Forced degradation studies of norepinephrine and epinephrine from dental anesthetics: Development of stability-indicating HPLC method and in silico toxicity evaluation.

Injectable solutions containing epinephrine (EPI) and norepinephrine (NE) are not stable, and their degradation is favored mainly by the oxidation of catechol moiety. As studies of these drugs under forced degradation conditions are scarce, herein, we report the identification of their degradation products (DP) in anesthetic formulations by the development of stability-indicating HPLC method. Finally, the risk assessment of the major degradation products was evaluated using in silico toxicity approach. HPLC method was developed to obtain a higher selectivity allowing adequate elution for both drugs and their DPs. The optimized conditions were developed using a C18 HPLC column, sodium 1-octanesulfonate, and methanol (80:20, v/v) as mobile phase, with a flow rate of 1.5 mL/min, UV detection at 199 nm. The analysis of standard solutions with these modifications resulted in greater retention time for EPI and NE, which allow the separation of these drugs from their respective DPs. Then, five DPs were identified and analyzed by in silico studies. Most of the DPs showed important alerts as hepatotoxicity and mutagenicity. To the best of our acknowledgment, this is the first report of a stability-indicating HPLC method that can be used with formulations containing catecholamines.

Melanin-concentrating hormone in the Locus Coeruleus aggravates helpless behavior in stressed rats.

Animal studies have shown that antagonists of receptor 1 of Melanin-Concentrating Hormone (MCH-R1) elicit antidepressive-like behavior, suggesting that MCH-R1 might be a novel target for the treatment of depression and supports the hypothesis that MCHergic signaling regulates depressive-like behaviors. Consistent with the evidence that MCHergic neurons send projections to dorsal and median raphe nuclei, we have previously demonstrated that MCH microinjections in both nuclei induced a depressive-like behavior. Even though MCH neurons also project to Locus Coeruleus (LC), only a few studies have reported the behavioral and neurochemical effect of MCH into the LC. We studied the effects of MCH (100 and 200 ng) into the LC on coping-stress related behaviors associated with depression, using two different behavioral tests: the forced swimming test (FST) and the learned helplessness (LH). To characterize the functional interaction between MCH and the noradrenergic LC system, we also evaluated the neurochemical effects of MCH (100 ng) on the extracellular levels of noradrenaline (NA) in the medial prefrontal cortex (mPFC), an important LC terminal region involved in emotional processing. MCH administration into the LC elicited a depressive-like behavior evidenced in both paradigms. Interestingly, in the LH, MCH (100) elicited a significant increase in escape failures only in stressed animals. A significant decrease in prefrontal levels of NA was observed after MCH microinjection into the LC. Our results demonstrate that increased MCH signaling into the LC triggers depressive-like behaviors, especially in stressed animals. These data further corroborate the important role of MCH in the neurobiology of depression.

Noradrenaline and dobutamine effects on the volume expansion with normal saline in rabbits subjected to hemorrhage

PURPOSE: To evaluate the effects of dobutamine (DB), noradrenaline (NA), and their combination (NADB), on volume retention in rabbits submitted to hemorrhage. METHODS: Thirty six rabbits were randomly divided into 6 groups: SHAM, Control, Saline, DB, NA, DB+NA. All the animals, except for SHAM, were subjected to hemorrhage of 25% of the calculated blood volume. Control animals were replaced with their own blood. The other groups received NSS 3 times the volume withdrawn. The intravascular retention, hematocrit, diuresis, central venous pressure, mean arterial pressure, NGAL, dry-to-wet lung weight ratio (DTWR) and the lung and kidney histology were analyzed. RESULTS: Replacement with NSS and NA, DB or NA+DB did not produce differences in the intravascular retention. After hemorrhage, the animals presented a significant decrease in the MAP and CVP, which were maintained until volume replacement. Regarding NGAL, dry-to-wet-lung-weight ratio, lung and kidney histology, there were no statistical differences between the groups. CONCLUSION: The use of noradrenaline, dobutamine or their combination did not increase the intravascular retention of volume after normal saline infusion.(AU)

The sympathetic nervous system affects the susceptibility and course of Trypanosoma cruzi infection.

Trypanosoma cruzi (T. cruzi) is an intracellular parasite that causes Chagas' disease, a major health problem in Latin America. Using a murine model of infection with this parasite, we have previously shown that corticosterone blood levels are markedly elevated during the course of the disease in C57Bl/6 male mice and that this increase is protective for the host by restricting the production of pro-inflammatory cytokines. Since the hypothalamus-pituitary-adrenal (HPA) axis usually operates in a concerted way with the sympathetic nervous system (SNS), we have now studied whether noradrenergic nerves can affect the course of T. cruzi infection and the sexual dimorphism observed in the disease. We found a decreased splenic noradrenaline concentration and content, paralleled by a reduction in noradrenergic nerve fibers in the spleen of infected mice, and increased HPA axis activity. These alterations were more marked in males than in females. When the spontaneous loss of noradrenergic nerve fibers was advanced by chemical sympathectomy prior to infection, males died earlier and mortality significantly increased in females. Chemical denervation did not significantly affect the concentration of specific IgM and IgG2a antibodies to T. cruzi, and did not worsen myocarditis, but resulted in increased parasitemia and IL-6 and IFN-γ blood levels. The results obtained in this model of parasitic disease provide further indications of the relevance of interactions between the immune system and the SNS for host defense.

Chronic stress improves NO- and Ca2+ flux-dependent vascular function: a pharmacological study.

BACKGROUND: Stress is associated with cardiovascular diseases. OBJECTIVE: This study aimed at assessing whether chronic stress induces vascular alterations, and whether these modulations are nitric oxide (NO) and Ca(2+) dependent. METHODS: Wistar rats, 30 days of age, were separated into 2 groups: control (C) and Stress (St). Chronic stress consisted of immobilization for 1 hour/day, 5 days/week, 15 weeks. Systolic blood pressure was assessed. Vascular studies on aortic rings were performed. Concentration-effect curves were built for noradrenaline, in the presence of L-NAME or prazosin, acetylcholine, sodium nitroprusside and KCl. In addition, Ca(2+) flux was also evaluated. RESULTS: Chronic stress induced hypertension, decreased the vascular response to KCl and to noradrenaline, and increased the vascular response to acetylcholine. L-NAME blunted the difference observed in noradrenaline curves. Furthermore, contractile response to Ca(2+) was decreased in the aorta of stressed rats. CONCLUSION: Our data suggest that the vascular response to chronic stress is an adaptation to its deleterious effects, such as hypertension. In addition, this adaptation is NO- and Ca(2+)-dependent. These data help to clarify the contribution of stress to cardiovascular abnormalities. However, further studies are necessary to better elucidate the mechanisms involved in the cardiovascular dysfunction associated with stressors.

Antidepressant-like behavioral, anatomical, and biochemical effects of petroleum ether extract from maca (Lepidium meyenii) in mice exposed to chronic unpredictable mild stress.

Maca has been consumed as a medical food in Peru for thousands of years, and exerts anxiolytic and antidepressant effects. Our present study aimed to evaluate the behavior and anatomical and biochemical effects of petroleum ether extract from maca (ME) in the chronic unpredictable mild stress (CUMS) model of depression in mice. Three different doses of maca extract (125, 250, and 500 mg/kg) were orally administrated in the six-week CUMS procedure. Fluoxetine (10 mg/kg) was used as a positive control drug. Maca extract (250 and 500 mg/kg) significantly decreased the duration of immobility time in the tail suspension test. After treatment with maca extract (250 and 500 mg/kg), the granule cell layer in the dentate gyrus appeared thicker. Maca extract (250 and 500 mg/kg) also induced a significant reduction in corticosterone levels in mouse serum. In mouse brain tissue, after six weeks of treatment, noradrenaline and dopamine levels were increased by maca extract, and the activity of reactive oxygen species was significantly inhibited. Serotonin levels were not significantly altered. These results demonstrated that maca extract (250 and 500 mg/kg) showed antidepressant-like effects and was related to the activation of both noradrenergic and dopaminergic systems, as well as attenuation of oxidative stress in mouse brain.

Stress alone or associated with ethanol induces prostanoid release in rat aorta via alpha2-Adrenoceptor.

BACKGROUND: Stress and ethanol are both, independently, important cardiovascular risk factors. OBJECTIVE: To evaluate the cardiovascular risk of ethanol consumption and stress exposure, isolated and in association, in male adult rats. METHODS: Rats were separated into 4 groups: Control, ethanol (20% in drinking water for 6 weeks), stress (immobilization 1h day/5 days a week for 6 weeks) and stress/ethanol. Concentration-responses curves to noradrenaline - in the absence and presence of yohimbine, L-NAME or indomethacin - or to phenylephrine were determined in thoracic aortas with and without endothelium. EC50 and maximum response (n=8-12) were compared using two-way ANOVA/Bonferroni method. RESULTS: Either stress or stress in association with ethanol consumption increased the noradrenaline maximum responses in intact aortas. This hyper-reactivity was eliminated by endothelium removal or by the presence of either indomethacin or yohimbine, but was not altered by the presence of L-NAME. Meanwhile, ethanol consumption did not alter the reactivity to noradrenaline. The phenylephrine responses in aortas both with and without endothelium also remained unaffected regardless of protocol. CONCLUSION: Chronic stress increased rat aortic responses to noradrenaline. This effect is dependent upon the vascular endothelium and involves the release of vasoconstrictor prostanoids via stimulation of endothelial alpha-2 adrenoceptors. Moreover, chronic ethanol consumption appeared to neither influence noradrenaline responses in rat thoracic aorta, nor did it modify the increase of such responses observed as a consequence of stress exposure.

Estresse Isolado ou Associado ao Etanol Libera Prostanóides em Aorta de Ratos via ?2-Adrenoceptores / Stress Alone or associated with Ethanol Induces Prostanoid Release in Rat Aorta via ?2-Adrenoceptor

Fundamento: Estresse e etanol são ambos, independentemente, importantes fatores de risco cardiovascular. Objetivo: avaliar o risco cardiovascular do consumo de etanol e exposição ao estresse, isolados e em associação, em ratos machos adultos. Métodos: Os ratos foram separados em quatro grupos: controle, etanol (20% na água de beber durante seis semanas), estresse (imobilização 1h dia/5 dias por semana/ 6 semanas) e estresse/etanol. As curvas de concentração-resposta à noradrenalina - na ausência e na presença de ioimbina, L-NAME ou indometacina - ou fenilefrina foram determinadas em aortas torácicas com e sem endotélio. EC50 e resposta máxima (n = 8-12) foram comparadas através de ANOVA de dois fatores (two-way) / método de Bonferroni. Resultados: Estresse ou estresse em associação com o consumo de etanol aumentaram as respostas máximas de noradrenalina em aortas intactas. Essa hiper-reatividade foi eliminada pela remoção do endotélio, ou pela presença da indometacina ou ioimbina, mas não foi alterada pela presença de L-NAME. Enquanto isso, o consumo de etanol não alterou a reatividade à noradrenalina. As respostas da fenilefrina em aortas com e sem endotélio também permaneceram inalteradas independentemente do protocolo. Conclusão: O estresse crônico aumentou as respostas aórticas dos ratos à noradrenalina. Esse efeito é dependente do endotélio vascular e envolve a liberação de prostanóides vasoconstritores através da estimulação de α-2 adrenoceptores endoteliais. Além disso, o consumo crônico de etanol pareceu não influenciar as respostas de noradrenalina em aorta de rato, nem modificar o aumento de tais respostas observadas em consequência da exposição ao estresse. .

Background: Stress and ethanol are both, independently, important cardiovascular risk factors. Objective: To evaluate the cardiovascular risk of ethanol consumption and stress exposure, isolated and in association, in male adult rats. Methods: Rats were separated into 4 groups: Control, ethanol (20% in drinking water for 6 weeks), stress (immobilization 1h day/5 days a week for 6 weeks) and stress/ethanol. Concentration-responses curves to noradrenaline - in the absence and presence of yohimbine, L-NAME or indomethacin - or to phenylephrine were determined in thoracic aortas with and without endothelium. EC50 and maximum response (n=8-12) were compared using two-way ANOVA/Bonferroni method. Results: Either stress or stress in association with ethanol consumption increased the noradrenaline maximum responses in intact aortas. This hyper-reactivity was eliminated by endothelium removal or by the presence of either indomethacin or yohimbine, but was not altered by the presence of L-NAME. Meanwhile, ethanol consumption did not alter the reactivity to noradrenaline. The phenylephrine responses in aortas both with and without endothelium also remained unaffected regardless of protocol. Conclusion: Chronic stress increased rat aortic responses to noradrenaline. This effect is dependent upon the vascular endothelium and involves the release of vasoconstrictor prostanoids via stimulation of endothelial alpha-2 adrenoceptors. Moreover, chronic ethanol consumption appeared to neither influence noradrenaline responses in rat thoracic aorta, nor did it modify the increase of such responses observed as a consequence of stress exposure. .

Prenatal stress induces up-regulation of glucocorticoid receptors on lymphoid cells modifying the T-cell response after acute stress exposure in the adult life.

It has been demonstrated that a short-duration stress (acute stress) may result in immunopreparatory or immunoenhancing physiological conditions. The aim of the present study was to investigate whether exposure to prenatal restraint stress (PRS) influences the impact of acute stress on the T-cell response in the adult life. We found that female mice exposed to PRS (PS mice) did not exhibit changes in the T-cell-dependent IgG antibody production with respect to prenatally non-stressed mice (no-PS mice). However, no-PS mice exposed to acute stress showed an increase of antibody production after antigen stimulation. In contrast, PS mice exhibited a decreased response after an acute situation. Spleen catecholamines and plasma corticosterone levels were increased in acute stress in both PS and no-PS mice. Nevertheless, lymphocyte response to hormones was altered in PS mice. Particularly, inhibitory effect of corticosterone was higher on lymphocytes from PS mice. In addition, an increase in protein levels and mRNA expression of glucocorticoid receptor was found in lymphoid cells from PS mice. These results show that prenatal stress alters the immune intrinsic regulatory mechanism that in turn induces an increased vulnerability to any stressful situation able to modify immune homeostasis.

Characterization of motor, depressive-like and neurochemical alterations induced by a short-term rotenone administration.

BACKGROUND: Rotenone exposure in rodents provides an interesting model for studying mechanisms of toxin-induced dopaminergic neuronal injury. However, several aspects remain unclear regarding the effects and the accuracy of rotenone as an animal model of Parkinson's disease (PD). In this study, we investigated the motor and depressive-like behaviors associated to neurochemical alterations induced by a novel protocol of rotenone administration. METHODS: In the first experiment, we adopted the paw test to characterize an effective dose of rotenone able to promote nigrostriatal toxicity. In the second experiment, control and rotenone 2.5 mg/kg groups were injected (ip) for 10 consecutive days. RESULTS: This test indicated that intraperitonial (ip) rotenone at 2.5 and 5.0 mg/kg promoted a significant neurotoxicity to striatum and nucleus accumbens. However, only 2.5 mg/kg of rotenone was associated to a negligible mortality rate. Open-field tests were conducted on 1, 7, 14 and 21 day after the last day of treatment and showed an important locomotor impairment, confined to 1 and 7 day. Besides, rotenone affected dopamine levels and increased its turnover in the striatum. Modified forced swim test (performed on 22 day) and sucrose preference test (performed on 14 and 21 day) demonstrated that rotenone produced impairments in the swimming and immobility. In parallel, increments in the serotonin and noradrenaline turnovers were observed in the striatum and hippocampus of the rotenone group. CONCLUSIONS: These data suggest important participations of serotonin and noradrenaline in depressive-like behaviors induced by rotenone. Thus, it is proposed that the current rotenone protocol provides an improvement regarding the existing rotenonemodels of PD.

Ativação adrenérgica intramiocárdica na cardiomiopatia chagásica e doença arterial coronariana / Intramyocardial adrenergic activation in chagasic cardiomyopathy and coronary artery disease / Activación adrenérgica intramiocárdica en la cardiomiopatía chagásica y enfermedad arterial coronaria

FUNDAMENTO: A norepinefrina miocárdica está alterada na disfunção ventricular esquerda. Em pacientes com cardiomiopatia chagásica (CC), essa questão ainda não foi discutida. OBJETIVO: Determinar o nível de norepinefrina (NE) miocárdica em pacientes com CC e compará-la em pacientes com doença arterial coronariana (DAC) e relacionar NE miocárdica com a fração de ejeção do ventrículo esquerdo (FEVE). MÉTODOS: Estudamos 39 pacientes com CC, divididos em grupo 1: 21 indivíduos com FEVE normal e grupo 2: 18 com FEVE diminuída. Dezessete pacientes com DAC foram divididos em grupo 3: 12 indivíduos com FEVE normal e grupo 4: 5 indivíduos com FEVE diminuída. Ecocardiografia bidimensional foi usada para medir a FEVE. A NE miocárdica foi determinada através de Cromatografia Líquida de Alta Eficiência (HPLC). RESULTADOS: A NE miocárdica na CC com e sem disfunção ventricular foi 1,3±1,3 e 6,1±4,2 pg/μg de proteína não-colagenosa, respectivamente (p<0,0001); na DAC com e sem disfunção ventricular, foi 3,3±3,0 e 9,8±4,2 pg/μg de proteína não-colagenosa, respectivamente (p<0,0001). Uma correlação positive foi observada entre a FEVE e a concentração de NE miocárdica em pacientes com CC (p<0,01; r = 0,57) e também naqueles com DAC (p<0,01; r=0,69). Uma diferença significante foi demonstrada entre as concentrações de NE em pacientes com FEVE normal (grupos 1 e 3; p = 0,0182), mas nenhuma diferença foi observada em pacientes com FEVE diminuída (grupos 2 e 4; p = 0,1467). CONCLUSÃO: Pacientes com CC e fração de ejeção global normal apresentam uma denervação cardíaca precoce, quando comparados à pacientes com doença arterial coronariana.

BACKGROUND: Myocardial norepinephrine is altered in left ventricular impairment. In patients with Chagas' cardiomyopathy (CC), this issue has not been addressed. OBJECTIVE: To determine the level of myocardial norepinephrine in patients with CC and compare it in patients with coronary artery disease, and to relate myocardial norepinephrine to left ventricular ejection fraction (LVEF). METHODS: We studied 39 patients with CC, divided into group 1: 21 individuals with normal LVEF and group 2: 18 individuals with decreased LVEF. Seventeen patients with coronary artery disease were divided into group 3: 12 individuals with normal LVEF and group 4: 5 individuals with decreased LVEF. Two-dimensional echocardiography was used to measure LVEF. Myocardial norepinephrine was determined by high-performance liquid chromatography. RESULTS: Myocardial norepinephrine in CC with and without ventricular dysfunction was 1.3±1.3 and 6.1±4.2 pg/μg noncollagen protein, respectively (p<0.0001); in coronary artery disease with and without ventricular dysfunction, it was 3.3±3.0 and 9.8±4.2 pgμg noncollagen protein, respectively (p<0.0001). A positive correlation was found between LVEF and myocardial norepinephrine concentration in the patients with Chagas' cardiomyopathy (p<0.01, r = 0.57) and also in those with coronary artery disease (p<0.01, r=0.69). A significant difference was demonstrated between norepinephrine concentrations in patients with normal LVEF (groups 1 and 3; p = 0.0182), but no difference was found in patients with decreased LVEF (groups 2 and 4; p = 0.1467). CONCLUSION: In patients with Chagas' cardiomyopathy and normal global ejection fraction there is an early cardiac denervation, when compared to coronary artery disease patients.

FUNDAMENTO: La norepinefrina miocárdica está alterada en la disfunción ventricular izquierda. En pacientes con cardiomiopatía chagásica (CC), esa cuestión aun no fue discutida. OBJETIVO: Determinar el nivel de norepinefrina (NE) miocárdica en pacientes con CC y compararla en pacientes con enfermedad arterial coronaria (EAC) y relacionar NE miocárdica con la fracción de eyección del ventrículo izquierdo (FEVI). MÉTODOS: 39 pacientes con CC, divididos en grupo 1: 21 individuos con FEVI normal y grupo 2: 18 con FEVI disminuida. Diecisiete pacientes con EAC fueron divididos en grupo 3: 12 individuos con FEVI normal y grupo 4: 5 individuos con FEVI disminuida. Ecocardiografía bidimensional fue usada para medir la FEVI. La NE miocárdica fue determinada a través de Cromatografía Líquida de Alta Eficiencia (HPLC). RESULTADOS: La NE miocárdica en la CC con y sin disfunción ventricular fue 1,3±1,3 y 6,1±4,2 pg/µg de proteína no colagenosa, respectivamente (p<0,0001); en la EAC con y sin disfunción ventricular, fue 3,3±3,0 y 9,8±4,2 pg/µg de proteína no colagenosa, respectivamente (p<0,0001). Una correlación positiva fue observada entre la FEVI y la concentración de NE miocárdica en pacientes con CC (p<0,01; r=0,57) y también en aquellos con EAC (p<0,01; r=0,69). Una diferencia significativa fue demostrada entre las concentraciones de NE en pacientes con FEVI normal (grupos 1 y 3; p = 0,0182), pero ninguna diferencia fue observada en pacientes con FEVI disminuida (grupos 2 y 4; p = 0,1467). CONCLUSIONES: Pacientes con CC y fracción de eyección global normal presentan una denervación cardíaca precoz, cuando son comparados a pacientes con enfermedad arterial coronaria.

Intramyocardial adrenergic activation in chagasic cardiomyopathy and coronary artery disease.

BACKGROUND: Myocardial norepinephrine is altered in left ventricular impairment. In patients with Chagas' cardiomyopathy (CC), this issue has not been addressed. OBJECTIVE: To determine the level of myocardial norepinephrine in patients with CC and compare it in patients with coronary artery disease, and to relate myocardial norepinephrine to left ventricular ejection fraction (LVEF). METHODS: We studied 39 patients with CC, divided into group 1: 21 individuals with normal LVEF and group 2: 18 individuals with decreased LVEF. Seventeen patients with coronary artery disease were divided into group 3: 12 individuals with normal LVEF and group 4: 5 individuals with decreased LVEF. Two-dimensional echocardiography was used to measure LVEF. Myocardial norepinephrine was determined by high-performance liquid chromatography. RESULTS: Myocardial norepinephrine in CC with and without ventricular dysfunction was 1.3±1.3 and 6.1±4.2 pg/µg noncollagen protein, respectively (p<0.0001); in coronary artery disease with and without ventricular dysfunction, it was 3.3±3.0 and 9.8±4.2 pg/µg noncollagen protein, respectively (p<0.0001). A positive correlation was found between LVEF and myocardial norepinephrine concentration in the patients with Chagas' cardiomyopathy (p<0.01, r = 0.57) and also in those with coronary artery disease (p<0.01, r=0.69). A significant difference was demonstrated between norepinephrine concentrations in patients with normal LVEF (groups 1 and 3; p = 0.0182), but no difference was found in patients with decreased LVEF (groups 2 and 4; p = 0.1467). CONCLUSION: In patients with Chagas' cardiomyopathy and normal global ejection fraction there is an early cardiac denervation, when compared to coronary artery disease patients.

Raman, IR, UV-vis and EPR characterization of two copper dioxolene complexes derived from L-dopa and dopamine.

The anionic complexes [Cu(L(1-))3](1-), L(-)=dopasemiquinone or L-dopasemiquinone, were prepared and characterized. The complexes are stable in aqueous solution showing intense absorption bands at ca. 605 nm for Cu(II)-L-dopasemiquinone and at ca. 595 nm for Cu(II)-dopasemiquinone in the UV-vis spectra, that can be assigned to intraligand transitions. Noradrenaline and adrenaline, under the same reaction conditions, did not yield Cu-complexes, despite the bands in the UV region showing that noradrenaline and adrenaline were oxidized during the process. The complexes display a resonance Raman effect, and the most enhanced bands involve ring modes and particularly the nuCC+nuCO stretching mode at ca. 1384 cm(-1). The free radical nature of the ligands and the oxidation state of the Cu(II) were confirmed by the EPR spectra that display absorptions assigned to organic radicals with g=2.0005 and g=2.0923, and for Cu(II) with g=2.008 and g=2.0897 for L-dopasemiquinone and dopasemiquinone, respectively. The possibility that dopamine and L-dopa can form stable and aqueous-soluble copper complexes at neutral pH, whereas noradrenaline and adrenaline cannot, may be important in understanding how Cu(II)-dopamine crosses the cellular membrane as proposed in the literature to explain the role of copper in Wilson disease.

Neonatal exposure to estradiol valerate programs ovarian sympathetic innervation and follicular development in the adult rat.

A single injection of estradiol valerate (EV) to 14-day-old rats (when the ovarian follicle population has been already established) disrupts cyclicity, increases the activity of key enzymes of androgen biosynthesis, and develops polycystic ovary by a causally related increase in ovarian noradrenaline (NA). The current study examined an early window of ovarian development to look for a specific stage of development at which estradiol can induce such changes in sympathetic activity and follicular development. A single dose of EV applied to rats before the first 12 h of life rapidly increases (after 24 h) the ovarian expression of nerve growth factor (Ngfb) and p75 low-affinity neurotrophic receptor (Ngfr) mRNAs. When adults, rats presented early vaginal opening, disrupted cyclicity, appearance of follicular cyst, absence of corpus luteum, and infertility. Total follicles decreased, mainly due to a reduced number of primordial follicles, suggesting that estradiol acts in the first stages of folliculogenesis, when primordial follicles are organizing. These changes paralleled a 6-fold increase in NA concentration. No changes in NA content were found in the celiac ganglia, suggesting a local, non-centrally mediated effect of estradiol. Surgical section of the superior ovarian nerve (the main source of sympathetic nerves to the ovary) to rats neonatally treated with EV decreased intraovarian NA, delayed vaginal opening, and blocked the development of follicular cyst and that of preovulatory follicles. Therefore, we can conclude that early exposure to estradiol permanently modifies ovarian sympathetic activity and causes profound changes in follicular development, leading to the polycystic ovary condition.

High fructose diet increases anterior hypothalamic alpha 2-adrenoceptors responsiveness.

Activation of alpha(2)-adrenoceptors in the anterior hypothalamic area (AHA) decreases sympathetic nervous system activity and blood pressure. The aim of the present study was to evaluate activity of pre- and postsynaptic alpha(2)-adrenoceptors in the AHA of fructose hypertensive rats (F), an animal model of insulin resistance and hypertension. The AHA of Control (C) and F anaesthetized rats was perfused with Ringer solution in the absence or presence of clonidine (100 or 300 microg ml(-1)) using reverse microdialysis. Clonidine effects on mean arterial pressure (MAP) and heart rate (HR), and on hypothalamic noradrenaline levels were measured along perfusion time. Noradrenaline extracellular levels in the AHA were significantly diminished in F hypertensive rats compared to C animals. The depressor effect of intrahypothalamic perfusion of clonidine on MAP was enhanced in F rats compared with C animals. Intrahypothalamic perfusion of clonidine reduced HR only in F rats. The effect of clonidine on noradrenaline hypothalamic extracellular levels was enhanced in F rats. These results suggest, in our experimental conditions, the existence of an increased responsiveness of pre- and postsynaptic alpha(2)-adrenoceptors in the AHA of F hypertensive rats. This fact could be a consequence of a compensatory supersensitivity of alpha-adrenoceptors due to a decrease in noradrenaline release from nerve terminals located in the AHA.

A quantitative study of rat uterine sympathetic innervation during pregnancy and post partum.

In mammals, pregnancy induces a transient and extensive degeneration of uterine sympathetic innervation. We used the models of unilateral oviduct ligation and in oculo myometrium transplant in pregnant rats to address the role of stretching forces and/or hormone milieu in the loss of sympathetic innervation. The sympathetic fibres of the uterine horn and in oculo myometrial transplants were quantified on tissue sections processed by the glyoxylic acid technique. In normal pregnant rats, the density of uterine horn innervation was significantly reduced at late pregnancy and recovery took place during post partum. The empty horn of pregnant rats showed no significant changes in density of myometrial innervation during pregnancy or post partum. In oculo myometrial transplants were organotypically reinnervated in virgin animals. When the transplants were exposed to gestational hormonal milieu, few or no fibres were observed to the end of pregnancy; however, a significant increase at post partum was observed. Results showed that both the effects of stretching and the hormone milieu derived from the fetus-placenta complex play a role as inductors of changes on sympathetic myometrial innervation during pregnancy and support the idea that immature muscular uterine fibres are more susceptible to the effects of pregnancy than those originating from adult animals.

A simple high-performance liquid chromatography assay for on-line determination of catecholamines in adrenal gland by direct injection on an ISRP column.

In the present research, assays were improved for the determination of catecholamines in adrenal gland. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The method involved direct injection of acid extract on to a serum albumin dimethylocadecyl-silane (HSA-C18) and the utilization of phosphate buffer (pH = 3.0): methanol (97:3 v/v), 0.025 g heptanosulfonic acid and 0.0025 g EDTA as mobile phase. The detection was obtained using an electrochemical detector L-ECD-6A-Shimadzu with a potential of the 85 mV. Identification was based on retention time. Quantification was performed by automatic peak-area determination. The detection limit is equal to 0.5 ng ml(-1). The HPLC method with electrochemical detection proposed here permits good separation of catecholamines in samples of adrenal gland from rats. The method has various advantages: fast, high precision and good selectivity and do not require sample treatment. The immobilization stress during 5 min did not provoke alteration in catecholamines contains in rat adrenal gland, due to the short time of the stress exposure. This study shows that the catecholamines (norepinephrine and epinephrine) adrenal increased significantly after the acute immobilization stress during 30 min as compared to control group. This increase probably is due to the emotional component of the immobilization stress. In conclusion, our studies suggest an effective participation of the adrenal glands to maintain the homeostasis of organism to the stressful conditions.

Influence of time of day on hypothalamic monoaminergic activity in early pregnancy: effect of a previous reproductive experience.

Several dopamine-related neurochemical and behavioral responses are influenced by the time of day. The light-dark shift is a major zeitgeber for various functionally important hypothalamic monoaminergic systems. However, these influences are modulated by reproductive state and by reproductive experience (RE) in females. Early pregnancy in rodents generates diurnal and nocturnal prolactin surges that are reduced in intensity in a second pregnancy. Dopamine (DA) is a major inhibitory factor of prolactin synthesis and secretion. Other neurotransmitters such as serotonin (5HT) and norepinephrine (NE) can modulate prolactin secretion as well. Previous works have demonstrated that RE induces changes in central concentrations of both dopamine and serotonin. In addition, RE modulates the responses of both dopaminergic and serotoninergic nerve terminals. The present investigation was designed to examine the possible effects of RE on hypothalamic concentrations of DA, NE, 5HT and their major metabolites homovanillic acid (HVA), 3-4-dihydroxyphenyl acetic acid (DOPAC), 3-methoxy mandelic acid (VMA) and 5-hydroxyindole 3-acetic acid (5HIAA), respectively. These parameters were measured in pregnant rats during the light-dark shift and the prolactin surges. Primi- and multigravid rats were sacrificed on the 7th-8th day of pregnancy between 1700 and 1900 h (light-dark shift and diurnal prolactin surge) or 0200 and 0400 h (nocturnal prolactin surge), and hypothalamic concentrations of DA, NE and 5HT and their metabolites were measured by high performance liquid chromatography coupled to an electrochemical detector (HPLC-ED). Trunk blood was collected and serum prolactin measured by radioimmunoassay. The prolactin surge was confirmed and multigravid rats showed significantly lower serum prolactin levels as compared to primigravid rats between 0200 and 0400 h. During the light-dark shift DA and NE concentrations increased while DOPAC/DA, HVA/DA and 5HIAA/5HT ratios decreased in multigravid rats compared to primigravid rats. Except for 5HIAA/5HT, these differences were not observed during the prolactin nocturnal surge. These results suggest that a previous reproductive experience induces central functional changes during pregnancy which are expressed differently according to the time of day.