Mechanical response of human thoracic spine ligaments under quasi-static loading: An experimental study.

PURPOSE: This study aimed to investigate the geometrical and mechanical properties of human thoracic spine ligaments subjected to uniaxial quasi-static tensile test. METHODS: Four human thoracic spines, obtained through a body donation program, were utilized for the study. The anterior longitudinal ligament (ALL), posterior longitudinal ligament (PLL), capsular ligament (CL), ligamenta flava (LF), and the interspinous ligament and supraspinous ligament complex (ISL + SSL), were investigated. The samples underwent specimen preparation, including dissection, cleaning, and reinforcement, before being immersed in epoxy resin. Uniaxial tensile tests were performed using a custom-designed mechanical testing machine equipped with an environmental chamber (T = 36.6 °C; humidity 95%). Then, the obtained tensile curves were averaged preserving the characteristic regions of typical ligaments response. RESULTS: Geometrical and mechanical properties, such as initial length and width, failure load, and failure elongation, were measured. Analysis of variance (ANOVA) revealed significant differences among the ligaments for all investigated parameters. Pairwise comparisons using Tukey's post-hoc test indicated differences in initial length and width. ALL and PLL exhibited higher failure forces compared to CL and LF. ALL and ISL + SSL demonstrated biggest failure elongation. Comparisons with other studies showed variations in initial length, failure force, and failure elongation across different ligaments. The subsystem (Th1 - Th6 and Th7 - Th12) analysis revealed increases in initial length, width, failure force, and elongation for certain ligaments. CONCLUSIONS: Variations of both the geometric and mechanical properties of the ligaments were noticed, highlighting their unique characteristics and response to tensile force. Presented results extend very limited experimental data base of thoracic spine ligaments existing in the literature. The obtained geometrical and mechanical properties can help in the development of more precise human body models (HBMs).

Reduced level of synapsin I protein in the rat striatum after intraventricular administration of proteasome inhibitors: preliminary studies.

BACKGROUND: We have recently described changes present in nigrostriatal terminals after intraperitoneal administration of MG-132 and changes that occur in the walls of the rat lateral ventricle after intraventricular administration of MG-132, lactacystin and epoxomicin - different classes of proteasome inhibitors. Substances that inhibit ubiquitin-proteasome system (UPS) activity, are intensively studied due to their potential role as novel therapeutic strategies in the treatment of cancer and ischaemia-reperfusion injury in the brain. The aim of this study is to determine the influence of intraventricular administration of MG-132, lactacystin and epoxomicin on the level in the rat striatum synapsin I - one of the most prominent neuron-specific phosphoproteins in the brain. MATERIALS AND METHODS AND RESULTS: Two weeks after administration of studied proteasome inhibitors, substantial reduction (up to 80%) of synapsin I was ob-served in the rat striatum. Because neurons, and especially dopaminergic ones, are sensitive to the depletion of proteasome function, we assume that observed synapsin I decrease may reflect changes in population of striatal neurons and/or nigrostriatal terminals. CONCLUSIONS: Understanding of cellular mechanisms standing behind our findings needs further studies, and could provide valuable contribution to the discussion on the mechanisms linking UPS inhibition and survival of neurons.

Pterostilbene induces accumulation of autophagic vacuoles followed by cell death in HL60 human leukemia cells.

Pterostilbene, a naturally occurring structural analog of resveratrol, has been reported to exert antiproliferative and proapoptotic effects in various cancer types. Recently, it has been demonstrated to induce both autophagy and apoptosis in human bladder and breast cancer cell lines. The aim of this study was to evaluate the effects of pterostilbene on HL60 human leukemia cells. Cell morphology was examined using confocal and electron microscopy. Cell viability was determined by MTT, neutral red uptake and trypan blue exclusion assays. LC3 processing was studied based on Western blotting and immunofluorescence analyses. Flow cytometry was used to study cell cycle distribution, phosphatidylserine externalization, caspase activation, disruption of mitochondrial membrane potential and intracellular production of reactive oxygen species. DNA degradation was examined by gel electrophoresis. We found that treatment of HL60 cells with pterostilbene at the IC90 concentration resulted in the G0/G1 cell cycle arrest. Pterostilbene induced conversion of cytosolic LC3-I to membrane-bound LC3-II and accumulation of large LC3-positive vacuolar structures. Pterostilbene also led to phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase activation and disruption of mitochondrial membrane potential. Moreover, it did not induce oxidative stress. Our results suggest that pterostilbene induces accumulation of autophagic vacuoles followed by cell death in HL60 cells.

Controlled cholesterol efflux from the aortic smooth muscle cells triggers microheterogeneity of plasma membrane lipids and induces modification of the mitochondrial topology.

It is generally accepted that phospholipids of plasma membrane display lateral segregation into small microdomains commonly known as lipid rafts. Such lateral lipid organization is under the control of cholesterol. Cholesterol depletion evolved by methyl-beta-cyclodextrin (MCD) has been found to induce further marked perturbation in lateral lipid organization, evidenced in the high field part of electron paramagnetic resonance spectra of plasma membranes labelled with a spectroscopic probe, namely 5-doxyl-stearic acid (5DOXS). Such perturbation of surface lipid topo-logy has been found to induce distinct changes in the mitochondrial morpho-logy, i.e. switch from filamentous form into small granular form.

NADH-generating substrates reduce peroxyl radical toxicity in RL-34 cells.

There is general agreement that oxidative stress may induce apoptotic and necrotic cell death. Recently it has been shown that NADH can be considered an important antioxidant as it reacts with peroxyl and alkoxyl radicals under in vitro conditions. Therefore, in the present study we hypothesized that an increase in intracellular NADH using specific substrates will protect RL-34 cells against cytotoxicity of 2'-azobis (2-amidinopropane) dihydrochloride (AAPH), which is a peroxyl radical generating compound. Cells treated for 24 hours with 6.0 mM AAPH were severely damaged: mitochondria were vacuolated, and the level of free radicals significantly increased. Both apoptotic and necrotic cells were detected (11.1% and 11.4%, respectively) even after 5 hours of treatment. Pretreatment of the cells with substrates which increase the intracellular level of NADH, such as lactate, beta-hydroxybutyrate, and ethanol, distinctly inhibited AAPH-induced reactive oxygen species (ROS) formation and cell death. On the other hand, acetoacetate (AcA), which decrease the intracellular level of NADH, had opposite effects. Interestingly, NADH-generating substrates augment, while AcA reduced superoxide radical formation induced by AAPH. These results may suggest that although NADH generating substrates may exert some deleterious effects within a cell by inducing reductive stress, they diminish alkoxyl or peroxyl radical cytotoxicity. The protection is associated with a decrease in ROS formation measured by dichlorofluorescein, but with an increase in superoxide radical formation.

Stress-induced changes of interleukin-1beta within the limbic system in the rat.

The aim of this study was to investigate the influence of two periods of life, namely P28 and P360, on the changes in interleukin-1beta (IL-1beta) immunoreactivity (-ir) in the hippocampus (CA1, CA3, DG) and amygdala (central-CeA, medial-MeA) caused by acute and repeated open field (OF), or by forced swim (FS) exposition. Rats were divided into groups: non-stressed, exposed to acute (one-time for 15 min) and chronic stressors (21 days for 15 min daily). We found IL-1beta-ir in the control group to be higher in P360 than in P28. In P28, under OF and FS exposure, IL-1beta-ir in the CeA remained unaltered but increased in the MeA and in the hippocampus after acute and chronic stress. In P360 no changes were observed in the IL-1beta-ir level after acute and chronic stimulation. These data demonstrate that only the levels of IL-1beta-ir in juvenile rat brains are affected by FS and OF. Additionally, there was no significant difference between FS and OF stimulation in IL-1beta-ir.

Distribution of neuronal nitric oxide synthase (nNOS)-immunoreactive elements in the rabbit piriform cortex.

The piriform cortex (PC), the primary olfactory cortex, is involved in the processes of learning and stress response and possibly plays an important role in epileptogenic activity. The results of several recent studies suggest that those PC neurons that contain neuronal nitric oxide synthase (nNOS) may play a key role during spatial learning and in the modulation of initiation, propagation and generalisation of seizures in various experimental models and may influence neuronal vulnerability after epileptic insults. The aim of this study was to characterise the pattern of distribution and morphology of nNOS-immunoreactive elements in PC of the adult rabbit. The co-localisation of nNOS and calretinin (CR) was also studied. The pattern of nNOS-ir within the rabbit PC is similar to that described previously in other mammals. The morphology of nNOS-ir elements, namely varicose fibres and Cajal-Retzius cells, suggest that NO has an important influence on PC function. Surprisingly, in the rabbit PC nNOS-ir elements show a very low level of co-localisation with CR-ir.

A case of multiple abnormalities of the azygos venous system: a praeaortic interazygos vein.

The posterior thoracic wall, an area drained by the azygos venous system, is a common site for surgical intervention. Since the venous part of the cardiovascular system is subject to most common variation, abnormalities in the azygos venous system are often reported. Some of the anatomical variants have significant clinical implications for computed tomography image assessment and mediastinal surgery. During dissection of the posterior mediastinum in a 76 year-old Caucasian male cadaver we found a rare variation in the azygos venous system. The hemiazygos vein drained the left 9th to 11th left posterior intercostal veins. While passing ventrally to the aorta at the level of the body of the eighth thoracic vertebra it was joined by two separate vessels found to be the continuations of the 7th and 8th left posterior intercostal veins. The resultant dilated vessel, termed the "interazygos vein", then opened into the azygos vein on the right side of the vertebral column. Variation in the azygos venous system has often been reported, but the abnormality observed by us appears to be extremely rare. The interazygos vein passing ventrally to the aorta may mimic enlarged lymph nodes and cause misinterpretation of a computed tomography image or, if accidentally damaged during mediastinal surgery, may lead to intraoperative haemorrhage. To the best of our knowledge this report provides new data of potential clinical significance.

The influence of acute and chronic open-field exposure on the hippocampal formation: an immunohistochemical study.

The hippocampus plays a role in new learning, memory and emotion and is a component of the neuroanatomical stress circuit. The structure is involved in terminating hypothalamic-pituitary-adrenocortical (HPA) axis responses to stress and attenuates stress responses by shutting off this axis. The immunoreactivity (-ir) of c-Fos, NGF and its receptor TrkA following acute and chronic open-field stress were studied in CA1-CA3 and the DG of the hippocampus. The material consisted of 21 male adult rats divided into three groups: nonstressed (control) animals and rats exposed to acute (15 min once) and chronic (15 min daily for 21 days) aversive stimulation (open-field exposure). The brains were stained with use of immunohistochemical methods for c-Fos, NGF or TrkA. In the animals exposed to acute open-field stress the number of c-Fos-, TrkA and NGF-ir cells was higher in all the structures studied than in the control animals. However they were differentiated only in c-Fos immunoreactivity. In the rats exposed to chronic open-field stress the number of c-Fos-ir cells in the structures of the hippocampal formation studied was smaller than in rats exposed to acute stress and was comparable to that in the control group. No differences were observed between the groups exposed to acute and chronic stress in the number of TrkA-ir cells in the structures under investigation. The number of NGF-ir neurons in CA1 and CA2 was lower after exposure to chronic than after exposure to acute stress but was still higher than that in the control group. Our findings indicate that neurons of CA1-CA3 and the DG are engaged in the stress response after acute as well as chronic open-field exposure. This is probably related to the important role of the hippocampus in processing new spatial information as well as in the habituation processes, although these appear to have different mechanisms.

Fluoride alters type I collagen expression in the early stages of odontogenesis.

Fluoride alters the expression and post-translational modifications of extracellular matrix proteins in dentin. The aim of our study was to determine the effects of fluoride on type I collagen expression during the early stages of tooth germ development in rats. Pregnant dams were divided into three groups and fed a standard diet. From the fifth day of pregnancy the three groups received tap water with, respectively, trace amounts of fluoride (C), a low fluoride concentration (FL) or and a high fluoride concentration (FH). Changes in type I collagen expression and distribution were evaluated. The expression of type I collagen was restricted to the extracellular spaces of cells of mesenchymal origin. In the youngest animals the most intense immunoreactivity for type I collagen was detected in predentin of the FL group. Although the intensity of immunostaining increased in proportion to the age of the animals, the largest increase in the groups investigated was detected in the FL group. We concluded that a low concentration of fluoride can act as a stimulator of type I collagen deposition in the extracellular matrix of dentin, while high concentrations of fluoride have an opposite effect, acting as an inhibitor of type I collagen formation in dentin.

Immunoreactivity of c-Fos, NGF and its receptor TrkA in the periventricular zone of the rat hypothalamus after open field exposure.

The immunoreactivity (ir) for c-Fos, NGF and TrkA, following an acute and chronic open field stress, were studied in the periventricular zone of rat hypothalamus. Adult rats were divided into three groups: control, exposed to acute (single exposure--15 minutes) and chronic (multiple exposures--15 minutes daily for 21 days) open field stress. In the control rats neurons immunoreactive to c-Fos, TrkA and NGF were found. The number of TrkA- and NGF-ir cells was high, whereas this of c-Fos-ir ones was low. In animals exposed to acute open field stress the number of c-Fos-ir cells in the examined nuclei varied, however it was much higher than that in the control animals. The number of TrkA-ir neurons in all the studied nuclei was also higher than that in the control animals, but the increase of the number of NGF-ir neurons was not observed in supraoptic nucleus. In the animals exposed to chronic open field stress the number of c-Fos-ir cells was increased in comparison to that in the control rats. After chronic stress exposure the number of TrkA-ir neurons in supraoptic nucleus remained high in comparison to that in animals exposed to acute stress, whereas it was decreased in other studied nuclei. No significant differences in the number of NGF-ir cells were observed between the groups exposed to the acute and chronic stress. Observed decrease of c-Fos- and TrkA-ir in the studied nuclei in the animals suffering from chronic stress in comparison with the acute one may indicate the occurrence of habituation phenomenon. This phenomenon does not concern NGF-ir.

The immunoreactivity of c-Fos, NGF and its receptor TrkA after open-field exposure in the central and medial nuclei of the rat amygdala.

The amygdala is a critical component of the neuroanatomical stress circuit. It plays a role in the generation of responses to emotional stimuli. The central (CeA) and medial (MeA) amygdaloid nuclei are implicated in activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. The immunoreactivity (-ir) of c-Fos, NGF and its receptor, TrkA, following acute and chronic open-field stress were studied in the CeA and MeA nuclei of the amygdala. The material consisted of 21 male adult rats divided into three groups: non-stressed (control) animals, rats exposed to acute (once only lasting 15 min) and chronic (15 min daily over 21 days) aversive stimulation (open-field exposure). The brains were stained with the use of immunohistochemical methods for c-Fos, NGF or TrkA. In the control rats c-Fos-, TrkA- and NGF-ir cells were observed in the nuclei studied, but the quantity varied, being moderate or high (immunoreactive to TrkA and NGF) or low (immunoreactive to c-Fos). In the animals exposed to acute open-field stress the number of c-Fos-ir, NGF-ir and TrkA-ir cells in the nuclei under examination was differentiated but higher than that in the control animals. In the animals exposed to chronic open-field stress the number of c-Fos-ir cells in the nuclei studied was similar and was smaller than those in animals exposed to acute stress. The number of TrkA-ir neurons was also lower in comparison to that in animals exposed to acute stress. However, no significant differences in the number of NGF-ir cells were observed between the groups exposed to acute and chronic stress. Diverse expression of c-Fos protein following both acute and chronic stress stimulation may prove the functional heterogeneity of the amygdaloid nuclei investigated. The decrease observed in both c-Fos- and TrkA-ir in MeA (only TrkA in CeA) of animals exposed to chronic stress may indicate the phenomenon of habituation.

Influence of the "open field" exposure on calbindin D28K, calretinin, and parvalbumin containing cells in the rat midbrain - developmental study.

The aim of our study was to analyze the influence of the open field (OF) exposure on: 1. Distribution of c-Fos positive nuclei in: ventral tegmental area, substantia nigra, periaqueductal gray. 2. Appearance of calbindin-D28k, calretinin and parvalbumin in midbrain neurons that are engaged in the stress response. 3. Changes of c-Fos and calcium-binding proteins expression during maturation. The material consisted of Wistar rats of age between 0 and 90 days. The OF exposure was applied throughout 10 min and 90 min before the death of the animals. The brain sections were double stained using the antibodies against c-Fos, CB, CR or PV. Our results showed that in all studied nuclei age-related increase of c-Fos expression (without changing of its distribution properties) was found. PV didn't show any co-localization with c-Fos in neurons of studied regions at any ages, however some PV-immunoreactive (PV-ir) basket-like structures around c-Fos-immunoreactive (c-Fos-ir) neurons were observed. In the youngest group of rats c-Fos-ir cells and cells immunoreactive for CB and CR constituted separate neuronal populations. During maturation increases in the level of their co-localization with c-Fos was observed. We may conclude that in adult rat midbrain structures CB-immunoreactive (CB-ir) and CR-immunoreactive (CR-ir) cells (probably projection neurons) are mainly activated in the stress response following OF exposure. In the contrary PV-ir cells has only an indirect (modulatory) influence upon the c-Fos-ir cells.

Changes in the morphology of the acinar cells of the rat pancreas in the oedematous and necrotic types of experimental acute pancreatitis.

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.

Mimicking of glutathione peroxidase deficiency by exposition of JAR cells to increased level of synthetic hydroperoxide.

A short chain synthetic analogue of lipid hydroperoxides was used to overload glutathione peroxidase (GPx) in human choriocarcinoma cell line JAR cells. Cells exposed to 100 microM tBuOOH displayed a 40% reduction in ATP level and significantly increased in membrane permeability, visualised by the lactate dehydrogenase (LDH) release into the extracellular medium. The intracellular level of oxygen free radicals measured as an oxidation of the dichlorodihydro-fluorescein diacetate (H2DCF-DA) significantly increased after 2 hours of cell exposition to 100 microM tBuOOH. Concomitantly MDA, 4-HNE level increased to 2 nmol/mg of cell protein after 2 hours. Mitochondria stained with MitoTracker Red CMXRos displayed a filamentous appearance in control cells but changed into granular less energised organelles after exposition to tBuOOH. Collectively, the above results indicate the importance of the contribution of oxidative stress in the development of pre-eclampsia.

The influence of storage time upon the real thickness of the histological brain sections.

The aim of the study was to assess the effect of storage time on the actual thickness of the histological sections. The study was performed on 5 brains of adult Wistar rats. The most rapid changes occur at the beginning of the storage process, after about one month the dynamics of changes decreases, but still the thickness of the sections diminishes. It is suggested that quantitative analyses should be performed in similar period of time and the critical care should be taken not only of the chemicals and procedures used, but also of the control of environmental factors.

Relation between the age of specimen and the shrinkage of brain frozen sections.

The aim of the study was to assess the effect of age of the animal upon the real thickness of the frozen sections. The study was performed on 19 rabbit brains. The thickness of the frozen sections regardless of their staining is age-dependent. The relation is proportional during the period from 7 to 180 postnatal day and characterizes both immunohistochemical as well as cresyl violet-stained sections; moreover, changes of the section thickness proceed parallelly. It is suggested that especially for some stereological parameters all required procedures should be standardized to achieve comparable and unbiasedly interpretable results.

Opposite effects of microtubule-stabilizing and microtubule-destabilizing drugs on biogenesis of mitochondria in mammalian cells.

Distribution of mitochondria as well as other intracellular organelles in mammalian cells is regulated by interphase microtubules. Here, we demonstrate a role of microtubules in the mitochondrial biogenesis using various microtubule-active drugs and human osteosarcoma cell line 143B cells and rat liver-derived RL-34 cells. Depolymerization of microtubules by nocodazole or colchicine, as well as 2-methoxyestradiol, a natural estrogen metabolite, arrested asynchronously cultured cells in G(2)/M phase of cell cycle and at the same time inhibited the mitochondrial mass increase and mtDNA replication. These drugs also inhibited the mitochondrial mass increase in the cells that were synchronized in cell cycle, which should occur during G(1) to G(2) phase progression in normal conditions. However, stabilization of microtubules by taxol did not affect the proliferation of mitochondria during the cell cycle, yet a prolonged incubation of cells with taxol induced an abnormal accumulation of mitochondria in cells arrested in G(2)/M phase of cell cycle. Taxol-induced accumulation of mitochondria was not only demonstrated by mitochondria-specific fluorescent dyes but also evidenced by the examination of cells transfected with yellow fluorescent protein fused with mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (pEYFP) and by enhanced mtDNA replication. Two subpopulations of mitochondria were detected in taxol-treated cells: mitochondria with high Delta(psi)(m), detectable either by Mito Tracker Red CMXRos or by Green FM, and those with low Delta(psi)(m), detectable only by Green FM. However, taxol-induced increases in the mitochondrial mass and in the level of acetylated (alpha)-tubulin were abrogated by a co-treatment with taxol and nocodazole or taxol and colchicine. These data strongly suggest that interphase microtubules may be essential for the regulation of mitochondrial biogenesis in mammalian cells.

Structural changes of mitochondria during free radical-induced apoptosis.

The initial proposal for apoptosis stressed nuclear change (condensation of chromatin) and the intactness of intracellular organelles, including mitochondria, based on light and electron microscopic observations. However, data have accumulated to demonstrate that the opening of megachannels of mitochondrial membranes, resulting in the swelling of the organelles, notably by Ca2+ and free radicals, is the crucial step in the apoptotic processes of the cell. Application of fluorescent dyes to mitochondria, combined with flow cytometry, has made it possible to detect subtle changes in the structure and function of the organelles related to apoptosis. The present article overviews structural aspects of mitochondria related to apoptosis, including the free radical-induced formation of megamitochondria.

The projection of the amygdaloid nuclei to various areas of the limbic cortex in the rat.

The connections of the amygdaloid body with the areas of the limbic cortex in the rat were studied by means of the method on the axonal retrograde transport of the fluorescent tracer FluoroGold. The tracer was injected into the anterior and posterior limbic cortices (cingulate gyrus, granular and agranular retrosplenial area, respectively. The localization of the corresponding amygdalar projection zones was investigated and the semiquantitative analysis of the connections was conducted. The projection zones in the rat amygdaloid body are organized topographically. Administration of the fluorescent tracer to the anterior and posterior part of the limbic cortex in the rat (cingulate gyrus and both retrosplenial areas, respectively) reveals labeling of cells only for injections to the former one. The labeled cells were present only in the major components of the basolateral amygdaloid complex. The main source of this projection was anterior part of basolateral nucleus (BLA). Some labeled neurons were found in the lateral nucleus and few in the ventral part of basolateral nucleus. No labeled cells were found within the basomedial nucleus.