Anxiolytic effects of repeated treatment with an essential oil from Lippia alba and (R)-(-)-carvone in the elevated T-maze

Lippia alba (Mill.) N.E. Brown (Verbenaceae) is widely used in different regions of Central and South America as a tranquilizer. The plant’s anxiolytic properties, however, merit investigation. The present study evaluated the effects of repeated daily (14 days) intraperitoneal (ip) treatment with an essential oil (EO) from a chemotype of L. alba (LA, chemotype II, 12.5 and 25 mg/kg; N = 6-8) and (R)-(-)-carvone (25 mg/kg; N = 8-12), the main constituent of this chemotype, on male Wistar rats (weighing 250 g at the beginning of the experiments) submitted to the elevated T-maze (ETM). The ETM allows the measurement of two defensive responses: inhibitory avoidance and one-way escape. In terms of psychopathology, these responses have been related to generalized anxiety and panic disorder, respectively. Treatment with the EO impaired ETM avoidance latencies, without altering escape, in a way similar to the reference drug diazepam (P < 0.05) (avoidance 2: control = 84.6 ± 35.2; EO 12.5 mg/kg = 11.8 ± 3.8; EO 25 mg/kg = 14.6 ± 2.7; diazepam = 7 ± 2.1). (R)-(-)-carvone also significantly altered this same response (P < 0.05; avoidance 1: control = 91.9 ± 31.5; carvone = 11.6 ± 1.8; diazepam = 8.1 ± 3.3). These results were not due to motor changes since no significant effects were detected in an open field. These observations suggest that LA exerts anxiolytic-like effects on a specific subset of defensive behaviors that have been implicated in generalized anxiety disorder, and suggest that carvone is one of the constituents of LA responsible for its action as a tranquilizer.

Effects of chronic corticosterone and imipramine administration on panic and anxiety-related responses

It is known that chronic high levels of corticosterone (CORT) enhance aversive responses such as avoidance and contextual freezing. In contrast, chronic CORT does not alter defensive behavior induced by the exposure to a predator odor. Since different defense-related responses have been associated with specific anxiety disorders found in clinical settings, the observation that chronic CORT alters some defensive behaviors but not others might be relevant to the understanding of the neurobiology of anxiety. In the present study, we investigated the effects of chronic CORT administration (through surgical implantation of a 21-day release 200 mg pellet) on avoidance acquisition and escape expression by male Wistar rats (200 g in weight at the beginning of the experiments, N = 6-10/group) tested in the elevated T-maze (ETM). These defensive behaviors have been associated with generalized anxiety and panic disorder, respectively. Since the tricyclic antidepressant imipramine is successfully used to treat both conditions, the effects of combined treatment with chronic imipramine (15 mg, ip) and CORT were also investigated. Results showed that chronic CORT facilitated avoidance performance, an anxiogenic-like effect (P < 0.05), without changing escape responses. Imipramine significantly reversed the anxiogenic effect of CORT (P < 0.05), although the drug did not exhibit anxiolytic effects by itself. Confirming previous observations, imipramine inhibited escape responses, a panicolytic-like effect. Unlike chronic CORT, imipramine also decreased locomotor activity in an open field. These data suggest that chronic CORT specifically altered ETM avoidance, a fact that should be relevant to a better understanding of the physiopathology of generalized anxiety and panic disorder.

Effects of reversible inactivation of the dorsomedial hypothalamus on panic- and anxiety-related responses in rats

The medial hypothalamus is part of a neurobiological substrate controlling defensive behavior. It has been shown that a hypothalamic nucleus, the dorsomedial hypothalamus (DMH), is involved in the regulation of escape, a defensive behavior related to panic attacks. The role played by the DMH in the organization of conditioned fear responses, however, is less clear. In the present study, we investigated the effects of reversible inactivation of the DMH with the GABA A agonist muscimol on inhibitory avoidance acquisition and escape expression by male Wistar rats (approximately 280 g in weight) tested in the elevated T-maze (ETM). In the ETM, inhibitory avoidance, a conditioned defensive response, has been associated with generalized anxiety disorder. Results showed that intra-DMH administration of the GABA A receptor agonist muscimol inhibited escape performance, suggesting an antipanic-like effect (P < 0.05), without changing inhibitory avoidance acquisition. Although a higher dose of muscimol (1.0 nmol/0.2 µL; N = 7) also altered locomotor activity in an open field when compared to control animals (0.2 µL saline; N = 13) (P < 0.05), the lower dose (0.5 nmol/0.2 µL; N = 12) of muscimol did not cause any motor impairment. These data corroborate previous evidence suggesting that the DMH is specifically involved in the modulation of escape. Dysfunction of this regulatory mechanism may be relevant in the genesis/maintenance of panic disorder.

Effect of Erythrina velutina and Erythrina mulungu in rats submitted to animal models of anxiety and depression

Erythrina velutina (EV) and Erythrina mulungu (EM), popularly used in Brazil as tranquilizing agents, were studied. The effects of acute and chronic oral treatment with a water:alcohol extract of EV (7:3, plant grounded stem bark; acute = 100, 200, 400 mg/kg; chronic = 50, 100, 200 mg/kg) were evaluated in rats (N = 11-12) submitted to the elevated T-maze (for avoidance and escape measurements) model of anxiety. This model was selected for its presumed capacity to elicit specific subtypes of anxiety disorders recognized in clinical practice: avoidance has been related to generalized anxiety and escape to panic. Additionally, animals were treated with the same doses of EV and EM (water:alcohol 7:3, inflorescence extract) and submitted to the forced swim test for the evaluation of antidepressant activity (N = 7-10). Both treatment regimens with EV impaired elevated T-maze avoidance latencies, without altering escape, in a way similar to the reference drug diazepam (avoidance 1, mean ± SEM, acute study: 131.1 ± 45.5 (control), 9.0 ± 3.3 (diazepam), 12.7 ± 2.9 (200 mg/kg), 28.8 ± 15.3 (400 mg/kg); chronic study: 131.7 ± 46.9 (control), 35.8 ± 29.7 (diazepam), 24.4 ± 10.4 (50 mg/kg), 29.7 ± 11.5 (200 mg/kg)). Neither EV nor EM altered measurements performed in the forced swim test, in contrast to the reference drug imipramine that significantly decreased immobility time after chronic treatment. These results were not due to motor alterations since no significant effects were detected in an open field. These observations suggest that EV exerts anxiolytic-like effects on a specific subset of defensive behaviors which have been associated with generalized anxiety disorder.

The inactivation of the basolateral nucleus of the rat amygdala has an anxiolytic effect in the elevated T-maze and light/dark transition tests

Pharmacological evidence indicates that the basolateral nucleus of the amygdala (BLA) is involved in the mediation of inhibitory avoidance but not of escape behavior in the elevated T-maze test. These defensive responses have been associated with generalized anxiety disorder (GAD) and panic disorder, respectively. In the present study, we determined whether the BLA plays a differential role in the control of inhibitory avoidance and escape responses in the elevated T-maze. Male Wistar rats (250-280 g, N = 9-10 in each treatment group) were pre-exposed to one of the open arms of the maze for 30 min and 24 h later tested in the model after inactivation of the BLA by a local injection of the GABA A receptor agonist muscimol (8 nmol in 0.2 æL). It has been shown that a prior forced exposure to one of the open arms of the maze, by shortening latencies to withdrawal from the open arm during the test, improves the escape task as a behavioral index of panic. The effects of muscimol in the elevated T-maze were compared to those caused by this GABA agonist in the avoidance reaction generated in the light/dark transition test. This defensive behavior has also been associated with GAD. In the elevated T-maze, intra-BLA injection of muscimol impaired inhibitory avoidance (control: 187.70 ± 14.90 s, muscimol: 37.10 ± 2.63 s), indicating an anxiolytic effect, without interfering with escape performance. The drug also showed an anxiolytic effect in the light/dark transition test as indicated by the increase in the time spent in the lighted compartment (control: 23.50 ± 2.45 s, muscimol: 47.30 ± 4.48 s). The present findings point to involvement of the BLA in the modulation of defensive responses that have been associated with GAD.

Risk factors for alloimmunization by patients with sickle cell disease

Blood transfusion in patients with sickle cell disease (SCD) is limited by the development of alloantibodies to erythrocytes. In the present study, the frequency and risk factors for alloimmunization were determined. Transfusion records and medical charts of 828 SCD patients who had been transfused and followed at the Belo Horizonte Blood Center, Belo Horizonte, MG, Brazil, were retrospectively reviewed. Alloimmunization frequency was 9.9 percent (95 percent CI: 7.9 to 11.9 percent) and 125 alloantibodies were detected, 79 percent of which belonged to the Rhesus and Kell systems. Female patients developed alloimmunization more frequently (P = 0.03). The median age of the alloimmunized group was 23.3 years, compared to 14.6 years for the non-alloimmunized group (P < 0.0001). Multivariate analyses were applied to the data for 608 hemoglobin (Hb) SS or SC patients whose number of transfusions was recorded accurately. Number of transfusions (P = 0.00006), older age (P = 0.056) and Hb SC (P = 0.02) showed independent statistical associations with alloimmunization. Hb SC patients older than 14 years faced a 2.8-fold higher (95 percent CI: 1.3 to 6.0) risk of alloimmunization than Hb SS patients. Female Hb SC patients had the highest risk of developing alloantibodies. In patients younger than 14 years, only the number of transfusions was significant. We conclude that an increased risk of alloimmunization was associated with older patients with Hb SC, specially females, even after adjustments were made for the number of transfusions received, the most significant variable.

An improved HPLC method for the quantitation of 6-mercaptopurine and its metabolites in red blood cells

A procedure is described for the rapid determination of the intra-erythrocyte concentration of 6-mercaptopurine (6-MP) and its metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). Erythrocytes (8 x 10(8) cells) in 350 æl Hanks solution containing 7.5 mg dithiothreitol were treated with 50 æl 70 percent perchloric acid. The precipitate was removed by centrifugation (13,000 g) and the supernatant hydrolyzed at 100§C for 45 min. After cooling, 100 æl was analyzed directly by HPLC using a Radialpack Resolve C18 column eluted with methanol-water (7.5:92.5, v/v) containing 100 mM triethylamine. 6-TG, 6-MP and the hydrolysis product of 6-MMP, 4-amino-5-(methylthio)carbonyl imidazole, were monitored at 342, 322 and 303 nm using a Shimadzu SPD-M10A diode array UV detector. The analytes eluted at 5.3, 6.0 and 10.2 min, respectively. The calibration curves were linear (rý > 0.998), and the analytical recoveries were 73.2 percent for 6-TG, 119.1 percent for 6-MP and 97.4 percent for 6-MMP. The intra- and inter-assay variations were highest for 6-MP (9.6 and 14.3 percent, respectively). The lowest detectable concentrations were 3, 3 and 25 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The quantification limits (coefficients of variation <15 percent) were 8, 10 and 70 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The method was applied to the analysis of 183 samples from 36 children under chemotherapy for acute lymphoblastic leukemia. The concentrations of the metabolites in the red cells of the patients ranged from 0 to 1934 pmol/8 x 10(8) erythrocytes for 6-TGN, and from 0 to 105.8 and 0 to 45.9 nmol/8 x 10(8) erythrocytes for 6-MP and 6-MMP, respectively. The procedure gave results that were in agreement with those obtained with other methods designed to detect cases of non-compliance with treatment, including patient interviews and medical evaluation, among others, demonstrating its applicability to monitoring the treatment of leukemic children.

Effect of acute treatment with a water-alcohol extract of Erythrina mulungu on anxiety-related responses in rats

We investigated the effect of acute oral treatment with a water-alcohol extract of the inflorescence of Erythrina mulungu (EM, Leguminosae-Papilionaceae) (100, 200 and 400 mg/kg) on rats submitted to different anxiety models: the elevated T-maze (for inhibitory avoidance and escape measurements), the light/dark transition, and the cat odor test. These models were selected for their presumed capacity to demonstrate specific subtypes of anxiety disorders as recognized in clinical practice. Treatment with 200 mg/kg EM impaired avoidance latencies (avoidance 1 - 200 mg/kg EM: 18 + or - 0 7 s, control group: 40 or - 9 s; avoidance 2 - 200 mg/kg EM: 15 + or - 4 s, control group: 110.33 + or - 38 s) in a way similar to the reference drug diazepam (avoidance 1: 3 + or - 0.79 s; avoidance 2: 3 + or - 0.76 s), without altering escape. Additionally, the same treatments increased the number of transitions (200 mg/kg EM: 6.33 + or - 0.90, diazepam: 10 + or - 1.54, control group: 2.78 + or - 0.60) between the two compartments and the time spent in the lighted compartment in the light/dark transition model (200 mg/kg EM: 39 + or - 7 s; diazepam: 61 + or - 9 s; control group: 14 + or - 4 s). The dose of 400 mg/kg EM also increased this last measurement (38 + or - 8 s). These results were not due to motor alterations since no significant effects were detected in the number of crossings or rearings in the arena. Furthermore, neither EM nor diazepam altered the behavioral responses of rats to a cloth impregnated with cat odor. These observations suggest that EM exerts anxiolytic-like effects on a specific subset of defensive behaviors, particularly those that have been shown to be sensitive to low doses of benzodiazepines

Prevalence of anemia among school-children from Rio-Acima (State of Minas Gerais, Brazil): use of the standardized prevalence method and evaluation of iron deficiency

The prevalence of anemia and iron deficiency was investigated in 332 children aged 7 to 15 years, 156 (47 per cent) boys and 176 (53 per cent) girls enrolled in the schools of the municipality of Rio Acima, MG. Seventy-four children were white (22.3 per cent), 218 were mulatto (65.7 per cent), and 40 were black (12 per cent). Mean hemoglobin level was 12.75 ñ 0.75 g/ dl. Lower values were determined for black children (12.32 + 0.87 g/dl) compared to white (l2.76 ñ 0.99 g/dl) and mulatto (12.81 ñ 0.94 g/dl) children. The prevalence of anemia was 16.6 per cent when determined on the basis of the percentage of children with hemoglobin values lower than the 3rd percentile for age and sex (standard method), and 36.2 per cent when determined by the standardized prevalence method for the evaluation of the prevalence of malnutrition in populations. Depletion of iron reserves was 8.13 per cent for the population in general and 20 per cent for the anemic children. This low prevalence of iron deficiency may have been the result of the value adopted as the lower normal limit (10 ng/ml) for serum ferritin values. The small percentage of anemic children with iron depletion may also be justified by the standard of normality adopted for hemoglobin values which was originally elaborated for the white population of North America and Finland and therefore may be inadequate for the population studied here, of diverse racial composition.

D-Fenfluramine selectively releases 5-HT from dorsal raphe terminals

The aim of this study was to investigate whether D-fenfluramine (FEN) releases 5-hydroxytryptamine (5-HT) selectively from dorsal raphe (DR) terminais. Male Wistar rats, 180-200 g, were implanted with microdialysis probes in the amygdala (Am; N = 5) and dorsal hippocampus (DH; N = 6) and 5-HT levels were measured by electrochemical detection. Under basal conditions, 5-HT levels were approximately 50 and 230 fmol per 30 min sample, in the Am and DH, respectively. FEN (1O mg/kg, ip) produced a 3-4-fold increase in 5HT relesse in the Am, but not in the DH. Since the Am is mainly innervated by DR fibers while the DH receives 5-HT input chiefly from the median raphe (MR), the present results support the view that FEN selectively releases 5-HT from DR terminals.

Morning steroid profile in children with congenital adrenal hyperplasia under different hydrocortisone schedules.

We studied 13 children with 21-hydroxyalse deficiency to explore the immediate potential suppressive effect of hydrocortisone dose schedule on the adrenal cortex. They were given 20 mg/m2 daily in a controlled trial. After random administration of a greater dose in the morning (7 patients) or at night (6 patients), we measured plasma levels of 17-hydroxyprogesterone, testosterone, and androstenedione at times-24, 0, 2, 4, and 6h. Considerable fluctuation of the steroid levels, unrelated to the drug intake, was observed. There was no statistically significant differences between the "morning dose" and "night dose" groups for any steroid. We conclude that; (i) the greater night dose did not avoid the 17-hydroxyprogesterone morning peaks, and (ii) the variation in plasma steroid levels is so marked that a single morning sample is unreliable to reflect the degree of adrenal suppression.

The elevated T maze, a new experimental model of anxiety and memory: effect of diazepam

In rats placed in a T maze consisting of an enclosed arm at right angles with two open arms elevated 50 cm above the ground, ip doses of 2 and 4 mg/kg diazepam (DZP) abolished the delay of withdrawal from the enclosed arm towards the open arms, measured by retesting in the presence of the drug soon after training, as well as by further retesting 72 h later, in the absence of the drug. Therefore, DZP had both anxiolytic and amnestic effects on this inhibitory avoidance task. In contrast, DZP did not affect the latency of withdrawal from one of the open arms towards the closed arm on the first day. Moreover, the latency of this escape response similarly decreased in all treatment groups in the retest performed 72 h later, indicating that memory of this task was resistant to DZP. These results support the view that the anxiolytic and amnestic effects of benzodiazepines are closely related, and suggest that this new elevated T maze model may be useful for simultaneous measurement of drug effects on anxiety and memory