Examination of Kynurenine Toxicity on Corneal and Conjunctival Epithelium: In vitro and in vivo Studies.

Kynurenine (KYN) is a metabolite of tryptophan, proposed for the treatment of corneal diseases. Our goal was to evaluate the effects of KYN on normal human corneal and conjunctival epithelial cells in vitro and the re-epithelization of corneal erosion in rabbits. In our study, we used corneal (10.014 pRSV-T) and conjunctival (HC0597) epithelium cell cultures. KYN was applied at a concentration range of 1-100 µM for 24 and 48 h. We examined the effects on cellular metabolism, viability, interleukin-1ß (IL-1ß), IL-6, IL-10 secretion, cytoskeleton organization and transwell migration ability. Following a bilateral corneal de-epithelialization, the rabbits received drops containing 1% KYN and a saline solution to the contralateral control eye, 5 times daily. Digital images were analyzed using the EPCO 2000 software. The metabolic activity of cells was slightly decreased by KYN in the corneal but not in the conjunctival epithelium. The viability of both epithelia was improved by KYN; it caused alterations in the secretion of IL-6 and IL-10 but not IL-1ß. It had no impact on both epithelia morphology and the organization of the cellular cytoskeleton. KYN stimulated the formation of pseudopodia projections in both epithelia in vitro, which may be important in terms of wound healing. However, there were no differences in the re-epithelization rate in vivo. At the tested concentrations, KYN was not toxic for the corneal and the conjunctival epithelium in vitro and did not affect corneal re-epithelization in rabbits in vivo. Our results suggest that KYN may be taken into consideration for the treatment of ocular disorders.

Tryptophan as a Safe Compound in Topical Ophthalmic Medications: In Vitro and In Vivo Studies.

BACKGROUND: To evaluate the effects of tryptophan (TRP) on normal human corneal and conjunctival epithelium in vitro and the re-epithelization of corneal erosion in rabbits. MATERIALS AND METHODS: Corneal epithelial cell (10.014 pRSV-T) and conjunctival epithelial cell (HC0597) cultures were used. The cellular metabolism, viability, secretion of IL-1ß, IL-6, IL-10, cytoskeleton organization, transwell migration were determined. Cells were incubated in the presence of TRP at 1-100 µM. After corneal de-epithelization rabbits received TRP drops (100 µM), 5 times a day. RESULTS: TRP increased conjunctival epithelium metabolism at 50 µM and increased the viability of corneal epithelium at 100 µM. TRP (10 µM) enhanced the production of IL-6 by the corneal epithelium and had no effect on IL-1ß and IL-10. CONCLUSIONS: TRP had no influence on the cellular cytoskeleton but induced a significant pseudopodia projection in both epithelia. TRP did not influence corneal re-epithelization in vivo. TRP was not toxic for corneal and conjunctival epithelia.

Kynurenic Acid Accelerates Healing of Corneal Epithelium In Vitro and In Vivo.

Kynurenic acid (KYNA) is an endogenous compound with a multidirectional effect. It possesses antiapoptotic, anti-inflammatory, and antioxidative properties that may be beneficial in the treatment of corneal injuries. Moreover, KYNA has been used successfully to improve the healing outcome of skin wounds. The aim of the present study is to evaluate the effects of KYNA on corneal and conjunctival cells in vitro and the re-epithelization of corneal erosion in rabbits in vivo. Normal human corneal epithelial cell (10.014 pRSV-T) and conjunctival epithelial cell (HC0597) lines were used. Cellular metabolism, cell viability, transwell migration, and the secretion of IL-1ß, IL-6, and IL-10 were determined. In rabbits, after corneal de-epithelization, eye drops containing 0.002% and 1% KYNA were applied five times a day until full recovery. KYNA decreased metabolism but did not affect the proliferation of the corneal epithelium. It decreased both the metabolism and proliferation of conjunctival epithelium. KYNA enhanced the migration of corneal but not conjunctival epithelial cells. KYNA reduced the secretion of IL-1ß and IL-6 from the corneal epithelium, leaving IL-10 secretion unaffected. The release of all studied cytokines from the conjunctival epithelium exposed to KYNA was unchanged. KYNA at higher concentration accelerated the healing of the corneal epithelium. These favorable properties of KYNA suggest that KYNA containing topical pharmaceutical products can be used in the treatment of ocular surface diseases.

Osteochondral Repair Using Porous Three-dimensional Nanocomposite Scaffolds in a Rabbit Model.

AIM: To evaluate the utility of a novel nanocomposite biomaterial consisting of poly-L/D-lactide, and hydroxyapatite bioceramics, enriched with sodium alginate in articular cartilage defect treatment. MATERIALS AND METHODS: The biomaterial was prepared using the method of solvent casting and particle leaching. The study was conducted on 20 New Zealand White rabbits. Experimental osteochondral defects were created in the femoral trochlear grooves and filled with biomaterials. In control groups, the defects were left to spontaneously heal. The quality of newly-formed tissue was evaluated on the basis of macroscopic and histological assessment. Additionally the level of osteogenic and cartilage degradation markers were measured. RESULTS: The majority of the defects from the treatment group were covered with tissue similar in structure and colour to healthy cartilage, whereas in the control group, tissue was uneven, and not integrated into the surrounding cartilage. CONCLUSION: The results obtained validate the choice of biomaterial used in this study as well as the method of its application.

A new model of detrusor overactivity in conscious rats induced by retinyl acetate instillation.

INTRODUCTION: A credible animal overactive bladder model used in basic research is an indispensable harbinger of safe and ethical clinical trials on human subjects. Our objective was to develop a new animal model of a hyperactive bladder that will be void of inflammatory urothelium lesions and display significant sensitivity to muscarinic receptor antagonists. METHODS: To examine the influence of 0.75% retinyl acetate solution on cystometric parameters, it was infused into the bladder for 5min. Cystometric studies with physiological saline were performed in conscious unrestrained rats 3days later. To examine the influence of retinyl acetate, acetic acid or cyclophosphamide on morphology of urinary bladders, the bladders were subjected to histopathological examination. RESULTS: We demonstrated that in rats subject to previous 5-minute bladder instillations with retinyl acetate, an increase of basal pressure, threshold pressure, micturition voiding pressure, bladder contraction duration, relaxation time, detrusor overactivity index, nonvoiding contraction frequency and amplitude occurs. On the other hand, a decrease in voided volume, post-void residual, volume threshold, voiding efficiency, intercontraction interval, bladder compliance and volume threshold to elicit nonvoiding contractions was observed. Administration of oxybutynin chloride (0.5mg/kg, i.v.) reversed changes of cystometric parameters evoked by retinyl acetate. Contrary to acetic acid and cyclophosphamide, bladders subjected to retinyl acetate infusion had no signs of bladder inflammation. DISCUSSION: The results obtained indicate that transient infusion of 0.75% retinyl acetate can induce detrusor overactivity, which is often observed in patients with overactive bladder syndrome (OAB). In addition, it was demonstrated that stimulating afferent C-fibres using retinyl acetate did not induce evident histopathological inflammatory lesions in the urinary bladder wall. It appears that in the future this model can prove useful in gaining more knowledge on the pathophysiology of OAB, and contribute to the preparation of new, more effective options of OAB pharmacotherapy.

Molecular diagnostics of promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) is characterised by proliferation of abnormal promyelocytes. The reciprocal translocation between the long arms of chromosomes 15 and 17, and the fusion between the retinoic acid receptor (RARa) gene, and PML gene, is unique to APL. Because of unsuccessful cytogenetic analysis of conventional G-banding technique (mitoses were not observed), we diagnosed three non-treatment patients with APL by following molecular methods: reverse transcription-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). At the time of diagnosis our patients showed reciprocal translocation t(15;17)(q22;q12) in all cases studied (66-85% of positive bone marrow cells). With the use of CGH we observed the unbalanced chromosomal aberrations: losses of 5q13.1, 5q31.3, 9p21 regions, gain of 5q32 region and trisomy of 18 chromosome.

[Evaluation of the morphology of the human placental cotyledon following dual in vitro perfusion in variable magnetic field]. / Ocena morfologii zrazika lozyska ludzkiego poddanego dwustronnej perfuzji in vitro w warunkach zmiennego pola magnetycznego.

UNLABELLED: Objectives and the aim of the study was electron-microscopy morphological estimation of the human placental cotyledon after 180 minutes of dual closed perfusion in vitro. MATERIALS AND METHODS: In the experimental group the cotyledons were exposed to variable magnetic field of 2 mT magnetic induction and 50 Hz frequency. The control group K (10 perfusions) was not subjected to magnetic field while the experimental group B (10 perfusions) was influenced by magnetic field. RESULTS: It was found that homogeneous variable magnetic field disturbs the ultrastructure of the nuclei and cytoplasma and it increases the density of the vascular-epithelial membrane of villi cells of human placenta in vitro. CONCLUSION: Variable sinusoildal, magnetic field of 2 mT magnetic induction and 50 Hz frequency disturbs the ultrastructure of the nuclei and cytoplasma and it increases the density of the vascular-epithelial membrane of villi cells of human placenta in vitro after 180 minutes of dual closed perfusion in vitro.

Experimental study of treatment effectiveness of the natural drug--Naran S in inflammatory conditions of the oral cavity in animals.

The aim of the study was the evaluation of the action of a new natural drug--Naran S, elaborated in the Department of Inorganic Chemistry of the Medical University of Lublin (patent registration P.332066) in the treatment of inflammatory conditions of the oral cavity mucous membrane. The experiment was conducted on male Wistar rats. Topical inflammation of the gum was induced by injecting a complete Freund adjuvant (AF) (Calbiochem) into the inter-dental papilla of the lower incisors. The material was taken, fixed and stained with hematoxylin and eosin and by the use of the Masson's method after 24 hours, 1, 2, and 4 weeks following the injection time. Morphology of the mucous membrane was examined and it was noticed that in the group treated with Naran S infusion the complete rebuilding of the epithelial strata, collagen fibres took place. The shape of the cells was regular and there occurred the elimination of the tissue fluid in the proper mucous membrane. The results obtained in the study allow to claim that the topically given plant drug thanks to its contents of biologically active substance such as: iridoids, flavonoids, phenol acids, tannins, displays curative activity in the therapy of the inflammatory conditions of the oral cavity mucous membrane.

Ultra-structure of the oral cavity epithelial cells in white rat after experimental application of Naran S preparation.

The effectiveness of the natural drug--Naran S was studied in the therapy of the topical inflammation of the oral cavity mucous membrane. The experiment was conducted on male Wistar rats that were subjected to induction of inflammatory condition by injecting Freund adjuvant and rinsing oral cavity with the infusion of the drug for the period of 28 days. The analysis of the ultra structure of the epithelium cells was conducted with the use of transmission electron microscope. It was observed that under the influence of the drug, the epithelium undergoes renewal, the cells are joined with thick desmosomes, the nuclei have the proper areolas and the mitochondrium matrix is not oedematic.

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 changes of white rat kidney after experimental administration of ethanol and cephalexin.

The experiment was carried out on Wistar rat males weighing about 200 g. Animals from the control group received water and standard granulated fodder ad libitum. Animals from control group II received 20% ethanol ad libitum instead of water. Animals from experimental group I received cephalexin in the single dose of 42 mg/24h, Animals from experimental group II received cephalexin in the dose of 42 mg/24h and 20% ethanol ad libitum. After 10 days animals were decapitated. Histological examinations (H+E staining and PAS reaction) were made on 7 micro thick paraffin slices. It was stated that ethanol causes hyperemia of the renal parenchyma and cephalexin stimulates diuresis. However, concomitant administration of ethanol and cephalexin causes functional changes (inhibition of diuresis) and degenerative changes in capillary loops of some renal corpuscles apart from hyperemia.

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.

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.

Ultrastructural changes of ciliary epithelial nucleus in experimental diabetes--an animal model.

The aim of this study was to observe in electron microscope the New Zealand rabbits ciliary epithelial nucleus structure changes, depending on the duration of experimental diabetes mellitus. The animals were administered alloxan in the form of 10% solution in 0.9% NaCl, at the dose of 100 mg/kg b.m. If the glycemia level was 11 mmol/l or higher the animal was included in the experimental group. 15 eyes of 15 adult New Zealand rabbits were studied. Conventional electron microscopy was used to show ultrastructural changes of ciliary epithelium nucleus. During the study we noticed severe changes in the structure of ciliary epithelial nucleus depending on diabetes mellitus duration. We found: deep indentations of nuclear membrane, open nuclear pores, irregularity in heterochromatin location and small sizes of the epithelial nucleus.

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.

Ultrastructural changes of pancreas of white rats after experimental administration of ethanol and cephalexin.

The experiment was carried out on Wistar rat males weighting about 200 g. Animals from experimental group I received 20% ethanol for drinking (ad libitum), animals from experimental group II--cephalexin in the dose of 42 mg/24 h, animals from experimental group III--cephalexin and ethanol in the mentioned doses. After 10 days the animals were decapitated and pancreases were collected for ultrastructural examinations in electron microscope. The performed experiments showed that 10-day administration of ethanol causes mainly the decrease of amount of ribosomes and zymogen granules in pancreatic exocrine cells, whereas cephalexin causes increase of amount of these organelles. Administration of both ethanol and cephalexin causes reversible degenerative changes including distinct decreasing of the number of ribosomes, swelling of many mitochondria and the presence of myelinic structures inside cells.