(1) Diadenosine tetraphosphate (AP4A). The purpose of this study was to determine whether AP4A is protective against methamphetamine (MA) -mediated toxicity in dopaminergic neurons in vitro and in vivo. Primary neuronal cultures were prepared from rat embryonic (E15) ventral mesencephalic tissue. Cells 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 also examined in vivo. Adult Sprague Dawley rats were injected with AP4A or vehicle intracerebroventricularly followed by 4 doses of MA (5mg/kg). 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. (2) Astaxanthin (ATX). We found that ATX has protective effects against ischemic brain injury. ATX or vehicle was administered intracerebroventricularly 10-20 minutes prior to a 60-min middle cerebral artery occlusion (MCAo) in adult rats. At 2 days after 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 -mediated 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. (3) pifithrin-a (PFT-a): (A) We found that PFTa has protective against MA mediated injury in dopaminergic neurons. High dose of MA reduced TH immunoreactivity and fiber density in primary dopaminergic neuronal culture. Co-treatment with PFTa significantly attenuated these degenerative changes. Our data suggest that treatment with a p53 inhibitor or suppression of p53 expression limits neuronal dysfunction and cell death induced by exposure to dopaminergic neurotoxins. (B) PFT-a enhanced the survival of neural progenitor cells (NPCs) both in vivo and in vitro. The enhancement of survival and proliferation of NPCs was first examined in SVZ neurospheres in tissue culture. PFT-a dose-dependently increased the number and size of new neurosphere formation. Delayed treatment (i.e. days 6 to 9 after injury) with PFT-a enhanced survival of endogenous neural progenitor cells (NPCs) in the subventricular zone in adult rats. PFTa enhanced proliferation, survival, migration, and differentiation of endogenous NPCs and enhanced the functional recovery in locomotor behavior in stroke animals. PFT- inhibited the expression of a p53-dependent pro-apoptotic gene, termed PUMA (p53-upregulated modulator of apoptosis), within the SVZ of stroke animals. Our data suggest that delayed treatment with a p53 inhibitor PFT-a is able to modify endogenous neurogenesis and improve the functional recovery after brain injury. (c) Systemic administration of PFT- enhanced the survival of dopaminergic neuronal transplants in vivo. PFT- treatment suppressed cell death, increased survival of TH (+) cells, enhanced TH neurite outgrowth from VM grafts, and augmented behavioral recovery in Parkinsonian rats. In conclusion, our data suggest that activation of p53 is an important mediator for cell death after injury or during neural repair. Transient suppression of p53 may increase the survival of neurons or NPCs after brain injury. (4) Retinoic acid (RA): Pretreatment with 9 cis RA (9cRA) increased locomotor activity, attenuated neurological deficits, and reduced cerebral infarction and TUNEL labeling in stroke rats. 9cRA increased the expression of bone morphogenetic protein 7 (BMP7) in brain. The protective response of 9cRA can be antagonized by BMP antagonist noggin. Our data suggest that RA can induce protective responses in cultured cells and in vivo, possibly through BMP7 mechanism. (5) (-)Naloxone: Neurodegeneration can occur through the activation of inflammatory processes. From experiments conducted this past year, we have observed improved behavioral recovery (body asymmetery and neurological score) following stroke when (+)naloxone was administered daily for 1 week starting one day after middle cerebral artery occlusion in rats. We are currently following up these studies comparing (-)naloxone as well as examining changes in markers of inflammation that are upregulated following ischemia.
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