Comparative study of the topographic localization of NADPH diaphorase positive cells in the rabbit and pheasant thymuses

Nicotinamide adenine dinucleotide hydrogen phosphatediaphorase (NADPHd) histochemistry was used as a marker for nitric oxide synthase (NOS). In the rabbit thymus, NADPHd staining was observed between the capsule and corticomedullary junction in radially oriented blood vessels in the cortex. The outer surface of the thymic lobule and interlobular septa showed adipocytes clumped together. There was a high density of NADPHd positive cells in the medulla, without a sharp boundary in corticomedullary space. In addition to radially oriented blood vessels in the cortex, they were also found as solitary profiles with well stained walls in the medulla. Neuronal plexuses were localized in perivascular topography. In the pheasant thymus, NADPHd positive cells were present as clusters which were distributed in the medulla and the corticomedullar area. NADPHd positive nerve fibres were localized in perivascular topography.     

Induction of micronuclei in plant cells after exposure to accelerated ion irradiation

The root apex cells ofPisum sativum were irradiated in an U-240 isochronous cyclotron at the Institute for Nuclear Research of the Ukrainian National Academy of Sciences, Kyiv. Several types of micronuclei, differing in structural peculiarities, were observed in the 84 h following exposure to beams of accelerated¹H,⁴He, and¹⁴N ions with linear energy transfer (LET) of 0.95, 9.34, and 221 keV/µym, respectively. The maximum micronucleus induction was observed after irradiation with helium ions. Results obtained show that the micronucleus assay is a responsive test for investigations of cytogenetic damage produced by high LET beams in dividing cell systems in vivo.     

Comparative effects of 60Co gamma-rays and neon and helium ions on cycle duration and division probability of EMT 6 cells. A time-lapse cinematography study

Exponentially growing cultures of EMT 6 cells were irradiated in vitro with neon ions, helium ions or 60Co gamma-rays. Time-lapse cinematography allowed the determination, for individual cells, of cycle duration, success of the mitotic division and the age of the cell at the moment of irradiation. Irradiation induced a significant mitotic delay increasing proportionally with the delivered dose. Using mitotic delay as an endpoint, the r.b.e. for neon ions with respect to 60Co gamma-rays was 3.3 +/- 0.2 while for helium ions it was 1.2 +/- 0.1. Mitotic delay was greatest in those cells that had progressed furthest in their cycle at the time of irradiation. No significant mitotic delay was observed in the post-irradiation generation. Division probability was significantly reduced by irradiation both in the irradiated and in the post-irradiated generation. The reduction in division probability obtained with 3 Gy of neon ions was similar to that obtained after irradiation with 6 Gy of helium ions or 60Co gamma-rays.     

Late ultrastructural effects of heavy ions and gamma irradiation in the gastrointestinal tract of the mouse

The irradiated gastrointestinal tract of LAF1 mice was examined one year following a single dose (1000 rad) of either 12C heavy ions or 60Co gamma rays. Qualitative ultrastructural analysis of the gastrointestinal tract of mice exposed to heavy ions or gamma irradiation did not show any discernible differences. In the stomach of irradiated mice, parietal cells contained numerous lysosomes; the gastric chief cells occasionally contained myelin figures. The epithelial cells of the small intestine, especially jejunum and ileum, showed several changes: (1) increased vacuolation was seen both inter- and intra-cellularly, (2) epithelial cell projections penetrated the basal lamina and were in contact with underlying mesenchymal cells, (3) occasional Paneth cells contained intracellular vacuoles consisting of fibrillar and granular material. In the large intestine occasional signs of degeneration were observed. Qualitative analysis of stromal elements of the gut in irradiated mice indicated the presence of damage to capillary endothelial cells, smooth muscle cells and some nerve processes. The amount of basement membrane (BM) around capillaries and small vessels was increased; the same phenomenon was observed to affect the nerve processes, but with less severity. Quantitative analysis of the basement membrane thickness around capillaries in irradiated vs. control mice showed significant differences. Basement membrane thickness around capillaries in the gastric mucosa and duodenum did not differ significantly in any of the treatment groups. In jejunum, the gamma treated animals exhibited significantly higher BM thickness when compared to unirradiated controls. In ileum, only 12C-heavy ion treated animals showed thicker BM when compared to their respective controls. In colon, both 12C- and 60Co-treated animals showed increased BM thickness when compared to controls.     

Heavy ion induced membrane damage: Hemolysis of erythrocytes and changes in erythrocyte membrane fluidity

Human erythrocytes were irradiated with heavy ions of energies between 4 and 18 MeV/u having linear energy transfer (LET) values between 92 and 14000 keV/µm. Hemolysis has been studied as a macroscopic parameter for membrane damage and changes of the fluidity as a more microscopic parameter. The membrane fluidity changed in a characteristic dose-dependent manner as detected by electron spin resonance employing 12-doxylstearic acid methyl ester spin label (SL 12). Lysis cross sections and RBE values were determined from dose effect curves. The results demonstrate a high hemolytic efficiency of heavy ions compared to X rays. With increasing LET values the measured relative biological efficiency (RBE) values increase continuously. In the complete LET range the cross sections formed one common curve as function of LET and no saturation effects are observed. This is in direct contrast to other biological endpoints such as cell inactivation or DNA damage.     

Initial yields of DNA double-strand breaks and DNA Fragmentation patterns depend on linear energy transfer in tobacco BY-2 protoplasts irradiated with helium, carbon and neon ions

The ability of ion beams to kill or mutate plant cells is known to depend on the linear energy transfer (LET) of the ions, although the mechanism of damage is poorly understood. In this study, DNA double-strand breaks (DSBs) were quantified by a DNA fragment-size analysis in tobacco protoplasts irradiated with high-LET ions. Tobacco BY-2 protoplasts, as a model of single plant cells, were irradiated with helium, carbon and neon ions having different LETs and with gamma rays. After irradiation, DNA fragments were separated into sizes between 1600 and 6.6 kbp by pulsed-field gel electrophoresis. Information on DNA fragmentation was obtained by staining the gels with SYBR Green I. Initial DSB yields were found to depend on LET, and the highest relative biological effectiveness (about 1.6) was obtained at 124 and 241 keV/microm carbon ions. High-LET carbon and neon ions induced short DNA fragments more efficiently than gamma rays. These results partially explain the large biological effects caused by high-LET ions in plants.     

Influence of emulsion type and atmospheric oxygen on the fading of latent image in electron microscopic radioautography

Summary The intensity of radioautographic reactions in Ilford L4, Sakura NR-H2 and Kodak NTE emulsions was compared after exposure in either dry air or dry helium gas at 4°C to test the stability of latent images in the presence or absence of oxygen. A light proof container is described in which slides bearing radioactive sections coated with the three emulsions were exposed in dry helium at a constant pressure of approximately 0.5 atm. The comparison of air and helium atmospheres during exposure of radioautographs was estimated qualitatively for ¹²⁵I-labeled thyroid sections stored for several years and, in addition, quantitative data was derived from ³H-labeled methacrylate sections stored from 21 days to 1 year.With the three emulsions under study, the background fog remains low under both exposure conditions at 4°C for as long as several years duration. Using L4 emulsion, similar high grain densities are obtained in air and helium and, therefore, the latent images in L4 emulsion remain stable in the presence of oxygen. In the case of NTE and NR-H2 emulsions, as the exposure time increases, substantially lower reaction intensities are observed in air than in helium. This difference in reaction intensity is evident by 3 weeks with NTE and after 4 weeks with NR-H2. Hence, there is fading of the latent images in the latter emulsions in the presence of oxygen.It is concluded that reliable results may be obtained with the L4 emulsion by exposure of radioautographs in dry air, whereas with the NR-H2 and NTE emulsions, exposure should be in an oxygen-free medium, such as is provided by a dry helium atmosphere.     

RADIOAUTOGRAPHIC STUDIES OF THE STEM CELL IN THE THYMUS OF THE IRRADIATED RAT

Radioautographic evidence is presented which characterizes the marrow derived stem cell which promotes thymic recovery following irradiation in the rat. These immigrant cells are similar in morphology to blood monocytes and have been called monocytoid, meaning monocyte-like in appearance. The typical cell had abundant pale staining cytoplasm and a nucleus with many invaginations and folds and a fine chromatin structure. There was no prominent nucleolus. The majority of these cells entered the thymus of the irradiated rat via the blood vessels into the septa and made their way through the connective tissue to the outer cortex. Three distinct morphological cell types appeared to be derived from the immigrant cells. These were fibrocyte-like cells which were located within the septa, macrophages located mainly within the medulla and septa, and large blast cells within the cortex, which proliferated giving rise to large thymocytes. The blast cells were characterized as having abundant moderately basophilic (and pyroninophilic) cytoplasm with a distinct cytoplasmic boundary, a large nucleus which still had invaginations and folds, a loose chromatin structure and one or more very prominent nucleoli. They were located in groups primarily within the outer cortex and often adjacent to blood vessels. They were found to be highly susceptible to damage in smear preparations. In contrast, their progeny, the large thymocytes were not highly susceptible to damage in smear preparations but teased out as large round cells with a highly basophilic rim of cytoplasm. The large thymocytes were precursors to medium and small cells. A radioautographic technique for 1 μ tissue sections is also described.     

Radiation-induced cardiomyopathy in the dog

Sequential necropsies and histologic evaluations of young adult beagle dogs were performed after irradiation of the thorax. Total doses to the heart were 36, 44, or 52 Gy given in 4-Gy fractions in 4 weeks. One month after irradiation there was little histologic evidence of damage visible by light microscopy. However, ventricular and septal weights were increased, probably due to edema. At 3 months damage to endothelial and mesothelial cells was evident. By 12 months the myocardium was thinned and focal degeneration and loss of muscle cells and Purkinje fibers were observed. There was extensive subendocardial and epicardial fibrosis as well as intimal proliferation in coronary arteries. Morphometric analyses were performed on the myocardium, pericardium, atria, and aorta. There was a slight increase in perivascular connective tissue in the myocardium. The pericardium was increased in thickness and the ratio of smooth muscle to elastin was decreased in the aorta. Severe fibrosis occurred only in the right atrium. At 1 year there was no clinical evidence of heart failure; however, evidence of myocardial damage was present histologically and functionally. Additional stress and continued aging are likely to enhance the damage and lead to serious complications. The interactions of irradiated lung and heart require further investigation.     

Effect of low-energy electron irradiation on DNA damage by Fe3+ ion

We investigated the combined effects of low-energy electron irradiation and Fe(3+) ion on DNA damage. We used lyophilized pBR322 plasmid DNA films with various concentrations (0 ~ 7 mM) of Fe(3+) ions and irradiation with monochromatic, low-energy 3 or 5 eV electrons for these studies. DNA-Fe(3+) films were recovered and analyzed by agarose gel electrophoresis to identify and compare the effects of Fe(3+) ions and/or low-energy electrons alone or in combination on DNA damage. In nonirradiated DNA-Fe(3+) films, there was little DNA damage observed (less than 10% of the total DNA loaded on the gel appeared damaged) for Fe(3+) ion up to 7 mM concentration. In irradiated DNA films without Fe(3+) ions, there was also very little DNA damage observed (less than 3% of the total DNA loaded on the gel appeared damaged). However, when DNA-Fe(3+) films, were irradiated with low-energy electrons, DNA damage was significantly increased compared to the sum of the damage caused both by either Fe(3+) ion or low-energy electrons irradiation alone. We proposed that both DEA and/or electron transfer processes might play a role in the enhanced DNA damage when DNA-Fe(3+) films were irradiated by low-energy electrons.     

A Monte Carlo code for the simulation of heavy-ion tracks in water

TILDA, a new Monte Carlo track structure code for ions in gaseous water that is valid for both high-LET (approximately 10(4) keV/microm) and low-LET ions, is presented. It is specially designed for a comparison of the patterns of energy deposited by a large range of ions. Low-LET ions are described in a perturbative frame, whereas heavy ions with a very high stopping power are treated using the Lindhard local density approximation and the Russek and Meli statistical method. Ionization cross sections singly differential with energy compare well with the experiment. As an illustration of the non-perturbative interaction of high-LET ions, a comparison between the ion tracks of light and heavy ions with the same specific energy is presented (1.4 MeV/nucleon helium and uranium ions). The mean energy for ejected electrons was found to be approximately four times larger for uranium than for helium, leading to a much larger track radius in the first case. For electrons, except for the excitation cross sections that are deduced from experimental fits, cross sections are derived analytically. For any orientation of the target molecule, the code calculates multiple differential cross sections as a function of the ejection and scattering angles and of the energy transfer. The corresponding singly differential and total ionization cross sections are in good agreement with experimental data. The angular distribution of secondary electrons is shown to depend strongly on the orientation of the water molecule.     

Structure and innervation of the pineal gland of the rabbit,Oryctolagus cuniculus (L.)

Summary A light microscopic investigation of the rabbit pineal gland with the aid of silver-stained sections gave the following results. In the gland a medulla and a cortex can be distinguished, the medulla containing so-called light and dark pinealocytes, the cortex only light ones. Autonomic nerve fibres reach the pineal organ by two routes: (1) via the perivascular spaces of pineal blood vessels and (2) via two distinct nerve bundles, the nervi conarii. Bilateral superior cervical ganglionectomy revealed that these pinealo-petal nerve fibres are mainly orthosympathetic postganglionic. Intramural pineal neurones with synaptic-like structures on their cell bodies and dendrites point to the presence of a parasympathetic innervation next to the orthosympathetic one. Direct afferent or efferent neural connections with the brain appeared to be absent. Acknowledgements. The author wishes to thank Professor Dr. J. Ariëns Kappers for encouragement and help, Mr. H. K. Koerten for his technical assistance and Miss A. M. Feddema for typing the manuscipt.     

Tissue changes in the rabbit periodontal ligament during orthodontic tooth movement

In this (semi) quantitative animal study the reaction of the periodontal ligament (PDL) to experimental tooth movement is described. To this end, rabbit first incisors were moved sideways with helical torsion springs for periods varying from 3-24 hours. The initial force of the springs was 50 gf. The histomorphology of the PDL was studied in 5 microns thick plastic sections. Comparison with control animals and animals wearing passive springs showed that tooth movement leads to an increased trauma in the PDL within only a few hours. This trauma is characterized by hyalinization, tears and ruptures in the fibres and blood vessels, and by the presence of extravascular erythrocytes and pyknosis. Tissue damage significantly increased with time. After 24 hours of tooth movement, the PDL fibers are compressed or stretched in 68% of the sections and the blood vessels in the PDL are compressed or stretched in 62% of the sections. Even in the controls, more than 15% of the sections displayed slightly stretched or compressed fibers, and about 10% showed slightly compressed or stretched blood vessels. This indicates that some damage is regularly present in a normally functioning PDL. Increases in the percentage of sections with blood vessel compression are found in all groups wearing passive springs, especially after 6 hours. A high concordancy in compression and tension patterns of blood vessels and fibers is present in 83% of the sections. Pyknotic cells are practically confined to areas with compressed PDL fibers in rabbits wearing active springs. Extravascular erythrocytes were found in sections with all types of fiber patterns. A significant majority of extravascular erythrocytes, however, was found in areas with compressed fibers.     

Three-dimensional organization of the developing vasculature of the kidney

Kidney function depends on a well-developed vascular system. Any impairment of the blood supply disturbs the integrity and function of the organ. The differentiation of renal vessels has been investigation for many years, but little is known about the relationship between nephrogenesis and vessel development. In the present work the spatial organization of the differentiating vessels was analyzed in precisely oriented tissue sections and in optical sections acquired by laser scan microscopy. Developing vessels as well as small capillaries were visualized with two endothelium-detecting antibodies. Small vessels running in parallel towards the organ capsule were detected in numerous cortico-medullary-oriented tissue sections. Cross-sections of the nephrogenic zone showed a regularly arranged network, which was composed of cells detected by both monoclonal antibodies. Parts of this network were localized in regions of the nephrogenic zone which have been assumed to be free of vessels or vessel-like structures for a long time. These results were confirmed by the laser-scan-microscopic analysis of complete cortex explants. The extraordinarily regular arrangement of the endothelial network in the nephrogenic zone allowed us to reconstruct the developing vascular system. The results presented here underline the close relationship between nephrogenesis and vessel development. Received: 20 May 1996 / Accepted: 11 July 1996     

Topographical relationships between catecholamine-and neuropeptide-containing fibers in the median eminence of the newt,Triturus alpestris

Summary Dopaminergic and peptidergic nerve fibers were simultaneously demonstrated with a double-labeling technique at the ultrastructural level. The first antibody, raised against tyrosine hydroxylase, was applied during the preembedding phase and visualized with the peroxidase method. The second antibody, raised against one of the peptides met-enkephalin, somatostatin or gonadotropin-releasing hormone (GnRH), was applied to the ultrathin sections and visualized with gold-labeled goat anti-rabbit IgG. The fibers of both categories were present in the zona externa of the median eminence, frequently contacting the basal lamina of the portal vessels. In addition, topographical relationships between different types of nerve fibers were observed in the perivascular areas, although there were no morphological signs of synaptic specializations. Using serial sections, it could be established that one GnRH-fiber contacted both a dopaminergic fiber and a fiber immunoreactive for met-enkephalin. The observations support earlier physiological data concerning the regulation of the hypothalamo-hypophyseal axis, with special emphasis on the release of neurohormones in the median eminence of the newt.     

Cortical and leptomeningeal cerebrovascular amyloid and white matter pathology in Alzheimer's disease

Alzheimer's disease (AD) is characterized by neurofibrillary tangles and by the accumulation of beta-amyloid (Abeta) peptides in senile plaques and in the walls of cortical and leptomeningeal arteries as cerebral amyloid angiopathy (CAA). There also is a significant increase of interstitial fluid (ISF) in cerebral white matter (WM), the pathological basis of which is largely unknown. We hypothesized that the accumulation of ISF in dilated periarterial spaces of the WM in AD correlates with the severity of CAA, with the total Abeta load in the cortex and with Apo E genotype. A total of 24 AD brains and 17 nondemented age-matched control brains were examined. CAA was seen in vessels isolated from brain by using EDTA-SDS lysis stained by Thioflavin-S. Total Abeta in gray matter and WM was quantified by immunoassay, ApoE genotyping by PCR, and dilatation of perivascular spaces in the WM was assessed by quantitative histology. The study showed that the frequency and severity of dilatation of perivascular spaces in the WM in AD were significantly greater than in controls (P< 0.001) and correlated with Abeta load in the cortex, with the severity of CAA, and with ApoE epsilon4 genotype. The results of this study suggest that dilation of perivascular spaces and failure of drainage of ISF from the WM in AD may be associated with the deposition of Abeta in the perivascular fluid drainage pathways of cortical and leptomeningeal arteries. This failure of fluid drainage has implications for therapeutic strategies to treat Alzheimer's disease.     

重离子射线照射对家蚕的生物影响

为解明重离子射线的生物影响,调查了氖、碳及氦(20Ne8+,LET=300keV/μm;12C5+,LET=116keV/μm和4He2+,LET=16.2keV/μm)等重离子射线照射家蚕(Bombyxmori)后的存活率及形态变化。重离子射线照射不同发育时期的幼虫后所引起的生物影响不同,幼虫的发育时期越早,照射后引起的生物影响越大;对同一时期的幼虫,随着剂量的增加,照射的生物影响加大;以化蛹率和羽化率为指标的放射线感受性在供试的3种射线间具有相似的变化倾向,只是射线的射程越长,照射的生物影响越大;对熟蚕卵巢存在部位的局部照射也显示相似的结果。同一射线的不同LET轨迹位置对家蚕的卵巢及真皮细胞的生物影响不同,用Mylar薄膜覆盖调节碳离子射线的射程,卵巢及真皮细胞越是接近射线高LET的Bragg峰,照射个体的鳞毛及卵的形成被强烈抑制。因此,重离子射线对家蚕的生物影响与细胞及植物种子等小个体不同,对于全体照射,重离子射线的射程长短所造成的生物影响比射线的LET大小所引起的生物影响要大;而对于局部照射,目的器官越是接近射线的高LET轨迹,照射的生物影响越大。     

Identification of pericytes in the central nervous system by silver staining of the basal lamina

Vibratome sections obtained from perfusion-fixed rat brains were stained by means of silver impregnation and physical development according to Gallyas (1970). Small pieces of the cerebral cortex were postfixed with buffered osmium tetroxide solution and processed for electron microscopy to examine the localization of the silver deposit at the cellular level. The cell surfaces of pericytes and smooth muscle cells were completely outlined by silver grains. Endothelial cells and perivascular astrocytes, however, showed an asymmetric distribution of the silver deposit, i.e., the deposit was restricted to the abluminal endothelial surface and to the astrocytic membrane adjacent to the vessel wall, respectively. The method allowed a clear-cut distinction between perikarya of endothelial cells and pericytes as well as glial cells in perivascular position, even at the light-microscopic level.     

80MeV/u20Ne10+离子对SMMC-7721细胞DNA损伤及细胞周期的影响

从辐照剂量和修复时间两个角度研究了重离子辐照对肿瘤细胞DNA损伤及细胞周期的影响,为重离子治癌的临床应用积累基础数据。不同剂量的80MeV／u^20Ne^10 辐照SMMC—7721细胞样品,利用单细胞凝胶电泳技术(Single Cell Gel Electrophoresis,SCGE)对细胞DNA损伤进行了检测,利用流式细胞技术(Flow Cytometry Methods,FCM)对细胞周期变化进行了分析。80MeV／u^20Ne^10 辐照后4小时内,SMMC—7721细胞的DNA损伤与辐照剂量呈线性关系,在0小时组其线性相关因子r为0．9621,4小时组为0．914;随着修复时间的增加,DNA损伤与辐照剂量不再线性相关,但0．5Gy,1Gy和2Gy三个剂量点的DNA损伤程度极为相近。另外,重离子辐照后SMMC—7721细胞发生S期和G2／M期阻滞现象,其随剂量变化及时间变化的规律不同于X、γ等低LET(Linear Energy Transfer)射线辐照。     

Superresolution Imaging of Aquaporin-4 Cluster Size in Antibody-Stained Paraffin Brain Sections

The water channel aquaporin-4 (AQP4) forms supramolecular clusters whose size is determined by the ratio of M1- and M23-AQP4 isoforms. In cultured astrocytes, differences in the subcellular localization and macromolecular interactions of small and large AQP4 clusters results in distinct physiological roles for M1- and M23-AQP4. Here, we developed quantitative superresolution optical imaging methodology to measure AQP4 cluster size in antibody-stained paraffin sections of mouse cerebral cortex and spinal cord, human postmortem brain, and glioma biopsy specimens. This methodology was used to demonstrate that large AQP4 clusters are formed in AQP4^−/− astrocytes transfected with only M23-AQP4, but not in those expressing only M1-AQP4, both in vitro and in vivo. Native AQP4 in mouse cortex, where both isoforms are expressed, was enriched in astrocyte foot-processes adjacent to microcapillaries; clusters in perivascular regions of the cortex were larger than in parenchymal regions, demonstrating size-dependent subcellular segregation of AQP4 clusters. Two-color superresolution imaging demonstrated colocalization of Kir4.1 with AQP4 clusters in perivascular areas but not in parenchyma. Surprisingly, the subcellular distribution of AQP4 clusters was different between gray and white matter astrocytes in spinal cord, demonstrating regional specificity in cluster polarization. Changes in AQP4 subcellular distribution are associated with several neurological diseases and we demonstrate that AQP4 clustering was preserved in a postmortem human cortical brain tissue specimen, but that AQP4 was not substantially clustered in a human glioblastoma specimen despite high-level expression. Our results demonstrate the utility of superresolution optical imaging for measuring the size of AQP4 supramolecular clusters in paraffin sections of brain tissue and support AQP4 cluster size as a primary determinant of its subcellular distribution.