Rachel Krolow

Neonatal hypoxia–ischemia induces sex-related changes in rat brain mitochondria

The effects of neonatal hypoxia-ischemia (HI) on energy metabolism in male and female rats were investigated, testing the hypothesis that HI-induced brain mitochondrial dysfunction could present in a dimorphic pattern. Impairment in electron transport chain complex activities at 2 and 18 h after HI was observed in cortex and hippocampus in rats of both sexes, with females presenting an overall activity higher than that of males. Females also showed loss of mitochondrial mass and membrane potential 18 h after HI, while males were only slightly affected. These findings suggest a dimorphism in mitochondrial dysfunction and provide information that may lead to new neuroprotection strategies.

Early biochemical effects after unilateral hypoxia–ischemia in the immature rat brain

Perinatal hypoxia–ischemia (HI) gives rise to inadequate substrate supply to the brain tissue, resulting in damage to neural cells. Previous studies at different time points of development, and with different animal species, suggest that the HI insult causes oxidative damage and changes Na+, K+–ATPase activity, which is known to be very susceptible to free radical‐related lipid peroxidation. The aim of the present study was to establish the onset of the oxidative damage response in neonatal Wistar rats subjected to brain HI, evaluating parameters of oxidative stress, namely nitric oxide production, lipoperoxidation by thiobarbituric acid reactive substances (TBA‐RS) production and malondialdehyde (MDA) levels, reactive species production by DCFH oxidation, antioxidant enzymatic activities of catalase, glutathione peroxidase, superoxide dismutase as well as Na+, K+–ATPase activity in hippocampus and cerebral cortex. Rat pups were subjected to right common carotid ligation followed by exposure to a hypoxic atmosphere (8% oxygen and 92% nitrogen) for 90 min. Animals were sacrificed by decapitation 0, 1 and 2 h after HI and both hippocampus and cerebral cortex from the right hemisphere (ipsilateral to the carotid occlusion) were dissected out for further experimentation. Results show an early decrease of Na+, K+–ATPase activity (at 0 and 1 h), as well as a late increase in MDA levels (2 h) and superoxide dismutase activity (1 and 2 h after HI) in the hippocampus. There was a late increase in both MDA levels and DCFH oxidation (1 and 2 h) and an increase in superoxide dismutase activity (2 h after HI) in cortex; however Na+, K+–ATPase activity remained unchanged. We suggest that neonatal HI induces oxidative damage to both hippocampus and cortex, in addition to a decrease in Na+, K+–ATPase activity in hippocampus early after the insult. These events might contribute to the later morphological damage in the brain and indicate that it would be essential to pursue neuroprotective strategies, aimed to counteract oxidative stress, as early as possible after the HI insult.

Early life stress interacts with the diet deficiency of omega-3 fatty acids during the life course increasing the metabolic vulnerability in adult rats.

Early stress can cause metabolic disorders in adulthood. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) deficiency has also been linked to the development of metabolic disorders. The aim of this study was to assess whether an early stressful event such as maternal separation interacts with the nutritional availability of n-3 PUFAs during the life course on metabolic aspects. Litters were randomized into: maternal separated (MS) and non-handled (NH). The MS group was removed from their dam for 3 hours per day and put in an incubator at 32°C on days 1° to 10° postnatal (PND). On PND 35, males were subdivided into diets that were adequate or deficient in n-3 PUFAs, and this intervention was applied during the subsequent 15 weeks. Animals body weight and food consumption were measured weekly, and at the end of the treatment tissues were collected. MS was associated with increased food intake (p = 0.047) and weight gain (p = 0.012), but no differences were found in the NPY hypothalamic content between the groups. MS rats had also increased deposition of abdominal fat (p<0.001) and plasma triglycerides (p = 0.018) when compared to the NH group. Interactions between early life stress and n-3 PUFAs deficiency were found in plasma insulin (p = 0.033), HOMA index (p = 0.049), leptin (p = 0.010) and liver PEPCK expression (p = 0.050), in which the metabolic vulnerability in the MS group was aggravated by the n-3 PUFAs deficient diet exposure. This was associated with specific alterations in the peripheral fatty acid profile. Variations in the neonatal environment interact with nutritional aspects during the life course, such as n-3 PUFAs diet content, and persistently alter the metabolic vulnerability in adulthood.

Effects of early life interventions and palatable diet on anxiety and on oxidative stress in young rats

Early life events can change biochemical, endocrine and behavioral aspects throughout the life of an animal. Since there is a strong relationship between stress, neonatal handling and feeding behavior, the aim of this study was to investigate the effects of these three factors on behavioral parameters (anxiety and locomotion), oxidative stress in brain structures (prefrontal cortex and hippocampus) and on plasma glucose. Nests of Wistar rats were handled (10 min/day), or not (control groups), on days 1-10 after birth. Males from these groups were divided into 4 subgroups: (1) stressed by isolation in childhood (pre-puberty) and with access to a highly palatable diet (2) stressed by isolation and receiving standard lab chow (3) not isolated and receiving a highly palatable diet and (4) not isolated and receiving standard chow. The animals were kept under these conditions for 7 days. Rats receiving the highly palatable diet consumed more food, more calories, gained more weight and had a higher plasma glucose level, but had a lower caloric efficiency than the standard chow groups. Both handling and palatable diet were able to increase food consumption on the first day of isolation. Isolation stress had an anxiogenic effect in the plus maze, which was counteracted by handling. Palatable diet increased time spent in the central area of the open field apparatus and in the open arms of the elevated plus maze, showing an anxiolytic effect. The use of both these conditions, however, does not appear to bring additional protection against the effects of stress during this particular period of life, i.e., pre-puberty. In the prefrontal cortex, handling reduced thiol content and appears to imbalance the antioxidant enzymes system, which is counteracted by a palatable diet. Hippocampus seems to be more sensitive than the prefrontal cortex to early interventions, especially to the highly palatable diet, and both handling and diet appear to imbalance the antioxidant enzyme system. Thus, measurements of antioxidant enzymes activities indicate that handling may endanger some brain structures and that the palatable diet was able to prevent some handling effects on antioxidant enzymes, depending on the brain structure.

Sex-specific effects of isolation stress and consumption of palatable diet during the prepubertal period on metabolic parameters

OBJECTIVES Social isolation during the prepubertal period may have long-term effects on metabolism. The exposure to stressful events is associated with increased palatable food intake, constituting reward-based eating. However, palatable food consumption in early life may lead to metabolic alterations later in life. We investigated whether isolation stress during early life can lead to metabolic alterations in male and female rats with or without exposure to a palatable diet. METHODS Animals were stressed by isolation during one week after weaning, with or without exposure to a palatable diet. RESULTS Stress and palatable diet induced increased caloric consumption. In females, there was a potentiation of consumption in animals exposed to stress and palatable diet, reflected by increased weight gain and triacylglycerol levels in juveniles, as well as increased adiponectin levels. Most of the effects had disappeared in the adults. Different effects were observed in males: in juveniles, stress increased unacylated ghrelin levels, and hypothalamic neuropeptide Y (NPY). Subsequently, adult males that were exposed to a palatable diet during prepuberty showed increased body weight and retroperitoneal fat deposition, increased glycemia, and decreased plasma adiponectin and hypothalamic NPY. Exposure to stress during prepuberty led to increased adrenals during adulthood, decreased LDL-cholesterol and increased triacylglycerol levels. CONCLUSION Isolation stress and consumption of palatable diet changes metabolism in a sex-specific manner. Prepuberty female rats were more prone to stress effects on food consumption, while males showed more long-lasting effects, being more susceptible to a metabolic programming after the consumption of a palatable diet.

Resveratrol treatment has neuroprotective effects and prevents cognitive impairment after chronic cerebral hypoperfusion

Abstract Objective: The present study investigated the neuroprotective effects of Resveratrol (RSV) in rats submitted to chronic cerebral hypoperfusion (CCH) in a model of permanent two-vessel occlusion (2VO). Methods: For this purpose, adult Wistar rats received daily i.p. injections of RSV (20 mg/kg) for 7 days, starting 1 hour after the 2VO procedure. Behavioral testing was run between the 30th and 45th days after the 2VO surgery. Accordingly, spatial working memory function in the Morris water maze was evaluated. At the end of the behavioral assessment (45th day post-surgery) part of experimental animals underwent transcardiac perfusion for histological analysis. Another group was euthanized on the 3rd, 14th, and 45th days post-surgery for nerve growth factor (NGF) evaluation. Results: Resveratrol treatment along 7 days after CCH significantly attenuated pyramidal cell death in the CA1 hippocampal subfield and prevented both spatial working and reference memory impairments. Our results revealed an enhancement of NGF expression 3 days after CCH in all ischemic animals. A late increase in hippocampal NGF levels was detected after 45 days only in CCH-RSV treated animals. Conclusions: Results presented here show morphological and functional neuroprotective actions of RSV treatment for CCH, as well as support the inducing effects of RSV on the expression of NGF and its possible association to the neuroprotective action in this rodent model of vascular dementia.

Effect of chronic administration of tamoxifen and/or estradiol on feeding behavior, palatable food and metabolic parameters in ovariectomized rats.

Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) used in the treatment of breast cancer; however many women complain of weight gain during TAM treatment. The anorectic effects of estradiol (E) and TAM are well known, although the effects of E on the consumption of palatable food are controversial and there is no information regarding the effects of TAM on palatable food consumption. The aim of this study was to investigate the effects of chronic treatment with estradiol and/or tamoxifen on feeding behavior in ovariectomized rats exposed to standard chow and palatable foods (Froot Loops® or chocolate). Additionally, parameters such as body weight, uterine weight, lipid profile and plasma glucose were also measured. Wistar rats were ovariectomized (OVX) and subsequently injected (ip.) for 40 days with: E, TAM, E+TAM or vehicle (OVX and SHAM - controls). Behavioral tests were initiated 25 days after the start of treatment. Froot Loops® consumption was evaluated in a novel environment for 3 min. Standard chow intake was evaluated for two days and chocolate intake for 7 days in the home cage in a free choice model (chocolate or standard chow). Rats injected with E, TAM and E+TAM groups showed a reduction in body weight and standard chow intake, compared with control groups. With regard to palatable food intake, the E, TAM and E+TAM groups demonstrated increased consumption of Froot Loops®, compared with the SHAM and OVX groups. In contrast, all groups increased their consumption of chocolate, compared with standard chow; however the E group consumed more chocolate than the OVX, TAM and E+TAM groups. Despite these differences in chocolate consumption, all groups showed the same caloric intake during the chocolate exposure period; however the TAM and E+TAM groups presented decreased body weight. Treatment with estradiol and tamoxifen showed a favorable lipid profile with low levels of TC, LDL, LDL/HDL ratio and lower levels of plasma glucose. The E group presented high levels of TG and HDL, when compared with the TAM and E+TAM groups. Taken together, results suggest that TAM acted in an estrogen-like manner on the majority of parameters analyzed. However, tamoxifen acts in a different manner depending on the type of palatable food and the exposure. In addition, the TAM group demonstrated weight loss, compared with other groups independently of the type of food presented (palatable food or standard chow), showing a low caloric efficiency.

Vulnerability to dietary n-3 polyunsaturated fatty acid deficiency after exposure to early stress in rats

The exposure to adverse events early in life may affect brain development. Omega-3 polyunsaturated fatty acid (n-3 PUFA) deficiency has been linked to the development of mood and anxiety disorders. The aim of this study was to examine the interaction between variations in the early environment (handling or maternal separation) and the chronic exposure to a nutritional n-3 PUFA deficiency on locomotor activity, sucrose preference, forced swimming test and on serum and hippocampal brain-derived neurotrophic factor (BDNF) levels. Rats were randomized into Non-handled (NH), Neonatal Handled (H) and Maternal Separated (MS) groups. Pups were removed from their dams (incubator at 32°C on postnatal days (PND) 1-10) during 10 min/day (H) or 3h/day (MS). On PND 35, males were subdivided into diets adequate or deficient in n-3 PUFA for 15 weeks. H and MS gained weight differently, and animals receiving the n-3 PUFA deficient diet gained less weight. MS displayed a higher food consumption and higher consumption of sucrose solution during the second hour of exposure to the sucrose preference test. No differences were observed in the swimming test. H group had increased locomotion and showed a higher response to amfepramone. No significant effect was observed on serum BDNF levels. BDNF protein levels were decreased in animals receiving the n-3 PUFA deficient diet. We observed that early life environment and a mild n-3 PUFA deficiency are able to affect several behavioral aspects (food and sucrose consumption and locomotor response), and lead to a differential hippocampal BDNF metabolism in adult life.

Isolation Stress During the Prepubertal Period in Rats Induces Long-Lasting Neurochemical Changes in the Prefrontal Cortex

Social isolation during postnatal development leads to behavioral and neurochemical changes, and a particular susceptibility of the prefrontal cortex to interventions during this period has been suggested. In addition, some studies showed that consumption of a palatable diet reduces some of the stress effects. Therefore, our aim is to investigate the effect of isolation stress in early life on some parameters of oxidative stress and energy metabolism (Na+,K+-ATPase activity, respiratory chain enzymes activities and mitochondrial mass and potential) in prefrontal cortex of juvenile and adult male rats. We also verified if the consumption of a palatable diet during the prepubertal period would reduce stress effects. The results showed that, in juvenile animals, isolation stress increased superoxide dismutase and Complex IV activities and these effects were still observed in the adulthood. An interaction between stress and diet was observed in catalase activity in juveniles, while only the stress effect was detected in adults, reducing catalase activity. Access to a palatable diet increased Na+,K+-ATPase activity in juveniles, an effect that was reversed after removing this diet. On the other hand, isolation stress induced a decreased activity of this enzyme in adulthood. No effects were observed on glutathione peroxidase, total thiols and free radicals production, as well as on mitochondrial mass and potential. In conclusion, isolation stress in the prepubertal period leads to long-lasting changes on antioxidant enzymes and energetic metabolism in the prefrontal cortex of male rats, and a palatable diet was not able to reverse these stress-induced effects.

Neonatal handling affects learning, reversal learning and antioxidant enzymes activities in a sex-specific manner in rats

Early life experiences have profound influences on behavior and neurochemical parameters in adult life. The aim of this study is to verify neonatal handling‐induced sex specific differences on learning and reversal learning as well as oxidative stress parameters in the prefrontal cortex and striatum of adult rats. Litters of rats were non‐handled or handled (10 min/day, days 1–10 after birth). In adulthood, learning and reversal learning were evaluated using a Y maze associated with palatable food in male and female rats. Morris water maze reversal learning was verified in males. Oxidative stress parameters were evaluated in both genders. Male neonatal handled animals had a worse performance in the Y maze reversal learning compared to non‐handled ones and no difference was observed in the water maze reversal learning task. Regarding females, neonatal handled rats had a better performance during the Y maze learning phase compared to non‐handled ones. In addition, neonatal handled female animals showed a decreased SOD/CAT ratio in the PFC compared to non‐handled females. We conclude that neonatal handling effects on learning and memory in adult rats are sex and task specific. The sex specific differences are also observed in the evaluation of antioxidant enzymes activities with neonatal handling affecting only females.