HEALING
CHRONIC WOUNDS PERFORMED
ON MOUSE EARS USING SILICA (SiO2)
AS A HOMEOPATHIC REMEDY
A
Pharmacological Study of Homeopathic High Dilutions
M. OBERBAUM, Z. WEISMAN, A. KALINKOVICH, Z. BENTWICH
Ruth Ben-Ari
Institute of Clinical Immunology, Kaplan Hospital, Rehovot;
The Hebrew University Hadassah Medical School,
Jerusalem, Israel
1 - Introduction
2 - Material and methods
3 - Results
4 - Discussion
5 - References
Homoeopathic medicine frequently uses potencies of silica,
especially as a treatment for abscesses, discharging fistulas, chronic ulcers,
discharging wounds and keloids and rejection of foreign bodies (Jouanny, 1985;
Metzger, 1981). It is also known as a deep-acting constitutional remedy. However,
one major advantage of experimenting with potencies of silica is the fact,
that it can be applied indicatively without the need to individualize. This
characteristic is not common in homeopathy, since individualization is one
of the basic requirements of homeopathy. Thus, the property of silica
for "anti-chronic inflammation" is exceptional and makes it possible
to prescribe it in most cases that suit this definition. It is also worth
nothing that unpotentized silica, has a use in conventional biomedical science,
as a substance with inhibitory and cytotoxic activity on macrophages (Moseley
et al., 1988; Gennari et al., 1987; Bowers et al., 1998; Souvannavong and Adams, 1990).
These are the reasons why we have decided to investigate the effect of various
dilutions of silica on wound healing. For this purpose we have developed a
model of a chronic wound in experimental animals that would simulate the situations
in which silica is used in homoeopathy and the results of the studies with
this model are hereby reported.
2.1. ANIMALS
AND WOUND PERFORMANCE
Female C57Bl/6 mice, 12-16 weeks old, were used in the
study. After an adaptation period of 2 weeks, the lobe of one ear of each
mouse was perforated with dental wire, weighing 110f,10 mg. The
ends of the wire were twisted together to form an "earring". The
constant friction of the wire along edges of the wound, resulted in a chronic
irritation. Without treatment, the wire caused a hole of variable size, which
was a function of the properties of the tissue, the weight of the hanging
wire and the detrimental effect of the constant irritation caused by the earring.
In some cases the wire "cut" with
time the ear, and fell off.
In the most cases the wound healed and
the hole closed around the wire, producing
granulation and later scar tissue.
The
animals were divided into three or four groups in experiments 1-9 and 10-13,
respectively, of ten animals per group (except for experiment no. 1, in which
there were four animals per group). The animals were treated with silica dilutions
of 5 C, 30 C and saline in experiments 1-9 and an additional dilution, namely,
200 C in experiments 10-13. The animals were given i.p. injections three times
a day in experiments 1-2, and oral administration, via drinking water, in
experiments 3-13.
2.2. PREPARATION OF DILUTIONS
The silica dilutions were prepared by Dolisos (France)
according to the official French Pharmacopoeia.
All the dilutions were centesimal dilutions (100-n). The first
two steps were performed by trituration of silicium oxide (SiO2) with lactose
powder at 1:100 relation (1 C and 2 C), followed by dissolving one part of
the triturated powder in 99 parts of 70% alcohol. This dilution (3 C) was
vigorously sucussed using a dynamizator. All the subsequent dilutions were
prepared using the same technique. In the last two dilutions, saline was used
as the diluent, instead of alcohol, these two dilutions were renewed once
a week, and kept in a refrigerator at 4° C. The control dilutions were prepared
in the same way, "diluting" alcohol in alcohol; the last two control
dilutions were done in saline.
2.3. METHOD OF EVALUATION
In experiments 1-3, a subjective evaluation of the hole
size was made, on a scale of 0 (closed hole, tight around the wire) to 4 (very
large hole). This was done by two independent observers. In the other experiments
we added also an objective evaluation of the hole size on the last day of
the experiment, using an image analysis system (Galai, Haifa, Israel). The
hole was photographed by an electronic camera and the data were transferred
to an IBM/AT computer, which calculated the hole size.The hole size of an
ear that was "cute" by the wire, was evaluated as if the border
of the hole were the edge of the ear.
3.1. PRELIMINARY
RESULTS
The preliminary experiment was performed on a small number
of animals per group, in order to find out whether there would be any reaction
when silica is used for a chronic wound. All the mice had been treated for
32 days with three i.p. injections of 0.5 ml of the dilutions, daily. On day
14, the earrings were hanged on the mouse ears.
On
the 5th day after the earrings were
inserted, a clear difference was observed in the hole size between the ears
of treated mice and those of the control group. This difference was maintained
until the end of the experiment (day 32). No sign of closure of the holes
around the wires in the control group was observed . In the treated group, there was a strong development
of granulation tissue, which closed the hole until it disappeared completely
(figure 1). An evaluation of the hole size, which was performed with the image
analyzer from the photograph of the holes, revealed a significant difference
between the groups (p < 0.01 in one-way ANOVA test) and between each of
the treated groups and the control group (p < 0.01 according to the Student
t- test).
Figure 1. Evaluation of hole size in wounded ears of mice receiving
intraperitoneal silica administration. Fourteen days after i.p. injection
of silica dilutions or saline were begun, one ear of each mouse was
perforated with dental wire, which was twisted to form an "earring."
Eighteen days after insertion of the earrings, the ears with holes
were photographed, and the hole size in the photograph was evaluated using
an image analysis system.
3.2. KINETICS OF THE THERAPEUTIC EFFECT
Encouraged by the dramatic effects reached in the preliminary
study, we attempted to evaluate the kinetics of the therapeutic effect. A
daily evaluation of the hole size was made, starting from the 3rd day after
the start of the experiment, until day 20. Contrary to the first experiment,
the dilutions were not injected but were added to the drinking water. In this
way, we reduced stress of holding down the animals and injecting them, three
times daily. The figure 2 shows the difference in hole size between the treated
groups and the control group which progressively increased, reaching its maximum
on day 9 (p < 0.05 in Kruskal-Wallis
one way analysis of variance and in Pearson chi square test). The figure also
shows a difference in hole size between the mice treated with the lowest dilution
(5 C) and those treated with the higher dilution (30 C). The healing process
was stronger at the high dilutions. After day 10, there was no difference
between the groups, and the hole size in the control group was equivalent
to that in the treated group. Contrary to the first experiment, the beneficial
therapeutic effect did not last until the end of the experiment.
Figure 2. Evaluation of hole sizes in wounded ears of mice receiving
oral silica administration. Dental wire was inserted into the ears of mice
as described in Figure 1. In parallel, either silica dilutions or saline was
added to the drinking water throughout the experiment. The hole size was subjectively
evaluated daily, beginning from the third day and continuing until the twentieth
day of the experiment.
3.3. THE INFLUENCE OF THE "EARRINGS" ON THE
THERAPEUTIC EFFECT
The question, whether there is really a need for the
earring, or, in other words, if there is a need for a chronic irritation in
order to obtain these results was examined in another experiment in which
we perforated an additional hole, in the second ear. The holes were made with
an instrument that made holes of a constant size. In these ears, no earrings
were inserted. In the attempt to improve the evaluation of the hole size,
we added a scanning with an image analysis system in order to measure the
hole size.
Table
1 represents a comparison of the subjective evaluation with the objective
measurement of the hole sizes, and shows a good correlation between the two.
In this experiment also, which was terminated after 5 days, a significant
effect of the silica dilutions on wound healing in the ears with earrings
was observed. The higher dilutions had a significantly stronger effect on
wound healing than did the low dilutions (p < 0.05 in one way ANOVA) (Figure
3).
TABLE 1. Comparison between objective and subjective evaluations
of hole sizes in the ears of treated and untreated mice
____________________________________________________________________
Treatment Group
Method of Evaluation
____________________________________________________________________
Subjective* Image Analyzer**
_________________________________________
Saline
1.59 37269
SiO2 10-10 0.5 23644
SiO2 10-60 0.4 16469
_____________________________________________
* Mean of the subjective evaluation using a scale of
0 = a hole closed tight around the wire, to 4 = a very large hole.
** Mean hole size (square microns)
Figure 3. Evaluation of hole size, with and without dental
wire, in wounded ears of mice. The protocol was the same as that in Figure 2, and in addition, a hole was made
in the other ear without inserting wire earrings. The experiment continued
for 5 days, and an objective evaluation of the results was made using an image
analysis system. Figure 3 shows the hole size in ears with dental wire earrings.
On
the perforated ear without the earring, there was no significant difference in hole size between the treated and the
control group (Figure 4).
Figure 4. See legend to Figure 3 for protocol. Hole size in ears
without dental wire earrings: no significant difference was seen between the
treated and untreated groups.
3.4. THE THERAPEUTIC EFFECT OF VERY HIGH DILUTIONS
In order to determine whether the observation that the
higher the dilution is, the stronger are its therapeutic properties is consistent,
we added another dilution to the experiment, namely a 200 C. There was a similar
finding as in the previous experiments, with a stronger effect of the 200
C dilution on the animals treated with it (on day 7 of the experiment): the
average area of the holes in the ears of the mice treated with saline was
72799 square microns whereas the average area of the holes in the mice treated
with Silica 5 C, 30 C and 200 C was 56195 square microns, 31484 square microns
and 28222 square microns, respectively (Figure 5), the difference between
hole areas being significant (p < 0.001 in one-way ANOVA).
3.5. SUMMARY OF REPETITIONS OF THE EXPERIMENTS
In three of the remaining nine experiments, a clear therapeutic
effect of silica dilutions was observed while in the six others, no significant
therapeutic effect could be shown; in
three of the six the reason was most probably technical: In the first, we
tried to hang silver rings in order to obtain a stronger inflammatory effect;
the silver caused a very strong irritation and large holes which did not show
a tendency to close. In the second experiment, we used very thin dental wire,
its weight was not heavy enough to cause sufficient chronic irritation, the
wounds remained too small and closed quickly and tightly around the wire.
In the third experiment we hung earrings on both ears of each animal in order
to see if there was a difference in hole size between ears; there was no significant
difference between groups or between the two ears of an individual. In the
remaining three experiments, the holes did not develop and closed immediately
around the wire; the reason for this was not clear.
Figure 5. The protocol was similar to the previous experiment except
that a group treated with a higher dilution of silica (10-400) was added.
The experiment continued for 7 days.
The present study represents the results of almost 2
years of work. It could be shown, that the healing of chronic irritated wounds,
caused by hanging a metal earring from mouse ears, could be enhanced, by treatement
with homoeopathic dilutions of silica (SiO2). This phenomenon was repeated
several times, checked carefully, and was statistically highly significant.
The process of wound healing reached its peak after a period of 3-10 days.
Subsequently there was an evening
up between the treated groups and the control group ( experiment b, Figure
2). In that experiment it seems that the wound healing proceeded in two phases
(Figure 2): in the first phase it led, most probably due to the chronic irritation
and the weight of the earrings, to an enlargement of the holes (the peak in
Figure 2 occurred at days 7-9). At the end of that phase the reparative process
started, in which the wound started to heal and its size diminished. This
phase reached its peak in all groups at the same time (day 10 in Figure 2).
According to this observation it seems that the silica was active mainly in
the first phase, in halting or slowing down the process of wound enlargement,
and less in diminishing the wound size.
It
is difficult to assume that these results are due to wrong evaluation methods.
In the first two experiments we evaluated the hole size subjectively. In the
third experiment we added an objective method and from the fourth experiment
on, we used only this objective method, namely evaluation with an image analysis
system. It should be noted that the results of this method, which are free
of an observer bias proved to be similar to the results of the subjective
evaluation (Table 1). It is also unlikely that the effects that were shown
are due to "mistakes" in the preparation of the dilutions, since
they could be shown in almost all the groups that were treated, as compared
with the control groups. These effects were
consistent (Figure 2). The mice in
the control group had been treated in the same way, kept in similar cages
and under the same conditions as the treated animals, and thus these factors
could not be the reasons for the significant differences between the groups.
The
argument that the "stress condition" of the injected animals could
be the reason for the difference between the groups, can hardly be supported
since there could not be any reason why the animals that were treated with
the active dilution should react differently to the "stress", from the mice that were treated with the control
dilution. The argument that there was a "difference" in the way
the injections were given between the groups, is also very unlikely since
in most experiments there was a clear advantage to the groups that were treated
with the active dilutions over the control groups, and also when these were
added to the drinking water.
Additional
support to the idea that the active dilutions of silica may have a curative
effect on healing of chronic wounds, can be obtained from the experiment that
showed a therapeutic effect only in the ears that had earrings hung on them.
There was no difference in the hole size of the ears that were just "perforated"
and did not have earrings hung from them, between the treated groups and the
control groups (experiment c, Figures 3 and 4).
Another
possibility that was raised was that some molecules of SiO2 remained in the
solution due to adhesion to the tube walls. This idea has to be rejected,
since in any dilution just 1 % of the previous dilution was used and besides,
it is hard to ignore the fact that the higher the dilution was, the stronger
the therapeutic effect was (10-400 > 10-60 >10-10 ). This observation
resembles the effects of homoeopathic remedies in the homoeopathic medicine
in which the higher the dilution is, the deeper, stronger and more long-lasting
the remedy acts.
There
is however a difficulty explaining the "failures" of some experiments:
six experiments out of 13 failed. In three of them, the reason was
technical: different wire (too thin, silver wires), and an experiment
in which we hanged a wire on both ears. In the three other experiments which
failed, the reasons for the failure are not clear. It is important to note
that the significance of the successful experiments was more than enough to
draw the conclusion that the effect was more than a random result. Moreover:
in none of the experiments was there a "therapeutic effect" of the
control dilution. The issue of the reproducibility
of homeopathic studies is certainly one of the big difficulties in
homoeopathic research.
An
interesting aspect of the study is the "conventional" aspects of
the influence of SiO2 on biological systems. As mentioned at the beginning
of this paper, there are many scientific
publications showing the influence of SiO2 on macrophages, and through them,
on the immunological reaction (Moseley et
al., 1988; Gennari et al., 1987;
Bowers et al., 1998; Souvannavong
and Adams, 1990). There are also reports on curative effects of SiO2 on experimental induced autoimmune diseases
such as myasthenia gravis and diabetes mellitus (Kinoshita et al., 1988; Charlton et al., 1988). As far as we know, there
has been only one controlled study examining the influence of very low doses
of SiO2 on an animal model (Davenas et
al., 1987). In that study it was shown that application of 10-19M SiO2
in the drinking water of C57BL/6 mice caused an increase in the activity of
the macrophages which were isolated from the treated animals, compared with
a control group. In that work, the dilutions still contained some molecules,
even though the concentrations used,
were several orders of magnitude lower than those used in the conventional
studies. In a part of our experiments, (experiments 10-13), we went even further
and used dilutions above the Avogadro number. To the best of our knowledge,
these dilutions do not contain any molecules of SiO2.
We
are aware of the problematic nature of the results described herein. Not only
do we claim that the lower the concentration, the stronger the reaction is,
but we found "dilutions" that do not contain anything that still
have a biological activity. We don't have any good explanation for this phenomenon.
There are today several attempts to explain the activity of such "dilutions"
(Bernard, 1965; Kumar and Jussal, 1979; Agnastostatos et al., 1991). Almost all of them claim
that the "drug information" is transferred to the diluent, and that
the diluent turns into the carrier of the "drug information". In
other words, if the drug/remedy is the "key" and the diluent is
the "lock", so the therapeutic
effect is attained with the "lock" and not the "key".
Be it as it may, the significance of the results we describe is in showing
that such effects do exist and that they cannot be accounted by placebo effect.
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