Grape powder intake prevents ovariectomy-induced anxiety-like behavior, memory impairment and high blood pressure in female Wistar rats

Gaurav Patki, Farida H Allam, Fatin Atrooz, An T Dao, Naimesh Solanki, Gaurav Chugh, Mohammad Asghar, Faizan Jafri, Ritu Bohat, Karim A Alkadhi, Samina Salim, Gaurav Patki, Farida H Allam, Fatin Atrooz, An T Dao, Naimesh Solanki, Gaurav Chugh, Mohammad Asghar, Faizan Jafri, Ritu Bohat, Karim A Alkadhi, Samina Salim

Abstract

Diminished estrogen influence at menopause is reported to be associated with cognitive decline, heightened anxiety and hypertension. While estrogen therapy is often prescribed to overcome these behavioral and physiological deficits, antioxidants which have been shown beneficial are gaining nutritional intervention and popularity. Therefore, in the present study, utilizing the antioxidant properties of grapes, we have examined effect of 3 weeks of grape powder (GP; 15 g/L dissolved in tap water) treatment on anxiety-like behavior, learning-memory impairment and high blood pressure in ovariectomized (OVX) rats. Four groups of female Wistar rats were used; sham control, sham-GP treated, OVX and OVX+GP treated. We observed a significant increase in systolic and diastolic blood pressure in OVX rats as compared to sham-controls. Furthermore, ovariectomy increased anxiety-like behavior and caused learning and memory impairment in rats as compared to sham-controls. Interestingly, providing grape powder treated water to OVX rats restored both systolic and diastolic blood pressure, decreased anxiety-like behavior and improved memory function. Moreover, OVX rats exhibited an impaired long term potentiation which was restored with grape powder treatment. Furthermore, ovariectomy increased oxidative stress in the brain, serum and urine, selectively decreasing antioxidant enzyme, glyoxalase-1 protein expression in the hippocampus but not in the cortex and amygdala of OVX rats, while grape powder treatment reversed these effects. Other antioxidant enzyme levels, including manganese superoxide dismutase (SOD) and Cu/Zn SOD remained unchanged. We suggest that grape powder by regulating oxidative stress mechanisms exerts its protective effect on blood pressure, learning-memory and anxiety-like behavior. Our study is the first to examine behavioral, biochemical, physiological and electrophysiological outcome of estrogen depletion in rats and to test protective role of grape powder, all in the same study.

Conflict of interest statement

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

Figures

Figure 1. General body parameters.
Figure 1. General body parameters.
Four groups of female Wistar rats were utilized in this study, control (sham surgery) rats provided with tap water for drinking or grape powder (GP) treated tap water, ovariectomized rats (surgical removal of ovaries, OVX) provided with drinking water, or with GP treated tap water. Daily food intake (A) water intake (B) body weight (C) was measured in all groups. Estradiol levels were measured in plasma using Estradiol EIA Kit (D). Bars are means ± SEM, n = 10 rats/group, P<0.05, (*) significantly different from control and GP.
Figure 2. Analysis of marker of oxidative…
Figure 2. Analysis of marker of oxidative stress 8-isoprostane in CON, GP, OVX and OVX+GP rats.
8-isoprostane was measured in serum (A) and urine (B) using EIA kit. Bars are means ± SEM, n = 10 rats/group, P<0.05, (*) significantly different from control and GP and (#) significantly different from OVX. Group designations: CON (control; sham operated rats provided with tap water for drinking), GP (grape powder: sham operated rats provided with GP treated tap water for drinking), OVX (ovariectomized: rats subjected to surgical removal of ovaries and provided with tap water for drinking), OVX+GP (rats subjected to surgical removal of ovaries and provided with GP treated tap water for drinking).
Figure 3. Analysis of protein carbonylation and…
Figure 3. Analysis of protein carbonylation and nitrotyrosinylation in CON, GP, OVX and OVX+GP rats.
The OxyBlot™ Protein Oxidation Detection Kit was used for immunoblot detection of carbonyl. Equal amount (20 µg) of protein from different brain regions were subjected to this kit based reaction by following manufacturer’s instructions. Brain homogenates from hippocampus were subjected to western blotting using anti-nitrotyrosine antibody. Bars are means ± SEM, n = 10 rats/group, P<0.05, (*) significantly different from control and GP. Group designations: CON (control; sham operated rats provided with tap water for drinking), GP (grape powder: sham operated rats provided with GP treated tap water for drinking), OVX (ovariectomized: rats subjected to surgical removal of ovaries and provided with tap water for drinking), OVX+GP (rats subjected to surgical removal of ovaries and provided with GP treated tap water for drinking).
Figure 4. Examination of anxiety-like behavior tests…
Figure 4. Examination of anxiety-like behavior tests including light-dark and open-field tests in CON, GP, OVX and OVX+GP rats.
Time spent in the lighted area in the light–dark test (A) The open-field test determined center time (B), rearing (C) and fecal boli (D). Bars are means ± SEM, n = 10 rats/group, P<0.05, (*) significantly different from control and GP and (#) significantly different from OVX. Group designations: CON (control; sham operated rats provided with tap water for drinking), GP (grape powder: sham operated rats provided with GP treated tap water for drinking), OVX (ovariectomized: rats subjected to surgical removal of ovaries and provided with tap water for drinking), OVX+GP (rats subjected to surgical removal of ovaries and provided with GP treated tap water for drinking).
Figure 5. Radial arm water maze (RAWM)…
Figure 5. Radial arm water maze (RAWM) memory tests in CON, GP, OVX and OVX+GP rats.
Short term (A) and long term (B) memory was assessed after a series of twelve radial arm water maze trials. The RAWM apparatus is shown as an insert containing a circular water pool with six swim paths. Bars are means ± SEM, n = 10 rats/group, P<0.05, (*) significantly different from control and GP. Group designations: CON (control; sham operated rats provided with tap water for drinking), GP (grape powder: sham operated rats provided with GP treated tap water for drinking), OVX (ovariectomized: rats subjected to surgical removal of ovaries and provided with tap water for drinking), OVX+GP (rats subjected to surgical removal of ovaries and provided with GP treated tap water for drinking).
Figure 6. Long-term potentiation (LTP) in CON,…
Figure 6. Long-term potentiation (LTP) in CON, GP, OVX and OVX+GP rats.
E-LTP evoked by a single train of HFS, was measured in the dentate gyrus (DG) as changes in the fEPSP slope (A) and pspike amplitude (B). Ovariectomized rats exhibited significantly lower values of both fEPSP slope and pspike amplitude compared to those of other groups. However, LTP of OVX+GP rats were similar to those of control rats as measured by fEPSP. Each point is the mean ± SEM of 3–4 rats. (*) indicate significant difference from all other groups. Group designations: CON (control; sham operated rats provided with tap water for drinking), GP (grape powder: sham operated rats provided with GP treated tap water for drinking), OVX (ovariectomized: rats subjected to surgical removal of ovaries and provided with tap water for drinking), OVX+GP (rats subjected to surgical removal of ovaries and provided with GP treated tap water for drinking).
Figure 7. Examination of blood pressure in…
Figure 7. Examination of blood pressure in CON, GP, OVX and OVX+GP rats.
Left panel: Diastolic blood pressure; Right panel: systolic blood pressure, P<0.05, (*) significantly different from control and GP and (#) significantly different from OVX. Bars are means ± SEM, n = 8–10 rats/group. Group designations: CON (control; sham operated rats provided with tap water for drinking), GP (grape powder: sham operated rats provided with GP treated tap water for drinking), OVX (ovariectomized: rats subjected to surgical removal of ovaries and provided with tap water for drinking), OVX+GP (rats subjected to surgical removal of ovaries and provided with GP treated tap water for drinking).
Figure 8. The levels of GLO-1 protein…
Figure 8. The levels of GLO-1 protein in the hippocampus, amygdala and cortex of CON, GP, OVX and OVX+GP rats.
Protein levels of GLO-1 (A-C) were determined by western blotting. Upper panels in (A-C) are representative blots for GLO-1 and protein loading control β-actin. Bar graphs are ratios of respective protein/β-actin. Bars are means ± SEM, n = 3 rats/group, P<0.05, (*) significantly different from control and GP. Group designations: CON (control; sham operated rats provided with tap water for drinking), GP (grape powder: sham operated rats provided with GP treated tap water for drinking), OVX (ovariectomized: rats subjected to surgical removal of ovaries and provided with tap water for drinking), OVX+GP (rats subjected to surgical removal of ovaries and provided with GP treated tap water for drinking).

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