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The direct and accurate estimation of nitric dioxide levels is an extremely laborious and technically demanding procedure in the molecular diagnostics of inflammatory processes. The aim of this work is to demonstrate that a stop-flow technique utilizing a specific spectroscopic biosensor can be used for detection of nanomolar quantities of NO(2) in biological milieu. The use of novel compound cis-[Cr(C(2)O(4))(AaraNH(2))(OH(2))(2)](+) increases NO(2) estimation accuracy by slowing down the rate of NO(2) uptake. In this study, an animal model of pancreatitis, where nitrosative stress is induced by either 3g/kg bw or 1.5 g/kg bw dose of L-arginine, was used. Biochemical parameters and morphological characteristics of acute pancreatitis were monitored, specifically assessing pancreatic acinar cell death mode, NO(2) generation and cellular glutathione level. The severity of the process correlated positively with NO(2) levels in pancreatic acinar cell cytosol samples, and negatively with cellular glutathione levels.

The arcuate nucleus of the hypothalamus (ARH) is involved in the control of energy homeostasis. This is the first study on the ultrastructural response of ARH neurons in aged rats after short-term fasting and subsequent refeeding. Male Wistar rats (24 weeks old) were fasted for 48 or 96 hours and were then refed for 24 hours. The controls were normally fed. The rats received water ad libitum. In both groups of fasting animals, we observed a rearrangement of the arcuate rough endoplasmic reticulum (RER) and Golgi complexes to form membranous whorls. Moreover, refeeding for 24 hours did not reverse this process. The RER was frequently found to be well organized into lamellar bodies composed of several cisternae. The membranous whorls were composed of concentric layers of endoplasmic reticulum and Golgi complexes. In addition, multiform lipofuscin granules were observed in close relationship with Golgi complexes and membranous whorls. Lipofuscin granules within the neurons of the arcuate nucleus are assumed to be a morphological manifestation of oxidative stress phenomena, which are presumably implicated in the formation of membranous whorls in both fasting and fasting/refed animals. This observation correlates with a significant increase in 8-isoprostane serum levels in the fasting and fasting/refed animals as compared to the fed control rats.

In this study, 15 kinds of powders with different compression mechanisms were used in the process of filling-binding substances in tablets with pellets. Applied substances possessed dominant brittle time-independent mechanism or time-dependent viscoplastic, viscoelastic mechanism of compression. Using 6 kN compression force in a single-stroke tablet press during 150 ms of compression, damage to the polymer film and pellet core was found in all formulations. As a result, the authors observed an increase of releasing rate of verapamil hydrochloride (VH). A larger contact area between powders and pellets and connected with this better protective properties were ensured by powders with time-independent compression mechanism (eg, D-sorbitol or D-mannitol). Unsymmetrically applied compression force was a reason for inconsistent densification and insufficient protection of the pellets. Taking into consideration the low rotation speed of the turret (10 rpm) in the rotary tablet press, the total compaction time was much longer than in the single-stroke tablet press. The compression time in the case of the rotary tablet press should be considered as the sum of the precompression (about 130 ms) and main compression (about 280 ms) phase times. Compression force applied by upper and lower punch in the precompression and main compression phase was affected uniformly on the pellets' surface, and when protected against fragmentation, allowed only some slight deformation. The powders in tablet formulation were fragmentized and rearranged independent of their compression mechanisms. It was found that the releasing rate of VH from pellets compressed by rotary tablet press with 6, 12, and 18 kN of compression force was similar to the releasing rate from uncompressed pellets.

Inhibitory effect of 1alpha,25dihydroxycholecalciferol (1,25D(3)=calcitriol) in different cell type is well recognized but its promoting effect on vascular smooth muscle cells (SMCs) is poor established. Therefore, the aim of this study was to determine stimulatory effect of calcitriol on aortal SMCs proliferation in culture. We used the cell division analysis procedure based on the quantitative sequential halving of the stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). This technique allowed the visualization of cycles of SMCs division by flow cytometry. Rat aortal SMCs were labeled with CFSE and cultured for up to 10 days with defined concentration of calcitriol in medium. Proliferative activity as the percentage of SMCs in different phases of the cell cycle using propidium iodide was determined. Apoptosis was assessed using Annexin-V/CFDA method. The results suggest that low concentrations of an active form of vitamin D-1alpha,25dihydroxycholecalciferol applied in supraphysiological concentration of 10 nmol/l is a mitogenic factor for aortal SMCs. None of the applied concentrations of calcitriol caused apoptosis. The findings well support our morphological (LM) and ultrastructural (TEM and SEM) observations.

Microcrystalline cellulose (MCC) and powdered cellulose (PC) are commonly used excipients for solid dosage forms e.g., pellets. The aim of this study was to compare the utility of the MCC and PC in the floating pellet cores comprising verapamil hydrochloride (VH) manufactured by extrusion and spheronization and influence on their physical properties like swelling, compressibility and VH release. It was found by scanning electron microscopy (SEM) investigation that porosity of surface of the pellets' cores increased with an increase of PC amount in composition. Differential scanning calorimetry (DSC) analysis indicated the lack of physicochemical interaction between PC and MCC either with VH or with any excipients in the pellet core. Formulation having the highest PC participation were characterized by the highest friability and compressibility and addition of MCC corresponded with a decrease of friability and compressibility. The results on pellets friability were not reflected by the results on the hardness test. It means that the PC contents growth contributes to the hardness growth. The swelling forces of physical mixture of powders containing PC and MCC was different and increased with increasing amount of PC in pellet's core. Pellets' cores were coated with Eudragit NE dispersion. It was found that VH release rate from coated pellets with higher amount of PC was considerably slower in comparison to the pellets containing highest MCC participation.

Limited experimental models of the oedematous and necrotic types of acute pancreatitis provide some understanding of the pathophysiology of this disease. Wistar rats were treated with cerulein at 10 mg/kg of body weight or with L-arginine at 1.5 or 3 g/kg of body weight in order to induce the oedematous or necrotic type of acute pancreatitis. After the induction period we examined samples of pancreata with light and electron microscopes. Morphological examination showed profound changes in the histology of the pancreas and its acinar cells and subcellular structures, especially in the group of rats which received a higher dose of L-arginine, amounting to 3 g/kg body weight. These included parenchymal haemorrhage and widespread acinar cell necrotic changes. 4-OH-TEMPO successfully prevented morphological deterioration as well as amylase release, suggesting that the severity of the two types of disease strongly depends on the intensity of the oxidative stress. Our results lend support to the assumption that reactive oxygen species play an axial role in the pathogenesis of both types of acute pancreatitis.

Objective: The purpose of this study was to examine protective and antioxidative effect of stilbene derivatives, resveratrol and diethylstilbestrol, in experimental acute pancreatitis (EAP)., Methods: EAP was induced in male Wistar rats by retrograde injection of tert-butyl hydroperoxide (ButOOH) solution, a well-known prooxidant agent, into the common bile pancreatic duct. After a 3-hour observation, the animals were killed. Blood samples were collected. Each pancreas was removed and weighed. Tissue samples were taken for microscopic studies. The carbonyl and sulfhydryl (SH) group levels were estimated in the homogenate., Results: Examination using light microscopy revealed morphologic changes in pancreata removed from EAP rats, namely focal edema, acinar cell vacuolization, and focal necrosis of pancreatic acini. The electron microscopic analysis also showed changes in their subcellular structures: dilated cisternae of the rough endoplasmic reticulum, swollen mitochondria, and "debris" of mitochondrial cristae. These changes corresponded with higher activities of serum amylase and tissue carbonyl groups levels and decreased SH group level compared with controls. Changes in pancreata were much less pronounced in the rats that received resveratrol or diethylstilbestrol for 8 days prior to ButOOH injection., Conclusion: Stilbene derivatives prevent pancreatic cells from structural changes during ButOOH-induced acute pancreatitis.

For the first time, a direct sensitive method of *NO(2) detection and measurement in biological material has been established. It is based on the interaction of this radical with the coordination compound of Cr(III) with aminodeoxysugar as biosensor. Our new method makes it possible to precisely assess *NO(2) level in experimental acute necrotizing pancreatitis induced by L-arginine, where oxidative and nitrosative stresses are supposed to play a key role in the pathomechanism of the disease. As much as 20 nmol of *NO(2)/mg protein was detected which correlated with severe deterioration of pancreatic acinar cell ultrastructure. Protective effect of superoxide radical scavenger 4-OH-TEMPO expressed as *NO(2) level decrease confirmed by preserved acinar cell ultrastructure and decreased pancreatic amylase release to blood serum is demonstrated. This study reveals a possible pathomechanism of L-arginine induced acute pancreatitis.

Our studies were carried out on the hearts of virgin female Wistar rats treated with 100,000 i.u. of vitamin D3 (calciol) per os for 3 consecutive days. Multifocal cardionecrosis was established macroscopically in 70% of the vitamin D-treated rats on the 7th day of the experiment when the rats were in the acute phase of intoxication. Using a scanning electron microscopy (SEM), we received three-dimensional information about the structural changes to the rat myocardium damaged by high doses of vitamin D3. The images of necrotic hearts revealed significant disruption of the structural integrity of the myocardium linked to fragmentation of the cardiac muscle bundles and a visible disruption of the extracellular matrix (ECM) components. In healthy hearts, the structural integrity of the myocardium and the dense network of the extracellular matrix were well preserved. In parallel, the effect of an increasing concentration of free Ca2+ on the total proteolytic activity of the heart muscle homogenate of the healthy and necrotic rats was investigated at neutral pH. These data showed that following vitamin D3 intoxication, the proteolytic processes in the rat hearts occurred in Ca2+ overload or saturation. On the basis of our morphological and biochemical results we can suggest that calcium-activated neutral proteinases may have contributed to the structural alteration of the extracellular matrix components and were in this way involved in vitamin D-induced cardionecrosis.

Thymocytes exposed to the pro-oxidant tert-butyl-hydroperoxide (ButOOH) display a number of dramatic changes in morphology similar to those observed in the case of dexamethasone-treated cells. Both reagents induce nuclear chromatin peripheral aggregation below the nuclear membrane. Some nuclei themselves break up producing two or more fragments. ButOOH-treated cells are morphologically characterised by cell shrinkage, extensive surface blebbing and, finally, fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and tightly packed with or without nuclear fragments. An increased level of lipid hydroxyperoxides was detected after exposure of thymocytes to ButOOH. Both oxidative stress markers and morphological damage to cells were prevented by the antioxidant 4-OH-TEMPO.

Many experimental models have been created to explain the pathophysiology of acute pancreatitis (AP). Investigations have been undertaken in this laboratory into the influence of strong oxidants introduced into the pancreas retrogradely through the bile-pancreatic duct. In these experiments a potentially toxic metabolite of ethanol-peracetic acid was used to induce AP. Wistar rats were treated with 1 mM and 40 mM peracetate and with a solvent as a control for 1 and 3 hours respectively. After a period of observation the samples of pancreata were examined in a light and electron microscope together with the content of sulphydryl groups as a marker of intracellular oxidative stress. The morphological examination showed profound changes in the histology of the pancreas and also in its subcellular structures, especially in groups 3 and 4 (with a higher concentration of peracetate). The changes included parenchymal haemorrhage and widespread acinar cell necrosis. The level of the sulphydryl groups decreased in the rats treated with peracetate. This suggests that the severity of the disease strongly depends on the intensity of the oxidative stress. The results confirmed the axial role of oxygen-derived free radicals in the pathogenesis of AP., (Copyright 2004 Via Medica)

The growth of solid tumours and their metastases is dependent on the development of new blood vessels (angiogenesis). Therefore angiogenesis inhibitors are potential antitumour drugs. In our previous studies it was found that the angiogenesis inhibitor TNP-470 given to transplantable melanoma-bearing hamsters can decrease the rate of the tumour growth, although the survival time of the animals treated was not significantly affected. It was found finally that TNP-470 given in the vicinity of the growing tumour can cause complete remission of the melanoma in hamsters treated in this way. To check what side-effects could be evoked by such treatment, an examination of the morphology of the blood vessels of the lungs, kidneys and livers of the treated animals was carried out. It was found that the angiogenesis inhibitor applied did not cause any changes which could be observed by light and electron microscopes in the structure of the examined blood vessels of the treated animals.

The smooth muscle cells (SMCs) of the arterial media play a predominant role in functional and structural alterations of the arterial wall. The transition from the "contractile" to the "synthetic" phenotype appears to be an early critical event in the development of atherosclerotic disease. A number of observations suggest that 1,25(OH)(2)D(3) (calcitriol) is of importance in maintaining normal cardiovascular function through its receptors in cardiac myocytes or aortal SMCs. The present study has focused on the microtubular (MT) network reorganisation after exposure to calcitriol. SMCs isolated by enzymatic digestion from the aortal media of neonatal rats were cultured on glass cover slips. 1 microM of 1,25(OH)(2)D(3) was added to the culture medium every second day. The cytoskeletal features of SMCs after calcitriol were visualised by the immunofluorescence staining of alpha-tubulin. The alterations in alpha-tubulin expression and the distribution of microtubules related to the activities of the vascular smooth muscle cells, namely adhesion, migration, multilayer formation and cell division, were observed. A spindle shape, decreased cell adhesion, low expression of alpha-tubulin and a longitudinally arranged microtubular network manifested the high rate of SMC differentiation in the calcitriol-treated culture. A flat stellate morphology, high expression of alpha-tubulin and a radially distributed three-dimensional microtubular network were observed in the SMCs of the control culture. Destructive changes in the microtubular architecture which altered the cellular shape were evident in SMCs undergoing apoptosis. Cells with apoptotic features were more frequent in calcitriol-exposed culture. In contrast to the regular SMC divisions observed in the control culture, some of the mitotic cells exposed to calcitriol contained broader bipolar, multipolar or disordered spindles. These alterations in the SMCs' microtubular cytoskeleton after calcitriol treatment were concomitant with changes in cell growth, differentiation and apoptosis, and may suggest a similarity to atherosclerotic plaque formation.

In order to explore the morphological basis of the altered feeding behaviour of old rats, an ultrastructural investigation of the magnocellular neurons of the hypothalamic paraventricular nucleus (PVN) was performed. Young and old male Wistar rats, 5 and 24 months old, respectively, and with each age group comprising 12 animals, were divided into 3 groups. The rats in Group I were used as controls (normally fed), the rats of Group II were fasted for 48 hours and in Group III the rats were fasted for 48 hours and then refed for 24 hours. The brains were fixed by perfusion and histological and ultrathin sections were obtained by routine methods. Common features of the magnocellular PVN neurons of young and old rats were abundant Golgi complexes and short fragments of RER localised at the cell periphery. In contrast to young rats, the PVN neurons of old animals showed deep indentations of the nuclear envelope and age-related residual bodies. In both age groups fasting for 48 hours led to the expansion of the Golgi complexes and dilatation of RER cisternae. In contrast to those in fed rats, RER cisternae in the neurons of old fasted animals were situated between the nuclear envelope and the Golgi zone. Prolonged RER cisternae were distributed in the peripheral cytoplasm of refed old rats. Our observations suggest that at the ultrastructural level the process of ageing does not change the responsiveness of magnocellular PVN neurons to fasting-refeeding.

Oestradiol-induced male Syrian hamster carcinogenesis is a well-known experimental model of human cancer of the breast, ovary and uterus. The pathomechanism postulated in this model is 4-hydroxylation of oestradiol and further free radical formation. The same process is suspected in human breast cancer. Dynamic changes in protein peroxidation were reported during the tumour induction. In this paper we try to correlate the protein peroxidation markers with the histopathological progression of the changes. The biochemical and histopathological evaluations were performed after 1, 3, 6 and 9 months of the hormone exposition. Significant protein peroxidation was observed as soon as after 1 month and increased further until the 6th month. After 9 months however, it was not significantly different from the control. The discrete histopathological changes after 1 month, progressed into tubular and interstitial hyperplasias after 3 and 6 months. After 9 months several dysplastic areas, sometimes with features of carcinoma in situ, were observed. The severe 9-month histopathological changes did not correlate with the protein peroxidation.

Many hypothalamic nuclei are involved in the regulation of food intake and energy homeostasis. An ultrastructural investigation of the hypothalamic ventromedial nucleus (VMN), a hypothetical "satiety centre" was performed to explore the morphological basis of altered feeding behaviour of old rats in an experimental model of fasting/refeeding. Young (5 months old, n=12) and old (24 months old, n=12) male Wistar rats were fasted for 48 hours, then refed for 24 hours and sampled thereafter. Brain tissue was fixed by perfusion, histological and ultrathin sections were obtained by routine methods. Although food intake was similar in control young and old rats, during refeeding old animals consumed less chow than young ones. The EM analysis of VMN neurones of old control rats revealed, besides typical age-related residual bodies, deep indentations of the nuclear envelope and the presence of long, undulating rough endoplasmic reticulum cisternae in the cell periphery. In both young and old rats fasting for 48 hours led to the expansion of Golgi complexes and increased folds of the nuclear envelope, which is suggestive of enhanced cellular activity of the VMN neurones. These fasting-induced alterations were sustained in the VMN neurones of refed rats in both age groups. The results showed that the VMN neurones of old control rats differ at the ultrastructural level from young ones. However, starvation and subsequent refeeding cause similar alterations in the hypothalamic neurones of "satiety centre" of both young and old rats.

Phenotypic modulation of smooth muscle cells (SMCs) from a contractile to a synthetic state characterised by active proliferation appears to be an early event in the pathogenesis of atherosclerosis. A similar transition occurs when SMCs are established in culture. In this study the phenotypic plasticity and surface structural changes of aortal smooth muscle cells during the transition from the contractile to the synthetic state and during maturation have been structurally assessed by scanning electron microscope (SEM). The experiments were performed on SMCs obtained from aorta of neonatal rats after enzymatic digestion and then cultured on glass coverslips. SEM observations revealed a three-dimensional appearance characteristic for different stages of SMCs. Intensively proliferating cells from monolayer region were large, polygonal in shape with lamellipodia and well spread. Long, uniform in diameter, finger-like microvilli were densely arranged on the surface of these cells. In the thickened region of culture, the cells were rather small, generally spindle-shaped, not well spread, with low density of short, bubble-like microvilli on the surface. Numerous plasma membrane structural alterations in apoptotic cells were observed by SEM: loss of cellular adhesion, smoothing, shrinkage and outpouching of membrane segments have been recognised as markers associated with the cell injury and death. It was concluded that scanning microscopy observations would allow a more complete understanding of SMCs and their changes in culture and atherosclerotic disease.

Introduction: A number of observations suggest that vitamin D plays an important role in maintaining normal cardiovascular function, through its receptors in cardiac muscle or in aortal smooth muscle cells (SMCs). The purpose of this study was to determine the effect of vitamin D active metabolite--calcitriol [1,25(OH)2D3] on aortal SMCs., Material and Methods: The cells were isolated from the aortal media of newborn rats by enzymatic digestion and maintained for 6 weeks in primary culture. 1.2 microM of calcitriol was added to the medium every second day., Results: Light microscopic investigations revealed that the control SMCs formed multilayer with characteristic 'hills and valleys'. Uniform cellular growth patterns were observed after calcitriol treatment. Ultrastructural studies indicated more rapid modulation of SMCs from the 'contractile' to the 'synthetic' phenotype following calcitriol treatment. Elastic fibers were more abundant in treated than in control cultures. The scanning electron microscope showed an increase of regular microvilli on the surface of SMCs. Morphometrical analysis of nuclei demonstrated phenotypic heterogeneity in populations of aortal SMCs. These results suggest that 1) calcitriol can induce changes in the phenotype and in the growth pattern of aortal SMCs, which may be associated with the onset or progression of the atherosclerosis process, 2) the basis of phenotypic changes is related to the stimulation of proliferative activity in these cells.

The amygdaloid body is a telencephalic structure belonging to the limbic system. The amygdaloid body consists of the two main nuclear groups: corticomedial and basolateral. The former-phylogenetically older group is composed of the central, medial, and cortical nuclei, while the latter, phylogenetically younger one, of the lateral, basolateral and basomedial ones. The results presented in our paper indicate differences in the structure and topography of the specific amygdaloid nuclei. Their subdivisions in the rabbit are not as evident as in the rat. Apart from structural differences, the cellular composition of specific nuclei does not differ distinctly. It can suggest that their intrinsic and extrinsic connections might be similar and the role and function of them is maintained (with few exceptions) through the phylogeny.

Data obtained by using ultrastructural and morphometric approaches revealed three types of neurons in the cat visual claustrum. The most numerous were medium-sized and large ones. They formed 3/4 of the cell population. The ultrastructural properties of those cell types were largely similar. Their cell bodies were oval, round, fusiform or triangular and contained more or less indented nuclear envelope. The cytoplasm of those cells was characterized by a high concentration of subcellular organelles and particularly rough endoplasmic reticulum. The characteristic feature of those cells was a low nucleus/cell body area ratio (47 +/- 1% and 43 +/- 1%, respectively). The proximal dendrites of medium-sized cells were usually wide at the base, relatively short and tapering, whereas, those arising in the large cells were often thick and had a short tapering base. The neurons described above stained by Golgi impregnation method showed spines on their distal dendrites both under the light and electron microscopy. The retrograde axonal transport of HRP and WGA-HRP following injections into the visual cortex confirm that they are mainly projection cells which form the ascending limb of the claustrocortical loop. The third type of neurons formed a less numerous group of small cells which differed from the larger ones in various respects. They possessed the large nuclei with deeply indented nuclear envelope and comparatively a thim layer of cytoplasm poor in subcellular organells among which free ribosomes and mitochondria were common. The nucleus/cells body area ratio high (59 +/- 2%). In Golgi preparations their dendrites did not show spines. The dendrites originating from that type of neurons were thin, long and did not posses a wide tapering base. They are mainly claustral intrinsic neurons.

A dark type degeneration of axon terminals was observed in the dorsocaudal part of the claustrum following lesions of the visual cortex. Most of the degenerating axon terminals had synapses mainly with dendritic spines (64.1%) and small dendrites (25.1%). Only 5.8% of degenerating axon terminals formed synaptic contacts with medium-sized dendrites, 1.0% with large dendrites and 0.5%--with other parts of the claustral neurons. 3.5% of degenerating axon terminals were observed to form a contact with more than one postsynaptic structure. Degenerating axon terminals displayed synaptic contacts exclusively of asymmetrical type with clearly identifiable postsynaptic densities which suggest corticoclaustral afferents to be excitatory. Our data strongly support the notion that there are direct synaptic contacts between the ascending arm of the corticoclaustral neuronal loop.