Karnovsky's Fixative Research Articles

Decreased Thickness and Integrity of the Macular Elastic Layer of Bruch's Membrane Correspond to the Distribution of Lesions Associated with Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. In its severest form, choroidal neovessels breach the macular Bruch's membrane, an extracellular matrix compartment comprised of elastin and collagen laminae, and grow into the retina. We sought to determine whether structural properties of the elastic lamina (EL) correspond to the region of the macula that is predilected toward degeneration in AMD. Morphometric assessment of the macular and extramacular regions of 121 human donor eyes, with and without AMD, revealed a statistically significant difference in both the integrity (P < 0.0001) and thickness (P < 0.0001) of the EL between the macular and extramacular regions in donors of all ages. The EL was three to six times thinner and two to five times less abundant in the macula than in the periphery. The integrity of the macular EL was significantly lower in donors with early-stage AMD (P = 0.028), active choroidal neovascularization (P = 0.020), and disciform scars (P = 0.003), as compared to unaffected, age-matched controls. EL thickness was significantly lower only in individuals with disciform scars (P = 0.008). The largest gaps in macular EL integrity were significantly larger in all categories of AMD (each P < 0.0001), as compared to controls. EL integrity, thickness, and gap length in donors with geographic atrophy did not differ from those of controls. These structural properties of the macular EL correspond spatially to the distribution of macular lesions associated with AMD and may help to explain why the macula is more susceptible to degenerative events that occur in this disease.

Neuropathological Studies on Cycloate-Induced Neuronal Cell Death in the Rat Brain

The herbicide cycloate (carbamothioic acid, ethyl(cyclohexyl)- S-ethyl ester) given as a single oral dose to rats, caused selective neuronal cell death in two regions in the rat forebrain, the pyramidal neurons of layers II–III throughout the pyriform cortex and in granule cells of the caudal ventro-lateral dentate gyrus. Male Alderley Park rats, 6–8-week-old, were given a single oral dose of either 0 or 2000 mg/kg cycloate and killed for neuropathological investigation 1, 2, 3, 7, 14 or 28 days after dosing, using a regime of perfusion fixation with modified Karnovsky's fixative, followed by routine paraffin embedding. Seven transverse levels of brain were examined from each rat. Cycloate-induced neuronal cell death was seen in the pyriform cortex 1 day after dosing and persisted through to Day 28, the lesion was more marked in the rostral compared to the caudal region of the pyriform cortex. Neuronal cell death was also observed in the ventro-lateral caudal dentate gyrus on Days 1–14, day after dosing. In the early stages, Days 1–3 and to a lesser extent Day 7, the neuronal cell death resembled apoptosis, characterized by condensation of nuclear material, cell shrinkage and strong cytoplasmic eosinophilia. By Days 14 and 28 and to a lesser extent Day 7, the cell death resembled necrosis, i.e. karyorrhectic nuclei with pale irregular cytoplasm. Microglial accumulation was associated with the neuronal cell injury. In control brains, an occasional apoptotic body was seen in both the pyriform cortex and dentate gyrus. Our results demonstrate that cycloate is a novel neurotoxicant, which following a single large oral dose induces a cell specific and highly localized forebrain lesion. The time course data analyzed temporally, suggests that cycloate may cause an up regulation of apoptosis in selected regions of the adult brain.

Immunoelectron microscopy study of polyamines using a newly prepared monoclonal antibody against spermidine: use of a mixture of glutaraldehyde and paraformaldehyde as a cross-linking agent in the preparation of the antigen.

We developed a mouse monoclonal antibody (ASPD-19, IgG3 sub-isotype mAb) against spermidine (Spd) conjugated to bovine serum albumin (BSA) using a mixture of glutaraldehyde (GA) and paraformaldehyde (PFA)-sodium borohydride for applications in immunoelectron microscopic studies. The antibody specificity was evaluated by an ELISA binding test simulating the immunocytochemistry (ICC) of tissue sections. The ASPD-19 mAb is highly specific for Spd and Spm, almost the same degree to each, and can distinguish alterations in the chemical structure of other polyamine (PA) analogs, showing less than 3.2% cross-reaction with N(1)-acetylspermidine, acetylspermine, or N(8)-acetylspermidine. By an indirect immunoperoxidase method using the ASPD-19 mAb, PA-like immunoreactivities were observed in different tissues fixed with Karnovsky fixative (a mixture of GA and PFA) in combination with borohydride reduction. In contrast, immunoreactivity was very low in tissues when the borohydride reduction step was omitted. The PA-like immunoreaction was completely abolished by the adsorption of the ASPD-19 mAb with 100 microg/ml of Spd or Spm, but was inhibited little or none by other PA-related compounds or amino acids. A light microscopic ICC method using ASPD-19 produced immunostaining of PAs in certain cells in rat tissues with high biosynthetic activities (small intestine, pancreas and spinal cord). A pre-embedding immunoelectron microscopic study using rat spinal cord showed PA immunoreactivity located predominantly on free (polysomes) and attached ribosomes of the rough endoplasmic reticulum (Nissl bodies) in the cytoplasm of motor neurons. These results are in complete agreement with the results obtained by our recent ICC method using another mAb (ASPM-29) produced against GA-conjugated Spm.

Sexual dimorphism and seasonal variation in submandibular gland histology of Bolomys lasiurus (Rodentia, Muridae).

Wild rodents (Bolomys lasiurus) of both sexes were caught in a cerrado grassland area during the dry (July-September) and rainy (January-March) seasons of Brazil. Fasted animals were perfused with Karnovsky fixative through the left ventricle, under ether anesthesia, and the submandibular gland was processed for embedding in historesin. Histological and histometric data show sexual dimorphism at both seasons. In the volume percentage of the granular convoluted tubules (GCT) and their secretory granules, the males exhibited higher values. The absolute volume occupied by these structures, however, was dimorphic only in the rainy season. The diameter of the GCT, the height of its epithelium, and its total length were also greater in males during the rainy season. The absolute volumes of the acini and of the ductal tree were identical in both sexes in the dry and rainy seasons but the acinar diameter increased in the males and females during the rainy season. The sexual dimorphism and the seasonal variations now described in the B. lasiurus submandibular glands could be explained by the augmented reproductive activity of the males in the rainy period.

Three-dimensional architecture of the connective tissue core and surface structures of the lingual papillae in the rabbit.

Three-dimensional characteristics of the epithelial cell layer and connective tissue interface of the tongue were studied using scanning electron microscopy. In this study, the fragments of tongue were fixed in modified Karnovsky's fixative solution. Subsequently, the specimens were treated with 10% NaOH solution for 4-7 days at room temperature and postfixed in 1% OsO4 in 0.1 M phosphate buffer (pH 7.4) for 2 hours at 4 degrees C. They were dehydrated through a graded ethanol series, and critical-point dried with CO2. The specimens were coated with gold and observed in a scanning electron microscope, JEOL JSM-6100. The results showed numerous papillae on the dorsal surface of the tongue divided into four groups (filiform, fungiform, foliate and vallate papillae). Filiform papillae are conically shaped; fungiform papillae have an irregular round surface; foliate papillae are oval in shape and have some parallel projections; and vallate papillae are located in the posterior part of the tongue and have a depression around the center. After the treatment with 10% NaOH solution, the original arrangements of connective papillae could be seen. This characteristic three-dimensional distribution of the collagen fiber bundles is typical for each superficial papillae depending on whether it is filiform, fungiform, foliate or vallate.

Changes in the distribution of anionic constituents in secretory granules of mouse pancreatic acinar cells after pilocarpine-induced degranulation.

We used cationized colloidal gold (CCG) to investigate the distribution of anionic sites in different secretory granules of mouse pancreatic acinar cell regranulation. Localization of anionic sites with CCG was carried out on ultrathin sections of a mouse pancreas, fixed in Karnovsky's fixative and OsO4 and embedded in Araldite. After pilocarpine-stimulated degranulation, there was a marked diminution in the anionic charge density of immature and mature granules of the 4-hr group (approximately 43.0 gold particles/microm2) compared to the 8-hr mature granules group (approximately 64.6 gold particles/microm2). Scattergram analysis to investigate the correlation between section profile size and cationized gold labeling density revealed a reverse correlation, the small granule profiles demonstrated a higher density compared to the larger profiles of the same group. On the basis of these observations, it appears that a post-translational processing of secretory content influences the granule anionic charge and thus may affect the intragranular buffer capacity.

Additional cell damage after transpupillary thermotherapy in choroidal malignant melanoma.

The aim of this study was to evaluate the effectiveness of transpupillary thermotherapy (TTT) in generating tumour necrosis by light and electron microscopy, as well as to evaluate additional cell damage in the area directly adherent to the necrotic zone. Four eyes of four patients diagnosed with intraocular malignant melanoma of the uvea were treated experimentally with diode laser TTT. In all cases a standard technique was used. All eyes were enucleated: one eye the day after TTT, two eyes 2 days after TTT, and one eye 6 weeks after TTT. Immediately after enucleation the eyes were immersed in standard Karnovsky's fixative with cocodylate buffer and prepared for light and electron microscopy. In the treated area of all four melanomas we found a dense band of necrotic tissue (zone A) consisting of an amorphous mass of dead cells sharply demarcated from the rest of the neoplastic tissue. Next to this zone was a more eosinophilic and also sharply demarcated band (zone B) that consisted of similar but less intensive changes. In the next band (zone C), marked injury to the cellular membrane and subcellular structures were seen on electron microscopy. The next band (zone D) consisted of changes mainly observed only within the cytoplasm of neoplastic cells and significantly less intensive than those in zone C. Outside zone D tumour cells that were normal in appearance were seen. No scleral alterations induced by heat were found. We concluded that after TTT the cytotoxic effect gradually decreases in proportion to the distance from the central point of the diode laser spot, with additional cell damage in the area adjacent to the necrotic zone. The interval between TTT and enucleation had no influence on the histological results.

Progressive accretion of amelogenin molecules during nanospheres assembly revealed by atomic force microscopy.

Amelogenin proteins, the principal components of the developing dental enamel matrix, self-assemble to form nanosphere structures that are believed to function as structural components directly involved in the matrix mediated enamel biomineralization. The self-assembly behavior of a recombinant murine amelogenin (rM179) was investigated by atomic force microscopy (AFM) for further understanding the roles of amelogenin proteins in dental enamel biomineralization. Recombinant rM179 amelogenin was dissolved in a pH 7.4 Tris-HCl buffer at concentrations ranging from 12.5 to 300 microg/ml. The solutions were adsorbed on mica, fixed with Karnovsky fixative and rinsed thoroughly with water for atomic force microscopy (AFM). At low concentrations (12.5-50 microg/ml), nanospheres with diameters varying from 7 to 53 nm were identified while at concentrations ranging between 100-300 microg/ml the size distribution was significantly narrowed to be steadily between 10 and 25 nm in diameter. These nanospheres were observed to be the basic building blocks of both engineered rM179 gels and of the developing enamel extracellular matrix. The stable 15-20-nm nanosphere structures generated in the presence of high concentrations of amelogenins were postulated to be of great importance in facilitating the highly organized ultrastructural microenvironment required for the formation of initial enamel apatite crystallites.

Migration and development of Sarcocystis neurona in tissues of interferon gamma knockout mice fed sporocysts from a naturally infected opossum

Migration and development of Sarcocystis neurona was studied in 50 gamma interferon knockout mice fed graded doses of S. neurona sporocysts from the intestine of a naturally infected opossum. Mice were examined at necropsy 1–62 days after feeding sporocysts (DAFS). All tissue sections were reacted with anti- S. neurona-specific polyclonal rabbit serum in an immunohistochemical (IHC) test. Between 1 and 3 DAFS, organisms were seen mainly in intestines. Between 4 and 11 DAFS, organisms were seen in several visceral tissues. Beginning with 13 DAFS, schizonts and merozoites were present in sections of brains of all infected mice. All regions of the brain were parasitized but the hind brain was most severely affected. S. neurona was found in the spinal cord of all 10 mice examined 22–30 DAFS. Of the 28 infected mice examined 20–62 DAFS, S. neurona was found in the brains of all 28, lungs of 14, hearts of 8 and eyes of 3. More organisms were seen in IHC-stained sections than in sections stained with hematoxylin and eosin. Treatment of tissues with glutaraldehyde, Karnovsky fixative, and ethylene diamino tetra acetic acid (EDTA, used for decalcification) did not affect staining of organisms by IHC.

Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B-labelled albumin in arterial tissue.

Measurements of the transport of circulating sulphorhodamine B-labelled albumin into the arterial wall, made by applying digital imaging fluorescence microscopy to sections of arteries fixed in situ, are limited in sensitivity by the low levels of tracer fluorescence and high levels of autofluorescence emitted from the tissue. Three attempts to improve these ratios are described. In the first, spectra of the tracer in solution and of arterial autofluorescence were used to design novel microscope filters for rhodamine-like dyes. By exciting with the rarely used yellow lines of the mercury arc lamp and detecting a narrow band of emission with Stokes shifts as small as 15 nm, the ratio of tracer fluorescence to autofluorescence was tripled. In the second, effects of different fixatives were investigated. Using a model system, it was confirmed that Karnovsky's fixative gives good tracer immobilization but elevates autofluorescence, whereas fixative-free buffer solutions give low autofluorescence but do not retain the tracer. It was further found that simple formaldehyde-based fixatives, hitherto considered to be poor fixatives of albumin, immobilized the tracer as well as the glutaraldehyde-based fixative, whilst giving autofluorescence levels comparable to those seen with buffer alone; they therefore give excellent tracer fluorescence to autofluorescence ratios. In the third, lowering specimen temperature by 50 degrees C was found to increase the intensity of tracer fluorescence by 30% whilst autofluorescence was unaffected. These data may have relevance to microscopical studies using other tissues and fluorescent tracers.

Biocompatibility of Two Current Adhesive Resins

The purpose of the study was to evaluate the biocompatibility of two current adhesive resins and a calcium hydroxide cement. Fifty-four polyethylene tubes were filled with these dental materials, which were hand-mixed or light-cured according to the manufacturer's directions: group 1--Clearfill Liner Bond 2 (Kuraray); group 2--Single Bond (3 M); and group 3--calcium hydroxide cement (Dycal-Dentsply). The materials were implanted into dorsal connective tissue of rats, which were killed 7, 30, and 60 days after the implantation procedure. The implant sites were excised, immersed in buffered Karnovsky's fixative, and processed using routine histological techniques. Sections of 6 microns thickness were stained with hematoxylin and eosin and assessed under light microscopy. Both adhesive resins at 7 days elicited a moderate/intense inflammatory reaction that decreased over time. Fibrous capsules surrounding the tubes were observed at 30 days. Half of the samples in groups 1 and 2 showed thin fibrous capsule formation containing macrophages, capillaries, lymphocytes, fibroblasts, and collagen fibers. Connective tissue healing was observed even though many specimens exhibited a persistent inflammatory reaction mediated by macrophages and giant cells at the 60-day evaluation. Dycal allowed complete healing at 30 days with only a thin fibrous capsule. In conclusion, all experimental materials were successfully walled off by the connective tissue of the rat. However the adhesive resins may release particulates that may, in turn, induce a persistent local inflammatory reaction. Consequently, in this specific condition, these materials cannot be regarded as biocompatible. Dycal was less irritating than the adhesive resins and was better tolerated by the connective tissue.

Mucin discharge from the isolated vascularity perfused rat colon in response to bile salts

The intestinal mucus layer plays an important role in the protection of the mucosa. This function is performed mainly by mucins, which are high molecular-weight glycoproteins produced by goblet cells. Luminal factors that govern mucin secretion are poorly known. The hypothesis that bile salts could induce colonic mucin discharge was thus tested in a model of isolated vascularly perfused rat colon. Methods : Ligated loops (I O-cm length) of rat colon were separated from adjacent tissue, transferred to a saline bath and perfused through the superior mesenteric artery with a Krebs Henseleit buffer containing 25% washed bovine erythrocytes, 3% bovine albumin, amino acids and glucose. After a lO-min control period, loops were filled with Iml of prewarmed saline containing stimulants. After 30-min period of stimulation, mucus was assayed in luminal content by enzyme-linked immunosorbent assay. Tissue samples from perfused rat colon were also immersed in Karnovsky's fixative for subsequent staining of mucus cells with alcian blue followed by the periodic acid schiff reagent. Results : Luminal administration of deoxycholate (I-lOmM) produced a dose-dependent release of mucins (maximal response at 550 % of control loops) in the isolated perfused rat colon. Hyodeoxycholate (IOmM) and Chenodeoxycholate (I0mM) also induced an increase in mucin discharge (response at 310 % and 290 % of controls,respectively) whereas IOmM cholate, ursodeoxycholate and tween-20 were without effect. Taurine conjugated bile salts (deoxycholate,hyodeoxycholate, and chenodeoxycholate) were less potent mucus secretagogues than unconjugated forms. Tetrodotoxin, atropine, indomethacin, a mast cells stabilizer (ketotifen), H I histamine antagonist(pyrilamine) and 5-HT receptors antagonists (ketanserin, SDZ, Zophren) did not significantly modify deoxycholate-induced mucin secretion. Conclusions : The present study conducted with the isolated vascularly perfused rat colon suggests that some bile salts may playa role in the regulation of colonic mucin secretion by way of a direct action on mucus cells

Fetal sciatic nerve growth is delayed following repeated maternal injections of corticosteroid in sheep.

A single course of prenatal corticosteroid reduces the mortality and morbidity of preterm birth. Repeated courses of prenatal corticosteroids are widely prescribed despite a lack of safety data. Repeated corticosteroids delay myelination in the ovine central nervous system at the time of preterm delivery but with catch-up at term. We aimed to evaluate their effect in the peripheral nervous system. Thirty date-mated ewes were administered either saline, a single injection of betamethasone, or four injections of betamethasone between 104 and 124 days' gestation, with delivery on day 125 or 145 (term = 150 days). Sciatic nerves were dissected and fixed in modified Karnovsky's fixative and prepared for light and electron microscopy to determine the proportion of myelinated axons and mean axon diameter. Repeated, but not single, corticosteroid administration resulted in significant decreases in the total cross-sectional and fascicle-containing areas of the sciatic nerve, and in the mean diameter of myelinated and unmyelinated axons. Deficits persisted at term. The proportion of myelinated axons was unaffected. Repeated prenatal corticosteroids have the capacity to affect the growth of peripheral nerve axons in sheep. Documentation of their effects in human pregnancy await randomized trials.

Microstructures of an Amelogenin Gel Matrix

The thermo-reversible transition (clear↔opaque) of the amelogenin gel matrix, which has been known for some three decades, has now been clarified by microstructural investigations. A mixed amelogenin preparation extracted from porcine developing enamel matrix (containing “25K,” 7.4%; “23K,” 10.7%; “20K,” 49.5%; and smaller peptides, 32.4%) was dissolved in dilute formic acid and reprecipitated by adjusting the pH to 6.8 with NaOH solution. Amelogenin gels were formedin vitroby sedimenting the precipitate in microcentrifuge tubes. The gels were fixed with Karnovsky fixative at 4 and 24°C, which was found to preserve their corresponding clear (4°C) and opaque (24°C) states. Scanning electron microscopy, atomic force microscopy, and transmission electron microscopy were employed for the microstructural characterization of the fixed clear and opaque gels. The amelogenin gel matrix was observed to possess a hierarchical structure of quasi-spherical amelogenin nanospheres and their assemblies. The nanospheres of diameters 8–20 nm assemble to form small spherical assemblies of diameters 40–70 nm that further aggregated to form large spherical assemblies of 70–300 nm in diameter. In the clear gel, most of the large assemblies are smaller than 150 nm, and the nanospheres and assemblies are uniformly dispersed, allowing an even fluid distribution among them. In the opaque gel, however, numerous spherical fluid-filled spaces ranging from 0.3 to 7 μm in diameter were observed with the majority of the large assemblies sized 150–200 nm in diameter. These spaces presumably result from enhanced hydrophobic interactions among nanospheres and/or assemblies as the temperature increased. The high opacity of the opaque (24°C) gel apparently arises from the presence of the numerous fluid-filled spaces observed compared to the low-temperature (4°C) preparation. These observations suggest that the hydrophobic interactions among nanospheres and different orders of amelogenin assemblies are important in determining the structural integrity of the dental enamel matrix.

Lung Morphometry and Collagen and Elastin Content: Changes During Normal Development and After Prenatal Hormone Exposure in Sheep

This study examined whether the improvement in lung function after prenatal hormone exposure coincided with changes in lung morphometry or in collagen and elastin content. Fetal lambs received a single intramuscular injection of betamethasone (0.5 mg/kg) plus L-thyroxine (T4) (15 micrograms/kg) or vehicle control 48 h before delivery at 121, 128, or 135 d gestational age (d 121, d 128, d 135, term = 150 d). T4 was administered in conjunction with betamethasone in an attempt to enhance the maturational response. The right-upper lobes were instillation fixed at 30 cm H2O by Karnovsky's fixative after a 40-min period of mechanical ventilation. A number of significant changes occurred between d 121 and d 135 in control animals: alveolar airspace volume increased by 270%; despite a 40% reduction in alveolar septal thickness, alveolar septal volume did not change appreciably, suggesting a "redistribution" of septal tissue into the formation of secondary alveolar septa, which doubled in number; and both parenchymal collagen and elastin volume increased significantly, whereas pleural collagen and elastin volume did not change. In contrast to the changes seen in control animals, exposure to betamethasone plus T4 led to alveolar septal thinning at each gestational age without an associated increase in secondary septal number, a 40% decrease in alveolar septal volume, and a proportionate reduction in parenchymal elastin at d 121. Although attenuation of alveolar septa coincides with redistribution of septal tissue into the formation of secondary septa during normal maturation, exposure to betamethasone plus T4 promotes thinning of alveolar septa in the absence of secondary septal formation, which results in a loss of alveolar septal tissue.

The planes of the utricular and saccular maculae of the guinea pig.

To establish a link between otolith anatomy and function it is necessary to know the regions of the utricular and saccular maculae, which are stimulated by any arbitrary linear acceleration stimulus. That requires accurate information about the location and orientation of the spatially extended maculae in head-fixed coordinates and referred to head-fixed landmarks (such as Reid's line). New data showing the location of the otolithic maculae in the guinea pig with respect to head-fixed stereotaxic coordinates are presented. Guinea pigs were perfused with Karnovsky's fixative and the maculae were exposed while the head was held in a guinea pig stereotaxic device. An electrolytically sharpened fine wire held in a calibrated micromanipulator was touched to points all over the surface of each macula under visual observation with the aid of a high-power operating microscope. The x, y, z coordinates of these points were plotted using a three-dimensional plotting program. Both maculae have pronounced curvature so that dorsoventral shear forces will stimulate regions of both the utricular and saccular maculae.

Simple method to isolate the intact duct system of the rat submandibular gland.

The excurrent duct system of the rat submandibular gland consists of a number of distinct segments. Using the direction of salivary flow as a reference point, these segments are, in order, intercalated duct, granular convoluted tubule, striated duct, excretory duct, main excretory duct (MED), and salivary bladder (which is an expanded portion of the MED). Because these ducts (with the exception of the MED and the salivary bladder) are encased in secretory endpieces, they are difficult to locate and to observe by scanning electron microscopy. A simple method has been devised to rid the gland of these obscuring endpieces so that the detailed architecture of the duct system can be examined. Rat submandibular glands were fixed initially by vascular perfusion with half-strength Karnovsky's fixative. The connective tissue capsule was removed from extirpated glands and the glands remained in fixative for varying lengths of time. For our purposes, a 30-minute immersion in the aldehyde mixture was optimum. After the sublingual gland was removed, the submandibular gland was softly struck with forceps having rounded tips, then shaken in fixative or buffer. The tissue that remained was postfixed in osmium tetroxide. This method results in the complete divestment of nonductular parenchyma from the rat submandibular gland, leaving the duct system clean and ready for microscopic examination.

In vitro Transplantation of Fetal Human Retinal Pigment Epithelial Cells onto Human Cadaver Bruch's Membrane

Retinal pigment epithelium transplantation has been proposed as adjunctive treatment for age-related macular degeneration following surgical excision of choroidal neovascular membranes. The goal of this study was to develop a model to evaluate retinal pigment epithelium transplantation onto human Bruch's membrane in vitro. We investigated the ability of cultured fetal human retinal pigment epithelium to colonize human cadaver Bruch's membrane, determined the incubation time needed to form a monolayer and to exhibit apical microvilli and tight junctions, and assessed the production of basement membrane. Freshly enucleated (less than 48 hours old) human eyes were cut through the pars plana, and the anterior segment, vitreous, and retina were removed. The native retinal pigment epithelium was debrided with a surgical sponge. Bruch's membrane and choroid at the macula were trephined with a 7.0 mm diameter trephine and then incubated with 1/2 ml of Dulbecco's modified Eagle's medium +15% fetal calf serum+basic fibroblast growth factor (1 ng ml−1), and fetal human retinal pigment epithelium at a concentration of 242,000 cells ml−1. Specimens were incubated for 1, 4, 6, 8, 12, or 24 hours. The specimens were fixed in half strength Karnovsky's fixative, processed, and analysed with scanning and transmission electron microscopy. The retinal pigment epithelium covered the debrided macular specimens to different degrees at different incubation times. After 1 hour, the cells started to attach and flatten (median percent coverage: 78%). The extent of Bruch's membrane coverage by fetal retinal pigment epithelium varied greatly between specimens. After 4–6 hours, the cells covered the entire debrided surface in a monolayer (median percent coverage: 97.2% at 4 hours, 99.8% at 6 hours). Tight junctions were observed, and the cells had few apical microvilli. The lateral cell borders were obliquely oriented with respect to Bruch's membrane, and the nuclei were elongated, exhibited prominent nucleoli, and were oriented parallel to Bruch's membrane. After 6–8 hours, cells started to become hexagonal (median percent coverage at 8 hours: 99.97%). Cells attached to the inner collagenous layer tended to be flatter than cells attached to residual native basement membrane. At 12 and 24 hours, expression of hexagonal shape, tight junctions, and apical microvilli were observed more frequently (median percent coverage: 99.87% at 12 and 100% at 24 hours). No newly formed basement membrane was observed at these time points. In separate experiments comparing attachment in the presence and absence of native RPE basement membrane, the presence of native retinal pigment epithelial basement membrane promoted the early attachment of the cells and more rapid expression of normal morphology. This in vitro system provides a reproducible way to study the adherence of retinal pigment epithelium to normal and diseased human Bruch's membrane.

The effects of freezing, storage, and thawing on cell compartment integrity and ultrastructure.

The effects of slow freezing and thawing on enzyme compartmentalization and ultrastructure were studied in rat liver slices frozen in dry ice, isopentane/ethanol-dry ice, or liquid nitrogen, and stored at -80 degrees C for 1-14 days. Non-frozen slices served as controls. Frozen liver slices were thawed in a Karnovsky fixative and processed for transmission electron microscopy (TEM). After all freezing protocols, the outer zone of frozen-thawed tissue was ultrastructurally very similar to that of non-frozen liver. Towards the center of the tissue, the ultrastructure progressively deteriorated. Comparison with 50-microm cryostat sections prepared for TEM showed that thawing and not freezing is the detrimental step for fair preservation of ultrastructure. After thawing, homogenization, and differential centrifugation, distribution patterns of soluble marker enzymes were analyzed (cytosol, lactate dehydrogenase; mitochondrial matrix, glutamate dehydrogenase; lysosomes, acid phosphatase). The enzyme activities were not affected by storage for 2 weeks and the activity distributions showed that protein leakage from compartments was only minimally increased in frozen-thawed tissue compared with that from non-frozen tissue, irrespective of the method of freezing. In conclusion, fairly large tissue slices (20x5x3 mm) may be frozen and stored at -80 degrees C for biochemical, ultrahistochemical or ultrastructural studies. For ultrastructural analysis, only the periphery of the tissue slice should be used.

Prolonged Storage of Fixative for Electron Microscopy: Effects on Tissue Preservation for Diagnostic Specimens

Prolonged storage of Karnovsky's fixative, at both 4 and -20d`C, is possible in the diagnostic electron microscopy setting. Ultrastructural detail was not compromised in specimens processed in fixative that had been stored for 6 months. Evidence of smooth muscle and neuroendocrine differentiation was present in the form of actin filaments/dense bodies and neurosecretory granules, respectively. No difference in preservation was detected between specimens fixed in freshly prepared Karnovsky's fixative and fixative that had been stored at either 4 or -20d`C for up to 6 months. Thus, community hospitals can be provided with Karnovsky's fixative on a semiannual basis for surgical pathology specimens that may require electron microscopy for diagnosis.