Development of cholinergic chronotropic control in chick (Gallus gallus domesticus) embryos.

In chick (Gallus gallus domesticus) embryos, instantaneous heart rate begins to fluctuate with the appearance of rapid, transient decelerations at around the end of the second week of incubation. Previously, it was shown that instantaneous heart rate decelerations were eliminated by administration of atropine and concurrently heart rate baseline was elevated in late embryos. Because the previous study lacked statistical treatment and there has been recent controversy over the development of tonic vagal control of the heart, we reexamine the hypothesis that transient decelerations of instantaneous heart rate are mediated by vagus nerve and the vagal tone begins to appear at around the end of the second week of incubation. Atropine administration tests were conducted for sixty-seven 11- to 14-day-old and 18-day-old embryos in total. Heart rate decelerations appeared sporadically in three out of ten 12-day-old embryos, but the difference of mode heart rate before and after administration of atropine was not significant. Seven out of nine 13-day-old embryos and all nine 14-day-old embryos showed heart rate decelerations and the difference of mode heart rate before and after atropine administration was significant. In late (18-day-old) embryos, magnitude and frequency of instantaneous heart rate decelerations further increased with additional appearance of transient, irregular accelerations. Administration of varying doses of atropine completely eliminated the heart rate decelerations and elevated the heart rate baseline more markedly than in young embryos, indicating the maturation of vagal tone late in incubation.

Effect of beta-blocker eyedrops on corneal epithelial barrier function.

To investigate the long-term effect of a topically applied beta-blocker on human corneal epithelium, the corneal epithelial barrier function and the superficial cell area of the corneal epithelium were evaluated. Seventeen normal healthy volunteers (without medication), 7 cataract patients (treated with pyrenoxine eyedrops) and 7 glaucoma or ocular hypertension patients (treated with 0.5% timolol maleate) were assigned to this study. The eyedrops had been used on a daily basis for at least 3 months. In the evaluation of corneal epithelial barrier function, fluorescein uptake was measured using a slitlamp fluorophotometer after application of 3 microl of 0.5% fluorescein for 10 min. In the evaluation of the superficial cell area, the central corneal epithelium was measured by tandem scanning confocal microscopy (TSCM). The healthy control and timolol groups were compared. Corneal fluorescein uptake in the healthy control, pyrenoxine and timolol groups was 20.3 +/- 3.2, 21.5 +/- 4.0 and 76.2 +/- 30.0 ng/ml (mean +/- standard error), respectively. There was a significantly higher fluorescein uptake in the timolol group compared to the pyrenoxine group (p = 0.0088) and the healthy control group (p = 0.0055). TSCM showed no significant difference in the superficial cell areas of the corneal epithelium between the healthy control and timolol groups. beta-Blocker eyedrops decreased the corneal epithelial barrier function. Their application was not accompanied by any biomicroscopic change in the superficial cell area.

Neovascularization from scleral wound as cause of vitreous rebleeding after vitrectomy for proliferative diabetic retinopathy.

PURPOSE: To determine the site of rebleeding into the vitreous after vitrectomy in patients with diabetic retinopathy. METHODS: We studied in detail 4 eyes of 4 patients in whom rebleeding into the vitreous followed successful vitrectomy for proliferative diabetic retinopathy. In addition, the fibrous membrane removed at surgery was studied by light and electron microscopy. RESULTS: In these 4 eyes, the second operation revealed that the source of the vitreous rebleeding was from a fibrovascular proliferation around the scleral wounds of the initial surgery, and no other neovascularization and/or reproliferation were observed in the whole retina. Rebleeding in these 4 eyes developed at an average of 9 weeks after initial surgery. The proliferative membrane was oval in shape and expanded from the residual vitreous that had been incarcerated in the scleral wound. The proliferative membrane removed during vitrectomy was poor in cellular components and contained extracellular matrix. Blood vessels of various sizes were also present. Electron microscopy showed the membrane was rich in extracellular components and contained high and low electron density cells. These cells often had microvilli and seemed to be of epithelial origin. CONCLUSIONS: These findings show that vitreous rebleeding may develop from fibrovascular proliferation from the scleral wound created during initial surgery. The proliferated membrane showed histological similarities with the fibrovascular proliferation usually seen in the diabetic retina and may represent a type of anterior proliferation secondary to retinal ischemia.

[Neovascularization from the scleral wound as the cause of vitreous rebleeding after vitrectomy for proliferative diabetic retinopathy].

Vitreous hemorrhage recurred in 11 eyes out of a series of 120 eyes treated by vitrectomy for proliferative diabetic retinopathy during a 10-month period. Neovascularization from the scleral wound, or fibrovascular proliferation, was identified as the cause of rebleeding in 4 eyes from findings during revitrectomy. None of the 4 eyes showed retinal disorders attributable as the cause of rebleeding. Rebleeding in these 4 eyes developed after an average of 9.0 weeks after initial surgery. The proliferative membrane was oval in shape and expanded from the residual vitreous which had been incarcerated in the scleral wound. The proliferative membrane obtained during revitrectomy was poor in cellular components and contained extracellular matrix. Blood vessels of various sizes were also present. Electron microscopically, the membrane was rich in extracellular components. It contained two types of cells, i.e., high and low electron density cells. These cells often had microvilli and seemed to be of epithelial origin. The findings show that vitreous rebleeding may develop from fibrovascular proliferation from the scleral wound after diabetic vitrectomy. The proliferated membrane showed histological similarities with the fibrovascular proliferation usually seen in the diabetic retina and may represent a type of anterior proliferation secondary to retinal ischemia.

Localization of Six4/AREC3 in the developing mouse retina; implications in mammalian retinal development.

The Six4/AREC3 gene was originally isolated as a regulatory factor which bound to the positive regulatory region of the Na, K-ATPase alpha 1 subunit. It is a murine homologue of the Drosophila sine oculis (so) gene, which is essential for the development of the entire insect visual system. In this study, we attempted to determine the localization of the Six4/AREC3 gene product in the developing mouse retina in order to examine its role in retinal cell differentiation. Immunohistochemistry with anti-SIX4/AREC3 and anti-Na, K-ATPase alpha 1 subunit antisera was performed on developing mouse retinas, and immunoblotting analysis with anti-SIX4/AREC3 was also performed. The localization of Six4-like immunoreactivity (Six4-LI) showed a temporally regulated pattern: During embryonic development, Six4-LI was found in the nuclei of cells located at the inner neuroblastic layer of the retina as early as on ED12, nearly corresponding to the onset of retinal cell differentiation. In the PD1 retina, Six4-LI was observed in the nuclei of the ganglion cells, and increased its intensity until PD4, and thereafter kept its intensity until PD7 when Six4-LI was often found in the cytoplasm. On PD4, the presumptive amacrine cells found in the inner portion of the inner nuclear layer appeared to be immunostained in their nuclei. On PD7, the presumptive bipolar cells located in the outer portion were immunostained in the nuclei. After that, Six4-LI gradually decreased, and in the mature retina no detectable Six4-LI was observed in the nuclei. This pattern of Six4-LI localization during retinal development seemed to correlate with retinal cell differentiation, but did not correlate with the distribution pattern of Na, K-ATPase alpha 1 subunit protein-like immunoreactivity. These results suggest that the Six4 gene may play a role in the differentiation of neural retinal cells during mouse retinal development, rather than regulating the expression of the Na, K-ATPase alpha 1 subunit gene.

Effects of aldose reductase inhibitor CT-112 on the corneal epithelial barrier of galactose-fed rats.

PURPOSE: To investigate whether the barrier function of the corneal epithelium is disrupted in galactosemic rats, and to assess the effects of the aldose reductase inhibitor CT-112, in the form of eyedrops, on the corneal epithelial barrier in galactosemic rats. METHODS: Forty rats were divided into 3 groups based on their diet: a control group, a galactose group and a CT-112 treated galactose group (CT-112 group). After 3 weeks, 31 rats from the 3 groups were subjected to fluorophotometry, in which fluorescein (F) was instilled into one eye and carboxyfluorescein (CF) was instilled into the other eye in a random fashion. The F and CF uptakes were then measured at the central cornea by a slit-lamp fluorophotometer. Three rats from each group were exposed to a horseradish peroxidase (HRP) solution for one hour, and the HRP-reactive substances within the corneal epithelium were also examined via electron microscopy. RESULTS: There was significantly higher F uptake in the galactose group than in the control (p = 0.003) and CT-112 groups (p = 0.028). There were no significant differences in CF uptake between the 3 groups. Histologically, HRP-reactive substances were found in much greater quantities within the superficial corneal cells of the galactose group than in the control or CT-112 groups. CONCLUSIONS: These results suggest that cell membrane disruption, as detected by F uptake and HRP penetration, was found in the superficial corneal cells of galactose-fed rats, and that intercellular junction integrity can be assayed by CF uptake and histological evaluation. Moreover, CT-112 eyedrops were effective in improving the corneal epithelial barrier dysfunction of galactose-fed rats.

A newly identified membrane protein localized exclusively in intracellular organelles of neurons.

We report here cloning of the cDNA of a novel membrane protein, termed p24, which, of the eight mouse tissues tested, was found only in brain where it is localized exclusively in neurons. The cDNA encodes 196 amino acids with a molecular weight of approximately 24000. P24 contains two putative membrane spanning domains and a sequence in the hydrophilic tail homologous to the microtubule-binding domain of microtubule-associated proteins, such as TAU and MAP-2. We prepared antibodies to p24 and demonstrated that the protein is rich in nerve fibers of the cerebral cortex, anterior cerebral nuclei and hypothalamus. When neuroblastoma Neuro 2a cells were treated with retinoic acid to induce differentiation, p24 mRNA increased but the p24 protein was not detected. The protein expressed from the p24 cDNA in non-neuronal Cos-7 cells was 24 kDa in size and were localized only in lysosomes. These findings indicate that p24 is a neuron-specific membrane protein localized in intracellular organelles of highly differentiated neural cells and suggest that it may play a role in the neural organelle transport system.

Distribution of endothelin and endothelin-A receptor in the lacrimal glands of the monkey (Macaca fuscata).

Endothelin (ET) is well known to be a potent vasoconstrictor peptide with autocrine and paracrine function. It has been documented that ET is also present in non-muscle tissues. The distribution of ET and ET-A receptor (ET-AR) in the monkey lacrimal gland was investigated by immunohistochemistry. Three adult male monkeys (Macaca fuscata) were perfused with a fixative. The lacrimal glands were then dissected and sectioned. Using rabbit anti-ET and anti-ET-AR antibodies, the immunohistochemical procedure was performed following an ABC technique. Some sections were treated with rhodamine-phalloidin, which selectively binds to actin filaments. ET immunoreactivity was present in stellate-shaped cells located around the alveoli. In sections double-stained with anti-ET antibody and rhodamine-phalloidin, ET immunoreactivity and abundant filamentous actin were identified in the same stellate cells. Immunostaining for ET-AR was also found in the stellate shape cells. The configuration of, and the abundance of actin filaments in the stellate-shaped ET- and ET-AR immunoreactive cells suggest that they are myoepithelial cells, which are contractile and may contribute to the process of lacrimal gland secretion or maintenance of the contour of the glandular endpieces. Our results indicate that endothelin is present in myoepithelial cells of the monkey lacrimal gland.

Collagen gel-embedding culture of conjunctival epithelial cells.

BACKGROUND: Collagen has effects on cell morphology, differentiation characteristics and function. Using collagen gel culture, several studies about cell differentiation were reported. In this study, the differentiation of rabbit conjunctival epithelial cells in a collagen gel-embedding culture system was investigated by electron microscope and lectin labeling. METHODS: Rabbit bulbar conjunctival epithelial cells were cultured in type I collagen gel. After 1 and 2 weeks of culture, some of these cells were stained with PAS and seven kinds of lectins, and others were examined by transmission electron microscopy. RESULTS: The conjunctival epithelial cells cultured within collagen gel formed stratified cell layers and globules with cavities. The inner layer cells facing the cavities showed PAS and lectin staining patterns similar to those of conjunctival goblet cells in vivo, whereas the staining patterns of the outer layer cells on the collagen matrices resembled the patterns of non-goblet epithelial cells. Microvilli on the surface of the innermost cells, basement membranes beneath the outermost cells, tight junctions, adherent junctions, interdigitating folds and desmosomes between cells were identified on electron microscopic examination. CONCLUSION: These results indicate that cell junction structures of the conjunctival epithelial cells are well developed in collagen gel-embedding culture systems, and that the inner layer cells have carbohydrates similar to those of conjunctival goblet cells. Culture of conjunctival epithelial cells within collagen gel is a useful model for examining differentiation of these cells.