(1) Diadenosine tetraphosphate (AP4A), two adenosine moieties bridged by 4 phosphates, is an endogenous purinergic ligand found in brain. Previous studies have shown that AP4A reduced neurodegeneration caused by dopaminergic neurotoxin 6-hydroxydopamine in rat striatum and substantia nigra. The purpose of this study was to determine whether AP4A is protective against methamphetamine (MA) mediated toxicity in dopaminergic neurons. Primary neuronal cultures were prepared from rat embryonic (E15) ventral mesencephalic tissue. On DIV8, cultures were treated with 2 mM MA for 48 hours. Application of MA increased LDH levels, decreased TH immunoreactivity, and increased TUNEL. All these changes were reduced by pretreatment with AP4A. The protective effect of AP4A was further examined in vivo. Adult Sprague Dawley rats were injected with AP4A or vehicle intracerebroventricularly followed by 4 doses of MA (5mg/kg), given subcutaneously every two hours. Administration of MA increased caspase-3 immunoreactivity in striatum and cortex. Pretreatment with AP4A significantly reduced the density of caspase-3 cells. Using microdialysis, dopamine (DA) release was monitored in dorsal striatum in freely moving rats. AP4A did not acutely alter MA-evoked DA release, suggesting that AP4A -induced protection is not directly mediated through a change in DA overflow. Taken together, these data show that AP4A has protective effects against MA-mediated neuronal injury both in vitro and in vivo. The mechanism of action may involve suppression of MA -induced apoptosis. (B) Astaxanthin (ATX) is a carotenoid antioxidant found in crustaceans. We found that ATX has protective effects against ischemic brain injury. Adult male Sprague-Dawley rats were anesthetized with chloral hydrate. ATX or vehicle was administered intracerebroventricularly 10-20 minutes prior to a 60-min middle cerebral artery ligation. At 2 days after MCA occlusion (MCAo), rats that received ATX had an increase in locomotor activity. ATX significantly reduced cerebral infarction and TUNEL labeling in ischemic brain. ATX antagonized ischemia Vmediated loss of aconitase activity, an indirect marker for the production of reactive oxygen species and reduced glutamate release, lipid peroxidation, translocation of chrome C, as well as TUNEL labeling in the ischemic cortex. ATX did not alter physiological parameters, such as blood gases, blood pH, blood pressure, body temperature, brain temperature and cerebral blood flow. Collectively, our data suggest that astaxanthin has protective effects against free radical toxicity and ischemia-related injury in vivo through the inhibition of oxidative stress, inhibition of glutamate release, and anti-apoptosis. (C) pifithrin-a (PFT-a): We found that delayed treatment with a p53 inhibitor PFT-a is able to modify stroke-induced endogenous neurogenesis and improve the functional recovery in stroke animals. Adult rats were subjected to a 90-min middle cerebral artery occlusion (MCAo). A p53 inhibitor pifithrin- (PFT-a) was administrated to stroke rats from days 6 to 9 after MCAo. Locomotor behavior was measured using an infra-red activity chamber. Proliferation, survival, migration, and differentiation of endogenous NPCs were examined using RTPCR, TUNEL, and immunohistochemistry. We found that PFT-a enhanced the functional recovery as assessed by a significant increase in multiple behavioral measurements. Delayed PFT-a treatment had no effect on the cell death process in the lesioned cortical region. However, it enhanced the survival of SVZ progenitor cells and promoted their proliferation and migration. PFT- inhibited the expression of a p53-dependent pro-apoptotic gene, termed PUMA (p53-upregulated modulator of apoptosis), within the SVZ of stroke animals. The enhancement of survival/proliferation of NPCs was further found in SVZ neurospheres in tissue culture. PFT-a dose-dependently increased the numberand size of new neurosphere formation. Our data suggest that delayed treatment with a p53 inhibitor PFT-a is able to modify stroke-induced endogenous neurogenesis and improve the functional recovery in stroke animals. (D) Trophic factors: Previous studies have indicated that methamphetamine (MA) potentiates neurodegeneration induced by ischemia in brain. We, and others, have reported that bone morphogenetic protein 7 (BMP7) is protective against MA and ischemic brain injury. The purpose of this study is to examine whether BMP7 reduces synergistic injury induced by both MA and cerebral ischemia. Adult CD-1 mice were treated with MA (10 mg/kg x 4, each dose two hours apart) or saline. Using the quantitative real time polymerase chain reaction, we found that MA suppressed the expression of BMP7 mRNA in the cerebral cortex one day after injection. Ischemic and reperfusional injuries were introduced by ligation of the right middle cerebral artery for 90 min after MA injection. Animals were sacrificed for caspase 3/7 activity assay and tri-phenyl-tetrazolium chloride staining at 1 hour and 2 days after reperfusion, respectively. Cerebral infarction and caspase-3/7 activity were enhanced in the stroke animals pretreated with MA;both responses were attenuated by pretreatment with BMP7. In conclusion, our data suggest that MA facilitates cerebral infarction after ischemia possibly mediated, in part, through the suppression of BMP7. (E) Retinoic acid (RA) is a biologically active derivative of vitamin A. Two isomers of RA, 9-cis-RA (9cRA) and all-trans-RA (atRA) have been identified. atRA is normally present at high levels in the developing spinal cord and at low levels in the forebrain of mouse embryos. RAs interact with two major groups of nuclear receptors: retinoic acid receptors (RAR) and retinoid X receptors (RXR). RXR forms heterodimers with RAR (RXR/RAR). 9cRA binds with high affinity to RXR , whereas both 9cRA and atRA activate RAR. Several physiological responses to RA have been identified. RA inhibited H2O2-induced apoptosis via suppression of c-fos/c-jun expression and JNK activation in mesangial cells and increased survival during anoxia/glucose deprivation in PC12 cells. Pretreatment with docosahexaenoic acid, a candidate ligand for RXR, reduced cerebral infarction induced by MCAo. We found that 9cRA, given one day before MCAo, increased locomotor activity, attenuated neurological deficits, and reduced cerebral infarction and TUNEL labeling in stroke rats. The protective response of 9cRA can be antagonized by bone morphogenetic protein antagonist noggin. These data suggest that RA can induce protective responses in cultured cells and in vivo. (F) (-)Naloxone:Neurodegeneration can occur through the activation of inflammatory processes. In the brain, microglia serve as the resident immune system cells and can be activated to respond to toxicity or damage that occurs in neurological diseases or drug-induced toxicity. A few days after a stroke, microglia become activated and produce signals that trigger an immune response which can exacerbate the deleterious effects of the stroke. The toll-like receptor 4 or TLR4 is present on microglia and activates them in response to toxicity. Recently, the drug naloxone has been shown to bind TLR4 and reduce microglial activation. The (-) naloxone form is used to treat opioid dependence but produces side effects. The (+)naloxone form does not interact with opioid receptor but does interact with TLR4. Therefore, we propose to determine whether the (+)naloxone form can improve outcome when administered following stroke in attempts to identify a novel therapy for stroke that would not have the side effects of (-)naloxone. Furthermore, these studies would investigate the molecular mechanism of naloxone, TLR4 and microglial activation by stroke.

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