Functional and morphological assessment of age-related changes in the choroid and outer retina in pigeons.

We sought to determine if choroidal and outer retinal deterioration occur with age in pigeons, as they do in other species, and investigated the relationship between age-related retinal and choroidal changes. In 64 pigeons ranging in age over the pigeon lifespan (0.5-20 years), we measured some or all among the following parameters: choroidal blood flow (ChBF) by laser Doppler flowmetry, choroidal thickness and choriocapillary vessel abundance by LM histology, choriocapillary endothelial cell transport specializations by EM histology, acuity by behavioral methods, and degenerating photoreceptor abundance and total photoreceptor abundance by LM histology. Regression and Receiver Operator Curve (ROC) analyses were used to characterize the pattern of age-related changes and determine the ages at or by which significant changes occurred. For the 45 birds for which we measured choroidal parameters, choriocapillary vessel abundance showed a curvilinear decline with age and half of this decline occurred by 3.5-4.6 years. The endothelial cell transport specializations called channels also declined curvilinearly with age. Choroidal thickness was slightly increased between the ages of 3-6 years, and thereafter declined steadily so that choroidal thickness in the oldest birds was half that in the youngest. ChBF showed an abrupt decline of about 20% at 4 years and a further 20% decline thereafter. In the 53 birds for which we obtained visual acuity and/or photoreceptor data, we observed a curvilinear decline in acuity (with half the decline having occurred by 8 years) and a prominent stepwise decline of about 20% in photoreceptor abundance at 4.7 years, followed by further decline thereafter. The period of major photoreceptor loss coincided with ages during which about 10% of photoreceptors appeared to show degenerative changes (4-8 years of age). Using partial correlation analysis with the common effect of age held constant, ChBF was found to have a positive correlation with acuity. Our results show that ChBF and choroidal vascularity decline significantly with age in pigeons, as do acuity and photoreceptor abundance. Our statistical analyses suggest that prominent choroidal vascular decline preceded the visual decline, and that there is a positive relationship between choroidal and visual functions. Thus, our findings are consistent with the view that age-related decline in choroidal function might contribute to age-related vision loss in pigeons.

Anterior stromal micropuncture electron microscopic changes in the rabbit cornea.

Anterior stromal micropuncture has become an effective treatment for recurrent erosion. The healing process in rabbit corneas was investigated. Following micropuncture of the corneal surface with a 27-gauge needle knife, electron microscopy was carried out at regular intervals from time 0 through 5 months. The corneal incisions began to fill with epithelium by day 1. Activated keratocytes were adjacent to the basement membrane defect by 7 days. The basement membrane appeared to be healed at 2 and 4 weeks. Epithelial projections into the stromal incisions with underlying mature basement membrane persisted at 5 months postsurgery. Basement membrane reproduction occurred much more rapidly following needle puncture than after microdiathermy. This was thought to occur because the corneal epithelial cell was immediately exposed to type I collagen, whereas following microdiathermy, new type I collagen must be secreted on the necrotic collagen before the corneal epithelium will secrete basement membrane.

Isolation and characterization of a mucosal triacylglycerol pool undergoing hydrolysis.

Absorbed and processed mucosal neutral lipid has been shown to be composed of at least two pools of triacylglycerol. One is likely to subserve chylomicron formation, and the other appears to be transported from the intestine via a nonlymphatic route. In the present study, 50 +/- 5% of the mucosal lipid pellets was centrifuged at 75,000 g.min [low-speed pellet (LSP)]. Discontinuous sucrose density gradient centrifugation of LSP showed that 61 +/- 7% of the lipid banded at the 0.25-0.86 M sucrose interface. Neutral lipid analysis showed that this subfraction was only 58% triacylglycerol, suggesting it was undergoing hydrolysis. Active lipolytic activity in vitro was found on incubation. The lipase had an alkaline pH optimum (pH 8.5) and persisted despite pancreatic ductular diversion. Lipolysis in vivo in a LSP fraction was shown by infusing [14C]glyceryltrioleate for 3.5 h followed by [3H]glyceryltrioleate for 30 min. Discontinuous sucrose density centrifugation of the LSP followed by an analysis of the lipids at the 0.25-0.86 M sucrose interface showed that 14C-neutral lipids were only 70 +/- 6% triacylglycerol, whereas 3H-neutral lipids were 88 +/- 2% triacylglycerol. 3H entered LSP slowly compared with the floating lipid in the same centrifuge tube. These studies suggest both in vivo and in vitro mucosal lipolysis by a specific, alkaline-active lipase. The turnover rate of LSP is likely to be slow by comparison with neutral lipid floating to the top of the centrifuge tube.

Fine structure observations on rat jejunal epithelial cells during fat processing and resorption following L-81 exposure and reversal.

In order to more easily follow membrane interaction following L-81 administration and subsequent lipid processing tannic acid was used to highlight cell membrane contrast in intestinal epithelial cells of Sprague-Dawley male rats. Jejunal epithelial cells were examined with the electron microscope following 4 h of L-81 administration and 45 min, 1, 1.5 and 2 h after the cessation of L-81 administration. After 4 h of L-81 administration the apical cytoplasm was seen to be filled with large, lipid droplets limited by a single, 4-5 nm thick, electron-dense rim. During the reversal process changes noted in the cytoplasm included the following. As early as 45 min clear and electron-dense-filled vesicles appeared scattered in the apical cytoplasm and juxtaposed to the lipid droplets. The large, non-membrane bound lipid droplets decreased in size and number over the two hour period while the number and size of electron-dense containing vesicles increased. The vesicles were subsequently found concentrated in the Golgi region where assimilation and production of chylomicron and VLDL size particles occurred. Eventually multiple groups of such particles were found intercellularly and basally. Thus the re-cycling of lipid and chylomicron production and secretion following L-81 reversal is similar to that following experimental studies on normal fat over-loading and following recovery after puromycin treatment. This data is discussed in light of previous data on fat processing and secretion in rat intestinal epithelial cells with particular reference to membrane events visible following tannic acid membrane highlighting.

Intraenterocyte distribution of absorbed lipid and effects of phosphatidylcholine.

The present studies were designed to investigate the distribution of absorbed lipid in intestinal mucosal cells and to identify the chylomicron precursor pool. Rats were infused intraduodenally with glyceryl tri[9,10(n)-3H]oleate (135 mumol/h); other rats were in addition infused with 9 mumol/h of phosphatidylcholine. After 5-h infusion the proximal one-half of intestine was removed and the mucosa obtained. It was found that 50% of the radioactivity in the whole homogenate pelleted on centrifugation at 75,000 g.min. The supernatant was further fractionated by high-speed centrifugation resulting in a floating lipid layer, a supernatant, and a microsomal pellet. The results showed that these subcellular fractions had a triacylglycerol specific activity 46-52% of the infusate's specific activity. Including phosphatidylcholine in the duodenal lipid infusion increased the triacylglycerol specific activity of all subcellular fractions (70%) resulting in a specific activity approaching that of the infusate, which would be expected of chylomicron triacylglycerol. These studies demonstrate 1) that considerable mucosal lipid is distributed into a low-speed pellet, 2) that mucosal triacylglycerol specific activity can be greatly increased by including phosphatidylcholine in a lipid infusion, and 3) that despite obtaining multiple subcellular fractions, the chylomicron precursor pool could not be clearly identified in the mucosa of control rats.

Isolation of the early phase of chylomicron formation in intestinal epithelial cells of rats.

Lipid is first observed electron microscopically in the rough endoplasmic reticulum of intestinal epithelial cells during active lipid absorption. We have been able to isolate this subcellular fraction by using discontinuous sucrose gradients of 0.25/0.86/1.11 M sucrose. A preliminary low speed centrifugation of mucosal homogenate removed the heavier subcellular organelles. The resulting supernatant was centrifuged at 5.25 x 10(6) x g.min. The pellet from this centrifugation was placed on top of the gradient and the fractions isolated at the density interfaces after centrifugation at 25.5 x 10(6) x g.min. The isolated fractions were characterized enzymatically and electron microscopically. Electron microscopically, the fractions were predominantly composed of rounded vesicles decorated with ribosomes. Most contained lipid droplets whose diameters were 453 nm in the lighter membranes and 245 nm in the membranes isolated from the heavier density region. The vesicles contained NADPH cytochrome c reductase and glucose-6-phosphatase activity indicative of the presence of microsomes. Contamination with other subcellular organelles was minimal. These studies demonstrate a method which enables the isolation of vesicles containing chylomicron-sized particles which are from the earliest phase of chylomicron formation. Isolation of chylomicrons from these vesicles will enable a better understanding of the maturation process of chylomicrons as they traverse the intestinal epithelial cell.

Dietary supplementation with Pluronic L-81 modifies hepatic secretion of very low density lipoproteins in the rat.

Supplementation of high fat/cholesterol-enriched diets with polyoxypropylene-polyoxyethylene copolymers containing 90% hydrophobic constituents has been found to impair enteric secretion of chylomicrons, lower plasma levels of very low density (VLDL) and low density (LDL) lipoprotein cholesterol and prevent diet-induced hypercholesterolemia and atherosclerosis. These agents are known to be absorbed from the gastrointestinal tract and excreted in bile. In order to determine whether dietary supplementation with this group of hydrophobic poloxalenes influences hepatic secretion of triglyceride-rich lipoproteins, groups of rats were maintained for 21-34 days on either standard chow, semisynthetic diet containing 10.0% safflower oil/1.0% cholesterol, or each of the above diets supplemented with the hydrophobic poloxalene Pluronic L-81. At the end of the feeding period, newly secreted hepatic VLDL were isolated from 2-hr recirculating liver perfusates, quantitated, and characterized. Compared to perfusions in chow-fed rats, perfusion experiments in rats fed the high fat/cholesterol-enriched semisynthetic diet revealed a 3.1-fold increased net hepatic VLDL secretion rate; enrichment of secretory VLDL in cholesteryl esters and in C18:2 core lipid fatty acids; and a shift in the size distribution of secretory VLDL towards larger particles. When the 0.5% Pluronic L-81 was included in the high fat/cholesterol-enriched semisynthetic diet, the net hepatic VLDL secretion rate fell significantly and the physicochemical properties of secretory VLDL in these rats were found to resemble those of chow-fed animals. Supplementation of the chow diet with L-81 resulted in a significant fall in the net hepatic VLDL secretion rate from that observed in rats fed chow alone. Compared to rats fed chow alone, perfusate VLDL from rats fed each of the other experimental diets contained markedly lower amounts of both apoB molecular weight variants, as analyzed by gradient gel electrophoresis and densitometric gel scanning. Since previous studies have demonstrated that VLDL are the major cholesterol transport lipoproteins following fat/cholesterol feeding; a precursor-product relationship exists between fat/cholesterol-induced hepatic VLDL and plasma VLDL; such particles are capable of delivering cholesterol to the arterial wall; and dietary supplementation with hydrophobic poloxalenes prevents both the increase in plasma VLDL-cholesterol and diet-induced atherosclerosis, it is possible that dietary supplementation with hydrophobic poloxalenes may influence the atherogenic process through direct and/or indirect effects on hepatic VLDL transport.

Electron microscopic studies of the assembly, intracellular transport, and secretion of chylomicrons by rat intestine.

A detailed ultrastructural investigation of the assembly, intracellular transport, and secretion of chylomicrons by rat proximal jejunal intestinal cells was performed in rats fed corn oil. Following fat feeding the smooth endoplasmic reticulum of the absorptive cells becomes laden with triglyceride droplets which are transported through channels of the endoplasmic reticulum to the Golgi apparatus. The Golgi zones become extremely prominent due to the accumulation of osmiophilic droplets, similar in size and configuration to chylomicrons, within proliferated Golgi vesicles. Golgi-derived secretory vesicles, containing nascent chylomicrons, migrate towards the lateral cell membrane. The secretory vesicle membranes fuse with the lateral plasmalemma and nascent chylomicrons are then discharged into the intercellular spaces. Alterations of specific domains of the secretory vesicles were prominent, appearing as coated pits. Coated pits were apparent in the lateral plasmalemma in areas of active chylomicron exocytosis suggesting their derivation from secretory vesicle-membrane fusion. Chylomicrons, within the intercellular spaces, pass through the basement membrane that lines the basal surfaces of the epithelial cells, traverse the cellular elements of the lamina propria, and finally gain access to the lymphatics entering these channels through gaps between adjacent endothelial cells. These observations indicate that nascent chylomicrons accumulate within Golgi vesicles as a pre-requisite to secretion and that secretion occurs by exocytosis resulting in the release of nascent chylomicrons from secretory vesicles.