Combined Effects of Early Life Stress and Prolonged Exposure to Western Diet on Emotional Responses and Gut Microbiota.

BACKGROUND: Exposure to early life stress (ELS) and maternal consumption of a high-fat and high-sugar diet can have detrimental effects on adult emotional responses. The microbiota and gut-brain axis have been proposed as playing a mediating role in the regulation of stress and emotion. METHOD: Young male rats were exposed to maternal separation (MS) together with maternal and postnatal consumption of a HFS diet (45%kcal saturated fat, 17%kcal sucrose). Anxiety-like behaviour was evaluated using an elevated zero-maze, and depression-like behaviour using the forced-swim and sucrose preference tests. Microbiota composition and derived metabolites were also analysed in faecal samples using a gas chromatograph and mass spectrometry. RESULTS: Combined exposure to MS and lifelong consumption of a HFS diet partially reversed the abnormal anxiety-like and depression-like behaviours in early adulthood caused by each adverse factor alone. Diet composition had a greater negative impact than ELS exposure on the gut microbiota, and both environmental factors interacted with microbiota composition partially counteracting their negative effects. CONCLUSIONS: The effects of exposure to early life stress and a HFS diet independently are partially reversed after the combination of both factors. These results suggest that ELS and diet interact to modulate adult stress response and gut microbiota.

A shift to a standard diet after exposure to a high-fat, high-sucrose diet from gestation to weaning restores brain metabolism and behavioral flexibility in adult rats.

Prolonged consumption of diets high in saturated fat and sugar has been related to obesity and overweight, which in turn are linked to cognitive impairment in both humans and rodents. This has become a current issue, especially in children and adolescents, because these stages are crucial to neurodevelopmental processes and programming of adult behavior. To evaluate the effects of gestational and early exposure to an obesogenic diet, three groups with different dietary patterns were established: high-fat and high-sucrose diet (HFS), standard diet (SD), and a dietary shift from a high-fat, high-sucrose diet to a standard diet after weaning (R). Spatial learning and behavioral flexibility in adult male and female Wistar rats were evaluated using the Morris water maze (MWM) at PND 60. Furthermore, regional brain oxidative metabolism was assessed in the prefrontal cortex and the hippocampus. Contrary to our hypothesis, the HFS diet groups showed similar performance on the spatial learning task as the other groups, although they showed impaired cognitive flexibility. The HFS group had increased brain metabolic capacity compared to that of animals fed the standard diet. Shifting from the HFS diet to the SD diet after weaning restored the brain metabolic capacity in both sexes to levels similar to those observed in animals fed the SD diet. In addition, animals in the R group performed similarly to those fed the SD diet in the Morris water maze in both tasks. However, dietary shift from HFS diet to standard diet after weaning had only moderate sex-dependent effects on body weight and fat distribution. In conclusion, switching from an HFS diet to a balanced diet after weaning would have beneficial effects on behavioral flexibility and brain metabolism, without significant sex differences.

Sexual dimorphism in spatial learning and brain metabolism after exposure to a western diet and early life stress in rats.

Prolonged daily intake of Western-type diet rich in saturated fats and sugars, and exposure to early life stress have been independently linked to impaired neurodevelopment and behaviour in animal models. However, sex-specific effects of both environmental factors combined on spatial learning and memory, behavioural flexibility, and brain oxidative capacity have still not been addressed. The current study aimed to evaluate the impact of maternal and postnatal exposure to a high-fat and high-sugar diet (HFS), and exposure to early life stress by maternal separation in adult male and female Wistar rats. For this purpose, spatial learning and memory and behavioural flexibility were evaluated in the Morris water maze, and regional brain oxidative capacity and oxidative stress levels were measured in the hippocampus and medial prefrontal cortex. Spatial memory, regional brain oxidative metabolism, and levels of oxidative stress differed between females and males, suggesting sexual dimorphism in the effects of a HFS diet and early life stress. Males fed the HFS diet performed better than all other experimental groups independently of early life stress exposure. However, behavioural flexibility evaluated in the spatial reversal leaning task was impaired in males fed the HFS diet. In addition, exposure to maternal separation or the HFS diet increased the metabolic capacity of the prefrontal cortex and dorsal hippocampus in males and females. Levels of oxidative stress measured in the latter brain regions were also increased in groups fed the HFS diet, but maternal separation seemed to dampen regional brain oxidative stress levels. Therefore, these results suggest a compensatory effect resulting from the interaction between prolonged exposure to a HFS diet and early life stress.

Early-life stress induces emotional and molecular alterations in female mice that are partially reversed by cannabidiol.

Gender is considered as a pivotal determinant of mental health. Indeed, several psychiatric disorders such as anxiety and depression are more common and persistent in women than in men. In the past two decades, impaired brain energy metabolism has been highlighted as a risk factor for the development of these psychiatric disorders. However, comprehensive behavioural and neurobiological studies in brain regions relevant to anxiety and depression symptomatology are scarce. In the present study, we summarize findings describing cannabidiol effects on anxiety and depression in maternally separated female mice as a well-established rodent model of early-life stress associated with many mental disorders. Our results indicate that cannabidiol could prevent anxiolytic- and depressive-related behaviour in early-life stressed female mice. Additionally, maternal separation with early weaning (MSEW) caused long-term changes in brain oxidative metabolism in both nucleus accumbens and amygdalar complex measured by cytochrome c oxidase quantitative histochemistry. However, cannabidiol treatment could not revert brain oxidative metabolism impairment. Moreover, we identified hyperphosphorylation of mTOR and ERK 1/2 proteins in the amygdala but not in the striatum, that could also reflect altered brain intracellular signalling related with to bioenergetic impairment. Altogether, our study supports the hypothesis that MSEW induces profound long-lasting molecular changes in mTOR signalling and brain energy metabolism related to depressive-like and anxiety-like behaviours in female mice, which were partially ameliorated by CBD administration.

Hippocampal neuropeptide Y2 receptor blockade improves spatial memory retrieval and modulates limbic brain metabolism.

INTRODUCTION: The neuropeptide Y (NPY) is broadly distributed in the central nervous system (CNS), and it has been related to neuroprotective functions. NPY seems to be an important component to counteract brain damage and cognitive impairment mediated by drugs of abuse and neurodegenerative diseases, and both NPY and its Y2 receptor (Y2R) are highly expressed in the hippocampus, critical for learning and memory. We have recently demonstrated its influence on cognitive functions; however, the specific mechanism and involved brain regions where NPY modulates spatial memory by acting on Y2R remain unclear. METHODS: Here, we examined the involvement of the hippocampal NPY Y2R in spatial memory and associated changes in brain metabolism by bilateral administration of the selective antagonist BIIE0246 into the rat dorsal hippocampus. To further evaluate the relationship between memory functions and neuronal activity, we analysed the regional expression of the mitochondrial enzyme cytochrome c oxidase (CCO) as an index of oxidative metabolic capacity in limbic and non-limbic brain regions. RESULTS: The acute blockade of NPY Y2R significantly improved spatial memory recall in rats trained in the Morris water maze that matched metabolic activity changes in spatial memory processing regions. Specifically, CCO activity changes were found in the dentate gyrus of the dorsal hippocampus and CA1 subfield of the ventral hippocampus, the infralimbic region of the PFC and the mammillary bodies. CONCLUSIONS: These findings suggest that the NPY hippocampal system, through its Y2R receptor, influences spatial memory recall (retrieval) and exerts control over patterns of brain activation that are relevant for associative learning, probably mediated by Y2R modulation of long-term potentiation and long-term depression.

Relationship between Prenatal or Postnatal Exposure to Pesticides and Obesity: A Systematic Review.

In recent years, the worldwide prevalence of overweight and obesity among adults and children has dramatically increased. The conventional model regarding the onset of obesity is based on an imbalance between energy intake and expenditure. However, other possible environmental factors involved, such as the exposure to chemicals like pesticides, cannot be discarded. These compounds could act as endocrine-disrupting chemicals (EDC) that may interfere with hormone activity related to several mechanisms involved in body weight control. The main objective of this study was to systematically review the data provided in the scientific literature for a possible association between prenatal and postnatal exposure to pesticides and obesity in offspring. A total of 25 human and 9 animal studies were analyzed. The prenatal, perinatal, and postnatal exposure to organophosphate, organochlorine, pyrethroid, neonicotinoid, and carbamate, as well as a combined pesticide exposure was reviewed. This systematic review reveals that the effects of pesticide exposure on body weight are mostly inconclusive, finding conflicting results in both humans and experimental animals. The outcomes reviewed are dependent on many factors, including dosage and route of administration, species, sex, and treatment duration. More research is needed to effectively evaluate the impact of the combined effects of different pesticides on human health.

Early life stress due to repeated maternal separation alters the working memory acquisition brain functional network.

Unfortunately, adverse environments in early life are frequently found in most human populations. Early life stress leads to diverse cognitive impairments, some of them related to learning and memory and executive functions such as working memory (WM). We employ an animal model of early stress using repeated maternal separation (MS) for 4 h a day on 21 consecutive days, pre-weaning. In adulthood, we tested their spatial WM using the Morris water maze. MS subjects showed a marked delay in the acquisition of the task. In addition, we explored brain energy oxidative metabolism and found an increase in cytochrome c oxidase (CCO) activity in the cingulate cortex, anterior thalamus, and supramammillary areas, indicating an intense effort to successfully solve the WM task. However, decreased CCO activity was found in the medial-medial mammillary nucleus in MS animals, which would partially explain the delayed acquisition of the WM task. Further studies are needed to explore the long-term alterations produced by early stress. LAY SUMMARY A stressful environment caused by the separation of baby rats from the mother for several hours a day in the first stages of postnatal life can be devastating to brain cells, making them look for alternative sources of energy, among other changes. These alterations in brain functional networks would lead to cognitive impairments such as the delayed acquisition of new learning and strategies.

Sex-Specific Effects of Early Life Stress on Brain Mitochondrial Function, Monoamine Levels and Neuroinflammation.

Sex differences have been reported in the susceptibility to early life stress and its neurobiological correlates in humans and experimental animals. However, most of the current research with animal models of early stress has been performed mainly in males. In the present study, prolonged maternal separation (MS) paradigm was applied as an animal model to resemble the effects of adverse early experiences in male and female rats. Regional brain mitochondrial function, monoaminergic activity, and neuroinflammation were evaluated as adults. Mitochondrial energy metabolism was greatly decreased in MS females as compared with MS males in the prefrontal cortex, dorsal hippocampus, and the nucleus accumbens shell. In addition, MS males had lower serotonin levels and increased serotonin turnover in the prefrontal cortex and the hippocampus. However, MS females showed increased dopamine turnover in the prefrontal cortex and increased norepinephrine turnover in the striatum, but decreased dopamine turnover in the hippocampus. Sex differences were also found for pro-inflammatory cytokine levels, with increased levels of TNF-α and IL-6 in the prefrontal cortex and hippocampus of MS males, and increased IL-6 levels in the striatum of MS females. These results evidence the complex sex- and brain region-specific long-term consequences of early life stress.

Environmental enrichment effects after early stress on behavior and functional brain networks in adult rats.

Early life stress is associated with long-term and pervasive adverse effects on neuroendocrine development, affecting normal cognitive and emotional development. Experimental manipulations like environmental enrichment (EE) may potentially reverse the effects of early life stress induced by maternal separation (MS) paradigm in rodents. However, the functional brain networks involved in the effects of EE after prolonged exposure to MS have not yet been investigated. In order to evaluate possible changes in brain functional connectivity induced by EE after MS, quantitative cytochrome c oxidase (CCO) histochemistry was applied to determine regional brain oxidative metabolism in adult male rats. Unexpectedly, results show that prolonged MS during the entire weaning period did not cause any detrimental effects on spatial learning and memory, including depressive-like behavior evaluated in the forced-swim test, and decreased anxiety-like behavior. However, EE seemed to alter anxiety- and depression-like behaviors in both control and MS groups, but improved spatial memory in the latter groups. Analysis of brain CCO activity showed significantly lower metabolic capacity in most brain regions selected in EE groups probably associated with chronic stress, but no effects of MS on brain metabolic capacity. In addition, principal component analysis of CCO activity revealed increased large-scale functional brain connectivity comprising at least three main networks affected by EE in both MS and control groups. Moreover, EE induced a pattern of functional brain connectivity associated with stress and anxiety-like behavior as compared with non-enriched groups. In conclusion, EE had differential effects on cognition and emotional behavior irrespective of exposure to MS. In view of the remarkable effects of EE on brain function and behavior, implementation of rodent housing conditions should be optimized by evaluating the balance between scientific validity and animal welfare.

Influence of environmental enrichment on the volume of brain regions sensitive to early life stress by maternal separation in rats.

BACKGROUND: Exposure to maternal separation (MS) in rodents may have long-lasting consequences for the structure and function of several brain regions, eventually associated with alterations in cognition and emotion later in life. Post-weaning environmental enrichment (EE) has been reported to ameliorate the detrimental effects of exposure to early life stress mainly in the hippocampus. METHOD: In vivo magnetic resonance imaging (MRI) was applied to evaluate possible volumetric changes in the dorsal and ventral hippocampus, the medial prefrontal cortex and the dorsal striatum of 90-day-old male rats after daily MS for 240 min from postnatal days 2-21. RESULTS: No significant volume changes were found in the selected brain regions in MS animals as compared with an age-matched control group. However, additional groups of control and MS animals with EE from days 21-60 showed significant volume increases in the medial prefrontal cortex and the ventral hippocampus as compared to the groups without EE. In addition, general hemispheric asymmetry was found in the volume of the brain regions measured. CONCLUSIONS: Our results demonstrate that EE could have differential effects depending on previous exposure to MS and on the development of brain lateralization.

Altered brain functional connectivity and behaviour in a mouse model of maternal alcohol binge-drinking.

Prenatal and perinatal alcohol exposure caused by maternal alcohol intake during gestation and lactation periods can have long-lasting detrimental effects on the brain development and behaviour of offspring. Children diagnosed with Foetal Alcohol Spectrum Disorders (FASD) display a wide range of cognitive, emotional and motor deficits, together with characteristic morphological abnormalities. Maternal alcohol binge drinking is particularly harmful for foetal and early postnatal brain development, as it involves exposure to high levels of alcohol over short periods of time. However, little is known about the long-term effects of maternal alcohol binge drinking on brain function and behaviour. To address this issue, we used pregnant C57BL/6 female mice with time-limited access to a 20% v/v alcohol solution as a procedure to model alcohol binge drinking during gestation and lactational periods. Male offspring were behaviourally tested during adolescence (30 days) and adulthood (60 days), and baseline neural metabolic capacity of brain regions sensitive to alcohol effects were also evaluated in adult animals from both groups. Our results show that prenatal and postnatal alcohol exposure caused age-dependent changes in spontaneous locomotor activity, increased anxiety-like behaviour and attenuated alcohol-induced conditioned place preference in adults. Also, significant changes in neural metabolic capacity using cytochrome c oxidase (CCO) quantitative histochemistry were found in the hippocampal dentate gyrus, the mammillary bodies, the ventral tegmental area, the lateral habenula and the central lobules of the cerebellum in adult mice with prenatal and postnatal alcohol exposure. In addition, the analysis of interregional CCO activity correlations in alcohol-exposed adult mice showed disrupted functional brain connectivity involving the limbic, brainstem, and cerebellar regions. Finally, increased neurogenesis was found in the dentate gyrus of the hippocampus of alcohol-exposed offspring, suggesting neuroadaptive effects due to early alcohol exposure. Our results demonstrate that maternal binge-like alcohol drinking causes long-lasting effects on motor and emotional-related behaviours associated with impaired neuronal metabolic capacity and altered functional brain connectivity.

The Psychoexposome: A holistic perspective beyond health and disease.

BACKGROUND: The concept of the exposome has emerged as a new strategy for studying all environmental exposures throughout an individual’s life and their impact on human health. Nowadays, electronic devices are available to collect data about an individual’s geolocation, biological function, or exposure biomarkers. The appearance of “omic” sciences and advances in bioinformatics have allowed massive data-gathering and analysis from various scientific fields. OBJECTIVE: to propose the term Psychoexposome in line with the concept of the exposome from the field of environmental sciences. METHOD: a literature review of psychological terms associated with the exposome concept was carried out and the rationale and benefits of a psychoexposme approach for psychological sciences is discussed. RESULTS: the terms psychology, psychiatry and neurological diseases are scarce in the exposome approach. A long tradition in psychology of performing epidemiological studies and in the study of multifactorial influences traits places psychologists at an advantageous starting point for conducting psychoexposome studies. CONCLUSION: psychology may take advantage from both exposome and omic sciences to create an integrated psychoexposome approach that may help in deciphering the etiology of psychological disorders and improving people’s mental health.

Spatial memory extinction differentially affects dorsal and ventral hippocampal metabolic activity and associated functional brain networks.

Previous studies showed the involvement of brain regions associated with both spatial learning and associative learning in spatial memory extinction, although the specific role of the dorsal and ventral hippocampus and the extended hippocampal system including the mammillary body in the process is still controversial. The present study aimed to identify the involvement of the dorsal and ventral hippocampus, together with cortical regions, the amygdaloid nuclei, and the mammillary bodies in the extinction of a spatial memory task. To address these issues, quantitative cytochrome c oxidase histochemistry was applied as a metabolic brain mapping method. Rats were trained in a reference memory task using the Morris water maze, followed by an extinction procedure of the previously acquired memory task. Results show that rats learned successfully the spatial memory task as shown by the progressive decrease in measured latencies to reach the escape platform and the results obtained in the probe test. Spatial memory was subsequently extinguished as shown by the descending preference for the previously reinforced location. A control naïve group was added to ensure that brain metabolic changes were specifically related with performance in the spatial memory extinction task. Extinction of the original spatial learning task significantly modified the metabolic activity in the dorsal and ventral hippocampus, the amygdala and the mammillary bodies. Moreover, the ventral hippocampus, the lateral mammillary body and the retrosplenial cortex were differentially recruited in the spatial memory extinction task, as shown by group differences in brain metabolic networks. These findings provide new insights on the brain regions and functional brain networks underlying spatial memory, and specifically spatial memory extinction. © 2016 Wiley Periodicals, Inc.

Brain functional network changes following Prelimbic area inactivation in a spatial memory extinction task.

Several studies suggest a prefrontal cortex involvement during the acquisition and consolidation of spatial memory, suggesting an active modulating role at late stages of acquisition processes. Recently, we have reported that the prelimbic and infralimbic areas of the prefrontal cortex, among other structures, are also specifically involved in the late phases of spatial memory extinction. This study aimed to evaluate whether the inactivation of the prelimbic area of the prefrontal cortex impaired spatial memory extinction. For this purpose, male Wistar rats were implanted bilaterally with cannulae into the prelimbic region of the prefrontal cortex. Animals were trained during 5 consecutive days in a hidden platform task and tested for reference spatial memory immediately after the last training session. One day after completing the training task, bilateral infusion of the GABAA receptor agonist Muscimol was performed before the extinction protocol was carried out. Additionally, cytochrome c oxidase histochemistry was applied to map the metabolic brain activity related to the spatial memory extinction under prelimbic cortex inactivation. Results show that animals acquired the reference memory task in the water maze, and the extinction task was successfully completed without significant impairment. However, analysis of the functional brain networks involved by cytochrome oxidase activity interregional correlations showed changes in brain networks between the group treated with Muscimol as compared to the saline-treated group, supporting the involvement of the mammillary bodies at a the late stage in the memory extinction process.

Effect of lighting conditions on brain network complexity associated with response learning.

Several studies have reported the brain regions involved in response learning. However, there is discrepancy regarding the lighting conditions in the experimental setting (i.e. under dark or light conditions). In this regard, it would be relevant to know if the presence/absence of visual cues in the environment has any effect in the brain networks involved in a response learning task. Animals were trained in a water T-maze under two different lighting conditions (light versus dark). All subjects reached the learning criterion of 80% correct arm choices. Quantitative cytochrome oxidase (CO) histochemistry was used as a metabolic brain mapping technique. Our results show that the ventral hippocampus and the parietal cortex are associated with the acquisition of a response learning task regardless of lighting conditions. In addition, when the same task is run in the dark, widespread recruitment of structures involving cortical, limbic and striatal regions was found.

Acute effects of 17 ß-estradiol and genistein on insulin sensitivity and spatial memory in aged ovariectomized female rats.

Aging is characterized by decline in metabolic function and insulin resistance, and both seem to be in the basis of neurodegenerative diseases and cognitive dysfunction. Estrogens prevent age-related changes, and phytoestrogens influence learning and memory. Our hypothesis was that estradiol and genistein, using rapid-action mechanisms, are able to modify insulin sensitivity, process of learning, and spatial memory. Young and aged ovariectomized rats received acute treatment with estradiol or genistein. Aged animals were more insulin-resistant than young. In each age, estradiol and genistein-treated animals were less insulin-resistant than the others, except in the case of young animals treated with high doses of genistein. In aged rats, no differences between groups were found in spatial memory test, showing a poor performance in the water maze task. However, young females treated with estradiol or high doses of genistein performed well in spatial memory task like the control group. Only rats treated with high doses of genistein showed an optimal spatial memory similar to the control group. Conversely, acute treatment with high doses of phytoestrogens improved spatial memory consolidation only in young rats, supporting the critical period hypothesis for the beneficial effects of estrogens on memory. Therefore, genistein treatment seems to be suitable treatment in aged rats in order to prevent insulin resistance but not memory decline associated with aging. Acute genistein treatment is not effective to restore insulin resistance associated to the early loss of ovarian function, although it can be useful to improve memory deficits in this condition.

Changes in brain oxidative metabolism induced by inhibitory avoidance learning and acute administration of amitriptyline.

The effects of antidepressant drugs on memory have been somewhat ignored, having been considered a mere side effect of these compounds. However, the memory impairment caused by several antidepressants could be considered to form part of their therapeutic effects. Amitriptyline is currently one of the most prescribed tricyclic antidepressants, and exerts marked anticholinergic and antihistaminergic effects. In this study, we evaluated the effects of inhibitory avoidance (IA) learning and acute administration of amitriptyline on brain oxidative metabolism. Brain oxidative metabolism was measured in several limbic regions using cytochrome oxidase (CO) quantitative histochemistry. Amitriptyline produced a clear impairment in the IA task. In animals exposed only to the apparatus, amitriptyline decreased CO activity in nine brain regions, without affecting the remaining regions. In animals that underwent the IA training phase, amitriptyline reduced CO activity in only three of these nine regions. In animals treated with saline, IA acquisition increased CO activity in the medial prefrontal cortex, the prelimbic cortex, and the medial mammillary body, and diminished it in the medial septum and the nucleus basalis of Meynert with respect to animals exposed only to the IA apparatus. In animals treated with amitriptyline, IA acquisition did not modify CO activity in any of these regions, but increased it in the anteromedial nucleus of the thalamus, the diagonal band of Broca, and the dentate gyrus. The results reveal a pattern of changes in brain oxidative metabolism induced by IA training in saline-treated animals that was clearly absent in animals submitted to the same behavioural training but treated with amitriptyline.

Induction of c-Fos expression in the mammillary bodies, anterior thalamus and dorsal hippocampus after fear conditioning.

The aim of the present study was to provide further evidence on the role of particular subdivisions of the mammillary bodies, anterior thalamus and dorsal hippocampus to contextual and auditory fear conditioning. We used c-Fos expression as a marker of neuronal activation to compare rats that received tone-footshock pairings in a distinctive context (conditioned group) to rats being exposed to both the context and the auditory CS without receiving footshocks (unconditioned group), and naïve rats that were only handled. Fos immunoreactivity was significantly increased only in the anterodorsal thalamic nucleus and the lateral mammillary nucleus of the conditioned group. However, the dorsal hippocampus showed the highest density of c-Fos positive nuclei in the naïve group as compared to the other groups. Together, our data support previous studies indicating a particular involvement of the mammillary bodies and anterior thalamus in fear conditioning.

Brain c-Fos immunocytochemistry and cytochrome oxidase histochemistry after a fear conditioning task.

The involvement of the basolateral and the medial amygdala in fear conditioning was evaluated using different markers of neuronal activation. The method described here is a combination of cytochrome oxidase (CO) histochemistry and c-Fos immunocytochemistry on fresh frozen brain sections. Freezing behavior was used as an index of auditory and contextual fear conditioning. As expected, freezing scores were significantly higher in rats exposed to tone-shock pairings in a distinctive environment (conditioned; COND), as compared to rats that did not receive any shocks (UNCD). CO labeling was increased in the basolateral and medial amygdala of the COND group. Conversely, c-Fos expression in the basolateral and medial amygdala was lower in the COND group as compared to the UNCD group. Furthermore, c-Fos expression was particularly high in the medial amygdala of the UNCD group. The data provided by both techniques indicate that these amygdalar nuclei could play different roles on auditory and contextual fear conditioning.

A SAS/IML program for implementing the modified Brown-Forsythe procedure in repeated measures designs.

In this paper, we present a computer program written in version 9.1 of SAS' interactive matrix language in order to implement a new approach for analyzing repeated measures data. Previous studies reported that the new procedure is as powerful as conventional solutions and generally more robust (i.e., insensitive) to violations of assumptions that underlie conventional solutions. The program also included a step-wise procedure based on the Bonferroni inequality to test comparisons among the repeated measurements. Both univariate and multivariate repeated measures data can be analyzed. Finally, the application of the SAS/IML program is illustrated with a numeric example.