Confocal microscopy of the bulbar conjunctiva in contact lens wear

Purpose: The aim of this study was to investigate the capabilities of laser scanning confocal microscopy (LSCM) for undertaking qualitative and quantitative investigations of the response of the bulbar conjunctiva to contact lens wear. Methods: LSCM was used to observe and measure morphological characteristics of the bulbar conjunctiva of 11 asymptomatic soft contact lens wearers and 11 healthy volunteer subjects (controls). Results: The appearance of the bulbar conjunctiva is consistent with known histology of this tissue based on light and electron microscopy. The thickness of the bulbar conjunctival epithelium of lens wearers (30.9 ± 1.1 μm) was less than that of controls (32.9 ± 1.1 μm) (P < 0.0001). Superficial and basal bulbar conjunctival epithelial cell densities in contact lens wearers were 91% and 79% higher, respectively, than that in controls (P < 0.0001). No difference was observed in goblet and Langerhans cell density between lens wearers and controls. Conjunctival microcysts were observed in greater numbers, and were larger in size, in lens wearers compared with controls. Conclusions: The effects of contact lens wear on the human bulbar conjunctiva can be investigated effectively at a cellular level using LSCM. The observations in this study suggest that contact lens wear can induce changes in the bulbar conjunctiva such as epithelial thinning and accelerated formation and enlargement of microcysts, increased epithelial cell density, but has no impact on goblet or Langerhans cell density.

Preparation, characterisation and in vivo osteogenesis of mesoporous bioactive glasses

Bone defects, especially large bone defects, remain a major challenge in orthopaedic surgery. Autologous bone transplantation is considered the most effective treatment, but insufficient donor tissue, coupled with concerns about donor site morbidity, has hindered this approach in large-scale applications. Alternative approaches include implanting biomaterials such as bioactive glass (BG), which has been widely used for bone defect healing, due to having generally good biocompatibility, and can be gradually biodegraded during the process of new bone formation. Mesoporous bioactive glass (MBG) is a newly developed bioactive glass which has been proven to have enhanced in-vitro bioactivity; however the in-vivo osteogenesis has not been studied. A critical problem in using the bone tissue engineering approach to restore large bone defects is that the nutrient supply and cell viability at the centre of the scaffold is severely hampered since the diffusion distance of nutrients and oxygen for cell survival is limited to 150-200µm. Cobalt ions has been shown to mimic hypoxia, which plays a pivotal role in coupling angiogenesis with osteogenesis in-vivo by activating hypoxia inducing factor-1α (HIF-1α) transcription factor, subsequently initiating the expression of genes associated with tissue regeneration. Therefore, one aim of this study is to investigate the in-vivo osteogenesis of MBG by comparison with BG and β-TCP, which are widely used clinically. The other aim is to explore hypoxia-mimicking biomaterials by incorporating Cobalt into MBG and β-TCP. MBG and β-TCP incorporated with 5% cobalt (5Co-MBG and 5CCP) have also been studied in-vivo to determine whether the hypoxic effect has a beneficial effect on the bone formation. The composition and microstructure of synthesised materials (BG, MBG, 5Co-MBG, 5CCP) were characterised, along with the mesopore properties of the MBG materials. Dissolution and cytotoxicity of the Co-containing materials were also investigated. Femoral samples with defects harvested at 4 and 8 weeks were scanned using micro-CT followed by processing for histology (H&E staining) to determine bone formation. Histology of MBG showed a slower rate of bone formation at 4 weeks than BG, however at 8 weeks it could be clearly seen that MBG had more bone formation. The in-vivo results show that the osteogenesis of MBG reciprocates the enhanced performance shown in-vitro compared to BG. Dissolution study showed that Co ions can be efficiently released from MBG and β-TCP in a controllable way. Low amounts of Co incorporated into the MBG and β-TCP showed no significant cytotoxicity and the Co-MBG powders maintained a mesopore structure although not as highly ordered as pure MBG. Preliminary study has shown that Co incorporated samples showed little to no bone formation, instead incurring high lymphocyte activity. Further studies need to be done on Co incorporated materials to determine the cause for high lymphocyte activity in-vivo, which appear to hinder bone formation. In conclusion, this study demonstrated the osteogenic activity of MBG and provided some valuable information of tissue reaction to Co-incorporated MBG and TCP materials.

The effect of the histological properties of tumors on transfection efficiency of electrically assisted gene delivery to solid tumors in mice

Uniform DNA distribution in tumors is a prerequisite step for high transfection efficiency in solid tumors. To improve the transfection efficiency of electrically assisted gene delivery to solid tumors in vivo, we explored how tumor histological properties affected transfection efficiency. In four different tumor types (B16F1, EAT, SA-1 and LPB), proteoglycan and collagen content was morphometrically analyzed, and cell size and cell density were determined in paraffin-embedded tumor sections under a transmission microscope. To demonstrate the influence of the histological properties of solid tumors on electrically assisted gene delivery, the correlation between histological properties and transfection efficiency with regard to the time interval between DNA injection and electroporation was determined. Our data demonstrate that soft tumors with larger spherical cells, low proteoglycan and collagen content, and low cell density are more effectively transfected (B16F1 and EAT) than rigid tumors with high proteoglycan and collagen content, small spindle-shaped cells and high cell density (LPB and SA-1). Furthermore, an optimal time interval for increased transfection exists only in soft tumors, this being in the range of 5-15 min. Therefore, knowledge about the histology of tumors is important in planning electrogene therapy with respect to the time interval between DNA injection and electroporation.

Spatial mapping of proteoglycan content in articular cartilage using near-infrared (NIR) spectroscopy

Diagnosis of articular cartilage pathology in the early disease stages using current clinical diagnostic imaging modalities is challenging, particularly because there is often no visible change in the tissue surface and matrix content, such as proteoglycans (PG). In this study, we propose the use of near infrared (NIR) spectroscopy to spatially map PG content in articular cartilage. The relationship between NIR spectra and reference data (PG content) obtained from histology of normal and artificially induced PG-depleted cartilage samples was investigated using principal component (PC) and partial least squares (PLS) regression analyses. Significant correlation was obtained between both data (R2 = 91.40%, p<0.0001). The resulting correlation was used to predict PG content from spectra acquired from whole joint sample, this was then employed to spatially map this component of cartilage across the intact sample. We conclude that NIR spectroscopy is a feasible tool for evaluating cartilage contents and mapping their distribution across mammalian joint

GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation. GATA-4 and GATA-5 are considered the ones with tumor suppressive functions, whereas GATA-6 is more related to tumor promotion. In the digestive system their roles in inflammation and tumor-related molecular pathways remain unclear. In this study, we examined the GATA-related molecular pathways involved in normal tissue organization and renewal and in inflammation-related epithelial repair in the gastrointestinal tract and liver. The overall purpose of this study was to elucidate the relation of GATA factors to gastrointestinal and hepatic disease pathology and to evaluate their possible clinical significance in tumor biology. The results indicated distinct expression patterns for GATA-4, GATA-5, and GATA-6 in the human and murine gastrointestinal tract and liver, and their involvement in the regulation of intestine-specific genes. GATA-5 was confined to the intestines of suckling mice, suggesting an association with postnatal enzymatic changes. GATA-4 was upregulated in bowel inflammation concomitantly with TGF-β signaling. In gastrointestinal tumors, GATA-4 was restricted to benign neoplasias of the stomach, while GATA-6 was detected especially at the invasive edges of malignant tumors throughout the gut. In the liver, GATA-4 was upregulated in pediatric tumors along with erythropoietin (Epo), which was detected also in the sera of tumor patients. Furthermore, GATA-4 was enhanced in areas of vigorous hepatic regeneration in patients with tyrosinemia type I. These results suggest a central role for GATA-4 in pediatric tumor biology of the liver. To conclude, GATA-4, GATA-5, and GATA-6 are associated with normal gastrointestinal and hepatic development and regeneration. The appearance of GATA-4 along with TGF-β-signaling in the inflammatory bowel suggests a protective role in the response to inflammation-related epithelial destruction. However, in extremely malignant pediatric liver tumors, GATA-4 function is unlikely to be tumor-suppressing, probably due to the nature of the very primitive multipotent tumor cells. GATA-4, along with its possible downstream factor Epo, could be utilized as novel hepatic tumor markers to supplement the present diagnostics. They could also serve a function in future biological therapies for aggressive pediatric tumors.

In vitro antioxidant and in vivo prophylactic effects of two gamma-lactones isolated from Grewia tiliaefolia against hepatotoxicity in carbon tetrachloride intoxicated rats

Grewia tiliaefolia is widely used in traditional Indian medicines to cure jaundice, biliousness, dysentery and the diseases of blood. Bioassay-guided fractionation of methanolic extract of the G. tiliaefolia bark has resulted in the isolation of D-erythro-2-hexenoic acid gamma-lactone (EHGL) and gulonic acid gamma-lactone (GAGL). Hepatoprotective activity of the methanolic extract and the isolated constituents were evaluated against CCl4-induced hepatotoxicity in rats. The treatment with methanolic extract, EHGL and GAGL at oral doses of 100, 150 and 60 mg/kg respectively with concomitant CCl4 intraperitoneal injection (I ml/kg) significantly reduced the elevated plasma levels of aminotransferases, alkaline phosphatase and the incidence of liver necrosis compared with the CCl4-injected group without affecting the concentrations of serum bilirubin and hepatic markers. EHGL and GAGL significantly inhibited the elevated levels of thiobarbituric acid reactive substances and glutathione in liver homogenates. Histology of the liver tissues of the extract and isolated constituents treated groups showed the presence of normal hepatic cords, absence of necrosis and fatty infiltration as similar to the normal control. The results revealed that the hepatoprotective activity of EHGL is significant as similar to the standard drug silymarin. To clarify the influence of the extract and isolated constituents on the protection of oxidative-hepatic damage, we examined in vitro antioxidant properties of the test compounds. The extract and the constituents showed significant free radical scavenging activity. These results suggest that the extract as well as the constituents could protect the hepatocytes from CCl4-induced liver damage perhaps, by their anti-oxidative effect on hepatocytes, hence eliminating the deleterious effects of toxic metabolites from CCl4, (C) 2009 Elsevier B.V. All rights reserved.

X-ray scattering and diffraction enhanced imaging studies of in vitro breast tissues

This thesis is a study of the x-ray scattering properties of tissues and tumours of the breast. Clinical radiography is based on the absorption of the x-rays when passing right through the human body and gives information about the densities of the tissues. Besides being absorbed, x-rays may change their direction within the tissues due to elastic scattering or even to refraction. The phenomenon of scattering is a nuisance to radiography in general, and to mammography in particular, because it reduces the quality of the images. However, scattered x-rays bear very useful information about the structure of the tissues at the supra-molecular level. Some pathologies, like breast cancer, produce alterations to the structures of the tissues, being especially evident in collagen-rich tissues. On the other hand, the change of direction due to refraction of the x-rays on the tissue boundaries can be mapped. The diffraction enhanced imaging (DEI) technique uses a perfect crystal to convert the angular deviations of the x-rays into intensity variations, which can be recorded as images. This technique is of especial interest in the cases were the densities of the tissues are very similar (like in mammography) and the absorption images do not offer enough contrast. This thesis explores the structural differences existing in healthy and pathological collagen in breast tissue samples by the small-angle x-ray scattering (SAXS) technique and compares these differences with the morphological information found in the DEI images and the histo-pathology of the same samples. Several breast tissue samples were studied by SAXS technique in the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Scattering patterns of the different tissues of the breast were acquired and compared with the histology of the samples. The scattering signals from adipose tissue (fat), connective tissue (collagen) and necrotic tissue were identified. Moreover, a clear distinction could be done between the scattering signals from healthy collagen and from collagen from an invasive tumour. Scattering from collagen is very characteristic. It includes several scattering peaks and scattering features that carry information about the size and the spacing of the collagen fibrils in the tissues. It was found that the collagen fibrils in invaded tumours were thinner and had a d-spacing length 0,7% longer that fibrils from healthy tumours. The scattering signals from the breast tissues were compared with the histology by building colour-coded maps across the samples. They were also imaged with the DEI technique. There was a total agreement between the scattering maps, the morphological features seen in the images and the information of the histo- pathological examination. The thesis demonstrates that the x-ray scattering signal can be used to characterize tissues and that it carries important information about the pathological state of the breast tissues, thus showing the potential of the SAXS technique as a possible diagnostic tool for breast cancer.

Biosynthesis of sulfated mucopolysaccharides in relation to vitamins A and K

1.(1) Incorporation of Na235SO4 into acid mucopolysaccharides of intestine and colon tissue has been studied in normal, vitamin A-deficient and excess vitamin A-fed rats. 2. (2) Vitamin A deficiency resulted in a significant decrease in [35S]sulfate incorporation into mucopolysaccharides isolated from intestines of male rats. There was no significant change in the total mucopolysaccharides per gram of fresh tissue. 3. (3) When rats are fed excessive amounts of retinyl acetate, increased [35S]sulfate incorporation into mucopolysaccharides of rat intestine and colon is observed. 4. (4) Supplementation of vitamin K1 to rats fed excessive amounts of vitamin A restores the incorporation of [35S]sulfate into the acid mucopolysaccharides to the normal level. 5. (5) The implications of these findings with special reference to the role of vitamins A and K in the synthesis of sulfated mucopolysaccharides are discussed.

A tropical oviparous lizard, Calotes versicolor, exhibiting a potentially novel FMFM pattern of temperature-dependent sex determination

Among squamate reptiles, lizards exhibit an impressive array of sex-determining modes viz. genotypic sex determination, temperature-dependent sex determination, co-occurrence of both these and those that reproduce parthenogenetically. The oviparous lizard, Calotes versicolor, lacks heteromorphic sex chromosomes and there are no reports on homomorphic chromosomes. Earlier studies on this species presented little evidence to the sex-determining mechanism. Here we provide evidences for the potential role played by incubation temperature that has a significant effect (P<0.01) on gonadal sex and sex ratio. The eggs were incubated at 14 different incubation temperatures. Interestingly, 100% males were produced at low (25.5 +/- 0.5 degrees C) as well as high (34 +/- 0.5 degrees C) incubation temperatures and 100% females were produced at low (23.5 +/- 0.5 degrees C) and high (31.5 +/- 0.5 degrees C) temperatures, clearly indicating the occurrence of TSD in this species. Sex ratios of individual clutches did not vary at any of the critical male-producing or female-producing temperatures within as well as across the seasons. However, clutch sex ratios were female- or male-biased at intermediate temperatures. Thermosensitive period occurred during the embryonic stages 3033. Three pivotal temperatures operate producing 1:1 sex ratio. Histology of gonad and accessory reproductive structures provide additional evidence for TSD. The sex-determining pattern, observed for the first time in this species, that neither compares to Pattern I [Ia (MF) and Ib (FM)] nor to Pattern II (FMF), is being referred to as FMFM pattern of TSD. This novel FMFM pattern of sex ratio exhibited by C. versicolor may have an adaptive significance in maintaining sex ratio. J. Exp. Zool. 317:3246, 2012. (c) 2011 Wiley Periodicals, Inc.

The biology and ecology of the invasive Indo-Pacific lionfish

The Indo-Pacific lionfishes, Pterois miles and P. volitans, are now established along the Southeast U.S. and Caribbean and are expected to expand into the Gulf of Mexico and Central and South America. Prior to this invasion little was known regarding the biology and ecology of these lionfishes. I provide a synopsis of chronology, taxonomy, local abundance, reproduction, early life history and dispersal, venomology, feeding ecology, parasitology, potential impacts, and possible control and management strategies for the lionfish invasion. This information was collected by review of the literature and by direct field and experimental study. I confirm the existence of an unusual supraocular tentacle phenotype and suggest that the high prevalence of this phenotype in the Atlantic is not the result of selection, but likely ontogenetic change. To describe the trophic impacts of lionfish, I report a comprehensive assessment of diet that describes lionfish as a generalist piscivore that preys on over 40 species of teleost comprising more than 20 families. Next, I use the histology of gonads to describe both oogenesis and reproductive dynamics of lionfish. Lionfish mature relatively early and reproduce several times per month throughout the entire calendar year off North Carolina and the Bahamas. To investigate predation, an important component of natural mortality, I assessed the vulnerability of juvenile lionfish to predation by native serranids. Juvenile lionfish are not readily consumed by serranids, even after extreme periods of starvation. Last, I used a stage-based, matrix population model to estimate the scale of control that would be needed to reduce an invading population of lionfish. Together, this research provides the first comprehensive assessment on lionfish biology and ecology and explains a number of life history and ecological interactions that have facilitated the unprecedented and rapid establishment of this invasive finfish. Future research is needed to understand the scale of impacts that lionfish could cause, especially in coral reef ecosystems, which are already heavily stressed. This research further demonstrates the need for lionfish control strategies and more rigorous prevention and early detection and rapid response programs for marine non-native introductions.

Temporal and spatial dynamics of spawning, settlement, and growth of gray snapper (Lutjanus griseus) from the West Florida shelf as determined from otolith microstructures

The goal of our study was to understand the spatial and temporal variation in spawning and settlement of gray snapper (Lutjanus griseus) along the West Florida shelf (WFS). Juvenile gray snapper were collected over two consecutive years from seagrass meadows with a benthic scrape and otter trawl. Spawning, settlement, and growth patterns were compared across three sampling regions (Panhandle, Big bend, and Southwest) by using otolith microstructure. Histology of adult gonads was also used for an independent estimate of spawning time. Daily growth increments were visible in the lapilli of snapper 11–150 mm standard length; ages ranged from 38 to 229 days and estimated average planktonic larval duration was 25 days. Estimated growth rates ranged from 0.60 to 1.02 mm/d and did not differ among the three sampling regions, but did differ across sampling years. Back-calculated fertilization dates from otoliths indicated that juveniles in the Panhandle and Big Bend were mainly summer spawned fish, whereas Southwest juveniles had winter and summer fertilization dates. Settlement occurred during summer both years and in the winter of 1997 for the southern portion of the WFS. Moon phase did not appear to be strongly correlated with fertilization or settlement. Histological samples of gonads from adults collected near the juvenile sampling areas indicated a summer spawning period.

Study on the intestinal bacteria of Labeo rohita (Ham.)

The quantitative and qualitative aspects of intestinal bacteria of rohu fish (Labeo rohita) showed that total viable count of bacteria ranged from 9.9 x 106 to 1.4 x 107 CFU/g of intestine in different age groups of fish. The bacterial load was highest in the month of July and lowest in January. The genera of the isolates from intestine included Coryneform, Micrococcus, Flavobacterium, Cytophaga, Achromobacter, Aeromonas Enterobacteriaceae and Vibrio. Coryneform was the dominant group throughout the study period followed by Micrococcus and Enterobacteriaceae. Marked variations in the bacterial load and generic composition of intestinal bacteria were evident during the study period in different age groups of rohu fish.

Food preference and intake pattern of Penaeus monodon (Fab.) in semi-intensive culture system

P. monodon juveniles prefer natural food than artificial feed in their initial stage of rearing. However, the percentage preference towards artificial feed increases as the days of culture/average body weight increases. Consumption of artificial feed was high during evening meal (1700h) and low during night meal (2200h). The index of intestine fullness was high (8.4±0.7) during initial stage of rearing, compared to that of final stage of rearing (4.9±0.4), probably due to low nutritional value of intake material and low water depth at initial stage. At all stage of rearing omnivorous feeding habit was established and no evidence of diet periodicity in feeding activity was observed.

A histological study of the spermatogenesis in Ompok pabda (Hamilton-Buchanan 1822)

The present study deals with the histological analysis of testicular development in Ompok pabda. For the study, male gonads were collected month wise from January to September at Freshwater Station, BFRI, Mymensingh. From the analysis, 4 stages of sperm formation, namely, spermatogonia, spermatocytes, spermatids and spermatozoa, were distinguished. The percent distribution of spermatozoa was highest in July (about 92%). Maximum GSI value was 1.129±0.271 found in July. By analyzing the histology of spermatogenesis it was established that this species breeds once in a year.

Reproductive biology, artificial propagation and larval rearing of two freshwater eels, Monopterus cuchia and Mastacembelus armatus

Studies on reproductive biology and artificial propagation including larval rearing of freshwater mud eel, Monopterus cuchia and spiny eel, Mastacembelus armatus were attempted. The gonadosomatic index (GSI) of mud eel ranged from 0.41 (August) to 5.52 (June) in males and 0.53 (August) to 7.61 (June) in females. In both cases the GSI showed a peak in June. Fecundity ranged from 228 (TL - 396 mm; W - 78g) to 5510 (TL - 865 mm; W - 630 g). In case of spiny eel, the GSI varied from 0.65 (August) to 8.30 (July) in males and 0.70 (August) to 10.46 (July) in females. GSI showed single peak in July. Fecundity ranged from 570 (TL - 240 mm; W - 30 g) to 10870 (TL - 601; W - 350g). Histology of the testes and ovaries of the eels were carried out to investigate the gonadal development stages during the reproductive months (August to November 2003). In case of male M. cuchia, the secondary primordial germ cells, primary spermatogonium, some spermatogonia A and clone of spermatogonium B in testis were observed in September. In October-males different sized lobules having spermatogonia, spermatocytes and spermatids were observed. In the ovary of M. cuchia, polygonal shaped oocytes were seen during September. The oogonia were reduced with dense and irregular shaped during October. Numerous pycnotic cells were visible during November. In male M. armatus numerous broken lobule walls were found in testes during September. In October, abundant primary germ cells, pycnotic nests of degenerating cells, spermatogonia and spermatids were observed. In females, ovaries had distinct yolk vesicles stage and yolk granules stages in August. In September, the follicular cells of the oogonia were ruptured, shrunk forming irregular shaped in October. Oogonia were also shrunk with thin, irregular shaped structure but broken parts of the ruptured follicular cells were scattered in case of M. armatus. Experimental attempts on artificial propagation indicated that both freshwater eels were difficult to breed using inducing agents like pituitary glands (PG) of 10, 20, 50, 100 and 150 mg per kg of body weight. Same doses were used for both sexes with equal sex-ratio. In both cases, brood fish died at higher doses of injection given at 100 and 150 mg PG/kg bodyweight. However, M. cuchia breed naturally in cisterns when provided with water hyacinths and tunnel in muddy bottom. M. cuchia fed with chopped cooked fish attained a mean weight of 18.75 ± 2.3 g and cent percent survival. While in case of M. armatus best growth by weight (12.0 ± 2.48 g) and cent percent survival were achieved using chopped raw fish. Car tyre was observed as best shelter for attaining the mean weight gain 22.53 ± 2.24 g and cent percent survival of M. cuchia. While PVC pipe was found to be the best shelter for M. armatus, where it attained the mean weight of 12.73 ± 1.88 g and cent percent survival.