Cognition in aged rhesus monkeys: effect of DHEA and correlation with steroidogenic gene expression.

Estradiol supplementation has been shown to enhance cognitive performance in old ovariectomized rhesus macaques (Macaca mulatta). To determine if similar benefits could be achieved in perimenopausal animals using alternative hormonal supplements, we administered dehydroepiandrosterone (DHEA) to old ovary-intact female rhesus macaques for ∼2.5 months. Using computerized touch screen memory tasks, including delayed response (DR) and delayed matching-to-sample (DMS), we observed improved performance with time in all of the animals but failed to detect a significant effect of DHEA. On the other hand, gene expression profiling disclosed a significant correlation between cognitive performance and the expression of several steroidogenic and steroid-responsive genes. The DR performance was positively correlated with hippocampal expression of AKR1C3 and STAR and negatively correlated with the expression of SDRD5A1. A positive correlation was also found between DMS performance and prefrontal cortical expression of AKR1C3 and a negative correlation with STAR, as well as a negative correlation with the hippocampal expression of HSD11B1 and NR3C1. Taken together, the results suggest that steroidogenic gene regulation within the brain may help to maintain cognitive function during the perimenopausal transition period, despite a decline in sex-steroid levels in the circulation.

Mitochondrial elongation in the macular RPE of aging monkeys, evidence of metabolic stress.

PURPOSE: This study was conducted to determine whether mitochondria of the macular retinal pigment epithelium (RPE) change with age in rhesus monkeys (Macaca mulatta). Mitochondria are the main instigators of oxidative stress, which has often been considered to play a role in the pathogenesis of age-related macular degeneration (AMD). Any pathological changes in the mitochondria of aging macular RPE, the main target of AMD, would be a clue to the pathogenesis of this common retinal degeneration afflicting both monkey and man. METHODS: Transmission electron microscopy was used to identify mitochondria and to determine their appearance, their density per unit area of RPE cytoplasm and their length. The eyes of seven monkeys, 1, 2, 6.5, 23, 26, 27 and 35 years of age, were studied. Measurements were kept separate for the basal, middle and apical third of each cell. The basal third of the macular RPE had many more mitochondria than the middle third, and the apical third was almost devoid of mitochondria. RESULTS: Mitochondrial number decreased and length increased with age. The increase in length was associated with an unusual clustering of mitochondria into parallel arrays of elongated mitochondria, with their long axis orthogonal to the basal membrane of the cell, structures not described before in RPE. CONCLUSIONS: Mitochondrial elongation is associated with metabolic and/or oxidative stress, which implies that age produces stress in macular RPE. The increased clustering of very elongated mitochondria suggests that pathological changes occur in mitochondrial organization with age. These changes support the hypothesis that age-related mitochondrial dysfunction plays a role in the pathogenesis of AMD.

Hormone supplementation during aging: how much and when?

Circulating levels of dehydroepiandrosterone, a major adrenal steroid, show a marked age-related decrease in both humans and nonhuman primates. Because this decrease has been implicated in age-related cognitive decline, we administered supplementary dehydroepiandrosterone to perimenopausal rhesus macaques (Macaca mulatta) to test for cognitive benefits. Although recognition memory improved, there was no benefit to spatial working memory. To address the limitations of this study we developed a hormone supplementation regimen in aged male macaques that more accurately replicates the 24-hr androgen profiles of young animals. We hypothesize that this more comprehensive physiological hormone replacement paradigm will enhance cognitive function in the elderly.

Bestrophin detected in the basal membrane of the retinal epithelium and drusen of monkeys with drusenoid maculopathy.

PURPOSE: To determine if bestrophin is present in the basal membrane of macular retinal pigment epithelium (RPE) and in drusen of rhesus monkeys with age-related drusenoid maculopathy. METHODS: The macular region of three rhesus monkeys (Macaca mulatta), 23-24 years of age, with drusenoid maculopathy was dissected from eyes fixed with 4% paraformaldehyde. The macula was sectioned into rectangular pieces. The sclera was removed from each segment and the remainder separated into segments of neural retina with retinal epithelium or choroid with retinal epithelium. These segments were incubated with a goat polyclonal antibody to human bestrophin 1, reacted with gold-labeled rabbit antibody to goat IgG, silver-enhanced, and processed for transmission electron microscopy. RESULTS: Bestrophin-labeled gold particles were found in quasi-linear arrays on the basal surface of the macular RPE and also within drusen where bestrophin was found in segments of membranous-like material. The array density of the bestrophin-linked gold particles on the basal membrane of the epithelium had a maximal value of about 5-100 bestrophin molecules/micron(2). Immuno-detection of bestrophin was most effective when examined in an RPE layer that remained attached to the neural retina, where the basal surface of the epithelium is more directly exposed to the antibodies. CONCLUSION: Bestrophin is present on the basal membrane of macular RPE of rhesus monkeys with age-related drusenoid maculopathy, and also found in the membranous-like structures of drusen. The latter finding provides insight into the pathogenesis of drusen by indicating that segments of the basal membrane of RPE contribute to the material that accumulates within drusen.

Circadian activity associated with spatial learning and memory in aging rhesus monkeys.

In rodents, spatial learning and memory tests require navigation, whereas in nonhuman primates these tests generally do not involve a navigational component, thus assessing nonhomologous neural systems. To allow closer parallels between rodent and primate studies, we developed a navigational spatial learning and memory task for nonhuman primates and assessed the performance of elderly (19-25 years) female rhesus monkeys (Macaca mulatta). The animals were allowed to navigate in a room containing a series of food ports. After they learned to retrieve food from the ports, a single port was repeatedly baited and the animals were tested until they learned the correct location. The location of the baited port was then changed (shift position). We also determined whether test performance was associated with circadian activity measured with accelerometers. Performance measures included trials to criterion, search strategies, and several indices of circadian activity. Animals learned the task as reflected in their search strategies. Correlations were found between the number of initial or shift trials and circadian activity parameters including day activity, dark:light activity ratio, sleep latency, and wake bouts. Thus, disruptions in circadian rhythms in nonhuman primates are associated with poorer performance on this novel test. These data support the usefulness of this spatial navigational test to assess spatial learning and memory in rhesus monkeys and the importance of circadian activity in performance.

The role of docosahexaenoic acid in retinal function.

An important role for docosahexaenoic acid (DHA) within the retina is suggested by its high levels and active conservation in this tissue. Animals raised on n-3-deficient diets have large reductions in retinal DHA levels that are associated with altered retinal function as assessed by the electroretinogram (ERG). Despite two decades of research in this field, little is known about the mechanisms underlying altered retinal function in n-3-deficient animals. The focus of this review is on recent research that has sought to elucidate the role of DHA in retinal function, particularly within the rod photoreceptor outer segments where DHA is found at its highest concentration. An overview is also given of human infant studies that have examined whether a neonatal dietary supply of DHA is required for the normal development of retinal function.

Growth and development in preterm infants fed long-chain polyunsaturated fatty acids: a prospective, randomized controlled trial.

OBJECTIVES: A randomized, masked, controlled trial was conducted to assess effects of supplementing premature infant formulas with oils containing the long-chain polyunsaturated fatty acids, arachidonic acid (AA; 20:4 n6), and docosahexaenoic acid (DHA; 22:6 n3) on growth, visual acuity, and multiple indices of development. METHODS: Infants (N = 470) with birth weights 750 to 1800 g were assigned within 72 hours of the first enteral feeding to 1 of 3 formula groups with or without long-chain polyunsaturated fatty acids: 1) control (N = 144), 2) AA+DHA from fish/fungal oil (N = 140), and 3) AA+DHA from egg-derived triglyceride (egg-TG)/fish oil (N = 143). Infants were fed human milk and/or Similac Special Care with or without 0.42% AA and 0.26% DHA to term corrected age (CA), then fed human milk or NeoSure with or without 0.42% AA and 0.16% DHA to 12 months' CA. Infants fed exclusively human milk to term CA (EHM-T; N = 43) served as a reference. RESULTS: Visual acuity measured by acuity cards at 2, 4, and 6 months' CA was not different among groups. Visual acuity measured by swept-parameter visual-evoked potentials in a subgroup from 3 sites (45 control, 50 AA+DHA [fish/fungal]; 39 AA+DHA [egg-TG/fish]; and 23 EHM-T) was better in both the AA+DHA (fish/fungal; least square [LS] means [cycle/degree] +/- standard error [SE; octaves] 11.4 +/- 0.1) and AA+DHA (egg-TG/fish; 12.5 +/- 0.1) than control (8.4 +/- 0.1) and closer to that of the EHM-T group (16.0 +/- 0.2) at 6 months' CA. Visual acuity improved from 4 to 6 months' CA in all but the control group. Scores on the Fagan test of novelty preference were greater in AA+DHA (egg-TG/fish; LS means +/- SE, 59.4 +/- 7.7) than AA+DHA (fish/fungal; 57.0 +/- 7.5) and control (57.5 +/- 7.4) at 6 months' CA, but not at 9 months' CA. There were no differences in the Bayley Mental Development Index at 12 months' CA. However, the Bayley motor development index was higher for AA+DHA (fish/fungal; LS means +/- SE, 90.6 +/- 4.4) than control (81.8 +/- 4.3) for infants

Long chain polyunsaturated fatty acids (LC-PUFA) and perinatal development.

UNLABELLED: This paper reports on the conclusions of a workshop on the role of long chain polyunsaturated fatty acids (LC-PUFA) in maternal and child health. The attending investigators involved in the majority of randomized trials examining LC-PUFA status and functional outcomes summarize the current knowledge in the field and make recommendations for dietary practice. Only studies published in full or in abstract form were used as our working knowledge base. CONCLUSIONS: For healthy infants we recommend and strongly support breastfeeding as the preferred method of feeding, which supplies preformed LC-PUFA. Infant formulas for term infants should contain at least 0.2% of total fatty acids as docosahexaenoic acid (DHA) and 0.35% as arachidonic acid (AA). Since preterm infants are born with much less total body DHA and AA, we suggest that preterm infant formulas should include at least 0.35% DHA and 0.4% AA. Higher levels might confer additional benefits and should be further investigated because optimal dietary intakes for term and preterm infants remain to be defined. For pregnant and lactating women we consider it premature to recommend specific LC-PUFA intakes. However, it seems prudent for pregnant and lactating women to include some food sources of DHA in their diet in view of their assumed increase in LC-PUFA demand and the relationship between maternal and foetal DHA status.

Infant vision and retinal function in studies of dietary long-chain polyunsaturated fatty acids: methods, results, and implications.

Animal and human studies have documented several effects of different dietary and tissue concentrations of long-chain polyunsaturated fatty acids (LCPUFAs) on retinal function and vision. The enhanced visual development associated with increased intakes of LCPUFAs, particularly docosahexaenoic acid (DHA), provides the strongest evidence for the importance of these fatty acids in infant nutrition. The 2 primary visual measures used to assess the efficacy of infant formula LCPUFA supplementation are the electroretinogram and visual acuity. This review briefly describes the methodology, neural basis, and interpretation of these measures, as well as other measures of visual development that may be used to extend the functional evaluation of infants fed formulas with different fatty acid compositions.

Polyunsaturated fatty acid status and neurodevelopment: a summary and critical analysis of the literature.

The rationale for randomized trials designed to measure the effects of variable docosahexaenoic acid (DHA) status on neurodevelopment in human infants came from earlier studies of neurodevelopment in animals that were deficient in DHA owing to diets low in alpha-linolenic acid. The session on neurodevelopment looked at the results of these animal studies and discussed outcomes that appear to be analogous in human infants with variable DHA status. Presentations focused mainly on measures of development that may be attributed to more specific developmental domains (e.g., visual attention, recognition memory, problem-solving), some of which have been shown to be affected by long-chain polyunsaturated fatty acid (LCPUFA) status. This paper derives from discussions that took place during the session and reviews subsequent developments in this area. Although more difficult to interpret, global measures of infant development (e.g., the Bayley Scales of Infant Development, and Brunet-Lezine) can only suggest a relationship to specific developmental domains, but they have been applied in some randomized trials of LCPUFA and infant development. Those results are also summarized here.

Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys.

Intracytoplasmic sperm injection has begun an era of considerable improvements in treating male infertility. Despite its success, questions remain about the dangers of transmitting traits responsible for male infertility, sex and autosomal chromosome aberrations and possible mental, physical and reproductive abnormalities. We report here the first births of rhesus monkeys produced by intracytoplasmic sperm injection at rates greater or equal to those reported by clinics. Essential assumptions about this process are flawed, as shown by results with the preclinical, nonhuman primate model and with clinically discarded specimens. Dynamic imaging demonstrated the variable position of the second meiotic spindle in relation to the first polar body; consequently, microinjection targeting is imprecise and potentially lethal. Intracytoplasmic sperm injection resulted in abnormal sperm decondensation, with the unusual retention of vesicle-associated membrane protein and the perinuclear theca, and the exclusion of the nuclear mitotic apparatus from the decondensing sperm nuclear apex. Male pronuclear remodeling in the injected oocytes was required before replication of either parental genome, indicating a unique G1-to-S transition checkpoint during zygotic interphase (the first cell cycle). These irregularities indicate that the intracytoplasmic sperm injection itself might lead to the observed increased chromosome anomalies.

Biochemical markers for puberty in the monkey testis: desmosterol and docosahexaenoic acid.

We previously reported that the sperm of rhesus monkeys and humans uniquely contain large amounts of desmosterol not found in other tissues and have a high concentration of the highly polyunsaturated n-3 fatty acid, docosahexaenoic acid (22:6 n-3). However, the lipid composition of the testis, from which sperm originate, is unknown. During puberty, the testis undergoes remarkable morphological changes as testosterone levels rise and sperm production begins. We hypothesized that testicular maturation might also involve dramatic changes in lipid composition. Accordingly, we characterized the sterol and fatty acid composition of the testis of rhesus monkeys throughout the lifespan, from birth to old age. Although the cholesterol content in the testis remained relatively unchanged throughout life, the desmosterol content first decreased from 59 microg/g in infants to 6 microg/g in prepubertal monkeys, increased to 83 microg/g during puberty, and reached a plateau of 248 microg/g in the young adult, where it remained into old age. The polyunsaturated fatty acid composition of the testis also changed markedly. Docosahexaenoic acid (22:6 n-3) increased from 5.1% of total fatty acids in infants and juveniles to 18.1% in postpubertal young adults. Although some n-6 fatty acids, arachidonic (20:4 n-6) and linoleic (18:2 n-6), decreased from 16.0% and 10.0% in prepubertal juveniles, respectively, to 7.1% and 3.3% in young adults; dihomogamma-linolenic acid (20:3 n-6), the precursor of 1 series PGs, increased greatly from 1.8% to 10.3%. Similar changes occurred in both membrane and storage lipids (phospholipids and triglycerides), respectively. After puberty, the testicular fatty acid pattern remained stable into old age. Our data demonstrated that puberty is accompanied by substantial changes in the lipid composition of the primate testis. These changes suggest that desmosterol and both n-3 and n-6 polyunsaturated fatty acids may have important roles in sexual maturation.

Visual attention in infant monkeys: effects of dietary fatty acids and age.

Effects of dietary essential fatty acids on visual attention were explored longitudinally in infant rhesus monkeys with a visual paired-comparison paradigm. Sets of primate faces and of patterns were presented at Weeks 2, 5, 9, and 13 to 9 infants deficient in omega-3 fatty acids and 8 fed a standard nursery diet. Familiarization to 1 member of each pair preceded simultaneous presentation of both stimuli. Infants fed the deficient diet showed longer individual looks in both immediate and 24-hr tests. Duration of looks decreased with age to familiar but not to novel stimuli. The proportion of time looking at the novel stimulus (% novel) increased with age but was not affected by diet. Look duration and % novel were differentially affected and may reflect different underlying processes.

Visual acuity, erythrocyte fatty acid composition, and growth in term infants fed formulas with long chain polyunsaturated fatty acids for one year. Ross Pediatric Lipid Study.

The CNS and the retina are enriched in long chain polyunsaturated (LCP) fatty acids, specifically docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6), which are present in human milk but not in most infant formulas. In the present study of 134 formula-fed and 63 breast-fed infants, we prospectively evaluated whether providing a source of DHA and AA or DHA alone in formula would increase red blood cell (RBC) phospholipid levels of these fatty acids, enhance visual function, or affect growth during the first year. Healthy term infants < 7 d old were randomized to be fed formulas containing linoleic acid (approximately 10% kcal) and alpha-linolenic acid (approximately 1% kcal) plus (1) no added LCP fatty acids (control formula), (2) DHA (0.12 wt% fatty acids) and AA (0.43 wt%) from egg yolk phospholipid (AA + DHA formula), or (3) DHA (0.2 wt%) from fish oil (DHA formula). A breast-fed group was studied concurrently and permitted formula supplementation after 3 mo. Visual acuity was measured using both the acuity card procedure and a visual evoked potential method at 2, 4, 6, 9, and 12 mo. Infants fed the control formula had 10-40% lower RBC levels of DHA and AA than infants in the breast-fed group. Infants fed the AA + DHA formula had levels of both LCP within approximately 10% of the values for infants in the breast-fed group, and infants fed the DHA formula had 25-55% higher DHA levels and 15-40% lower AA levels. There were no differences in growth or in visual function during this 12-mo feeding study.

The role of n-3 fatty acids in visual and cognitive development: current evidence and methods of assessment.

Low levels of dietary n-3 fatty acids during development lead to changes in the electroretinogram and visual acuity in rats, monkeys, and human infants. Additional aspects of vision can be measured in infants and may provide further useful information about the nature of the changes induced by n-3 fatty acid deficiency and their potential long-term relevance for formula-fed human infants. Several tests are available to assess the development of specific cognitive functions such as visual recognition and short-term memory. These measures can provide information that is not now available to test the hypothesis that n-3 fatty acids are important for other aspects of neural function, including intellectual or cognitive development.

Effect of dietary N-3 fatty acids upon the phospholipid molecular species of the monkey retina.

PURPOSE: To characterize the molecular species composition of ethanolamine glycerophospholipids (EGP) in the primate retina and to examine the effects of different dietary fats, the authors fed rhesus monkeys diets containing widely ranging amounts of n-3 fatty acids. METHODS: From birth, infant monkeys were fed either a control soybean oil diet, containing 8% of total fatty acids as 18:3 (n-3), or a safflower oil-based n-3 fatty acid deficient diet containing < 0.4% 18:3 (n-3). A subset of the n-3 deficient group was later repleted with 1.6% ethyl docosahexaenoate, 22:6 (n-3), starting at 10 months of age. Tissues were taken from all monkeys upon termination at 21 to 51 months of age. The diacyl, alkenylacyl, and alkylacyl EGPs were quantitated by high-pressure liquid chromatography (HPLC). RESULTS: Twenty-eight molecular species were identified in the retina of control monkeys. Ether phospholipids comprised 36% of the retinal ethanolamine glycerophospholipids. Species containing polyunsaturated fatty acids in both the sn-1 and sn-2 positions (dipolyenes) were present only in the diacyl subclass and comprised 16% of the total species. Species having n-3 fatty acids in the sn-2 position contributed 59%, 36%, and 70% of total species in the diacyl, alkenylacyl, and alkylacyl subclasses, respectively. In the molecular species of the n-3 fatty acid deficient monkeys, the major change was the loss of most of the 18:0-22:6(n-3) species and its partial replacement with 18:0-22:5(n-6). In contrast, the species 18:1-22:6(n-3) decreased only slightly, from 6.2% to 4.8% of total diacyl species. Although the total concentration of dipolyenes (15% to 20% of the total species) was not affected by diet, their fatty acid compositions were changed drastically. The dipolyene species 22:6(n-3)-22:6(n-3) nearly disappeared in the n-3 deficient monkeys. Concomitantly, two new species, 22:5(n-6)-22:6(n-3) and 22:5(n-6)-22:5(n-6), appeared at 2.6% and 2.0%, respectively. Deficient monkeys given the ethyl ester of 22:6(n-3) in the diet recovered to a near-normal molecular species composition, except in the ether lipids, in which 16:0-20:4 remained low. CONCLUSION: Diets of differing n-3 fatty acid content had profound qualitative and quantitative effects on the molecular species of retinal phospholipids, and the replacement of 22:6(n-3) by 22:5(n-6) in the retinas of n-3 deficient monkeys was asymmetric and functionally incomplete.

Home cage behavior of rhesus monkeys with long-term deficiency of omega-3 fatty acids.

In an observational study with a blind observer, rhesus monkeys deficient in omega-3 (omega-3 or n-3) fatty acids initiated more bouts of stereotyped behavior in their home cages than monkeys fed a matched control diet abundant in omega-3 fatty acids. Locomotion bouts were also more frequent in deficient monkeys, but nonstereotyped locomotion did not differ. Both stereotyped behavior and the sum of all behavioral bouts were more frequent in 4-5-year-old than in 2-3-year-old monkeys, and stereotype decreased after meals in males but not females. The stereotyped behaviors associated with a deficit in omega-3 fatty acids were those typical of rhesus monkeys raised as partial social isolates or those whose surroundings have been disrupted.