Host response to mycobacterial infection in the alcoholic rat: male and female dimorphism

BACKGROUND: The intravitreal injection of ornithine produces selective damage to the retinal pigment epithelium (RPE) and results in a loss of RPE, choriocapillaris and photoreceptor cells. To elucidate the mechanism of secondary retinal atrophy, we investigated the presence of apoptotic cells in a rat model of ornithine-induced retinopathy. METHODS: At 6 and 12 h and 1, 2, 4, 7, 14 and 28 days after an intravitreal injection of L-ornithine hydrochloride in rat eyes, we removed the eyes and subjected them to histopathological examination. We detected apoptotic cells by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick end labeling (TUNEL) assay, which stains the 3'-OH ends of fragmented DNA. We used electron microscopy to detect the apoptotic cells morphologically. RESULTS: RPE cells were selectively damaged immediately after ornithine administration. TUNEL-positive photoreceptor cells appeared exclusively in the photoreceptor cell layer 12 h after ornithine administration. The number of TUNEL-positive cells increased throughout the 2 days following the injection, then decreased markedly. TUNEL-positive cells remained until 28 days, when the photoreceptor cells had disappeared. The ganglion cell layer, inner nuclear layer and damaged RPE cells were negative for TUNEL staining during all stages. The electron microscopic study also revealed the pyknotic nuclei of apoptotic photoreceptor cells. CONCLUSION: An intravitreal injection of ornithine caused primary damage to the RPE, and subsequently some of the photoreceptor cells revealed apoptosis by TUNEL assay. These findings suggest the dysfunction of the RPE causes photoreceptor cell death according to the intrinsic program of an apoptotic mechanism.     

Immunoreactivity for calcium-binding proteins in the claustrum of the monkey

The sequence of degenerative changes in the retinal pigment epithelium (RPE) and the choroid of retinal degeneration (rd)-mice was studied in correlation with photoreceptor changes. Three weeks to 26-month-old animals were investigated using light and transmission electron microscopy, enzyme histochemistry and quantitative morphology. Changes in the choriocapillaris (CC) were additionally studied by scanning electron microscopy of corrosion cast preparations. In 3-week-old mice, in which most of the outer segments of photoreceptors in the central portion of the retina had disappeared but remnants of the cells were still present, the RPE was enlarged and showed elongated microvilli. In 8-week-old animals, the photoreceptors were completely absent in large areas of the posterior pole region. In these areas the RPE was also completely lost. Quantitative evaluation performed in histological serial sections showed that loss of RPE measured as length of RPE-free Bruch's membrane, continuously increased up to the age of 20 months. In 8-week-old animals, CC adjacent to degenerating RPE showed loss of fenestration. In 10-week-old animals, the CC disappeared in those areas where the RPE was already lacking. The loss of CC increased with increasing age and in 20-month-old animals 5-10% of the entire CC was lacking. Loss of the related arterioles and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)-positive nerve fibers occurred only in approximately 2-year-old rd-mice. Compared to other animal models, RPE and CC defects in rd-mice are relatively large. The rd-mice might therefore provide a good tool to study factors involved in CC degeneration.     

Ophthalmologic findings in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency caused by the G1528C mutation: a new type of hereditary metabolic chorioretinopathy

OBJECTIVE: The purpose of the study was to determine the nature and course of ophthalmic abnormalities in long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, a recently discovered disorder of mitochondrial fatty acid beta-oxidation. STUDY DESIGN: The study design was a cohort (case series). PARTICIPANTS: A retrospective review of the records of 15 children who had died during their first 2 years was performed. Also performed were a longitudinal reanalysis and cross-sectional clinical examination of four long-term survivors aged 5 to 31 years. MAIN OUTCOME MEASURES: Visual acuity, refraction, visual fields, ophthalmoscopy, fluorescein angiography, biometry, corneal topography, electroretinography (ERG), visual-evoked potentials (VEPs), color vision, and dark adaptation were measured. RESULTS: In seven children, ophthalmoscopic findings were within normal limits at 3 days to 13 months of age (median, 4.8 months). In 11 children, a granular retinal pigment epithelium (RPE), with or without pigment clumping in the macula, was seen at 4 months to 5 years of age (median, 9 months). Two long-term survivors, 16 and 31 years of age, eventually had circumscribed atrophy of the choroid, RPE, and retina, which coincided with a posterior staphyloma type 1. They had progressive axial myopia starting at 6 and 12 years of age and later paracentral scotomas leading to poor central vision. They suffered from early difficulty with mesopic vision, glare, and a severe generalized color vision deficiency that started as a tritanomaly. A third survivor was mildly myopic at 5 years of age. All four surviving patients had visually insignificant, flake-like supranuclear opacities in the lens. The ERG initially was normal but deteriorated during the first decade and later was unrecordable. The VEP responses remained fairly normal. Initially, angiography showed no blockade of the choroidal fluorescence because of the thin RPE. Filling of choroidal vessels was delayed, and the choriocapillaris and, later, larger choroidal vessels in the posterior pole became nonperfused. CONCLUSIONS: In LCHAD deficiency, the fundus is normal at birth (stage 1). Soon, however, pigment dispersion occurs in the RPE (stage 2), followed by circumscribed chorioretinal atrophy, occlusion of choroidal vessels, and deterioration of central vision, often with relative sparing of the peripheral fundus (stage 3). Finally, posterior staphylomas and central scotomas may develop (stage 4). Developmental cataract, progressive myopia, and deterioration of visual fields and color vision are new findings in LCHAD deficiency. The chorioretinopathy and abnormal ERG precede the development of myopia and posterior staphyloma, which, in turn, coincide with the loss of macular vision. The authors postulate that the RPE or choriocapillaris is primarily affected. Awareness of the characteristic ocular features is important because of an opportunity for dietary treatment, genetic counseling, and prenatal diagnosis.     

A long-term course of experimentally produced choroidal neovascularization in the rat

We studied morphologically a long-term course of experimental choroidal neovascularization (ChNV) induced by krypton laser photocoagulation in the rat retina. Fifty-two weeks after photocoagulation, ChNV was enveloped completely by the retinal pigment epithelium. Vascular endothelial cells of ChNV were thin, with many fenestrations and wide lumen. The ChNV maintained the morphological characteristics of mature leaky capillaries similar to choriocapillaris. The lumen of the neovascularizations tended to be compressed by massive collagen fibers produced by the retinal pigmented epithelium. We found that experimental ChNV in the rat retina retains the characteristics of leaky capillaries for a long time unlike that in the monkey ChNV.     

Transplanted and repopulated retinal pigment epithelial cells on damaged Bruch's membrane in rabbits

AIMS: The authors studied how artificially damaged Bruch's membrane influenced growth and differentiation of transplanted embryonic retinal pigment epithelial (RPE) cells and of host RPE cells in rabbits. METHODS: Embryonic RPE cells obtained from pigmented rabbits were transplanted into the subretinal space of adult albino rabbits. The host RPE was removed with a silicone cannula, and Bruch's membrane was damaged by scratching with a microhooked 27 gauge needle under the detached retina in closed vitrectomy. The transplantation sites were examined 3, 7, and 14 days after surgery by light and electron microscopy. RESULTS: Varying degrees of damage in Bruch's membrane were observed. Pigmented and hypopigmented RPE cells showed a normal polarity and tight junctions were seen at the sites of mild to moderate damage 3-7 days after the surgery. In contrast, fibroblast-like cells with no such features of RPE cells formed multiple layers at the sites of severe damage involving the full thickness of Bruch's membrane and the choriocapillaris even 14 days after the surgery. Without transplantation, host RPE cells repopulated the damaged areas in the same way as transplanted RPE cells. CONCLUSIONS: Transplanted embryonic RPE cells as well as host RPE cells grew and differentiated on the moderately damaged Bruch's membrane, while the severely damaged Bruch's membrane did not allow differentiation of RPE cells although these cells could grow and cover the damaged areas.     

Cellular response in subretinal neovascularization induced by bFGF-impregnated microspheres

PURPOSE: To determine the sequence of cellular changes associated with a new rabbit model of subretinal neovascularization (SRN) induced by subretinal injection of basic fibroblast growth factor (bFGF)-impregnated microspheres. METHODS: bFGF-impregnated gelatin microspheres, prepared by forming a polyion complex between gelatin and bFGF, were subretinally implanted into rabbit eyes. The eyes were studied by immunochemistry at 3 days to 8 weeks after implantation. Antibodies to CD4, CD8, cytokeratin, CD31, glial fibrillary acidic protein (GFAP), and RAM11 were used. RESULTS: Cytokeratin-positive retinal pigment epithelial (RPE) cells appeared on day 3 and continued to increase in number in the subretinal space throughout the growth of the SRN membrane, becoming the predominant cell type. Macrophages (RAM11-positive) appeared early, but most disappeared within 7 days. GFAP-positive Müller cells were evident early in the retina but migrated into the subretinal space after 7 days; the gliotic adhesion they formed between the retina and the SRN membrane was prominent at 8 weeks. CD31-positive endothelial cells were first evident at 14 days and formed neovascular channels that were still present for up to 8 weeks. CD4- and CD8-positive lymphocytes appeared in the early stages but were few in number. CONCLUSIONS: SRN membranes are primarily composed of RPE cells and vascular endothelial cells. The membrane adheres to the retina by a gliotic band. The cellular components involved in the membrane of this model resemble those found in SRN membranes removed from patients with age-related macular degeneration.     

Intrauterine infection with Ehrlichia phagocytophila in a cow

An infection with Ehrlichia phagocytophila, the agent of tickborne fever, can cause abortion or stillbirth in cows in late pregnancy but, to the authors' knowledge, there have been no reports of intrauterine infection in cows followed by clinical signs in the calf. To study the effect of E phagocytophila on the fetus, a cow was infected experimentally after 270 days of pregnancy. It developed the clinical and haematological signs characteristic of tickborne fever six days after infection. At 287 days of pregnancy the cow gave birth to a live calf, which became ill at 13 days of age. The general condition and behaviour of the calf were only mildly affected but it had a high temperature and swollen prescapular lymph nodes. Its appetite and suck reflex remained normal. E phagocytophila inclusion bodies were visible, predominantly in neutrophils and eosinophils, for seven days. The calf seroconverted, as detected by indirect immunofluorescence, 14 days after it became ill.     

Clinicopathologic correlation of localized retinal pigment epithelium debridement

PURPOSE: To characterize changes in the retina, retinal pigment epithelium (RPE), and choriocapillaris with fluorescein angiography (FA) and histology after hydraulic or abrasive RPE debridement in 26 domestic short-haired cats. METHODS: Hydraulic debridement was produced by injecting balanced salt solution forcefully into the subretinal space. For abrasive debridement, RPE were removed with a silicone-tipped cannula after creating a localized retinal detachment. The FAs were performed after surgery, and tissue was prepared for light microscopy (LM) and scanning electron microscopy (SEM). RESULTS: Sixty-seven blebs were examined by FA 1 hour after surgery, and RPE debridement was confirmed by SEM or LM in 15 blebs from 10 animals. Hyperfluorescence and variable central fluorescein leakage were seen 1 week after surgery in 52 of 53 blebs (which includes all 27 blebs from the 1-week timepoint and 26 of 29 blebs from the 4-week timepoint that were studied by FA 1 week after surgery). Choriocapillary filling delays were seen in no hydraulic debridements, but in 11 of 14 abrasive blebs, especially in areas showing leakage late in the angiogram. In 1 of 13 hydraulic and 12 of 14 abrasive debridements, areas of late dye leakage had no RPE with outer retinal degeneration. At the 4-week timepoint, 1 of 17 hydraulic and 10 of 12 abrasive debridements had foci of delayed or absent choriocapillary perfusion by FA, with degenerated outer retina, no RPE, and choriocapillary atrophy by histologic analysis. CONCLUSIONS: Abrasive debridement is more commonly associated with abnormal FAs and with incomplete RPE repopulation, choriocapillaris atrophy, and outer retinal degeneration than is hydraulic debridement. This clinicopathologic study may give insight into FA interpretation after choroidal neovascular membrane removal in human patients.     

Peripheral component in the enhanced antinociceptive effect of systemic U-69,593, a kappa-opioid receptor agonist in mononeuropathic rats

To evaluate the ability of cilostazol, an antiplatelet and vasodilating agent, to promote axonal regeneration in streptozotocin-induced diabetic rats, the time until beginning of regeneration (initial delay) and the axonal regeneration rate of the sciatic nerve were estimated using the pinch test, and ornithine decarboxylase activity was measured in dorsal root ganglia. At 5 weeks of diabetes, axonal regeneration rate remained unchanged but the initial delay was prolonged and ornithine decarboxylase induction was delayed in diabetic rats compared with those in normal rats. Cilostazol had little effect on these parameters in normal or diabetic rats. At 10 weeks of diabetes, diabetic rats showed both prolongation of initial delay and a decrease in axonal regeneration rate. Cilostazol markedly increased axonal regeneration rate in diabetic rats. Ornithine decarboxylase induction following nerve injury disappeared almost completely in diabetic rats but was maintained by cilostazol treatment. The effect of cilostazol in diabetic rats is thought to be mediated through its preventive effect on circulatory disorders. The active site of the drug appears to be early processes in nerve regeneration before ornithine decarboxylase induction. Further, the results suggest that the both axonal regeneration and this induction are sensitive to circulatory defects in diabetes.     

Sudden death in implantable cardioverter-defibrillator recipients: clinical context, arrhythmic events and device responses

Reports regarding the effect of all-trans-retinoic acid (RA) on the cell growth of retinal pigment epithelial cells (RPE) have been contradictory. The aims of this study are to clarify the in vitro effect of RA on RPE cells and to examine polyamine metabolism after RA stimulation. A 4-day incubation of fetal-calf-serum (FCS)-stimulated RPE cells with 10 or 25 microM RA significantly increased both cell number and [3H]thymidine incorporation. RPE cells grown over an extended period for 8 days also increased in number and reached full confluency. However, if the incubation was further extended to 12 days, no further increase in cell number was detected. RA treatment of FCS-stimulated RPE cells shifted the peak of ornithine decarboxylase (ODC) activity from 16 to 4 h. S-adenosylmethionine decarboxylase (SAMDC) activity and spermidine/spermine N1-acetyltransferase (SAT) activity of RA-treated RPE cells were significantly greater until 8 and 16 h after incubation, respectively. The putrescine content was significantly increased in RA-treated RPE cells up until 24 h, while spermidine, spermine and N1-acetylspermidine contents were significantly increased until 16 h. Our findings suggest that RA treatment increases the intracellular polyamine concentration of RPE cells via activation of ODC, SAMDC and SAT and that this results in the promotion of RPE cell growth until the cells reach full confluency.     

Blood-spinal cord barrier function and morphometry after single doses of x-rays in rat spinal cord

PURPOSE: The effects of irradiation on blood-spinal cord barrier (BSCB) function and ultrastructure were evaluated using a rat spinal cord model. METHODS AND MATERIALS: Rats received a single dose of 25 Gy to the cervical spinal cord (C2-T2). At various times following irradiation and before the onset of paralysis, BSCB function was assessed using horseradish peroxidase (HRP) as a vascular tracer, and barrier-related structural changes in the capillaries were evaluated using morphometric techniques. RESULTS: Focal extravasation of HRP was seen at 93 days after irradiation, and extensive extravasation was apparent by 114 days in white matter, but not in gray matter. At 93 days, pathologic changes apparent by light microscopy were very minor in the white matter of the irradiated segment. By 107 days, myelin beading, Wallerian degeneration, edema, and histiocytes were apparent in white matter, and these features became increasingly prominent over the following weeks. No noteworthy changes were seen in gray matter at these times. Electron microscopic examination showed that, during the first 93 days following irradiation, more than half of the endothelial cells in white matter had disappeared (p < 0.05). In terms of the putative vascular pores, no abnormalities in endothelial junctions (the presumed small pore) were found, but there was an increase in the density of endothelial vesicles (a putative form of the large pore) in irradiated white matter (p < 0.001), but not in gray matter. Pericytes, thought to act as a second line of defence in the blood-brain barrier, increased in size but not in number in the irradiated white matter of the spinal cord. CONCLUSION: We suggest that radiation damage to endothelial cells, which form the BSCB prior to the onset of neurological deficit, may play an important role in the pathogenesis of white matter necrosis.     

Serum-free media for culturing and serial-passaging of adult human retinal pigment epithelium

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm-2. Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9+/-2.7% and 47.1+/-4.1%, respectively) and DMEM with serum (87.2+/-5.6% and 52.9+/-10.5%, respectively) than DMEM without serum (59.2+/-5.6% and 33.1+/-6.9%, respectively; P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12+/-3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21+/-5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.     

Calpain localization changes in coordination with actin-related cytoskeletal changes during early embryonic development of Drosophila

Calpain, a calcium-dependent intracellular protease, was identified in Drosophila melanogaster. Drosophila calpain has an amino acid sequence highly homologous to those of mammalian calpains and exhibits a distinct domain structure consisting of cysteine protease and calcium-binding domains. Specific antibodies raised against a recombinant calpain fragment were used to identify the localization of calpain in developing Drosophila embryos. Calpain was first detected near the anterior pole and in posterior region of the embryo just after fertilization. The anterior calpain disappeared during the cleavage cycles. On the other hand, the posterior calpain moved to the posterior pole when polar buds were formed, and condensed just below the pole cells. At cleavage cycles 8 and 9, when nuclei reached the egg surface, calpain was localized between the nuclei at the surface beneath the precleavage furrows. Co-staining experiments with anti-actin antibody revealed that calpain condenses specifically at the edge of and between actin caps that underlie the plasma membrane immediately above each nucleus. These results indicate that calpain is involved in the dynamic changes in the embryonic cytoskeleton, especially actin-related structures, during early embryogenesis prior to cellularization.     

Tissue response to nano-hydroxyapatite/collagen composite implants in marrow cavity

The formation of otoconia in the endolymphatic sac (ES) of the larval newt, Cynops pyrrhogaster, has been studied by light and transmission electron microscopy. Some of the epithelial cells of the ES contain an abundance of swollen vesicles, Golgi complexes, rough endoplasmic reticula and ribosomes at the late larval stages 50 and 51, approximately 26-30 days after eggs are laid. Five days later, at stage 52, crystals are present in the vacuoles between the epithelial cells. Serial sections indicate that these vacuoles actually form small canals which lie in the wall and join the lumen of the ES. Reconstruction of the ES shows that several canals are contained in the ES wall. At stage 56, about 72 days after eggs are laid, a large number of otoconia are present in the ES lumen, while the otoconia disappear from the canals. It appears that the otoconia are first produced in the canals and then released to the lumen. Some epithelial cells of the ES are thought to expel the organic and inorganic material to the canals to form the otoconia in situ. The process of formation of the otoconia in the ES is discussed.     

Characterisation of the infarct-limiting effect of delayed preconditioning: timecourse and dose-dependency studies in rabbit myocardium

BACKGROUND: Although the choroidal neovascularization (CNV) is a common pathologic feature of a number of different eye diseases, its pathological mechanisms have not been fully elucidated. We investigated the expression of vascular endothelial growth factor (VEGF) in CNV using an experimental primate model. METHOD: CNV was induced by intense laser photocoagulation in four monkey eyes. Single eyes were enucleated at 1, 3, 7 or 14 days after photocoagulation and examined immunohistochemically for VEGF, macrophage antigen, von Willebrand factor and glial fibrillary acidic protein (GFAP). Expression of VEGF mRNA was examined by in situ hybridization. RESULTS: One day after photocoagulation, the normal structure of the outer portion of the retina and the inner portion of the choroid was destroyed. Three days after photocoagulation, choroidal vascular endothelial cells migrated into the subretinal space through the defect in Bruch's membrane. Increased expression of VEGF was detected in the accumulating macrophages, migrating retinal pigment epithelial (RPE) cells and Müller cells. Maximal expression of VEGF was observed between 3 and 7 days after wounding, and many newly formed vessels extended into the subretinal space 7-14 days after photocoagulation. CONCLUSION: VEGF derived from RPE cells, macrophages and Müller cells may play a role in the formation of CNV.     

Changes in choroidal innervation in Royal College of Surgeons rats with hereditary retinal degeneration

In Royal College of Surgeons (RCS) rats with hereditary retinal degeneration loss of retinal pigmented epithelium (RPE) and choriocapillaris is most pronounced in the upper-temporal quadrant. To investigate whether changes in choroidal vasodilative innervation might be involved in the RPE degeneration, we analyzed whole mount preparations of the retina and choroid stained for nitric oxide synthase and for NADPH-diaphorase (d) of 19 dystrophic RCS rats and 24 age-matched congenic controls of different age groups. Density of NADPH-d-positive nerve fibers was quantitatively evaluated in the upper-temporal and lower-nasal quadrant. Our results revealed that even in control animals there were much less positively stained nerve fibers in the upper-temporal than in the lower-nasal quadrant. Nerve fiber density in both quadrants increased for up to 3 months and remained nearly constant throughout life. In the dystrophic animals up to 3 months of age nerve fiber density was similar to that seen in the controls. In dystrophic animals older than 3 months nerve fiber density in the upper-temporal quadrant decreased significantly, whereas density in the lower-nasal quadrant revealed nearly the same values as in the age-matched controls. Decrease of NADPH-d stained nerve fibers in this quadrant occurred prior to the vascular changes in the choriocapillaris. In the retina of RCS dystrophic rats an increase of NADPH-d-positive amacrine cells was found only in 3-month-old animals. Most of these cells were located in the vicinity of irregularly arranged branches of the central retinal artery. In animals 5 months of age and older the number of cells decreased to the same values found in controls, so that we assume that increase of NADPH-d-positive amacrine cells is involved in capillary degeneration or sprouting.     

Fine structure of the retinal pigment epithelium in the Port Jackson shark (Heterodontus phillipi)

The structure of the retinal epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) has been studied by light and electron microscopy in the Port Jackson shark (Heterodontus phillipi). In this elasmobranch the RPE consists of a single layer of low cuboidal cells which show basal (scleral) infolding and apical (vitreal) processes that enclose photoreceptor outer segments. Laterally these epithelial cells are joined by a series of apically located tight junctions. The RPE cells display a large vesicular nucleus, abundant smooth endoplasmic reticulum as well as numerous polysomes and mitochondria. Phagosomes are present, rough endoplasmic reticulum is scarce and myeloid bodies were not observed. Melanosomes are absent over the choroidally located tapetum lucidum, but are not abundant even in extratapetal areas. This paucity of melanosomes probably makes retinomotor movements unimportant. Bruch's membrane or complexus basalis is a pentalaminate structure. The endothelium of the choriocapillaris is thin but minimally fenestrated.     

Tissue effects of subclinical diode laser treatment of the retina

OBJECTIVE: To determine whether consistent tissue effects are obtained when laser lesions are produced with a commercially available diode laser that are near the limit of clinical detection at the time of treatment. METHODS: Continuous-wave or micropulse diode laser was used to produce clinically undetectable (subthreshold) or barely detectable (threshold) retinal lesions in pigmented rabbits. Tissue effects at intervals after treatment were determined in retinal pigment epithelial (RPE) whole mounts by fluorescence microscopy, and in sections of retina and RPE by light and electron microscopy. RESULTS: Continuous-wave and micropulse laser lesions that were originally clinically undetectable were detectable as zones of pigment mottling after 5 days. By microscopy, compaction and/or swelling was seen in the outer retina, and cells in the RPE monolayer became heterogeneous in size, shape, and pigmentation, but the tissue responses in the outer retina and RPE were variable even within and among lesions in the same eye. CONCLUSIONS: Subthreshold energies used to create both continuous-wave and micropulse laser lesions produced variable effects on the RPE and the overlying neurosensory retina. It appears that, near the minimum effective dose of laser irradiation, individual RPE cell heterogeneity becomes detectable as variability in sensitivity to laser injury. CLINICAL RELEVANCE: As laser energy is reduced to limit collateral tissue damage in clinical applications, it may be difficult to generate reproducible lesions because of heterogeneity among individual cells.     

Ultrastructural studies of experimental acute pancreatitis

Acute pancreatitis was studied by electron microscopy after retrograde infusion of either trypsin, and/or beta-glucuronidase into the canine pancreatic duct. Marked changes were induced by the mixture of trypsin and beta-glucuronidase. (1) The acinar cells were initially excavated from the acinar lumen and formed cystic bodies in themselves. The cystic bodies were then disrupted at their marginal membranes, and the acinar cells were filled with a large amount of fibrillar materials which originated from the contents of the cystic bodies. At this time, the luminal margin of the acinar cells completely disappeared. (2) The cellular organellas and the intracellular fibrillar materials in the acinar cells were discharged into the interstitial space through the disrupted basal lamina. Infection in the pancreatic ductal system was considered to play an important role in the pathogenesis of acute hemorrhagic pancreatitis.     

Polarity and the development of the outer blood-retinal barrier

The retinal pigment epithelium (RPE) is a monolayer that separates the outer surface of the neural retina from the choriocapillaris. Because the choriocapillaris is fenestrated, it is the RPE that forms the outer blood-retinal barrier and regulates the environment of the outer retina. Like all epithelia and endothelia, the ability of RPE to regulate transepithelial transport depends upon two properties: apical tight junctions to retard diffusion through the paracellular spaces of the monolayer, and an asymmetric distribution of proteins to regulate vectorial transport across the monolayer. During development, these properties form gradually. Initially, the tight junctions are leaky, and the RPE exhibits only partial polarity. As the neural retina and choriocapillaris develop, there are progressive changes in the composition of the apical junctional complexes, the expression of cell adhesion proteins, and the distribution of membrane and cytoskeletal proteins. Development can be used to dissect the multiple mechanisms that establish and maintain polarity and barrier function. These mechanisms are regulated by the interactions that develop between the RPE and its neighboring tissues. This review discusses the remodeling of the apical, lateral and basal plasma membranes of RPE that occurs during normal development, and establishes a framework to integrate the data obtained from multiple species. It examines the progress in understanding how environmental interactions regulate this development.