Ultrastructural findings in feline corneal sequestra

OBJECTIVES: (1) To describe the ultrastructural features of corneal sequestra in cats; and (2) to enhance our understanding regarding the pathogenesis of feline corneal sequestration. METHODS: Nine corneal sequestra were harvested via keratectomy from globes of nine cats. The sequestra were routinely fixed then postfixed for high resolution light and transmission electron microscopy (HR-LM and TEM, respectively). The tissues were embedded in Epon/Araldite. Sections of 0.5-microm thickness were cut and stained with 1% toluidine blue in 1% sodium tetraborate solution for HR-LM. Ultrathin sections were collected on copper grids and stained with uranyl acetate and Sato's lead stain for TEM. Ultrathin sections were examined and the images were captured on an Advantage HR CCD camera using a Hitachi 7500 electron microscope operated at 80 kV. Two healthy corneas from two cats were harvested immediately following euthanasia. These corneal tissues (control samples) were processed in the same manner as the corneal sequestra for HR-LM and TEM. A portion of each sequestrum was also submitted for polymerase chain reaction (PCR) testing for infectious agents including feline herpesvirus-1 (FHV-1), Toxoplasma gondii, Chlamydophila felis and Mycoplasma spp. RESULTS: Ultrastructure of healthy corneal tissues revealed basal corneal epithelial cells aligned adjacent to a thin acellular layer similar to Bowman's layer with underlying tightly packed, regularly arranged, collagen fibrils oriented in different planes. Keratocytes were elongated and had long and irregularly shaped nuclei, and cytoplasm contained rough endoplasmic reticulum and abundant membrane-bound vesicles. In contrast, corneal sequestra contained varying amounts of an amorphous, electron-dense substance, continuous with intact basal epithelial basement membranes peripherally, and overlying corneal ulceration and loosely packed collagen fibrils. Remnants of necrotic keratocytes were seen in spaces between disarranged collagen layers. In all samples, occasional keratocytes exhibited morphology indicative of apoptosis including clumping and margination of chromatin, and shrunken cytoplasm. Varying degrees of inflammation were noted on HR-LM and TEM of affected corneas including peri- and intralesional neutrophils, lymphocytes, plasma cells, and macrophages. Corneal sequestra were FHV-1-positive (n = 3), FHV-1- and T. gondii-positive (n = 1), T. gondii-positive (n = 3), or negative for DNA of these infectious agents (n = 2) using PCR. All corneal sequestra were negative for DNA of Chlamydophila felis and Mycoplasma spp. using PCR. CONCLUSIONS: Apoptosis may play a role in the pathogenesis of feline corneal sequestration independent of the presence of DNA of these infectious organisms. Prospective clinical studies are warranted to further understand the significance of T. gondii in relation to feline corneal sequestration.     

Morphological and functional alterations in adult boar epididymis: Effects of prenatal and postnatal administration of flutamide

Background

The dynamic cross-talk between epididymal cells is hormonally regulated and, in part, through direct cell-to-cell interactions. To date, no information is available regarding possible impact of anti-androgens on the proteins involved in the gap junctional communication within the boar epididymis. Thus, a question arised whether prenatal or postnatal exposure to an anti-androgen flutamide alters the expression of gap junction protein - connexin43 (Cx43) and androgen receptor (AR) expression in the caput, corpus and cauda epididymis and leads to delayed effects on morphology and function of adult pig epididymis.

Methods

First two experimental groups received flutamide prenatally on gestational days 20-28 and 80-88 (GD20 and GD80) and further two groups were exposed to flutamide postanatally on days 2-10 and 90-98 after birth (PD2 and PD90). Epididymides were collected from adult boars. Routine histology was performed using hematoxylin-eosin staining. The expression of Cx43 and AR were analyzed using immunohistochemistry and Western blotting. Both analyses were supported by quantitative approaches to demonstrate the variations of the expression levels following the treatment. Apoptotic cells were identified using TUNEL assay.

Results

Histological examination revealed differences in epididymal morphology of flutamide-exposed boars when compared to controls. Scarce spermatic content were seen within the corpus and cauda lumina of GD20, PD2 and PD90 groups. Concomitantly, frequency of epididymal cell apoptosis was significantly higher (p < 0.05) after exposure to flutamide at GD20. Moreover, in GD20, PD2, and PD90 groups, significantly lower AR expression (p < 0.05) was found in the principal and basal cells of the corpus and cauda regions, while in the stromal cells AR expression was significantly reduced (p < 0.05) along the epididymal duct. Concomitantly, a decrease in Cx43 expression (p < 0.05) was noticed in the stromal cells of the cauda region of GD20 and PD2 groups. This indicates high sensitivity of the stromal cells to androgen withdrawal.

Conclusions

The region-specific alterations in the epididymis morphology and scarce spermatic content within the lumina of the corpus and cauda indicate that flutamide can induce delayed effects on the epididymal function of the adult boar by decrease in AR protein levels that results in altered androgen signaling. This may cause disturbances in androgen-dependent processes including Cx43 (de)regulation, however, we can not exclude the possibility that in response to flutamide decreased Cx43 expression may represent one mechanism responsible for functional disturbance of the boar epididymis.     

Polyphenols from evening primrose ( Oenothera paradoxa ) defatted seeds induce apoptosis in human colon cancer Caco-2 cells

Polyphenols extracted from evening primrose seeds (industrial waste product) were studied as apoptosis inducers in human colorectal adenocarcinoma Caco-2 and HT-29 cell lines and in rat normal intestinal IEC-6 cells. The extract dose-dependently inhibited the growth of Caco-2, HT-29, and IEC-6 cells. However, nuclear DNA fragmentation characteristic of apoptosis was observed only in Caco-2. After 72 h of incubation with the extract at 150 μM gallic acid equivalents (44.1 μg extract/mL), Caco-2 cell numbers decreased to 19% of control and 48.8% of the cells were identified by flow cytometry as apoptotic. Under the same conditions only 8% of HT-29 cells and 12.6% of IEC-6 cells exhibited hypodiploid DNA content. The effects of the extract and its fractions on phosphatidylserine exposure and cell membrane integrity were assessed by high content screening image cytometry. The fractions strongly and dose-dependently reduced Caco-2 cell numbers, whereas HT-29 and IEC-6 cells were affected to lesser extents.     

Quantitative trait loci affecting intensity of violet flower colour in potato

Anthocyanins occur in potato tuber skin and flesh, sprouts, leaves, stems and flowers. The goal of this study was to identify genomic regions and candidate gene alleles key for accumulation of anthocyanins in potato corolla in various quantities. QTL analyses were performed in two mapping populations segregating for flower colour intensity and candidate genes were identified on the basis of function and location (chalcone isomerase, chi; chalcone synthase, chs) or location (RNA-dependent RNA polymerase 1, RDR1). We detected three and four QTL affecting the violet flower colour intensity using the two mapping populations, respectively. In both populations a locus F, necessary for violet flower colour, segregated and we used different approaches to differentiate the qualitative effect of this locus and to detect the genetic factors affecting the quantitative flower colour intensity. The strongest QTL and the only one common for the two mapping populations was located on chromosome V. The role of all three candidate genes, chi, chs and RDR1, in control of flower colour intensity is supported to different extents by the performed genetic analyses. The most important QTL on chromosome V is most likely in the same position as the QTL for anthocyanin tuber flesh coloration described previously, which indicates that the natural variation in some biosynthetic and/or regulatory genes may influence anthocyanin levels in multiple tissues.     

Effect of heat treatment on the flavor of oat flakes

In order to assess the effect of heat treatment procedure on the flavor and volatile compounds of oats, raw oats, kiln and dried oats, dehulled oats and oat flakes were analysed. A sensory profile method was used to monitor changes in flavor. It changed from hay-like for raw oats into nutty, bread-like for oat flakes. Headspace solid phase microextraction (SPME) and solvent assisted flavor evaporation (SAFE) techniques were applied for isolation of aroma compounds. The most abundant compound in headspace was hexanal – its concentration varied from 176 to 1671 μg/kg depending on the processing stage. The key aroma compounds of oat flakes identified using gas chromatography – olfactometry and aroma extract dilution analysis (AEDA) were 2-methyl-3-furanthiol with roast/cooked oatmeal flavor together with methional, dimethyl trisulfide, 1-octen-3-ol, 2-methyl-3,5-diethylpyrazine.     

Expression of Interleukin (IL)‐1β and IL‐1 Receptors Genes in the Hypothalamus of Anoestrous Ewes after Lipopolysaccharide Treatment

This study was designed to determine the effect of intravenous lipopolysaccharide (LPS) administration on the secretion of interleukin (IL)‐1β and IL‐1 receptors (IL‐1Rs) gene expression in the hypothalamus of anoestrous ewes. Gene expression of IL‐1β and its receptors was assayed by the real‐time polymerase chain reaction. The expression of IL‐1β in the hypothalamus was detected using Western blot. Our results showed that IL‐1β mRNA is transcribed in the ovine hypothalamus. Lipopolysaccharide increased (p ≤ 0.01) the IL‐1β gene expression in the pre‐optic area 2.4‐fold, the anterior hypothalamus (AHA) 3.4‐fold, the medial basal hypothalamus 3.7‐fold and the medial eminence 3.9‐fold. The pro‐form and mature form of IL‐1β protein were found in the hypothalamus after endotoxin injection. In general, the endotoxin also increased more than two times (p ≤ 0.01) the expression of IL‐1 receptor type I (IL‐1R1) and type II (IL‐1R2) genes in the hypothalamus, except the AHA, where the number of IL‐1R2 mRNA was extremely low and not sufficient to the quantitative analysis. These results demonstrate that the peripheral immune/inflammatory challenge increases the IL‐1β expression in the hypothalamus. This endogenous IL‐1β seems to be involved in the modulation of processes which are regulated at the hypothalamic level. One of these processes could be a reproduction.     

Changes in the quality of meat (Longissimus thoracis et lumborum) from Kamieniec lambs during long‐term freezer storage

The aim of this study was to determine changes in the quality of lamb meat (Longissimus thoracis et lumborum), which was vacuum‐packaged and freezer‐stored (?26°C) for 6 and 12 months. The experiment was performed on 12 male lambs of the Kamieniec Longwool breed, raised to 106 days of age. In comparison with fresh meat, thawed meat was characterized by lower ash content, higher pH, greater natural drip loss and cooking loss, and lower scores for taste intensity. Vacuum packaging and low‐temperature storage protected lamb meat against oxidative changes, and alleviated the adverse effects of oxidation on the color, aroma and taste of meat. It can be concluded that freezer storage (?26°C) of vacuum‐packaged meat can help meet consumer demand for lamb meat products in periods when fresh meat is unavailable. However, it should be noted that long‐term frozen storage induces undesirable changes in meat quality, including a decrease in water‐holding capacity and taste intensity.