To study this high heterogeneity in the inhibitory effects of NSAIDs, we have developed an in silico approach to evaluate NSAID action on targets under different PGHS-1 microenvironmental conditions, such as arachidonic acid, reducing cofactor, and peroxide concentrations. The designed technique permits translating the drug IC50, obtained in one experimental setting to another, and predicts in vivo inhibitory effects based on the relevant in vitro data. For the aspirin case, we elucidated the mechanism underlying the enhancement and reduction (aspirin resistance) of its efficacy, depending on PGHS-1 microenvironment in in vitro/in vivo experimental settings. We also present the results of the in silico screening of the combined action of sets of two NSAIDs , and study the mechanism of the experimentally observed effect of the suppression of aspirin-mediated PGHS-1 inhibition by selective and nonselective NSAIDs. Furthermore, we discuss the applications of the obtained results to the problems of standardization of NSAID test assay, dependence of the NSAID efficacy on cellular environment of PGHS-1, drug resistance, and NSAID combination therapy.The detailed kinetic model of Prostaglandin H Synthase-1 (PGHS-1) was applied to  The E2 state can be converted back to the resting state E1 by two consecutive reactions with an external reducing cosubstrate, RC, with a release of oxidised cosubstrate, OC (reactions 10 and 12). Alternatively, the E2 state can undergo a one-electron internal reduction via intramolecular electron transfer from the tyrosine 385 residue (Tyr385) to a protoporphyrin group (reaction 13), resulting in the formation of the E5 state,  containing the Tyr385* radical and a ferryl heme (Intermediate II). The E5 state can also complete the POX cycle and return to the resting state E1 via reactions 9 and 10 with cosubstrate. According to the Branched Chain Mechanism, the presence of the Tyr385* radical is ultimately required for the activation of AA oxidation reaction in the COX-site  (E9), containing AA in the COX-site. Reaction 2 corresponds to the abstraction of a hydrogen atom from AA by Tyr385* radical and formation of the E13 enzyme state containing AA* radical in the COX-site, . This AA* radical then reacts with two oxygen molecules and the intermediate product, PGG2* radical, is produced (reaction 3). The final step of the COX1 cycle in our model is the irreversible reaction 4, which describes the process of E5 state regeneration with Tyr385* radical, accompanied by the release of the intermediate product, PGG2 from the COX-site . Th. Th29]. e enzyme  and SimBin silico screening of the inhibition actions of different NSAIDs on PGHS-1 in in vitro experimental conditions. To simulate the in silico experiments on the NSAID interaction with PGHS-1 under in vivo conditions , we have upgraded our model in such a way that allows taking into account the synthesis and degradation of PGHS-1 in the cells , while in in vivo experiments with intact cells, the type of intracellular reducing cosubstrate and its level are unknown. Also, the type and the concentration of the reducing substrate, RC, in 50, depending on the experimental settings. In particular, it is known from the enzymatic theory that the dependence of the competitive inhibitor IC50 on the substrate concentration, S, may be written as:mK—Michaelis constant for the substrate, dK—the dissociation constant of inhibitor. Equation (1) shows that less competition of inhibitor with substrate at low substrate concentration (AA) causes the NSAID IC50 to decrease. Thus, the increase of NSAID effect in in vivo conditions may be expected. It has been shown that relation (1) holds in the wide range of the substrate concentrations for PGHS-1 [50 on AA concentration (1) is complicated by the complex kinetics of PGHS-1, particularly due to the existence of activation threshold in the range of low AA concentration [The factors mentioned above may change the value of NSAID’s ICr PGHS-1 . Howeverntration . Furthermore, reducing cosubstrate exerts a complex influence on the enzyme activity. Cosubstrate has been established to be an activator at low concentration and a suppressor of PGHS-1 activity at high concentrations of cosubstrate . in silico screening of oxygen consumption of PGHS-1 over a wide range of substrate and cosubstrate concentrations. To study the dependence of PGHS-1 activity on AA and RC concentrations in detail, we carried out an The obtained results show that the activation threshold of PGHS-1 depends not only on the internal properties of the enzyme , but alson,aspirink =10−5 μM−1 s−1[2 to PGH2 .According to the simulation of the dose dependence for aspirin shown in ls 50 μM . The expM–200 μM ,14. The 50, measured in vivo experiments, is much lower (1–3 μM), than the IC50 obtained in in vitro assays. To analyse this discrepancy, we carried out the in silico experiments, simulating the experiments on aspirin effect on platelets. Generally in such experiments, platelets are treated with the different concentrations of aspirin during the preincubation period of 25–30 minutes, and then the production of TxA2 is stimulated by calcium ionophore. The resulting concentration of TxB2 is then measured in 10–15 minutes [Extensive experimental studies of aspirin effect on intact cells  ,13,14,17 minutes . in silico experiment under in vivo conditions, we adjusted our method to the simulation of PGHS-1 catalysis in the platelet environment. The following assumptions were used in the calculation: (1) There is no de novo synthesis of PGHS-1 in platelets (synk = 0), but platelets contain a PGHS-1 depot. We estimated this initial concentration of PGHS-1 equal to 2.5 μM [2, produced after platelet stimulation [degk, was set as 10−5 s−1 based on the experimental estimation of PGHS half-life [in vitro experiments, as a cosubstrate.In the o 2.5 μM , based omulation . (2) PGHalf-life . (3) Int2 production. Thus we assumed that PGHS-1 functions in platelets at AA, RC concentrations close to its activation threshold , as compared to the IC50 estimate, derived from the in vitro one (100 μM). An analysis of the results obtained in in vitro and in vivo conditions allowed us to draw a conclusion on the possible mechanism, underpinning the stronger inhibitory effect of aspirin in vivo.As can be seen from the comparison of two theoretically derived dose dependencies shown in in silico experiment showed that this phenomenon originates from some complex enzyme dynamics, observed in the AA-RC concentration range close to the activation threshold of PGHS-1. In this range the interaction between catalytic activities of acetylated and non-acetylated (intact) forms of the enzyme contributes significantly to the inhibition of PGHS-1 (see scheme in 2 from the system (see 2[2 by the acetylated enzyme. Note that a kinetic interaction of acetylated and non-acetylated PGHS-1 results in a more pronounced effect on the enzyme inhibition at the concentrations of AA and RC close to the activation threshold (in vivo conditions), rather than far from one (in vitro conditions).The stem see . In thisem  [We applied the developed method to  PGHS-1) ,18, and  PGHS-1) . in silico the in vitro experiments on the inhibitory actions of celecoxib and ibuprofen on purified PGHS-1 [on,celecoxk = 1.6 μM−1 s−1, d,celecoxK = 0.2 μM and ibuprofen: on,ibuprofenk = 7 μM−1 s−1, d,ibuprofenK = 0.1 μM. Both the theoretical and experimental [50 of celecoxib and ibuprofen are in the range of 30 μM. The experimental values of the IC50 of ibuprofen lay in the wide range of the concentrations, 3.3 μM–100 μM [To estimate the affinities of these drugs to PGHS-1, we reproduced d PGHS-1 , and obtM–100 μM ,48,49,50in vitro conditions to the in vivo ones. The in silico screening of the drug action on platelet PGHS-1 was carried out at the concentrations of RC and AA near the activation threshold of the enzyme . According to this dependence, the fall in the AA concentration leads to the decrease in the IC50 value. Note that this mechanism, caused by the drug and AA competition, does not show up in the case of in vitro and in vivo experiments with platelets treated by slow irreversible inhibitor, aspirin. This effect is due to the fact that enzyme binds with aspirin in the absence of competitive substrate at the preincubation stage.The second reason for the decrease in IC50 (1 μM) is in agreement with the experimental values [50 are most likely to be explained by the variations in the experimental conditions used in different studies. For example, some experiments were performed on washed platelets [50 value [The obtained value of the celecoxib ICl values ,11,18, wl values ,11,18. Tlatelets . The metin vivo condition than in vitro one. The obtained value of the ibuprofen IC50 is 2 μM, which is in agreement with the experimental data, measured in the different experimental conditions: 7.6 μM (WBA) [The results of our calculations also showed that ibuprofen produces a stronger inhibitory effect on PGHS-1 μM (WBA) , 1.4 μM–μM (WBA) , 5 μM (bμM (WBA) . in silico screening of celecoxib and ibuprofen actions on PGHS-1 showed that these two drugs with different selectivity to PGHS-1 possess approximately equal abilities to inhibit PGHS-1 in platelets and in in vitro conditions. Below we continue to compare these NSAIDs by using in silico experiments in the different experimental conditions.The in vitro and in vivo experiments, as well as clinical studies, demonstrate that some nonselective NSAIDs  [The problem of NSAID co-administration is being actively discussed in literature in the context of uncertainty of the resulting therapeutic and side effects which arise from such combinations ,26,27. Baproxen) ,26 and Paproxen)  can undeaproxen) . But, thin silico screening of the resulting effect of two NSAIDs in combination on PGHS-1 in vivo and in vitro conditions. In this section, we demonstrate the application of our method to in silico study of the combined effects of aspirin and ibuprofen, as well as aspirin and celecoxib. To elucidate the mechanism of the interference of NSAIDs, we adapted our method to the in silico experiments we reproduced the conditions of the experiments on a combined action of aspirin with celecoxib, and aspirin with ibuprofen on PGHS-1 in vitro conditions and platelets [p-phenylenediamine), and the rate of cosubstrate oxidation was monitored spectrophotometrically [in silico experiment we used kinetic parameters of aspirin, ibuprofen, and celecoxib identified previously  faster than aspirin , and therefore tends to replace aspirin molecule in the COX-site at the high concentration of ibuprofen (celecoxib). It leads to saving some part of PGHS-1 from irreversible acetylation by aspirin. The substitution of aspirin by ibuprofen (celecoxib) starts when the drug concentration is in the range of the dissociation constants of ibuprofen, d,ibuprofenK = 0.1 μM or celecoxib, d,celecoxK = 0.2 μM  and arachidonic acid—all competing for the same COX binding site of the enzyme. These three small molecules differ by their kinetic properties—the rate of binding and the affinity to the COX-site of the enzyme, which results in an antagonistic effect between these species in the wide range of their concentrations. As has been established experimentally  and theo2 μM see . The sim= 0.1 μM ). Consid= 0.1 μM , AA inte50 (≅30 μM). The rise in the inhibition dose dependence curve observed in the range of 10 μM–100 μM of ibuprofen (celecoxib) concentrations is determined by the inhibition action of ibuprofen (celecoxib) in the region of its ICin vitro and in vivo experimental conditions, we carried out a computational simulation of the combined action of aspirin and ibuprofen (celecoxib) using the experimental conditions of the work [2 produced was measured after 10 min of the cyclooxygenase reaction. In the in silico experiment the intracellular concentration of AA and RC were chosen near the activation threshold of PGHS-1 (see discussion in The suppression effect of ibuprofen (celecoxib) on the aspirin-mediated inhibition of PGHS-1 has been confirmed in experiments on platelets  see exp. To analthe work . In thisin vivo case as well. However, there is the noticeable difference between these two cases. There is no restoration of the initial inhibition level in the range of ibuprofen (celecoxib) concentrations higher than 10 μM. The activation of platelets at these drug concentrations is determined by complete removal of ibuprofen (celecoxib) from intercellular environment of platelets [The theoretical dose dependencies for ibuprofen and celecoxib in the presence of 100 μM aspirin are shown in latelets . in silico experiment under the conditions of partial removal of celecoxib after the preincubation stage. The resulting dose dependence, given in in vitro case  is less than in in vitro case (30 μM) and close to the minimum of the dose dependence curve (at 1 μM). In this case the dip in the dose dependence curve is not formed. Note that the same effect of the disappearance of the blocking effect was observed in the case of the combination of aspirin and ibuprofen (data not shown). Thus, the in silico experiments showed that blocking effects of ibuprofen and celecoxib on the aspirin-mediated inhibition of platelet PGHS-1 depend significantly on the drug concentration in the intercellular environment. So the further in silico study of the conditions, at which NSAIDs block aspirin inhibitory effect on PGHS-1 in platelets, needs the consideration of pharmacokinetic data.Under in silico experiments on NSAID action on PGHS-1 was shown to be a promising tool to be applied to the following problems of in silico pharmacology:In silico screening of inhibition effects of different types of NSAIDs on PGHS-1 in various environmental conditions of the enzyme;in vitro assays, platelets, endothelia and other cells;Dissection of the key factors, which determine the variability of PGHS-1 response to NSAID action in In silico comparison of the different NSAIDs and the establishment of bioequivalence of the various drugs in the different experimental conditions;in vitro experimental conditions to in vivo settings;Translation of the results on NSAIDs action on PGHS-1, obtained in In silico study of the drug interference, underlying the combined action of two NSAIDs on PGHS-1 to predict the potential risks and benefits of NSAID combination therapy.The developed method of the in silico screening of PGHS-1 inhibition by NSAIDs in the intracellular environments corresponding to the inflammation progression in different cells may be a useful technique for predicting the anti-inflammatory and adverse effects of NSAIDs and their combinations in the changeable intracellular conditions. The proposed method of the


Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR). The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers’ oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris.Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac  Tribulus terrestris (TT), is used in the industrial production of medical preparations and feed additives based on its saponin fraction  sh shTribulactivity –17. It sIt is well known that two oocyte-specific genes, growth differentiation factor 9 (GDF9) and bone morphogeneticprotein 15 (BMP15), play a key role in the regulation of folliculogenesis in many species. These factors induce the proliferation and differentiation of the follicular cells during follicular development from the primordial stage –19. In aThere are only few in vivo studies available in this field. A previous study , showed TT affect their expression through metabolic pathways. To test this hypothesis, we aimed to evaluate the growth factors, GDF9 and BMP15, at mRNA and protein levels to determine: a) how TT affects folliculogenesis and ovulation rates in rabbit-mothers and b) how TT impacts the ovaries of F1 female offspring born to the treated mothers.Considering the pivotal role of the paracrine factors GDF9 and BMP15 in folliculogenesis from the primordial stage until ovulation, we hypothesize that the biological active compounds of New Zealand rabbits in the animal facility of the Institute of Biology and Immunology of Reproduction, Sofia, Bulgaria. The experiment started with 14 sexually mature does (mothers generation) randomly divided in the control and experimental groups of 7 females each. The animals from the experimental group were individually provided the feed additive, VemoHerb-T , with their drinking water, in daily doses of 3.5 mg/kg body weight for 45 days prior to insemination. The VHT preparation contained a dry extract of Tribulus terrestris with the following active substances: 60% furostanol saponins, determined as protodioscin; flavonoids—10.0%; tannic compounds—tannin—10.0%. Both groups were fed with standard diet for mature rabbits. At the end of the experimental period, female rabbits from both groups were mated with one control male rabbit. The number and survivability of offspring were estimated. The animals from the parental generation were slaughtered, after weaning of the offspring, for ovary collection.The investigations were conducted with 28 female white Among the female newborns , two new groups were formed, control and experimental, of 7 females each to the origin from the mothers’ generation. The animals were fed standard diet and kept in the same conditions as the mothers’ generation, but without VHT additive, until puberty onset (6 months old) and were slaughtered thereafter for ovary collection.All animals were reared and handled in accordance with the Bulgarian Veterinary Law (25/01/2011), in terms of the life conditions and welfare of experimental animals, adapted to the European Union regulation 86/609. In accordance with the Bulgarian Veterinary Law , only the National Ethics commission for animals can approve the use of animas for scientific purposes. The present study is one of the planned institutional projects for the period 2013–2018 and its protocol has been approved by the National Ethics commission for animals within the delivered permission of use of the animals in the experiments" . The responsible person for animal welfare at the Institute, working directly with the animals, is licensed by Bulgarian veterinary services for animal experimentation. The slaughtering of animals was performed after previously anesthetizing with 2% xylazine. All efforts were made to minimize suffering.Ovaries removed from the slaughtered animals were kept in PBS and brought to the laboratory immediately. One ovary from each animal was used for the collection of oocytes and cumulus cells. The cumulus-oocyte complexes (COCs) were aspirated by puncturing the follicles (diameter ≥2 mm) and collected under the stereomicroscope SMZ-10 . Firmly compacted COCs with multilayered cumulus cells and a uniformly granulated cytoplasm were separated into oocytes and cumulus cells by repeated aspiration using a narrow-bore Pasteur pipette. Final denudation of the oocytes was performed by hyaluronidase treatment , followed by washing thrice in PBS. The collected samples were stored at -80°C until RNA extraction.Total RNA was isolated from cumulus cells and oocytes separately for each animal, using a commercial RNA purification kit . A 1-μL volume of the solution was used for a concentration test and examination of the RNA integrity with a Qubit RNA Assay Kit . The extracted RNA was kept at –70°C.The first strand of cDNAs was synthesized by the kit “Easy Script Plus Reverse Transcriptase“, according to the manufacturer’s instructions using the oligo dT and random primers mix included in the kit.AAAGACCAGCTGCAGCATCC-3′and reverse primer: 5′-TGGTGTGAACTGGAGAGCCA-3′; BMP15-forward primer: 5′-GCTGGGATCACTGGATCATT-3′ and reverse primer: 5′- TGTACAGGACTGGGCAATCA -3′. All primers were commercially synthesized .The analysis of the GDF9 and BMP15 gene expressions in oocytes and cumulus cells was performed by RT-PCR using two sets of primers, GDF9-forward primer: 5′-PCR amplification was performed under the following conditions: predenaturation at 95°C for 10 min; 40 cycles for each sample that comprised an initial denaturation step at 95°C for 5 s, annealing at 60°C for 20 s, and extension at 60°C for 30 s. Rabbit glyceraldehyde-3-phosphate dehydrogenase  was used as a control gene for PCR. At the same time, negative controls (without cDNA template), with cDNA substituted for water, were set up for comparison. Each sample was run in triplicate. The cycle threshold values (CT) indicated the quantity of the target gene in each sample and the sequence of the target gene was determined in realtime using an Mx3005P QPCR system .In order to correct the inter-run fluctuations, standard curves were generated for each gene by serial dilution of the mix cDNA samples . A 2-μL volume of each concentration was used as a sample separately for the construction of GDF9, BMP15, and GAPDH gene standard curves.The second ovary of each animal was fixed with 10% formaldehyde PBS and subsequently dehydrated and embedded in paraffin. Serial sections of 5-μm thickness were cut by using a microtome type 2125RT . The histological estimation of the folliculogenesis was done by counting the number of the different classes of follicles per μm.For the detection and analysis of GDF9 and BMP15 proteins, the immunohistochemical technique described by  was perft-test was applied. In all other cases, the Wilcoxon rank-sum test was used to estimate the significance of results.Depending on the sample number and normality of distribution, parametric and non-parametric statistics were used. For n > 30 and normal distributions, Student’s p value ≤ 0.05 were considered as an up-regulation; FC ≤ 0.5 and p ≤ 0.05 were considered as downregulation. Genes with a FC between 0.5 and 2 were considered to indicate no significant change.The data from PCR were developed by DataAssist Sft.  using the ΔCt method . The qua2), using the software program ImageJ. A t-test was used to compare the results between control and experimental groups. Statistical significance was set at p < 0.05.The immunostaining reaction was evaluated on representative fields of the ovary slices. Immunoreactivity of GDF9 and BMP15 was assessed as a percent of the staining intensity of the positive cells inside a defined area (μmS1 Table). However, the survivability of newborns of treated mothers was higher than that of the control group . The density of the primary and secondary follicles in the ovaries of the treated mothers’ generation and the density of the preantral follicles in the ovaries of F1 females born to the treated mothers were higher than those in the controls (S2 Table).Supplementation of VHT in the rabbit mothers’ diet led to an increase in ovary weight in both generations of female rabbits. VHT did not affect fertility; the number of offspring born was similar to that of the control group . A remarkable difference was observed in the expression of GDF9; the mRNA level of GDF9 in the oocytes of the F1 generations’ ovaries was several times higher than those in the mothers’ ovaries. In contrast, the expression of BMP15 and GDF9 in the cumulus cells of rabbit-mothers’ ovaries and F1 females was not significantly different .In the oocytes and cumulus cells collected from all does, both BMP15 and GDF9 transcripts were detected. There were the differences in the expression of the investigated genes between generations. BMP15 and GDF9 expression in the oocytes of control F1 females’ ovaries was higher than that in the control mothers’ ovaries . The significant increase in GDF9 mRNA levels in both oocytes and cumulus cells was observed in the ovary of mothers treated with VHT .Supplementation of the rabbit-mothers’ diet with the feed additive VHT led to a decrease in the BMP15 mRNA level in the oocytes and an increase in its level in the cumulus cells . We noticed down-regulation of BMP15 in oocytes of the offspring  and GDF9 in cumulus cells .The expression level of mRNA in the investigated genes was lower in both oocytes and cumulus cells in the ovaries of F1 females’ offspring born to experimental mothers compared to the F1 females born to the controls (Table 1).The direct effect of the VHT supplementation on the expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of the treated mothers was several times higher than that in the F1 female offspring born to them  from both groups, control and experimental. In addition, both growth factors were detected in the theca, interstitial, and corpus lutein cells.Immunopositive signals of BMP15 and GDF9 were detected in the oocytes and cumulus cells of the follicles in the ovaries of mothers and F1 females’  and GDF9  in the oocytes from primordial and primary follicles and the lowest in the oocytes from antral follicles in all groups. Significantly higher signals for both proteins were observed in the primary and secondary oocytes from ovaries of the mothers treated with VHT. The increased secretion of GDF9 in the oocytes from antral follicles of treated mothers’ ovaries corresponded with the results of mRNA level changes obtained by PCR, whereas the results related to the BMP15 signals in the oocytes are somewhat contradictory. Although the level of BMP15 mRNA in the oocytes of treated mothers decreased, there were no differences in the intensity of protein signals compared to the control oocytes. It is likely that a reverse of the protein BMP15 from cumulus cells to oocytes occurred due to a deficit in mRNA production in the oocytes. Thus, the BMP15 protein expression was significantly higher in the cumulus cells of the antral follicles in both experimental groups F0 mothers and F1 female offspring generations .The computer estimated immune staining intensity of the investigated proteins had shown the most intensive signals of BMP15 .In the cumulus cells, the intensity of BMP15 and GDF9 signals increased from primary to secondary and preantral follicles and decreased again to antral follicles. The more intense GDF9-signals were detected in the cumulus cells from the secondary follicles of the treated mothers ; despite the suppression of FSH production, the secretion of estradiol and progesterone did not decrease in women treated with Tribestan [Our results show the stimulating effect of the additive VemoHerb-T on the weight of rabbit mothers’ ovary and weight of the offspring. Similar results were observed in mice  and in Gs system , our ress system , which dus study . It is kus study . An enharibestan .TT treated rat was found in a previous study [TT treatment.However, it is impossible to explain all morphological changes in the ovaries of experimental animals, particularly the increase in the density of secondary follicles, by the hormonal changes only, because at this stage they are not sensitive to hormones. Similar to the present study, an enhanced number of secondary follicles in the ovaries of us study . These rCurrently existing literature related to rodents mainly provides information on the expression of GDF9 and BMP15 genes in the oocytes , 26. In We established the presence of BMP15 and GDF9 proteins in the follicles at all stages of development as well as in the interstitial and corpus lutein cells, which strongly suggest their pivotal role in the whole process of folliculogenesis in rabbits and confirms the previous investigations in other rodents , 32.T. terrestris caused the alteration of mRNA levels of both genes, but in different ways. Although we noticed a downregulation of BMP15 in the oocytes of treated mothers, we did not observe any failure in their fertility. This corresponds with the conclusion of [In the present study, the alteration in the expression of the BMP15 and GDF9 genes, as a result of the supplementation of feed additive VHT to the mothers’ diet, indicated their high sensitivity to metabolic changes in the ovaries. These findings are consistent with those of previous studies , 37, in usion of  that in Additionally, we found a significant upregulation of the GDF9 gene in the oocytes and cumulus cells of treated mothers. GDF9 is the closest homolog to BMP15 (known as GDF9B). Both bind to the same type II receptor, BMPRIIB, which is essential for GDF9/BMP15 signaling in granulosa cells , 19 and TT. In addition, the diameter of secondary and preantral follicles was higher than that in controls (unpublished data). This is likely due to the ability of GDF9 to regulate granulosa cell mitosis through both, Smad-dependent and independent signaling pathways [The morphological changes in the treated rabbits’ ovaries followed the changes in the expression of growth factors. There is substantial evidence in several mammalian species that GDF9 is essential for the early stages of follicle development . The incpathways .T. terrestris causes a significant increase in GDF9 at the mRNA and protein levels in the oocytes and cumulus cells of rabbits. These results provide additional value to the explanation of the therapeutic effect of TT described in previous studies [TT should be the main reason for the success of TT in removing ovarian cysts [To our knowledge, this is the first study to demonstrate that  studies , 16 in f studies . In addi studies  and cann studies . The incan cysts , 52.TT acts like the LH-hormone, a high level of GDF9 is also essential for modulating the LH receptors to ensure an adequate reaction to the hormone, because the oocyte-secreted factors, GDF9 and BMP15, have an effect on FSH and LH through the expression of their receptors on target cells [TT explains the consequences of TT action. However, the exact mechanism underlying the increase in GDF9 level remains unclear and requires further investigations.If we accept the mainly discussed hypothesis that et cells –59. TherTT during 45 days of the periconceptual period was sufficient to induce changes in the oocytes-secreted gene BMP15. It is likely that the pathways of TT action on the investigated growth factors are different. The upregulation of GDF9 mRNA in the oocytes and cumulus cells stimulated by TT was not inherited in F1 females; moreover, we observed a lower level of GDF9 mRNA in the oocytes and cumulus cells of these animals compared to the F1 females born to the control mothers.The analysis of mRNA levels of BMP15 and GDF9 in the oocytes and cumulus cells of F1 female offspring showed heritable changes in the BMP15 gene function. The down regulation of this gene was expressed also in the oocytes of F1 females born to treated mothers. Our results strongly suggest that rabbit’s oocytes are susceptible to epigenetic modulations during folliculogenesis and that the bioactive supplement VHT should be one of the factors causing this transgenerational epigenetic effect related to BMP15. Epigenetic processes can occur due to the direct exposure to external factors at critical periods, particularly during sensitive developmental windows for the germ cells, such as folliculogenesis . NutritiTT on the expression of GDF9 and BMP15 at mRNA and protein levels in the oocytes and cumulus cells of rabbits in two generations. The results indicate that these growth factors are sensitive to the bioactive compounds of TT in different ways at different follicle structures. At the same time, GDF9 and BMP15 showed their interchangeability in rabbits. Therefore, overexpression of GDF9 in the case of BMP15 suppression ensured normal development and ovulation of the follicles and high fertility at the first insemination after the treatment.The present study provides for the first time the evidence for the stimulating effect of T. terrestris on female reproduction at the molecular level. Further studies are required to elucidate the exact pathways of TT action on growth factors expression, as well as on the epigenetic mechanisms of the influence of TT on F1 female offspring.The present study provides insights into the therapeutic effect of the plant S1 TableTribulus terrestris, producer Vemo-Ltd) 45 days prior to insemination. Both groups were mated with the same male rabbit. Data are presented as a mean ± SEM; Wilcoxon rank-sum test was used for the significance consideration.The experimental group of mothers was treated with the feed additive VemoHerb-T (dry extract of the plant (DOCX)Click here for additional data file.S2 Tablet-test was applied, and the differences were considered significant at p < 0.05.Serial 5-μm sections in 25 μm depth of each ovary were analyzed. Data are presented as mean ±SEM, (DOCX)Click here for additional data file.S1 FigTribulus terrestris, producer Vemo-Ltd) 45 days prior to insemination. Both groups were mated with the same male rabbit. A significantly higher survivability of F1 offspring born to experimental mothers was observed.The experimental group of mothers was treated with the feed additive VemoHerb-T (dry extract of plant the (TIF)Click here for additional data file.


Prostate cancer is one of the most prevalent oncological diseases in males worldwide, and the mortalities resulting from this type of cancer are mainly due to metastasis. The most common models for the study of metastasis are transgenic and immunocompromised mice, which enable the study of the metastatic process in a controlled way by the injection of prostate cancer cells into the mice. In the present study, NOD-SCIDγ mice were injected orthotopically with PC3 cells in the anterior prostate in order to establish a metastatic model. The results demonstrated the development and growth of a primary tumor that preceded the formation of micrometastases in the lung, liver and pancreas, followed by macrometastases in the liver. This model adequately represents the dynamics of the metastatic process, and may be useful for novel therapeutic assays and post-surgical relapse studies. In vivo models of cancer enable the study of tumor development and the dissemination of cancer cells in a complex organism, thereby simulating the process observed in human  is the preferred animal for in vivo models of cancer, due to its homogeneous biological features and the short time over which cancer develops in this organism , ventral, dorsal and lateral lobes. The latter 2 are jointly referred to as the dorsolateral lobe . This isWith regards to research into PCa, there is a limited number of studies that have investigated the anterior prostate as a site for the injection of cells . In the 2 atmosphere.The PC3 cell line , a human prostate carcinoma cell line established from a bone metastasis, was used in the present study. When cultured, these cells present adherent growth and epithelial morphology. This line is negative for androgen receptor and posesses a high tumorigenic capacity. The PC3 cells were cultured in DMEM/F12  supplemented with 10% fetal calf serum (Life Technologies), and maintained at 37°C in a humidified incubator with 5% CONOD-SCIDγ mice  were obtained from the high safety animal facility, Faculty of Medicine, University of Chile , and maintained in a laminar flow room under specific pathogen-free conditions. All the food, water and litter were sterilized prior to use. The temperature and humidity were controlled at 20–21°C and 50–60%, respectively. Daily light cycles consisted of 12 h light and dark. The cages were fully cleaned once or twice per week. The animals were manipulated under sterile conditions.6 PC3 cells suspended in PBS , as indicated in A total of 7 groups of 2 mice each were injected with 50 µl phosphate-buffered saline (PBS) or 10The animals were anesthetized with an intraperitoneal injection of 10 mg/kg xylazine  combined with 100 mg/kg ketamine  in a total volume of 200 ml of sterile water. The implantation was performed under surgically sterile conditions. For the orthotopic injection, the abdomen was cleaned with iodine solution , and a 1-cm midline incision was created to expose the prostate gland. A 30-gauge needle and a 1-ml disposable syringe were used for the injection of the cell suspension. The needle was inserted into the middle portion of the right anterior lobe, and then was directed towards the cranial portion of the lobe, to reduce the risk of accidental implantation in the opposite lobe. The abdominal wound was closed in 2 layers with 6/0 absorbable surgical suture.6 PC3 cells in PBS) were injected into the subcutaneous tissue of the right lateral abdomen of the mice without anesthesia, using a 30-gauge needle and a 1-ml disposable syringe.Subcutaneous implantation is an easy and secure way for injecting tumor cells. However, it may not be representative of prostate cancer and may not result in metastatic disease . In orde2 chamber. A post-mortem examination was performed to observe macroscopic changes, presence and localization of tumors and to obtain tissues for the subsequent microscopic and molecular analyses.At the end of the experiment, the animals were euthanized in a COThe tissues were fixed by submersion in neutral-buffered formalin  for 24 h, and then trimmed and placed in histological cassettes for dehydration, inclusion in paraffin (Sigma-Aldrich) and staining with hematoxylin and eosin . The tissues were subjected to an indirect immunoperoxidase method as follows: Antigen retrieval was achieved by exposing the samples to 90°C for 30 min in citrate buffer , followed by overnight incubation at 4°C with the mouse monoclonal anti-human mitochondria antibody . Next, the samples were treated using a streptavidin-biotin detection method  followed by hematoxylin counterstaining. The images of the tissues were obtained with a DM3000 laboratory microscope .All the procedures involving animals were approved by the Bioethical Committee for Animal Research of the Faculty of Medicine, University of Chile (protocol CBA#0487 FMUCH).The animals in group 1 (PBS injection) presented no signs of tumor development. Their prostatic lobes were moderately translucent, with small blood vessels .In the animals of groups 2–6, tumors developed at the site of injection. The tumors were white, firm and multinodular . The datMacroscopic metastases were observed in the 2 mice of group 6. Metastatic tumors (2 mm in diameter) were identified in the liver (2 tumors in one mouse and 3 tumors in the other) and kidney (1 tumor in each mouse) . These tIn the mice of group 7 (subcutaneous injection), tumors developed in the site of injection. These tumors adhered to the dermis and the subcutaneous tissue, but not to the underlying muscle . The tumNo changes were observed in the tissues from the mice of group 1. The immunohistochemical analysis for human mitochondria was negative in all the tissues tested from these mice.2), including atypical mitotic figures characterized by asymmetric or non-bipolar metaphases.Tumors developed in the anterior prostate of the mice of groups 2–6. The tumors had well-defined, unencapsulated margins, with invasion foci into the surrounding stroma . In the The immunohistochemical analysis for human mitochondria demonstrated the human origin of the tumors , despiteIn the lungs of the mice of groups 4 and 5, small aggregates of tumor cells were observed, located near the end of terminal bronchioles and under the visceral pleura . In the In the animals of group 6, similar aggregates of tumor cells were observed in the lung and pancreas. No macroscopic tumors had developed in these organs by the end of the experiment.The hepatic tumors were formed by cells  arranged in a solid pattern . Renal tin vivo models of cancer with the aim of studying the dynamics of progression and dissemination of tumors  were localized, well-delimitated and non-encapsulated . These tIn the mice in groups 2–4, the prostate tumors grew slowly during the first 30 days post-injection, retaining a well-delimitated and non-encapsulated nodular morphology. This is similar to the growth rate observed in the subcutaneous tumors . This grThe cell morphology was similar in all the tumors, and 2 basic morphological patterns were identified . In the The second pattern consisted of fusiform cells arranged in poorly-defined bundles . In the The 2 patterns (nests and bundles) combined are morphologically similar to the highest grade in the Gleason score used in human PCa (grade 5), which represents an anaplastic, poorly differentiated pattern, usually associated with local invasion and metastasis .Metastases were initially observed in the lung, at 30 days post-injection (group 4), and were consistently identified in all the animals from groups 5 and 6. These tumors were microscopic, and consisted of clusters of 5 and ≤25 cells in groups 4 and 6 (55 days post injection), respectively. The lung tumors were smaller than the metastases observed in the liver, pancreas and kidney of the mice in these groups. The lung is a frequent target for metastases, due to biomechanical factors, since it is the first capillary bed encountered by the disseminated cells that have passed through the right ventricle ,25. ThisThe 2 histological patterns described for the primary tumors were present in certain liver and renal metastases, but in a less prominent manner. Differences in the cytological morphology between primary tumor cells, circulating tumor cells and disseminated tumor cells in a similar model have been reported previously, but the study involved surgical orthotopic implantation of fragments of tumors, which may account for the variability observed . In the In conclusion, the orthotopic injection of PC3 cells in the anterior prostate of NOD-SCIDγ mice results in the development of a primary tumor that has a growth rate that varies from slow at the beginning of tumor formation, to fast at the end. The prostatic tumor precedes the development of micrometastases in the lung, liver and pancreas. The tumor cells are capable of forming macroscopic tumors (macrometastases) in the liver and kidney. These cells may be identified, either individually or in groups, using a human mitochondrial marker. The primary and metastatic tumors present similar histological features, with 2 main identifiable morphological patterns.The modification of the orthotopic model described in the present study may represent a valuable tool for the study of cytoreductive surgery and the recurrence of PCa.


Objectives: This study investigated the effectiveness of low power red and infrared lasers and that of Er:YAG laser, in association with CPP-ACPF cream, on remineralization of white spot lesions.Study Design: Fifty intact premolars were immersed in a demineralization solution for 10 weeks to induce caries like lesions and then were divided into five groups. In group 1, the teeth were covered with a CPP-ACPF cream for 3 minutes and then irradiated with a low power red laser  for 1 minute through the cream. In group 2, the treatment was the same as that in group 1, but an infrared laser  was employed. The specimens in group 3 were irradiated with an Er:YAG laser  combined with CPP-ACPF. In group 4, the CPP-ACPF cream was applied for 4 minutes and group 5 was submitted to neither laser nor CPP-ACPF. The micro Vickers hardness was compared at 20, 60 and 100 µ from the enamel surface among the groups.Results: The highest microhardness was observed in the low power red and Er:YAG laser groups and the lowest one belonged to the CPP-ACPF alone and control groups. However, no significant difference was found in microhardness of the experimental groups at any of the evaluation depths (p>0.05). Conclusions: With the laser parameters used in this study, neither the combined application of Er:YAG laser with CPP-ACPF nor the combination of low power lasers with CPP-ACPF provided a significant increase in remineralization of enamel caries. Key words:Low level laser, Er:YAG, laser, enamel caries, CPP-ACP, microhardness, white spot lesion. Dental caries is still the most common oral disease throughout the world . Enamel Recently, casein phosphopeptide amorphous calcium phosphate (CPP-ACP) products have been introduced for prevention of dental caries and induction of remineralization in tooth tissues ,5. RecenIt has been demonstrated that the application of high power lasers such as CO2 and erbium lasers  are effective in caries prevention -13. As tThe aim of this study was to investigate the effectiveness of low power red and infrared lasers and that of the Er:YAG laser, in association with CPP-ACPF cream, on remineralization of white spot lesions of enamel.Fifty freshly extracted maxillary premolar teeth were collected and stored in saline solution at room temperature until the time of the experiment. Teeth with caries, cracks and defects in enamel structure were discarded. The specimens were immersed in a demineralizing solution containing 2.2 mM CaCl2, 2.2 mM NaH2PO4, and 50 mM acetic acid (PH 4.8)  for 10 wGroup 1 : In this group, the teeth were covered with CPP-ACPF cream for a total period of 4 minutes. After 3 minutes of cream application, the enamel was irradiated with a low power red laser  for 1 minute, and the cream was allowed to remain on the surface during irradiation. The laser apparatus emitted a wavelength of 660 nm and operated at the maximum power of 200 mW, continuous wave mode and at the approximate distance of 5 mm from the tooth surface. The total energy delivered to the enamel was 6 J.Group 2 : The demineralized enamel was covered with CPP-ACPF cream for four minutes and was irradiated with a low power infrared laser at the last one minute of cream application, similar to that described for group 1. The device was a gallium aluminum arsenide (Ga-Al-As) diode laser , emitting a wavelength of 810 nm and operating at the maximum output power of 200 mW in continuous wave mode. Each treatment window received 6 J of energy.Group 3 (Er:YAG laser): The treatment windows in this group were first covered with a thin layer of CPP-ACPF cream for 30 seconds, and then the Er:YAG laser  was irradiated through the cream over a period of 20 s. The handpiece was held manually at an approximate distance of 5 mm (focused mode) and perpendicular to the tooth surface, and the area was irradiated uniformly using scanning movements. The laser beam was emitted with energy of 100 mJ, frequency of 10 Hz, and with the use of minimum air/water spray. The beam diameter at the focal area of the handpiece was 1 mm, providing the energy density (fluence) of 12.7 J/cm2 per pulse. After laser irradiation, the thickness of the CPP-ACPF cream was increased as necessary and it was allowed to remain for a total period of 4 minutes on the enamel surface.Group 4 : The demineralized enamel in this group was only treated with application of a sufficient thickness of CPP-ACPF cream for 4 minutes.Group 5 (Control): The teeth in the control group were not subjected to either CPP-ACPF cream or laser irradia-tion.After the treatment procedures in the experimental groups, the CPP-ACPF cream was thoroughly rinsed from the enamel surfaces, and the teeth were then kept in distilled water until microhardness assessment.For microhardness analysis, the crowns were separated from the roots and then were embedded in epoxy resin. The specimens were sectioned occlusogingivally through the center of the treatment windows with a diamond disk at low speed, and the enamel surfaces were polished with sandpaper discs. After rinsing with distilled water, microhardness was assessed by a micro Vickers hardness apparatus  using a vertical load of 100 g for 5 seconds. The indenter of the apparatus was placed at 20 µ, 60 µ and 100 µ from the enamel surface to determine Vickers hardness number (VHN) at different depths.- Statistical analysisp<0.05.The normal distribution of the data was confirmed by the Kolmogorov-Smirnov test and the homogeneity of variances by the Levene’s test. A one-way ANOVA was run to determine the difference in microhardness of the experimental groups at each enamel depth. The statistical analysis was done using Statistical Package for the Social Sciences  and the significance level was determined at p>0.05; Although fluoride is the most effective agent in prevention and regression of dental caries, its repeated applica-tion may be associated with the risk of dental fluorosis . Caries In this study, the microhardness of the specimens treated with the CPP-ACPF cream was almost the same as those untreated. This means that one time application of this agent had no effect on remineralization of white spot lesions. It has been demonstrated that CPP-ACP products bind to dental plaque and provide a source of readily available calcium that restricts mineral loss and assists remineralization ,18. SomeDifferent theories have been explained for laser-induced changes in tooth structure. Regarding the high power lasers, it is believed that the photothermal effects of lasers are responsible for the modification in dental hard tissue structure including reduction of carbonate content and alterations in organic matters which lead to increase in the acid resistance . The mecet al.  with CPP-ACPF cream provided a significant increase in remineralization of enamel white spot lesions.


The most important known metabolic complication of the newborns from diabetic mothers, including diabetes type 1 and 2 and gestational diabetes, is the postnatal hypoglycemia. If unrecognized and undiagnosed, hypoglycemia in this particular high-risk group can determine severe neurological lesions and even death, in a significantly higher proportion than those from the general population. The present paper brings arguments for the crucial importance of screening for post-natal hypoglycemia in the early hours after birth, and discusses the management strategies and the topics that still remain in debate. Newborns from diabetic mothers, including diabetes type 1 and 2 and gestational diabetes, represent the group with the highest risk of developing symptomatic hypoglycemia in the immediate hours after birth. This metabolic risk is believed to be due to the relative fetal hyperinsulinism, manifested as a feedback mechanism for the balance of the high glucose levels induced by the maternal diabetes, and is particularly severe in those cases of poorly controlled maternal pre-existent diabetes with high levels of HbA1c [3]. Compared to controls from non-diabetic mothers, the prevalence of hypoglycemic episodes in IDM is as high as 40% [4], and subsequently the risk of long-term neurological consequences, but also the immediate risk of convulsions, coma and even death is also significantly higher . Neonatal hypoglycemia screening in all newborns from diabetic mothers (IDM-infants from diabetic mothers) became mandatory from the first hour of life, because of the short- and long-term consequences of these episodes, independent of the type of maternal diabetes or of the initial good and balanced presentation at birth .Episodes of hypoglycemia in IDM can persist up to one week after birth, with an increased frequency in the first 4 days of post-natal life, until the metabolic levels of endogenous insulin become balanced .A consensus on the definition of hypoglycemia is still in debate in the recent literature, and also the current management of asymptomatic hypoglycemia and the necessities of exogenous carbohydrate intake are still under discussion, for the first hours of post-natal life . The threshold levels of different studies for the definition of neonatal hypoglycemia are: under 45mg/d l (9), or 40 mg/dl  or 36mg/dl(2 mmol/l) . These variations are usually in the margins of the assay methods used or because of the actual source of the sample . The use of peripheral sampling with glucometers is efficient and user-friendly to screen for hypoglycemia, but for a certified diagnosis, a central sampling still needs to be done, as we know that the central levels are actually below the measurement of glucometers, and the relationship is not deductible from a formula calculus.Most studies describe hypoglycemia as levels under 30-50mg/dl  in the first 24 hours of post-natal life and 45-50mg/dl  after 24 hours [3] is that, in the context of maternal diabetes, neonates should be screened immediately after birth and then at 30 min, 1 h, 2 hrs, 4 hrs, 8 and 12 hrs and at any moment when symptoms that suggest hypoglycemia occur [3]. Also, after the initiation of glucose-therapy, 30 minutes after the iv perfusion starts, and after every significant modification of the infused dose [11-13].One important topic under discussion is the moment to start the screening and the frequency of measurements in the first hours of post-natal life. The algorithm advocated by the National Guideline on Neonatal Hypoglycemia Screening [4]. Cycles of glycemic control should start immediately at birth from the umbilical cord sampling at birth. A high level of glucose should trigger more attention as rebound hyperinsulinism could appear with subsequent severe hypoglycemia [3-4]. If, after the initiation of iv treatment, the glucose levels are still under 40mg/dl, treatment can be incremented with 2mg/kg/min or the concentration of the glucose solution to be changed to 12,5%, on a central way, until normal glucose levels are achieved . Hydrocortisone can also be used as an adjuvant in very severe and resistant cases. The aim of the treatment is to obtain normal glucose levels for 12 hours and then a decrement of the supplements can be started with 1-2 mg/kg/min and the discontinuation of the iv supplementation when the enteral feeding is considered sufficient to maintain a correct glycemic control .In order to prevent long-term neurological lesions, a quick response with glucose treatment should be initiated for all neonates who show symptoms of hypoglycemia. A bolus of 2ml/kg glucose 10% iv should be administered, followed by a continuous glucose perfusion of 6-8 mg/kg/min, in order to maintain glycemic levels over 40mg/dl  [13]. An alternative efficient method to keep normal glucose levels is to use dextrose-gel by oral massage in the first 48 hours of life.More debates concern the newborn with asymptomatic hypoglycemia. In these cases, the most important is the initiation of enteral feeding immediately after birth, during the first hour of life, breastfeeding or with assistance [3].The threshold levels at which iv glucose is judged necessary are also different between different protocols, variations are from 25mg/dl to 36mg/dl, or even as high as 40mg/dl, for those more protective. It is recommended to ample for glucose 30 minutes after the first breast-fed meal. In the first 4 hours of post-natal life, values under 25mg/dl in asymptomatic IDM, under enteral nutrition, will be resampled after 1 h. If the levels are still in the interval 25-40mg/dl, the newborn needs continuous monitoring. Values under 25mg/dl, at resampling, request the initiation of iv glucose therapy even in the absence of clinical symptoms .The purpose of monitoring and therapy after the first 4 hours is to maintain glucose levels above 45 mg/dl in pre-prandial sampling, checked up to 12 hours of post-natal life. Under the threshold of 35mg/dl, in asymptomatic IDM, hourly checks are required. Borderline values between 35-45mg/dl trigger continuing monitoring. At any point in time values under 35mg/dl, initiate iv glucose therapy .In this respect, all Neonatal Care Units should develop local protocols following the recommendations of the National Clinical Guide for the Screening and Treatment of Neonatal Hypoglycemia developed by consensus in 2010 and adopted by the Ministry of Health and the National Professional Boards [It is very important to stress that the correct and early diagnosis and prevention of hypoglycemic episodes, in these fragile high-risk newborns, prevents long-term neurological effects and altered outcomes.


Approximately 35% of patients with confirmed HER2 breast cancer progress to metastases of the central nervous system (CNS). Total cerebral radiotherapy is considered as standard treatment for these cases; however, studies have shown that some chemotherapy drugs can be used during radiotherapy without significantly increasing its toxicity. In this article, we report the case of a patient with HER2-positive breast cancer who showed isolated progression of the illness in the CNS, which was observed during the treatment period using T-DM1 concomitantly with radiotherapy of the CNS without apparent toxicity of the combination and keeping the illness controlled. Through a review of the literature on the use of radiotherapy and chemotherapy with T-DM1 for the treatment of cerebral metastases in HER2-positive breast cancer, we describe the efficacy and tolerance of the concomitant application of these treatments. Breast cancer is one of the most common neoplasia in Western countries and one of the main causes of death among women, as well as the most important among women from 40 to 59 years of age . Of the Generally, among patients with breast cancer, approximately 25% progress to metastases of the central nervous system (CNS) . MeanwhiIt is known that the blood–brain barrier limits the penetration of the anti-neoplasic agents into the CNS. Nevertheless, the true functioning of this system in metastatic illness is not completely understood, given the capacity of these lesions to take up contrast in imaging studies, suggesting a breach in this barrier . ConsequIn this study, we describe the efficacy and tolerance of the treatment of a patient with HER2-positive breast cancer with metastases of the CNS subjected to total cerebral radiotherapy simultaneously with systemic therapy with trastuzumab emtansine (T-DM1).A 62-year-old female patient with leucoderma, with a diagnosis of carcinoma of the breast, was subjected to modified radical left mastectomy and axillary lymphadenectomy, followed by breast reconstruction with a prosthesis.An anatamopathological study showed a 2 cm invasive ductal carcinoma in the left breast, the presence of micrometastases in a lymph node, positivity for oestrogen and progesterone receptors and a score of 3 + for the product of the HER2 oncogene. The pathological staging observed was pT1cpN1miM0.2 of adriamycin and 600 mg/m2 of cyclophosphamide within a period of 2 months. Afterward, trastuzumab and paclitaxel were initiated, but the patient completed only two cycles, as this was interrupted by an allergic reaction to taxane. The use of trastuzumab and tamoxifen was continued for one year.Chemotherapy was performed with four cycles of 60 mg/mDue to bone pain 7 months after the start of treatment, a bone scan was requested, which revealed increased osteoblastic activity in the spinal column at T6 and T7.Computed tomograpghy (CT) scans of the thorax, abdomen, pelvis, and spine showed pulmonary micronodules and a lytic lesion with a component of soft areas affecting the pedicle and elements of the posterior arch of T6, compatible with neoplastic implant.The patient developed symptoms compatible with medullary compression. Magnetic resonance imaging (MRIs) of the cervical and thoracic spine showed secondary implants in T6 and at the level of C3 and C5, with components of soft areas projecting into the epidural space, reducing the width of the spinal canal and discreetly compromising the medulla in this region.The anatamopathological study of the bone lesion confirmed metastatic adenocarcinoma in the bone tissue. The immunohistochemical analysis indicated the breast as the probably site of origin, with positive oestrogen receptors and a score of 3 + for the product of the HER2 oncogene.Thus, treatment with radiotherapy and fulvestrant associated with zoledronic acid was initiated and maintained for 4 months.With the clinical progression, evidenced by an increase in the pain intensity, weekly docetaxel was initiated, followed by capecitabine and lapatinib for a period of 1 month; however, the symptoms did not improve.She was referred to protocol NCT01419197, a randomised, open (TH3RESA), Phase-III, multicentric study with two branches, which assessed the efficacy and safety of T-DM1 in comparison with other treatments in patients with HER2-positive metastatic or locally advanced/recurrent breast cancer. The patients in question were randomised into the group which would be treated with T-DM1.Then, chemotherapy cycles with T-DM1 every 21 days were started. In the following visits and throughout the cycles, the bone pain was unchanged with the use of morphine.During the treatment with T-DM1, imaging studies were done every 6 weeks for control. From the 12th week of treatment with the protocol, CT scans of the thorax, abdomen, and spine showed a partial response, with the disappearance of the pulmonary implants, and only the bone lesions remaining. There was no evidence of progression of the illness.In addition, Doppler echocardiograms were taken every 3 months which showed no cardiac toxicity on the treatment with T-DM1.In the first cycle, mild nausea was reported, possibly related to the treatment with T-DM1, which was treated with 10 mg bromopride orally 3× per day.In the fourth cycle, there were signs of moderate neuropathy, possibly related to T-DM1, and 75 mg Lyrica orally per day was started to treat this condition.Eleven months after the start of treatment with T-DM1, she had a convulsive episode. A cranial MRI was performed, which showed isolated progression in the CNS. The results were discussed with the study team and it was decided to continue the treatment with T-DM1, as the patient showed controlled illness outside the CNS. Thus, total cerebral radiotherapy was initiated, applied to the CNS with a dose of 3 Gy/dau, concomitant with the treatment with T-DM1. The use of 10 mg hidantal orally 3× per day was instituted for the control of convulsive symptoms.Following 6 months of monitoring, it was observed that the patient had a good tolerance for radiotherapy, as no adverse reactions or clinically significant toxicity were observed. A control MRI was performed, which showed a lack of progression in the CNS. Other imaging studies continued to show a partial response outside the CNS. No sequelae appeared following the treatment.This case suggests that in a patient with isolated progression of metastatic illness in the CNS, a treatment option would be the administration of radiotherapy concomitantly with treatment with T-DM1, as no significant toxicity was shown and there was control of the progression of the illness.Cerebral metastases are the most common form of intracranial neoplasia, as they are present in up to 25% of all patients with advanced cancer. In breast cancer, the incidence is approximately 25%, and it is the second most common type to evolve with metastases in the CNS, after only pulmonary neoplasias. HER2 positivity is recognised as a risk factor for cerebral metastases in patients with breast cancer, increasing the incidence of these metastases to 37.3% , 7, 8.Due to the accuracy and sensitivity of current diagnostic methods, an increase in the number of cases of metastases from breast cancer has been observed. The principal sites for breast cancer metastases are the CNS, with the parenchyma being the most affected area, followed by the cerebellum and the encephalic trunk. The supratentorial region is more affected than the infratentorial, with a predilection for perivascular areas and connections between white and grey matter. Metastatic lesions located in the infratentorial region generally originate from malignancies located in the abdominal or pelvic regions, once they gain access through the venous drainage of these areas through the internal vertebral venous plexus–Batson plexus .HER-2 positivity confers an increased risk of recurrence of distant metastases and a decrease in the survival of patients with breast cancer without anti-HER2 treatment, while there is an increase in survival with the institution of this treatment. Another point is the association with the aggressiveness of metastases, which is elevated in HER2-positive patients. In most cases, dissemination follows the identification of other systemic lesions. Trastuzumab, an anti-HER2 antibody, has a limited ability to cross the blood–brain barrier due to its high molecular weight. Due to this difficulty, approximately 10% of patients taking trastuzumab exclusively as a treatment for metastatic breast cancer develop isolated involvement of the CNS .Multiple cerebral metastases of breast cancer exist in two-thirds of patients at the time of diagnosis, and they generally have a random distribution, and can produce any neurological sign or symptom, or a combination of these. The principal clinical manifestations are headaches, behavioural changes, cognitive changes, and clinical signs such as aphasia, difficulty walking, focal deficits and epileptic crises. Nevertheless, 20% of cerebral metastases remain asymptomatic .The most effective method for complementary assessment is magnetic resonance imaging (MRI) with gadolinium contrast. This has greater sensitivity than computerised tomography (CT) with contrast for the identification of lesions in the parenchyma or leptomeninges. Approximately 20% of patients with a single lesion detected by CT are found to have multiple lesions when assessed by MRI. Treatment options for patients with cerebral metastases of breast cancer are the use of systemic chemotherapy, total or localised cerebral radiotherapy, conventional surgery, and radiosurgery . SystemiThe use of chemotherapy in the treatment of advanced breast cancer depends on multiple factors. In addition to a good doctor–patient relationship, in which both must be in agreement on the best course of treatment within their expectations and risk factors, the characteristics of the patient and his or her type of cancer are important to the decision on what drug should be used. The greatest difficulty in cancer management is in establishing the best time for the use of medication. In this case, trastuzumab–emtansine was used, better known by the term T-DM1. The drug is made up of a combination of three parts: a specific humanised antibody for the extracellular region of the HER2 receptor – trastuzumab – an antimicrotubule agent derived from maytansine – DM1 – and a linking thioether responsible for connecting the two structures already mentioned. As for its mechanism, trastuzumab links to the HER2 receptor, allowing for the internalisation of the T-DM1 molecule by the endosomes, mediated by antigens. Then, the DM1 portion is released in the intracellular environment. Then, this reacts by inhibiting the polymerisation of beta-tubuline. This occurs through the linking process, which occurs in a competitive manner with the linking alkaloid. Following this process, cellular death occurs .As it is a relatively new drug, its interactions with other medications are still being investigated. In a study of the association of T-DM1 with pertuzumab, the risk of complications was shown to be low. As for the association of T-DM1 with taxanes (taclitaxel and docetaxel), studies are being carried out to find possible adverse reactions of clinical importance. Until then, it is demonstrated that there is no relevant difference between the isolated administration of T-DM1 and its association with taxanes in relation to the severity and types of adverse reactions. As a monotherapy, T-DM1 showed an increase in survival and lower toxicity in relation to the use of lapatinib plus capecitabine in patients with advanced HER2-positive breast cancer in prior treatment with trastuzumab and taxanes , 14.While it has shown good clinical results, T-DM1 is not a drug which is free of side effects. Among the main side effects were fatigue (64.5), nausea (48.4%), headache (30.4%), fever (28.4%), nosebleeds (28.9%), and constipation (28.6). These events were classified with grade 1 or 2 in intensity. It is also important to indicate the events classified with grade 3 or higher, of which the main effects were thrombocytopenia (7.7%), hypocalcaemia (4%), increased fatigue (4%), increased AST (3.3%), shortness of breath (2.9%), anaemia (2.6%), cellulite (2.6%), hyperglycaemia (2.2%), and low back pain (2.2%) .The published studies still show controversies relating to the efficacy of treatment with isolated trastuzumab in advanced breast cancer with cerebral metastasis. What has been discussed is that the increase in the survival rate may be related to control of the systemic illness caused by the drug and not by the control of the cerebral metastasis .et al, a regression of the size of multiple asymptomatic metastases of the CNS was observed, affecting cerebral and cerebellar hemispheres and the cerebral trunk in a young patient with advanced inflammatory mammary neoplasia following treatment with Ado-trastuzumab emtansine [et al, an unexpected reduction in the size of metastases in the right parietal lobe and adjacent leptomeninges and cerebellum were observed bilaterally following additional total cerebral radiotherapy and treatment with T-DM1 for the control of the systemic illness [In the case study done by Torres mtansine . Similar illness .We conclude from this study that there was a good response to the association of total cerebral radiotherapy to the use of T-DM1, keeping the patient free from the progression of the illness in the CNS, with good tolerance and with no evidence of adverse reactions, clinically significant toxicity, or sequelae.We declare no conflicts of interest of any type in the preparation of this manuscript.


Empirical Mode Decomposition (EMD), due to its adaptive decomposition property for the non-linear and non-stationary signals, has been widely used in vibration analyses for rotating machinery. However, EMD suffers from mode mixing, which is difficult to extract features independently. Although the improved EMD, well known as the ensemble EMD (EEMD), has been proposed, mode mixing is alleviated only to a certain degree. Moreover, EEMD needs to determine the amplitude of added noise. In this paper, we propose Phase Space Ensemble Empirical Mode Decomposition (PSEEMD) integrating Phase Space Reconstruction (PSR) and Manifold Learning (ML) for modifying EEMD. We also provide the principle and detailed procedure of PSEEMD, and the analyses on a simulation signal and an actual vibration signal derived from a rubbing rotor are performed. The results show that PSEEMD is more efficient and convenient than EEMD in extracting the mixing features from the investigated signal and in optimizing the amplitude of the necessary added noise. Additionally PSEEMD can extract the weak features interfered with a certain amount of noise. The inner product transformation can be adopted to obtain features of the mechanical system by measuring the correlation between vibration signal and a pre-defined kernel function. However, a description of continuous and instantaneous variation of a dynamic system with non-linear and non-stationary properties may be generally absent due to the lack of adaptivity. On the other hand, the low intelligence and flexibility attributed to the non-adaptivity may lead to the requirement of a large amount of apriori knowledge or new kernels for a suitable match of the inner product. Signal processing techniques, which discover the essential rules contained in a mechanical dynamic system, are widely used to realize the vibration analysis, the fault identification, and the status estimation of systems ,4,5,6,7.Empirical Mode Decomposition (EMD) , proposeIt is obvious that the extraction results from non-linear and non-stationary signals, combined with noise, approach linear and stationary information. Consequently, the nature of a dynamic system may be reconstructed with these brief signal components approaching linear and stationary information through an appropriate expression, Phase Space Reconstruction (PSR) ,23. ThenStudies on PSR were conducted as early as 1980 ,23. SincThe rest of this paper is organized as follows. In As a pioneer of the adaptive data analysis method, EMD was proposed by Huang . This weIn order to satisfy the needs of comparison in the subsequent improvement, the whole flow of the EEMD algorithm and the description ,21 are s(1)M, the statistical property of added white noise, and m = 1.Initialize the ensemble number (2)M times in a cycle.(2-1)mth white noise with the given statistical property to the signal,mn(t) represents the mth added white noise, and mx(t) represents the noise-added signal of the mth trial.Add the (2-2)mx(t) into IMFs icm,  and a residual series mr by EMD:icm, denotes the ith IMF of the mth trial, I is the number of IMFs, and mr represents the only residual volume.Decompose the noise-added signal (2-3)m < M, the flow performs back to (2-1), accompanying m = m + 1. Then, perform circulation from (2-1) to (2-3) until m = M.If Perform trails (3)ic of the M trials for each IMF and the residual r using the following equation.Calculate the ensemble mean (4)Output all of the true IMFs which should satisfy the formula:Steps:nε is the deviation between the original signal and the reconstruction result, ε represents the amplitude of added noise that we need to find out, and M is the ensemble number.According to the flow chart and the description above, we can understand that mode mixing can be restrained by adding white noise to the investigated signal to change the distribution of extremes. In this process, the critical portion of the amplitude of added noise determines the restraining level. This parameter mainly follows the statistical regularity as follows :(5)εn=εε and the largest M can ensurethe smallest εn. However, in fact, the smallest ε will lead totoo small change of extreme distribution and the largest M will cause the higher complexity of the algorithm. To solve the above problem in practice, ε can be designed as 0.2 times of the standard deviation of the investigated signal generally according to noise added experiments. On the other hand, Wu, et al., also presented some empirical rules for complement: if the high frequency components are dominant in the investigated signal, ε can be slightly smaller while the low frequency components are dominant, ε can be slightly greater. Several researchers have conducted the investigation into the relationship between the high or low frequency components and ε  represents the mathematical expectation, α is the projection direction included in A.Seung es in ML . As a rees in ML ,41 and aiY as an example, its variance can be written asC is the covariance matrix of X.Therefore, taking one of the new variables αTα = 1 can be converted to an unconstrained extreme problem asv is the Lagrangian multiplier.Thus, the extreme problem under the condition of Calculating the differential of Equation (9) and setting the result to be zero, the MSE can be deduced as a solution to the characteristic Equation (10).The main features can be selected to be the original signal and then the goal of feature extraction can be achieved accordingly. Note that PCA is able to verify the feasibility of extracting the orthogonal features of the investigated signals. However, other embedding and manifold learning methods would be adopted instead of PCA if the investigated signal with certain non-linear and non-stationary property were difficult to analyze.On the basis of the noise decoupling, EEMD is expected to decompose the signal into a set of IMFs with frequencies arranged from high frequency to low one. In vibration analyses of rotating machinery, IMFs with too high frequency would be the stochastic noise usually, and IMFs with too low frequency would be the trends, the false components, and the residual components. IMFs that represent the vibration property of rotating machinery can be obtained by eliminating the useless components.ε will be a proper value that is greater than 0.2 times of the standard deviation of the investigated signals, and the ensemble number M will be less than 100.The vibration signals of rotating machinery are mainly concentrated in the low frequency region. We remark that, according to the instruction of parameter criteria in Different PSR methods are suitable for the different purposes. For the linear and stationary IMFs, we adopt the Hankel matrix mentioned in Through the extended expression of IMFs in the multi-phase-space, manifold learning methods may be used to extract features of the investigated signal further. Non-linear manifold learning methods proposed recently seem to be fussy and inappropriate for feature extraction in multi-phase space with approximately linear and stationary characteristics. Consequently, the multi-manifold-learning (MML) with PCA, instead of complex and conventional SVD, is adopted here, tentatively, for the single elements extraction in multi-phase-space. A new set of “element storage” would be constructed with these single elements. By inspecting the relationship of these elements between spaces properly and combining the same kind of elements, the modified intrinsic mode function (MIMF) would be reconstructed. Thus the independent extraction of signal components would be realized accordingly. The overview of PSEEMD and its details are given in Step 1: Implement EEMD with the investigated signal and obtain the preliminary IMFs.Step 2: Select required IMFs and construct MPS by the multi-Hankel-matrix.Step 3: Extract elements from MPS by MML of Multi-PCA and develop the element storage.Step 4: Inspect the element storage and combine the same kind of elements.Step 5: Reconstruct MIMFs.Implementation procedure of PSEEMD is described as follows and the detailed computation flow is shown in No.1: The amplitude of added noise in EEMD is set to be 0.2 times of the investigated signal standard deviation or an optimized value. The following verification with PSEEMD in No.2: Required IMFs should be selected according to some prior knowledge or indicators of the investigated signal.No.3: The element storage may be enormous and some prior knowledge may be also used to inspect and combine the same kind of elements.Notes:A simulation signal is adopted to explain the proposed PSEEMD in this section. Since rubbing is a typical fault in rotating machinery, the simulation signal mainly includes the fundamental frequency and harmonics of rubbing. Rubbing is defined as the impact when the interval between static part and spindle is tiny. This fault signal is commonly characterized with evident fundamental frequency, along with the integer harmonics and the fractional harmonics. The fundamental frequency accompanying integer frequency less than or equal to the quadruplicated frequency is a dominant feature presented by the running status of our present rotor test bench. A simulation signal constructed by the four frequency components is shown in With the original amplitude of added noise equal to 0.77, the ensemble number of 100 and the sifting number of 20, the PSEEMD method is employed to analyze the simulation signal and the decomposed first six Modified IMFs, both in the time-domain and the frequency-domain, are illustrated in ε in a proper interval whose lowerbound is greater than 0.2 times of the standard deviation of investigated signal. These two kinds of decompositions are shown in It can be seen from the two figures that MIMFs 1–6 correspond to the passband noise, the quadruplicated vibration frequency (1X), the triple vibration frequency (2X), the double vibration frequency (3X), the fundamental vibration frequency (4X), and the low frequency noise, respectively. It can also be observed from ε only enables some weak information transfer rather than eliminating mode mixing. Moreover, the information transfer may be more obvious if the complexity of investigated signal increases. Thus, these results imply that neither the original EEMD nor the optimized EEMD can accurately extract independent information of the simulation signal.The comparison between the original IMFs in On the basis of the results of the simulation and comparison, it appears that PSEEMD is able to obtain more accurate and independent IMFs than the original EEMD and the optimized EEMD.The rotating machinery is a kind of critical equipment in the fields of electrical power, petrochemical complex, metallurgical industry and aerospace engineering. Contemporary rotating machinery is confronted with severe production conditions, complex and diverse equipment structure, and quick updating of physical and virtual function, which has raised a higher reliability requirement for the versatile and critical units of rotor systems. For the security and stability of the rotating machinery, it is expected to acquire breakthrough on the feature extraction and the identification of the running status, especially on the weak feature extraction for the rotor vibration signal with non-linear and non-stationary properties under the strong noise condition.Consequently, a Bently RK4 rotor test bench is adopted in our research. The main framework is given in A typical fault of rubbing is designed in our research to verify the feasibility of PSEEMD. Rubbing is defined as the impact when the interval between the static part and spindle is tiny, and this fault can be yielded by a supported design. The installation of the rubbing support is shown in The rubbing rotor vibration signal in the time-domain and the frequency-domain are shown in According to the waveform of the rubbing signal in the time-domain, it is difficult to find detailed information of features due to the interference of the strong noise, except the global oscillatory amplitude. Although the primary component distribution can be seen in the frequency-domain, 2X, 3X and 4X are exactly the weak features, especially 4X. This information indicates non-linear and non-stationary complexity of the rubbing features. In order to solve this feature extraction problem and manifest the feasibility of PSEEMD, decomposition of the rubbing signal by PSEEMD has been computed, as shown in From the two figures, it can be noted that MIMF1 and MIMF2 represent high frequency noises lying in the first two regions. MIMF3, MIMF4, MIMF5 and MIMF6 are corresponding to 4X, 3X, 2X and 1X, respectively, when MIMF6 and MIMF5 exhibit great smoothness of sinusoidal wave. Furthermore, MIMF4 and MIMF3 show inferior smoothness but still represent primary features. This distinction is due to the noise strength. Taking MIMF6 and MIMF3 as an example, the 1X of MIMF6 is from those of IMF5 and IMF4, which are far away from the strong noise region. Nevertheless, the 4X of MIMF3 is directly from that of IMF2, which is characterized with strong noise. Note that MIMFs 3–6 are almost equal to the four marked components in For comparison, the rubbing signal is also analyzed, both by the original EEMD and the optimized EEMD, where the original amplitude of added noise is equal to 1.09 and the optimized one is 1.26. These two kinds of decompositions are shown in ε only enables some information transfer rather than eliminating mode mixing. Neither the original EEMD nor the optimized EEMD can accurately extract the independent information from the investigated signal.Comparing the original IMFs in ON the basis of the analyses above, it seems that PSEEMD is more accurate than the original EEMD and the optimized EEMD in extracting the independent and weak information. For final confirmation of PSEEMD, MIMFs 3–6 are applied for reconstructing the rubbing signal. The result depicted in Compared with the original rubbing signal, the reconstructed signal retains the rubbing features of 1X, 2X, 3X and 4X; it eliminates the annoying noise and needless compositions due to the independent and weak information extraction capability of PSEEMD. The four components of 1X, 2X, 3X and 4X contained in the investigated signal are truly represented by the four single, intact and independent MIMFs. As a result, PSEEMD can still accurately extract the features independently and eliminate mode mixing although the complexity resulting from the non-linear and non-stationary features increases significantly compared with that of the simulation signal.Style mixing can be eliminated.The change in the amplitude of added noise is robust.The weak features can be extracted independently.The advantages of (1)–(3) are realized under the condition of suffering from strong noise.PSEEMD may have the generalization capability. It is expected to apply PSEEMD in analyzing non-linear and non-stationary signals in other fields.The independent extraction model of mixing features reconstructed in phase space named PSEEMD has been proposed to improve mode mixing of EEMD and realize the independent extraction of the frequency information. PSEEMD has been applied in vibration analysis of rotating machinery. Moreover, the theoretical analyses, the model construction, and the application have been performed. The following advantages of PSEEMD can be obtained.Further study on the verification of PSEEMD application in rubbing coupled with other faults should be conducted.


Nigella sativa seeds, on morphine dependence and tolerance were investigated in mice. Dependence and tolerance are major restricting factors in the clinical use of opioid analgesics. In the present study, the effects of thymoquinone, the major constituent of Male adult NMRI mice were made tolerant and dependent by repeated injections of morphine  during a 3-day administration schedule. The hot-plate test was used to assess tolerance to the analgesic effects of morphine. Naloxone  was injected to precipitate withdrawal syndrome in order to assess the morphine dependence. To evaluate the effects of thymoquinone on tolerance and dependence to morphine, different single or repeated doses of thymoquinone were administered in mice. Rotarod was used to assess the motor coordination.Administration of single or repeated doses of thymoquinone  significantly decreased the number of jumps in morphine dependent animals. Repeated administration of thymoquinone  and also single injection of thymoquinone  attenuated tolerance to the analgesic effect of morphine. None of the thymoquinone doses  produced any antinociceptive effects on their own. Motor coordination of animals was impaired by the high dose of thymoquinone (40 mg/kg).Based on these results, it can be concluded that thymoquinone prevents the development of tolerance and dependence to morphine. Physical dependence to opioids and tolerance to their analgesic effects, are major problems limiting the clinical use of opioids , a member of the Ranunculaceae family, is emerging as a unique herb with outstanding therapeutic effects. N. sativa, has been traditionally used for the treatment of respiratory, gastrointestinal, kidney, and liver diseases. Many medicinal properties of N. sativa have been attributed to its seeds extracts and/or oil . The animals were housed in plastic cages and kept at 23±2 °C on a 12 hours light/dark cycle at least 7 days prior to testing. Commercial food pellets and tap water were freely available at all times except during the experiments.All procedures and experiments were carried out in accordance with institutional guidelines for laboratory animal care and use.DrugsThe following drugs were used in this study: thymoquinone (Sigma-Aldrich), morphine sulphate , naloxone hydrochloride , and diazepam . Drugs were dissolved in normal saline. Thymoquinone was suspended in 0.8% (v/v) tween 80. All compounds were prepared freshly and administered intraperitoneally (i.p.) in a volume of 0.1 ml/20 g body weight. Control animals received the same volume of vehicle . Induction of dependence and toleranceIn order to induce dependence and tolerance in mice, morphine was administered intraperitoneally (i.p.) 3 times per day at 9 a.m. (50 mg/kg), 1 p.m. (50 mg/kg), and 5 p.m. (75 mg/kg) for 3 consecutive days. The dose of 75 mg/kg at 5 p.m. was administered to prevent overnight withdrawal syndrome in animals. The last dose of morphine (50 mg/kg) was administered on the fourth day at 9 a.m. : Group 1 (negative control) received morphine and vehicle . Groups 2, 3, and 4 received morphine and different single doses of thymoquinone  on day 4. Groups 5, 6, and 7 received morphine and different doses of thymoquinone  three times per day on days 1, 2, and 3, but not on day 4. Groups 8 and 9 (positive controls) received morphine and diazepam (4 mg/kg).Two hours after the injection of the last dose of morphine on the fourth day, naloxone , the selective antagonist of opioid receptors, was injected to the animals (groups 1-9) to precipitate withdrawal syndrome. Mice were immediately placed individually in a plexiglass box and the number of jumps, as the major sign of withdrawal syndrome, was recorded during a 30-min period  were administered 30 min before the injection of naloxone  in mice on the fourth day. These animals (groups 2-4) did not receive thymoquinone before the injection of morphine during the 3-day administration schedule.In order to assess the effects of thymoquinone on the induction of physical dependence to morphine, different doses of thymoquinone  were administered in mice 30 min before the injection of morphine during the 3-day administration schedule. These animals (groups 5-7), did not receive thymoquinone on the fourth day.Evaluation of tolerance to morphineThe animals were divided into seven groups (n=7 each): Group 10 (control) received morphine and vehicle . Groups 11, 12, and 13 received morphine and different single doses of thymoquinone  on day 4. Groups 14, 15, and 16 received morphine and different doses of thymoquinone  three times per day on days 1, 2, and 3, but not on day 4. Moreover, the analgesic effect of different single doses of thymoquinone  was evaluated on 3 more groups of mice (groups 17-19) to determine whether or not, thymoquinone possesses antinociceptive effect on their own. These animals did not receive any other drugs.In order to assess the development of tolerance to analgesic effects of morphine in control or thymoquinone-treated mice, the hot-plate test was used. Thirty minutes after the injection of morphine  on the days 1 and 4, the mice were placed on a metal plate maintained at 55±1 °C and the latency of nociceptive responses including licking or flicking of the limbs or jumping were measured in the animals. A cut-off time of 45 s was selected to prevent tissue damage. The latency of nociceptive responses in mice was expressed as the hot-plate latency  were administered 30 min before the injection of the last dose of morphine  on the fourth day. These animals (groups 11-13) did not receive thymoquinone before the injection of morphine during the 3-day administration schedule. Moreover, to assess the effects of thymoquinone on the development of morphine tolerance, different repeated doses of thymoquinone  were administered 30 min before the injection of morphine during the 3-day administration schedule. These animals (groups 14-16) did not receive thymoquinone before the injection of morphine on the fourth day. Evaluation of motor coordination The effects of thymoquinone and diazepam on motor coordination was assessed using the rotarod apparatus . In this test, separate groups of animal were used. On the first day, mice were trained to remain for 5 min on a rod rotating at the initial and final speed of 10 and 20 rpm, respectively (the acceleration time was 20 sec). On the next day, 60 and 90 min after the administration of the different doses of thymoquinone  or diazepam , the animals were placed on the rotating rod and the length of time each animal remained on the rotating rod (time-on-rod) was recorded . In the evaluation of motor coordination, none of the animals received morphine. Statistical analysisThe data were expressed as mean values ± S.E.M for 7 mice and tested with analysis of variance (ANOVA) followed by the multiple comparison test of Tukey. Differences between means were considered statistically significant if p<0.05. Effects of thymoquinone on morphine dependenceRepeated administration of morphine induced physical dependence in mice in control group. These animals (group 1) received vehicle  30 min before each dose of morphine during the 3-day administration schedule. Administration of naloxone  two hours after the injection of the last dose of morphine  on the fourth day, precipitated the withdrawal syndrome in control group so that the jumping behavior was significantly increased in mice , 2. Administration of single doses of thymoquinone  30 minutes before the injection of naloxone  on the fourth day, significantly decreased the number of jumps in morphine-dependent animals in groups 3 and 4  . MoreoveEffects of thymoquinone on morphine toleranceAnalgesic effects of morphine following repeated administration during 3 days, decreased significantly in the hot-plate test (p<0.001). However, repeated administration of thymoquinone  30 minutes before the injection of morphine during 3 days, significantly attenuated the development of tolerance to analgesic effects of morphine in groups 15 and 16  .Moreover, administration of single dose of thymoquinone  30 minutes before the injection of the last dose of morphine  on the fourth day, prolonged the latency of nociceptive response in hot-plate test  . HoweverThe attenuating effect of thymoquinone on morphine tolerance was not attributed to the analgesic effects of thymoquinone, because none of the thymoquinone doses  produced any antinociceptive effects on their own . Effects of thymoquinone on motor coordination In rotarod test, 30 minutes after the administration of thymoquinone (40 mg/kg), the remaining time of animals on the rotating rod (time-on-rod) decreased significantly (p<0.05). Thymoquinone at the doses of less than 40 mg/kg (10 and 20 mg/kg) did not have any effects on motor coordination of mice in rotarod test . MoreoveThe results of the present study showed that thymoquinone attenuated the development of tolerance to analgesic effects of morphine. Thymoquinone also prevented the induction of physical dependence to morphine and attenuated the severity of the signs of withdrawal syndrome in morphine dependent mice. N. sativa oil prevented tolerance and dependence induced by morphine and tramadol in mice (Abdel-Zaher et al., 2010N. sativa oil, it seems that the inhibitory effects of N. sativa on opioid-induced tolerance and dependence arise from the beneficial effects of thymoquinone. It has recently been reported that In the present study, repeated administration of morphine over the course of 3 days, induced physical dependence in mice so that the injection of naloxone elicited the signs of withdrawal syndrome . This prThe results of the present work showed that the pretreatment of animals with thymoquinone during the 3-day administration of morphine, attenuated the signs of withdrawal syndrome precipitated by naloxone on the fourth day . MoreoveThe results of this study showed that thymoquinone could also be efficacious against tolerance to analgesic effect of morphine. Following repeated administration of morphine over the course of 3 days, the pain threshold in animals decreased in hot-plate test. As it is shown in Tolerance to opioid analgesics, defined as a loss of analgesic effect following repeated treatments such that a higher dose of the drug is required for equivalent effect. This may also lead to the opioid dependence and further complicate the process of pain treatment (Alldredge et al., 2013A receptors (Hosseinzadeh and Parvardeh, 2004A receptors and muscimol as a selective agonist of GABAA receptors, prevent morphine dependence and suppress the signs of withdrawal syndrome in morphine-dependent mice (Tejwani et al., 1993A receptors. Various studies have also shown that some pharmacological effects of thymoquinone including antianxiety and anticonvulsant effects may be attributed to its stimulating effect on the central GABAOn the other hand, the role of calcium currents through voltage dependent calcium channels has been established in tolerance and dependence to opioids (Dogrul et al., 2002N. sativa, are among the most well-studied antioxidant compounds. Many lines of studies have indicated that thymoquinone functions as a potent antioxidant and radical scavenger (Khan et al., 2012N. sativa oil attenuates tolerance and dependence to morphine and tramadol through inhibiting the activity of inducible NOS enzyme (Abdel-Zaher et al., 2010Thymoquinone and its parent herbal medicine, As it is shown in Based on the results of this study, it can be concluded that thymoquinone attenuates dependence and tolerance to morphine in mice. The current study provides evidence for the effectiveness of applying thymoquinone to prevent tolerance to analgesic effects of morphine and to attenuate the acute signs of morphine withdrawal syndrome.