Sabina Glozman

Intraamniotic Ethyl Docosahexaenoate Administration Protects Fetal Rat Brain from Ischemic Stress

Abstract: Studies were conducted on the prenatal rat given a single intraamniotic injection of ethyl docosahexaenoate (Et‐DHA; 9.6–12 mmol per fetus) or subjected to an n‐3 fatty acid‐deficient diet to assess the role of docosahexaenoate on oxidative stress during episodes of ischemia. A time‐dependent decrease in the ability of brain slices from animals treated with Et‐DHA to produce thiobarbituric acid‐reactive substance (TBARS), most pronounced after 1 day (from 58.1 ± 4.22 to 15.9 ± 1.6 nmol/mg of DNA), was noticed on stimulation with Fe2+. Brain slices from fetuses treated for 1 day with Et‐DHA and those from untreated fetuses produced TBARS levels of 46.7 ± 6.5 and 114.8 ± 10.8 nmol/mg of DNA, respectively, after a 20‐min occlusion of the fetal‐maternal circulation at embryonic day 20, suggesting a protective effect of Et‐DHA. The protective effect of a single dose of Et‐DHA in utero remained high up to 3 days after injection (p < 0.001) and was long‐lasting, yet not significant, up to 3 days following birth. In agreement with a reduction in TBARS production by slices, the endogenous levels of TBARS in brains of Et‐DHA‐treated animals were lower than in the controls. Et‐DHA‐injected fetuses exhibited significantly higher levels of esterified DHA than the non‐injected controls. n‐3‐deficient diet given to dams for 2 weeks before birth did not affect the levels of TBARS production in control fetal brain slices but abolished the increase caused by ischemia. Et‐DHA administration for 24 h to n‐3‐deficient fetuses reduced the amount of TBARS produced by the fetal brain slices from 49.1 ± 8.5 to 31.7 ± 4.1 nmol/mg of DNA. A protective effect from oxidative damage after postischemic oxidative stress in fetal brain following DHA supplements is suggested, whereas the effect of n‐3 fatty acid deficiency in this regard is more ambiguous.

Docosahexaenoic acid accumulation in the prenatal brain: prooxidant and antioxidant features.

Docosahexaenoic acid (DHA; 22:6n-3) is the major polyunsaturated fatty acid (FA) in the adult rat brain and it accumulates significantly more than any other FA prior to birth. Under normal nutritional conditions, fetal-brain DHA accumulation is substantial, with a “DHA accretion spurt” being demonstrated in the last period of gestation. Under stress conditions, this spurt may be harmful owing to an in crease in multiple double-bond targets for lipid peroxidation. The “DHA accretion spurt” is supported by the maternal supply of DHA or its precursor. Under maternal dietary n-3 FA deficiency, DHA content in the fetal brain can be restored by direct intraamniotic injection of mM concentrations of ethyl-DHA (Et-DHA). This approach may hold a potential advantage in the event of maternal-fetal insufficiency, a stress that may cause intrauterine growth retardation. It also revealed a potential beneficial effect after in utero ischemic stress; brain slices from Et-DHA-treated fetuses formed less oxidation products, as detected by thiobarbituric acid (TBA), compared to controls. Furthermore, brain-lipid extracts from Et-DHA but not ethyl-oleate treated fetuses, exhibited hydroxyl radical scavenging activity, as demonstrated by electron spin-resonance technique. Part of the beneficial effect of Et-DHA administration on the fetal brain may be attributed to enhanced free-radical scavenging capability, a phenomenon not directly related to vitamin E or lipid-soluble antioxidant levels.

Ethyl docosahexaenoate-associated decrease in fetal brain lipid peroxide production is mediated by activation of prostanoid and nitric oxide pathways

Previously we have shown that intraamniotic administration of ethyl docosahexaenoate (Et-DHA) to pregnant rats resulted in decreased lipid peroxidation in the fetal brain, under a variety of conditions (S. Glozman, P. Green, E. Yavin, J. Neurochem. 70 (1998) 2482-2491). In the present study we examine the potential mechanisms to explain this effect. This was done by a pharmacological approach, utilizing brain slice preparations from Et-DHA treated or control rats in the presence of various agents and examining the formation of products in the tissue slices or incubation medium. Et-DHA treated brains produced 2-3-fold more prostanoids (PN) than control brains, indicating cyclooxygenase (COX) activation. Indomethacin at 50 microM inhibited PN formation and also abolished Et-DHA induced decrease in lipid peroxides, as evident by the levels of thiobarbituric acid reactive substances (TBARS) released in the medium. The phospholipase A2 inhibitors quinacrine and p-bromophenacyl bromide added at 0.1 mM concentration each to either slices from controls or Et-DHA treated fetal brains, decreased TBARS production. Et-DHA treated brains released 2.2-fold more nitric oxide (NO) than control brains and NO synthase (NOS) inhibitors abolished this effect. Increasing the concentration of NO by the addition of an NO donor greatly decreased the concentration of the TBARS in the medium. These results suggest that at least some of the effect of Et-DHA on decreased lipid peroxidation may be explained by a shift of oxygen species utilization via enzymatically regulated, therefore metabolically controlled, COX and NOS activities.

Lipid Peroxides are Generated by the Fetal Rat Brain After Episodes of Global Ischemia in Utero

Complete arrest of maternal-fetal blood supply for up to 30 min caused a time-dependent increase in the endogenous levels of lipid peroxides (LPO) in fetal brain and liver extracts and fetal blood and amniotic fluids as indicated by the appearance of thiobarbituric acid reactive substances (TBARS), A steady increase of TBARS from 48.0 ± 2.2 pmol/g wet weight to 75.0 ± 5.6 pmol/g wet weight up to 30 min restriction was noticed in the fetal brain. The fetal liver TBARS values increased by approximately 69% after 5 min restriction and remained high, above the control level, for 30 min. After two days reperfusion following 30 min restriction, the TBARS levels in the fetal brain were 1.8 fold higher above the control, while those of the liver returned to control values. The levels of the lipid-soluble antioxidant α-tocopherol were reduced by about 40% and 50% in the placenta and brain tissues after 5 min restriction, respectively. Slices of fetal brain incubated at 37°C in DMEM under oxygen in the presence of 50 μM Fe2+ were able to generate LPO in a time- and tissue concentration-dependent manner. After 15 min incubation, about 6.3 fold increase in total TBARS levels could be measured in the presence of 50 μM Fe2+, most of which was released in the medium. The iron chelator desferrioxamine (25 μM) and the antioxidant α-tocopherol (10 μM) added to the incubation medium, each inhibited by about 88% TBARS production. After 20 min episode of ischemia, fetal brain slices released into the medium 138.5 ± 9.8 nmol/15 min/mg DNA compared to 75.9 ± 4.5 nmol/15 min/mg DNA released by the sham preparations. After 3 h reperfusion, brain slices from fetuses exposed to 20 min ischemia continued to produce TBARS above control levels, whereas those of brief ischemia (5 min) returned to control levels. The data indicate that the limited resistance of the fetal brain to brief, rather than prolonged, periods of ischemia, is likely due to a lack of free FA for LPO generation, rather than the levels of tissue lipid antioxidants.

Docosahexaenoic Acid Sources for the Developing Brain during Intrauterine Life

Docosahexaenoic acid (DHA, 22: 6n-3) and arachidonic acid (AA, 20: 4n-6) provision to the developing fetus, with emphasis towards brain and vascular system growth, is a subject of increasing concern particularly under pathological conditions associated with premature birth or in utero growth restriction following obstruction of the maternal-fetal blood flow. Most of DHA, but also AA accretion under physiological conditions, is maternally dependent and requires adequate maternal nutrition and normally functioning placental-fetal circulation. It has been demonstrated that unlike other fatty acids (FA), DHA is preferentially transported across the placenta into the fetal circulation. The selective transplacental DHA transfer is probably mediated by specific carrier proteins. While some of the latter may be acting in fetal organs, the mechanism(s) for the selective accumulation of DHA in brain is still unknown. The fetal brain and also the fetal liver are capable of producing DHA from linolenic (LnA, 18:3 n-3) acid. How effective this local elongation-desaturation mechanism for DHA provision is and to what degree this route is activated in premature births is not clear. Transfer of DHA via the fetal gastrointestinal tract is an additional route to provide DHA to other fetal organs. As indicated by animal model studies, it holds the potential for DHA supply when the maternal pathway is compromised.

Ischemic reperfusion brain injury in fetal transgenic mice with elevated levels of copper-zinc superoxide dismutase

Abstract Aim: To examine the effect of overexpression of human intracellular copper-zinc superoxide dismutase (CuZnSOD1) gene on fetal mice brain exposed to in-utero ischemic reperfusion injury. Design: Transient in-utero ischemia (7 min) was induced in pregnant transgenic mice overexpressing human CuZnSOD1 and wild-type mice by occluding the blood supply to the uterine artery on day 17 of pregnancy, followed by 24 hours of reperfusion. The level of lipid peroxidation in fetal mice brains was compared between the transgenic and non-transgenic (control) fetal mice. Motor and coordination skills of transgenic and control adult mice (six to eight months old) which were exposed to ischemic reperfusion injury in-utero were compared by the rope grip test and visible platform task. Results: We first measured CuZnSOD1 activity in the brains of the transgenic fetal mice and confirmed that the enzyme activity is 4.2-fold higher than control. We also established that ischemia reperfusion on day 17 of pregnancy led to increased level of TBARS (Thiobarbituric acid reactive substance) in brains of wild-type fetal mice when compared to sham operated mice (72.5 ± 3.4 vs. 49.4 ± 1.5 nmol/mg. p < 0.001). The increase was markedly accentuated in the CuZnSOD1 transgenic mice, and significantly higher compared to control mice exposed to ischemia-reperfusion (85.6 ± 4.0 vs. 69.5 ± 2.3 nmol/mg, p < 0.001). Moreover, we found that the transgenic mice that were subjected to in-utero ischemia reperfusion exhibited a significantly higher rate of failures in the rope grip test and poorer performance in the visible platform task, when compared to non-transgenic mice exposed to identical insult. Conclusions: Oxygen free radicals play an important role in the pathogenesis of perinatal hypoxia. Overexpression of the enzyme CuZnSOD1 in transgenic mice exposes their brains to increased damage during ischemic-reperfusion insult.

Docosahexaenoic acid-deficient phosphatidyl serine and high α-tocopherol in a fetal mouse brain over-expressing Cu/Zn-superoxide dismutase

The over-expressed Cu/Zn-superoxide dismutase (Cu/Zn-SOD) gene has been found in some circumstances phenotypically deleterious and associated with oxidative injury-mediated aberrations while in other studies it was considered neuroprotective. In this work we examine a number of biochemical markers in fetal and adult brain from transgenic (tg) mice expressing the human Cu/Zn-SOD gene, which may determine this dual characteristic. These markers include the polyunsaturated fatty acid (PUFA) profile in discrete phospholipid species, the alpha-tocopherol levels, a marker for lipid anti-oxidant status, and thiobarbituric acid reactive substance (TBARS), a marker for the tissue oxidative status. The PUFA profile in choline- and ethanolamine-phosphoglycerides was similar in tg and nontransgenic (ntg) animals of either fetal or adult brain. Serine-phosphoglycerides, however, showed a marked decrease from 20. 07+/-0.53 to 14.92+/-0.87 wt% and 14.52+/-1.15 wt% in docosahexaenoic acid (DHA; 22:6 n3), in the tg 51 and tg 69 fetal brains, respectively, but not in the comparable adult tissues. The alpha-tocopherol levels were significantly higher in the fetal compared to the adult brain. There were no differences in the anti-oxidant levels between the ntg and tg fetal brains, but there were differences in the adult animals; the tg mice were higher by at least two-fold than the control animals. The basal TBARS in the tg 51 fetal brain was 35% lower than that of ntg mouse and in the presence of Fe(2+), brain slices from the former released less TBARS (57% reduction) into the medium than the latter. These results suggest that higher dosages of Cu/Zn-SOD gene are compatible with increased alpha-tocopherol levels, reduced basal TBARS levels and a DHA deficiency in the fetal, but not the adult, tg brain.