Relatively few pre-clinical studies have been carried out in toxicological research in order to verify the effects of homeopathic remedies under standardized conditions and their results are not incontrovertible [2–17, 19, 21].
The present study was designed to explore the possibility to test in a controlled way the effects of homeopathic remedies on two known experimental models of acute inflammation in the rat. To this aim, the study considered six different remedies indicated by homeopathic practice for this type of symptom, in two experimental edema models (carrageenan- and autologous blood-induced edema), using two treatment administration routes (sub-plantar injection and oral administration).
On the overall the study involved more than 700 rats in one of the largest pre-clinical study of the effects of homeopathic remedies ever performed.
In the first series of experiments (phase A) some statistically significant effects of homeopathic remedies were observed in two experimental conditions: oral administration in carrageenan-induced edema and sub plantar administration in blood-induced edema (reduction in paw volume increase up to 28% and 21% compared to the saline control, respectively). These effects were more evident and statistically significant in the initial and/or final phases of inflammation, when it is less marked and probably easier to control. The most relevant results concerned Apis, Lachesis and Phosporus in the oral treatment of carrageenan-induced edema (with a range of edema reduction from 11% to 28%). The anti-inflammatory effects of the homeopathic remedies were approximately 50% less than those of the reference drug indomethacin.
When retested in phase B, where a double-blinding procedure and coding of remedies was performed, the three tested homeopathic remedies (at different dilutions) did not show any anti-inflammatory effect. The lack of reproducibility of the results in the second experiment may be explained by the different experimental protocol used. In particular, two experimental conditions were modified in the second phase: the generation of allocation sequences to the treatments and the blinding of these sequences. Moreover, the blinding process could have altered the storage of homeopathic remedies (glass in A, plastic in B), and this was an additional source of difference in the protocols between experiments A and B.
Indeed, in the first experiment animals were randomly allocated to cages and all animals hosted in the same cage received the same treatment. Although it is unlikely that a "cage-effect" has occurred during the first experiment, in the second one, animals were randomly distributed in different cages and in each cage animals received the different treatments at random. It is conceivable, at least in theory, that some unknown "cross-effect" among animals in the same cage, treated with different remedies, could have taken place, thus reducing the (already low) net difference between verum and placebo. It is important to underline that the activity of indomethacin was reproduced in all phases of the experiment, suggesting that if some cross-contamination occurred, this would have affected only saline-treated and homeopathy-treated rats reducing all responses and decreasing a possible (small) effect of homeopathic drugs.
Experiment B differs from A also by inclusion of higher potencies (D30), which were hypothesized to "radiate" through sealed ampoules . This elusive effect could leave open the possibility of some cross-contamination during some stages of the experimental process that we have not considered in the protocol.
Furthermore, it is important to consider the possibility of an (unconscious) effect of the researchers, due to the absence of blinding of treatment allocation in the first phase. This may be the case if the induction of edema, the administration of treatments or the collection of the response variable were performed in different ways for animals treated with verum, with placebo or with the active reference drug. This confounding effect is rarely controlled in conventional animal research (thus constituting a possible weak point of this discipline), while it is appropriately taken into account almost always in clinical research. However, even if it is difficult to conceive how this confounding effect could have acted in our experimentation, it cannot be totally excluded [23, 24]. The fact that the effects of the homeopathic dilutions studied in pre-clinical tests carried out by different research groups, when observed, are often small and difficult to reproduce , emphasizes the relevance of the experimental conditions in which significant effects can be observed.
The experimental models we used to evaluate anti-inflammatory treatments have explored what is conventionally denoted as pharmacological "activity" on one symptom (e.g. foot swelling). While conventional anti-inflammatory drugs are designed to suppress the underlying enzymatic mechanism of inflammation (e.g. prostaglandin production), homeopathic treatment is supposed to regulate the pathological excess of inflammation because the phenomenon by itself is seen as an expression of natural healing dynamics (the so called Hahnemann's "life force"). According to classical homeopathic theory, an "anti-edema" effect could not reflect the full potential of the homeopathic treatments of inflammatory diseases. On the other hand, other experimental approaches and/or different formulations showed consistent anti-inflammatory effects of homeopathic remedies such as Arnica compositum [2, 3], Apis  and Arnica Montana  using the rat-paw edema model. So, other technical factors, such as the composition of the medicines (e.g. single remedies versus complex formulations), and the type of solvent used (water, saline, water/alcohol mixtures) , may explain the observed discrepancies. If a homeopathic treatment acts by influencing the natural healing dynamics of the whole treated subject by means of small doses or highly diluted administrations, this action could be, at least in theory, highly sensitive to even small changes in experimental conditions . Moreover, when used in humans, a homeopathic treatment is also chosen on the basis of the global pathophysiological characteristics of the individual, and not only in relation to local symptoms.