Ron P. Ward

Dietary N-6 and N-3 fatty acids and salt-induced hypertension in the borderline hypertensive rat.

This study examined the effects of salt-loading on blood pressure (BP) development in the borderline hypertensive rat (BHR) and its modification by dietary n−3 and n−6 fatty acids. In experiment 1, 4 groups (n=10/group) of male BHR receiving 1% NaCl as a replacement for tap water were placed on chow enriched with either olive oil (OL), sunflower oil (SUN), evening primrose oil (EPO) or fish oil for 6 weeks. BP, heart rate, body weight, water, Na+ and K+ intake and urinary output were measured weekly. SUN and fish oil reduced the pressor response to salt seen vs the OL group by 50%, and EPO abolished the pressor response, reducing BP below control levels. The BP response was unrelated to either food intake or water and electrolyte intake and excretion. In experiment 2, male BHR received water +/− 18∶3n−6 (0.04 mg/hr in OL via ip pump) or 1% NaCl +/− 18∶3n−6 (n=12/group) for 12 weeks, followed by 2 weeks recovery on tap water. Salt increased BP, and 18∶3n−6 decreased this response, but had no effect on animals receiving tap water. In experiment 3, effects of 3 doses of 18∶3n−6 (0.04, 0.08, and 0.12 mg/hr) on the pressor response to 1% NaCl were examined. All doses reduced the BP response to salt vs controls with no dose-response. These data suggest that the BHR is genetically salt-sensitive, and that dietary n−3 and n−6 fatty acids can attenuate the cardiovascular response to salt in this model.

EFFECTS OF PRENATAL AND EARLY POSTNATAL FATTY ACID SUPPLEMENTATION ON BEHAVIOR

Abstract This study examined the effects of n-6 and n-3 fatty acid supplementation, during gestation and nursing, on offspring behavior and learning. Female Wistar Kyoto rats (n=6/group) received 8 week osmotic pumps releasing olive oil (OL), or OL plus linoleic acid, gamma linolenic acid, arachidonic acid, or alpha linolenic acid (0.04 mg/hr). A sixth group received dummy pumps. Animals were then bred. On day 2 postpartum, 3 females/group had pumps removed; the rest underwent sham surgery. Offspring were weaned and placed on normal diets on day 24. Offspring activity was evaluated in the open field on days 26 and 60. Two pups/litter were sacrificed on days 2 and 26 postpartum for brain lipid analysis. Animals underwent active avoidance conditioning between days 63–80 postpartum. Dietary interventions significantly affected locomotor activity on both test days. On day 60, all n-3 and n-6 fatty acids reduced the number of locomotor efforts, while n-6 fatty acids reduced total squares crossed. OL reduced rearing on both test days. Pre-vs. pre-plus-postnatal supplementation produced similar results. The proportion of animals successfully completing the conditioning paradigm was significantly increased by alpha linolenic supplementation. Supplementation produced no changes in brain phospholipids. Open field behavior and learning were unrelated to brain phosphatidylethanolamine levels of 22:6n-3. These data suggest that dietary composition of n-3, n-6, and n-9 fatty acids during development may produce long term changes in behavior and learning ability. These changes occur in the absence of demonstrable alterations of brain membrane lipid composition, and may be due to changes in prostanoid levels and brain growth during development.

Interactive effects of prenatal ethanol and N−3 fatty acid supplementation on brain development in mice

This study assesses the combined effects on brain and behavioral development of ethanol administration and supplementation of the maternal diet with long chain n−3 polyunsaturated fatty acids. From day 7 to 17 of gestation, pregnant mice were fed equivalent daily amounts of isocaloric liquid diets; 20% of the energy was provided by either ethanol or maltose-dextrin, and a further 20% by either safflower oil (rich in linoleic acid, 18∶2n−6), or a combination of safflower oil with a fish oil concentrate (rich in eicosapentaenoic acid, 20∶5n−3, and docosahexaenoic acid, 22∶6n−3). On day 18 the liquid diets were replaced by lab chow; a fifth group was maintained on lab chow throughout the experiment. Measures on the pups included brain weight and the fatty acid composition of the brain phospholipids on days 22 and 32 post-conception (birth=day 19), as well as behavioral development. Maternal weight gain during gestation was decreased by ethanol relative to maltose-dextrin, and increased by fish relative to safflower oil. On day 32, the brain weight of ethanoltreated animals fed fish oil was greater than their safflower oil controls, whereas the reverse was true in the two maltose-dextrin groups; a similar trend was apparent on day 22. The brain phospholipid content of the longer chain fatty acids (20∶4n−6, 22∶4n−6, 22∶5n−6, 20∶5n−3, 22∶5n−3, 22∶6n−3) on day 22 reflected that of the prenatal diet, with the proportion of n−3 compounds being higher and that of n−6 floer in the fish oil than safflower oil groups. Prenatal dietary effects were absent by day 32, with the exception of lower 22∶5n−6 in fish oil groups. Dietary supplementation with n−3 fatty acids increased the ratio of 20∶3n−6 to 20∶4n−6, which is consistent with a blockade of the activity of Δ-5 desaturase. On day 22 the incorporation of dietary long chain n−3 fatty acids into the brain phosphatidylcholine fraction was enhanced in the ethanol-treated animals; by day 32 the animals treated prenatally with ethanol also showed increased levels of long chain n−6 compounds. Behavioral development was retarded by ethanol, but there was no effect of the dietary oils. These results support the hypothesis that effects of ethanol on the developing brain may be modified by the availability of an exogenous supply of long chain fatty acids.

Attenuation of Psychosocial Stress-Induced Hypertension by Gamma-Linolenic Acid (GLA) Administration in Rats

Abstract This study investigated a model of psychosocial stress-induced hypertension in the rat, and examined effects of the prostaglandin E precursor, gamma-linolenic acid (GLA) on the development of hypertension during psychosocial stress. In the first study, male rats were housed four/cage for an acclimation period of 21 days, followed by a 14-day control period. An experimental group (N = 12) was then placed in isolation cages for 14 days, then regrouped for a 7-day recovery period. Controls (N = 12) remained group-housed. Eight animals per group were sacrificed after the experimental period, and four per group after recovery for organ weight analysis. Mean systolic blood pressure (BP) was similar between groups during the control period (126 ± 2 and 125 ± 2 mm Hg), but increased during isolation, reaching 140 ± 2 mm Hg (P < 0.001) by Day 14. During recovery BP returned to control levels. No changes in heart rate, heart weight/body weight or adrenal weight were seen. The second study utilized a protocol similar to that of the experimental group of the first study, minus the recovery period. On Day 1 of the control period 28-day osmotic pumps were implanted ip, releasing olive oil or GLA in olive oil. Four groups of rats (N = 8/group) received either (i) olive oil (controls), (ii) 0.018 mg GLA/hr, (iii) 0.040 mg GLA/hr, or (iv) 0.040 mg GLA/hr with no stress. Organ weights were obtained following stress in groups 1-3. Controls developed a sustained elevation in BP within 24 hr of isolation. Animals receiving 0.018 mg GLA/hr developed elevated BP upon isolation, but the BP was less than that of controls on Days 1 (P < 0.05) and 14 (P < 0.001) of isolation. Animals receiving 0.040 mg GLA/hr demonstrated a greatly attenuated rise in BP vs controls (P < 0.001) on all isolation days. GLA in unstressed rats had no effect on BP. Heart rate, heart weight/body weight, and adrenal weight were unchanged in all groups. These data suggest that (i) isolation is a useful tool for investigating reversible psychosocial stress-induced hypertension, and (ii) GLA, while not affecting BP in unstressed animals, produces a dose-dependent attenuation of the BP response to chronic stress.

Effects of essential fatty acid administration on cardiovascular responses to stress in the rat

This study examined the effects of 18∶2(n−6), 18∶3(n−6), 20∶4(n−6) and 18∶3(n−3) on cardiovascular responses to isolation stress in male rats. Group-acclimated rats were fasted for 2 days, then placed on a fat-free diet. Two wk later animals were divided into six groups (six animals per group) and given eight-wk intraperitoneal osmotic pumps releasing 1.47×10−7 mol/hr of either olive oil (OL), or of 18∶2(n−6), 18∶3(n−6), 20∶4(n−6) or 18∶3(n−3) in OL. Another group received dummy pumps. Two wk after pump implantation, animals were isolated for four wk. Blood pressure (BP), heart rate and body weight were followed before and during stress. Following the stress period, animals were assessed for cardiovascular reactivity to norepinephrine (NOR) and angiotensin (ANG).Prior to isolation, 18∶3(n−6) lowered BP vs OL (p<0.01). Stress increased BP within 24 hr in all groups except 18∶3(n−6) and 20∶4(n−6). Treatment with 20∶4(n−6) vs OL prevented the BP rise (p<0.001) only for the first two wk of stress. Administration of 18∶3(n−6) vs OL prevented any BP increase over the four-wk stress period (p<0.001). Stress increased heart rate in all groups except 20∶4(n−6). Heart rate was lowered by 18∶3(n−6) vs OL (p<0.01) before and during stress. Vascular reactivity to NOR was unaffected by treatment, but OL and 18∶3(n−6) decreased responses to ANG infusion. These data suggest that 18∶3(n−6) supplementation attenuates cardiovascular responses to chronic stress, and that Δ6- and Δ5-desaturase activity are inhibited during chronic psychological stress.

Effects of Eicosapentaenoic Acid (20:5ω3) on Stress Reactivity in Rats:

Abstract This study examined the effects of eicosapentaenoic acid (EPA) on cardiovascular responses to isolation stress in male rats. Group-reared rats, on a fat-free diet, were given olive oil (OL), or EPA in OL (1.47 x 10−7 mol/hr) via 8 week osmetic pumps, or a dummy pump (DUM), 2 weeks prior to a 4 week isolation period. Blood pressure (BP) , heart rate, and body weight were monitored weekly and pressor responses to i.a. norepinephrine and angiotensin were assessed at the end of the study. BP increased during stress in all animals vs. pre-stress conditions, but was attenuated by EPA (p<0.001). Heart rate also increased during stress in all groups, but was greater in the EPA group (p<0.001). In contrast, body weight gain during stress was similar in DUM and EPA groups, but depressed by OL (p<0.001). Vascular response to norepinephrine was enhanced by EPA vs. DUM and OL, whereas the response to angiotensin was similar in EPA and DUM groups, but reduced by OL. These data suggest that EPA may attenuate cardiovascular responses to psychological Stress.

Attenuation of cyclosporine-induced hypertension by dietary fatty acids in the borderline hypertensive rat

The effects of dietary (10% calories) safflower (SAF), evening primrose (EPO), and fish oil (F) as sources of linoleic acid (control), gamma-linolenic acid, and long-chain n-3 fatty acids, respectively, on cardiovascular and renal responses to chronic (5 weeks) cyclosporine administration were studied in male borderline hypertensive rats. In one experiment (n = 9/group), oral administration of CsA at 0.1 mg/kg.day significantly increased awake systolic blood pressure vs. placebo in SAF-fed rats (P less than 0.01). This increase was prevented by both EPO (P less than 0.001) and F (P less than 0.01), in the absence of group differences in body weight gain or plasma electrolyte levels. In a second experiment, CsA also increased blood pressure vs. placebo in SAF-fed rats (P less than 0.001). While this increase was prevented by EPO (P less than 0.001), F had no significant effect. Differences in group blood pressure responses were not explained by group differences in body weight gain or trough levels of blood CsA. Renal function, assessed in anesthetized rats after week 5, demonstrated a CsA-related (10 mg/kg.day) decrease in whole-kidney GFR in SAF-fed animals vs. placebo (P less than 0.05) that was prevented by EPO and attenuated by F. EPO and F also tended to reduce the CsA-induced elevation in renovascular resistance, but this difference did not reach statistical significance. These findings suggest the potential of dietary EPO and F to offset nephrotoxic effects of CsA administration, and suggest that EPO may also be useful in countering CsA-induced hypertension.

Gamma linolenic acid attenuates cardiovascular responses to stress in borderline hypertensive rats

The purpose of the present study was to investigate the effects of gamma linolenic acid (GLA) on cardiovascular responses to psychosocial stress (isolation) and to pressor hormones in the genetically borderline hypertensive rat (SHR×WKY). Adult male SHR×WKY were divided into two groups following five weeks of group housing. One group (GLA) received eight weeks constant flow osmotic pumps releasing 0.04 mg GLA in olive oil/kg-hr, while the second group received dummy pumps (DUM). One week following pump implantation, each group was divided into two subgroups and exposed to a four-week experimental period of either continued group housing (no stress) or isolation (stress). A two-week recovery period of group housing followed the experimental period. Blood pressure and heart rate were determined weekly by the tail cuff technique. At the end of the recovery period, animals in the no stress condition were anesthetized and received an arterial cannula for NOR and ANG infusion and direct BP recording. Then the responses to an ED50 of NOR and ANG were determined. All animals were then killed for determination of heart weight and adrenal weight. All groups had mean control period systolic BP values ranging from 143–146 mm Hg. In the no stress condition, neither GLA nor DUM altered BP over the course of the study. However, BP increased in the DUM group durign all four weeks of the isolation period vs the control period (p<0.01), whereas BP increased only in week 1 in the GLA group (p<0.05). Heart rate increased during stress in the DUM group (p<0.05), but not in the GLA group. Vascular reactivity to NOR was unaffected by GLA administration. In contrast, GLA increased the duration of the pressor response to ANG (p<0.01), but tended to decrease the magnitude of the pressor response (p<0.09) vs DUM. GLA had no effect on heart weight/body weight ratio. Adrenal weight/body weight ratio was lower in the DUM/no stress group than all other treatment groups.These data indicate that GLA administration attenuates the cardiovascular responses to chronic stress in animals with a genetic predisposition to hypertension, in the absence of an effect on resting BP. They also demonstrate a limitation of GLA effect, in mature animals, to epigenetic pressor factors. Furthermore, GLA action may involve an alteration of the cardiovascular responses to ANG, but not NOR. These findings suggest that GLA may be useful in preventing the neurogenic triggering of hypertension by chronic stress in genetically stress-sensitive animals.

Effect of essential fatty acid depletion on tissue phospholipid fatty acids in spontaneously hypertensive and normotensive rats

Weanling male spontaneously hypertensive (SHR) and normotensive (WKY) rats were maintained on a fat-free semisynthetic diet and killed at various intervals. The effects of fat-depletion on the appearance of essential fatty acid (EFA) deficiency symptoms, the progressive changes of major fatty acids in plasma, liver, heart, and kidney phospholipids (PL), and in skin total lipids were compared between these two strains. After five weeks on the diet, the slower growth and the appearance of EFA deficiency symptoms became evident in SHR. In general, fat-depletion reduced the levels of n−6 fatty acids, whereas it increased those of 20∶3n−9. However, the fat-depletion induced reduction of 18∶2n−6 in heart PL and 20∶4n−6 in kidney, while the elevation of 20∶3n−9 in plasma, heart, and kidney PL were greater in WKY than in SHR. As a result, the elevation of biochemical EFA deficiency index—20∶3n−9/20∶4n−6 ratio—was greater in WKY than in SHR. In comparison with WKY, the concentrations of liver triacylglycerols and the weights of adipose tissues in SHR were reduced to a greater extent, indicating an active catabolism of triacylglycerols in SHR. This study suggests that the earlier appearance of morphological symptoms of EFA deficiency in SHR was not associated with the reducing n−6 EFA levels or with an elevation of triene/tetraene ratio, but possibly to a reduced supply of n−6 EFA for skin prostaglandin synthesis.

Attenuation of stress-induced hypertension by exercise independent of training effects: an animal model.

The present study attempted to determine if exercise, in the absence of physical training, could alter development of hypertension during chronic exposure to a psychosocial stressor. Two groups of genetically normotensive rats were exposed to social stress for 7 days, following 5 weeks of acclimation to social isolation. One group had access to exercise in a running wheel during the stress period, while the second group did not. Blood pressure, heart rate, body weight, and running activity were monitored throughout the study, and heart and adrenal gland weights were obtained following sacrifice of the animals after exposure to stress. Blood pressure increased significantly to hypertensive levels on days 4 and 7 in the group denied access to exercise but was unchanged in the exercise group. Degree of attenuation of stress-induced hypertension was unrelated to amount of running activity. There were no differences in body weight, heart rate, or organ weight between groups. Exercise appeared to act specifically via diversional, or coping, mechanisms to buffer the response of the body to stress.