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. 2012;7(12):e51324.
doi: 10.1371/journal.pone.0051324. Epub 2012 Dec 12.

Neuroprotective and anti-inflammatory effects of the flavonoid-enriched fraction AF4 in a mouse model of hypoxic-ischemic brain injury

Paul G W Keddy  1 , Kate Dunlop, Jordan Warford, Michel L Samson, Quinton R D Jones, H P Vasantha Rupasinghe, George S Robertson
Affiliations

Neuroprotective and anti-inflammatory effects of the flavonoid-enriched fraction AF4 in a mouse model of hypoxic-ischemic brain injury

Paul G W Keddy et al. PLoS One. 2012.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Motor performance scores.
Animals were treated orally with vehicle (10 ml/kg/day for 3 days) or AF4 (50 mg/kg/day for 3 days) and subjected to 50 min of unilateral forebrain hypoxia-ischemia (HI). The amount of time spent on the rotarod was recorded as a measure of performance, with longer times indicative of better motor performance. An overall score was calculated by taking the difference between the average of 3 trials on the rotarod day 14 post-HI and 24 h pre-HI. Relative to mice that were given vehicle (10 ml/kg/day for 3 days, n = 20), animals which received AF4 (50 mg/kg/day for 3 days, n = 20) displayed superior motor performance 2 weeks after HI. *p<0.001 relative to vehicle, Mann Whitney U test (two tailed).
Figure 2
Figure 2. Brain injury and hemispheric volume loss.
Representative Nissl-stained brain sections from two animals in the motor performance study (A and B). Note the loss of tissue (arrows) in brain sections from animals treated with vehicle (A and B, left panels) and protection produced by AF4 (A and B, right panels). Volumetric measurements from the mice used in the motor performance study (C). A Mann-Whitney U test revealed that mice which received AF4 (50 mg/kg, p.o.; once a day for 3 days) displayed considerably less brain damage than vehicle (10 ml/kg, p.o.; once a day for 3 days) treated mice. *p<0.001 relative to vehicle.
Figure 3
Figure 3. Dose-dependent reductions of HI-induced hippocampal neuron loss produced by oral administration of AF4.
Five groups of adult male C57Bl/6 mice received either water (10 ml/kg, p.o.) or AF4 (5, 10, 25 or 50 mg/kg, p.o.) once daily for 3 days followed by 50 min of unilateral forebrain hypoxia-ischemia (HI) (left hemisphere, panels A, C, E, G, I) 24 h after the last dose. Animals were killed 2 weeks later and brain sections processed for immunohistochemical detection of the neuron specific marker NeuN. Cell counts revealed that neuroprotection was achieved by the 25 mg/kg dosing regime of AF4 and that increasing the dose of AF4 to 50 mg/kg did not produce a further reduction in neuronal loss in this structure (F). *p<0.05 versus vehicle and AF4 (5 mg/kg). No other comparisons were significantly different. AVONA followed by Bonferroni tests.
Figure 4
Figure 4. Dose-dependent reductions of HI-induced striatal neuron loss produced by oral administration of AF4.
Five groups of adult male C57Bl/6 mice received either water (10 ml/kg, p.o.) or AF4 (5, 10, 25 or 50 mg/kg, p.o.) once daily for 3 days followed by 50 min of unilateral forebrain hypoxia-ischemia (HI) (left hemisphere, panels A, C, E, G, I) 24 h after the last dose. Animals were killed 2 weeks later and brain sections processed for immunohistochemical detection of the neuron specific marker NeuN. Cell counts revealed that neuroprotection was achieved by the 25 mg/kg dosing regime of AF4 and that increasing the dose of AF4 to 50 mg/kg did not produce a further reduction in neuronal loss in this structure (F). *p<0.05 versus vehicle and AF4 (5 mg/kg). No other comparisons were significantly different. AVONA followed by Bonferroni tests.
Figure 5
Figure 5. Effects of 1, 3 or 7 pre-doses of AF4 on HI-induced neuron loss in the striatum and hippocampus.
Four groups, composed of 8–10 adult male C57Bl/6 mice each, were dosed orally (p.o.) once a day with water (vehicle, 10 ml/kg) or AF4 (25 mg/kg) for 1, 3, or 7 days. Twenty-four hours after the final dose of AF4 or vehicle all animals were subjected to 50 min of unilateral hypoxia-ischemia and sacrificed 2 weeks later. Brains sections from these animals were processed immunohistochemically to visualize the neuron specific marker NeuN in the striatum and hippocampus. Cell counts revealed that neuroprotection was achieved by 3 pre-doses of AF4 and that increasing the number of pre-doses to 7 did not produce a further reduction in brain injury. *p<0.05 versus vehicle and 1 pre-dose. No other comparisons were significantly different. AVONA followed by Bonferroni tests.
Figure 6
Figure 6. Effects of 7 days of AF4 pre-dosing on HI-induced pro-inflammatory gene expression in the hippocampus.
Four groups of mice were pre-dosed with vehicle (Veh, 10 ml/kg/day for 7 days) or AF4 (AF4, 25 mg/kg/day for 7 days) and subjected to sham treatment (Sham; Veh-Sham, AF4-Sham) or unilateral forebrain hypoxia-ischemia (HI) (Veh-HI, AF4-HI) (A–F). The ipsilateral dorsal hippocampus was harvested 6 hours later. Fold increases in mRNAs encoding TNF-α, IL-1β, IL-6, IκBα, TLR2 and TLR4 were determined by qRT-PCR, n = 6 for each group. *p<0.05 relative to all other groups. ★p<0.05 relative to Veh-Sham and AF4-Sham. No other comparisons were significantly different. AVONA followed by Bonferroni tests.
Figure 7
Figure 7. Effects of 7 days of AF4 pre-dosing on anti-apoptotic gene expression in the hippocampus following HI.
Four groups of mice were pre-dosed with vehicle (Veh, 10 ml/kg/day for 7 days) or AF4 (AF4, 25 mg/kg/day for 7 days) and subjected to sham treatment (Sham; Veh-Sham, AF4-Sham) or unilateral forebrain hypoxia-ischemia (HI) (Veh-HI, AF4-HI) (A–D). The ipsilateral dorsal hippocampus was harvested 6 hours later. Fold increases in mRNAs encoding Bcl-2, cIAP1, cIAP2 and XIAP were determined by qRT-PCR, n = 6 for each group. A,*p<0.05 relative to all other groups. C and D, *p<0.05 relative to Veh-Sham and AF4-Sham. No other comparisons were significantly different. AVONA followed by Bonferroni tests.
Figure 8
Figure 8. Effects of 7 days of AF4 pre-dosing on erythropoietin gene expression in the hippocampus and striatum 1 or 6 h following HI.
Four groups of mice were pre-dosed with vehicle (Veh, 10 ml/kg/day for 7 days) or AF4 (AF4, 25 mg/kg/day for 7 days) and subjected to sham treatment (Sham; Veh-Sham, AF4-Sham) or unilateral forebrain hypoxia-ischemia (HI) (Veh-HI, AF4-HI) (A–D). The ipsilateral dorsal hippocampus (A and B) and striatum (C and D) were harvested from these animals 1 h (A and C) or 6 h (B and D) later. Fold increases in mRNAs encoding erythropoietin (EPO) were determined by qRT-PCR, n = 6 for each group. B and D, *p<0.05 relative to all other groups. ★p<0.05 relative to all other groups. No other comparisons were significantly different. AVONA followed by Bonferroni tests.
Figure 9
Figure 9. AF4 attenuated OGD-induced death of mouse primary cortical neuron cultures.
Percentage of total possible LDH release from mouse primary cortical cultures treated with vehicle (0.1% DMSO), quercetin, quercetin-3-O-glucoside (Q3G) or quercetin metabolites [Q-3′-O-sulphate (Q3′S), Q-3-O-glucuronic acid (Q3GluA), isorhamnetin-3-glucuronic acid (IR3GluA)] or AF4 (n = 12) at a concentration of 1.0 µg/ml and subjected to 12 h of OGD. Treatment with AF4 but not quercetin, quercetin metabolites or cyclopentyl adenosine (CPA) showed a significant reduction in % LDH release in comparison to the vehicle treatment group at a concentration of 1.0 µg/ml. *p<0.05 versus all other groups. No other comparisons were significantly different. AVONA followed by Bonferroni tests.
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