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Biomedical subjects

M J Hollenberg

Publications and source records attributed to M J Hollenberg.

At least 19 recordsLinked to original sources
PubMed1993

Growth factor-induced retinal regeneration in vivo.

It is apparent from a number of studies that the RPE has a remarkable ability to regenerate neural retina. While retinal regeneration from the RPE has not been reported in adult vertebrates, with the exception of the newt, there is evidence that many vertebrate species have the ability to regenerate a new neural retina during the early development. Studies of retinal regeneration in the chicken embryo have provided some insight into the requirements for this process. Recent investigations using copolymer implants as an intraocular delivery system for growth factors have demonstrated that the state of differentiation of RPE cells in the stage 22-24 chicken embryo can be altered in vivo by specific growth factors, aFGF and bFGF. These results raise the distinct possibility that variations in the local production of FGFs and their receptors in the eye during development may, in part, regulate the pathway of differentiation of RPE and neural retina precursors. Further research on the role of FGFs and their receptors in retinal development and regeneration will not only contribute to our understanding of how the differentiated state is achieved and maintained but may provide a foundation for future attempts to develop methods of treatment for various degenerative and proliferative diseases of the eye.

Animals↗
PubMed1992

Chemical extraction of the cytosol using osmium tetroxide for high resolution scanning electron microscopy.

Detailed examination of subcellular structures in three dimensions (3D) by high resolution scanning electron microscopy (HRSEM) is now possible due to improvements in the design of the scanning electron microscope and the introduction of methods of specimen preparation using chemical removal of the cytosol and cytoskeleton by dilute osmium tetroxide. Cells which have been fixed, frozen, cleaved, thawed, and subjected to cytosol extraction display intact intracellular structures in 3D including nuclear chromatin, endoplasmic reticulum, mitochondria, and the Golgi complex at a resolution close to that of conventional biological transmission electron microscopy (TEM). Small changes in the 3D structure of subcellular components can be conveniently examined in this way in development, in a variety of physiological processes and in disease. Broad areas of the specimen can be quickly surveyed by HRSEM since sectioning is not required and specimens of comparatively large size (up to 5 mm3) can be placed in the microscope. Extraction of the cytosol with dilute osmium tetroxide (OsO4) exposes subcellular structures in relief, permitting their examination in 3D from several aspects. However, the OsO4 extraction technique is limited, since significant intracellular structures, such as the cytoskeleton, vesicles, and antibody binding sites can be removed or inactivated during the cytosol removal steps.

Animals↗
PubMed1992

Improved techniques for successful neonatal rat surgery.

Problems encountered in neonatal rat surgery include mortality due to anesthesia and postoperative mortality due to cannibalism or neglect by the dam. We required a method of anesthesia which would enable us to perform complicated, lengthy, recovery eye surgery on day-old rat pups. Because ethical concerns have been raised regarding hypothermia, the currently recommended procedure for anesthesia of newborn rats, we adapted two effective techniques for anesthetizing adult rats for use in neonates. In the first of these methods, halothane was administered via a gas anesthetic machine which allowed for precise regulation of anesthetic levels. The second method employed diluted Innovar-Vet, a neuroleptanalgesic drug combination that is easily administered by injection, with oxygen supplementation. Because each surgical procedure required 30 to 45 minutes and was technically demanding, it was important to minimize the loss of experimental animals due to cannibalism. To accomplish this, we developed an easy, noninvasive method to encourage acceptance of surgically manipulated pups by the dam, which included hand gentling and olfactory conditioning of pregnant females. All pups (63/63) survived eye surgery under halothane anesthesia and of those examined 7 days later, 55/57 (97%) were alive and appeared normal. Of the pups treated with Innovar-Vet, 16/16 (100%) survived anesthesia and all were normal in appearance when examined 7 days later. Our results suggest that using these anesthetic methods coupled with appropriate conditioning of the dam and handling of the pups contribute to successful neonatal rat surgery.

Anesthesia, General↗
PubMed1991

Tridimensional ultrastructure of perfusion fixed gastrointestinal epithelial cells by high resolution scanning electron microscopy.

Improvements in the design of modern scanning electron microscopes (SEM) and new methods of specimen preparation incorporating chemical removal of the cytosol and cytoskeleton, now make it possible to view cells and their organelles in three dimensions (3D) at high magnification. In this experiment, high resolution SEM (HRSEM) utilizing new methods of tissue preparation was used to study the intracellular structures of the mouse ileum. In addition, in vivo intestinal perfusion was used to further enhance cellular preservation. Using these modifications it was possible to visualize, in 3D, the fine structure of intestinal epithelial cells and intracellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi complex, as well as microvilli and cell membrane. Whole mitochondria appeared as irregularly shaped organelles which contained tubular cristae. Plate-like cristae were not observed. The brush border was found to be closely packed array of cylindrical projections. The extensive folding and structural intricacy of lateral cell membranes between absorptive cells could only be appreciated by viewing this tissue with 3D HRSEM. The use of HRSEM to study 3D ultrastructure of cells and their organelles will improve our understanding of the structure-function relationships in both the healthy and diseased gastrointestinal tract.

Animals↗
PubMed1991

The structure of the developing chick retinal pigment epithelium revealed by high resolution scanning electron microscopy.

The retinal pigment epithelium (RPE) in the developing eye of chick embryos has been studied during the early stages of development by high resolution scanning electron microscopy (HRSEM). Specimen preparation techniques which involve removal of the cytoplasmic matrix permitted visualization of organelles and other subcellular structures within RPE cells in detail and in three dimensional (3-D) stereo HRSEM. Using this technique, we were able to examine changes in melanosome structures during development and demonstrate that pigmentation in the RPE was present by day 4 of development. RPE plasma cell membranes showed extensive folding of the apical portion of the membrane closest to the developing neural retina by day 9. Examination of RPE photoreceptor junction revealed photoreceptor inner segments by day 6 and an outer segment by day 9. Mitochondria in the RPE were found to contain tubular cristae only. The ultra-structure in 3-D of the Golgi apparatus, smooth and rough endoplasmic reticulum, lysosomes and nuclear chromatin of the RPE, and Bruch's layer was revealed by the HRSEM method.

Animals↗
PubMed1991

Resolution limit of serial sections for 3D reconstruction of tubular cristae in rat liver mitochondria.

Following stereo, high resolution scanning electron microscopy (HRSEM) of various rat tissue mitochondria prepared by mild glutaraldehyde fixation followed by freeze cleavage and extraction of the cytosol (Lea and Hollenberg, 1989a), a new model for the ultrastructure of mitochondrial cristae has been developed (Lea and Hollenberg, 1989b). The cristae mitochondriales, previously considered in most cells to have a shelf like structure, were found in many cell types including hepatocytes by HRSEM to be tubes which spanned the mitochondrial matrix and were continuous with the inner mitochondrial membrane at both ends. Despite computer aided, serial, reconstruction of serial thin sections examined in the transmission electron microscope (TEM), tubular cristae in mitochondria have not been resolved entirely, even though circular cross sectional profiles of cristae have been observed in the TEM. The current study was undertaken to probe this deficiency and to understand its cause. In this study, the problems of inherent contrast and resolution loss in the digitized image have been reduced by using a computer based, background, extraction process analogous in the computer to the chemical cytosol extraction process used for HRSEM (Lea and Hollenberg, 1988; Hollenberg et al., 1989). Despite the use of this membrane enhancement algorithm, it was still not possible to reconstruct entire tubular cristae, as observed by HRSEM, even when the micrograph negatives were digitized and reprocessed (Lea and Hollenberg, 1989b). A comparison of serial section thickness deduced from the diffraction colour of the respective section (60 nm) to the measured diameter of tubular cristae profiles (30 nm), with membrane thickness of 10 nm, suggested that this size disparity is responsible for the apparent loss of resolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Induction of retinal regeneration in vivo by growth factors.

We have previously reported that basic fibroblast growth factor (bFGF) can induce retinal regeneration in the stage 22-24 chicken embryo. The present study was undertaken to identify the cellular source of the regenerate and to determine whether other growth factors also elicit regeneration in this animal model. Polymer implants containing bFGF were inserted into eyes of chicken embryos immediately after extirpation of the neural retina. The retinal pigment epithelium (RPE) was left intact. Evaluation by light microscopy revealed that in bFGF-treated eyes the new neural retina arose by transdifferentiation of the entire RPE layer. Differentiation of the new neural retina occurred in a sequence similar to that of normal development but proceeded in a reverse (vitread) direction. All retinal laminae had differentiated by Day 15. However, the regenerate displayed reversed polarity, with photoreceptors closest to the lens. The RPE, pecten, and optic nerve were absent. Focal areas of degeneration in the retinal regenerate became evident for the first time on Day 10. Retinal regeneration was also observed after treatment with higher doses of acidic fibroblast growth factor, but not with nerve growth factor-beta, transforming growth factor-beta 1, insulin, or insulin-like growth factors I or II. These results raise the possibility that FGFs may play a role in retinal differentiation during development.

Animals↗
PubMed1989

Mitochondrial structure revealed by high-resolution scanning electron microscopy.

Mitochondrial structure has been examined in three dimensions using high-resolution scanning electron microscopy in cells from rat liver, retina (photoreceptors and retinal pigment epithelium), and kidney (proximal convoluted tubular cells and podocytes). Tissues were prepared by aldehyde-osmium fixation and freeze cleavage using a cryoprotectant, followed by removal of the cytosol by immersion in a dilute osmium tetroxide solution. The microscope used (Hitachi S-570) was equipped with a secondary electron detector located in the column above the specimen, situated within the objective lens. Mitochondria in all tissues examined were found to have only tubular cristae, which in some instances could be seen to span the entire diameter of the organelle. The walls of the tubular cristae, when unfractured, were in contact with the inner mitochondrial membrane; and their lumens were open to the intermembranous space. We hypothesize that in cells of many, perhaps most tissues, mitochondrial cristae are not shelf-like but are, in fact, tubes which span the mitochondrial matrix and are continuous with the inner mitochondrial membrane at both ends.

Animals↗
PubMed1989

Basic fibroblast growth factor induces retinal regeneration in vivo.

In the present study, we have investigated the effect of basic fibroblast growth factor (bFGF) on retinal regeneration in the stage 22-24 chick embryo. The neural retina was surgically removed in ovo leaving the retinal pigment epithelium (RPE) intact and then slow-release, plastic implants containing bFGF were inserted into the eye. Light microscopic examination of eyes 7 days later revealed that bFGF induced retinal regeneration in a dose-dependent manner. The absence of the RPE in these eyes and the reversed polarity of the regenerated neural retina is consistent with the hypothesis that this process occurs by transdifferentiation of the RPE. This represents the first time that a known molecule has been shown to induce retinal regeneration in vivo.

Animals↗
PubMed1989

Tridimensional ultrastructure of glomerular capillary endothelium revealed by high-resolution scanning electron microscopy.

Recent advances in specimen preparation techniques and scanning electron microscope (SEM) design have permitted ultrastructural examination of the glomerular capillary wall in three dimensions using high-resolution scanning electron microscopy (HRSEM). Specimens in which the cytosol and cytoskeleton have been extracted, but cell membranes nuclear structures and organelles left in place, were studied using a Hitachi SEM with a resolution of approximately 3 nm. Each HRSEM micrograph displayed a depth of field and information content equivalent to 15-30 consecutive, ultrathin, transmission electron microscope (TEM) sections viewed simultaneously in perfect serial alignment. The results have confirmed previous ultrastructural observations obtained by use of TEM and, in addition, have revealed new ultrastructural features of the normal rat glomerulus. A morphometric analysis of glomerular endothelium carried out using the HRSEM micrographs revealed that the endothelial cell processes, which lie between the fenestrae, are nearly circular in cross section and that they, as well as the fenestrae, have a diameter of approximately 60 nm. The potential functional role of the fenestrae in controlling access to the underlying basement membrane requires further study.

Animals↗
PubMed1989

High resolution scanning electron microscopy of rabbit corneal endothelium to show effects of UV-visible irradiation in the presence of chlorpromazine.

The ultrastructure of rabbit cornea endothelial cells was examined by scanning electron microscopy (SEM) in freeze-cleaved corneas using a Hitachi S-570 scanning electron microscope in the high resolution mode (HRSEM). In order to study phototoxic effects in vitro, rabbit corneas (experimental) were cultured as organ culture in the presence of 5 micrograms/ml chlorpromazine (CPZ) and irradiated. For comparison, control 1 corneas were not irradiated but incubated in the dark without CPZ in the medium; control 2 corneas were also kept in the dark but in the presence of CPZ; control 3 corneas were irradiated with no CPZ in the medium. Cellular damage was not seen in the three types of control corneas, but in the experimental corneas the endothelial cells showed extensive disruption of the cell membrane and some deterioration of the intracellular components. Our study confirmed that HRSEM is a satisfactory new technique for visualizing damage of the intracellular organelles of corneal endothelium.

Animals↗
PubMed1988

High resolution scanning electron microscopy of the retinal pigment epithelium and Bruch's layer.

The application of a new technique in vision research for investigating the three-dimensional ultrastructure of extra- and intracellular organelles and components is presented, based on the use of high resolution scanning electron microscopy (HRSEM). This technique has been made possible by advances in scanning electron microscope design which permit resolution of particles less than 3 nm in diameter and new tissue preparation techniques which remove the cytosol and leave cell membranes and organelles in relief. Application of these methods to the ultrastructure of the retinal pigment epithelium and Bruch's layer in the albino rat has revealed a number of new structural features and confirmed others by using a different approach in tissue preparation and examination. It is now possible to obtain HRSEM micrographs with an information content equivalent to at least 15 ultrathin, serial, transmission electron microscope sections, all perfectly aligned, in one micrograph without having to resort to accepted reconstruction techniques. It is clear that this new method has the potential to rapidly advance our knowledge of the structures involved in vision in both health and disease and in a variety of experimental conditions.

Animals↗
PubMed1983

X-ray microscopy using a scanning electron microscope for the purpose of imaging central nervous system structures.

A simple modification of an Hitachi S.450 specimen stage permits point source X-ray microscopy with a scanning electron microscope. Point source X-ray microscopy was applied to nervous tissue to determine the feasibility of utilizing this technique instead of a light microscope mounted camera lucida to produce 2- and 3-dimensional images of whole structures such as neurons. Various target materials, radio-opaque materials and photographic films were examined in this study.

Animals↗
PubMed1978

Morphology of the rectinal pigment epithelium in the vitamin A deficient rat.

Ultrastructural changes in the retinal pigment epithelium (RPE) and adjacent photoreceptor cells have been followed in the Wistar rat during the course of long-term vitamin A deficiency. Of particular interest has been the discovery of unusual concentric aggregates within the photoreceptor outer segments and the inner cytoplasm of the RPE. The aggregates were present throughout the course of the retinal degeneration induced by vitamin A deficiency and could be identified in the RPE either by themselves in the apical cytoplasm or within phagolysosomes. It is postulated that the concentric aggregates result initially from abnormal formation or condensation of outer segment membranes and are then slowly degraded by lysosomal action in the RPE cytoplasm. In addition, acid phosphatase activity and typical phagosomes (shed outer segment packets) have been demonstrated in the cytoplasm of the RPE of the vitamin A deficient rats. The latter findings indicate that, at least to some extent, the normal phagocytic and lytic processes of the RPE are retained in this nutritional disorder.

Acid Phosphatase↗
PubMed1977

Complicated colobomatous microphthalmos in the BW rat: a new form of inherited retinal degeneration.

A new model of inherited retinal degeneration has been found in the rat. It is inherited in association with a number of other ocular defects, including microphthalmos, coloboma, retinal dysplasia, optic nerve hypoplasia and/or aplasia, as well as medullation of the nerve fiber layer of the retina. Together, these abnormalities constitute a condition referred to as complicated colobomatous microphthalmos. This condition was originally discovered in the Bmn strain of rats but subsequently transferred to a new genetic background in the Bmn-wys strain of rats (BW). This facilitated the histological evaluation of both the developmental and degenerative ocular defects in the adult animals. A well defined pattern emerged relating eye size, optic nerve size and retinal histology. Normal-sized eyes had normal-sized optic nerves and normal retinal histology while intermediate-sized eyes with no optic nerves had uniformly thin retinas. In contrast, intermediate-sized eyes with small optic nerves had areas of both normal thickness and thin retina. All of these eyes developed retinal degeneration characterized by a late onset and slow progression associated with normal phagocytic activity in the pigment epithelium and a tendency for the rod outer segments to fragment into very thin structures rather than accumulate as lamellar debris. This indicates that the retinal degeneration in the BW model differs in many respects from the well studied RCS model.

Animals↗