The opposite roles of glucocorticoid and α1-adrenergic receptors in stress triggered apoptosis of rat Leydig cells.

The stress-induced initiation of proapoptotic signaling in Leydig cells is relatively well defined, but the duration of this signaling and the mechanism(s) involved in opposing the stress responses have not been addressed. In this study, immobilization stress (IMO) was applied for 2 h daily, and animals were euthanized immediately after the first (IMO1), second (IMO2), and 10th (IMO10) sessions. In IMO1 and IMO2 rats, serum corticosterone and adrenaline were elevated, whereas serum androgens and mRNA transcription of insulin-like factor-3 in Leydig cells were inhibited. Reduced oxygen consumption and the mitochondrial membrane potential coupled with a leak of cytochrome c from mitochondria and increased caspase-9 expression, caspase-3 activity, and number of apoptotic Leydig cells was also observed. Corticosterone and adrenaline were also elevated in IMO10 rats but were accompanied with a partial recovery of androgen secretion and normalization of insulin-like factor-3 transcription coupled with increased cytochrome c expression, abolition of proapoptotic signaling, and normalization of the apoptotic events. Blockade of intratesticular glucocorticoid receptors diminished proapoptotic effects without affecting antiapoptotic effects, whereas blockade of intratesticular α(1)-adrenergic receptors diminished the antiapoptotic effects without affecting proapoptotic effects. These results confirmed a critical role of glucocorticoids in mitochondria-dependent apoptosis and showed for the first time the relevance of stress-induced upregulation of α(1)-adrenergic receptor expression in cell apoptotic resistance to repetitive IMOs. The opposite role of two hormones in control of the apoptotic rate in Leydig cells also provides a rationale for a partial recovery of androgen production in chronically stressed animals.

Complexity reduction of chromatin architecture in macula densa cells during mouse postnatal development.

AIM: To determine whether complexity of chromatin structure in kidney macula densa cells (MDC) decreases during postnatal development in mice. METHODS: The levels of chromatin structural complexity were measured by determining fractal dimension of MDC nuclei. Kidney tissue was obtained from the total of 32 male Swiss albino mice divided into four age groups (n = 8): newborn (0 days), 10 days old, 20 days old and 30 days old. For a total of 640 MDC chromatin structures, fractal dimension, lacunarity, as well as parameters of Grey level co-occurrence matrix (GLCM) texture were determined. RESULTS: Chromatin fractal dimension in animals aged 10 days, 20 days and 30 days was significantly lower (P < 0.05, P < 0.01 and P < 0.001, respectively), compared with newborn mice. This complexity reduction of chromatin architecture is in accordance with previously published studies, which detected generalized and sustained loss of both tissue and cell complexity during aging. The loss of complexity was texture-independent, since there was no statistically significant difference (P > 0.05) in both chromatin angular second moment and inverse difference moment between the age groups. CONCLUSION: Our results indicate that age-related nuclear intrinsic factors which do not influence chromatin texture may have an important role in MDC postnatal development.

Replacing animal use in physiology and pharmacology teaching in selected universities in Eastern Europe--charting a way forward.

The aims of this study were to explore the use of animals in teaching and the implementation of innovative technology-based teaching practices across a small sample of universities in Eastern Europe. The research methods used were a questionnaire circulated four weeks before a workshop took place (in October 2009, in Belgrade, Serbia), as well as focused, face-to-face group discussions, led by one of the authors during the workshop. Twenty-two faculty (physiologists and pharmacologists), from 13 Eastern European countries, attended the meeting. Fourteen of the eighteen schools represented at the workshop were making use of animals, in some instances in quite large numbers, for their teaching. For example, a single department at a Romanian university used over 250 animals per annum, and at least 1130 animals were used, per annum, across all of the institutions. The species used in largest numbers were the rat (34%), frog/toad (29%), mouse (22%), rabbit (10%), guinea-pig (4%) and dog (1%). None of the universities sampled had implemented institution-wide virtual learning environments (VLEs), although there were isolated instances of local use of VLEs. There was relatively little current use of technology-based teaching and learning resources, but there was considerable enthusiasm to modernise teaching and to introduce innovative learning and teaching methods. The major perceived barrier to the introduction of replacement alternatives was the lack of versions in local languages. There was a consensus view that developing local language exemplars and evaluating their usefulness was likely to have the greatest impact on animal use, at least in the short-term.

Aging-Related Increase of cGMP Disrupts Mitochondrial Homeostasis in Leydig Cells.

Since mitochondria play an essential role in the testosterone biosynthesis, serve as power centers and are a source of oxidative stress, a possible mitochondrial dysfunction could be connected with decreased activity of Leydig cells and lowered testosterone production during aging. Here we chronologically analyzed age-related alterations of mitochondrial function in Leydig cells correlated by the progressive rise of cGMP signaling and with respect to testosterone synthesis. To target cGMP signaling in Leydig cells, acute or long-term in vivo or ex vivo treatments with sildenafil (phosphodiesterase 5 [PDE5] inhibitor) were performed. Aging-related accumulation of cGMP in the Leydig cells is associated with mitochondrial dysfunction illustrated by reduced ATP and steroid production, lowered O2 consumption, increased mitochondrial abundance and mtDNA copies number, decreased expression of genes that regulate mitochondrial biogenesis (Ppargc1a/PGC1a-Tfam-Nrf1/NRF1), mitophagy (Pink1), fusion (Mfn1, Opa1), and increased Nrf2/NRF2. Acute in vivo PDE5 inhibition overaccumulated cGMP and stimulated testosterone but reduced ATP production in Leydig cells from adult, middle-aged, and old rats. The increased ATP/O ratio observed in cells from old compared to adult rats was diminished after stimulation of cGMP signaling. Opposite, long-term PDE5 inhibition decreased cGMP signaling and improved mitochondrial function/dynamics in Leydig cells from old rats. Mitochondrial abundance in Leydig cells decreased while ATP levels increased. Chronic treatment elevated Tfam, Nrf1, Nrf2, Opa1, Mfn1, Drp1, and normalized Pink1 expression. Altogether, long-term PDE5 inhibition prevented age-related NO and cGMP elevation, improved mitochondrial dynamics/function, and testosterone production. The results pointed on cGMP signaling in Leydig cells as a target for pharmacological manipulation of aging-associated changes in mitochondrial function and testosterone production.

Dependence of Leydig Cell's Mitochondrial Physiology on Luteinizing Hormone Signaling.

Knowledge about the relationship between steroidogenesis and the regulation of the mitochondrial bioenergetics and dynamics, in steroidogenic cells, is not completely elucidated. Here we employed in vivo and ex vivo experimental models to analyze mitochondrial physiology in Leydig cells depending on the different LH-cAMP environments. Activation of LH-receptor in rat Leydig cells ex and in vivo triggered cAMP, increased oxygen consumption, mitoenergetic and steroidogenic activities. Increased mitoenergetic activity i.e., ATP production is achieved through augmented glycolytic ATP production and a small part of oxidative phosphorylation (OXPHOS). Transcription of major genes responsible for mitochondrial dynamics was upregulated for Ppargc1a (regulator of mitogenesis and function) and downregulated for Drp1 (main fission marker), Prkn, Pink1 and Tfeb (mitophagy markers). Leydig cells from gonadotropin-treated rats show increased mitogenesis confirmed by increased mitochondrial mass, increased mtDNA, more frequent mitochondria observed by a transmission electron microscope and increased expression of subunits of respiratory proteins Cytc/CYTC and COX4. Opposite, Leydig cells from hypogonadotropic-hypogonadal rats characterized by low LH-cAMP, testosterone, and ATP production, reduced markers of mitogenesis and mitofusion (Mfn1/2, Opa1) associated with reduced mtDNA content. Altogether results underline LH-cAMP signaling as an important regulator of mitochondrial physiology arranging mitochondrial dynamics, bioenergetic and steroidogenic function in Leydig cells.

The irritative property of alpha-tricalcium phosphate to the rabbit skin.

The aim of this study was to assess the irritant properties of a new developed calcium phosphate ceramic, alpha-tricalcium phosphate (alpha-TCP) after single application to intact skin of the rabbit. The test substance, alpha-TCP was produced by modified hydrothermal method and prepared in two different forms, as a solid material (disc 5 x 2 mm) and paste. Both, solid material and paste of alpha-TCP were evaluated for primary skin irritation to the ISO 10993-10:2002/Amd 1:2006 Biological Evaluation of Medical Devices - Part 10. At the end of the study macroscopic examination of the skin was performed. In this model, general and local tolerances were good. Score of primary irritation (SPI) and primary irritation index (PII) of alpha-TCP for both, solid material and paste, revealed that there was no significant toxicity/irritability (PII = 0.0) as compared to the negative control (PII = 0.0). Positive control did cause significant skin irritation in acute irritation test using Draize technique in rabbit model (PII = 2.11). Based on present results, it can be concluded that the the irritation potential of the tested material is negligible. However, other procedures for preclinical safety assessments of the alpha-TCP material are needed in order to completely elucidate its toxic potential.

The impact of introducing computer-based alternatives to the use of animals in the teaching of physiology and pharmacology at Balkan universities - a pilot study.

Balkan universities use a substantial number of small mammals and amphibians in the teaching of physiology and pharmacology. This project investigated whether making computer-based alternatives readily available, and combining this availability with a staff development workshop focusing on methods of integrating such resources into undergraduate curricula, would have any effect on animal use. Teachers from 20 Institutes (from five Balkan countries) participated in the workshop. They presented information about animal use in teaching in their universities, and agreed to introduce at least one computer-based alternative into their teaching in the following year. They were surveyed by questionnaire before, during, and one year after, attending the workshop, in order to estimate any changes in animal use. The results showed a significant (P < 0.01) reduction in animal use and a high level of implementation of the alternatives provided at the workshop. Teachers recognised the potential benefits of using computers to support their teaching. They lacked knowledge about what computer-based alternatives are available and how to find information about them, including published evidence of their educational effectiveness. In this pilot study, a combination of staff development and making alternatives readily available to teachers had a significant impact on animal use in the teaching of physiology and pharmacology.

Pathophysiology of migraine--from molecular to personalized medicine.

INTRODUCTION: Understanding of migraine pathophysiology has substantially improved over the last two decades. As a result, migraine is now mainly considered to be a disorder of the brain, rather than one of the vasculature or the meninges. PATHOPHYSIOLOGY: Although it remains speculative how exactly they relate to each other, the following three processes are important in migraine: 1. Cortical spreading depression is a wave of intense depolarization, it starts in the occipital lobe, propagates through the brain and is followed by a period of suppressed activity. 2. Activation of the trigemonovascular system causes the release of neuropeptides (e.g. calcitonin gene-related peptide, substance P) from the peripheral trigeminal nerve endings. These neuropeptides are thought to play a role in causing and maintaing headache. 3. Sensitization of peripheral and central brain areas, it is thought that pulsating quality of migraine headache is caused by a process of peripheral sensitization. Cutaneous allodynia is a marker of central sensitization. LINK BETWEEN AURA AND HEADACHE: The view that the aura is caused by cortical spreading depression has become generally accepted, and the same is true for the view that activation of the trigemonovascular system underlies migraine headache. However, the relationship between the aura and the activation of the trigemonovascular system and the start of headache remains elusive. GENETICS OF MIGRAINE: One of the most important aspects of the pathophysiology of migraine is the hereditary nature of the disorder. CONCLUSION: Identification of polymorphisms and genetic biomarkers should help us to understand migraine pathophysiology better and thus enable the development of specific, effective "individually-tailored treatment" for each particular migraine patient (personalized medicine).

Comparative analysis of the animal model and results of the clinical research of the aneurysm inclination angle as the predisposing factor for the occurrence of rupture.

INTRODUCTION: Natural course of aneurysms that occur on blood vessels of the brain singles out the need for understanding the mechanism of the occurrence of aneurysm wall rupture and identification of anatomic characteristics as predictive factors for hemorrhage to occur. OBJECTIVE: In this study we comparatively present results of our researches and experimental models on animals. METHODS: We made a comparative analysis of anatomical characteristics of blood vessels of the brain and aneurysms obtained on the basis of digital subtraction angiography and intraoperative finding. In this article we review recent research in the anatomic characteristics of intracranial aneurysms and parent blood vessels. We present a series of 185 aneurysms (ruptured and unruptured) dissected at the Neurosurgical Clinic of Clinical Center of Serbia in Belgrade. RESULTS: Inclination angle may be considered as the vital predesposing factor for intracranial aneurysm rupture. In aneurysms that ruptured it was 139.748+/-27.242 degrees, while in unruptured aneurysms it was considerably smaller and amounted to 100.882+/-22.001 degrees (p<0.01). CONCLUSION: Inclination angle may be regarded as the vital predisposing factor since it differs considerably in unruptured and ruptured aneurysms. Aneurysms with blood stream angle smaller than 115 degrees have very small probability of rupture, while blood stream angle bigger than 150 degrees presents a high risk of rupture.

Predictable morphometric parameters for rupture of intracranial aneurysms - a series of 142 operated aneurysms.

AIM: Intracranial aneurysm rupture is followed by high mortality and morbidity. In order to understand the aneurysm's natural course, it is necessary to recognize the predisposing factors for the rupture. MATERIAL AND METHODS: Analysis included 142 operated aneurysms (94 hemorrhaging and 48 unruptured) in the period from 2008 to 2010. RESULTS: The ratio between the width of the aneurysm neck and diameter of the carrying blood vessel - artery in ruptured aneurysms (OR) was 1.58 ± 0.61, and in unruptured aneurysms 1.14 ± 0.52 (p < 0.01). Aspect ratio of ruptured aneurysm was 1.89 ± 0.59, and in unruptured 1:33 ± 0.17. The angle of inclination of ruptured aneurysms was 139.22 ± 21.53, while in unruptured aneurysms it was 101.73 ± 21.26. CONCLUSION: Based on the results of our research, a predictive model of morphometric characteristics of the vessel bearing the aneurysm to rupture can be identified: an irregular shape of the aneurysm, AR > 1.6, OR > 1.5 and inclination angle > 135 deg.

The effect of grain size on the biocompatibility, cell-materials interface, and mechanical properties of microwave-sintered bioceramics.

The effect of decreasing the grain size on the biocompatibility, cell-material interface, and mechanical properties of microwave-sintered monophase hydroxyapatite bioceramics was investigated in this study. A nanosized stoichiometric hydroxyapatite powder was isostatically pressed at high pressure and sintered in a microwave furnace in order to obtain fine grained dense bioceramics. The samples sintered at 1200°C, with a density near the theoretical one, were composed of micron-sized grains, while the grain size decreased to 130 nm on decreasing the sintering temperature to 900°C. This decrease in the grain size certainly led to increases in the fracture toughness by much as 54%. An in vitro investigation of biocompatibility with L929 and human MRC-5 fibroblast cells showed noncytotoxic effects for both types of bioceramics, while the relative cell proliferation rate, cell attachment and metabolic activity of the fibroblasts were improved with decreasing of grain size. An initial in vivo investigation of biocompatibility by the primary cutaneous irritation test showed that both materials exhibited no irritation properties.

Immobilization stress reduces oxygen consumption of the isolated interstitial rats' testes cells.

The aim of this study was to investigate the effects of acute and repeated immobilization stress on oxygen consumption (QO2) of the isolated interstitial rats' testes cells (ISC). The oxygen consumption by ISC testes was measured in vitro with a Clark-type oxygen electrode. Acute immobilization stress (2 h) induced decrease in QO2 (-49% V4, -31% V3) which was statistically significant (p<0.01). Repeated immobilization stress (2 hours daily for 10 consecutive days) induced a fall in QO2 (-10% V4, -4% V3) but this inhibition of respiration was not statistically significant (p>0.05). The mechanisms by which immobilization stress induces mitochondrial dysfunction as well as mechanisms which develop an adaptive response to repeated immobilization remain unclear, so that further investigations of this mechanisms are required.

[Possible inhibitory effect of nitric oxide donor, dipropylenetriamine nonoate (DPTA/NO), on oxygen consumption of isolated rats' testis interstitial cells].

INTRODUCTION: The understanding of testicular physiology, pathology, and male fertility issues requires knowledge of male germ cell death, energy production and oxygen consumption. The aim of this study was to examine the effect of a new donor of exogenous nitric oxide, di-propylen-three amin-NONOate (DPTA/NO), on the oxygen consumption by freshly isolated rats' testis interstitial cells. MATERIAL AND METHODS: Rats' testis interstitial cells (ISC) were isolated according to Anakwe method and oxygen consumption was measured in an ex vivo bath. The ISC were stimulated with glutamate (0.5 M, 20 microl). DPTA (10(-3) - 10(-6) M) was added and its effect was tested in the absence of ADP (state 4 of respiration) and after that in the presence of ADP (state 3). The reaction was followed for 10 minutes and for each concentration the measurement was done in duplicate. The same measurements without treatment of DPTA/NO were done for control group. RESULTS: DPTA/NO (10(-4) - 10(-6) M) reduced the oxygen consumption rate compared with the control group. The inhibition of respiration was not noticed in concentration of 10(-3) M. In V3 respiratory phase, all tested concentrations of DPTA/NO (10(-3) - l0(-6) M) decreased the oxygen consumption rate in comparison with the control group. CONCLUSION: Our results demonstrate that a NO donor, DPTA/NO (10(-3) - 10(-6) M) inhibits oxygen consumption in isolated rat testis interstitial cells in a manner that could be concentration dependent. Because of limited number of measurements this was not fully proved and additional experiments are needed.

[Labor pain--physiologal basis and regulatory mechanisms].

Clinically, labor (visceral) pain is extremely prevalent in general population, yet its mechanisms have been poorly understood. With development of new electrophysiological techniques and molecular biology technologies, our understanding of physiological bases of labor pain has been markedly improved; in that way possibilities for therapeutic modulation of labor pain are expanded. The aim of this study was to describe the new insight into this topic. In this paper, the theory was exposed that the reason for labor pain had been found in sensitization at the levels of the uterus, dorsal root neurones and phychologic factors. Peripheral sensitization occurs due to cervical inflammatory reaction, associated with cervical ripening and remodeling. Chemical inflammatory mediators (notably prostaglandins, cytokines, granulocytes, enzymes such as metalloproteinases, integrines) activate nociceptive nerve fibers. Nociceptive threshold is reduced (resulting in primary hyperalgesia) and because of that there occurs the total number of action potentials generated and propagated by nociceptive peripheral nerves (visceral hypersensitivity). Central sensitization arises due to phosphorylation of N-methyl-D-aspartate (NMDA) receptors of dorsal root neurones. Numerous receptors, ion channels and signaling molecules, which, together with opioid peptides participate in spinal pain control, represent, at the level of central sensitization, possible therapeutic goals for labor pain modulation. Some of them are: DREAM which constitututively suppresses transcription of mRNA for opioid peptides, oncostatin M, COX-2 inhibitors, cFOS protein, tachykinins, gamma-butyric acid agonist, L-type Ca++ channels. Fear, as one of the most frequent phychologic factors, does (not) provide good control in transmision of pain sensitization by descendent nerve fibers. Some of the candidates for objective pain marker are also described. This article outlines the factors that, based on the contemporary viewpoint, could reduce transmission of pain signals, and thus broaden therapeutic possibilities for modulation of labor pain.

[Central venous catheter-related infections: risk factors and effects of glycopeptide antibiotics].

INTRODUCTION: Central venous catheters (CVC) are used in the treatment of critically ill patients. Indications for placement of CVCs include hemodynamic monitoring, administration of intravenous fluids, medications and total parenteral nutrition. MATERIAL AND METHODS: We investigated risk factors and effects of glycopeptide antibiotics on the development of central venous catheter-related injections in 300 patients treated in intensive care units. A semiquntitative culture technique was used. The investigation included: age, diagnosis on admission, catheter insertion site, catheter duration, the first or next catheter and using of glycopeptide drugs. RESULTS: 91 catheters (30.3%) were colonised, catheter-related infection was found in 50 catheters (16.7%). Infections were more frequent in catheters inserted through the internal jugular vein than in subclavian venous catheters, they were also more frequent if duration of catheterization was longer than seven days, but less frequent in patients who received glycopeptide antibiotics. The isolated microorganism was Staphylococcus aureus. Discussion According to the literature, a number of catheter-related risk factors for infections include: insertion site, type of catheter, the number of manipulations, inadequat asepsis, lumen number, type of antiseptic. The relative importance of one risk factor over another is difficult to assess, given that studies have no priority report. CONCLUSION: The duration of catheterization and the insertion site were the most frequent risk factors for infection. The use of glycopeptide antibiotics during catheterization has protective effects.

[Plasma levels of cortisol and opioid peptide beta-endorphin during spontaneous vaginal delivery].

INTRODUCTION: Labor pain is very frequent in clinical practice, but the underlying mechanisms as well as numerous neuroendocrine responses activated by such pain have not been fully explained yet. OBJECTIVE: The objective of the study was to determine the influence of labor pain on plasma levels of cortisol and opioid peptide beta-endorphin. METHOD: Cortisol and beta-endorphin levels were measured in blood plasma of: health, non-pregnant women (group 1, n=8), health pregnant women (group 2, n=8) and in parturitions, through fourth ages (group 3, n=8). Plasma level ofcortisol was measured by radioimmunoassay, and beta-endorphin by enzyme immunoassay. Data were expressed as mean +/- standard error of mean and were analyzed by Student's t test and Mann Whitney test. RESULTS: Plasma level of cortisol in group 2 was significantly increased compared to the group 1. During labor progression, plasma level of cortisol was rising till the third labor age. Plasma level of cortisol in fourth labor age was not significantly different from the age one and group 1. Plasma level of beta-endorphin was (ng/L): in group 1: 64 +/- 20, group 2: 70 +/- 22, group 3: the first labor age: 75 +/- 15, the second labor age: 193 +/- 54, the third labor age: 346 +/- 97 and the fourth labor age: 114 +/- 31. CONCLUSION: These results indicate that both beta-endorphin and cortisol are involved in regulation and modulation of labor pain and stress.

[Animal models in the study of atherosclerosis]. / Ogledne zivotinje u izucavanju ateroskleroze.

When selecting an animal species for atherosclerosis research, the most important issue is matching the model to the experiment. In choosing the atherosclerosis model there is a wide variety of choices. Genetic hyperlipidemic disorders are best studied in Watanabe rabbits and in transgenic (knockout or overexpressed) mice. Interaction between clotting disorders and atherosclerosis can be evaluated in von Wilebrand's disease swine. If hypo- or hyper-responsiveness to atherogenic stimuli is to be investigated, one should consider the pigeons. Interactions between atherosclerosis and hypertension can be studied in rabbits and monkeys. Macaca nigra has proven to be very valuable in studies concerning the interactions between atherosclerosis and diabetes. Using suitable manipulation and other advantages, rabbits will yield significant insight into specific aspects of hypercholesterolemic effects and atherosclerosis.

Endothelial NO formation does not control myocardial O2 consumption in mouse heart.

To test whether endothelium-derived nitric oxide (NO) regulates mitochondrial respiration, NO was pharmacologically modulated in isolated mouse hearts, which were perfused at constant flow to sensitively detect small changes in myocardial O2 consumption (MVO2). Stimulation of NO formation by 10 microM bradykinin (BK) increased coronary venous nitrite release fivefold to 58 +/- 33 nM (n = 17). Vasodilatation by BK, adenosine (1 microM), or papaverine (10 microM) decreased perfusion pressure, left ventricular developed pressure (LVDP), and MVO2. In the presence of adenosine-induced vasodilatation, stimulation of endothelial NO synthesis by BK had no effect on LVDP and MVO2. Also, inhibition of NO formation by NG-monomethyl-l-arginine (l-NMMA, 100 microM) did not significantly alter LVDP and MVO2. Similarly, intracoronary infusion of authentic NO 2 microM were contractile dysfunction and MVO2 reduction observed. Because BK-induced stimulation of endothelial NO formation and basal NO are not sufficient to impair MVO2 in the saline-perfused mouse heart, a tonic control of the respiratory chain by endothelial NO is difficult to conceive.