Differential Age-Dependent Mitochondrial Dysfunction, Oxidative Stress, and Apoptosis Induced by Neonatal Hypoxia-Ischemia in the Immature Rat Brain.

Neonatal hypoxia-ischemia (HI) is among the main causes of mortality and morbidity in newborns. Experimental studies show that the immature rat brain is less susceptible to HI injury, suggesting that changes that occur during the first days of life drastically alter its susceptibility. Among the main developmental changes observed is the mitochondrial function, namely, the tricarboxylic acid (TCA) cycle and respiratory complex (RC) activities. Therefore, in the present study, we investigated the influence of neonatal HI on mitochondrial functions, redox homeostasis, and cell damage at different postnatal ages in the hippocampus of neonate rats. For this purpose, animals were divided into four groups: sham postnatal day 3 (ShP3), HIP3, ShP11, and HIP11. We initially observed increased apoptosis in the HIP11 group only, indicating a higher susceptibility of these animals to brain injury. Mitochondrial damage, as determined by flow cytometry showing mitochondrial swelling and loss of mitochondrial membrane potential, was also demonstrated only in the HIP11 group. This was consistent with the decreased mitochondrial oxygen consumption, reduced TCA cycle enzymes, and RC activities and induction of oxidative stress in this group of animals. Considering that HIP3 and the sham animals showed no alteration of mitochondrial functions, redox homeostasis, and showed no apoptosis, our data suggest an age-dependent vulnerability of the hippocampus to hypoxia-ischemia. The present results highlight age-dependent metabolic differences in the brain of neonate rats submitted to HI indicating that different treatments might be needed for HI newborns with different gestational ages.

Early-life stress affects behavioral and neurochemical parameters differently in male and female juvenile Wistar rats.

Neonatal handling is an early life stressor that leads to behavioral and neurochemical changes in adult rats in a sex-specific manner and possibly affects earlier stages of development. Here, we investigated the effects of neonatal handling (days 1-10 after birth) on juvenile rats focusing on biochemical parameters and olfactory memory after weaning. Male neonatal handled rats performed more crossings on the hole-board task, increased Na+ /K+ -ATPase activity in the olfactory bulb, and decreased acetylcholinesterase activity in the hippocampus versus non-handled males. Female neonatal handled animals increased the number of rearing and nose-pokes on the hole-board task, decreased glutathione peroxidase activity, and total thiol content in the hippocampus versus non-handled females. This study reinforces that early life stress affects behavioral and neurochemical parameters in a sex-specific manner even before the puberty onset.

Preventive and therapeutic effects of environmental enrichment in Wistar rats submitted to neonatal hypoxia-ischemia.

Environmental enrichment (EE) at early stages of neurodevelopment attenuates HI-induced behavioral, histological and cellular damage. However, the effects of EE exposure during gestational or early postnatal period and the possible influence of sexual dimorphism on EE protection are not fully understood. Present study evaluated the effects of pre-natal and postnatal EE, as well as their combination, in male and female rats submitted to neonatal HI at postnatal day (PND) 3. Wistar rats were housed in EE or in standard condition (SC) during all pregnancy. At PND1, the litters were randomly allocated to the same prenatal environment during lactation (SC + SC or EE + EE) or housed in a new environment until weaning (SC + EE or EE + SC). Behavioral tasks were performed from PND 60-75. Then, animals were euthanized for biochemical and histological analysis. Prenatal and early postnatal EE alone improved performance of HI males in the Water Maze spatial memory task, while HI females were most benefited from early postnatal stimulation. Moreover, EE attenuated HI-induced lower anxiety-like behavior in rats of both sexes and decreased hyperlocomotion in HI females. Hippocampus tissue preservation and higher VEGF and TrkB levels were observed in all HI groups exposed to EE. Interestingly, HI males exposed to prenatal or postnatal EE alone exhibited higher GFAP levels and additional tissue preservation. Therefore, both prenatal and early postnatal environmental enrichment cause attenuation of HI-induced impairments, revealing their preventive and therapeutic actions, possibly due to VEGF and astrocyte activity; some of these effects are sex-specific.

Locomotor Training Promotes Time-dependent Functional Recovery after Experimental Spinal Cord Contusion.

Locomotor training (LT) has been exhaustively investigated as a treatment for the spinal cord injury (SCI), however the literature reports both positive and negative effects over the functional recovery. The initiation period of LT following SCI is one of the major variables that needs attention. To investigate the better period, three different starting times were investigated after SCI in rats. Methods: Wistar rats were randomly divided into groups: control, SCI (rats with spinal cord contusion), and SCI groups exposed to LT starting 7, 14 or 28 days after the injury (SCI-T7, SCI-T14 and SCI-T28). LT was performed on a treadmill, five days a week, 20 minutes per day, for ten weeks. Basso, Breattie and Bresnahan (BBB) scale and Horizontal Ladder walking test were used to evaluate the motor function; at the end, morphological and biochemical analyses of the spinal cords, tibialis anterior and soleus muscles were performed. Results: SCI-T14 and SCI-T28 groups had an improvement in both behavioral tests, while SCI-T7 presented a worsening in the functional performance. Late training groups preserved motoneurons in the spinal cord, showed larger muscle fiber areas and higher BDNF expression in tibialis anterior muscle. SCI-T7 group had higher lesion volume after LT in comparison with the SCI group. Late onset of LT promoted an increment of the hindlimb function, while early onset of training worsened the functional recovery of the SCI animals. These results demonstrate a critical LT starting time after the injury, contributing to define the best therapeutic window for rehabilitation.

Energy drinks and their component modulate attention, memory, and antioxidant defences in rats.

PURPOSE: This study aimed to evaluate the effects of the subchronic consumption of energy drinks and their constituents (caffeine and taurine) in male Wistar rats using behavioural and oxidative measures. METHODS: Energy drinks (ED 5, 7.5, and 10 mL/kg) or their constituents, caffeine (3.2 mg/kg) and taurine (40 mg/kg), either separately or in combination, were administered orally to animals for 28 days. Attention was measured though the ox-maze apparatus and the object recognition memory test. Following behavioural analyses, markers of oxidative stress, including SOD, CAT, GPx, thiol content, and free radicals, were measured in the prefrontal cortex, hippocampus, and striatum. RESULTS: The latency time to find the first reward was lower in animals that received caffeine, taurine, or a combination of both (P = 0.003; ANOVA/Bonferroni). In addition, these animals took less time to complete the ox-maze task (P = 0.0001; ANOVA/Bonferroni), and had better short-term memory (P < 0.01, Kruskal-Wallis). The ED 10 group showed improvement in the attention task, but did not differ on other measures. In addition, there was an imbalance in enzymatic markers of oxidative stress in the prefrontal cortex, the hippocampus, and the striatum. In the group that received both caffeine and taurine, there was a significant increase in the production of free radicals in the prefrontal cortex and in the hippocampus (P < 0.0001; ANOVA/Bonferroni). CONCLUSIONS: Exposure to a combination of caffeine and taurine improved memory and attention, and led to an imbalance in the antioxidant defence system. These results differed from those of the group that was exposed to the energy drink. This might be related to other components contained in the energy drink, such as vitamins and minerals, which may have altered the ability of caffeine and taurine to modulate memory and attention.

Prenatal and Early Postnatal Environmental Enrichment Reduce Acute Cell Death and Prevent Neurodevelopment and Memory Impairments in Rats Submitted to Neonatal Hypoxia Ischemia.

Environmental enrichment (EE) is an experimental strategy to attenuate the negative effects of different neurological conditions including neonatal hypoxia ischemia encephalopathy (HIE). The aim of the present study was to investigate the influence of prenatal and early postnatal EE in animals submitted to neonatal HIE model at postnatal day (PND) 3. Wistar rats were housed in EE or standard conditions (SC) during pregnancy and lactation periods. Pups of both sexes were assigned to one of four experimental groups, considering the early environmental conditions and the injury: SC-Sham, SC-HIE, EE-sham, and EE-HIE. The offspring were euthanized at two different time points: 48 h after HIE for biochemical analyses or at PND 67 for histological analyses. Behavioral tests were performed at PND 7, 14, 21, and 60. Offspring from EE mothers had better performance in neurodevelopmental and spatial memory tests when compared to the SC groups. HIE animals showed a reduction of IGF-1 and VEGF in the parietal cortex, but no differences in BDNF and TrkB levels were found. EE-HIE animals showed reduction in cell death, lower astrocyte reactivity, and an increase in AKTp levels in the hippocampus and parietal cortex. In addition, the EE was also able to prevent the hippocampus tissue loss. Altogether, present findings point to the protective potential of the prenatal and early postnatal EE in attenuating molecular and histological damage, as well as the neurodevelopmental impairments and the cognitive deficit, caused by HIE insult at PND 3.

Metabolic and feeding behavior alterations provoked by prenatal exposure to aspartame.

The use of artificial sweeteners has increased together with the epidemic growth of obesity. In addition to their widespread use in sodas, artificial sweeteners are added to nearly 6000 other products sold in the US, including baby foods, frozen dinners and even yogurts. It has been suggested that the use of nonnutritive sweeteners can lead to body weight gain and an altered metabolic profile. However, very few studies have evaluated the effects of maternal consumption of artificial non-caloric sweeteners on body weight, feeding behavior or the metabolism of offspring in adult life. In this study, we found that animals exposed to aspartame during the prenatal period presented a higher consumption of sweet foods during adulthood and a greater susceptibility to alterations in metabolic parameters, such as increased glucose, LDL and triglycerides. These effects were observed in both males and females, although they were more pronounced in males. Despite the preliminary nature of this study, and the need for further confirmation of these effects, our data suggest that the consumption of sweeteners during gestation may have deleterious long-term effects and should be used with caution.

Litter size reduction alters insulin signaling in the ventral tegmental area and influences dopamine-related behaviors in adult rats.

Postnatal overfeeding is a well-known model of early-life induced obesity and glucose intolerance in rats. However, little is known about its impact on insulin signaling in specific brain regions such as the mesocorticolimbic system, and its putative effects on dopamine-related hedonic food intake in adulthood. For this study, rat litters were standardized to 4 (small litter - SL) or 8 pups (control - NL) at postnatal day 1. Weaning was at day 21, and all tests were conducted after day 60 of life in male rats. In Experiment 1, we demonstrated that the SL animals were heavier than the NL at all time points and had decreased AKT/pAKT ratio in the Ventral Tegmental Area (VTA), without differences in the skeletal muscle insulin signaling in response to insulin injection. In Experiment 2, the standard rat chow intake was addressed using an automated system (BioDAQ, Research Diets(®)), and showed no differences between the groups. On the other hand, the SL animals ingested more sweet food in response to the 1 min tail-pinch challenge and did not develop conditioned place preference to sweet food. In Experiment 3 we showed that the SL rats had increased VTA TH content but had no difference in this protein in response to a sweet food challenge, as the NL had. The SL rats also showed decreased levels of dopamine D2 receptors in the nucleus accumbens. Here we showed that early postnatal overfeeding was linked to an altered functioning of the mesolimbic dopamine pathway, which was associated with altered insulin signaling in the VTA, suggesting increased sensitivity, and expression of important proteins of the dopaminergic system.

Gender-dependent effect on nociceptive response induced by chronic variable stress.

It has previously been reported that exposure to repeated restraint stress induces hyperalgesia in male rats, an effect that was not observed in females. The aim of the present study was to investigate the effects of chronic variable stress over 40days on nociception threshold indexed by tail-flick latency in male and female adult rats. The results showed different behavior in chronically stressed animals when compared to the control group: male rats showed a decrease in tail-flick latency while females presented an increase in this parameter. For female rats this effect was independent of the phase of the estrous cycle. Several sources of data indicate that behavioral and physiological responses to stress are sexually dimorphic, including in nociception, and the estrous cycle appears to be a factor that influences opioid analgesia in female. These effects are modulated by the strain and conditions of nociception assay. Additional studies concerning the mechanisms involved in the hyperalgesic response in males and the differences on nociceptive response in females chronically exposed to stress are needed.

Brief daily postpartum separations from the litter alter dam response to psychostimulants and to stress.

Neonatal handling induces several behavioral and neurochemical alterations in pups, including decreased responses to stress and reduced fear in new environments. However, there are few reports in the literature concerning the behavioral effects of this neonatal intervention on the dams during the postpartum period. Therefore, the aim of the current study was to determine if brief postpartum separation from pups has a persistent impact on the dam's stress response and behavior. Litters were divided into two neonatal groups: 1) non-handled and 2) handled [10 min/day, from postnatal day (PND) 1 to 10]. Weaning occurred at PND 21 when behavioral tasks started to be applied to the dams, including sweet food ingestion (PND 21), forced swimming test (PND 28), and locomotor response to a psychostimulant (PND 28). On postpartum day 40, plasma was collected at baseline for leptin assays and after 1 h of restraint for corticosterone assay. Regarding sweet food consumption, behavior during the forced swimming test or plasma leptin levels did not differ between dams briefly separated and non-separated from their pups during the postpartum period. On the other hand, both increased locomotion in response to diethylpropion and increased corticosterone secretion in response to acute stress were detected in dams briefly separated from their pups during the first 10 postnatal days. Taken together, these findings suggest that brief, repeated separations from the pups during the neonatal period persistently impact the behavior and induce signs of dopaminergic sensitization in the dam.

Brief daily postpartum separations from the litter alter dam response to psychostimulants and to stress

Neonatal handling induces several behavioral and neurochemical alterations in pups, including decreased responses to stress and reduced fear in new environments. However, there are few reports in the literature concerning the behavioral effects of this neonatal intervention on the dams during the postpartum period. Therefore, the aim of the current study was to determine if brief postpartum separation from pups has a persistent impact on the dam's stress response and behavior. Litters were divided into two neonatal groups: 1) non-handled and 2) handled [10 min/day, from postnatal day (PND) 1 to 10]. Weaning occurred at PND 21 when behavioral tasks started to be applied to the dams, including sweet food ingestion (PND 21), forced swimming test (PND 28), and locomotor response to a psychostimulant (PND 28). On postpartum day 40, plasma was collected at baseline for leptin assays and after 1 h of restraint for corticosterone assay. Regarding sweet food consumption, behavior during the forced swimming test or plasma leptin levels did not differ between dams briefly separated and non-separated from their pups during the postpartum period. On the other hand, both increased locomotion in response to diethylpropion and increased corticosterone secretion in response to acute stress were detected in dams briefly separated from their pups during the first 10 postnatal days. Taken together, these findings suggest that brief, repeated separations from the pups during the neonatal period persistently impact the behavior and induce signs of dopaminergic sensitization in the dam.

Neonatal morphine administration leads to changes in hippocampal BDNF levels and antioxidant enzyme activity in the adult life of rats.

It is know that repeated exposure to opiates impairs spatial learning and memory and that the hippocampus has important neuromodulatory effects after drug exposure and withdrawal symptoms. Thus, the aim of this investigation was to assess hippocampal levels of BDNF, oxidative stress markers associated with cell viability, and TNF-α in the short, medium and long term after repeated morphine treatment in early life. Newborn male Wistar rats received subcutaneous injections of morphine (morphine group) or saline (control group), 5 µg in the mid-scapular area, starting on postnatal day 8 (P8), once daily for 7 days, and neurochemical parameters were assessed in the hippocampus on postnatal days 16 (P16), 30 (P30), and 60 (P60). For the first time, we observed that morphine treatment in early life modulates BDNF levels in the medium and long term and also modulates superoxide dismutase activity in the long term. In addition, it was observed effect of treatment and age in TNF-α levels, and no effects in lactate dehydrogenase levels, or cell viability. These findings show that repeated morphine treatment in the neonatal period can lead to long-lasting neurochemical changes in the hippocampus of male rats, and indicate the importance of cellular and intracellular adaptations in the hippocampus after early-life opioid exposure to tolerance, withdrawal and addiction.

Isolation stress exposure and consumption of palatable diet during the prepubertal period leads to cellular changes in the hippocampus.

Social isolation is one of the most potent stressors in the prepubertal period and may influence disease susceptibility or resilience in adulthood. The glucocorticoid response and, consequently, the adaptive response to stress involve important changes in mitochondrial functions and apoptotic signaling. Previous studies have shown that consumption of a palatable diet reduces some stress effects. Therefore, the aim of the present study was to investigate whether isolation stress in early life can lead to cellular alterations in the hippocampus. For this, we evaluated oxidative stress parameters, DNA breakage index, mitochondrial mass and potential, respiratory chain enzyme activities, apoptosis, and necrosis in the hippocampus of juvenile male rats submitted or not to isolation stress during the pre-puberty period. We also verified whether consumption of a palatable diet during this period can modify stress effects. Results show that stress led to an oxidative imbalance, DNA breaks, increased the mitochondrial potential and early apoptosis, and decreased the number of live and necrotic cells. In addition, the palatable diet increased glutathione peroxidase activity, high mitochondrial potential and complex I-III activity in the hippocampus of juvenile rats. The administration of a palatable diet during the isolation period prevented the stress effects that caused the reduction in live cells and increased apoptosis. In conclusion, the stress experienced during the pre-pubertal period induced a hippocampal oxidative imbalance, DNA damage, mitochondrial dysfunction, and increased apoptosis, while consumption of a palatable diet attenuated some of these effects of exposure, such as the reduction in live cells and increased apoptosis, besides favoring an increase in antioxidant enzymes activities.

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.

Consumption of a palatable diet by chronically stressed rats prevents effects on anxiety-like behavior but increases oxidative stress in a sex-specific manner.

It has been proposed that animals subjected to chronic stress show a stress response that can be reduced by the intake of highly palatable foods ("comfort foods"). However, a palatable diet, rich in sugar or fat, can also lead to oxidative damage and neuronal injury. So, the aim of this study is to verify, in male and female rats, the effects of exposure to chronic stress during free access to regular chow and to a highly palatable diet, on exploratory and anxiety-like behavior, on oxidative stress and on DNA breaks in two structures of the nervous system, hippocampus and striatum. The results showed stress- and diet-induced DNA breaks and an imbalance in the activity of antioxidants enzymes, such as CAT, GPx and SOD in the both structures. In addition, we observed that female rats appear to have higher susceptibility to the stress effects evaluated, and that access to a palatable diet was able to counteract some behavioral effects of stress. However, this same diet-induced oxidative stress and increased DNA breaks, especially in males. Replication of these results with larger sample sizes would further reinforce these conclusions.

Early life handling decreases serotonin turnover in the nucleus accumbens and affects feeding behavior of adult rats.

In our previous studies, we reported that neonatally handled rats have an increased ingestion of sweet food but are resistant to the damaging effects of a chronic exposure to a highly palatable diet. Accumbal serotonin (5-HT) is important for feeding behavior and plays a role in the vulnerability to diet-induced obesity. Therefore, our hypotheses were (1) 5-HT turnover in the nucleus accumbens is altered in neonatally handled animals and plays a role in their differential feeding behavior and (2) if this is so, a chronic pharmacological treatment affecting 5-HT reuptake (chronic imipramine) would be able to revert the behavioral findings. Litters were divided into nonhandled and handled (10 min/day, Days 1-10 after birth). In Experiment 1, we demonstrated that a decreased 5-HT metabolism in the nucleus accumbens was observed in adult handled animals. In Experiment 2, the two previous groups were subdivided and assigned to receive imipramine diluted in water or water alone. After 30 days of treatment, we evaluated their weight gain and feeding behavior. Handled rats weighed less than nonhandled rats, and all imipramine-treated rats showed a reduction in weight gain after 60 days of treatment. Imipramine reverted the increased sweet food consumption seen in neonatally handled rats. We conclude that serotonin is involved in the altered feeding behavior of neonatally handled rats, and this protocol is an important tool for studying the mechanisms by which early life events have a long-term impact on feeding preferences.

Early life experience alters behavioral responses to sweet food and accumbal dopamine metabolism.

Neonatal handling in rats persistently alters behavioral parameters and responses to stress. Such animals eat more sweet food in adult life, without alterations in lab chow ingestion. Here, we show that neonatally handled rats display greater incentive salience to a sweet reward in a runway test; however they are less prone to conditioned place preference and show less positive hedonic reactions to sweet food. When injected with methylphenidate (a dopamine mimetic agent), non-handled rats increase their sweet food ingestion in the fasted state, while neonatally handled rats do not respond. We did not observe any differences regarding baseline general ambulatory activity between the groups. A lower dopamine metabolism in the nucleus accumbens was observed in handled animals, without differences in norepinephrine content. We suggest that early handling leads to a particular response to positive reinforcers such as palatable food, in a very peculiar fashion of higher ingestion but lower hedonic impact, as well as higher incentive salience, but diminished dopaminergic metabolism in the nucleus accumbens.

Fluoxetine alters feeding behavior and leptin levels in chronically-stressed rats.

Stress-induced alterations in feeding behavior are sexually dimorphic and have been related to changes in monoamine levels. Fluoxetine is commonly used as an antidepressant and has also been suggested as an adjunct to other strategies to treat obese individuals. Leptin may interact with stress hormones and with the brain serotonergic system, possibly affecting the feeding behavior of stressed rats. The aim of this study is to evaluate the interaction between chronic fluoxetine treatment and leptin levels in adult female Wistar rats submitted to chronic variable stress. After 30 days of stress, control and stressed groups were subdivided into two groups that received daily injections of vehicle or fluoxetine (8 mg/kg, i.p.). Body weight was evaluated before and after fluoxetine treatment. The animals gained weight with time, signifying that there is a difference in weight gain over time when fluoxetine-treated animals are, or not, subjected to the stress model. Both fluoxetine and stress induced a decrease in sweet food consumption. On the 60th day of fluoxetine treatment, leptin levels were decreased in fluoxetine-treated animals and there was no effect of stress. We conclude that chronic fluoxetine treatment induced a decreased intake of sweet food, as well as a reduction in leptin levels, and that this result could represent a compensatory response to reduced food intake rather than a direct anorectic mechanism. No interaction with chronic stress was observed.

Both infantile stimulation and exposure to sweet food lead to an increased sweet food ingestion in adult life.

We have reported that neonatal handling leads to increased sweet food preference in adult life. Our aim was to verify if these differences in feeding behavior appear before puberty, and whether other types of intervention in periadolescence (such as exposure to toys) could interfere with sweet food consumption later in life. Nests of Wistar rats were (1) non-handled or (2) handled (10 min/day) on days 1-10 after birth. Males from these groups were subdivided in two subgroups: one was habituated to sweet food (Froot Loops-Kellogs) in a new environment for 4 days and tested for sweet food preference at age 27 days, before submitting to a new habituation and test for sweet food ingestion again in adult life. The other subgroup was habituated and tested only in adulthood. In another set of experiments, neonatally non-handled rats were exposed or not to a new environment with toys in periadolescence, and tested for sweet food ingestion as adults. Neonatal handling increases sweet food consumption only if the habituation and tests are performed after puberty. Interestingly, infant exposure to sweet food had a similar effect as neonatal handling, since controls that were exposed to sweet food at age 22 to 27 days increased their ingestion as adults. Exposure to toys in periadolescence had the same effect. We suggest that an intervention during the first postnatal days or exposure to an enriched environment later in the pre-pubertal period leads to behavioral alterations that persist through adulthood, such as increased sweet food ingestion.

Relationship between polymorphisms in thrombophilic genes and preeclampsia in a Brazilian population.

The purpose of this study was to evaluate the thrombophilic genes in pregnant women with and without preeclampsia independently or in combination. In a prospective case-control study, we investigated four polymorphisms in thrombophilic genes in 75 women with mild or severe preeclampsia and 145 women with normal pregnancy. The genotype frequencies were assessed and the odds ratio (OR) calculated. When we analyzed the polymorphisms independently and the development of preeclampsia, no association was observed [methylenetetrahydrofolate reductase (MTHFR) 677TT genotype, OR 2.07, 95% confidence interval (CI) 0.99-4.30; prothrombin mutation (F II) (GA or AA genotypes) OR 8.11, 95% CI 0.89-73.92; factor V Leiden (FV Leiden) OR 3.94, 95% CI 0.35-44.23; plasminogen activator inhibitor (PAI-1) 4G/4G genotype, OR 1.63, 95% CI 0.87-3.05] not even with severe preeclampsia subgroup analysis. However, when we investigated a possible interaction among these polymorphisms on the development of the preeclampsia, the OR for having one risk genotype, one or two genotype risk factors and two genotype risk factors compared to those without genotype risk factors were 1.97 (95% CI 1.08-3.59), 2.21 (95% CI 1.25-3.92) and 4.27 (95% CI 1.3-13.9), respectively. In conclusion, in the population analyzed, the presence of the genotype risk factors alone does not seem to be associated with the development of preeclampsia even in the severe presentation form. However, an interaction among the MTHFR, F II, FV and PAI-1 gene polymorphisms on the development of the preeclampsia was indicated.