10 Horrific Animal Experiments
Robinson-AnellierungIm orthotopen murinen Blasentumormodell erfolgte eine intravesikale Tumortherapie mit BCG bei gleichzeitiger Antibiotikatherapie. Nach Primärinfektion überlebten die mit Chinolonen behandelten Mäuse signifikant länger als Mäuse der Kontrollgruppe, unabhängig von einer Steroid-Gabe. Experimdnts intravesikale Therapie mit BCG beim orthotopen Blasentumor führte zu einem signifikant verringerten Tumorgewicht im Vergleich zur unbehandelten Kontrollgruppe. Steroid experiments with Effekt wurde durch zusätzliche Gabe von Antibiotika experimehts kompromittiert. Die alleinige Dauergabe von Steroid experiments with während der intravesikalen BCG-Therapie welches zink fur testosteron oyuncular? zu einer signifikant erhöhten Absterberate durch chronische Immunsuppression.
Experimentelle Untersuchungen zur optimalen Therapie der systemischen BCG-Infektion in vivo
Toyka; T-cell apoptosis in situ in experimental autoimmune encephalomyelitis following methylprednisolone pulse therapy, Brain , Volume , Issue 7, 1 July , Pages —, https: The apoptosis-inducing effects of i.
Two pulses of methylprednisolone were given at the peak of mild and of severe disease. We also studied the expression of bcl-2, a typical anti-apoptotic regulatory protein, in T cells, and found no change after methylprednisolone treatment compared with controls. Methylprednisolone did not induce apoptosis of oligodendrocytes, which would have been an unwanted side effect in CNS cells. Our results may have implications for the use of glucocorticosteroids at very high doses in the treatment of inflammatory disorders of the CNS, such as multiple sclerosis, or of other target organs.
Glucocorticosteroids GS are potent anti-inflammatory drugs, and are commonly used in the treatment of autoimmune disorders. Their therapeutic efficacy has been established in immune neuropathies Dyck et al. The pharmacological effects of GS are based on several mechanisms, leading to the modulation of cell activation, cytokine expression, the secretion of inflammatory mediators, leucocyte migration and the reduction of local oedema Goldstein et al.
Furthermore, GS induce apoptotic cell death in susceptible cell types. Wyllie was the first to describe this effect for cultured thymocytes Wyllie, T-cell apoptosis in situ after i. GS treatment was shown recently in experimental autoimmune neuritis Zettl et al. At higher doses, GS can exert proapoptotic effects by other mechanisms, such as binding to membrane receptors with the consecutive activation of a second messenger system, and by affecting the physicochemical properties of the cell membrane Brann et al.
GS-mediated apoptosis was shown to be blocked by the antiapoptotic protein bcl-2 in vitro in thymocytes Ivanov and Nikolic-Zugic, and partially blocked in human leukaemia cells Hartmann et al. Several clinical trials have investigated high-dose GS treatment in inflammatory diseases of the nervous system. Milligan demonstrated a clinical benefit from a 5-day course of i. In acute optic neuritis, a 3-day course of mg i.
There is now evidence that high-dose GS therapy with mg MP i. Oestrogens, another group of steroid hormones, have attracted interest as possible therapeutic agents since higher concentrations of these hormones during pregnancy have been linked to lessened disease activity in female multiple sclerosis patients, and possibly a milder course of the disease Damek and Shuster, Here we formally investigated whether, in terms of induction of T-cell apoptosis in situ , maximal doses of MP are superior to the high doses previously used in the rat adoptive transfer-experimental autoimmune encephalomyelitis AT-EAE model and also in human therapy.
For all experiments, female Lewis rats, aged 6—8 weeks and with a body weight of — g, were obtained from Charles River, Sulzfeld, Germany. Animals were housed in plastic cages without grid floors and were given commercial food pellets and water ad libitum.
All experiments were carried out in accordance with Bavarian state regulations for animal experimentation and were approved by the responsible authorities.
In vitro studies were performed on rat thymocytes. Apoptosis was studied in vitro following a previously reported method Gold et al. Encephalitogenic T cells for in vivo experiments were generated and maintained as described in detail previously Gold et al.
Seventy-two hours later, activated T-cell blasts were separated from cell debris by centrifugation on Ficoll gradients Nycomed AS, Oslo, Norway. Disease onset with first clinical signs was on day 2 severe or 3 mild ; a maximum of grade 2—3 was reached in mild EAE and grade 4—5 in severe EAE.
The time schedule essentially followed a protocol that had been established in earlier studies Zettl et al. All experiments [except high-performance liquid chromatography HPLC analysis] were performed in groups of six animals each. At the peak of the disease, on day 4 or 5, the animals were injected i. The spinal cord was removed, postfixed for 1 h in the same fixative, cut into 5 mm slices, dehydrated and embedded in paraffin.
Five micrometre cross-sections were deparaffinized with xylene and rehydrated. Immunohistochemistry was performed using a monoclonal mouse antibody to a pan-T-cell antigen B —1; Holland Biotechnology, Leiden, Netherlands diluted 1: To detect disruption of the blood—brain barrier, we used an anti-albumin antibody Nordic, Bochum, Germany diluted 1: Apoptotic T cells were identified by in situ tailing as described Gold et al.
Tissue sections were deparaffinized, rehydrated and treated with chloroform for 1 s. To label apoptotic cells in the tissue, we used the Boehringer detection kit, following the instructions given by the supplier.
For T-cell immunohistochemistry we followed the protocol given above, except that alkaline phosphatase-based detection was used with New-fuchsin DAKO as the chromogenic substrate.
Sections were mounted in an aqueous mounting medium Aquatex; Merck, Darmstadt, Germany. Preincubation in the microwave oven W was performed by boiling in 10 mM sodium citrate buffer, pH 6. After they had been cooled to room temperature, sections were washed thoroughly with distilled water followed by TBS.
As the secondary antibody, we used a biotinylated goat anti-rabbit IgG antibody Vector, via Linaris, Wertheim, Germany , diluted 1: Analysis of inflammatory infiltrates and of apoptosis was performed by an observer blinded to the treatment, who rated 10 visual fields 1.
Apoptosis was assessed by morphological criteria Wyllie, or labelling by in situ tailing. Apoptotic oligodendrocytes were determined in 0. Semiquantitative analysis of disruption of the blood—brain barrier was performed as described recently Morrissey et al. For the analysis of MP concentration in serum, CSF and spinal cord, groups of three or four rats were injected once, 2 h before lethal anaesthesia with CO 2.
First, blood was drawn by cardiac puncture. Then CSF was collected by puncture with a 1 ml syringe and 0. With few exceptions, samples were clear and not contaminated with blood.
The spinal cord was removed for tissue analysis. All probes were placed immediately on dry ice. Two calibration ranges were used: In vitro experiments showed a rate of For in vivo studies, cells were taken from the same restimulation cycle and the cell concentration was adjusted beforehand to standardize the severity of disease. This experiment in mild EAE was repeated with similar results data not shown. The following experiments were done to study very high doses, i.
These results were reproduced in another set of experiments in animals with severe EAE data not shown. Next, we studied high-dose therapy in mild EAE Fig. The reduction in T-cell infiltration did not reach statistical significance. Similar results were obtained when the experiment was repeated data not shown. The efficacy of oestrogens was studied in severe EAE.
Since this treatment was not well tolerated and animals exhibited signs of cardiorespiratory dysregulation, we performed no further experiments with higher doses of fosfestrol or with other oestrogens data not shown. In order to show that the effects on T-cell apoptosis were indeed related to tissue concentrations of MP, we measured ex vivo concentrations in different compartments. For these studies, the rats were injected only once and sampling was performed 2 h later to achieve the presumed peak concentration Kaiser and Kley, In both experiments, controls received i.
In the first experiment we compared three groups: The rates of bcl-2 positive T-cell infiltration in the spinal cord or spleen were similar for the different treatments Table 2 and Fig. To investigate the effects of MP on other cells in the CNS that are susceptible to apoptosis, we studied oligodendrocytes.
The rate of apoptosis was assessed on the basis of morphological criteria using CNPase immunohistochemistry. There was no increase in oligodendrocyte apoptosis 18 or 48 h after two i.
Here we have demonstrated that high-dose i. This effect was dependent on the dose of GS and the severity of the disease: Also, the synthetic oestrogen fosfestrol did not increase the rate of apoptosis. Probably as a consequence of increased T-cell apoptosis, inflammatory T-cell infiltration was markedly reduced. A study of the regulatory protein bcl-2 did not show a notable change in the number of bclpositive T cells shortly after injection.
There were no unwanted side-effects on glial cells such as oligodendrocytes. We might have seen effects of the decrease in inflammation on the clinical course if we had followed the animals for a longer period into recovery, but this was not possible because of the experimental design.
No beneficial effect on the severity of the relapse was observed. A possible pharmacological mechanism to explain our findings is the higher level of MP that we found in the CSF and spinal cord, which could lead to qualitatively different effects of GS.
The effects of MP on T-cell apoptosis could have been due to interaction with apoptosis regulatory proteins Ivanov and Nikolic-Zugic, Investigation of bclexpressing T cells by immunohistochemical double labelling revealed no change for the antiapoptotic bcl-2 at any time after administration of i. These results are in accordance with a recent observation in which hydrocortisone-induced apoptosis in the murine thymus was not associated with altered bcl-2 expression Denis et al.
This may indicate the existence of factors regulating GS-induced T-cell apoptosis, such as the direct activation of caspases. To test the possibility that MP would increase the apoptosis not only of T cells but also of glial cells, we analysed oligodendrocytes immunohistochemically.
We noted that MP did not induce apoptosis of oligodendrocytes as an unwanted side-effect, even when we looked at late time points after MP administration. GS have been shown to exert their multiple actions via different pathways. In general, these mechanisms act at the genomic or non-genomic level. To date, it is unclear whether GS-mediated T-cell apoptosis can also be exerted by non-genomic actions. Non-genomic mechanisms of GS, such as the direct alteration of membrane fluidity, binding to specific cell membrane receptors and the use of cyclic adenosine monophosphate as a second messenger Rosner et al.
Another possible mediator of GS-induced apoptosis is calcium. At present it is unclear whether this mechanism also contributes to the effects of very high doses of MP. It remains unclear whether non-genomic actions of GS can contribute to their anti-inflammatory effects in the therapy of autoimmune diseases Buttgereit et al.
In humans, GSR are saturated at a dose of 2. Because of the different surface areas of humans and rats, in rats some drugs might need a dose up to 7-fold higher compared with humans Klaassen et al.
This depends on the drug as well as on the pharmacological effect investigated. In this respect, the dose-dependent induction of apoptosis shown here can help to understand the superior efficacy of very high dose MP therapy in multiple sclerosis.
Thieme E-Journals - Aktuelle Urologie / Abstract
Many translated example sentences containing "steroids" – German-English dictionary and search engine for German translations. relatively small dose of adrenal steroid can increase dramatically the survival of including lethal shock of varying etiology, both in experimental animals and . The patients can be on steroids or other additional compounds like creatine, .. University of California in Los Angeles reported on experiments to counteract.