Four-day pulse of sodium cromoglycate modulates pulmonary vessel wall remodeling during 21-day hypoxia in rats.

AIM OF THE STUDY: Remodeling of pulmonary resistance arteries in rats due to 4-day hypoxia could be successfully suppressed by sodium cromoglycate. In this study, we tested the difference in the suppression between two distinct time patterns of cromoglycate administration during 21-day hypoxia. In the experiment, we focused on some details in both smooth muscle cells and extracellular matrix of pulmonary arterial walls. METHODS: During 21-day hypoxia, rats were treated with sodium cromoglycate either in the first four days or in the last four days. The first four days were chosen to test efficiency of an initial pulse of cromoglycate to suppress pulmonary vascular remodeling. The last four-day administration tested possibility to block remodeling post hoc. RESULTS: Initial pulse reduced and modified remodeling in all levels of pulmonary arteries, which comprises neomuscularization of prealveolar arteries, asymmetrical hypertrophy of tunica media in muscular pulmonary arteries and hypertrophy of tunica media and tunica adventitia in large conduit arteries. Terminal pulse had only negligible effect. CONCLUSIONS: Only the initial cromoglycate therapy led to significant morphological suppression of remodeling. We therefore assume important role of initial remodeling influencing during long time hypoxia experiment.

Mast cell stabilization with sodium cromoglycate modulates pulmonary vessel wall remodeling during four-day hypoxia in rats.

AIM OF THE STUDY: In rats, the environment with low content of oxygen induces hypoxic pulmonary hypertension. Remodeling of pulmonary resistance arteries is particularly triggered by the mast cell degranulation products, e.g., rodent-like interstitial collagenase (matrix metalloproteinase 13). Administration of sodium cromoglycate leads to stabilization of mast cell granules, and thus to the modified remodeling process. MATERIALS AND METHODS: During four-day hypoxia, we treated rats with sodium cromoglycate. Pulmonary vascular remodeling was assessed as well as counts of periarterial pulmonary mast cells, both total and matrix metalloproteinase 13-positive ones. RESULTS: Four-day hypoxia induced remodeling of both resistance arteries and large conduit arteries. We have found increase in the tunica media thickness of resistance arteries. Tunica adventitia thickness of both resistance arteries and large conduit arteries with a diameter of over 300 µm increased as well; the latter ones revealed increase in the number of vasa vasorum in their walls. Mast cell stabilization suppressed hypoxic pulmonary vascular remodeling in resistance pulmonary arteries. Four-day hypoxia led to changes in distribution of toluidine blue-detected and MMP-13 positive periarterial mast cells; this redistribution was also influenced by the administration of sodium cromoglycate. CONCLUSIONS: The number of pulmonary periarterial mast cells seemingly decreases during hypoxia due to their degranulation, which disables their identification. Large conduit arteries do not affect final blood pressure in the pulmonary vascular bed; however, their structure changes substantially under hypoxia. Such remodeling changes are not mediated by mast cell products only since they have occurred in spite of stabilization of mast cell granules.

Airway wall remodeling in young and adult rats with experimentally provoked bronchial asthma.

BACKGROUND: Airway wall remodeling is a typical finding in patients suffering from bronchial asthma. While morphological changes have been thoroughly described in adults, less is known about such changes in children because of the limited accessibility of relevant material. To overcome this constraint, animal asthma models may be used instead of human specimens. This study examined rats with artificially stimulated chronic asthma-like symptoms. METHODS: Brown Norway rats of two age categories (young and adult) were sensitized by ovalbumin (OA), and their intrapulmonary airways (IA) were studied using morphometric and histochemical methods. RESULTS: OA administration induced a significant increase in lung resistance in young animals but not in adults. The total IA wall area was significantly increased in both young and adult OA rats. In young animals, thickening of the adventitia played a more crucial role in this increase than it did in adults, in which the mucosa and the submucosa participated to a higher degree. The IA walls of young OA rats had significantly higher levels of infiltrating eosinophils than those of adult OA animals. The multiplication of goblet cells was more pronounced in adult rats, which was associated with a tendency to produce a higher proportion of acidic glycoconjugates. CONCLUSIONS: OA stimulation affected the IA of young rats differently than those of adult animals. Changes in the outer IA layer of young rats can be triggered by activated eosinophils; however, stimulated airway epithelium can be a source of factors that influence the inner IA layers in adult rats.

Generation of hydrogen peroxide in the developing rat heart: the role of elastin metabolism.

Reports describing production of reactive oxygen species in neonatal heart are missing. As lysyl oxidase is potentially important source of H(2)O(2), we studied its role during ontogenic development of rat heart. H(2)O(2) was detected in thin sections of developing rat heart by fluorescence microscopy with the use of fluorescence probe 2'-7'-dichlorofluorescin. The experimental design comprised foetuses 21 days after conception, and then the animals sampled on the 1st, 4th, 7th, 10th, 15th, 30th and 60th day after birth. We also used 7-month-old animals as an example of ageing effects. Since the day 4 on, H(2)O(2) was produced only extracellularly up to the day 15, between days 30 and 60 intracellular production was detected as well, and in 7-month-old animals only extracellular production was observed. The specific inhibitors of lysyl oxidase almost completely quenched the H(2)O(2)-dependent fluorescence. Starting from day 7, blue autofluorescence specific to oxidized proteins developed in the vessel wall. Intracellular blue autofluorescence specific to autoxidation products developed after day 30. Chloroform extraction diminished the intracellular blue fluorescence, leaving the extracellular fluorescence intact. This confirmed the protein nature of the fluorophores. Lysyl oxidase is significant source of H(2)O(2) in the heart vessel wall during development and H(2)O(2) oxidatively modifies elastin producing protein blue autofluorescence.

Intravitreal carboplatin concentration and area under concentration versus time curve after intravitreal and periocular delivery.

PURPOSE: To determine platinum (Pt) concentrations and area under the concentration versus time curve (AUC) of the vitreous humor after periocular or transcorneal intravitreal administration of carboplatin in rabbits. METHODS: Eighteen albino rabbits were included in an in vivo experiment. Each animal received a single dose of either 30 mg of carboplatin by periocular injection (POI30 group: n = 6) or 15 mg by periocular injection (PI15 group: n = 6), or 0.05 mg by transcorneal intravitreal injection (TII group: n = 6), respectively, into the right eye. Vitreous humor from the right eyes and plasma samples were collected post dose at 1, 2, 6, 24, 48, 168, and 336 hours or 448 hours, respectively. Flameless atomic absorption spectroscopy was employed to analyze total platinum concentrations in blood and vitreous humor. AUC was calculated using the trapezoidal rule. RESULTS: Pt concentration was mostly < 1 mg/L (0-3.15 mg/L) in the vitreous humor samples and > or = 2 mg/L (2.33-7.3 mg/L) in the blood samples 1 hour after administration in POI groups. Markedly higher Pt concentrations were found 1 hour after intravitreal (TII) administration (10.285-66.759 mg/L) and decreased below 1 mg/L no less than 168 hours after administration. The mean AUC for Pt in vitreous humor was significantly lower (p = 0.0001) after both POI30 and P0I15 administration compared to TII route (8.955 +/- 2.464 mg/L/min). CONCLUSIONS: These findings proved that intravitreal carboplatin delivery enables the achievement of relatively stable concentrations and AUC of platinum in the rabbit vitreous humor. This moreover suggests that transcorneal intravitreal delivery of carboplatin aiming to treat retinoblastoma vitreous seeding is a promising mode of chemotherapy.

Evaluation of different methods detecting intracellular generation of free radicals.

Reactive oxygen species (ROS) play several biological roles. We investigated the applicability of fluorescent probes for their detection (i) in rabbit lens epithelial cells during ageing in culture, and (ii) in thin sections of rat heart. We used dihydroethidium (DHE), dichlorofluorescin (DCFH), and dihydrorhodamine 123 (DHR) together with detection of autofluorescence both in cells and in chloroform extracts. Superoxide production was confirmed by a specific histochemical method using Mn(2+). All methods demonstrated higher production of ROS in older cells. All probes revealed different sites of ROS production in young and old cells and could be used for investigation of ROS generation during cell ageing. In the thin sections of rat heart DCFH was not suitable for intracellular ROS detection. The results indicate that the potential of fluorescent dyes in ROS detection is not usually fully exploited, and that blue autofluorescence is associated with oxidative damage.

Novel contribution to clubfoot pathogenesis: The possible role of extracellular matrix proteins.

Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the feet and lower legs. Clubfoot belongs to a group of fibro-proliferative disorders but its origin remains unknown. Our study aimed to achieve the first complex proteomic comparison of clubfoot contracted tissue of the foot (medial side; n = 16), with non-contracted tissue (lateral side; n = 13). We used label-free mass spectrometry quantification and immunohistochemistry. Seven proteins were observed to be significantly upregulated in the medial side (asporin, collagen type III, V, and VI, versican, tenascin-C, and transforming growth factor beta induced protein) and four in the lateral side (collagen types XII and XIV, fibromodulin, and cartilage intermediate layer protein 2) of the clubfoot. Comparison of control samples from cadavers brought only two different protein concentrations (collagen types I and VI). We also revealed pathological calcification and intracellular positivity of transforming growth factor beta only in the contracted tissue of clubfoot. Most of the 11 differently expressed proteins are strongly related to the extracellular matrix architecture and we assume that they may play specific roles in the pathogenesis of this deformity. These proteins seem to be promising targets for future investigations and treatment of this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Effect of inhalation of single dose of beclomethasone on airway epithelium.

Inhaled corticosteroids are being recommended for the treatment of bronchial asthma for their anti-inflammatory properties and reduction of airway hyperreactivity. The first tissue coming to the contact with all inhaled substances is the airway epithelium. In this experiment, the immediate effect of a single MDI dose of beclomethasone on the ultrastructure of the tracheal and bronchiolar epithelium was studied. Due to the beclomethasone administration, the secretory elements were highly affected. The tracheal goblet cells were damaged, mucus release was significantly accelerated, and the mechanism of secretion was influenced. The bronchiolar Clara cells revealed signs of the pathological alteration. Their secretory granules were usually stored in the cytoplasm. Occasionally, degenerating Clara cells were found after the beclomethasone administration. The injury of ciliated cells in both locations was only mild and this fact was reflected in slight impairment of the tracheal ciliary border. As a morphological sign of impaired self-cleaning ability, inspissated secretion was discovered among cilia. According to this evaluation, the inhalation of the single dose of beclomethasone caused a moderate damage to the tracheal epithelium and a mild one to the epithelium of terminal bronchioles. The results draw attention to the adverse effects of otherwise therapeutically beneficial inhaled glucocorticosteroids.

Oxidative Stress in the Developing Rat Brain due to Production of Reactive Oxygen and Nitrogen Species.

Oxidative stress after birth led us to localize reactive oxygen and nitrogen species (RONS) production in the developing rat brain. Brains were assessed a day prenatally and on postnatal days 1, 2, 4, 8, 14, 30, and 60. Oxidation of dihydroethidium detected superoxide; 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate revealed hydrogen peroxide; immunohistochemical proof of nitrotyrosine and carboxyethyllysine detected peroxynitrite formation and lipid peroxidation, respectively. Blue autofluorescence detected protein oxidation. The foetuses showed moderate RONS production, which changed cyclically during further development. The periods and sites of peak production of individual RONS differed, suggesting independent generation. On day 1, neuronal/glial RONS production decreased indicating that increased oxygen concentration after birth did not cause oxidative stress. Dramatic changes in the amount and the sites of RONS production occurred on day 4. Nitrotyrosine detection reached its maximum. Day 14 represented other vast alterations in RONS generation. Superoxide production in arachnoidal membrane reached its peak. From this day on, the internal elastic laminae of blood vessels revealed the blue autofluorescence. The adult animals produced moderate levels of superoxide; all other markers reached their minimum. There was a strong correlation between detection of nitrotyrosine and carboxyethyllysine probably caused by lipid peroxidation initiated with RONS.

The effects of intravenously administered methylxanthine preparations on the glycoconjugate composition of goblet cells in rabbit tracheal epithelium.

Effects of methylxanthine derivatives, which are inhibitors of phosphodiesterases I-IV used against bronchial asthma, on the composition of glycoconjugates in goblet cells were evaluated in tracheal epithelium of rabbits at 15 and 30 min after intravenous administration of aminophylline (Syntophyllin) and a mixture of etophylline and theophylline (Oxantil), respectively. Percentages of tracheal goblet cells containing neutral, total acidic, sulphated acidic, and sialylated acidic glycoconjugates were assessed using both conventional and lectin histochemistry. No significant changes were found in both experimental groups at 15 min after exposure. A significant decrease in percentage of alpha(2-3)-sialylated glycoconjugate-containing goblet cells occurred at 30 min after administration of Syntophyllin only. It is concluded that the mucus composition of tracheal goblet cells has been affected by the bronchospasmolytic drug Syntophyllin but not by the vasodilator drug Oxantil.

The effects of intravenously administered methylxanthines on the proportion of goblet cells containing fucosylated glycoconjugates in rabbit tracheal epithelium.

The proportion of goblet cells containing various fucosylated glycoconjugates was evaluated with the use of lectin histochemistry in rabbit tracheal epithelium at 15 and 30 min after intravenous administration of either aminophylline (Syntophyllin) or a mixture of etophylline and theophylline (Oxantil). Methylxanthine derivatives are nonspecific inhibitors of phosphodiesterases that are used to treat bronchial asthma; the proportion of fucosylated glycoconjugates strongly affects rheologic properties of respiratory tract mucus. It is concluded that administration of Syntophyllin dramatically lowered the proportion of goblet cells containing fucosylated glycoconjugates in rabbit tracheal epithelium, especially at 30 min after exposure. This decrease was strongest in the levels of alpha(1-2)-fucosylated glycoconjugates. Therefore, Syntophyllin substantially altered the composition and viscoelastic properties of mucus of the upper respiratory tract. The vasodilator Oxantil exerted less pronounced changes in the proportion of goblet cells, but the strongest effect was again found in the levels of alpha(1-2)-fucosylated glycoconjugates.

Retinal toxicity after repeated intravitreal carboplatin injection into rabbit eyes.

BACKGROUND: The aim of this study was to assess retinal toxicity in a rabbit model after carboplatin delivered as repeated transcorneal intravitreal injection, in order to determine the highest possible safe dose for use in human retinoblastoma "seeding" tumor chemotherapy. METHODS AND RESULTS: We used six albino rabbits in an in vivo experiment and injected 0.008 mg of carboplatin intravitreally (iv) 4 times at two-week intervals. 0.08 mL saline was injected into the left eye. We recorded electroretinograms (ERGs) before the first carboplatin injection and after the fourth injection. Platinum concentration was measured 1 h after the fifth additional injection. We found reduced dark-adapted b-wave amplitudes and, light-adapted b-wave and a-wave amplitudes. The differences between right and left eyes was significant but we found no histopathologic retinal changes. CONCLUSIONS: 0.008 mg of carboplatin is probably the highest possible safe dose for the treatment of retinoblastoma patients. Questionable is direct extrapolation of retinal toxicity from the rabbit eye model to the human eye.

A cell-free nanofiber composite scaffold regenerated osteochondral defects in miniature pigs.

The aim of the study was to evaluate the effect of a cell-free hyaluronate/type I collagen/fibrin composite scaffold containing polyvinyl alcohol (PVA) nanofibers enriched with liposomes, basic fibroblast growth factor (bFGF) and insulin on the regeneration of osteochondral defects. A novel drug delivery system was developed on the basis of the intake effect of liposomes encapsulated in PVA nanofibers. Time-controlled release of insulin and bFGF improved MSC viability in vitro. Nanofibers functionalized with liposomes also improved the mechanical characteristics of the composite gel scaffold. In addition, time-controlled release of insulin and bFGF stimulated MSC recruitment from bone marrow in vivo. Cell-free composite scaffolds containing PVA nanofibers enriched with liposomes, bFGF, and insulin were implanted into seven osteochondral defects of miniature pigs. Control defects were left untreated. After 12 weeks, the composite scaffold had enhanced osteochondral regeneration towards hyaline cartilage and/or fibrocartilage compared with untreated defects that were filled predominantly with fibrous tissue. The cell-free composite scaffold containing PVA nanofibers, liposomes and growth factors enhanced migration of the cells into the defect, and their differentiation into chondrocytes; the scaffold was able to enhance the regeneration of osteochondral defects in minipigs.

Topotecan vitreous and plasma levels and retinal toxicity after transcorneal intravitreal delivery in healthy albino rabbits: alternative retinoblastoma treatment.

AIM: To determine intravitreal and plasma concentrations and retinal toxicity after transcorneal intravitreal injection of 1 µg and 2 µg of topotecan (Hycamtin). METHOD: Twelve healthy albino rabbits were included in this in vivo experiment. Six anesthetized albino rabbits received a single transcorneal intravitreal injection of 1 µg (group A) or 2 µg (group B) of topotecan. Vitreous and blood samples were collected until 168 h. Left eyes were treated with the same volume of saline. Plasma and vitreous levels of topotecan were determined by high-performance liquid chromatography. Area under the plasma concentration time curve (AUC) was calculated using trapezoidal rule. Clinical evidence of toxicity was classified into four grades according to anatomical structures. Electroretinograms (ERGs) were recorded. RESULTS: Time to maximum concentration was observed up to 2 h after drug injection in group A whereas up to 1 h in group B. Low levels of topotecan were detected in plasma in both groups and in the vitreous humor of the contralateral eye in group B. Topotecan levels (mean vitreous AUC in group A 2.55 µg/mL.h and in group B 5.338 µg/mL.h) were detectable up to 6 h in both groups. We observed following structural changes in rabbit eyes: corneal vascularization, cataract, hemophthalmus, choroidal edema and focal retinal atrophy. Abnormal ERGs were obtained. CONCLUSION: Our findings proved that transcorneal intravitreal administration of 1 µg and 2 µg of topotecan results in potentially cytotoxic intraocular concentrations. More studies are needed to establish the safety of topotecan for retinoblastoma in children.

Intracranial pressure and experimental model of diffuse brain injury in rats.

OBJECTIVE: In this study, we present a simple closed head injury model as a two-stage experiment. The height of the weight drop enables gradation of head trauma severity. METHODS: The head injury device consists of three parts and there are three adjustable parameters-weight (100-600 g), height of fall (5-100 cm) and elasticity of the springs. Thirty male Wistar rats underwent monitoring of intracranial pressure with and without induction of the head injury. RESULTS: The weight drop from 45 to 100 cm led to immediate seizure activity and early death of the experimental animals. Severe head injury was induced from 40 cm weight drop. There was 50% mortality and all surviving rats had behavioral deterioration. Intracranial pressure was 9.3 +/- 3.76 mmHg. Moderate head injury was induced from 35 cm, mortality decreased to 20-40%, only half of the animals showed behavioral pathology and intracranial pressure was 7.6 +/- 3.54 mmHg. Weight drop from 30 cm caused mild head injury without mortality and neurological deterioration. Intracranial pressure was slightly higher compared to sham group- 5.5 +/- 0.74 mmHg and 2.9 +/- 0.81 mmHg respectively. CONCLUSION: This model is an eligible tool to create graded brain injury with stepwise intracranial pressure elevation.

Fibrin/hyaluronic acid composite hydrogels as appropriate scaffolds for in vivo artificial cartilage implantation.

Hydrogels prepared from a mixture of fibrin and high-molecular weight (MW) hyaluronic acid (HA) were found to be suitable scaffolds for chondrocyte seeding and pig knee cartilage regeneration. Collagen in the hydrogels is not necessary for the formation of biomechanically stable tissue. Regenerated cartilage showed very good biomechanical and histological properties only 6 months after implantation. Notably, the quality of the healing process was dependent on the initial chondrocyte concentration of the scaffolds. These experiments were performed according to good laboratory practice (GLP).

Local administration of 2% trimecaine affects the content of fucosylated glycoconjugates in goblet cells in rabbit tracheal epithelium.

The proportion of fucosylated glycoconjugate-containing rabbit tracheal goblet cells after intratracheal application of trimecaine was studied to evaluate its possible unfavourable effects. This lapine model is comparable with diagnostic findings in humans because airway epithelia in humans and rabbits are similar; tracheal epithelium is also practically identical to bronchial epithelium in both species. Local trimecaine anaesthesia caused a proportional decrease in percentage of the tracheal goblet cells containing both alpha(1-2)- and alpha(1-6)-, alpha(1-3)- and alpha(1-4)-fucosylated glycoconjugates as revealed 10 min postexposure using lectin histochemistry. In previous studies, only mild ultrastructural damage to the airway's epithelium was revealed, but a conspicuous decrease in sialylated glycoconjugate-containing tracheal goblet cells and the dominance of acidic sulphated glycoconjugates were observed as after-effects of the same treatment. Glycoconjugate changes can influence the inner environment of airways (e.g. viscoelastic properties of the airways' mucus and mucosal barrier functions) and thus the patient's defence barriers in airways may be weakened. Concurrently, the histochemical properties of goblet cells can be altered in bronchoscopic specimens. Since trimecaine is widely used as local anaesthesia in airways in bronchoscopy, it is necessary to heed these aforementioned effects.

Acute and chronic hypoxia as well as 7-day recovery from chronic hypoxia affects the distribution of pulmonary mast cells and their MMP-13 expression in rats.

Chronic hypoxia results in pulmonary hypertension due to vasoconstriction and structural remodelling of peripheral lung blood vessels. We hypothesize that vascular remodelling is initiated in the walls of prealveolar pulmonary arteries by collagenolytic metalloproteinases (MMP) released from activated mast cells. Distribution of mast cells and their expression of interstitial collagenase, MMP-13, in lung conduit, small muscular, and prealveolar arteries was determined quantitatively in rats exposed for 4 and 20 days to hypoxia as well as after 7-day recovery from 20-day hypoxia (10% O2). Mast cells were identified using Toluidine Blue staining, and MMP-13 expression was detected using monoclonal antibody. After 4, but not after 20 days of hypoxia, a significant increase in the number of mast cells and their MMP-13 expression was found within walls of prealveolar arteries. In rats exposed for 20 days, MMP-13 positive mast cells accumulated within the walls of conduit arteries and subpleurally. In recovered rats, MMP-13 positive mast cells gathered at the prealveolar arterial level as well as in the walls of small muscular arteries; these mast cells stayed also in the conduit part of the pulmonary vasculature. These data support the hypothesis that perivascular pulmonary mast cells contribute to the vascular remodelling in hypoxic pulmonary hypertension in rats by releasing interstitial collagenase.