Expression of caspase 1 and histomorphology of lung after cladribine treatment.

BACKGROUND: Cladribine is a useful immunosuppressive drug for the treatment of autoimmune diseases, leukemias and multiple sclerosis (MS). Despite the drug having low toxicity, side effects have been reported connected with myelosuppression, neutropenia and severe anemia. OBJECTIVES: The objective of this study was to investigate the influence of cladribine on lung pathomorphology and the expression of caspase 1 using immunohistochemistry method. MATERIAL AND METHODS: The study was conducted on Wistar rats, which were divided into a control group (C) and an experimental group (E). In group C, the rats were given a 0.9% NaCl solution by a subcutaneous injection, at the same dose as the dose of drug used in the experiment. In group E, the animals received cladribine at a dose of 0.07 mg/kg/24 h by a subcutaneous injection. The animals were decapitated 24 h following the last dose. To detect collagen deposition, we utilized Masson's trichrome staining. To evaluate the intensity of the inflammatory process in the lung, an immunohistochemistry reaction was carried out with the use of caspase 1. RESULTS: In group E, we observed an increase in the thickness of space between the alveoli. A statistically significant (p < 0.017243) difference between the thicknesses of the interalveolar septum was seen between the research groups. In E group, we observed regions with collagen deposition, alveolar epithelial cell hyperplasia, hyperemia and inflammatory cell infiltration. Caspase 1 activity was higher in group E. The immunohistochemical reaction with caspase 1 was positive in 49% of all the interalveolar cells in group E; however, in group C about 13% of the interalveolar cell showed positive immunohistochemistry (IHC) response. CONCLUSIONS: Cladribine-based therapy might have negative influence on lung morphology. The interstitial changes in the lung tissue suggest that cladribine is a drug that may be the cause of drug-induced lung disease and may lead to several respiratory disorders.

CB2R agonist prevents nicotine induced lung fibrosis.

INTRODUCTION: Nicotine stimulates fibroblast proliferation while increasing inflammation and fibrosis of tissues. The cannabinoid receptor 1 (CB1R) is mainly located in the CNS, while cannabinoid receptor 2 (CB2R) is located in the immune cells within the body. CB2R regulates inflammatory processes and fibroblast function. PURPOSE: We investigated the impact of CB2R agonist, JWH 133 and the antagonist, AM630 on lung tissue, applied directly before nicotine application. MATERIAL AND METHODS: 40 mice were placed into 4 groups. The experimental groups received nicotine intraperitoneally at a dose of 0.05 mg/kg of body weight (BW) for 14 days. Group B also received AM630 (0.5mg/kg of BW), while Group A was administered with JWH133 (1 mg/kg of BW). Group N received nicotine alone. The Control group C received 0.9% NaCl. After decapitation, lung tissues were stained with H&E, Trichrome Masson's method, and IHC against CTGF and α-SMA. The digital image processing system Image J with the IHC profiler plugins was then employed, optical density and IHC optical density score were calculated. RESULTS: In the N group, an increase in the thickness of alveolar spaces (9.16 SD4.95µm vs. 4.77SD2.99µm in the C group), leukocytes infiltration and collagen deposition has been observed(OD: 0.20 SD0.0vs 0.07SD0.04 in the C group). In the B group, the alveolar space thickness has been the highest (11.57SD8.13µm). Furthermore, in this group, hyperaemia, destruction of lung structure, hyperplasia of II type pneumocyte and interstitial fibrosis has been observed (OD: 0.23 SD0.08). In contrast, the lung tissue of the A group has had normal structure and the thinnest alveolar septum (3.88 SD2.64µm). The expression of CTGF and α-SMA has been the highest in the B group. CONCLUSION: Nicotine induces interstitial lung fibrosis that is enhanced by the CB2R antagonist and diminished by the CB2R agonist. Therefore, the CB2R agonist may offer a protection against fibrosis.

Intrinsic apoptosis pathway in fallopian tube epithelial cells induced by cladribine.

Cladribine is a purine nucleoside analog which initiates the apoptotic mechanism within cells. Moreover, the available data confirms that cladribine, with the participation of the p53 protein, as well as the proapoptotic proteins from the Bcl-2 family, also induces the activation of the intrinsic apoptosis pathway. However, while there has been a lot of research devoted to the effect of cladribine on lymphatic system cells, little is known about the impact of cladribine on the reproductive system. The aim of our study was to evaluate apoptosis in oviduct epithelial cells sourced from 15 different female rats. In so doing, the sections were stained with caspases 3, 9, and 8. Results suggest that cladribine also induces apoptosis in the oviduct epithelial cells by way of the intrinsic pathway. Indeed, the discontinuing of the administration of cladribine leads to a reduction in the amount of apoptotic cells in the oviduct epithelium.

P53 protein in proliferation, repair and apoptosis of cells.

The p53 protein is an important factor of many intra- and extracellular processes. This protein regulates the repair of cellular DNA and induces apoptosis. It is also responsible for the regulation of the senescence and the cell entering the subsequent stages of the cellular cycle. The protein p53 is also involved in inhibiting angiogenesis and the induction of oxidative shock. In our study, we examined the activity of p53 protein in the uterine epithelial cells in rats treated with cladribine. Its action is mainly based on apoptosis induction. We compared the activity of p53 protein in cells with a high apoptosis index and in cells with active repair mechanisms and high proliferation index. We observed stronger p53 protein expression in the epithelial cells of the materials taken 24 h after the last dose of 2-CdA associated with the active process of apoptosis and inhibition of proliferation. After 4 weeks from the last dose of cladribine, the stronger expression of p53 protein was associated with both the existing changes in the cell's genome, the effects of the ongoing repair mechanisms, as well as the high proliferation activity.

Histomorphometry of nuclei of proximal convoluted tubules epithelium of kidney of experimental animals after Cladribine (2-CdA) administration.

The area of the section of nuclei of the epithelium cells lining the proximal convoluted tubules of the kidney of white Wistar rats was examined. The animals were given Cladribine (2-CdA) subcutaneously at the dosages of 0.07 mg/kg b.w./24 h for seven days and 0.1 mg/kg b.w./24 h for 6 days in three courses with 5 weeks' break between each. The animals were killed in each instance, 24 hours after the last dose of the drug and 4 weeks after the last dose. The mean area of the section of the cell's nuclei was measured in projection microscope. Results of the examination were counted statistically. In experimental groups I and III the surface areas of the proximal tubules epithelium nuclei cross-sections are smaller than in the control grous. In the experimental groups II and IV the surface areas of the proximal tubules epithelium nuclei cross-sections are bigger than in the control group.

Histomorphology of renal corpuscles of kidney of experimental animals after Cladribine (2-CdA) administration.

The renal corpuscles of the kidney of white Wistar rats was examined. The animals were given Cladribine (2-CdA) subcutaneously at the dosages of 0.07 mg/kg b.w./24 h for 7 days and 0.1 mg/kg b.w./24 h for 6 days in 3 courses with 5 weeks' break between each. The animals were killed in each instance, 24 hours after the last dose of the drug and 4 weeks after the last dose. The kidney samples were taken for histological and histochemical examination, then stained with hematoxylin and eosin, using the Masson's, PAS, and Feulgen's methods. In all of the groups, changes were observed but the intensity differed, with widening or narrowing of the urinary space, thickening of the basement membrane of the parietal layer of the Bowman's capsule and the basement membrane of capillaries, and density changes in capillary vessels. Hyperaemia in renal glomeruli and in all parenchyma, and infiltrations around the tubules and renal glomeruli, were also observed.

Histomorphology of renal proximal convoluted tubules of kidney of experimental animals after Cladribine (2-CdA) administration.

The proximal convoluted tubules of the kidney of the white Wistar rats were examined. The animals were given Cladribine (2-CdA) sub-cutaneously at the dose: 0.07 mg/kg b.w./24 h for 7 days and 0.1 mg/kg b.w./24 h for 6 days in 3 courses, with 5 weeks' break between each. The animals were killed in each instance 24 hours after the last dose of the drug, and 4 weeks after the last dose. The kidney's samples were taken for histological and histochemical examination and were stained with hematoxylin and eosin, using the Masson's, the PAS's, and the Feulgen's method. In experimental group I, we observed few changes (in comparison to the control group): cells of the epithelium of some of the proximal convoluted tubules were however, puffy. In a few tubules we observed some unknown substance and the lumen of some of these tubules was narrowed. In experimental group II, a few proximal convoluted tubules and their lumen were wider with an unknown substance within. We also observed hydropic degeneration. The brush border of these tubules was a little lower than in control group. In experimental group III, the cells of the epithelium of proximal convoluted tubules rested on a thicker basement membrane, the lumen of most of the proximal convoluted tubules being narrow and filled with some unknown substance. In experimental group IV, the lumen of the tubules was a little wider, and the epithelial cells were smaller than in the control group, thus the lumen of the tubules was wider. In the cytoplasm of epithelial cells we observed numerous PAS(+) granules. The low brush border appeared damaged.

The ultrastructure of kidney renal corpuscles of experimental animals after Cladribine (2-CdA) administration.

The renal glomeruli of the kidney of white Wistar rats were examined. The animals were given Cladribine (2-CdA) subcutaneously at dosages of 0.07 mg/kg b.m./24 h for 7 days and 0.1 mg/kg b.m./24 h for 6 days in 3 courses with 5 weeks' break between each. The animals were killed in each instance, 24 hours after the last dose of the drug or 4 weeks after the last dose. The kidney samples were taken for ultrastructural examination. In all of the groups, changes were observed but the intensity differed: widening or narrowing of the urinary space, thickening of the basement membrane of the parietal layer of the Bowman's capsule and the basement membrane of capillaries, and density changes in capillary vessels as well as infiltrations around the renal glomeruli. Most changes were observed in experimental group IV: the picnotic nuclei of epithelium, widening and fusion of the foot processes of podocytes (the narrowing of the filtration spaces) and numerous invaginations of the nuclear envelope of damaged podocytes.

Ultrastructure of kidney renal proximal convoluted tubules of experimental animals after Cladribine (2-CdA) administration.

The proximal convoluted tubules of the kidney of white Wistar rats were examined. The animals were given Cladribine (2-CdA) subcutaneously at dosages of 0.07 mg/kg b.m./24 h for 7 days and 0.1 mg/kg b.m./24 h for 6 days in three courses with 5 weeks' break between each. The animals were decapitated 24 hours after the last dose of the drug and 4 weeks after the last dose. The kidney samples were taken for ultrastructural examination. Giving Cladribine at the dose of 0.1 mg/kg b.m./24 h for 7 successive days does not lead to instant changes in the ultrastructure of the proximal convoluted tubules. Changes noticed 4 weeks after Cladribine administration are the following: decrease in amount of mitochondria, the presence of numerous vacuoles, changes in the structure of the brush border, presence of numerous glycogen granules, and the presence of a diluted cytoplasm around the nucleus. Giving 2-CdA at the dose of 0.07 mg/kg b.m./24 h for 6 days in three courses leads to similar changes in proximal convoluted tubules with more extensive damages of the brush border. These were no more intensive after 4 weeks' break in Cladribine administration.

Histological and ultrastructural changes in cross-striation muscle cells, under the influence of atorvastatin-reductase HMG-CoA inhibitor.

The investigation was carried out on 15 Wistar rats--males weighing about 200 mg each. The animals were divided into three groups: one control group and two experimental groups, with five animals in each. In experimental group I the animals received emulsion of Atorvastatin in distilled water at the therapeutical dose of 80 mg/kg of body mass, by stomach tube for 6 weeks. In experimental group II the animals received atorvastatin at the maximal dose of 800 mg/kg of body mass. Skeletal muscles of experimental animals (rats) after 6 weeks' administration of atorvastatin in therapeutical and maximal dosages did not show any essential differences in comparison with the control group, when examined under light microscope. Degenerative changes were observed after Atorvastatin administration, when examined under electron microscope. These changes were dependent upon dosage and were directly proportional to dosage rate. Six-week administration of Atorvastatin in the therapeutical dose (80 mg/kg b. m.) produced invagination of the nuclear envelope into the cell nucleus, and within the cytoplasm, numerous vacuoles, some of which included the myelin structures, were evident. Atorvastatin administration in maximal dosage (800 mg/kg) under electron microscope examination, showed the following differences: the appearance of numerous vacuoles in the perinuclear spaces, and between myofibrils; dilation of mitochondria; disintegration of sacomers; fibrinosis within the intercellular spaces.

Histological picture of intestinal mucosa of rats after simultaneous administration of atorvastatin and ethyl alcohol.

The rats received atorvastatin in the therapeutical dose and 10x higher, 20% solution of ethanol and ethanol with atorvastatin for 6 weeks. The microscopic investigations (H + E staining, Masson's method, PAS method by McManus) showed no changes in histological picture of intestinal mucosa after giving atorvastatin. The rats receiving alcohol and alcohol with atorvastatin showed similar changes, but different than the first groups. Under the epithelium of intestinal villi there were observed light, vesicle-like spaces, separation of epithelium from connective tissue stroma, also lack of the epithelium of the top of villi and hyperaemia of stroma of villi. The observed changes were stronger in rats receiving simultaneously alcohol and atorvastatin in the dose 10x higher than therapeutical. Hyperaemia of connective tissue of stroma of villi was still visible after month period of administration.

Investigations of ultrastructure of damaged and regenerated skeletal muscle fibers.

Our investigations concerned the head of the parietal part of quadriceps femoris, and we based our investigation on observations of the ultrastructure of muscle fibers using an electron microscope. We observed tissue samples taken from patients (10 men) 25-35 years old, who had old damage of knee joint ligament (after about 6 week's immobilization). In the first group, segments of tissue of parietal head of quadriceps femoris were taken inter-operationally from patients in whom there was found old damage of knee joint ligament. The second group was of tissue segments of this muscle after surgical repair of knee and rehabilitation, which consisted in power training using resistance machines. The muscle fiber samples of quadriceps femoris which were taken from patients during the first operation, showed big changes in their ultrastructure. These changes included: myofibrils disintegration; disturbance of regularly arranged striation in sarcomers; dissappearance of Z line. In the sarcoplasm, we observed large vacuolisation, and in the interfibrillar spaces--an accumulation of exudate and morphotic elements of blood outside the capillary vessels. Observations of muscle tissue after regeneration, showed a big improvement in the muscle cell's ultrastructure--the myofibrils were regularly arranged, and the sarcomers striations showed no deviations from normal structure. We also observed a considerable increase in the number of properly formed ultrastructure mitochondria when compared with the first group.

The proximal convoluted tubule of rats' nephron after experimental administration of gentamicin.

The rats in this experiment received gentamicin in intraperitoneal injections in a dose ten times bigger than therapeutical. After ten days of drug administration, in the cells of proximal convoluted tubules of kidney there were observed the dilution of cytoplasm of various intensity, increases in amount and size of lysosomes, widening of endoplasmic reticulum tubules and swelling of mitochondria. The microscopic and ultrastructural pictures, after three weeks' break in gentamicin administration, showed fast regeneration of renal tubules. A large diversity of intensification of changes in particular individuals suggested individual reaction to gentamicin nephrotoxicity.

Histological examination of the submandibular gland following experimental administration of Metizol.

The submandibular gland of the white Wistar rats was examined. The animals were given Metizol for 21 days and 42 days at the dose of 1 mg/kg b.m./24 h. The submandibular gland samples were taken for histological and histochemical examination. Then they were stained with hematoxylin and eosine as well as by Masson's, PAS's and Feulgen's method. The mean area of section of cell nuclei was measured. The results of examination were counted statistically. The following changes were noticed: After 21 days of administration of Metizol in the submandibular gland the mean area of the tubules was increased. The quantity of tubules increased as well. In the tubules cells some more secretion was noticed. The follicles shrank. After 42 days of administration of Metizol the appearance, number and stainability of the tubules and follicles were similar to control group.

Histological examination of the Loeventhal gland after experimental administration of Metizol.

The Loeventhal gland of the white Wistar rats was examined. The animals were given Metizol for 21 days and 42 days at the dose of 1 mg/kg b.m./24 h. The Loeventhal gland's samples were taken for histological and histochemical examination. Then they there stained with hematoxylin and eosin, Masson's, PAS's, and Feulgen's method. The mean area of section of cell nuclei was measured. Results of examination were counted statistically. The following changes were noticed: after 21 days of administration of Metizol in the Loeventhal gland the mean area of the section of cells nuclei was decreased; after 42 days of administration of Metizol the mean area of the section of cell nuclei was decreased as well, but to a lesser degree than in group 1. New follicles appeared which can be the expression of cell mitotic activity.