We report here neuroprotective and anti-inflammatory effects of a flavonoid-enriched fraction isolated from the peel of Northern Spy apples (AF4) in a mouse of model of hypoxic-ischemic (HI) brain damage. Oral administration of AF4 (50 mg/kg, once daily for 3 days) prior to 50 min of HI completely prevented motor performance deficits assessed 14 days later that were associated with marked reductions in neuronal cell loss in the dorsal hippocampus and striatum. Pre-treatment with AF4 (5, 10, 25 or 50 mg/kg, p.o.; once daily for 3 days) produced a dose-dependent reduction in HI-induced hippocampal and striatal neuron cell loss, with 25 mg/kg being the lowest dose that achieved maximal neuroprotection. Comparison of the effects of 1, 3 or 7 doses of AF4 (25 mg/kg; p.o.) prior to HI revealed that at least 3 doses of AF4 were required before HI to reduce neuronal cell loss in both the dorsal hippocampus and striatum. Quantitative RT-PCR measurements revealed that the neuroprotective effects of AF4 (25 mg/kg; p.o.; once daily for 3 days) in the dorsal hippocampus were associated with a suppression of HI-induced increases in the expression of IL-1β, TNF-α and IL-6. AF4 pre-treatment enhanced mRNA levels for pro-survival proteins such as X-linked inhibitor of apoptosis and erythropoietin following HI in the dorsal hippocampus and striatum, respectively. Primary cultures of mouse cortical neurons incubated with AF4 (1 µg/ml), but not the same concentrations of either quercetin or quercetin-3-O-glucose or its metabolites, were resistant to cell death induced by oxygen glucose deprivation. These findings suggest that the inhibition of HI-induced brain injury produced by AF4 likely involves a transcriptional mechanism resulting from the co-operative actions of various phenolics in this fraction which not only reduce the expression of pro-inflammatory mediators but also enhance pro-survival gene signalling.

STOP (stable tubule only polypeptide) null mice display neurochemical and behavioral abnormalities that resemble several well-recognized features of schizophrenia. Recent evidence suggests that the hematopoietic growth factor erythropoietin improves the cognitive performance of schizophrenics. The mechanism, however, by which erythropoietin is able to improve the cognition of schizophrenics is unclear. To address this question, we first determined whether acute administration of the erythropoietin analog known as darbepoetin alpha (D. alpha) improved performance deficits of STOP null mice in the novel objective recognition task (NORT). NORT performance of STOP null mice, but not wild-type littermates, was enhanced 3 h after a single injection of D. alpha (25 microg/kg, i.p.). Improved NORT performance was accompanied by elevated NADPH diaphorase staining in the ventral hippocampus as well as medial and cortical aspects of the amygdala, indicative of increased nitric oxide synthase (NOS) activity in these structures. NOS generates the intracellular messenger nitric oxide (NO) implicated in learning and memory. In keeping with this hypothesis, D. alpha significantly increased NO metabolite levels (nitrate and nitrite, NOx) in the hippocampus of both wild-type and STOP null mice. The NOS inhibitor, N (G)-nitro-L- arginine methyl ester (L-NAME; 25 mg/kg, i.p.), completely reversed the increase in hippocampal NOx levels produced by D. alpha. Moreover, L-NAME also inhibited the ability of D. alpha to improve the NORT performance of STOP null mice. Taken together, these observations suggest D. alpha enhances the NORT performance of STOP null mice by increasing production of NO.

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Erythropoietin has been reported to improve the behavioral performance of healthy mice in tests thought to depend on synaptic plasticity in the CA1 region of the hippocampus. We show here for the first time that a single injection of the erythropoietin analog darbepoetin alfa reverses pre-existing cognitive deficits in adult rats that had been subjected to transient global ischemia produced by four-vessel occlusion (4-VO). Quantification of neuronal density demonstrated that 12 min of 4-VO selectively killed more than 90% of CA1 neurons in the dorsal hippocampus. Rats that had sustained a bilateral loss of hippocampal CA1 neurons in this range (4-VO rats) displayed more errors and longer escape latencies in the Barnes maze compared with sham-operated controls. A single injection of darbepoetin alfa (5000 U/kg i.p.) 4 h before behavioral testing decreased deficits in escape latency for 4-VO rats but not sham-operated controls. This improvement in spatial working memory performance was correlated with increased levels of nitric-oxide metabolites in the ventral hippocampus. Systemic administration of the nitric-oxide synthase inhibitor N(G)-nitro-L-nitro-arginine methyl ester reversed the increase in nitric-oxide metabolites and improvements in spatial working memory produced by darbepoetin alfa (5000 U/kg, i.p.) at a dose (10 mg/kg, i.p.) that did not impair the spatial working memory performance of intact rats. Taken together, these findings suggest that darbepoetin alfa reverses pre-existing spatial working memory deficits resulting from transient global ischemia by increasing the activity of nitric-oxide synthase, an enzyme implicated in synaptic plasticity.

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