The purpose of this investigation was to observe the sequential ultrastructural changes in tumor implants of a well established tumor line in isologous mice.

The purpose of this study was to determine whether defoliation, a condition of stress caused by Choristoneura fumiferana herbivory, induces a disturbed pathologic condition in presumably normal tissue produced by the affected balsam fir trees. Techniques used in this investigation included dry weight determination, light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and biochemical and physical analyses. Terminal branch sections that contained shoots with buds and needles were cut from the upper crown on opposite sides of the tree. Collections were made from several previously selected trees in Vermont during April, June and July. The collected materials were labelled, placed in plastic bags and returned to the laboratory for histological and electron microscopic studies. The shoots derived from buds of stressed trees were shorter and produced fewer needles than shoots derived from equivalent position in nondefoliated buds. Unlike July-collected buds, the April buds did not exhibit distinct apical meristem zonation. The ultrastructural study of defoliated April-collected buds differed from nondefoliated buds, in that nondefoliated buds, were very active prior to bud burst and shoot elongation. This activity was indicated by the substantial increase in the number of Golgi-associated vesicles, several large vacuoles with heavy electron dense granular inclusions, abundant rough endoplasmic reticulum, proplastids and cell plate formation. Defoliated bud cells, unlike the nondefoliated bud cells did not contain a high population of cells exhibiting Golgi-associated rich regions, mitochondria, and apparent ce 11 p 1 ate formation. The absence or reduced presence of these metabolic indicators, in defoliated Aprilcollected buds suggest that the defoliated buds are metabolically disturbed. The eventual elongation of shoots of reduced length and number of needles as observed in June-collected buds indicates that activity was not completely inhibited in defoliated buds. The smaller bud size in defoliated balsam fir could also mean that prior accumulation of substances by the bud was effectua1 in that bud development may have been delayed. These findings indicate that Choristoneura fumiferana herbivory alters bud development.

In order to investigate the early ultrastructural lesions at the first sign of lameness in the oligofructose (OF) model of laminitis, the disease was induced in four horses, while another four horses were sham-treated controls. Minor lesions were detected in lamellar samples examined by light microscopy. Examination by transmission electron microscopy (TEM) showed excessive waviness, breaks and separation of portions of lamellar basement membrane (BM) in the treated horses. There was also disintegration and disappearance of hemidesmosomes (HD) and epidermal basal cell (EBC) cytoskeleton, and an increase in the distance between the EBC plasmalemma and the centre of the BM. A link was thus established between the first clinical signs of lameness and ultrastructural changes in the lamellar dermo-epidermal interface. This implied that pathogenesis was underway well before clinical signs (24 h) and that successful therapy would need to be instituted earlier than previously considered. Earlier therapy may be facilitated if delivery of efficacious drugs to the foot was achievable. A treatment modality that delivered effective concentrations of anti laminitic drugs to the target organ (the epidermal lamellae) was thus an objective of this study. Hoof lamellar tissue from five ponies treated with prolonged euglycaemic hyperinsulinaemia and four control (sham-treated) ponies were harvested and processed for TEM. Lamellae from treated ponies showed attenuation and elongation of secondary epidermal lamellae (SEL), HD number reduction and infiltration of leukocytes. Unlike carbohydrate induced laminitis in horses, there was no global separation at the lamellar dermal/epidermal interface in ponies. Two unique lamellar lesions found in this induction model was mitosis among EBCs and thickening of the BM, not normally characteristic of acute laminitis. The pathophysiology of hyperinsulinaemic laminitis remains unresolved but if insulin, delivered directly to the foot, induced laminitis several pathophysiological questions would be answered. In particular, it would emphasise the laminitogenic potential of insulin alone in the pathogenesis of laminitis. It also allows the treatment foot to be compared with the remaining three that act as internal controls. A modality that delivered drugs like insulin to the target organ (the epidermal lamellae) was needed and was an objective of this study. A microdialysis (MD) method, based on continuous sampling of the lamellar extracellular fluid (ECF), was developed to monitor lamellar drug concentrations. MD probes were implanted in the hoof lamellar tissue of six normal Standardbred horses under local anaesthesia. A bolus intravenous (IV) dose (5 mg/kg BWT) of gentamicin sulfate was injected into the jugular vein. MD and blood samples were collected at different time points during 24 h, and calibrated and analyzed using an ELISA method for gentamicin. During the first 8 h, the concentration of gentamicin was significantly higher in blood than lamellar ECF, a result that is reversed when lamellar MD is repeated during IO infusion of gentamicin. The results showed that this modestly invasive method was a useful tool to monitor changes in the lamellar ECF during drug delivery or during laminitis development. Knowledge of the anatomy and dynamics of blood circulation in the equine foot is fundamental to understand laminitis pathophysiology. Using histology, decalcification, diaphanization, computed tomography (CT), micro CT and gelatin-India Ink vascular perfusion, the normal anatomy of the dorsal part of distal phalanx (DP) and its vascular relationship to hoof lamellae was characterised. The results showed a close relationship between the distal phalangeal and lamellar circulations and raised the possibility of accessing the lamellar circulation via the DP and the possibility that IO perfusion (IOP) of the DP could deliver drugs to the lamellae. IOP of the DP with methyl methacrylate (MMA) corrosion casting material resulted in filling of the lamellar and sublamellar vascular network and incomplete filling of lamellar capillaries. Perfusion of common digital artery with a suspension of barium sulfate resulted in filling of lamellar arteries but not capillaries. Perfusion of the common digital vein resulted in filling of lamellar veins but not capillaries. Perfusion with barium sulfate partitioned veins from arteries because particle size prevented entry into capillaries. IOP with barium sulfate filled only veins revealing that vascular egress from the DP was venous. This study showed that a retrograde venous connection exists between the DP and lamellar circulations with the potential for lamellar drug delivery. Intra-arterial (IA) and IO infusion results using gelatin-India Ink were markedly improved when cadaver limbs were subjected to cyclic loading within the physiological range. Without loading lamellar capillaries failed to fill no matter what the injection pressure. Cyclic loading cadaver limbs 6 times resulted in complete lamellar capillary filling and suggested that cyclic limb loading contributed to perfusion of lamellar capillaries normally in horses. To evaluate IO delivery of drugs to hoof lamellae in the standing, conscious horse, gentamicin solution (25 mg/mL) was slowly infused (20 µL/min) through an IO bone screw. Lamellar ECF was collected via a lamellar MD probe and blood was collected from the jugular vein. Gentamicin was 50-100 times more concentrated in lamellar ECF than in blood. This study introduces a potential method for delivery of drugs into the lamellar tissue in the standing, conscious horse. Laminitis pathology occurs before clinical signs and can be induced by insulin as well as enteric OF overload. Thus therapy delivered to the target of laminitis, the hoof lamellae, has an improved chance of success if delivered promptly, safely and at high concentrations. A validated drug delivery and lamellar analysis system that achieves these criteria, was the discovery of this project and is now available to combat laminitis.

The present experiments were designed to evaluate the effects of pifithrin-α (PFT-α), which is a p53 inhibitor, on doxorubicin (DOX)-induced apoptosis and cardiac injury. Administration of DOX (22.5 mg/kg ip) in mice upregulated the mRNA levels of Bax and MDM2, whereas PFT-α attenuated those levels when administered at a total dose of 4.4 mg/kg at 30 min before and 3 h after DOX challenge. DOX treatment led to an upregulation of p53 protein levels, which was preceded by elevated levels of phosphorylated p53 at Ser15. PFT-α had no effect on the level of p53 or its phosphorylated form. The protein levels of Bax and MDM2 were elevated by DOX and attenuated by PFT-α. DOX gave rise to increased apoptosis-positive nuclei in cardiac cells, elevated serum creatine phosphokinase, ultrastructural alterations, and cardiac dysfunction. PFT-α offered protection against all of the aforementioned changes. Finally, PFT-α did not interfere with the antitumor potency of DOX. This study demonstrates that PFT-α effectively inhibits DOX-induced cardiomyocyte apoptosis, which suggests that PFT-α has the potential to protect cancer patients against DOX-induced cardiac injury.

歯科基礎医学会, 高橋茂 = Shigeru Takahashi, YAGレーザー照射によるラット顎下腺の損傷と修復に関する形態学的研究 = Morphological studies on the regeneration of rat submandibular gland following YAG laser irradiation, 歯科基礎医学会雑誌, APR 1993, 35(2), pp.115-146 / Hokkaido University (北海道大学) / 博士 / 歯学

Solanine, the green substance in potato, Solanum tuberosum L., tubers, is a toxic glycoalkaloid that is a potential human health hazard. To control the formation of this glycoalkaloid a greater understanding of its site of synthesis is needed. Labelling of solanidine, a direct precursor in the biosynthesis of solanine, with digitonin may indirectly locate the site of solanine synthesis in tubers. A study using ultrastructural cytochemical techniques was initiated to explore this possibility. Sprouted tips and peridermal complex (periderm and cortex) tissue were fixed three different ways: (1) glutaraldehyde only, (2) osmium tetroxide and glutaraldehyde, and (3) glutaraldehyde-osmium tetroxide-digitonin mixture. The glutaraldehyde-osmium tetroxide-digitonin mixture provided the best fixation for this study and was used throughout. An alkaloid extract of potato tissues incubated with digitonin resulted in a precipitate being formed. Application of the alkaloid extract to TLC plates before and after incubating with digitonin indicated only solanidine was removed by the digitonin. In the electron micrographs the solanidine-digitonin complex was recognized as darkly stained needles or spicules. These spicules were observed mostly in the vacuoles in the sprouted tips. However, a few spicules were also noted in the cytoplasm. Relatively few spicules were observed in the peridermal tissue.

Plant parasitic nematodes cause billons of dollars in annual crop losses. One of the most damaging is the root-knot nematode, Meloidogyne incognita, which is known to attack more than 3000 plants. This research will contribute to the understanding of host-plant resistance through characterization of the early infection processes of Meloidogyne incognita race 3 in susceptible (Lahontan) and resistant (Moapa) alfalfa cultivars by light microscopy and transmission electron microscopy. Neither differential penetration of M. incognita J2 into Lahontan, nor migration of J2 from Moapa, played a significant role in the resistance mechanism(s). Coiled nematodes in the cortex were observed in greater numbers in the Moapa 48 hours after inoculation. This position was interpreted as a sign of disorientation and starvation. By 96 hours after inoculation, no coiled nematodes were observed in Lahontan. In Moapa, resistance probably depends not only on the failure of the J2 to identify a suitable feeding site and initiate giant cells, but also on its inability to maintain the giant cells, once they are initiated. At the ultrastructural level, 48 hours after inoculation, the most evident change in both cultivars was the appearance of a uniform interstitial material (IM) between the nematode cuticle and the root cell wall. At 96 hours, IM in Moapa was completely agglutinated while in Lahontan it was still uniform or only slightly agglutinated. Due to these clear differences between both cultivars I propose that the IM plays a role in the resistance of Moapa to M. incognita. Immunolabeling techniques were employed to determine if the distribution of the nematode's surface coat, deposited in host tissues, differs in resistant and susceptible alfalfa cultivars. At 72 hours after inoculation, labeling of surface coat epitopes in Moapa was stronger than at 24 and 48 hours after inoculation. Labeling was observed on the nematode's cuticle, the plant cell wall, and the IM. In Lahontan, 72 and 96 hours after penetration, labeling of the surface coat epitopes was observed on the nematode's cuticle, the root cell walls, and the cell wall junctions of cells near the nematode, but not in direct contact with the cell.

No presente estudo realizamos uma análise ultraestrutural e imuhistoquímica da organização das fibrilas de colágeno no endométrio durante a gestação de camundongos silvestres e deficientes em decorim. Os resultados mostraram que as fibrilas de colágeno em ambos os genótipos sofrem grande variação de forma e tamanho. Observou-se variação significante, na percentagem de distribuição dos diâmetros das fibrilas de colágeno existentes na região decidualizada em ambos os genótipos, porém foi maior nos animais Dcn-/-. Estes animais também apresentam maior percentagem de fibrilas finas quando comparados aos animais Dcn+/+. Observamos ainda que biglicam é expresso no endométrio não decidualizado dos animais Dcn-/-, no 3º dia de gestação. A expressão de lumicam mostrou-se nítida no estroma decidualizado e não decidualizado nos animais Dcn-/- no 7º dia de gestação e foi ausente nos animais Dcn-/-. Estes resultados mostraram que a ausência do decorim promove distúrbios no processo de agregação lateral das fibrilas espessas de colágeno. / The present study is an ultrastructural investigation into the organization of collagen fibrils in the pregnant endometrium of wild-type and decorin-deficient mice. Our results showed that collagen fibrils from both genotypes present a great variability of shape and size in cross section. Significant variation in the diameter of collagen fibrils exists in the decidualized endometrium from both groups of animals. In the decidualized endometrium, the diameter of collagen fibrils increases in both genotypes were higher in Dcn-/- than in Dcn+/+ animals. In the Dcn-/- animals the percentage of thin fibrils with diameter is also higher, when compared with Dcn+/+ animals. We also showed that Bgn is expressed in the non decidualized endometrium in the Dcn-/- animals, on day 3 of pregnancy. The expression of lumican showed a very sharp labeling in the decidualized stroma from day 7, and in the non decidualized estroma from Dcn-/- animals. These results suggest that the deficience of decorin may play a role on collagen fibrillogenesis in different stages of pregnancy.

Made available in DSpace on 2016-04-28T20:16:27Z (GMT). No. of bitstreams: 1 2008 - Jessika Mara Martins Ribeiro.pdf: 4505620 bytes, checksum: 3ace512cbb45ea3a26cc5d88a93ac2b2 (MD5) Previous issue date: 2008-06-24 / Irradiation is a physical process efficiently used in the conservation of foods. The purpose of this study was to investigate the effects of irradiation on the natural mycoflora of poultry feeds and on strains of Aspergillus spp. Maize flour samples, soy crumb and feed were collected directly from the production line of a poultry farm in Avelar, RJ, and exposed to doses of 0, 3.5, 8 and 15 kGy of gamma radiation. Counting, isolation and identification of the contaminant mycoflora were performed before and after irradiation. The radiosensitivity of strains of reference of Aspergillus spp. was determined in CYA medium and in corn for doses ranging from 0 to 8 kGy. Comparisons between the morphologies of control and irradiated strains were performed by using macroscopy, optical microscopy and transmission electron microscopy. Toxigenic profile determination and genetic evaluation by RAPD were also carried out. Higher doses have been found to reduce the number of active colonies, causing elimination of the mycoflora at 8 kGy. A larger radiosensitivity of yeasts was observed in comparison with filamentous fungi. A significant reduction in fungi population occurred at 3.5 kGy to levels below the limit that ensures the hygienic quality of ingredients and poultry feeds. The residual mycoflora was found to decrease with post-irradiation time and included mostly Cladosporium spp., Curvularia spp., Fusarium spp. and Aspergillus spp. and sterility of mycelium prevented further identification of the surviving species of Aspergillus spp. Differences in radioresistance were found among species of Aspergillus and the highest tolerance to radiation was observed for A. parasiticus. Initial morphologic changes were found to be more severe during the first isolation after irradiation than in later ones, with the fungi gradually recovering their normal growth rate. Ultrastructural changes in the irradiated strains were observed mostly in the plasmatic membrane and membranous organelles of nuclei and mitochondria. An increase in the rate of production of toxins by the irradiated strains has been found, however no significant alterations have been observed in their genotypes. Such findings apparently indicate that irradiation stress affected the metabolic response of the fungi, leading to a larger production of toxins. In addition, when appropriate conditions of feeding and growth were restored, the physiologic damages were gradually repaired. Therefore, under such circumstances, irradiated strains may resume growing, thus further deteriorating the substratum. / Irradia??o ? um processo f?sico eficazmente utilizado na preserva??o de alimentos. Este estudo teve como objetivo investigar os efeitos da exposi??o ? radia??o gama sobre a micobiota natural de ra??es av?colas e cepas de Aspergillus spp. Amostras de fub?, farelo de soja e ra??o foram coletadas diretamente da linha de produ??o de uma granja av?cola, no munic?pio de Avelar, RJ e, submetidas ?s doses de 0; 3,5; 8 e 15 kGy de radia??o gama. Foram realizados: contagem, isolamento e identifica??o da micobiota contaminante antes e ap?s a irradia??o. A radiossensibilidade de cepas de refer?ncia do g?nero Aspergillus spp. foi estudada em meio CYA e em milho, com doses de 0 a 8 kGy. Foi comparada a morfologia de cepas controle e irradiadas por macroscopia, microscopia ?ptica e microscopia eletr?nica de transmiss?o. Estudou-se o perfil tox?geno e realizou-se uma avalia??o gen?tica pela t?cnica de polimorfismo de ADN amplificado ao acaso. Observou-se redu??o na contagem com o aumento da dose, tendo sido verificada a elimina??o da micobiota com 8 kGy. Constatou-se tamb?m uma maior radiossensibilidade de leveduras, em rela??o aos fungos filamentosos. Ocorreu efeito do tempo de armazenamento, sendo que as contagens ap?s 45 dias foram inferiores ?s encontradas aos 7 dias ap?s a irradia??o. A dose 3,5 kGy proporcionou significativa redu??o da contagem f?ngica, a n?vel abaixo do limite estipulado para a garantia da qualidade higi?nica de ingredientes e ra??o av?cola. No entanto, h? uma micobiota residual, formada principalmente pelos g?neros: Cladosporium spp., Curvularia spp., Fusarium spp. e Aspergillus spp., esses ?ltimos ap?s serem repicados, apresentaram apenas o desenvolvimento de mic?lio est?ril, o que n?o permitiu a identifica??o da esp?cie. Diferen?as em radiossensibilidade foram observadas entre as cepas de refer?ncia de Aspergillus avaliadas, sendo A. parasiticus o mais radiorresistente em ambos os substratos avaliados: meio CYA e milho. Observou-se menor conidiog?nese e aumento de estruturas de resist?ncia, como os escler?cios. As altera??es morfol?gicas foram mais intensas no isolamento inicial ap?s irradia??o sendo que, gradualmente, os isolados irradiados voltaram a apresentar crescimento semelhante ao padr?o nos repiques sucessivos. Altera??es ultraestruturais nas cepas irradiadas foram observadas principalmente em n?vel de membrana plasm?tica e de organelas, em especial n?cleo e mitoc?ndrias. Essas cepas tiveram aumentada sua produ??o de toxinas. Por outro lado, n?o foram observadas altera??es significativas no gen?tipo, ao menos com os tr?s marcadores utilizados. Esses relatos refor?am a hip?tese de que situa??es de estresse induzem uma maior produ??o de toxina pelos fungos. A irradia??o produz um estresse, afetando metabolicamente o fungo. Contudo, ao serem fornecidos nutrientes e condi??es adequadas de crescimento, essa altera??o fisiol?gica ? superada. Esses achados sugerem que cepas sobreviventes ? irradia??o podem voltar a se desenvolver, deteriorando o substrato, desde que sejam fornecidas condi??es favor?veis ao para seu crescimento.

O estresse parece favorecer a hiperalgesia e alodinia, podendo estar associados à alteração da função muscular mastigatória. Alterações morfofisiológicas em músculos da mastigação induzidos pela alteração oclusal associado ao estresse crônico ainda são escassas na literatura. Este estudo investigou os efeitos do estresse crônico repetido em músculo masseter superficial e profundo de ratos submetidos ou não à exodontia unilateral no ganho do peso dos animais, nas alterações morfológicas (HE, MET), histoquímicas (NADH, SDH e ROS), imunoistoquímicas (laminina e CD31), atividade de MMP-2, -9 e infiltração de neutrófilos (MPO). Vinte ratos (machos, 200g) foram alocados em quatro grupos (n=5): controle (GC), exodontia unilateral (GM), estresse crônico repetido (GE), exodontia associado ao estresse crônico repetido (GME). GE e GME foram submetidos a 10 dias de protocolo de estresse crônico repetido (2 horas diárias) a partir do 14º dia após a exodontia. Houve uma diminuição significativa no ganho de peso dos animais GE e GME. Não foram observadas alterações nos níveis de MMPs e na infiltração de neutrófilos no feixe superficial dos diferentes grupos. GE, GM e GME demostraram alterações morfofisiológicas, ultraestruturais e histoquímicas no feixe profundo, com características específicas e distintas de GC; GE apresentou as maiores alterações. Conclui-se que a exodontia e sua associação ao estresse foram responsáveis por discretas alterações morfofisiológicas no músculo masseter de ratos, contudo o estresse crônico repetido causou modificações morfofisiológicas e ultraestruturais significantes, sendo responsável também pela alteração no peso dos animais. / Stress seems to favor the hyperalgesia and allodynia, which may be related with altered masticatory muscle function. Morphological and physiological changes in the masticatory muscles induced by occlusal alteration associated with chronic stress are still scarce in the literature. This study investigated the effects of repeated chronic stress in superficial and deep masseter muscle of rats with or without the extraction unilateral weight gain of animals and morphological changes (HE MET), histochemical (NADH, SDH and ROS), immunohistochemical (laminin and CD31), MMP-2, -9 activities and neutrophil infiltration (MPO). Twenty rats (male, 200g) were allocated into four groups (n=5): control group (CG), unilateral exodontia (MG), repeated chronic stress (EG), extodontia and repeated chronic stress (MEG). EG and MEG were submitted to 10 days of repeated chronic stress protocol, 2 hours daily, from the 14th day after the extraction. There was a significant decrease in weight gain of animals EG and MEG. No changes were observed in the levels of MMPs and neutrophil infiltration among different groups. EG, MG and MEG have shown morphophisyological, ultrastructural and histochemical changes with specific characteristics and distinct GC GE presenting the higher changes. We conclude that the exodontia and its association to stress were responsible for discrete morphophysiological changes in the masseter muscle of rats, however repeated chronic stress caused significant morphophysiological and ultrastructural changes, being also responsible for change in weight of the animals.