Microplicae: characteristic ridge-like folds of the plasmalemma.

Scanning electron microscopy reveals that the free surfaces of stratified squamous epithelial cells lining the alimentary tract, cornea, and conjunctiva exhibit characteristic ridge-like folds of plasmalemma. These microplicae are approximately 0.1-0.2 micronm in width, of variable height (0.2-0.8 micronm) and length, may followstraight or winding paths, often branch, and exhibit a wide variety of patterns over the surfaces of cells. Transmission electron microscopy reveals that microplicae often have a fine (100-150 A) electron-dense zone subjacent to their plasmalemma and an intracellular matrix characterized by a disorderly arrary of fine filaments (40-60 A in diameter). Microplicae appear to arise from plasmalemmal fold which once provided for intercellular interdigitation and desmosome abhesion between adjacent cells. Ruthenium red staining demonstrates that microplicae and interplical grooves are covered with a polyanionic glycocalyx. Although free surface microplicae may merely represent the renmants of intercellular interdigitations or a modified expression of microvillous-like extensions, it is also possible that they serve another specific function. In this regard it is speculated that microplical and interplical grooves may function to hold a layer of lubricating and cushioning mucin designed to protect the underlying plasmalemma from abrasive abuse.

Noninvasive vital microscopy to monitor tubular necrosis of cold-stored kidneys.

This study evaluated the ability of a new form of vital microscopy, termed tandem scanning confocal microscopy (TSCM), to determine the histopathological status of kidneys while they are being preserved in cold storage preservation solutions. The TSCM observations were performed on rat kidneys stored at 0-2 degrees C in one of several cold-storage preservation solutions (Euro-Collins, UW solution, phosphate-buffered dextran), and correlated with conventional light microscopic observations of these same kidney samples. The present investigation provides histopathological guidelines for the use of TSCM in determining the extent of necrosis that a kidney suffers during its cold-storage preservation, and demonstrates that TSCM can be used to observe cold-stored living kidneys repeatedly over time and provide immediate and artifact-free informative images regarding their histopathological status. The combined TSCM and conventional light microscopic observations also provided information regarding the ability of the various cold storage solutions being studied to preserve the normal histology status of cold-stored kidneys.

An evaluation of the ability of dextrans to reduce acute tubular necrosis during cold storage preservation.

In this study, the ability of low molecular dextrans to prevent morphologically detectable acute tubular necrosis during cold storage was evaluated. Rat kidneys were flushed with a sodium phosphate buffer (pH 7.2) containing different concentrations of dextran 10 (m.w. of 10,000 or less) and stored at 0-2 degrees C for up to 5 days (samples taken at 24-hr intervals). It was found that solutions containing 20% or more of dextran 10 provided significantly improved morphological preservation of kidney nephrons when compared with currently popular kidney cold storage preservation solutions (i.e. University of Wisconsin and Euro-Collins solutions). Adding smaller amounts (i.e., 15%) of dextran 10 to a cold storage solution already containing another effective osmotic agent (i.e., sucrose) also resulted in superior morphological preservation, indicating a beneficial additive effect of using more than one osmotic agent. Dextran 40 (m.w. 40,000) did not provide as good morphological preservation as did a similar concentration of dextran 10. It is concluded that the use of the proper kind and proper amount of low molecular weight dextrans in preservation solutions can significantly reduce the morphologically detectable acute tubular necrosis during cold storage.

Ultrastructure of kidney preservation: varying the amount of an effective osmotic agent in isotonic and hypertonic preservation solutions.

Currently the level of effective (i.e., impermeant) osmotic agent necessary to prevent cell swelling during the cold storage of kidneys is not known. In the present investigation, we used light microscopy and transmission electron microscopy to evaluate the amount of the osmotic agent sucrose which is needed to protect the rat kidney parenchyma from damage over 24 and 48 hr of cold storage. Our observations indicate that when sucrose contributes 140 mOsmol or more to the total osmolality of phosphate-buffered solutions, most cell swelling and associated ultrastructural damage can be prevented over 48 hr of cold storage. We also found little difference between the quality of kidney ultrastructural preservation which results when kidneys are stored in isotonic (300 mOsmol) versus hypertonic (400 mOsmol) solutions that contain the same amount of sucrose. The overall quality of preservation seen with solutions which contain 140 or 200 mOsmol of sucrose is dramatically better than that which we previously observed with such clinically popular kidney preservation solutions as Collins, Euro-Collins, and Sacks.

The spatial organization of corneal endothelial cytoskeletal proteins and their relationship to the apical junctional complex.

PURPOSE: To determine the spatial organization of the major cytoskeletal proteins and their relationship to the apical junctional complex (AJC) in the normal rabbit corneal endothelium. METHODS: Normal endothelial cytoskeletal structure in three dimensions was studied in rabbit eyes by laser scanning confocal microscopy after en bloc immunocytochemical staining of whole corneal tissue with various antibodies and fluorescent probes; specificity of antibodies to rabbit corneal endothelial cell proteins was established by Western blot analysis. RESULTS: Normal actin microfilament network organization was seen predominantly as a complex apical array forming a circumferential bundle. The tight junction-associated protein ZO-1 was positive at the apical junctions, forming a hexagonal pattern that was localized between and just proximal to the circumferential actin microfilament bundles. The distribution of ZO-1 was discontinuous around the cell, with the largest gaps (1 micron in diameter) occurring at the Y-junction between adjacent endothelial cells; transmission electron microscopy of the apical face of the endothelium confirmed the existence of 1-micron diameter gaps in the adherens junctions located at the Y-junction. Antivimentin antibodies showed a ring of intermediate filaments located just below the circumferential actin microfilament band. This ring appeared to be continuous with a basal mat of filaments, which together formed a basketlike structure within endothelial cells. An intricate cytoplasmic, perinuclear network of microtubules was observed by antitubulin antibodies that appeared unrelated either to the apical circumferential actin microfilament bundle or to intermediate vimentin filament ring. Staining of endothelial cells with NBD-ceramide identified a prominent, perinuclear Golgi complex suggesting an association between microtubules and Golgi. CONCLUSIONS: The organization of cytoskeletal elements and the tight junction-associated protein ZO-1 is similar to the classical AJC of transporting epithelia, comprised of a zonulae occludens (ZO) located apical to a zonulae adherens (ZA) and desmosomes. The organizational pattern seen in corneal endothelial cells, however, is distinct from transporting epithelia in that the ZO and ZA are discontinuous, with large gaps in the ZO-1 distribution at the Y-junction between adjacent endothelial cells. The authors propose that the structural differences in the AJC underlie the functional differences between classical transporting epithelia, which actively pump fluid from the lumen to the mucosa, and the corneal endothelium, which has a "pump-leak" fluid transport mechanism.

Actin filament organization during endothelial wound healing in the rabbit cornea: comparison between transcorneal freeze and mechanical scrape injuries.

PURPOSE: To compare and contrast the in vivo mechanism of wound healing after mechanical scrape and transcorneal freeze (TCF) injury in a rabbit eye model by examining changes in the cytoskeletal organization of contractile, filamentous actin (f-actin) microfilaments as relates to differences in cell migration or translocation during endothelial repair. METHODS: Endothelial wound healing after mechanical scrape and transcorneal freeze injury was studied in rabbit eyes using laser scanning confocal microscopy (LSCM). Central corneal mechanical scrape injury was made using an olive tip cannula, and TCF injury was made using a 3-mm diameter stainless steel probe cooled with liquid nitrogen. Cytoskeletal changes in f-actin stained with phalloidin-FITC were observed during wound healing using LSCM. RESULTS: At 6 hours after mechanical scrape, the leading edge of the migrating sheet showed a decrease in the intensity of phalloidin-FITC staining, suggesting a decrease in cortical f-actin. Migrating endothelial cells in vivo did not appear to develop stress fibers after mechanical scrape, which is consistent with an in vitro cell spreading mechanism of endothelial wound healing. By 24 hours, the denuded area was almost fully resurfaced by migrating endothelial cells. On the other hand, TCF injury produced fibroblastic changes in the endothelial cells with extension and elongation of spindle-shaped endothelial cells at the leading edge by 24 hours after injury. Fibroblastic endothelial cells developed prominent actin stress-fibers, which is consistent with an in vitro cell migration mechanism of endothelial wound healing. Three days after TCF, the wounded area was resurfaced with two cell types: rough, fibroblast-like cells forming a retrocorneal fibrous membrane having prominent f-actin bundles or stress fibers with few cell-cell junctions, and smooth, polygonal-shaped endothelial cells having tight cell junctions with a cortical distribution of f-actin. After 28 days the retrocorneal fibrous membrane was posteriorly covered with normal endothelium. CONCLUSIONS: These data support the hypothesis that endothelial wound healing involves two separate, injury-dependent, mechanisms--cell spreading and cell migration.

Regional chemotherapy in an experimental model of Wilms' tumor in rats.

A s.c. experimental model of Wilms' tumor in rats was used to compare the effects of intratumoral treatment with vincristine plus actinomycin D to i.v. treatment with these chemotherapeutic drugs. The Wilms' tumor model is a fast-growing solid tumor that has been shown to be resistant to traditional clinical treatment procedures used for Wilms' tumor in man. Injection of the chemotherapeutic drugs directly into the tumor mass was found to be more effective than i.v. therapy in causing long-term remission of the tumor. Intratumoral therapy was also less toxic to the animals than i.v. therapy when measured by post-treatment survival rates and weight loss during the 1st week following treatment. However, intratumoral treatment caused an initial fibrosis of the tumor tissue, which resulted in a slower rate of absorption of the resultant fibrotic tumor mass than was seen in tumors treated i.v. Also, intratumoral injection resulted in necrosis of the overlying skin, which healed as the fibrotic tumor tissue was absorbed. Intratumoral treatment of a cervical tumor was found to cause the remission of a second major tumor mass located at some distance from the initial injection (i.e., in the lumbar region). No significant benefits were noted when dimethylsulfoxide (DMSO) was used in place of aqueous mannitol as a vehicle to deliver the chemotherapeutic agents. There was a significant correlation between the drug dose-to-tumor-size ratio (D/T ratio) and the effectiveness of the chemotherapy. When this ratio was high enough, a single treatment with a combination of vincristine and actinomycin D usually resulted in total remission of the experimental Wilms' tumor in response to either intratumoral or i.v. therapy.

In vivo osmotic pertubation of intercellular fluid channels in the rabbit corneal endothelium.

An in vivo rabbit corneal model was used to evaluate morphological changes in the corneal endothelium associated with osmotically increasing fluid movement from the anterior chamber into the stroma. When the corneal stroma is rendered more hypertonic than normal by immersing the scraped epithelial side of the cornea in a hypertonic sucrose solution, intercellular channels and apical pores at the Y-junctions between endothelial cells become greatly enlarged. The foregoing changes are reversible and do not appear to result in damage to the corneal endothelium. These observations suggest that specific intercellular channels in the corneal endothelium may provide pathways for the movement of fluid from the aqueous humor into the stroma.

In vivo confocal microscopic studies of endothelial wound healing in rabbit cornea.

Corneal endothelial wound healing in living rabbit eyes after mechanical scrape (MS) and transcorneal freeze (TCF) injury was studied using tandem scanning confocal microscopy (TSCM). MS injury was created on the central corneal endothelium with an olive tip cannula; TCF injury was created using a 3-mm-diameter stainless steel probe cooled with liquid nitrogen. In vivo observation of wound healing using TSCM was correlated with scanning electron microscopy (SEM) for fixed tissues. At 6 h after MS, migrating endothelial cells at the leading edge showed lamellipodial processes on in vivo TSCM and SEM. After 24 h, the denuded area was almost fully resurfaced by migrating endothelial cells showing wide spaces between nuclei by TSCM. After 28 days, resurfaced endothelial cells showed normal hexagonal mosaic appearance with enlarged cells by TSCM and SEM. TCF injury produced fibroblastic changes in the endothelial cells with elongation and spreading by 24 h after injury. After 3 days, the wounded area was resurfaced with two cell types: (a) migrating endothelial cells at the peripheral area, which appeared polygonal in shape with wide intracellular spaces and (b) fibroblast-like cells at the center of the wound, which formed a retrocorneal fibrous membrane (RCFM). The RCFM was posteriorly covered with normal endothelium after 28-60 days. TSCM of the stroma demonstrated spindle-shaped, activated keratocytes migrating into the wounded stroma at 3-14 days. In conclusion, TSCM allows viewing of dynamic four-dimensional morphologic changes (x, y, z, and time) during in vivo cellular repair of corneal wound healing after either MS or TCF injury.

The histopathology of kidney uriniferous tubules as revealed by noninvasive confocal vital microscopy.

Tandem scanning confocal microscopy (TSCM) permits the noninvasive microscopic viewing of unstained, living, solid organs in real time. This article provides an overview of recent studies conducted by the author and other scientists, using TSCM and demonstrating its advantages in evaluating the histopathology of uriniferous tubules associated with normothermic renal ischemia, the nephrotic syndrome, and extracorporeal cold-storage preservation of kidneys.