuroprotective Agents; Phenanthrenes; Poly(ADP-ribose) Polymerase Inhibitors; Proteolysis; Recombinant Proteins; Reperfusion; Stroke; Thrombosis; Tissue Plasminogen Activator; Treatment Outcome; Vasospasm, Intracranial

Stroke is the leading cause of neurological disability in humans. Middle cerebral artery occlusion (MCAO) followed by reperfusion is widely accepted to mimic stroke in basic medical research. Triptolide is one of the major active components of the traditional Chinese herb Tripterygium wilfordii Hook F, and has been reported to have potent anti-inflammatory and immunosuppressive properties. Since its preclinical effects on stroke were still unclear, we decided to study the effects of Triptolide on focal cerebral ischemia/reperfusion injury in this study. The results showed that Triptolide treatment significantly attenuates brain infarction volume, water content, neurological deficits, and neuronal cell death rate, which were increased in the MCAO model rats. Immunohistochemistry was used to analyze the expression of glial fibrillary acidic protein (GFAP), Cyclooxygenase-2 (COX-2), inducible nitric oxide (iNOS), and NF-κB in the ischemic brains. The administration of Triptolide showed down-regulation of the iNOS, COX-2, GFAP, and NF-κB expression in MCAO rats. It also increased the expression of bcl-2, and suppressed levels of bax and caspase-3 compared with the MCAO group. Our findings revealed that Triptolide exerts its neuroprotective effects against inflammation with the involvement of inhibition of NF-κB activation.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents, Alkylating; Apoptosis; Blotting, Western; Cyclooxygenase 2; Disease Models, Animal; Diterpenes; Epoxy Compounds; Glial Fibrillary Acidic Protein; Immunoenzyme Techniques; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Male; Neuroprotective Agents; NF-kappa B; Nitric Oxide; Phenanthrenes; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Severity of Illness Index; Signal Transduction

Transient receptor potential melastatin 4 (TRPM4) is a voltage-dependent, nonselective cation channel. Under pathological conditions, sustained activation of TRPM4 leads to oncotic cell death. Here, we report the upregulation of TRPM4 in vascular endothelium following hypoxia/ischemia in vitro and in vivo. In human umbilical vein endothelial cells, TRPM4 expression was increased at both the mRNA and protein levels following oxygen-glucose deprivation. Blocking TRPM4 with 9-phenanthrol greatly enhanced tube formation on Matrigel. In a rat permanent middle cerebral artery occlusion model, TRPM4 was upregulated in the vascular endothelium within the penumbra region after stroke. TRPM4 expression peaked 1 day post-occlusion and gradually decreased. In vivo siRNA-mediated TRPM4 silencing enhanced angiogenesis and improved capillary integrity. A twofold reduction in infarct volume and a substantial recovery of motor function were observed in animals receiving the siRNA treatment. Interestingly, the protective effect of TRPM4 suppression disappeared 5 days after stroke induction, indicating that TRPM4 upregulation is critical for cerebral damage during the acute phase of stroke. TRPM4 could be a potential therapeutic target for ischemic stroke.

Topics: Animals; Cell Hypoxia; Endothelium, Vascular; Genetic Therapy; Human Umbilical Vein Endothelial Cells; Humans; Infarction, Middle Cerebral Artery; Locomotion; Male; Neovascularization, Physiologic; Phenanthrenes; Rats; Rats, Wistar; RNA Interference; RNA, Messenger; RNA, Small Interfering; TRPM Cation Channels; Up-Regulation

The receptor for advanced glycation end products (RAGE) has a potential role as a damage-sensing molecule; however, to date, its involvement in the pathophysiology of stroke and its modulation following neuroprotective treatment are not completely understood. We have previously demonstrated that expression of distinct RAGE isoforms, recognized by different antibodies, is differentially modulated in the brain of rats subjected to focal cerebral ischemia. Here, we focus on the full-length membrane-bound RAGE isoform, showing that its expression is significantly elevated in the striatum, whereas it is reduced in the cortex of rats subjected to transient middle cerebral artery occlusion (MCAo). Notably, the reduction of cortical levels of full-length RAGE detected 24 h after reperfusion is abolished by systemic administration of a neuroprotective dose of the poly(ADP-ribose) polymerase (PARP) inhibitor, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34). More interestingly, a significant reduction of plasma soluble RAGE (sRAGE) occurs 24 h after reperfusion and this effect is reverted by a neuroprotective dose of PJ34. Soluble forms of RAGE, generated either by alternative splicing or by proteolysis of the full-length form, effectively bind advanced glycation end products, thereby competing with the cell surface full-length RAGE, thus providing a 'decoy' function that may counteract the adverse effects of receptor signaling in neurons and may possibly exert cytoprotective effects. Thus, our data confirm the important role of RAGE in ischemic cerebral damage and, more interestingly, suggest the potential use of sRAGE as a blood biomarker of stroke severity and of neuroprotective treatment efficacy.

Topics: Animals; Brain; Brain Ischemia; Enzyme Inhibitors; Glycation End Products, Advanced; Infarction, Middle Cerebral Artery; Male; Neurons; Neuroprotective Agents; Phenanthrenes; Poly(ADP-ribose) Polymerase Inhibitors; Rats; Rats, Wistar; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Stroke

Recombinant tissue-type plasminogen activator (rt-PA) is presently the only pharmacological treatment approved for thrombolysis in patients suffering from ischemic stroke. Although reperfusion of ischemic tissue is essential, the use of rt-PA is limited due to its narrow therapeutic window and risk of hemorrhagic transformations. Recent studies have shown that rt-PA amplifies the post-ischemic activation of the nuclear enzyme poly(ADP-ribose)polymerase (PARP). This enzyme has been shown to contribute to both the breakdown of the blood brain barrier and spontaneous hemorrhagic transformations after ischemia. We therefore examined the capacity of PJ34 (N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino) acetamide hydrochloride), a potent inhibitor of PARP, to reduce the hemorrhagic transformations that occur after rt-PA in mice with permanent focal cerebral ischemia. Ischemia was produced by intraluminal occlusion of the left middle cerebral artery and treated with vehicle, rt-PA (10 mg/kg, i.v., 6 h after occlusion) or rt-PA plus PJ34 (3, 6 or 12 mg/kg, i.p., at ischemia onset and 4 h later). Hemorrhagic transformations, neurological examination, and infarct volumes were evaluated 48 h after the onset of ischemia. Delayed administration of rt-PA resulted in increased hemorrhagic transformations and aggravated the neurological deficit. Giving PJ34 (3 mg/kg) markedly reduced the hemorrhagic transformations, an effect not owing to a modification of matrix metalloprotease activity. Furthermore, PJ34 improved the neurological functions of rt-PA-treated ischemic mice. To conclude, the PARP inhibitor PJ34 makes rt-PA safer in experimental ischemic stroke.

Topics: Animals; Brain Ischemia; Enzyme Inhibitors; Hemorrhage; Infarction, Middle Cerebral Artery; Male; Matrix Metalloproteinases; Mice; Middle Cerebral Artery; Phenanthrenes; Poly(ADP-ribose) Polymerase Inhibitors; Recombinant Proteins; Tissue Plasminogen Activator

Microglia are the major immune cells in the central nervous system and play a key role in brain injury pathology. However, the role of activated microglia after subacute cerebral ischemia (SCI) remains unknown. To address this issue, we established a permanent middle cerebral artery occlusion (pMCAO) rat model and treated pMCAO rats with N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide (PJ34) (an inhibitor of microglial activation), or with vehicle alone. Finally, we determined the differences between the PJ34-and vehicle-treated rats with respect to neurological deficits, infarct volume, neuronal loss and the expression of CD11b (a marker of microglial activation), glial cell line-derived neurotrophic factor (GDNF) and tumor necrosis factor-α (TNF-α) at 1, 3 and 7 days after treatment. We found that the PJ34-treated rats had more severe neurological deficits and a larger infarct volume and exhibited a decreased CD11b expression, more neuronal loss, decreased expression of GDNF mRNA and protein but increased expression of TNF-α mRNA and protein compared with the vehicle-treated rats at 3 and 7 days after treatment. These results indicate that activated microglia provide a neuroprotective role through balancing GDNF and TNF-α expression following SCI.

Topics: Animals; Brain Ischemia; CD11b Antigen; Disease Models, Animal; Glial Cell Line-Derived Neurotrophic Factor; Infarction, Middle Cerebral Artery; Male; Microglia; Phenanthrenes; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

Disruption of blood-brain barrier (BBB) and edema formation play a key role in the development of neurological dysfunction after cerebral ischemia. In this study, the effects of Tanshinone IIA (Tan IIA), one of the active ingredients of Salvia miltiorrhiza root, on the BBB and brain edema after transient middle cerebral artery occlusion in rats were examined. Our study demonstrated that Tan IIA reduced brain infarct area, water content in the ischemic hemisphere. Furthermore, Tan IIA significantly decreased BBB permeability to Evans blue, suppressed the expression of intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-9 (MMP-9), inhibited the degradation of tight junction proteins zonula occludens-1 (ZO-1) and Occludin. These results demonstrated that Tan IIA was effective for attenuating the extent of brain edema formation in response to ischemia injury in rats, partly by Tan IIA's protective effect on the BBB. Our results may have implications in the treatment of brain edema in cerebral ischemia.

Topics: Abietanes; Animals; Blood-Brain Barrier; Brain; Edema; Evans Blue; Infarction, Middle Cerebral Artery; Intercellular Adhesion Molecule-1; Ischemic Attack, Transient; Matrix Metalloproteinase 9; Membrane Proteins; Occludin; Permeability; Phenanthrenes; Phytotherapy; Plant Extracts; Plant Roots; Rats; Reperfusion Injury; Salvia miltiorrhiza; Tight Junctions; Water

The objective of this study was to evaluate whether Tanshinone IIA (TSA) was neuroprotective in permanent focal cerebral ischemia and to determine the possible mechanisms of its neuroprotection. Mice were subjected to permanent middle cerebral artery occlusion. The neuroprotection of TSA was investigated with respect to neurological deficit scores and infarct volume. Biochemical analyses for malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in serum, and nitric oxide (NO) content and the inducible nitric oxide synthase (iNOS) activity in brain tissue were performed at 24 h after ischemia. Immunohistochemistry was used to measure the expression of iNOS. In vitro, the effects of TSA were tested in the cultured astrocytes exposed to hydrogen dioxide (H2O2). TSA (5, 10 and 20 mg/kg, i.p.) significantly reduced the infarct volume and improve neurological deficit. TSA also significantly increased the activity of SOD after 24 h of ischemia and decreased the MDA level, NO content, and iNOS expression. In vitro, the translocation of NF-kappaB was inhibited by TSA and the survival rate of astrocytes was markedly increased and the NO production was decreased. In conclusion, these results illustrated that TSA protected the brain from ischemic injury by suppressing the oxidative stress and the radical-mediated inflammatory insult.

Topics: Abietanes; Animals; Astrocytes; Brain; Brain Ischemia; Cells, Cultured; Cerebral Infarction; Drugs, Chinese Herbal; Hydrogen Peroxide; Infarction, Middle Cerebral Artery; Male; Malondialdehyde; Mice; Mice, Inbred ICR; Neuroprotective Agents; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Phenanthrenes; Rats; Rats, Sprague-Dawley; Superoxide Dismutase

Tanshinone IIB (TSB) is a major active constituent of the root of Salvia miltiorrhiza (Danshen) used in the treatment of acute stroke. Danshen extracts and TSB have shown marked neuron-protective effects in mouse studies but there is a lack of clinical evidence for the neuron-protective effects of Danshen and its active ingredients. This study investigated the neuron-protective effects of TSB in experimentally stroked rats. TSB at 5 and 25 mg/kg by intraperitoneal injection significantly reduced the focal infarct volume, cerebral histological damage and apoptosis in rats subjected to middle cerebral artery occlusion (MCAO) compared to MCAO rats receiving vehicle. This study demonstrated that TSB was effective in reducing stroke-induced brain damage and may represent a novel drug candidate for further development. Further mechanistic studies are needed for the neuron-protective activity of TSB.

Topics: Abietanes; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Brain; Brain Infarction; Disease Models, Animal; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Infarction, Middle Cerebral Artery; Injections, Intraperitoneal; Male; Nerve Degeneration; Neurons; Neuroprotective Agents; Phenanthrenes; Plant Extracts; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Stroke; Treatment Outcome

Cryptotanshinone (CTS), a major constituent from the roots of Salvia miltiorrhiza (Danshen), is widely used in the treatment of coronary heart disease, stroke and less commonly Alzheimer's disease. Our recent study indicates that CTS is a substrate for P-glycoprotein (PgP/MDR1/ABCB1). This study has investigated the nature of the brain distribution of CTS across the brain-blood barrier (BBB) using several in vitro and in vivo rodent models. A polarized transport of CTS was found in rat primary microvascular endothelial cell (RBMVEC) monolayers, with facilitated efflux from the abluminal side to luminal side. Addition of a PgP (e.g. verapamil and quinidine) or multi-drug resistance protein 1/2 (MRP1/2) inhibitor (e.g. probenecid and MK-571) in both luminal and abluminal sides attenuated the polarized transport. In a bilateral in situ brain perfusion model, the uptake of CTS into the cerebrum increased from 0.52 +/- 0.1% at 1 min to 11.13 +/- 2.36 ml/100 g tissue at 30 min and was significantly greater than that of sucrose. Co-perfusion of a PgP/MDR1 (e.g. verapamil) or MRP1/2 inhibitor (e.g. probenecid) significantly increased the brain distribution of CTS by 35.1-163.6%. The brain levels of CTS were only about 21% of those in plasma, and were significantly increased when coadministered with verapamil or probenecid in rats. The brain levels of CTS in rats subjected to middle cerebral artery occlusion and rats treated with quinolinic acid (a neurotoxin) were about 2- to 2.5-fold higher than the control rats. Moreover, the brain levels in mdr1a(-/-) and mrp1(-/-) mice were 10.9- and 1.5-fold higher than those in the wild-type mice, respectively. Taken collectively, these findings indicate that PgP and Mrp1 limit the brain penetration of CTS in rodents, suggesting a possible role of PgP and MRP1 in limiting the brain penetration of CTS in patients and causing drug resistance to Danshen therapy and interactions with conventional drugs that are substrates of PgP and MRP1. Further studies are needed to explore the role of other drug transporters in restricting the brain penetration of CTS and the clinical relevance.

Topics: Alzheimer Disease; Animals; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Biological Transport; Blood-Brain Barrier; Brain; Capillary Permeability; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Endothelial Cells; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Knockout; Microcirculation; Multidrug Resistance-Associated Proteins; Neuroprotective Agents; Neurotoxicity Syndromes; Phenanthrenes; Plant Roots; Quinolinic Acid; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Stroke; Tissue Distribution; Triterpenes

Tanshinone IIA (TSA) is a major constituent of Salvia miltiorrhiza Bunge widely used in the treatment of stroke. This current study aimed to investigate the nature of brain penetration of TSA using several in vitro and in vivo models. The uptake and efflux of TSA in primary rat brain microvascular endothelial cells (RBMVECs) were altered in the presence of a PgP inhibitor or multidrug-resistance-associated protein (Mrp1/2) inhibitor. A polarized transport of TSA was found in RBMVEC monolayers with facilitated efflux from the abluminal to the luminal side. The polarized transport of TSA was attenuated by PgP or Mrp1/2 inhibitors. In an in situ rat brain perfusion model, TSA crossed the blood-brain barrier at a greater rate than that for sucrose, and the brain penetration was increased in the presence of a PgP or Mrp1/2 inhibitor. The brain levels of TSA were only about 31% of that in the plasma and it increased to 74-77% of plasma levels when verapamil or quinidine was coadministered in rats. The entry of TSA to the central nervous system (CNS) significantly increased in rats subjected to middle cerebral artery occlusion or treatment with quinolinic acid. The normalized brain penetration of TSA in mdr1a((-/-)) mice was much higher than the wild-type mice. Taken collectively, these findings provide evidence that TSA has limited brain penetration through the blood-brain barrier owing to the contribution of PgP and possibly Mrp1/2.

Topics: Abietanes; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Blood-Brain Barrier; Brain; Cells, Cultured; Endothelial Cells; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Knockout; Multidrug Resistance-Associated Proteins; Phenanthrenes; Plant Roots; Quinidine; Quinolinic Acid; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Verapamil

Overactivation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) contributes to ischemic brain injury. Because PARP upregulates proinflammatory genes, we investigated whether inducible nitric oxide synthase (iNOS), a gene involved in the deleterious effects of postischemic inflammation, participates in the mechanisms by which PARP activation contributes to cerebral ischemic injury.. The middle cerebral artery (MCA) was occluded in mice for 20 minutes using an intravascular filament, and injury volume was measured 72 hours later in Nissl-stained brain sections. mRNA expression was assessed in the postischemic brain by the quantitative "real-time" polymerase chain reaction.. The PARP inhibitor PJ34 reduced infarct volume and attenuated postischemic iNOS mRNA upregulation by 72%. To determine whether iNOS contributes to the toxicity of PARP, the iNOS inhibitor aminoguanidine was co-administered with PARP inhibitors. Unexpectedly, co-administration of PARP and iNOS inhibitors, or treatment of iNOS-null mice with PARP inhibitors, abrogated the protective effect afforded by iNOS or PARP inhibition alone. The loss of neuroprotection was associated with upregulation of the inflammatory genes iNOS, intercellular adhesion molecule-1, and gp91(phox).. The results suggest that iNOS expression contributes to the deleterious effects exerted by PARP activation in cerebral ischemia. However, iNOS activity is required for the protective effect of PARP inhibition and, conversely, PARP activity must be present for iNOS inhibition to be effective. The findings unveil a previously unrecognized deleterious interaction between iNOS and PARP that is relevant to the development of combination therapies for ischemic stroke.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Guanidines; Infarction, Middle Cerebral Artery; Intercellular Adhesion Molecule-1; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; NADPH Oxidase 2; NADPH Oxidases; Neuroprotective Agents; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phenanthrenes; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

To study effect of triptolide (TL) on neuronal apoptosis in cerebral tissue of rat after ischemia-reperfusion.. Triptolide at dose 0.2 or 0.4 mg x kg(-1) was intraperitoneally injected once a day for 4 d. The focal cerebral ischemia-reperfusion model was established with thread embolism in middle artery before triptolide injection on the fourth day. Neurological deficit score of rats was evaluated; and immunohistochemical techniques were used to count positive cells of express of MPO and TUNEL in cerebraltissue.. Compared with the control group, the deficit of neural function was significantly improved, and the number of infiltrate of neutrophil and neuronal apoptosis in cerebral tissue was remarkably reduced in two TL-treated groups.. The results suggested that TL can inhibit infiltration of neutrophil and decrease the degree of neuronal apoptosis in cerebral tissue.

Topics: Animals; Apoptosis; Brain Ischemia; Diterpenes; Epoxy Compounds; Female; Infarction, Middle Cerebral Artery; Male; Neuroprotective Agents; Neutrophil Infiltration; Phenanthrenes; Plants, Medicinal; Rats; Rats, Wistar; Reperfusion Injury; Tripterygium

Focal cerebral ischemia activates the nuclear protein poly(ADP-ribose) polymerase (PARP) by single DNA strand breaks which leads to energy depletion and cell necrosis. Deletion or inhibition of PARP protects against ischemic brain injury. Here we examined the neuroprotective effect of PJ34, a novel potent inhibitor of PARP in vitro and in vivo. Serum-free primary neuronal cultures derived from rat cortex (E15-17) and kept in culture for 10 days were exposed to oxygen glucose deprivation (OGD) in vitro. Neuronal injury was quantified by LDH release after 24 h. Pretreatment with 30-1000 nM PJ34 significantly protected from OGD-induced cell injury in a dose-dependent manner. For in vivo experiments SV/129 mice were treated with PJ34 (50 microg) by intraperitoneal injection 2 h before 1 h middle cerebral artery occlusion (MCAo) and again 6 h later. Twenty-three h after reperfusion ischemic injury was significantly decreased compared to vehicle-treated controls (infarct volume reduction of 40%, p<0.05). Similarly, in a rat model of MCAo (2 h occlusion followed by up to 22 h reperfusion), PJ34 administration (10 mg/kg i.v.) significantly reduced infarct size, and the effect of the drug was maintained even if it was given as late as 10 min prior to reperfusion time. PJ34 significantly protected in a 4 h, but not in a 24 h permanent occlusion model. In conclusion, PJ34, a novel, potent inhibitor of PARP exerts massive neuroprotective agents, with a significant therapeutic window of opportunity. The present work strengthens the concept that pharmacological PARP inhibition may be a suitable approach for the treatment of acute stroke in man.

Topics: Animals; Brain; Brain Ischemia; Cells, Cultured; Culture Media, Serum-Free; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Infarction, Middle Cerebral Artery; Mice; Neurons; Neuroprotective Agents; Phenanthrenes; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar; Stroke