Defocused CO2 laser on equine skin: a histological examination.

REASONS FOR PERFORMING STUDY: No studies have been published on effects of treatment with a defocused beam carbon dioxide (CO2) laser on equine skin histology. A better understanding of this will help to define how lasers should be used, in order to reduce potential side effects. OBJECTIVE: To describe the acute effects of different doses of defocused CO2 laser, ranging from therapeutic to surgical levels, on equine skin. METHODS: Defocused CO2 laser was administered to the skin in the hamstrings (91 J/cm2), fetlock (137 J/cm2) and loin (450 J/cm2) areas of 13 Standardbred horses. The acute effects on skin histology were examined 90 min after the end of therapy. RESULTS: Mild changes with focal spongiosis and subepidermal clefts were found after 91 J/cm2 irradiation and more severe changes with diffuse subepidermal clefts after the 137 J/cm2 dose. A homogeneous eosinophilic acellular zone of dermis and destruction of adnexal structures, and significant thinning of the epidermis was observed after the 450 J/cm2 dose. CONCLUSIONS: The present study indicates acute dose-dependent changes in equine skin histology after laser treatment Severe tissue damage was induced using a 450 J/cm2 dose. POTENTIAL RELEVANCE: To reduce the potential side effects of defocused CO2 laser treatment, the laser parameters must be carefully evaluated. Caution should be taken if doses higher than 91 J/cm2 (16 W, 4 min, and 42 cm2) are used in irradiation of equine skin.

Functional receptors for atrial natriuretic peptide in the rat mammary gland during lactation.

The present study was undertaken: 1) to localize and characterize atrial natriuretic peptide (ANP) receptors in the rat mammary gland; and 2) to elucidate ANP-induced cellular formation of cyclic GMP (cGMP) and alterations in alveolar morphology during both early and late lactation. Receptor autoradiography, employing rat-specific [125I]ANP as radioligand, demonstrated binding sites in the secretory tissue and larger blood vessels of the mammary gland. Binding of [125I]rANP to membrane fractions was completely displaced by unlabeled ANP and brain natriuretic peptide. C-type natriuretic peptide and cANP(4-23) revealed limited competition with radiolabeled ANP only during early lactation, indicating a more heterogeneous receptor population at that time. Systemically administered ANP induced cGMP formation in the alveolar epithelium, as shown with immunohistochemistry, and increased mammary tissue cGMP concentrations in vivo throughout the lactation period. Image analysis revealed enlargement of alveolar (but not epithelial) cell area after ANP stimulation in late lactation, suggesting altered alveolar filling or myoepithelial cell relaxation. These results indicate that ANP induces biological effects in the rat mammary gland through specific ANP-A receptor interaction with subsequent intracellular cGMP formation. ANP may therefore play a regulatory role in the control of mammary gland blood supply and secretory function.

Membrane-associated carbonic anhydrase activity in the kidney of CA II-deficient mice.

Carbonic anhydrase II-deficient mice offer a possibility to study the localization along the nephron of membrane-associated carbonic anhydrase (CA) activity without interference from the cytoplasmic enzyme. We studied the localization of CA in kidneys from CA II-deficient and control mice by immunocytochemistry (CA II) and histochemistry. Cytoplasmic staining was found in convoluted proximal tubule, thick limb of Henle, and principal and intercalated cells of collecting duct in the control animals but was absent in the CA II-deficient mice. In cells with cytoplasmic staining the cell nuclei were stained. Intense histochemical activity was associated with apical and basolateral membranes of convoluted proximal tubule, first part of thin limb, thick limb, and basolateral membranes of late distal tubule. In collecting ducts of control animals, the basolateral cell membranes of intercalated cells were the only clearly stained membranes. In CA II-deficient animals one type of intercalated cell was stained most intensely at the apical membranes and another only at the basolateral. We suggest that the former corresponds to Type A intercalated cells secreting H+ ions to the luminal side and the latter to Type B cells secreting H+ ions to the basolateral side.

Membrane-associated CA activity in the eye of the CA II-deficient mouse.

PURPOSE: Membrane-associated carbonic anhydrase (CA) activity is probably of great importance for transepithelial transport of ions and fluid. Histochemical studies have indicated its presence in the eye, but such histochemical data are difficult to evaluate because of interference from cytoplasmic CA isozymes, of which CA II is predominant. CA II-deficient mice offered the possibility to study the localization of membrane-associated CA activity, without influence from CA II: METHODS: The localization of CA in the eyes of CA II-deficient mice and of normal mice was studied by the cobalt-phosphate histochemical method. RESULTS: In both types of mice, intense histochemical CA activity was associated with the apical and basolateral membranes of the pigmented and nonpigmented ciliary epithelium, of the corneal endothelium, and of the pigmented epithelium of the retina. It also was localized at the cell borders of the Müller cells and of the lens epithelium and fibers. There also was CA activity in the endothelium of the capillaries of the choroid and retina but not in that of the larger vessels. CONCLUSIONS: Membrane-associated CA activity is found in many ocular cells known to transport fluid and ions. Inhibition of the CA activity of the basolateral membranes of the ciliary nonpigmented epithelium probably explains the reduction of aqueous humor flow seen after the administration of CA inhibitors.

Carbonic anhydrase activity in different placenta types: a comparative study of pig, horse, cow, mink, rat, and human.

The placenta has multiple functions, being the organ which provides oxygen and nutrients to the developing conceptus. In the placenta, the enzyme carbonic anhydrase (CA) may provide ions for exchange with Na+, K+, and Cl- in transepithelial movement of ions and fluid, as well as facilitating carbon dioxide diffusion. It can also be active in intermediary metabolism, such as gluconeogenesis, urea, and fatty acid synthesis. Placental material from pig, horse, cow, mink, rat, and human was therefore investigated, representing placenta types with variations in shape, internal architecture, and nature of the interhemal barrier. After glutaraldehyde fixation, sections were stained by a histochemical CA-method demonstrating all active isozymes. The most striking feature in common was a positive reaction in the maternal capillaries, when present, as in pig, horse, cow, and mink. In the maternal epithelium, the activation of CA was only observed in the pig, which also exhibited the strongest activity at the maternal interface, which reacted moderately in rat, weakly in horse, and was not visible in cow and human. The trophoblast was positive in pig and rat, whereas it was negative in horse, cow, human, and mink placentae except for few scattered trophoblast cells in pig, horse, and cow, which showed very intense activity. In the fetal capillaries, a positive reactivity was only observed in mink and human. The utilization of CA in placental transfer and metabolism is thus highest in the pig, rat, and mink, compared with horse, cow, and human. It can therefore be concluded that the activation and localization of CA in the placental interhemal barrier varies considerably among species.

Carbonic anhydrases in cytosol, nucleus, and membranes of rat liver.

The relative contribution of each functional carbonic anhydrase (CA) isozyme to liver CA activity of fed or starved adult male rats has been determined. The functional isozymes are CA II, CA III, CA IV, and CA V. Total CA, CA III, CA II, CA IV, and CA V activities (in mumol CO2 converted.min-1.liver-1), as measured by mass spectrometric assay using NaHC18O16O in aqueous solution at pH 7.4 and 37 degrees C, were 94,867, 38,621, 37,000, 14,515, and < 5,000 in fed rats and 40,630, 10,498, 9,137, 18,338, and < 2,600 in starved rats, respectively. CA II was unevenly distributed throughout the liver. In perivenous and periportal cytosols, as determined by the digitonin-pulse perfusion technique, CA II activity was (in mg cytosolic protein-1) 325 and 69 in fed rats and 167 and 33 in starved rats, respectively. CA III was more evenly distributed and less affected by starvation: CA III activity in perivenous and periportal cytosols was (in mg cytosolic protein-1) 84 and 55 in fed rats and 113 and 52 in starved rats, respectively. Evidence that CA III was concentrated in the nucleus was obtained histochemically by the Ridderstråle cobalt-precipitation technique in 2-microns-thick glutaraldehyde-fixed sections from adult fed rats. Liver CA activity was higher in the perivenous hepatocytes in cytosols and nuclei, whereas CA IV was homogeneously distributed. Incubation of the 2-microns sections with 1 microM acetazolamide resulted in inhibition of all membrane-associated CA, 50% of cytosolic CA, and no nuclear CA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of phytoestrogen supplementation in the feed on the shell gland of laying hens at the end of the laying period.

Shell quality decreases as laying hens age and the aim of present study was to investigate how a supplement of daidzein, a natural phytoestrogen in soya, affects key factors in the shell gland and eggshell quality in late-stage laying hens. Hybrids of Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB), received either a daidzein diet (50 mg/kg feed) or a control diet from 60 to 72 weeks of age. Both the total number of capillaries and capillaries with carbonic anhydrase (CA) activity were higher in the LSL hybrid than in the LB. After daidzein supplementation the number of CA positive capillaries was unaffected in the LSL but increased in the LB hybrid indicating a higher sensitivity to daidzein in this hybrid. Estrogen receptor alpha and beta (ERα, ERß) were localized and the complete picture of the two ERs can now be described in shell gland of domestic hens. Nuclear and cytoplasmic staining was generally stronger for ERß, while membrane associated staining was present only for ERα. Interestingly, capillary endothelium contained only ERß and since estrogen regulation of CA is well documented, the presence of an endothelial ER provides one possible route for the increase in CA positive capillaries found in LB hybrids. Eggshell quality or egg production was not affected by daidzein supplementation. The hybrids used in this study showed anatomical differences and reacted differently to daidzein supplementation, but if this can be explained by the divergences in ERß localization noted between the hybrids remains to be clarified.

Defective reproductive organ morphology and function in domestic rooster embryonically exposed to o,p'-DDT or ethynylestradiol.

Environmental pollutants with estrogenic activity have a potential to disrupt estrogen-dependent developmental processes. The objective of this study was to investigate if embryonic exposure to the environmental estrogens o,p'-DDT (1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane; 37, 75, 150 or 300 microg/g egg) and EE2 (17alpha-ethynyl estradiol; 60 ng/g egg) affects the reproductive system in domestic roosters. Following egg injection on Embryonic Day 4, the newly hatched chicks were sexed by cloacal inspection. A skewed phenotypic sex ratio with overrepresentation of chicks deemed as females was observed in the groups exposed to the three highest doses of o,p'-DDT but not in the EE2-exposed group. Normal sex ratios were observed in all groups at adulthood. However, a cloacal deformation seemed to remain in the adult roosters, causing an abnormal semen flow upon semen collection. Semen yield was significantly reduced in both o,p'-DDT-exposed and EE2- exposed birds, whereas semen quality was unaffected. When killed, deformations of the left testis were found in all treatment groups. Image analysis revealed a reduced seminiferous tubular area in the roosters exposed to the two highest doses of o,p'-DDT. Embryonic exposure to o,p'-DDT caused decreased comb weight and right-spur diameter, while EE2 only affected right-spur diameter. In conclusion, this study shows that embryonic exposure to estrogenic compounds can induce permanent effects in male birds. The effects of the two studied compounds were partly similar but o,p'-DDT also induced alterations not seen in the EE2-treated birds.

Intracellular localization of carbonic anhydrase in some vertebrate nephrons.

The Hansson histochemical method for carbonic anhydrase has been modified by embedding of the tissue in a resin before sectioning and incubation. While retaining the now generally accepted validity and specificity of the original method it has several advantages. It is simple and rapid and appears to give unusually sharp localization of the stain. The section thickness may be varied to suit light microscopy and electron microscopy with resolution down to 25 A. The method has been discussed especially with regard to false localization. The intracellular distribution of carbonic anhydrase in the kidneys of frogs, rats and birds has been examined. As a basis for the histochemical investigation an ultrastructural analysis of the avian nephron has been performed, using mainly young swifts. Thin tubules are found exclusively in medullary nephrons, and their cells are of an unusual structure combining high cell thickness with strong development of the paracellular route. The avian initial collecting tubule contains a high proportion of dark cells containing mainly baso-lateral carbonic anhydrase. The distribution and morphology of dark cells in vertebrate nephrons has been surveyed. These cells are also compared with oxyntic cells and it is suggested that the cells are responsible for the terminal acidification of urine taking place in the kidney. A simplified terminology for the vertebrate nephron is suggested.

Intracellular localization of carbonic anhydrase in the frog nephron.

The intracellular distribution of carbonic anhydrase has been studied in the frog nephron by a histochemical method. The study of the detailed intracellular localization was possible by sectioning tissue embedded in plastic (Sorwall, JB-4), before staining essentially according to Hansson. This procedure preserves the intracellular structure well and the resolution is high. For light microscopy sections from 1 to 10 mum were used. For electron microscopy 0.25 mum thick sections were stained and examined in the electron microscope using an accelerating voltage of 100 kV. The highest concentrations of the enzyme seem to be localized to the cell membranes or their immediate neighbourhood. A faint cytoplasmic staining may sometimes be observed. In distal tubule cells the apical part of the cell membrane was heavily stained. Weaker staining was found at the lateral membranes and their infoldings and to some extent the basal parts of the cell membrane. In the canaliculi cells only the lateral and basal parts of the membranes were stained. This latter localization is similar to that in the parietal cells of the mammalian stomach.

Histochemical study of the distribution of carbonic anhydrase in the cat brain.

The distribution of carbonic anhydrase (CA) in cerebrum, cerebellum and medulla oblongata of the cat brain has been examined by a histochemical method. Neuron cell bodies and dendrites are stained in some locations. Many axons are distinctly stained and different intensities of staining can be seen even in adjacent axons. One of the most intensely stained structures is the capillary endothelium and stained capillaries are found in all parts examined. Glial cells are intensely stained in agreement with biochemical and earlier histochemical works. Myelin sheaths are never stained, possibly due to enzyme loss during embedding. The localization of the enzyme shows regional differences. In this respect, the medulla oblongata has been examined in more detail. A small area close to the ventral surface, medial to the roots of the hypoglossal nerve, is characterized by a high CA staining of the neuropil. The cell membrane of some large neurones and the capillary endothelium in this area were also stained. With regard to position and CA content, this area corresponds well to the characteristics of the medullary chemosensitive area as defined by previous experimental studies.

Carbonic anhydrase localization in normal and osteochondrotic joint cartilage of growing pigs.

The histochemical localization of carbonic anhydrase in the normal and osteochondrotic epiphyseal growth cartilage from 15 growing pigs (6 to 18 weeks old) was studied. All animals were clinically normal. The entire thickness of the articular-epiphyseal cartilage complex from the femoral condyles was fixed in 2% glutaraldehyde and embedded in a water-soluble glycolmethacrylate. Sections (1-2 microns) were incubated on the surface of a medium containing cobalt, phosphate, and bicarbonate. A black precipitate formed at sites of enzymatic activity. This method shows the activity of all different isoenzymes of carbonic anhydrase. The specificity was checked by adding the carbonic anhydrase inhibitor acetazolamide to the incubation medium. Osteochondrosis in the epiphyseal growth cartilage was characterized by chondronecrotic areas in resting, proliferative, hypertrophic, and calcifying regions. When the hypertrophic and calcifying regions were involved, insufficient cartilage calcification and focally impaired ossification were seen. The chondronecrotic areas were surrounded by groups of morphologically viable cells, or so-called "clusters." Carbonic anhydrase was present in chondrocytes of hypertrophic and calcifying regions of the normal growth cartilage and in osteoclasts and erythrocytes. No evidence of carbonic anhydrase activity was found in the articular cartilage or in the resting region of normal growth cartilage in any of the pigs. No enzyme activity was found in the osteochondrotic cartilage, either in clusters or dead cells. The lack of carbonic anhydrase in the osteochondrotic cartilage demonstrated in this study may result in an inability to produce the alkaline matrix necessary for calcification and could be one reason for the insufficient calcification typical of this cartilage.

Histochemical localization of carbonic anhydrase in the testis and epididymis of the rabbit.

The distribution of carbonic anhydrase (CA) was studied in the testis and epididymis of mature, male rabbits using a cobalt precipitation method. CA was found only in the endothelium of the capillaries in the testis. The epididymal duct was divided into initial, middle and terminal segments. Strong cytoplasmic CA was present in the apical cells in the initial and middle segments. Vacuoles with CA staining in the membranes were found in the principal cells in the middle segment. Intensely stained basal cells were present in the terminal segment. In the last part of the terminal segment and the first of the ductus deferens the basolateral cell membranes were also stained. The function of the enzyme is discussed especially in relation to acidification of the epididymal fluid and facilitation of CO2 diffusion.

Intracellular distribution of carbonic anhydrase in the rat kidney.

The rat kidney was studied by light and electron microscope after it was histochemically stained for carbonic anhydrase activity. Glomeruli and Bowman's capsule were inactive. Convoluted proximal tubules showed intense activity at the brush border and the basolateral membranes. Cytoplasmic activity also was found. Straight proximal tubules had considerable enzyme activity at basolateral membranes but only low activity at the brush border and in the cytoplasm. In nephrons with long loops, the descending thin limb contained cytoplasmic enzyme activity, whereas the ascending thin limb was inactive. Thin limbs of short loops showed a varying enzyme pattern. In the thick limb of Henle's loop, most enzyme activity was found at the luminal cell border. Distal convoluted tubules showed enzyme activity only at basal infolded membranes. In the late distal tubule, intercalated cells appear among the "ordinary" distal cells, and they contained abundant cytoplasmic enzyme. Many highly active intercalated cells were found also in the cortical and outer medullary segments of the collecting duct. The chief cells in these segments also showed some cytoplasmic enzyme activity. In the inner medullary segment of the collecting duct, enzyme activity disappeared gradually, and the tip of the papilla lacked activity. Acetazolamide (10 microM) completely abolished visible staining, whereas Cl 13850 (10 microM), an inactive acetazolamide analogue, did not interfere with the staining.

Localization of carbonic anhydrase in the sperm-storing regions of the turkey and quail oviduct.

The localization of carbonic anhydrase in the sperm storage regions of turkey and quail was investigated using a histochemical method showing the activity of all the isozymes present. Intense carbonic anhydrase activity was found in the turkey sperm storage tubules and infundibular storage glands, whereas no activity could be detected in the quail at these sites. Both species did, however, show strong membrane-bound and cytoplasmic activity in the non-ciliated cells of the utero-vaginal surface epithelium and scattered cells of the vaginal epithelium. The enzyme catalyses the reaction CO2 + H2O <--> H+ + HCO3-, and the presence of carbonic anhydrase in these regions makes rapid changes in pH possible. It is suggested that increasing pH and/or the addition of bicarbonate stimulates sperm motility needed during transfer of the oviducal lumen. A lowering of the pH would keep the sperm quiescent during storage. The duration of sperm storage is considerably longer in the turkey than in the quail. The high quantity of carbonic anhydrase in the turkey sperm storage tubules may, thus, play a role in the duration of sperm storage.

Membrane-associated carbonic anhydrase activity in the brain of CA II-deficient mice.

Membrane-associated carbonic anhydrase (CA) activity is of importance for transepithelial transport of ions and fluid. Histochemical studies have indicated its presence in the brain, but the data are difficult to evaluate because of interference from cytoplasmic CA isozymes, of which CA II is the predominant one. CA II-deficient mice offer a possibility to study the location of membrane-associated CA-activity, without interference from CA II. The location of CA activity in the brain of CA II-deficient and normal mice was studied by the cobalt-phosphate histochemical method, and that of CA I, CA II and CA III by an immunocytochemical method. The brains of both types of mice lacked cytoplasmic isozymes CA I and CA III, and the CA II-deficient mice also lacked CA II. In the normal mice, oligodendrocytes and choroid epithelium stained for CA II in the cytoplasm. In normal and CA (II)D-mice there was an intense membrane associated histochemical CA activity in neuronal processes. Neuronal perikarya were not stained. Endothelial membranes of brain capillaries showed strong histochemical CA-activity. Choroid epithelial cells had histochemical CA activity in the cytoplasm and along apical and baso-lateral cell membranes. The results suggest that membrane-associated CA-activity found along neuronal processes probably modulates pH of the extracellular fluid and thus neuronal activity. CA II and the membrane-associated CA of choroidal epithelium are probably involved in the secretion of cerebrospinal fluid.

Localization of carbonic anhydrase in the goat mammary gland during involution and lactogenesis.

The aim of this study was to determine whether carbonic anhydrase (CA) activity in goat mammary capillaries is regulated mainly by local or systemic mechanisms. One gland was dried before the contralateral gland, and after parturition only one gland was milked. Biopsies were taken from the mammary glands of three goats at 14 d intervals during involution and the start of the following lactation. A histochemical method was used to visualize sites of CA activity. To follow the involution process, milk (liquid) samples were taken from both teats each week and analysed for pH and composition. The time course of CA activity disappearance and reappearance in the capillaries was related to changes in milk composition and alveolar area. A dense network of capillaries showing membrane-bound staining for CA was found surrounding the alveoli in the lactating gland. CA activity gradually decreased in the drying gland, although the other gland was being milked. After 8 weeks involution the dried gland had a significantly lower number of stained capillaries than the milked gland. Almost no stained capillaries were found during late pregnancy, when both glands were dried and the tissue growth maximal. During lactation milk pH was 6.6 +/- 0.3 and this increased to 7.0 +/- 0.1 in the course of involution. In the last trimester of pregnancy the pH returned to its lower value, while the mammary gland was devoid of stained capillaries. Therefore, the capillary CA could not have been directly involved in the pH regulation of milk. The CA activity reappeared in the capillaries directly after delivery, but only in the milked gland. Clearly the regulation of CA activity is influenced more by local than by systemic factors and is associated with the metabolic activity of milk secretion.

Histochemical localization of carbonic anhydrase in the testis and epididymis of the boar.

The testis and epididymis of sexually mature, fertile boars were studied for localization of carbonic anhydrase (CA) using a cobalt precipitation technique. In the testis, cytoplasmic CA was found in the Sertoli cells and in the capillaries surrounding the seminiferous tubules. The epididymal duct was divided into initial, middle and terminal segments, and regional differences in CA activity were observed. The cell membranes of the basal cells were stained in the initial and middle segments. Strong cytoplasmic CA staining was present only in the apical cells in the initial and middle segments. The basolateral cell membranes were stained in the principal cells of the terminal segment and the ductus deferens. As a rule the capillaries surrounding the epididymal duct were unstained. The enzyme, specifically localized in regions of the male genitalia acting as sperm reservoirs, might be related to the quiescence of the stored spermatozoa by influencing the acid-base status of the epididymal fluid.