As little information about the effect of ultra high dilutions of glucocorticoid in reproduction is available in the literature, pregnant female Wistar rats (
In the attempt to characterize the high-dilution action as a biological phenomenon, several experimental studies have been performed in the last years using cell or animal models [
In face to this contemporaneous scenario, the aim of this study was to create an experimental model to check if the exposure of mothers to high dilutions of a biological active substance is able to produce changes in reproduction and hereditary features in the offspring.
In previous studies made by our group, the isopathy principle was checked in laboratory animals using dexamethasone as an experimental model. Dexamethasone was chosen because of its well-known pharmacological action, whose results could be easily identified. Also, as a corticoid hormone, it could be used in a wide variety of experimental models and results could be compared to each other [
In this study, the dexamethasone model was used because of the availability of its background data and because of its known action on reproduction [
Adult 60 days old female Wistar rats were supplied by the UNITOX-Royal Technology and Development Institute. One week before the beginning of the experiment, they were randomly organized in conventional polypropylene cages, receiving water and food
P (parents) generation females were randomly distributed in four groups of 12 animals each. During CR1: treated with dexamethasone 4 mg/kg diluted into dexamethasone 15 cH (MIX), CR2: treated with dexamethasone 4 mg/kg (DEX, positive control), CR3: treated with dexamethasone 15 cH (UHD, ultra-high dilution), CR4: vehicle or water (CONT, negative control).
Group codes were made by a person who was not directly related to the experimental manipulation and were maintained closed in a sealed cover up to the final statistics, after all procedures of cell counting.
Treatments were done three times a week, by subcutaneous injection [
(a) (b) (c)
The dilution 15 cH was chosen because, in clinical practice, it can be used to treat a wide range of symptoms, from those of behavioral nature up to those related to functional and structural features. In fact, in this protocol, the behavior development and the inflammation process of subcutaneous tissue were both analyzed and related in the F1 (first descendent) generation. Also, data about the effects of dexamethasone 15 cH in acute experimental inflammation were well described before [
The pure water (vehicle) was chosen as negative control because, as observed in a systematic review [
From the birth, the F1 generation was analyzed according the following variables: daily weight individual control, day of fur appearance, day of eyes opening, day of pinna detachment, day of first incisive tooth outbreak, day of testicles dehiscence in males, day of vagina opening in females, day of complete development of postural reflex.
The postural reflex was evaluated by the capacity of newborn to return immediately to the quadruped position after been placed in dorsal
After the weaning, the P generation females were euthanized by anesthesia overdose and submitted to necropsy.
After 60 days of life, 12 newborns (F1 generation), from different mothers of the same group, were randomized in two groups, UHD and CONT, and submitted to subcutaneous injection of 0.1 mL of 1%
The quantification of mast cells/basophiles was made by counting 200 cells per slide, from predetermined contiguous microscopic fields.Mast cells were classified as “degranulated” and “not degranulated” according to the presence or not of histamine granules outside the cell, despite the degree of cytoplasm emptiness. To avoid interpretative bias, two independent observers did this procedure in blind. Results were treated together in statistical analysis.
The immunohistochemical procedure had the aim to put in evidence the adhesion molecules expressed by leukocytes (CD18) and endothelium (CD54), as well as to differentiate mononuclear phagocytes to other mononuclear cells into the infiltrate, including complementary evidence of their different stages of maturation, by using the CD163 marker.
Slices of 3 to 5 microns were cut and adhered on commercial silane-treated slides. Then, slides were deparaffinized in both, xylol and absolute alcohol bath, and washed in current tap water. All slides were heated in a citrate buffer bath (SIGMA, pH = 6.0), during 20 minutes, in ebullition, for antigen unmasking. After that, slides were washed in PBS, pH = 7.2 (SIGMA), dried with a soft paper, and cuts were delineated with Pap Pen (AbCam).
The endogenous peroxidase blocking was done by incubating slides in 5% H2O2 1 : 4 in methanol PA (ISOFAR). Slides were washed in PBS (sigma) and incubated with normal horse serum (VECTOR) during 20 minutes, for blocking unspecific protein adsorption sites, then immediately incubated with the primary antibody (see Table
Immunohistochemistry used markers (adapted from KAWAKAMI, 2011) [
Marker | Cell | Source | Molecular target | Clone | Origin/target (species) | Dilution |
---|---|---|---|---|---|---|
Anti-CD54 | Activated endothelial cell | Serotec | ICAM 1 | 1A29 | Mouse-rat | 1 : 10 (5 |
Anti-CD18 | Leukocytes | Serotec | Beta-2 integrin | WT.3 | Mouse-rat | 1 : 10 (5 |
Anti-CD163 | Activated macrophages | Serotec | Surface glycoprotein (ED2) | ED2 | Mouse-rat | 1 : 10 (5 |
Anti-MAC 387 | Monocytes, macrophages, and polymorphonuclear | AbCAM | Intracytoplasmatic protein (calprotectin) | polyclonal | Rabbit-rat | 1 : 20 (50 |
In the next day, cuts were washed in PBS and incubated during 30 minutes with the secondary antibody conjugated to micropolymers coupled to peroxidase (VECTOR). Then, they were washed again and incubated with DAB (DAKO) for 3 seconds. After being washed in current tap water, all slides were stained with the Harris hematoxylin and mounted under a glass cover. Slides stained by hematoxylin-eosin method have done as reference to specific slides areas and to count polymorphonuclear cells (PMN), specifically neutrophils. In this case, the number of PMN cells per field was determined in 10 randomized fields, using 100x lens.
In the case of CD18, CD163, and MAC 387 markers, 10 randomized microscopic fields were observed per slide, using immersion objective (100x). The number of positive cells per field was recorded. For MAC 387, only mononuclear cells were considered. For CD18, counting procedures were done by two independent observers, in blind, considering both the total of positive cells and only the mononuclear positive cells. This proportion indicates the rate of mononuclear cell migration. For CD 163, different degrees of staining were considered, because the ED protein is expressed progressively, according to monocyte maturation into the inflammation site [
Photomicrographs of inflamed footpad of the offspring born to mothers treated with dexamethasone 15 cH. Animals were inoculated with 1% carrageenan into the footpad. Material stained by immunohistochemistry using polyclonal anti-CD163 (Serotec). (a) Negative mononuclear cells, (b) monocyte marked on the membrane, (c) monocyte marked on the cytoplasm, (d) macrophage marked on the cytoplasm. 100x objective.
The MANOVA test was used to compare the fourth initial groups, considering the basic toxicological parameters. The Student’s
The method first employed to evaluate results was the Bartlett test, in order to determinate the homogeneity of samples. Then, Student’s
Values of
The protocol was approved by the Research Bioethical Committee of UNIP (number 008/2009) and was according to the Brazilian Laws for Animal Protection.
Data obtained from the first part of the study show smaller weigh gain in P generation females treated with dexamethasone 4 mg/kg, independently of the cotreatment with dexamethasone 15 cH (MIX and DEX groups). This result reflects the output of the gestation interruption in these groups (Figure
(a) Female weight evolution during pregnancy (grams); (b) food consumption during pregnancy (grams); (c) water consumption during gestation (mL). MIX = dexamethasone 15 cH + dexamethasone 4 mg/kg (UHD + DX); DEX = dexamethasone 4 mg/kg (DX); UHD = dexamethasone 15 cH; CONT = control (water). *MANOVA,
At the end of the treatment, both groups presented no significant mild reduction in food intake compared to UHD and CONT groups (Figure
At the end of gestation, no birth was observed in MIX and DEX groups, it means, despite the cotreatment with dexamethasone 15 cH, all females exposed to dexamethasone 4 mg/kg exhibited its classical toxic effect. At the necropsy, these females presented incidence of 100% of adrenal fascicular zone atrophy and 100% of embryo absorption. Pulmonary hemorrhage was seen in 17 to 42% of females from these groups (
Percentage of macroscopic lesions in MIX = dexamethasone 15 cH + dexamethasone 4 mg/kg; DEX = dexamethasone 4 mg/kg; UHD = dexamethasone 15 cH; CONT = control (water). *
Macroscopic lesions | MIX Dexa 4 mg/kg + Dexa 15 cH | DEX Dexa 4 mg/kg | UHD Dexa 15 cH | CONT vehicle |
---|---|---|---|---|
Embryo absorption | 100** | 100** | 10 | 20 |
Cannibalism evidence (percentage of dead newborns found in the cage) | — | — | 13 | 6 |
Cannibalism evidence (percentage of mothers that showed this behavior) | — | — | 41 | 25 |
Adrenal fascicular zone atrophy | 83 | 100 | 08 | 0 |
Diffuse lung hemorrhage | 42* | 17 | 0 | 0 |
The results show a discrete protective effect of dexamethasone 15 cH on toxic effects of pharmacological doses of the same drug in pregnant females, regarding of water consumption. The other parameters were according to the expected effect of corticoid intoxication during pregnancy. Thus, 100% of females treated with pharmacological doses of dexamethasone, despite the concomitant treatment with dexamethasone 15 cH, presented embryo absorption. In this case, only the F1 generation born to UHD and CONT mothers was analyzed for postnatal parameters.
The homeopathic dexamethasone exposition did not alter parameters related to post-natal development of newborns (Table
Parameters of individual postnatal development (days). MIX = dexamethasone 15 cH + dexamethasone 4 mg/kg; DEX = dexamethasone 4 mg/kg; UHD = dexamethasone 15 cH; CONT = control (water). Student’s
Parameters (days) | UHD | CONT |
---|---|---|
Pinna (ear) opening | 2.43#/0.55## | 2.38/0.49 |
Eyes opening | 12.32/1.39 | 12.16/0.98 |
Vagina opening | 40.05/0.23 | 40.00/0.00 |
Superior incisive teeth dehiscence | 9.79/1.56 | 9.98/1.53 |
Superior incisive teeth dehiscence | 10.63/1.21 | 10.93/1.53 |
Fur covering | 1.01/0.11 | 1.00/0.00 |
Testicle dehiscence | 4.00/0.00 | 4.00/0.00 |
Postural reflex learning | 12.31/2.15 | 12.85/2.30 |
F1 generation weight gain evolution (grams). The 4 selected rats of each offspring were weighed together; so, there is no standard deviation and statistics for each time. UHD = dexamethasone 15 cH; CONT = control (water).
After 60 days, the F1 generation was inoculated with 1% carrageenan into the footpad, to evaluate its patterns of inflammation response face to an irritant stimulus. The edema curve was measured each hour, during 4 hours, and no difference between groups was seen, considering this macroscopic parameter (Figure
Paw edema after 1% kappa carrageenan (0.1 mL, sc). Millimeters. UHD = dexamethasone 15 cH; CONT = control (water). Student “
After 4 hours of edema evaluation, rats were sacrificed and the histopathological analysis of inflamed subcutaneous tissue was done. The toluidine blue staining followed by histomorphometry revealed a significant difference in mast cell/basophile degranulation percentage between groups. Thus, rats born to mothers treated with dexamethasone 15 cH (UHD) presented more degranulated mast cells per field than control (Table
Number of inflammatory cells in the subcutaneous tissue of the footpaw inoculated with 1% kappa carrageenan. PMN cells were counted using HE stained slides. Immuno-histochemistry was used to identify different stages of phagocyte maturation (CD 163) and the number of mononuclear phagocytes per field (MAC 387). Adhesion molecules CD18 (beta integrin) and CD54 (ICAM 1) were marked in leukocytes and endothelial cells. The intensity of CD54 marked cells was measured by pre-defined scores measured by three independent observers. The proportion of degranulated mast cells (N/200 total counted cells) is represented by percentage. The values are represented by mean (
Dexamethasone 15 cH | Control | |
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Eosin/hematoxylin (PMN) |
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MAC 387 + (Macrophages) |
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CD163 + (negative monocytes) |
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CD163 + Membrane-marked monocytes |
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CD163 + cytoplasm marked monocytes |
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CD163 + cytoplasm marked macrophages |
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CD 18 + (positive mononuclear cells) |
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CD 18 + (negative mononuclear cells) |
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CD 18 + (positive total leukocytes) |
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CD 18 + (negative total leukocytes) |
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CD 54 + (scores) |
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Degranulated mast cells ( |
9.5/200 (4.75%)# | 5.9/200 (2.95%) |
Photomicrographs of inflamed footpad of the offspring inoculated with 1% carrageenan (magnitude 400x). ND = not degranulated cell; D = degranulated cells.
After the HE staining method, a nonsignificant increase of neutrophils per field was seen in UHD group, associated to a significant increase in MAC 387 + mononuclear cells per field. A greater proportion between CD163 + monocytes and negative monocytes (Table
CD 163 positive monocytes and macrophages per slide (10 fields per slide), representing inflamed subcutaneous tissue of the carrageenan inoculated footpad from rats born to mothers treated with dexamethasone 15 cH (UHD) or water (CONT). Quantification was based on 4 categories of staining intensity: (a) negative monocytes; (b) irregular positivity of monocyte membranes; (c) intense cytoplasm positivity of monocytes; (d) intense cytoplasm positivity of macrophages. Columns represent mean. (#)
No significance was seen in relation to specific CD18+ mononuclear cells per field, but a significant increase of total CD18+ leukocytes was seen in UHD group, in relation to control (CONT), indicating higher expression of beta-2 integrin in PMN cells than in mononuclear cells (Table
The results show that no developmental variation is seen in rats born to mothers treated with dexamethasone 15 cH (
The whole data indicated changes of the pattern of inflammatory response in the high-diluted prenatal exposed offspring, in a sense of showing the “acute trend” in migrated leukocyte profile after an irritant stimulus, what is not seen in the control. This result reinforces the hypothesis of some kind of “vertical” (mother-fetus) transmission of information from high-diluted active substances, although no macroscopic parameter has been touched.
This study is pioneer in this field of knowledge and has some fundamental and applied implications. Under the fundamental research about high dilutions, these data point to some important aspects of the way of action of high-diluted products; it means changing the global configuration pattern of physiological parameters, instead of changing one or another specific marker involved in the process. Under the applied point of view, these results give useful information about putative consequences after the use of homeopathic preparations in breeding animals, especially in organic farms. According to the common sense, since there is no chemical residue in homeopathic remedies, the safety of these products would be absolute, regarding animal-derived food products. However, no trustful conclusion can be done “a priori,” whatever is the theme of study. In this case, an important window is opened, mainly related to the unknown long-term consequences of the use of homeopathic products in herds. This is not a simply toxic effect but a biological underexplored phenomenon that begins to be investigated. The scientific studies made under rigorous and systematic methodology are a sine-qua-non condition to the scientific development of the high-dilution field, as well in human, as well in veterinary medicine.
Although innovative and preliminary, the present study reflects original results and the plausibility to state the mother-fetus transmission of biological information from the theoretical above Avogadro’s number high-diluted dexamethasone. The only previous result found in the literature about that is a paper published by our own group, using mice in a similar model [
Abrupt changes in results according to the experimental context and variables are quite common in experimental studies about highly diluted endogenous substances. In [
The effect of 0.4 mg/kg dexamethasone in young rat development is well known and is related to bone growth and calcification delay [
The choice of using CD163 as a phagocyte maturation marker, beside MAC 387, has allowed the observation of detailed dynamics in migration and maturation of mononuclear cells into inflammatory site, since the maturation of monocytes in active macrophages is followed by the 15-fold increase in CD163 expression by these cells. The CD163 is a type I transmembrane glucoprotein (175 kD), also known as ED2. Its expression is related to the endocytosis of hemoglobin complexes and to the followed metabolism. Positive cells for ED2 protein have modulation role in inflammation and they can secret autocrine IL-10, which makes positive feedback with IL-4 and corticoids [
In this study, the dilutions of dexamethasone used were very high, above Avogadro’s number—15 cH means 10-33 M—and, even though, subtle modulation effects could be seen, regarding mast cell/basophile degranulation and inflammation cells infiltrate profile and maturation. This new perspective adds more information about the role of glucocorticoid in pregnancy and opens a wide range of research possibilities about homeopathy and its use in pregnant animals.
As conclusion, the proposed experimental model was able to demonstrate the mother-fetus transmission of homeopathic information, opening a new field of studies about homeopathy in pregnancy and its particular features in organic animal production.
No conflict of interests is identified in this study.
This study was supported by the CNPq—Productivity Research Program, by FAPESP (2007/59661-5), by UNISA, and by UNIP grants. The authors thank Paulo Ailton Vedovato for technical support.