Tyrosine hydroxylase and ß2-adrenergic receptor expression in leukocytes of spontaneously hypertensive rats: putative peripheral markers of central sympathetic activity

The sympathetic nervous system (SNS) plays a fundamental role in the pathophysiology of cardiovascular diseases, including primary arterial hypertension. In this study, we aimed to investigate whether the expression of the rate-limiting enzyme in catecholamine synthesis, tyrosine hydroxylase (TH), and the β2-adrenergic receptor (β2-AR) in immune cells from peripheral blood, reflect central SNS activity in spontaneously hypertensive rats (SHR). TH expression in the lower brainstem and adrenal glands and β2-AR expression in the lower brainstem were analyzed by western blot analyses. In the leukocytes, TH and β2-AR expression was evaluated by flow cytometry before and after chronic treatment with the centrally-acting sympathoinhibitory drug clonidine. Western blot analyses showed increased TH and β2-AR expression in the lower brainstem and increased TH in adrenal glands from SHR compared to normotensive Wistar Kyoto rats (WKY). Lower brainstem from SHR treated with clonidine presented reduced TH and β2-AR levels, and adrenal glands had decreased TH expression compared to SHR treated with vehicle. Flow cytometry showed that the percentage of leukocytes that express β2-AR is higher in SHR than in WKY. However, the percentage of leukocytes that expressed TH was higher in WKY than in SHR. Moreover, chronic treatment with clonidine normalized the levels of TH and β2-AR in leukocytes from SHR to similar levels of those of WKY. Our study demonstrated that the percentage of leukocytes expressing TH and β2-AR was altered in arterial hypertension and can be modulated by central sympathetic inhibition with clonidine treatment.

Involvement of catecholamines in the myocardium of rats submitted to experimental model of portal hypertension / Envolvimento das catecolaminas no miocárdio de ratos submetidos a modelo experimental de hipertensão portal

ABSTRACT Background: The role of autonomic nervous system in the development and maintenance of portal hypertension is not fully elucidated. It is known that the gene expression of norepinephrine in the superior mesenteric artery varies with time, and it may contribute for splanchnic vasodilation and its consequent hemodynamic repercussions. It is still not known exactly how the adrenergic expression behaves at the heart level in the initial stages of this process. Aim: To evaluate the immunohistochemical expression of the enzyme tyrosine hydroxylase (tyrosine 3-monooxygenase), involved in the synthesis of norepinephrine, in the myocardium of rats submitted to partial ligation of the portal vein. Methods: Twenty-four Wistar rats were divided into two groups: Sham Operated and Portal Hypertension. The partial ligation was performed in the Portal Hypertension group, and after 1/6/24 h and 3/5/14 days the animals were euthanized. Immunohistochemical analysis was performed to quantify the expression of the stained enzyme using the ImageJ program. Results: The Portal Hypertension group expressed percentages between 4.6-6% of the marked area, while the Sham Operated group varied between 4-5%. Although there was no statistical significance, the percentage stained in the Portal Hypertension group followed an increasing pattern in the first 6 h and a decreasing pattern after 24 h, which was not observed in the Sham Operated group. Conclusion: The expression of noradrenaline in rat myocardium during the first two weeks after partial ligation of the portal vein, with tyrosine hydroxylase as marker, did not show differences between groups over time.

RESUMO Racional: O papel do sistema nervoso autônomo na hipertensão portal não está completamente elucidado. Sabe-se que, nessa condição, a expressão gênica da norepinefrina na artéria mesentérica superior modifica-se com o tempo, podendo ser importante contribuinte para a vasodilatação esplâncnica e suas repercussões hemodinâmicas. Apesar dos estudos sobre as repercussões cardiovasculares na hipertensão portal, ainda não se sabe como a expressão adrenérgica se comporta a nível cardíaco nas etapas iniciais desse processo. Objetivo: Avaliar a expressão imunoistoquímica da enzima tirosina hidroxilase (tirosina 3-mono-oxigenase), relacionada à síntese da norepinefrina, no miocárdio de ratos submetidos à ligadura parcial da veia porta. Métodos: Foram utilizados 24 ratos, distribuídos em dois grupos: Sham Operated e Hipertensão Portal. A ligadura parcial da veia porta foi realizada apenas no grupo Hipertensão Portal e, após 1/6/24 h e 3/5/14 dias, os animais foram eutanasiados. Foi feita a análise imunoistoquímica para quantificar a expressão da enzima corada, utilizando o programa ImageJ. Resultados: No grupo Hipertensão Portal, o miocárdio expressou percentuais entre 4,6-6% de área marcada, enquanto que no grupo Sham Operated variou entre 4-5%, sem significância estatística. Apenas no grupo Hipertensão Portal, a porcentagem corada pela enzima seguiu padrão crescente nas primeiras 6 h e decrescente após 24 h. Conclusão: A expressão da noradrenalina no miocárdio de ratos durante as primeiras duas semanas após a ligadura parcial da veia porta, tendo como marcador a enzima tirosina hidroxilase, não apresentou diferenças entre grupos ao longo do tempo.

Effects and Mechanisms of Radiofrequency Ablation of Renal Sympathetic Nerve on Anti-Hypertension in Canine / Efeitos e Mecanismos da Denervação Simpática Renal por Radiofrequência na Anti-Hipertensão em Cães

Abstract Background: Radiofrequency ablation of renal sympathetic nerve (RDN) shows effective BP reduction in hypertensive patients while the specific mechanisms remain unclear. Objective: We hypothesized that abnormal levels of norepinephrine (NE) and changes in NE-related enzymes and angiotensinconverting enzyme 2 (ACE2), angiotensin (Ang)-(1-7) and Mas receptor mediate the anti-hypertensive effects of RDN. Methods: Mean values of systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) were assessed at baseline and follow-up. Plasma and renal norepinephrine (NE) concentrations were determined using highperformance liquid chromatography with electrochemical detection, and levels of NE-related enzyme and ACE2-Ang(1-7)- Mas were measured using real time PCR, Western blot and immunohistochemistry or Elisa in a hypertensive canine model fed with high-fat diet and treated with RDN. The parameters were also determined in a sham group treated with renal arteriography and a control group fed with normal diet. Results: RDN decreased SBP, DBP, MAP, plasma and renal NE. Compared with the sham group, renal tyrosine hydroxylase (TH) expression was lower and renalase expression was higher in the RDN group. Compared with the control group, renal TH and catechol-o-methyl transferase (COMT) were higher and renalase was lower in the sham group. Moreover, renal ACE2, Ang-(1-7) and Mas levels of the RDN group were higher than those of the sham group, which were lower than those of the control group. Conclusion: RDN shows anti-hypertensive effect with reduced NE and activation of ACE2-Ang(1-7)-Mas, indicating that it may contribute to the anti-hypertensive effect of RDN.

Resumo Fundamentos: A denervação simpática renal por radiofrequência (DSR) mostra redução eficaz da pressão arterial (PA) de pacientes hipertensos, ainda que os mecanismos específicos permaneçam obscuros. Objetivo: Fizemos a hipótese de que níveis alterados de noradrenalina (NA) e mudanças nas enzimas relacionadas à NA e enzima conversora de angiotensina 2 (ECA-2), angiotensina (Ang)-(1-7) e receptor Mas são mediadores dos efeitos antihipertensivos da DSR. Métodos: Foram avaliados os valores médios de pressão arterial sistólica (PAS), pressão arterial diastólica (PAD) e pressão arterial média (PAM) no início e durante o seguimento. Foram medidas as concentrações plasmática e renal de noradrenalina (NA) por cromatografia líquida de alta eficiência com detecção eletroquímica, e os níveis de enzima relacionada à NA e ECA2-Ang-(1-7)-Mas através de PCR em tempo real, Western blot e imunohistoquímica ou Elisa em um modelo canino de hipertensão que recebeu ração rica em gordura e foi tratado com DSR. Os parâmetros também foram determinados em um grupo de cirurgia simulada submetido à arteriografia renal e em um grupo controle que recebeu dieta normal. Resultados: DSR causou diminuição da PAS, PAD, PAM e das concentrações plasmática e renal de NA. Em comparação ao grupo placebo, a expressão da tirosina hidroxilase (TH) renal foi menor e a da renalase foi maior no grupo DSR. Em comparação ao grupo controle, os níveis de TH renal e de catecol-o-metil-transferase (COMT) foram maiores e os de renalase foram menores no grupo cirurgia simulada. Além disso, os níveis renais de ECA2, Ang-(1-7) e Mas foram maiores no grupo DSR do que no grupo cirurgia simulada, que, por sua vez, foram menores do que no grupo controle. Conclusões: A DSR mostra efeitos anti-hipertensivos com redução da NA e ativação da ECA2-Ang-(1-7)-Mas, o que indica que pode contribuir com o efeito anti-hipertensivo da DSR.

Nocardia otitidiscaviarum (GAM-5) induces parkinsonian-like alterations in mouse

Parkinson's disease, a major neurodegenerative disorder in humans whose etiology is unknown, may be associated with some environmental factors. Nocardia otitidiscaviarum (GAM-5) isolated from a patient with an actinomycetoma produced signs similar to Parkinson's disease following iv injection into NMRI mice. NMRI mice were infected intravenously with a non-lethal dose of 5 x 10(6) colony forming units of N. otitidiscaviarum (GAM-5). Fourteen days after bacterial infection, most of the 60 mice injected exhibited parkinsonian features characterized by vertical head tremor, akinesia/bradykinesia, flexed posture and postural instability. There was a peak of nocardial growth in the brain during the first 24 h followed by a decrease, so that by 14 days nocardiae could no longer be cultured. At 24 h after infection, Gram staining showed nocardiae in neurons in the substantia nigra and occasionally in the brain parenchyma in the frontal and parietal cortex. At 21 days post-infection, tyrosine hydroxylase immunolabeling showed a 58 percent reduction of tyrosine hydroxylase in the substantia nigra, and a 35 percent reduction of tyrosine hydroxylase in the ventral tegmental region. Dopamine levels were reduced from 110 ± 32.5 to 58 ± 16.5 ng/mg protein (47.2 percent reduction) in brain from infected mice exhibiting impaired movements, whereas serotonin levels were unchanged (191 ± 44 protein in control and 175 ± 39 ng/mg protein in injected mice). At later times, intraneuronal inclusion bodies were observed in the substantia nigra. Our observations emphasize the need for further studies of the potential association between Parkinson's disease or parkinsonism-like disease and exposure to various nocardial species.

Iatrogenic Creutzfeldt-Jakob disease following human growth hormone therapy: case report

We report the case of a 41-year-old man with iatrogenic Creutzfeldt-Jakob disease (CJD) acquired after the use of growth hormone (GH) obtained from a number of pituitary glands sourced from autopsy material. The incubation period of the disease (from the midpoint of treatment to the onset of clinical symptoms) was rather long (28 years). Besides the remarkable cerebellar and mental signs, the patient exhibited sleep disturbance (excessive somnolence) from the onset of the symptoms, with striking alteration of the sleep architecture documented by polysomnography. 14-3-3 protein was detected in the CSF, and MRI revealed increased signal intensity bilaterally in the striatum, being most evident in diffusion-weighted (DW-MRI) sequences. This is the second case of iatrogenic CJD associated with the use of GH reported in Brazil

Neonatal handling and the expression of immunoreactivity to tyrosine hydroxylase in the hypothalamus of adult male rats

Neonatal handling has long-lasting effects on behavior and stress reactivity. The purpose of the present study was to investigate the effect of neonatal handling on the number of dopaminergic neurons in the hypothalamic nuclei of adult male rats as part of a series of studies that could explain the long-lasting effects of neonatal stimulation. Two groups of Wistar rats were studied: nonhandled (pups were left undisturbed, control) and handled (pups were handled for 1 min once a day during the first 10 days of life). At 75-80 days, the males were anesthetized and the brains were processed for immunohistochemistry. An anti-tyrosine hydroxylase antibody and the avidin-biotin-peroxidase method were used. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted bilaterally in the arcuate, paraventricular and periventricular nuclei of the hypothalamus in 30-æm sections at 120-æm intervals. Neonatal handling did not change the number of TH-IR neurons in the arcuate (1021 + or - 206, N = 6; 1020 + or - 150, N = 6; nonhandled and handled, respectively), paraventricular (584 + or - 85, N = 8; 682 + or - 62, N = 9) or periventricular (743 + or - 118, N = 7; 990 + or - 158, N = 7) nuclei of the hypothalamus. The absence of an effect on the number of dopaminergic cells in the hypothalamus indicates that the reduction in the amount of neurons induced by neonatal handling, as shown by other studies, is not a general phenomenon in the brain

Perspectivas de la terapia génica en la enfermedad de Parkinson / Perspectives of the genic therapy in the Parkinson's disease

En la enfermedad de Parkinson idiopática, así como en algunos otros parkinsonismos, la actual estrategia terapéutica sigue basándose en el uso de fármacos y otros procedimientos destinados a "normalizar" la neurotransmisión dopaminérgica central. Cuando un enfermo parkinsoniano debuta con su sintomatología clínica, ya ha perdido aproximadamente un 80 por ciento de las neuronas dopaminérgicas nigrales, lo cual se manifiesta en una reducción de aproximadamente dos órdenes de magnitud de las concentraciones estriatales de dopamina

Mazindol: efeitos anorético e neurotóxico em ratos jovens e idosos / Mazindol: anoretic and neurotoxic effects in young old rats

Os efeitos da administraçäo crônica do mazindol (5 e 10 mg/kg i.p. e 60 mg/kg s.c) sobre a evoluçäo ponderal e a atividade da tirosina hidroxilase cerebral foram determinados em ratos machos jovens e idosos. O potencial anorético da droga foi observado através da avaliaçäo comparativa do peso corporal dos animais. A atividade da tirosina hidroxilase no estriato foi utilizada como parâmetro indireto da possível neurotoxicidade do mazindol. Nos ratos jovens notou-se uma perda significativa de peso aos 15 dias de tratamento, com recuperaçäo ascendente deste ao longo do tempo. Nos ratos idosos houve também perda de peso significativa; no entanto, tal recuperaçäo näo foi observada. Näo foram detectadas alteraçöes significativas da atividade enzimática em relaçäo aos grupos controles para ambas as idades. Estes resultados säo analisados a partir de dados encontrados na literatura

Morphology and distribution of dopaminergic neurons in the ground squirrel retina

Tyrosine hydroxylase (TH), the rate limiting enzyme in the conversion of tyrosine to DOPA, is a reliable marker for catecholaminergic (dopaminergic) neurons. To investigate the distribution of dopamine in the retina of the thirteen-lined ground squirrel (Spermophilus tridecemlineatus), retinal sections and wholemounts were incubated with an antiserum directed against TH and then processed using the avidin-biotin immunohistochemical method. TH-like immunoreactivity was exhibited by amacrine and interplexiform-like cells in the innermost portion of the inner nuclear layer (INL) and by cells we presume to be displaced amacrines in the ganglion cell layer (GCL). Their somata were 12 to 20 microns in diameter, with the majority measuring approximately 18 microns. In transverse sections the processes of the three types of neurons were seen to extend into lamina 1 of the inner plexiform layer (IPL). In horizontal sections 2-3 primary dendrites were seen to ramify and the branches extended for considerable distances, with overlap between the dendritic fields of neighboring TH cells. A distance to the nearest neighbor analysis suggests the TH-neurons in the INL are distributed in a non-random fashion

Influence of 6-hydroxydopamine on ethanol narcosis in mice

Se estudian los efectos de la administración de 6-hidroxidopamina (6-OHDA0, 100 Y 200 microng i.c.v., en la duración de la narcosis por etanol en ratones sin o con tratamiento de alfa-metil-p-tirosina (AMPT), p-cloro-fenilalanina (PCPA) o 5-hidroxitriptófano (5-HTP). La narcosis por etanol fue significativamente más prolongada en los ratones que recibieron 200 microng de 6-OHDA i.c.v. que en los testigos. La duración de la narcosis en los ratones tratados con ambas dosis de 6-OHDA fue significativamente más larga cuando éstos recibieron previamente AMPT (inhibidor de la biosíntesis de seotonina). En 5-HTP (precursor de la biosíntesis de serotonina). En cambio, en los pretratados con PCPA (inhibidor de la triptófano hidroxilasa), la 6-OHDA no modificó la duración de la narcosis por etanol. Las alcoholemias al momento de despertar no cambiaron significativamente por la 6-OHDA. Estos resultados son consistentes con la hipótesis de que la disminución de noradrenalina así como el aumento de serotonina cerebrales prolongan la narcosis por etanol y, en cambio, el aumento de noradrenalina o la disminución de serotonina en el cerebro reducen la narcosis por etanol

Involvement of a serotonergic control in the regulation of plasma levels of immunoreactive beta-endorphin

Se midió la concentración de beta-endorfina inmunoreactiva (B-E i.r.) en plasma e hipotálamo de ratas tratadas con alfa-metil-p-tirosina ( alfa -MT) (dos dosis de 200 mg/kg) o p-cloro-fenil-alanina (PCPA) (dos dosis de 150 mg/kg). Se determinó además la concentración de B-E i.r. en plasma después de una única dosis (5 mg/kg) de anfetamina o luego de la estimulación eléctrica del núcleo mediano del rafe (NMR). Los niveles plasmáticos de B-E i.r. disminuyeron por la administración de PCPA o aumentaron por la estimulación eléctrica del NMR. Tanto la adminsitración de alfa -MT como la de anfetamina fue inefectiva en modificar la concentración plasmática de B-E i.r. Los resultados sugieren un papel para la serotonina en la regulación del nivel plasmático de B-E i.r.