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.

Amphibians and ultra high diluted thyroxine--further experiments and re-analysis of data.

BACKGROUND: A model of thyroxine and metamorphosis of highland amphibians is frequently mentioned as an example of experiments on extremely diluted substances in discussions around 'homeopathy'. METHODS: The model was scrutinized by reanalysing the results of the initial researcher A and a second researcher B as well as of 5 external researchers C between 1990 and 2013. Rana temporaria larvae were taken from an alpine highland biotope. The test solution was thyroxine 10(-30) (T30x), tetra-iodo-thyronine sodium pentahydrate diluted with pure water in 26 steps of 1:10, being agitated after each step. Analogously prepared water (W30x) was used for control. Tadpoles were observed from the 2-legged to the 4-legged stage. Experiments were performed in different years, at different times of season, and their duration could vary. Frequencies of 4-legged animals, effect sizes and areas under the curves (AUCs) were calculated and regression analyses were performed to investigate possible correlations between year, season, duration etc. Experiments were in line with animal protection guidelines. RESULTS: The total set of data A + B + C as well as subsets A (initial researcher, N=286+293), B (second centre, 965 + 965) and C (5 external researchers, 690 + 690) showed an effect of extremely diluted agitated thyroxine reverse to that known of molecular thyroxin, i.e. test values were below control by 11.4% for A, 9.5% for B and 7.0% for C (p<0.001 for each of the subsets). The effect size (Cohen's d) was >0.8 (large) for both A and B and 0.74 (medium) for C. CONCLUSION: Although a perfect reproducibility was not obtained, this paradoxical phenomenon was generally consistent in different observations. Correlations were found between details of laboratory handling, as well as environment temperature, and the size of the results.

Replication of an experiment on extremely diluted thyroxine and highland amphibians.

INTRODUCTION: The purpose of this study was to reproduce an experiment with diluted thyroxine and amphibians. A detailed account of the difficulties of this line of research has been published by the initial team (Endler and Scherer-Pongratz). One experiment which has been reported reproducible by the initial team and independent researchers is the effect of extremely diluted agitated thyroxine (T30x) versus analogously prepared water (W30x) in amphibians from biotopes above the tree line (highland amphibians). METHODS: (A) The author replicated the experiment. Rana temporaria were taken from an alpine biotope and the methods given in the original protocols were followed. Animals were treated from the 2-legged stage on. (B), the author reanalyzed the results reported by the initial team and by independent researchers (van Wijk, Lassnig, Zausner-Lukitsch, Bach, Harrer). RESULTS: (A) In the author's own experiment, there was a clear trend of T30x animals developing more slowly (i.e. up to 6 h within 3 days) than W30x animals. This is in line with the previous experiments. Due to small numbers of animals, the differences in the frequency of larvae reaching the 4-legged stage and the stage with reduced tail were not statistically significant (p > 0.05). The effect size was large (d > 0.08). (B) In the analysis of all available data with regard to the 4-legged stage, pooled T30x values from the initial team were 10.1% smaller than W30x values (100%) and pooled T30x values from the 5 independent researchers were 12.4% smaller (p < 0.01 and d > 0.08). Analogously, the number of animals entering the juvenile stage with reduced tail was smaller for T30x than for W30x.

Treatment of highland frogs from the two-legged stage with homeopathically prepared thyroxin (10-11 - 10-21).

The influence of moderately diluted, agitated, i.e., homeopathically prepared, thyroxin solutions (10-11 - 10-21, final concentration in the basin water 0.6 10-15 - 0.6 10-25 parts by weight after the first application) on metamorphosis in highland Rana temporaria from the two-legged stage was studied. In accordance with the homeopathic idea of effects of specially prepared dilutions being inverse to those of their mother substances, animals were treated either with thyroxin 10-11 - 10-21 or analogously prepared blank solution (water). Development was monitored by documenting the number of animals that had entered the four-legged stage. It has been found that animals treated with the thyroxin solutions metamorphosed more slowly than the control animals, i.e., the effect of the homeopathically prepared thyroxin was opposed to the usual effect of molecular thyroxin. The number of test animals that reached the four-legged stage at defined points in time was smaller (2-13.5%) in the group treated with homeopathically prepared thyroxin at the points in time, compared to control. The results in this study sustain the previous multiresearcher findings that show that diluted homeopathically prepared thyroxin is able to slow down metamorphosis of R. temporaria.

The effect of homeopathically prepared thyroxine on highland frogs: influence of electromagnetic fields.

BACKGROUND: Previous experiments show that amphibian larvae are responsive to homeopathically prepared thyroxine. METHODS: We studied the effect of a highly diluted and agitated thyroxine solution exposed to various electromagnetic fields on metamorphosis in highland Rana temporaria. The devices tested were: microwave oven, mobile phone, airport X-ray, and a red light barcode scanner. Animals were treated either with homeopathically prepared thyroxine (10(-30) parts by weight, 10(-35) in the water in which the animals were kept), or analogously prepared blank solution, or analogously prepared thyroxine exposed to the electromagnetic field of one of the devices tested. Solutions were administered at 48h intervals according to a standardized protocol. RESULTS: Animals treated with the standard test solution thyroxine 10(-30) metamorphosed more slowly than the control animals, ie the effect of the homeopathically prepared thyroxine was opposed to the usual physiological effect of molecular thyroxine. The cumulative number of test animals that had reached the four-legged stage at defined points in time was smaller in the group treated with homeopathically prepared thyroxine at most of the points in time. This was found independently by all three research teams involved. In contrast, this effect did not occur when the thyroxine solution had been exposed to the field of the early model microwave oven, or mobile phone. There was no difference between aqueous or alcoholic solutions were used, and there was, if any, only a small protective effect from aluminum foil. Airport X-ray and red light barcode scanning did not diminish the effect of the homeopathic solution.

Pretreatment with thyroxine (10-(8) parts by weight) enhances a 'curative' effect of homeopathically prepared thyroxine (10-(13)) on lowland frogs.

We studied the influence of a moderate homeopathically prepared thyroxine dilution (final concentration in the basin water 10-(13) parts by weight) on the metamorphosis of lowland Rana temporaria which had been hyperstimulated with thyroxine. Two groups of animals were pretreated by immersing them in a molecular thyroxine dilution (10-(8) parts by weight). This pretreatment speeds up development, as is well known. In accordance with the homeopathic/isopathic idea of detoxication or cure, the same hormone was then diluted and agitated in successive steps for further treatment. This homeopathically prepared dilution was administered at 24-hour intervals to one of the groups. An analogously prepared blank solution was used for the control group. Our hypothesis, which was derived from earlier studies, was that animals treated with the test solution would metamorphose more slowly than the control animals, i.e. that the homeopathically prepared thyroxine would have a 'curative' effect. In this new series of experiments this hypothesis was examined by 3 independent researchers. In the experiments carried out by 2 of the 3 researchers the number of animals that reached the four-legged stage at defined points in time was smaller in the group treated with homeopathically prepared thyroxine. In the third laboratory no difference was found between the groups. However, the overall inhibiting effect was statistically significant and more pronounced than in earlier, less promising studies and in parallel experiments in which nonprestimulated animals had been used. Other studies carried out by the 3 researchers involved animals from highland biotopes, where the natural environment probably induces a greater sensitivity towards thyroxine or higher thyroxine levels. These animals reacted to the homeopathically prepared thyroxine with a slowing down of metamorphosis, even when they had not been prestimulated with a molecular dose of the hormone. This effect was observed in all 3 laboratories and is consistent with the results of previous studies.

Pretreatment with thyroxin 10(-8) and the effect of homeopathically prepared thyroxin 10(-30) on highland frogs--a multi-researcher study.

BACKGROUND: Studies performed in 5 laboratories have shown that homeopathically prepared highly diluted thyroxin (10(-30)=30x) slowed down metamorphosis of highland amphibians. Metamorphosis of lowland amphibians, however, could be slowed down by a low dilution of thyroxin (10(-8)=8x) if animals had been artificially pretreated with thyroxin. OBJECTIVE: To combine the advantages of using animals from highland biotopes and hyperstimulation prior to treatment. ANIMALS AND METHODS: Rana temporaria from an alpine biotope were pretreated in an aqueous molecular thyroxin dilution (10(-8) parts by weight, hyperstimulation). This is supposed to accelerate metamorphosis. In accordance with the homeopathic idea of detoxification or cure, one group of these hyperstimulated animals was then treated with thyroxin 30x, and another group with water 30x. Experiments were performed by 4 independent researchers. RESULTS: As a trend, the thyroxin-30x animals metamorphosed more slowly than the water-30x animals. The number of thyroxin-30x animals that reached the 4-legged stage at defined points in time was slightly smaller at some but not all points in time, compared to control. This is in line with previous findings and can be discussed as an interesting result. Contrary to our working hypothesis, however, differences were not bigger than in the previous experiments in which animals had not been pretreated with thyroxin 10(-8). CONCLUSION: This study supports previous findings but does not prove the assumption that pretreatment of highland animals with molecular thyroxine improves the original protocol.

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.