Pathological changes in the central nervous system following exposure to ionizing radiation.

Experimental studies in animals provide relevant knowledge about pathogenesis of radiation-induced injury to the central nervous system. Radiation-induced injury can alter neuronal, glial cell population, brain vasculature and may lead to molecular, cellular and functional consequences. Regarding to its fundamental role in the formation of new memories, spatial navigation and adult neurogenesis, the majority of studies have focused on the hippocampus. Most recent findings in cranial radiotherapy revealed that hippocampal avoidance prevents radiation-induced cognitive impairment of patients with brain primary tumors and metastases. However, numerous preclinical studies have shown that this problem is more complex. Regarding the fact, that the radiation-induced cognitive impairment reflects hippocampal and non-hippocampal compartments, it is highly important to investigate molecular, cellular and functional changes in different brain regions and their integration at clinically relevant doses and schedules. Here, we provide a literature review in order support the translation of preclinical findings to clinical practice and improve the physical and mental status of patients with brain tumors.

Early cardiac injury in acute respiratory distress syndrome: comparison of two experimental models.

Acute respiratory distress syndrome (ARDS) is characterized by diffuse lung damage, inflammation, oedema formation, and surfactant dysfunction leading to hypoxemia. Severe ARDS can accelerate the injury of other organs, worsening the patient´s status. There is an evidence that the lung tissue injury affects the right heart function causing cor pulmonale. However, heart tissue changes associated with ARDS are still poorly known. Therefore, this study evaluated oxidative and inflammatory modifications of the heart tissue in two experimental models of ARDS induced in New Zealand rabbits by intratracheal instillation of neonatal meconium (100 mg/kg) or by repetitive lung lavages with saline (30 ml/kg). Since induction of the respiratory insufficiency, all animals were oxygen-ventilated for next 5 h. Total and differential counts of leukocytes were measured in the arterial blood, markers of myocardial injury [(troponin, creatine kinase - myocardial band (CK-MB), lactate dehydrogenase (LD)] in the plasma, and markers of inflammation [tumour necrosis factor (TNF)alpha, interleukin (IL)-6], cardiovascular risk [galectin-3 (Gal-3)], oxidative changes [thiobarbituric acid reactive substances (TBARS), 3-nitrotyrosine (3NT)], and vascular damage [receptor for advanced glycation end products (RAGE)] in the heart tissue. Apoptosis of heart cells was investigated immunohistochemically. In both ARDS models, counts of total leukocytes and neutrophils in the blood, markers of myocardial injury, inflammation, oxidative and vascular damage in the plasma and heart tissue, and heart cell apoptosis increased compared to controls. This study indicates that changes associated with ARDS may contribute to early heart damage what can potentially deteriorate the cardiac function and contribute to its failure.

Effects of nitric oxide donor on the lung functions in a saline lavage-induced model of ARDS.

Acute respiratory distress syndrome (ARDS) is characterized by acute hypoxemia, neutrophil-mediated inflammation, and lung edema formation. Whereas lung damage might be alleviated by nitric oxide (NO), goal of this study was to evaluate if intratracheal NO donor S-nitroso-N-acetylpenicillamine (SNAP) can positively influence the lung functions in experimental model of ARDS. New Zealand rabbits with respiratory failure induced by saline lavage (30 ml/kg, 9+/-3 times) were divided into: ARDS group without therapy, ARDS group treated with SNAP (7 mg/kg i.t.), and healthy Control group. During 5 h of ventilation, respiratory parameters (blood gases, ventilatory pressures) were estimated. After anesthetics overdosing, left lung was saline-lavaged and cell count, cell viability and protein content in bronchoalveolar lavage fluid (BALF) were measured. Right lung tissue was used for estimation of wet/dry weight ratio, concentration of NO metabolites, and histomorphological investigation. Repetitive lung lavage induced lung injury, worsened gas exchange, and damaged alveolar-capillary membrane. Administration of SNAP reduced cell count in BALF, lung edema formation, NO metabolites, and histopathological signs of injury, and improved respiratory parameters. Treatment with intratracheal SNAP alleviated lung injury and edema and improved lung functions in a saline-lavaged model of ARDS suggesting a potential of NO donors also for patients with ARDS.

Reduction of lung inflammation, oxidative stress and apoptosis by the PDE4 inhibitor roflumilast in experimental model of acute lung injury.

Damage of alveolar-capillary barrier, inflammation, oxidative injury, and lung cell apoptosis represent the key features of acute lung injury (ALI). This study evaluated if selective phosphodiesterase (PDE)-4 inhibitor roflumilast can reduce the mentioned changes in lavage-induced model of ALI. Rabbits with ALI were divided into 2 groups: ALI without therapy (A group) and ALI treated with roflumilast i.v. (1 mg/kg; A+R group). One group of healthy animals without ALI served as ventilated controls (C group). All animals were oxygen-ventilated for further 4 h. At the end of experiment, total and differential counts of cells in bronchoalveolar lavage fluid (BALF) and total and differential counts of white blood cells were estimated. Lung edema formation was assessed from determination of protein content in BALF. Pro-inflammatory cytokines (TNFalpha, IL-6 and IL-8) and markers of oxidation (3-nitrotyrosine, thiobarbituric-acid reactive substances) were detected in the lung tissue and plasma. Apoptosis of lung cells was investigated immunohistochemically. Treatment with roflumilast reduced leak of cells, particularly of neutrophils, into the lung, decreased concentrations of cytokines and oxidative products in the lung and plasma, and reduced lung cell apoptosis and edema formation. Concluding, PDE4 inhibitor roflumilast showed potent anti-inflammatory actions in this model of ALI.

Effects of phosphodiesterase 5 inhibitor sildenafil on the respiratory parameters, inflammation and apoptosis in a saline lavage-induced model of acute lung injury.

Acute lung injury (ALI) is associated with deterioration of alveolar-capillary lining and transmigration and activation of inflammatory cells. Sildenafil, phosphodiesterase 5 (PDE5) inhibitor, inhibits degradation of cyclic guanosine monophosphate (cGMP) by competing with cGMP for binding site of PDE5. Positive effects of sildenafil treatment result from influencing proliferation of regulatory T cells and production of proinflammatory cytokines and autoantibodies as well as from modulation of platelet activation, angiogenesis, and pulmonary vasoreactivity. This study evaluated if intravenous sildenafil can influence inflammation, edema formation, apoptosis, and respiratory parameters in rabbits with a model of ALI induced by repetitive lung lavage by saline (30 ml/kg). animals were divided into 3 groups: ALI without therapy (ALI), ALI treated with sildenafil intravenously (1 mg/kg; ALI + Sil), and healthy ventilated controls (Control) which were oxygen-ventilated for 4 hours following treatment administration. during this period, respiratory parameters (ventilator pressures, lung compliance, blood gases, oxygenation indexes etc.) were regularly measured. at the end of experiment, animals were overdosed by anesthetics. The left lung was saline-lavaged and total and differential cell counts and protein content in the bronchoalveolar lavage fluid (BAL) were estimated. The right lung was used for determination of lung edema formation expressed as wet/dry lung weight ratio, for detection of inflammation and oxidative stress markers by ELISA methods, and for detection of lung epithelial cells apoptosis by TUNEL methods and level of caspase-3. Sildenafil treatment reduced leak of cells (P < 0.05), particularly of neutrophils (P < 0.001) into the lung, release of pro-inflammatory mediators (TNF-α, P < 0.001; IL-8 and IL-6, P < 0.01), level of nitrite/nitrate (P < 0.001), markers of oxidative damage (3-nitrotyrosine and malondialdehyde, both P < 0.01), lung edema formation (P < 0.01), protein content in BAL (P < 0.001), and apoptosis of epithelial cells (P < 0.01), and improved respiratory parameters. Concluding, the results indicate a future potential of PDE5 inhibitors also for the therapy of ALI.

Intravenous dexamethasone attenuated inflammation and influenced apoptosis of lung cells in an experimental model of acute lung injury.

Acute lung injury (ALI) is characterized by diffuse alveolar damage, inflammation, and transmigration and activation of inflammatory cells. This study evaluated if intravenous dexamethasone can influence lung inflammation and apoptosis in lavage-induced ALI. ALI was induced in rabbits by repetitive saline lung lavage (30 ml/kg, 9+/-3-times). Animals were divided into 3 groups: ALI without therapy (ALI), ALI treated with dexamethasone i.v. (0.5 mg/kg, Dexamed; ALI+DEX), and healthy non-ventilated controls (Control). After following 5 h of ventilation, ALI animals were overdosed by anesthetics. Total and differential counts of cells in bronchoalveolar lavage fluid (BAL) were estimated. Lung edema was expressed as wet/dry weight ratio. Concentrations of IL-1beta, IL-8, esRAGE, S1PR3 in the lung were analyzed by ELISA methods. In right lung, apoptotic cells were evaluated by TUNEL assay and caspase-3 immunohistochemically. Dexamethasone showed a trend to improve lung functions and histopathological changes, reduced leak of neutrophils (P<0.001) into the lung, decreased concentrations of pro-inflammatory IL-1beta (P<0.05) and marker of lung injury esRAGE (P<0.05), lung edema formation (P<0.05), and lung apoptotic index (P<0.01), but increased immunoreactivity of caspase-3 in the lung (P<0.001). Considering the action of dexamethasone on respiratory parameters and lung injury, the results indicate potential of this therapy in ALI.

Effects of S-Nitroso-N-Acetyl-Penicillamine (SNAP) on Inflammation, Lung Tissue Apoptosis and iNOS Activity in a Rabbit Model of Acute Lung Injury.

Acute lung injury is characterized by lung edema, surfactant dysfunction, and inflammation. The main goal of our study was to evaluate effects of S-nitroso-N-acetyl-penicillamine (SNAP) on migration of cells into the lung and their activation, inducible NO synthase (iNOS) activity, and apoptosis in experimental acute lung injury (ALI) in rabbits. ALI was induced by repetitive lung lavage with saline. The animals were divided into the following groups: (1) ALI without therapy, (2) lung injury treated with SNAP (ALI + SNAP), and (3) healthy animals (Control). After 5 h of ventilation, total and differential counts of cells in the bronchoalveolar lavage fluid (BALF) were assessed. Concentrations of interleukins (IL)-1ß, IL-6, and IL-8, endogenous secretory receptor for advanced glycation endproducts (esRAGE), sphingosine-1-phosphate receptor (S1PR)3, caspase-3, and mRNA expression of inducible NO synthase (iNOS) in lung tissue and nitrite/nitrate in plasma were analyzed. In the right lung, apoptotic cells were evaluated by TUNEL assay. In the animals with ALI, higher counts of cells, mainly neutrophils, in BALF and increased production of pro-inflammatory substances were observed compared with controls. SNAP therapy reduced a leak of cells into the lung and decreased concentrations of pro-inflammatory and apoptotic markers, reduced mRNA expression of iNOS, and decreased apoptotic index in the lung.

Effects of budesonide on the lung functions, inflammation and apoptosis in a saline-lavage model of acute lung injury.

Diffuse alveolar injury, edema, and inflammation are fundamental signs of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Whereas the systemic administration of corticosteroids previously led to controversial results, this study evaluated if corticosteroids given intratracheally may improve lung functions and reduce edema formation, migration of cells into the lung and their activation in experimentally-induced ALI. In oxygen-ventilated rabbits, ALI was induced by repetitive saline lung lavage, until PaO2 decreased to < 26.7 kPa in FiO2 1.0. Then, one group of animals was treated with corticosteroid budesonide (Pulmicort susp inh, AstraZeneca; 0.25 mg/kg) given intratracheally by means of inpulsion regime of high-frequency jet ventilation, while another group was non-treated, and both groups were oxygen-ventilated for following 5 hours. Another group of animals served as healthy controls. After sacrifice of animals, left lung was saline-lavaged and protein content was measured and cells in the lavage fluid were determined microscopically. Right lung tissue was used for estimation of edema formation (expressed as wet/dry weight ratio), for histomorphological investigation, immunohistochemical determination of apoptosis of lung cells, and for determination of markers of inflammation and lung injury (IL-1ß, IL-6, IL-8, TNF-α, IFNγ, esRAGE, caspase-3) by ELISA methods. Levels of several cytokines were estimated also in plasma. Repetitive lung lavage worsened gas exchange, induced lung injury, inflammation and lung edema and increased apoptosis of lung epithelial cells. Budesonide reduced lung edema, cell infiltration into the lung and apoptosis of epithelial cells and decreased concentrations of proinflammatory markers in the lung and blood. These changes resulted in improved ventilation. Concluding, curative intratracheal treatment with budesonide alleviated lung injury, inflammation, apoptosis of lung epithelial cells and lung edema and improved lung functions in a lavage model of ALI. These findings suggest a potential of therapy with inhaled budesonide also for patients with ARDS.

Effect of Fractionated Irradiation on the Hippocampus in an Experimental Model.

BACKGROUND: Ionizing radiation induces altered brain tissue homeostasis and can lead to morphological and functional deficits. The aim of the present study was to investigate the short-term and long-term effect of ionizing radiation on cell population resides adult rat hippocampus. MATERIALS AND METHODS: Adult male Wistar rats received whole- brain irradiation with fractionated doses of gamma rays (a total dose of 20 Gy) and were investigated 30 and 100 days later. A combination of Fluoro-Jade C histochemistry for visualization of degenerating neurons, immunohistochemistry for detection of astrocytes and confocal microscopy were used to quantify the neurodegenerative changes in the hippocampal dentate gyrus and CA1 subfield. RESULTS: A significant increase of Fluoro-Jade C labelled neurons was seen in both of investigated areas through the whole experiment, predominantly 30 days after irradiation. Non- significant decrease of GFAP- immunoreactive astrocytes was found in the hippocampal dentate gyrus and CA1 subfield until 100 days after irradiation. CONCLUSION: Our recent results showed that radiation response of cell types resides the adult hippocampus may play contributory role in the development of adverse radiation-induced late effects.

Proliferation activity in the adult rat brain following exposure to ionizing radiation.

BACKGROUND: The aim of our study was to investigate radiationinduced shortterm effects on the rat forebrain. MATERIAL AND METHODS: Adult male Wistar rats received whole body exposure with fractionated doses of gamma rays (a total dose of 3 Gy) and were investigated seven and 14 days later. Immunohistochemistry and confocal microscopy were used to determine proliferating cells derived from anterior subventricular zone (SVZa) and distributed along the subventricular zone olfactory bulb axis (SVZ OB axis). Cell counting was performed in four anatomical parts along the welldefined pathway, known as the rostral migratory stream (RMS) represented by the SVZa, vertical arm, elbow and horizontal arm. RESULTS: Different rate of cell overdistribution was found in all counted parts through the entire experiment, mostly detectable in the elbow and horizontal arm. CONCLUSION: Results suggested that radiation response of proliferating cells resides the SVZa may a play contributory role in the development of more adverse radiationinduced: late effects.

Radiation-induced long-term alterations in hippocampus under experimental conditions.

BACKGROUNDS: The aim of the present study was to investigate the effect of ionizing radiation on the cell population that co-forms hippocampal formation in an adult rat brain. MATERIALS AND METHODS: Adult male Wistar rats were exposed to whole-body irradiation with fractionated doses of gamma rays (the total dose of 4 Gy). Thirty, 60 and 90 days after irradiation the cell-specific types housed in the CA1, CA3 subregions and adjacent layers were labelled using immunohistochemistry for specific cell phenotypes; Ki-67 marker was used for proliferating cells and GFAP for detection of astrocytes. RESULTS: During the 30th day post-exposure, a considerable increase in the numbers of Ki-67-positive cells was seen. Moreover, significant decline in the density of neurons, mostly in the CA1 subregion, was observed on the 60th day. Slight overaccumulation of Ki-67-positive cells was seen in CA1 area 90 days after radiation treatment. Temporary decrease of GFAP-positive astrocytes was seen thirty days after irradiation, followed by their subsequent increase 60 days after exposure. Secondary decrease of GFAP-positive cells in both of regions was found in the group surviving 90 days post-irradiation. CONCLUSION: Results showed that radiation response of neurons and astrocytes that form the adult hippocampus may play contributory role in the development of prognostically unfavourable adverse radiation-induced late effect.

Expression pattern of anti-apoptotic protein survivin in dysplastic nevi.

UNLABELLED: The anti-apoptotic protein survivin was detected in a panel of 27 dysplastic nevi. From each representative paraffin block 4 mm sections were cut and stained with anti-survivin antibody (DAKO, Clone 12C4). In each section, the labeling intensity, the subcellular location of survivin antigen, the percentage of labeled cells and the degree of dysplasia were assessed. Survivin was present in 23 out of 27 cases (85.2%), but absent in 4/27 cases (14.8%). Positive staining was confined to the cytoplasm (C) of nevus cells only in 18 cases (66.7%), while cytoplasmic as well as nuclear positivity (NC) was found in 5 cases (18.5%). In no case solely nuclear staining could be seen. Furthermore, in 4 out of 5 cases (80%) with NC staining, severe dysplasia was found. Our data point at usefulness of survivin staining, otherwise rarely performed in dysplastic nevi. We confirm the importance of nuclear location of the survivin antigen, which may be helpful for assessing the possible progression to melanoma. KEYWORDS: survivin, immunohistochemistry, nevi, dysplasia, melanoma.

Effect of paternal rat irradiation transmitted to the progeny during prenatal development.

The transgenerational transmission of radiation damage was investigated on the basis of quantitative changes of nucleic acids and histones, the integral index of tissue and organ cellularity. Male Wistar rats, whole-body irradiated with the dose of 3 Gy of gamma rays, were mated with non-irradiated females 25 or 80 days after exposure and their progeny were investigated on the 15th (embryos), 17th (embryos), or 19th (embryonic brain) day of prenatal development (E15, E17, and E19Br, respectively). A significant increase in DNA and RNA concentration and content was found on the 15th day and predominantly on the 17th day of gestation in the progeny of males irradiated 80 days before mating. On the contrary, in the progeny of the same males, concentration of histones was decreased in groups E15 and E19Br. Finally, the radiation alterations in the progeny arisen from irradiated spermatogonia (by paternal exposure 80 days before mating) were more profound in nucleic acids than in histones. Our findings suggest an incidence of radiation-induced genome instability manifested as enhanced proliferating activity of cells in response to DNA damage in the progeny of males, mated at later intervals after exposure.

Effect of low-dose irradiation on proliferation dynamics in the rostral migratory stream of adult rats.

Ionizing radiation as one of the strongest cytogenetic factors can induce significant injury to adult brain. In the present study, adult male Wistar rats were exposed to single whole-body gamma irradiation with the dose of 3 Gy. One, 5, 10, 25, 40 or 80 days after irradiation, proliferating cells were labelled using BrdU immunohistochemistry. BrdU-positive cells were counted individually in the three anatomical parts of the brain RMS: the vertical arm, elbow, and horizontal arm. The number of BrdU+ cells decreased rapidly during the 1st day after exposure in the whole extent of the RMS. In course of the following days, considerable increase was observed in the elbow and vertical arm of the RMS with the maximal values on the 5th and 10th days, respectively; until the 40th day after irradiation, the numbers of BrdU+ cells returned to the control level. Contrary to the two previous parts of the RMS, in the horizontal arm, no statistically significant increase was found and the decrease under control values occurred at the longest survival time. Our results suggest that the whole-body irradiation of rats with the sublethal dose of gamma irradiation can induce acute as well as long-lasting changes in the brain regions where proliferation activity is retained during adulthood.