A morphometric and molecular study of the apoptosis observed on tadpoles' tail explants under the exposition of triiodothyronine in different homeopathic dilutions.

BACKGROUND: As a therapeutic system, homeopathy is supported by: i) similitude and experimentation in healthy individuals, ii) potentization. A challenge for researchers consists in looking for signals in water (or vehicle) to explain the storage of information in extremely high dilutions and the transfer of such information to the living systems. Anuran amphibian metamorphosis is controlled by thyroid hormones (TH), including the resorption of the tadpole tail. Apoptosis is a genetically regulated form of cell death that can be triggered by various extracellular and intracellular stimuli resulting in coordinated activation of a family of cysteine proteases called caspases. METHODS: This study was blind and randomized. It performed in three stages: I) the identification of the most effective T3 homeopathic dilution to induce apoptotic reactions in Rana (Lithobates) catesbeianus tadpole tail explants stimulated by T3 in substantial, II) study of different controls and III) detection in explants under the action of the most effective dilution of T3, as established in Stage I. RESULTS: There was no statistically significant difference between tail macroscopic dimensions between the groups. T3 10cH decreased the expression of caspase 3/7 mRNA, in explants treated with T3 20 nM. CONCLUSION: The present experiment is in agreement with the hypothesis that T3, at a 10cH homeopathic dilution, changes the metamorphosis molecular network.

Ultra high dilution of triiodothyronine modifies cellular apoptosis in Rana catesbeiana tadpole tail in vitro.

BACKGROUND: Ultra High Dilutions (UHD) are diluted beyond the Avogadro limit with dynamization (dilution with succussion). The process of anuran amphibian metamorphosis is controlled by thyroid hormones, including the resorption of the tadpole tail. METHODS: A randomized and blinded study was performed to investigate the influence of triiodothyronine (T3) 5·10(-24)M (10cH) on apoptosis induced by T3 100 nM in Rana catesbeiana tadpoles' tail tips, in vitro. Explants were randomized to three groups: control: no T3 in pharmacological or UHD dose; test: T3 100 nM and challenged with T3 10cH (UHD); positive control: T3 100 nM, treated with unsuccussed ethanol. The apoptotic index and the area of explants of test and control groups at the first and final day of the experiment were compared by t-test. RESULTS: There was no difference in tail tip area between test and control groups, but a significantly higher (p<0.01) index of apoptosis in explants of the test group. CONCLUSION: This data suggest that T3 10cH modifies the effect of T3 at pharmacological dose, opening new perspectives for further studies and investigation of the dose-effect curve.

Luffa operculata effects on the epithelium of frog palate: histological features.

UNLABELLED: Luffa operculata is the botanical name of buchinha-do-norte or cabacinha, which is a medicinal plant widely used for the treatment of rhinitis and rhinosinusitis. In Europe and USA, it is available in homeopathic medicines. In Brazil, Luffa operculata dry fruit infusion is inhaled or instilled into the nose releasing profuse mucous secretion, thus relieving nasal symptoms. Nevertheless, this often may cause irritation, epistaxis or anosmia. STUDY DESIGN: Experimental. MATERIAL AND METHOD: The effects of Luffa operculata were evaluated in different concentration infusions, in isolated frog palate preparation, testing 46 palates after immersion. Four groups (n = 10) were tested with the infusion prepared with frog Ringer (isotonic): control; 60 mg/L; 600 mg/L; and 1200 mg/L. An additional group was tested using the infusion with water (600 mg/L H2O, n = 6). Epithelial samples were harvested to be studied under light microscopy and electron transmission microscopy. RESULTS: In treated palates, light microscopy findings were dose-dependent standard toxic changes. Electron transmission microscopy showed enlargement of intercellular spaces and tight junctions disruption, pointing to ion-fluid transport abnormalities. CONCLUSIONS: Luffa operculata infusion in currently used doses can promote significant structural and ultrastructural changes in the epithelium of this ex vivo model of respiratory mucosa.

Antagonists discriminate muscarinic excitation and inhibition in sympathetic ganglion.

The effect of muscarinic antagonists was studied on the muscarinic slow IPSP (inhibitory postsynaptic potential) and slow EPSP (excitatory postsynaptic potential) in bullfrog sympathetic ganglia using the sucrose-gap recording method. Pirenzepine, alcuronium and atropine reduced slow IPSP amplitude more than slow EPSP amplitude. The most selective antagonists studied were pancuronium and gallamine which blocked or substantially reduced the slow IPSP without significantly affecting slow EPSP amplitude. The results suggest that the muscarinic inhibitory response may involve a different muscarinic receptor subtype, and/or receptor-ion-channel complex, than the muscarinic excitatory response.

Luffa operculata affects mucociliary function of the isolated frog palate.

BACKGROUND: Luffa operculata is a medicinal plant used in homeopathic and alternative medicine. In the United States, it is sold in a purified spray form, whereas a homemade L. operculata dry fruit infusion (DFI) is commonly used in Latin America. The L. operculata DFI is applied intranasally, inducing profuse mucous secretion and relieving nasal symptoms. Nevertheless, this medication may cause irritation of the nasal mucosa, as well as epistaxis or anosmia. Given the growing popularity of alternative medicine, a decision was made to evaluate the effects of this substance on mucous membranes. METHODS: The effects of L. operculata DFI on mucociliary transport velocity, ciliary beat frequency, and transepithelial potential difference (PD) were evaluated in an isolated frog palate preparation. We tested 46 palates immediately before immersion and again at 5 and 20 minutes after immersion. Four groups (n = 10) were tested in frog Ringer: control; L. operculata DFI, 60 mg/L; 600 mg/L; and 1200 mg/L. An additional group was tested using L. operculata DFI prepared with water (600 mg/L of H2O, n = 6). Epithelial samples were harvested for ultrastructural study. RESULTS: In treated palates, mucociliary transport velocity and ciliary beat frequency decreased significantly (p < 0.001 and p < 0.008, respectively). There was a dose-dependent decrease in PD modulus (p < .007). Our PD findings indicated ion-fluid transport abnormalities, which were confirmed by transmission electron microscopy that showed enlargement of interepithelial spaces. CONCLUSION: In this ex vivo model, the L. operculata DFI infusion promoted significant changes in the mucociliary function of the epithelium, suggesting that it is potentially noxious to human nasal mucosa.

Effects of verapamil on indirect muscle twitch responses.

The effects on indirectly elicited muscle twitch amplitude associated with the calcium (slow) channel blocker, verapamil, with or without pancuronium were investigated using isolated bullfrog sciatic nerve-sartorius muscle preparations. Verapamil (2-8 mM) produced a dose-related depression of indirect muscle twitch height (P less than 0.05). Twitch response was depressed 11% below control by the lowest concentration employed and 86% by the highest concentration. Pancuronium (0.07 mM) depressed neuromuscular function 35% below control (P less than 0.05). The combination of 5 mM or 8 mM verapamil with 0.07 mM pancuronium caused significantly greater degrees of depression than either drug alone. Verapamil produced significant depression of twitch height in vitro in relatively high concentrations. The mechanism of action remains unknown. Verapamil possesses pharmacologic properties that may be unrelated to slow (calcium) channel inhibition. The reduction of muscle twitch height caused by verapamil alone (5 mM) could not be antagonized by neostigmine, calcium, or frequent washings.

Homeopathically prepared dilution of Rana catesbeiana thyroid glands modifies its rate of metamorphosis.

One strand of research on the scientific basis of homeopathy is based on inversion effects of dilutions and the biophysical properties of information transfer. A model developed by Endler, was the basis for the study of the influence of high-diluted solution (1:1026 part by weight) of thyroid glands on the rate of metamorphosis of the frog Rana catesbeiana from the no legged to four-legged stage. The glands were obtained from tadpoles and prepared according by (dilution and succussion). Similar pure hydroalcoholic solution (unsuccussed) was used as control. In order to identify significant differences in the frequencies of four-legged tadpoles, in homeopathic and control group, we used a chi-square goodness-of-fit test (P<0.01) and the cumulative risk for metamorphosis by Cox's Proportional Hazards model (P<0.05). The number of animals that reached the four-legged stage is generally smaller in the treated group, than in the hydroalcoholic control group. It was postulated that thyroid hormones transmitted information' specific to the molecules used to prepare the solution, even though the molarity was beyond Avogadro's number.

Inhibitory actions of vecuronium bromide on acetylcholine and glutamate responses at the frog and crayfish neuromuscular junction.

Effects of vecuronium bromide, an analog of pancuronium, on the cholinergic and glutamatergic neuromuscular junction were investigated. Vecuronium depressed the postsynaptic response of the frog end-plate at lower concentrations than 10(-6) g/ml without affecting the presynaptic events. Vecuronium decreased the amplitude of the double ACh potential, but the second potential was more markedly reduced than the first. In analogy with d-tubocurarine, this suggests that vecuronium may act in part as an open channel blocker at the frog end-plate. Vecuronium depressed both the glutamate response and the excitatory junctional potential at the crayfish neuromuscular junction, although high concentrations were required. The drug increased the decay rate of extracellularly recorded excitatory junctional potentials at the crayfish neuromuscular junction. The reduction of the crayfish synaptic response caused by vecuronium can be explained by the open channel blocking action at this junctional site. The problem that cholinergic antagonists possess a property of channel blocking at the other transmitter system was discussed.

Efeitos da Luffa operculata sobre o epitélio do palato de rã: aspectos histológicos / Luffa operculata effects on the epithelium of frog palate: histological features

Luffa operculata é o nome botânico da buchinha-do-norte ou cabacinha, uma planta medicinal usada popularmente no tratamento das rinites e rinossinusites. Na Europa e nos EUA, está em medicamentos homeopáticos. No Brasil, a infusão (chá) do fruto seco de Luffa operculata é utilizada para inalacão ou instilacão nasal, resultando em liberacão profusa de muco que alivia os sintomas nasossinusais, mas há relatos freqüentes de irritacão nasal, epistaxe e anosmia. FORMA DE ESTUDO: Experimental. MATERIAL E MÉTODO: Avaliamos os efeitos da infusão de Luffa operculata em diferentes concentracões, no modelo experimental do palato isolado de rã, examinando 46 palatos após imersão. Quatro grupos (n=10) foram testados com infusão feita em Ringer-rã (solucão isotônica): controle; 60mg/l; 600mg/l e 1200mg/l. Um grupo foi testado em água (600mg/l H2O, n=6). Coletamos amostras do epitélio para estudo histológico à microscopia-de-luz e microscopia eletrônica de transmissão. RESULTADOS: Nos palatos tratados, os achados à microscopia-de-luz mostram lesões epiteliais de padrão tóxico, dose-dependentes. Na microscopia eletrônica, aumento dos espacos intercelulares e ruptura de tight junctions apontam para anormalidade no transporte iônico e de fluidos. CONCLUSÕES: A infusão de Luffa operculata, nas concentracões utilizadas popularmente, promove alteracões significantes na estrutura e ultraestrutura epitelial deste modelo ex vivo de mucosa respiratória.

Ultradiluição homeopática de triiodotironina altera a apoptose celular da cauda de girinos de Rana catesbeiana: in vitro / Ultra high dilution of triiodothyronine modifies the cellular apoptosis of Rana catesbeiana tadpole tail: in vitro

Ultradiluição (UHD) é o efeito de uma solução, diluída acima do número de Avogrado, que na dependência da sua dinamização (diluição com sucussão) induz um efeito celular supressivo ou estimulante, com conseqüente obtenção de uma curva dose-efeito oscilatória. Por outro lado, a 3,3,5 Triiodo-L-Tironina (T3) é o hormônio mais importante na indução e manutenção das mudanças metamórficas dos girinos, nelas incluídas a absorção da cauda. O presente estudo, cego e randomizado, tem como objetivo comprovar que o T3 5.10-24M (10ª cH) altera a apoptose induzida pelo T3 100 nM na cauda de girinos de Rana catesbeiana, in vitro. Foram distribuídos 60 explantes em três grupos: Grupo A: sem o estímulo do T3 em dose farmacológica e em UHD; Grupo B (teste): sob a ação de T3 100 nM e T3 10ª cH (5.10-24 M); Grupo C (controle): sob a ação do T3 100 nM e etanol 70% sem sucussão. A análise estatística da área dos explantes, no primeiro e ultimo dia do experimento, e do índice apoptótico foi realizado através do teste t Student e foi considerado estatisticamente significante quando p<0,05. Embora sem diferenças significativas na área dos explantes do grupo teste e no grupo controle, um maior e significante índice apoptótico foi identificado nos explantes do grupo teste. Este resultado confirma que o T3 na 10ª cH altera a ação do T3 em dose farmacológica. Futuros experimentos serão realizados, com diferentes dinamizações, com o objetivo da parametrização da curva dose-efeito.

Ultra High Dilution (UHD) is the effect of a solution, beyond the Avogadro limits, that in the dependence of the applied dinamization (dilution with succussion) elicits a suppressive or a stimulant effect on a living cell, with a consequent generation of an oscillatory dose-effect curve. The entire process of anuran amphibian metamorphosis is under thyroid hormones control, included the complete resorption of the tadpole tail. A random and blind study was performed, with the intent to prove that T3 5.10-24 M (10ª cH) modifies the apoptosis induction of T3 100 nM in Rana catesbeiana tadpoles tail tips, in vitro. 60 Explants were distributed in three ways: Group A: without T3 action, at pharmacological and UHD dose; Group B (test): under the action of T3 100 nM and treated with T3 10ª cH (UHD); Group C (control): under the action of T3 100 nM and treated with ethanol 70% unsuccussed. In order to identify significant differences in the area of the remainder explants, at the first and final day of the experiment, and in the apoptotic index we used a student t-test. Although we didnt find statistical difference in macroscopic tadpoles tail tips area from test and control groups, a high and significant (p<0,05) index of apoptosis in histology was found in explants of test group. This data confirms that T3 10 cH modifies the effect of T3 at pharmacological dose. More studies will be necessary, using different dinamizations, to the parameterization of the dose-effect curve proceeding from these experiments.