Metabolic and neurological consequences of the treatment with polyphenols: a systematic review in rodent models of noncommunicable diseases.

BACKGROUND: Noncommunicable diseases (NCDs) lead to drastic metabolic alterations with associated energy balance and body weight changes, two related physiological processes regulated by the brain. Polyphenol-based treatments for NCDs have emerged as a promising therapy, which seems to involve the energy balance modulation. However, it remains unclear what the most effective polyphenols-based treatment is to attenuate adverse effects in the energy balance of NCDs. OBJECTIVES: This systematic review aimed to evaluate the literature on the metabolic and neurological effects of polyphenols-based treatment in rodent models of NCDs. METHODS: Literature search was carried out in the following databases: CINAHL, Medline/PubMed, SCOPUS, and Web of Science. For title and abstract screening, original papers with polyphenols exposure in rodents were selected. For full-text screening, studies with models of NCDs that reported metabolic and neurological outcomes when treated with polyphenols were selected for inclusion in this review. RESULTS: 23 articles, using individual compound (11 articles) or polyphenols extracts (12 articles), were included in this review: 5 articles using tea polyphenols, 12 articles using grape-derived polyphenols, 3 articles using the polyphenol quercetin, and 3 articles using other polyphenol sources. Most results agree on the beneficial effect of polyphenols in attenuating alterations in energy balance and body weight. Such effects were associated with neuroprotective responses in different brain areas including hippocampus and hypothalamus. CONCLUSION: In conclusion, this review shows that the treatment with polyphenols, especially resveratrol or quercetin, attenuates the adverse effects of NCDs on energy balance and are associated with neuroprotective effects.

Oro-facial functions in experimental models of cerebral palsy: a systematic review.

Children who suffer from cerebral palsy (CP) often present comorbidities in the form of oro-facial dysfunctions. Studies in animals have contributed to elaborate potential therapies aimed at minimising the chronic disability of the syndrome. To systematically review the scientific literature regarding the possible effects that experimental models of CP can have on oro-facial functions. Two independent authors conducted a systematic review in the electronic databases Medline, Scopus, CINAHL, Web of Science and Lilacs, using Mesh and Decs terms in animal models. The motor and sensory parameters of sucking, chewing and swallowing were considered as primary outcomes; reactivity odour, controlled salivation, postural control, head mobility during feeding and the animal's ability to acquire food were secondary outcomes. Ten studies were included in the present review. Most studies used rabbits as experimental models of CP, which was induced by either hypoxia-ischemia, inflammation or intraventricular haemorrhage. Oro-facial functions were altered in all experimental models of CP. However, we found more modifications in hypoxia-ischemia models overall. On the other hand, the model of inflammation was more effective to reproduce higher damage for coordinating sucking and swallowing. All of the CP experimental models that were assessed modified the oral functions in different animal species. However, further studies should be conducted in order to clarify the mechanisms underlying oro-facial damage in order to optimise treatment strategies for children who suffer from CP.

Fluoxetine induces lean phenotype in rat by increasing the brown/white adipose tissue ratio and UCP1 expression.

The serotonergic system plays a crucial role in the energy balance regulation. Energy balance is mediated by food intake and caloric expenditure. Thus, the present study investigated the mechanisms that might be associated with fluoxetine treatment-induced weight reduction. Wistar male rat pups received daily injections with subcutaneous fluoxetine (Fx-group) or vehicle solution (Ct-group) from day 1 until 21 days of age. Several analyses were conducted to verify the involvement of mitochondria in weight reduction. We found that body weight in the Fx-group was lower compared to control. In association to lower fat mass in the Fx-group (25%). Neither neonatal caloric intake nor food intake reveals significant differences. Evaluating caloric expenditure (locomotor activity and temperature after stimulus), we did not observe differences in locomotor activity. However, we observed that the Fx group had a higher capacity to maintain body temperature in a cold environment compared with the Ct-group. Since brown adipose tissue-(BAT) is specialized for heat production and the rate of heat production is related to mitochondrial function, we found that Fx-treatment increases respiration by 36%, although after addition of GDP respiration returned to Ct-levels. Examining ROS production we observe that Fx-group produced less ROS than control group. Evaluating uncoupling protein (UCP) expression we found that Fx-treatment increases the expression by 23%. Taken together, our results suggest that modulation of serotonin system results in positive modulation of UCP and mitochondrial bioenergetics in brown fat tissue.

Differential developmental programming by early protein restriction of rat skeletal muscle according to its fibre-type composition.

AIMS: Differences in fibre-type composition of skeletal muscle have been associated with obesity and insulin resistance. As a poor nutrient environment early in life is a predisposing factor for the development of obesity and related metabolic diseases at adulthood, this study aimed at determining the long-term consequences of maternal undernutrition on the structural and metabolic properties of two skeletal muscles characterized by their different fibre-type composition and metabolic properties. METHODS: The fibre-type composition and enzymatic activities of hexokinase (HK), beta-hydroxyacyl-CoA dehydrogenase (ß-HAD) and citrate synthase (CS) were measured in soleus and extensor digitorum longus (EDL) muscles from adult rats born to dams fed a control (17% protein) or a low-protein [8% protein (PR)] diet throughout pregnancy and lactation. In addition, the expression levels of several genes regulating glycolysis, fatty acid oxidation and mitochondrial biogenesis were determined by real-time PCR. RESULTS: Protein rats exhibited enhanced density of type II fibres along with decreased rate of fatty acid oxidation and glycolysis in soleus but not EDL. Malnourished rats exhibited also a different gene expression profile in soleus and EDL. Altogether, these alterations correspond to a state of energy deficiency and are present in animals which do not show yet any sign of obesity or glucose intolerance. CONCLUSION: We conclude that maternal protein restriction alters in the long term the structural and enzymatic properties of offspring skeletal muscle in a fibre-type-dependent manner. These alterations might have a causative role in the development of obesity and related metabolic disorders later in life.

Effects of a moderate physical training on the leptin synthesis by adipose tissue of adult rats submitted to a perinatal low-protein diet.

The aim of the study was to verify if moderate physical training affects leptin content in visceral and subcutaneous adipose tissue of adult rats subjected to a low-protein diet during the perinatal period. Male Wistar rats were divided into 2 groups according to their mother's diet during gestation and lactation: control (17% casein, C, n=12) and low-protein (8% casein, LP, n=12). On postnatal day 60, half of each group was submitted to moderate physical training (8 wks, 5 d · wk - 1, 60 min · d - 1, at 70% of VO2max, T) or not. After the physical training period, visceral and subcutaneous adipose tissues were removed. Leptin content was evaluated by western blotting. Starting from the fifth week on, T pups showed a reduction in the body weight. Similarly, LP+T offspring showed a lower body weight starting from the sixth week on. Western blotting analysis showed that leptin content in the visceral tissue was higher in the LP rats (p<0.01) and it was reversed in LP+T. No difference was found in the subcutaneous tissue. Moderate physical training attenuated the effects of a perinatal low-protein diet on the leptin content in visceral adipose tissue in adult offspring.

Differential adaptations during growth spurt and in young adult rat muscles.

During the post-weaning growth and maturation period (25/90 days after birth), rat limb muscles are submitted to specific adaptations. Our aim was to characterize the mechanical properties of two muscles that are opposite in terms of fibre-type distribution, the soleus and the extensor digitorum longus (EDL) muscles of male Wistar rats. Results showed a fast-to-slow fibre-type transition in soleus while no modification in fibre-type distribution was observed in EDL. A growth-induced increase in muscle force was observed. Soleus underwent an increase in twitch kinetics, but EDL showed no modification. Resistance to fatigue was higher in 90-day-old soleus but not modified in the EDL. Surprisingly, analysis of maximal shortening velocity showed a decrease in both soleus and EDL. Finally, tension/extension curves indicated a growth-induced increase in series elastic stiffness in the two muscles. These results suggest that during this growth period, skeletal muscles are submitted to differential adaptations. Moreover, whereas adaptation of biomechanical properties observed can be explained partly by an adaptation of fibre profile in soleus, this is not the case for EDL. It is suggested that changes in muscle architecture, which are often disregarded, could explain some variations in mechanical properties, especially when muscles undergo an increase in both mass and length.

Is the maturation of monosynaptic stretch reflex in rats affected by neonatal malnutrition?

We compared the maturation of the monosynaptic stretch reflex in control rats and in rats submitted to neonatal malnutrition. Electrical stimulations of the sciatic nerve were applied in wakeful rats of different ages (21-90 days) to record, by surface electrodes, the maximal direct motor response (M(max)) or the maximal Hoffmann reflex (H(max)). Percussion on the Achilles tendon induced the T-reflex. Animals submitted to neonatal malnutrition showed significant reductions in H-reflex latency and in velocity index of nervous conduction. The H- or T-reflex amplitudes were lower for malnourished rats of 21 days but the difference was significant only for the T(max)/M(max) ratios. The reflexes evoked at older ages did not present differences between control and malnourished rats. In conclusion, rats submitted to neonatal malnutrition present long-term alteration in reflex latency and nervous conduction velocity. Neonatal malnutrition also alters the reflex excitability at weaning but, since the rat were submitted to a normal diet after weaning, a normal reflex excitability was rapidly recovered which indicates a remarkable plasticity of the reflex pathway.

Sertraline delays the somatic growth and reflex ontogeny in neonate rats.

This study investigated the somatic maturation and ontogeny of reflexes in neonate rats treated with sertraline (Sert) during the suckling period. The animals were divided into four groups; three that received daily doses of Sert (5, 10 or 15 mg/kg s.c.; groups Sert5, Sert10, and Sert15, respectively), and a fourth group that received distilled water (Dw) (1 ml/kg/b.w.). Growth indicators (body weight, axis of the head and tail length) were measured daily, from the 1st to the 21st postnatal day. The reflexes (righting, free-fall righting, negative geotaxis, cliff avoidance, auditory startle response, vibrissa placing and palm grasp) and physical-feature maturation (ear unfolding, auditory conduit opening, irruption of the lower incisors and eye opening) were recorded each day of the animal's life. All groups were compared to the Dw group. The body weight gain was reduced in all the Sert groups. Moreover, a delay in the growth of the body length was observed in all the Sert groups. Higher Sert doses reduced the speed of growth in the tail length. The medio-lateral head axis reduced in Sert15 and Sert5 doses. Otherwise, Sert10 had a temporary acceleration in this growth, but the growth of the anteroposterior head axis had a delay in all the Sert groups. The highest doses induced a delay in physical-feature maturation. The palm grasp reflex (disappearance) was retarded in Sert10; cliff avoidance advanced in Sert10; negative-geotaxis and free-fall righting retarded in Sert15. The findings suggest that altered serotonergic system activity induced by sertraline early in life could play a role in the retardation of the somatic growth ontogeny as well as a delay in the maturation of some reflexes.

Thie expression of an intraspecific aggressive reaction in the face of a stressor agent alters the immune response in rats.

The repercussion on the immune response of the expression of intraspecific aggressiveness in the face of a stressor agent was investigated in rats. Ninety-day-old animals were divided into three groups: the control group (only immunological measurements were performed), the foot-shock (FS) (animals individually receiving FS), and the intraspecific aggressive response (IAR) group (animals receiving FS and presenting IAR). For immunological measurements, blood samples were collected promptly at 7 and 15 days after FS or IAR. The FS reduced the total leukocyte amount presented. However, aggressiveness triggered not only reduction of the leukocytes, but also lymphocyte decrease and neutrophil increase. Moreover, an elevation in total leukocytes associated with an increase in the humoral immune response was also observed one week after IAR. In this study, the expression of intraspecific aggressiveness in the face of a stressor seemed to activate the immune system and to potentiate the antigen specific humoral response.

The expression of an intraspecific aggressive reaction in the face of a stressor agent alters the immune response in rats

A repercussão sobre a resposta imune da expressão da agressividade intra-específica diante de um estressor foi investigada em ratos. Aos 90 dias de vida, os animais foram divididos em três grupos: grupo-controle (foram realizadas apenas mensurações imunológicas), choque nas patas (FS) (os animais receberam FS individualmente) e grupo resposta agressiva intra-específica (IAR) (os animais receberam FS e apresentaram IAR). Para as medições imunológicas, amostras de sangue foram coletadas imediatamente, 7 e 15 dias após FS ou IAR. O FS reduziu a quantidade total de leucócitos. Contudo, a agressividade foi acompanhada, além da redução do número de leucócitos, por diminuição de linfócitos e aumento de neutrófilos. Além disso, também foi observada elevação no número de leucócitos associada a aumento na resposta imune humoral uma semana após as IAR. Neste estudo, a expressão da agressividade intra-específica diante de um estressor parece ativar o sistema imune e potencializar a resposta humoral antígeno específica.

The expression of an intraspecific aggressive reaction in the face of a stressor agent alters the immune response in rats

The repercussion on the immune response of the expression of intraspecific aggressiveness in the face of a stressor agent was investigated in rats. Ninety-day-old animals were divided into three groups: the control group (only immunological measurements were performed), the foot-shock (FS) (animals individually receiving FS), and the intraspecific aggressive response (IAR) group (animals receiving FS and presenting IAR). For immunological measurements, blood samples were collected promptly at 7 and 15 days after FS or IAR. The FS reduced the total leukocyte amount presented. However, aggressiveness triggered not only reduction of the leukocytes, but also lymphocyte decrease and neutrophil increase. Moreover, an elevation in total leukocytes associated with an increase in the humoral immune response was also observed one week after IAR. In this study, the expression of intraspecific aggressiveness in the face of a stressor seemed to activate the immune system and to potentiate the antigen specific humoral response.

A repercussão sobre a resposta imune da expressão da agressividade intra-específica diante de um estressor foi investigada em ratos. Aos 90 dias de vida, os animais foram divididos em três grupos: grupo-controle (foram realizadas apenas mensurações imunológicas), choque nas patas (FS) (os animais receberam FS individualmente) e grupo resposta agressiva intra-específica (IAR) (os animais receberam FS e apresentaram IAR). Para as medições imunológicas, amostras de sangue foram coletadas imediatamente, 7 e 15 dias após FS ou IAR. O FS reduziu a quantidade total de leucócitos. Contudo, a agressividade foi acompanhada, além da redução do número de leucócitos, por diminuição de linfócitos e aumento de neutrófilos. Além disso, também foi observada elevação no número de leucócitos associada a aumento na resposta imune humoral uma semana após as IAR. Neste estudo, a expressão da agressividade intra-específica diante de um estressor parece ativar o sistema imune e potencializar a resposta humoral antígeno específica.

Neonatal administration of citalopram delays somatic maturation in rats

We investigated the somatic maturation of neonate rats treated during the suckling period with citalopram, a selective serotonin reuptake inhibitor. Groups with 6 male neonates were randomly assigned to different treatments 24 h after birth. Each litter was suckled by one of the dams until the 21st postnatal day. Body weight, head axis and tail length were measured daily from the 1st to the 21st postnatal day. Time of ear unfolding, auditory conduit opening, incisor eruption, and eye opening was determined. Pups received 5 mg (Cit5), 10 mg (Cit10) or 20 mg/kg (Cit20) citalopram sc, or saline (0.9 percent NaCl, w/v, sc). Compared to saline, body weight was lower (24.04 percent, P < 0.01) for Cit10 from the 10th to the 21st day and for Cit20 from the 6th to the 21st day (38.19 percent, P < 0.01). Tail length was reduced in the Cit20 group (15.48 percent, P < 0.001) from the 8th to the 21st day. A reduction in mediolateral head axis (10.53 percent, P < 0.05) was observed from the 11th to the 21st day in Cit10 and from the 6th to the 21st day in Cit20 (13.16 percent, P < 0.001). A reduction in anteroposterior head axis was also observed in the Cit20 group (5.28 percent, P < 0.05) from the 13th to the 21stday. Conversely, this axis showed accelerated growth from the 12th to the 21stday in the Cit5 group (13.05 percent, P < 0.05). Auditory conduit opening was delayed in the Cit5 and Cit20 groups and incisor eruption was delayed in all citalopram groups. These findings show that citalopram injected during suckling to rats induces body alterations and suggest that the activity of the serotoninergic system participates in growth mechanisms.

Neonatal administration of citalopram delays somatic maturation in rats.

We investigated the somatic maturation of neonate rats treated during the suckling period with citalopram, a selective serotonin reuptake inhibitor. Groups with 6 male neonates were randomly assigned to different treatments 24 h after birth. Each litter was suckled by one of the dams until the 21st postnatal day. Body weight, head axis and tail length were measured daily from the 1st to the 21st postnatal day. Time of ear unfolding, auditory conduit opening, incisor eruption, and eye opening was determined. Pups received 5 mg (Cit5), 10 mg (Cit10) or 20 mg/kg (Cit20) citalopram sc, or saline (0.9% NaCl, w/v, sc). Compared to saline, body weight was lower (24.04%, P < 0.01) for Cit10 from the 10th to the 21st day and for Cit20 from the 6th to the 21st day (38.19%, P < 0.01). Tail length was reduced in the Cit20 group (15.48%, P < 0.001) from the 8th to the 21st day. A reduction in mediolateral head axis (10.53%, P < 0.05) was observed from the 11th to the 21st day in Cit10 and from the 6th to the 21st day in Cit20 (13.16%, P < 0.001). A reduction in anteroposterior head axis was also observed in the Cit20 group (5.28%, P < 0.05) from the 13th to the 21st day. Conversely, this axis showed accelerated growth from the 12th to the 21st day in the Cit5 group (13.05%, P < 0.05). Auditory conduit opening was delayed in the Cit5 and Cit20 groups and incisor eruption was delayed in all citalopram groups. These findings show that citalopram injected during suckling to rats induces body alterations and suggest that the activity of the serotoninergic system participates in growth mechanisms.

Malnutrition during brain growth spurt alters the effect of fluoxetine on aggressive behavior in adult rats.

Malnutrition effect during the suckling period on aggressive behavior was investigated in adult rats treated and not treated with fluoxetine, a selective serotonin reuptake inhibitor. Sixty-four Wistar male rats were allocated in two groups, according to their mothers' diet during lactation. The well-nourished group was fed by mothers receiving a 23% protein diet; the malnourished one by mothers receiving a 8% protein diet. Following weaning, all rats received the 23% protein diet. On the 90th day after birth, each nutritional group was divided into two subgroups, one receiving a single daily injection of fluoxetine (10 mg/kg) and the other of a saline solution (0.9% NaCl) for 14 days. Treatment with Fluoxetine reduced aggressive response in well-nourished but not in malnourished rats. These findings suggest that the serotoninergic system was affected by malnutrition during the critical period of brain development, and persisted even after a long period of nutritional recovery.

Early malnourished rats are not affected by anorexia induced by a selective serotonin reuptake inhibitor in adult life.

The effect of early postnatal malnutrition upon food intake and its modulation by the selective serotonin reuptake inhibitor (SSRI) citalopram, was investigated in adult rats. Sixty four Wistar rats were allocated to two groups, according to their mother's diet during lactation. Mothers receiving a 23% protein diet fed the well-nourished group; mothers receiving 8% protein diet fed the malnourished. After weaning, all rats received the 23% protein diet ad libitum. On the 120th day after birth, each nutritional group was divided in two subgroups (each one, n = 16) which received a single daily injection of citalopram (10 mg/kg) or saline (0.9% NaCl) for 14 days. Chronic treatment with citalopram decreased both the food intake and weight gain in the well-nourished rats, but not in the malnourished ones. These data are consistent with findings concerning the nutritional manipulation of the nervous system during its higher vulnerable phase, suggesting that early malnutrition alters the effect of treatment of SSRI in adult rats, and that malnutrition during the critical period of brain development affects the serotoninergic system.

Citalopram has an antagonistic action on cortical spreading depression in well-nourished and early-malnourished adult rats.

Adult, well-nourished (W) and early-malnourished (M) male Wistar rats were injected intraperitoneally for 7 days with 20 mg/kg CIT and cortical spreading depression (CSD) was recorded for 4 h on the day following the treatment. M-animals presented lower body weights, as well as higher CSD velocities of propagation, than the W ones, as previously reported. Compared to saline-injected controls, rats treated with CIT for 7 days presented comparable body weights and lower mean CSD velocities, per hour of recording, the differences being significant at the second hour (3.29+/-0.31 versus 3.56+/-0.40 mm/min; P < 0.05). Topical, cortical application of CIT (1- and 5 mg/ml solutions over the intact dura-mater) reduced dose-dependently the CSD velocity (maximal reductions of 16.3 and 55.8% for the 1 and 5 mg/ml solutions, respectively; P < 0.05), as well as the amplitude of the CSD-slow potential change (58.2 and 88.3%). In three out of seven W-rats and in one out of seven M-rats, topical CIT (5 mg/ml) blocked CSD propagation. The effects were reverted by flushing the treated region with saline. In the M-groups, CIT affected CSD in the same manner as in the W ones. The results reinforce previous evidence for an antagonistic influence of the serotoninergic activity on CSD.

Reduction of intraspecific aggression in adult rats by neonatal treatment with a selective serotonin reuptake inhibitor.

Most studies suggest that serotonin exerts an inhibitory control on the aggression process. According to experimental evidence, this amine also influences growth and development of the nervous tissue including serotoninergic neurons. Thus, the possibility exists that increased serotonin availability in young animals facilitates a long-lasting effect on aggressive responses. The present study aimed to investigate the aggressive behavior of adult rats (90-120 days) treated from the 1st to the 19th postnatal day with citalopram (CIT), a selective serotonin reuptake inhibitor (20 mg/kg, s.c., every 3 days). Aggressive behavior was induced by placing a pair of rats (matched by weight) in a box (20 x 20 x 20 cm), and submitting them to a 20-min session of electric footshocks (five 1.6-mA - 2-s current pulses, separated by a 4-min intershock interval). When compared to the control group (rats treated for the same period with equivalent volumes of saline solution), the CIT group presented a 41.4% reduction in the duration of aggressive response. The results indicate that the repeated administration of CIT early in life reduces the aggressive behavior in adulthood and suggest that the increased brain serotoninergic activity could play a role in this effect.

Reduction of intraspecific aggression in adult rats by neonatal treatment with a selective serotonin reuptake inhibitor

Most studies suggest that serotonin exerts an inhibitory control on the aggression process. According to experimental evidence, this amine also influences growth and development of the nervous tissue including serotoninergic neurons. Thus, the possibility exists that increased serotonin availability in young animals facilitates a long-lasting effect on aggressive responses. The present study aimed to investigate the aggressive behavior of adult rats (90-120 days) treated from the 1st to the 19th postnatal day with citalopram (CIT), a selective serotonin reuptake inhibitor (20 mg/kg, sc, every 3 days). Aggressive behavior was induced by placing a pair of rats (matched by weight) in a box (20 x 20 x 20 cm), and submitting them to a 20-min session of electric footshocks (five 1.6-mA - 2-s current pulses, separated by a 4-min intershock interval). When compared to the control group (rats treated for the same period with equivalent volumes of saline solution), the CIT group presented a 41.4 percent reduction in the duration of aggressive response. The results indicate that the repeated administration of CIT early in life reduces the aggressive behavior in adulthood and suggest that the increased brain serotoninergic activity could play a role in this effect

Permanent and transitory morphometric changes of NADPH-diaphorase-containing neurons in the rat visual cortex after early malnutrition.

We investigated the histochemical positivity to NADPH-diaphorase, which reveals nitric oxide synthase activity, in area 17 of rats malnourished early in life, both in the post-weaning period (group M1), and in adulthood after nutritional recovering (group M2). Control pups (C1 and C2 groups) received ad libitum after weaning the same diets as their mothers. Rats of group M2 were nutritionally recovered by receiving the control diet from post-natal day 42 until adulthood. Aldehyde-fixed sections (200-microm thick) through area 17 were processed for NADPH-diaphorase histochemistry following the malic enzyme indirect method. The features of NADPH-diaphorase-containing neurons of area 17 of malnourished young (M1) and adult (M2) rats were analyzed quantitatively in comparison to the matched groups C1 and C2. Permanent changes, represented by increase in the density and dendritic field areas of NADPH-diaphorase-positive cells, and transitory ones, represented by decreased values of soma areas, were observed in area 17 of the M1 and M2 cases. However, some other features, such as dendritic branch angle and number of dendrites per cell in the gray matter, remained unchanged after malnutrition. Thus, the findings indicate a possible relationship between early malnutrition and alterations in nitric oxide synthase-containing cells in the visual cortex. Physiological implications of these data may be related to synaptic plasticity and refinement of developmental brain circuits.

Effect of stress on the production of O(2)(-) in alveolar macrophages.

Alveolar macrophages (AM) of male rats (200-250 g), stressed or not, were evaluated with relationship to superoxide production (SP). Plasma levels of corticosterone were measured. The control group showed larger SP than the stressed group in all intervals of time. Exposure in vitro of AM to a synthetic glucocorticoid for 40 min (the same time of restraint stress) inhibits SP. Therefore, it seems under stress situations there is an increase of plasma levels of corticosterone and a decrease of SP in AM after stimulation with PMA. O(2)(-) is a potent microbicide and its reduction could cause the loss of microbicidal activity of AM.