Jenny Chou

Dietary supplementation with blueberries, spinach, or spirulina reduces ischemic brain damage

Free radicals are involved in neurodegenerative disorders, such as ischemia and aging. We have previously demonstrated that treatment with diets enriched with blueberry, spinach, or spirulina have been shown to reduce neurodegenerative changes in aged animals. The purpose of this study was to determine if these diets have neuroprotective effects in focal ischemic brain. Adult male Sprague-Dawley rats were fed with equal amounts of diets (blueberry, spinach, and spirulina) or with control diet. After 4 weeks of feeding, all animals were anesthetized with chloral hydrate. The right middle cerebral artery was ligated with a 10-O suture for 60 min. The ligature was later removed to allow reperfusional injury. Animals were sacrificed and brains were removed for caspase-3 enzymatic assays and triphenyltetrazolium chloride staining at 8 and 48 h after the onset of reperfusion. A subgroup of animals was used for locomotor behavior and biochemical assays. We found that animals which received blueberry, spinach, or spirulina enriched diets had a significant reduction in the volume of infarction in the cerebral cortex and an increase in post-stroke locomotor activity. There was no difference in blood biochemistry, blood CO2, and electrolyte levels among all groups, suggesting that the protection was not indirectly mediated through the changes in physiological functions. Animals treated with blueberry, spinach, or spirulina had significantly lower caspase-3 activity in the ischemic hemisphere. In conclusion, our data suggest that chronic treatment with blueberry, spinach, or spirulina reduces ischemia/reperfusion-induced apoptosis and cerebral infarction.

Astaxanthin reduces ischemic brain injury in adult rats

Astaxanthin (ATX) is a dietary carotenoid of crustaceans and fish that contributes to their coloration. Dietary ATX is important for development and survival of salmonids and crustaceans and has been shown to reduce cardiac ischemic injury in rodents. The purpose of this study was to examine whether ATX can protect against ischemic injury in the mammalian brain. Adult rats were injected intracerebroventricularly with ATX or vehicle prior to a 60‐min middle cerebral artery occlusion (MCAo). ATX was present in the infarction area at 70‐75 min after onset of MCAo. Treatment with ATX, compared to vehicle, increased locomotor activity in stroke rats and reduced cerebral infarction at 2 d after MCAo. To evaluate the protective mechanisms of ATX against stroke, brain tissues were assayed for free radical damage, apoptosis, and excitoxicity. ATX antagonized ischemia‐mediated loss of aconitase activity and reduced glutamate release, lipid peroxidation, translocation of cytochrome c, and TUNEL labeling in the ischemic cortex. ATX did not alter physiological parameters, such as body temperature, brain temperature, cerebral blood flow, blood gases, blood pressure, and pH. Collectively, our data suggest that ATX can reduce ischemia‐related injury in brain tissue through the inhibition of oxidative stress, reduction of glutamate release, and antiapoptosis. ATX may be clinically useful for patients vulnerable or prone to ischemic events.—Shen, H., Kuo, C.‐C., Chou, J., Delvolve, A., Jackson, S.N., Post, J., Woods, A.S., Hoffer, B.J., Wang, Y., Harvey, B.K. Astaxanthin reduces ischemic brain injury in adult rats. FASEB J. 23, 1958–1968 (2009)

Delayed treatment with a p53 inhibitor enhances recovery in stroke brain

Cerebral ischemia can activate endogenous reparative processes, such as proliferation of endogenous neural progenitor cells (NPCs) in the subventricular zone (SVZ). Most of these new cells die shortly after injury. The purpose of this study was to examine a novel strategy for treatment of stroke at 1 week after injury by enhancing the survival of ischemia‐induced endogenous NPCs in SVZ.

Neuroregenerative effects of BMP7 after stroke in rats.

Previous reports have indicated that the expression of bone morphogenetic protein-7 (BMP7) is enhanced after ischemic injury in brain. This upregulation may induce endogenous neurorepair in the ischemic brain. The purpose of this study was to examine neuroregenerative effects of BMP7 after ischemia-reperfusion injury. Adult Sprague-Dawley rats were anesthetized with chloral hydrate. Right middle cerebral artery (MCA) was transiently ligated with 10-O suture for 1 h. One day after MCA occlusion, vehicle or BMP7 was infused to the contralateral cerebral ventricle. To identify possible neurogenesis, bromodeoxyurindine (BrdU) was systemically injected on the fourth and fifth days after MCA occlusion. Animals treated with BMP7 showed a rapid correction of body asymmetry and neurological deficits, suggesting BMP7 facilitates recovery after stroke. Animals were sacrificed at 1 month after stroke and brains were analyzed using immunohistological techniques. BMP7 treatment enhanced immunoreactivity of BrdU in the subventricular zone, lesioned cortex, and corpus callosum. These BrdU-positive cells co-labeled with nestin and NeuN. Our behavioral and anatomical data suggest that BMP7 promotes neuroregeneration in stroke animals, possibly through the proliferation of new neuronal precursors after ischemia.

Activation of adenosine A3 receptors reduces ischemic brain injury in rodents

Adenosine A3 receptor (A3R) agonists have been shown to reduce cardiac and lung injury, but the protective roles of A3R agonists in the CNS are not well characterized. The protective effect of selective A3R agonist chloro‐N6‐(3‐iodo‐benzyl)‐adenosine‐5′‐N‐methyluronamide (Cl‐IB‐MECA) was first examined in primary cortical cultures. In cortical culture, Cl‐IB‐MECA pretreatment antagonized the hypoxia‐mediated decrease in cell viability. In vivo, Cl‐IB‐MECA or vehicle was given intracerebroventricularly or intravenously to anesthetized rats. Animals were subjected to focal cerebral ischemia induced by transient middle cerebral artery (MCA) ligation. Intracerebroventricular or repeated intravenous administration (i.e., at 165 min and 15 min before MCA ligation) of Cl‐IB‐MECA did not alter blood pressure during ischemia but increased locomotor activity and decreased cerebral infarction 2 days after. In these animals, Cl‐IB‐MECA also reduced the density of TUNEL labeling in the lesioned cortex. The possibility of endogeneous neuroprotection was further examined in A3R knockout mice. After MCA ligation, an increase in cerebral infarction was found in the A3R knockouts compared with the A3R wild‐type controls, suggesting that A3Rs are tonically activated during ischemia. Additionally, intracerebroventricular pretreatment with Cl‐IB‐MECA decreased the size of infarction in the wild‐type controls, but not in the A3R knockout animals, suggesting that Cl‐IB‐MECA‐induced protection was mediated through the A3 receptors. Collectively, these data suggest that Cl‐IB‐MECA reduced cerebral infarction through the activation of A3Rs and suppression of apoptosis.

CDNF Protects the Nigrostriatal Dopamine System and Promotes Recovery after MPTP Treatment in Mice

Cerebral dopamine neurotrophic factor (CDNF) is a recently discovered protein, which belongs to the evolutionarily conserved CDNF/MANF family of neurotrophic factors. The degeneration of dopamine neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment is well characterized, and efficacy in this model is considered a standard criterion for development of parkinsonian therapies. MPTP is a neurotoxin, which produces parkinsonian symptoms in humans and in C57/Bl6 mice. To date, there are no reports about the effects of CDNF on dopamine neuron survival or function in the MPTP rodent model, a critical gap. Therefore, we studied whether CDNF has neuroprotective and neurorestorative properties for the nigrostriatal dopamine system after MPTP injections in C57/Bl6 mice. We found that bilateral striatal CDNF injections, given 20 h before MPTP, improved horizontal and vertical motor behavior. CDNF pretreatment increased tyrosine hydroxylase (TH) immunoreactivity in the striatum and in the substantia nigra pars reticulata (SNpr), as well as the number of TH-positive cells in substantia nigra pars compacta (SNpc). Posttreatment with CDNF, given 1 week after MPTP injections, increased horizontal and vertical motor behavior of mice, as well as dopamine fiber densities in the striatum and the number of TH-positive cells in SNpc. CDNF did not alter any of the analyzed dopaminergic biomarkers or locomotor behavior in MPTP-untreated animals. We conclude that striatal CDNF administration is both neuroprotective and neurorestorative for the TH-positive cells in the nigrostriatal dopamine system in the MPTP model, which supports the development of CDNF-based treatment for Parkinsons disease.

Bone morphogenetic proteins are involved in fetal kidney tissue transplantation-induced neuroprotection in stroke rats

Both bone morphogenetic proteins (BMPs) and glial cell line-derived neurotrophic factor (GDNF) reduce ischemia-induced cerebral injury in rats. Intracerebral transplantation of fetal kidney tissue, which normally expresses BMPs and GDNF during development, reduces ischemic injury in cerebral cortex. In this study, we tested the hypothesis that BMP is involved in this neuroprotective response. Fetal kidney tissue was cut into small pieces and transplanted into cortical areas adjacent to the right middle cerebral artery (MCA) in adult rats. In situ hybridization of brain indicated that these fetal kidney transplants contained high levels of BMP-7 mRNA three days after grafting. Immunohistochemical analysis of grafted brain showed co-localization of BMP-7 and PAX-2 immunoreactivity in the graft, suggesting that these transplants contained BMP protein. Some animals were grafted with fetal kidney tissue after intraventricular administration (ICV) of the BMP antagonist noggin (1 micro g) or after vehicle, followed by MCA ligation for 60 min. Animals receiving fetal kidney tissue transplantation developed significantly less body asymmetry, as compared to stroke animals that either did not receive transplantation or received fetal kidney grafts and noggin pretreatment. Analysis of these brains after triphenyltetrazolium chloride staining showed that fetal kidney tissue transplantation reduced the volume of infarction in the cerebral cortex. Noggin pretreatment reduced the protection induced by fetal kidney grafting, although noggin itself did not cause increase in cerebral infarction. Eight hours after ischemia, brain homogenates were obtained from grafted and control animals to assay caspase-3 enzymatic activity. This analysis demonstrated that fetal kidney grafts significantly reduced ischemia-induced caspase-3 activity. Reduction of caspase-3 activity could also be antagonized by noggin pretreatment. In conclusion, our data suggest that fetal kidney transplantation reduces ischemia/reperfusion-induced cortical infarction and behavioral deficits in adult rats, which are, at least partially, mediated through the effect of BMPs from the transplants.

Bone morphogenetic protein-7 reduces toxicity induced by high doses of methamphetamine in rodents.

Methamphetamine (MA) is a drug of abuse as well as a dopaminergic neurotoxin. We have previously demonstrated that pretreatment with bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinsons disease. In this study, we examined the neuroprotective effects of BMP7 against MA-mediated toxicity in dopaminergic neurons. Primary dopaminergic neurons, prepared from rat embryonic ventral mesencephalic tissue, were treated with MA. High doses of MA decreased tyrosine hydroxylase immunoreactivity (THir) while increasing terminal deoxynucleotidyl transferase-mediated dNTP nick end labeling. These toxicities were significantly antagonized by BMP7. Interaction of BMP7 and MA in vivo was first examined in CD1 mice. High doses of MA (10 mg/kgx4 s.c.) significantly reduced locomotor activity and THir in striatum. I.c.v. administration of BMP7 antagonized these changes. In BMP7 +/- mice, MA suppressed locomotor activity and reduced TH immunoreactivity in nigra reticulata to a greater degree than in wild type BMP7 +/+ mice, suggesting that deficiency in BMP7 expression increases vulnerability to MA insults. Since BMP7 +/- mice also carry a LacZ-expressing reporter allele at the BMP7 locus, the expression of BMP7 was indirectly measured through the enzymatic activity of beta-galactosidase (beta-gal) in BMP7 +/- mice. High doses of MA significantly suppressed beta-gal activity in striatum, suggesting that MA may inhibit BMP7 expression at the terminals of the nigrostriatal pathway. A similar effect was also found in CD1 mice in that high doses of MA suppressed BMP7 mRNA expression in nigra. In conclusion, our data indicate that MA can cause lesioning in the nigrostriatal dopaminergic terminals and that BMP7 is protective against MA-mediated neurotoxicity in central dopaminergic neurons.

Neurotrophic effects of bone morphogenetic protein-7 in a rat model of Parkinson's disease.

Previous studies have demonstrated that pretreatment with bone morphogenetic protein-7 (BMP7) reduces ischemic neuronal injury in vivo. Moreover, exogenous application of BMP7 increases both the number of tyrosine hydroxylase (+) cells and dopamine (DA) uptake in rat mesencephalic cell cultures. The purpose of this study was to investigate the in vivo effects of BMP7 on 6-hydroxydopamine (6-OHDA) induced lesioning of midbrain DA neurons. Adult Fischer 344 rats were anesthetized and injected with BMP7 or vehicle into the left substantia nigra, followed by local administration of 9 microg of 6-OHDA into the left medial forebrain bundle. The lesioned animals that received BMP7 pretreatment, as compared to vehicle/6-OHDA controls, had a significant reduction in methamphetamine-induced rotation 1 month after the surgery. BMP7-pretreatment partially preserved KCl-induced dopamine release in the lesioned striatum and significantly increased TH immunoreactivity in the lesioned nigra and striatum. In summary, our data suggest that BMP7 has neuroprotective and/or neuroreparative effects against 6-OHDA lesioning of the nigrostriatal DA pathway in an animal model of Parkinsons disease (PD).

Methamphetamine-induced dopamine-independent alterations in striatal gene expression in the 6-hydroxydopamine hemiparkinsonian rats.

Unilateral injections of 6-hydroxydopamine into the medial forebrain bundle are used extensively as a model of Parkinsons disease. The present experiments sought to identify genes that were affected in the dopamine (DA)–denervated striatum after 6-hydroxydopamine-induced destruction of the nigrostriatal dopaminergic pathway in the rat. We also examined whether a single injection of methamphetamine (METH) (2.5 mg/kg) known to cause changes in gene expression in the normally DA-innervated striatum could still influence striatal gene expression in the absence of DA. Unilateral injections of 6-hydroxydopamine into the medial forebrain bundle resulted in METH-induced rotational behaviors ipsilateral to the lesioned side and total striatal DA depletion on the lesioned side. This injection also caused decrease in striatal serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels. DA depletion was associated with increases in 5-HIAA/5-HT ratios that were potentiated by the METH injection. Microarray analyses revealed changes (± 1.7-fold, p<0.025) in the expression of 67 genes on the lesioned side in comparison to the intact side of the saline-treated hemiparkinsonian animals. These include follistatin, neuromedin U, and tachykinin 2 which were up-regulated. METH administration caused increases in the expression of c-fos, Egr1, and Nor-1 on the intact side. On the DA-depleted side, METH administration also increased the expression of 61 genes including Pdgf-d and Cox-2. There were METH-induced changes in 16 genes that were common in the DA-innervated and DA-depleted sides. These include c-fos and Nor-1 which show greater changes on the normal DA side. Thus, the present study documents, for the first time, that METH mediated DA-independent changes in the levels of transcripts of several genes in the DA-denervated striatum. Our results also implicate 5-HT as a potential player in these METH-induced alterations in gene expression because the METH injection also caused significant increases in 5-HIAA/5-HT ratios on the DA-depleted side.