O24: THE PANCREATIC EPSILON-CELLS RECOVER THE EMBRYONIC GHRELIN SECRETION IN RESPONSE TO BARIATRIC SURGERY

Introduction Many surgical techniques are employed in the treatment of obesity. A main consequence of these techniques is the severe improvement of Type 2 Diabetes mellitus. Many hypotheses had been exposed to know the intrinsic mechanism developed in this relationship. The enterohormones seems to be a definitive effector. The ghrelin is an enterohormone released for the gastric fundus and it has been related to the mentioned improvement. We hypothesized about the role of pancreatic epsilon cells, which have the capacity to release ghrelin during the embryonic stages. We studied the changes in the ghrelin immunostaining in endocrine pancreas of rats which underwent bariatric surgery. MATERIAL AND Method We employed 16 non obese euglycemic male Wistar rats, randomised in the surgical groups. These groups were the surgical techniques (Sleeve gastric –SG- and Roux en Y Gastric Bypass –RYGB-), and two controls (fasting and surgical). After three months, rats were sacrificed; the pancreas were obtained and processed for the immuno-cytochemical technique. Result We reported a significant increase of epsilon cells (ghrelin positive/mm2 pancreatic area) in the pancreas of SG versus the control groups (vs FC, P<0.01 and vs sham, P<0.05). CONCLUSION. SG and RYGB are surgical techniques broadly employed in humans and both reduce severely the fundus. Paradoxically, the serum level of ghrelin in patients are preserved. We reported that the total suppression of the fundus gastric produced the recovery of an embryonic pancreatic function. This mechanism could be related with the complex physiologic mechanism that improve T2DM after bariatric surgery. Take-home message After bariatric surgery, the pancreas release of ghrelin increased as the response to gastric reduction.

Ultrastructure and cytochemistry of the mature spermatozoon of Khawia armeniaca (Cholodkovsky, 1915) (Caryophyllidea: Lytocestidae), a parasite of Capoeta capoeta sevangi (De Filippi, 1865) (Teleostei, Cyprinidae)

SummaryThe mature spermatozoon of Khawia armeniaca, a monozoic caryophyllidean parasite of templar fish Capoeta capoeta sevangi (De Filippi, 1865) from the Lake Sevan, Armenia, has been studied using transmission electron microscopy and cytochemical technique of Thiéry (1967) for the first time. The mature spermatozoon of K. armeniaca consists of a single axoneme with the 9+‘1’ trepaxonematan structure, cortical microtubules and nucleus which are situated parallel to the longitudinal axis of the spermatozoon, and a moderately electrondense cytoplasm with glycogen particles. The cortical microtubules are arranged in one continuous semicircle beneath the plasma membrane in Region II and anterior part of Region III of the mature spermatozoon. The two opposite rows of cortical microtubules are observed in the remaining nuclear and at the beginning of the postnuclear part (Regions III, IV) of the male gamete The number of cortical microtubules is remarkably variable in the spermatozoa of various Khawia species. K. armeniaca exhibits the highest number of cortical microtubules in comparison with K. sinensis and K. rossittensis. Glycogen was detected in the cytoplasm of prenuclear (II), nuclear (III) and postnuclear (IV) regions with different ultrastructural organization of the mature spermatozoon of K. armeniaca. Variations of sperm ultrastructural characters within caryophyllideans and other cestodes are discussed.

Seasonal Alteration of the Cytosolic and Nuclear Ca2+ Concentrations in Overwintering Woody and Herbaceous Perennials in Relation to the Development of Dormancy and Cold Hardiness

Seasonal alteration of the cytosolic and nuclear Ca2+ concentrations of spruce (Picea engelmannii Parry) and brome grass (Bromus inermis Leyss) was investigated by the antimonate precipitation cytochemical technique. Electron microscopic (EM) observations revealed that electron-dense Ca2+ antimonate deposits, an indication of Ca2+ localization, were seen mainly in the vacuole, the cell wall and the intercellular space in samples of both species, collected on 14 July 1997. Few deposits were found in the cytosol and nuclei, showing a low resting level during summer months. On 8 Aug. 1997 following a decrease in daylength of 1 hour and 12 minutes, Ca2+ accumulation was initiated in spruce with increased cytosolic and nuclear Ca2+ deposits, but not in brome grass. On 8 Sept. 1997, Ca2+ accumulation occurred in the cytosol of brome grass. This followed a drop in ambient temperature to 12 °C. Cytosolic and nuclear Ca2+ deposits continued to increase in spruce. Controlled experiments confirmed that it was the low temperature, not shortening daylength, that triggered Ca2+ accumulation in brome grass. High cytosolic and nuclear Ca2+ concentrations lasted about three months in spruce from early August to early November. However, the high cytosolic and nuclear Ca2+ concentrations in brome grass lasted only about 20 days from early September to the end of the month. During winter and spring, both species had low resting cytosolic and nuclear Ca2+ concentrations. The relationship between the duration of the high cytosolic and nuclear Ca2+ concentrations and the status of the developed dormancy/cold hardiness is discussed in light of current findings.

Cytochemical Demonstration of Oxidative Damage in Alzheimer Disease by Immunochemical Enhancement of the Carbonyl Reaction with 2,4-Dinitrophenylhydrazine

Formation of carbonyls derived from lipids, proteins, carbohydrates, and nucleic acids is common during oxidative stress. For example, metal-catalyzed, “site-specific” oxidation of several amino acid side-chains produces aldehydes or ketones, and peroxidation of lipids generates reactive aldehydes such as malondialdehyde and hydroxynonenal. Here, using in situ 2,4-dinitrophenylhydrazine labeling linked to an antibody system, we describe a highly sensitive and specific cytochemical technique to specifically localize biomacromolecule-bound carbonyl reactivity. When this technique was applied to tissues from cases of Alzheimer disease, in which oxidative events including lipoperoxidative, glycoxidative, and other oxidative protein modifications have been reported, we detected free carbonyls not only in the disease-related intraneuronal lesions but also in other neurons. In marked contrast, free carbonyls were not found in neurons or glia in age-matched control cases. Importantly, this assay was highly specific for detecting disease-related oxidative damage because the site of oxidative damage can be assessed in the midst of concurrent age-related increases in free carbonyls in vascular basement membrane that would contaminate biochemical samples subjected to bulk analysis. These findings demonstrate that oxidative imbalance and stress are key elements in the pathogenesis of Alzheimer disease.

Freeze-fracture Cytochemistry: A New Method Combining Immunocytochemistry and Enzyme Cytochemistry on Replicas

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphatase activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.

Study of the action of tamoxifen on the mammary gland epithelium of premenopausal patients by lysosome quantification

Tamoxifen is an antiestrogen drug widely utilized for the adjuvant hormonal treatment of breast carcinoma. Its use in the primary prophylaxis of this disease is currently being proposed. Although the drug has few side effects, its precise action on breast tissue that has not undergone neoplastic transformation has not been fully elucidated. This prospective, randomized study assessed the estrogen activity of tamoxifen on the mammary gland epithelium of premenopausal patients using a quantitative analysis of mammary epithelium lysosomes identified by the cytochemical technique of GOMORI for acid phosphatase and by light microscopy. Tamoxifen significantly increased the number of lysosomes only during the secretory phase of the menstrual cycle. We concluded that the early effect of the drug on normal mammary tissue is synergistic with the effect of estrogen during the premenopausal period.

Free-radical-derived oxidants and ocular pathology in a model of chronic hypoxia

Hypoxia, whether systemic or localized, plays a role in a number of ocular pathologies. Systemic hypoxia results from cardiovascular or pulmonary abnormalities; localized hypoxia can result from regional vascular blockage or chemical hypoxia as observed in diabetes. Free radical derived oxidants have been implicated in the pathogenesis of vascular and other complications of hypoxia. We studied eyes to determine if there were similarities between the morphological components of diabetic retinopathy and chronic hypoxic retinopathy. Using the cerium NADH-oxidase cytochemical technique to localize the free radical derived oxidant, hydrogen peroxide (H2O2), we investigated the sites and role of oxidative injury in eyes from a rabbit model of chronic systemic hypoxia.New Zealand albino rabbits, reared in a normobaric, hypoxic chamber (65±3 mm Hg) [normoxia = 149 mm Hg], were removed at ages five weeks (immature) or twelve weeks (mature). Animals were euthanized with pentobarbital; eyes were enucleated, fixed and processed for NADH-oxidase localization.

Cytochemical detection of superoxide in cerebral inflammation and ischemia in vivo

We used a cytochemical technique for the detection of superoxide in cerebral inflammation and ischemia-reperfusion in anesthetized cats. The technique is based on the oxidation of Mn2+ to Mn3+ by superoxide; Mn3+, in turn, oxidizes diaminobenzidine. The oxidized diaminobenzidine forms an osmiophilic electron-dense product that is detected by electron microscopy. The reagents, manganese chloride (2 mM) and diaminobenzidine (2 mg/ml), were placed topically on the brain surface of anesthetized cats equipped with cranial windows. Inflammation was induced by topical carrageenan with or without phorbol 12-myristate 13-acetate to activate leukocytes. In inflammation, superoxide was detected in the plasma membrane and in the phagocytic vacuoles of leukocytes. In ischemia-reperfusion, superoxide was identified in the meninges in association with blood vessels. It was located primarily in the extracellular space and occasionally in endothelial and vascular smooth muscle cells. In both inflammation and ischemia, the reaction product was eliminated by superoxide dismutase or by the omission of either manganese or diaminobenzidine. It was unaffected by sodium azide, which inhibits peroxidases. No superoxide was detected in the brain parenchyma. The findings confirm the generation of superoxide is cerebral ischemia-reperfusion and show that it is produced in cerebral vessels.

Use of the tannic acid/para-phenylenediamine lipid cytochemical technique for SEM of atherosclerotic endothelium

A cytochemical technique for the preservation and visualization of neutral lipid in atherosclerotic tissue was developed by Guyton and Klemp.This tannic acid/para-phenylenediamine (TA-PDA) procedure prevented the extraction of extracellular lipid by dehydrating solvent, which permitted it to be imaged in the aortic subendothelium by TEM. We applied the technique to enable documentation by SEM of lipid particles associated with the luminal surface of aortic endothelium during experimental atherogenesis. Preliminary results in a porcine model had suggested that a lipid exocytic, or retro-endocytic, process might occur in chronic hyperlipidemia.

An immunofluorescent cytochemical technique applying micromanipulation to detect microtubules in plant tissues inoculated with fungal spores

An immunofluorescent cytochemical technique to detect microtubules was developed to examine the involvement of microtubules in incipient responses of barley coleoptile cells to fungal attacks. Infiltration of antibodies into target cells was promoted by scratching cell walls of chemically-fixed coleoptile cells with a micromanipulator. In uninoculated control cells, cortical microtubules were arranged obliquely or transversely to the longitudinal axis of the cells. On the other hand, in coleoptile cells which had been fixed 8–10 h after inoculation with a nonpathogen, Erysiphe pisi, microtubules gathered in coleoptile cells beneath mature appressoria of the fungus. When coleoptiles had been fixed 12 h after inoculation, many of the microtubules gathered around an incipient, small papilla, giving a network appearance. The present technique would be helpful for studying the role of microtubules in host cell responses to fungal attack. Key words: microtubules, immunofluorescent cytochemistry, barley coleoptile, Erysiphe pisi.