The sensory portion of the retina is composed of multiple cell layers with complex functions and interactions vital to ocular homeostasis. Xenobiotics can induce physiological and morphological perturbations of these cell layers, and subsequently compromise vision. We describe an ion channel activator (ICA)-induced retinopathy observed in a preclinical drug safety study in dogs. Purebred beagle dogs received either ICA in vehicle (0.75% Methocel® and 0.05% Tween 80) or vehicle alone (control) for nine months. During the study, visual deficits and fundic changes were observed clinically, leading to premature termination of the study at eight months. The posterior globe of the left eye was fixed by the method of Acland and Aguirre, dehydrated in a series of graded methanol baths, and embedded in epoxy resin. Semi-thin (l-μm thick) sections of the retina were stained with toluidine blue, and examined by light microscopy. Ultrathin (60-90-nm thick) sections of the retina were mounted on copper grids,

Wound healing is coordinated by the diverse activities of cells of the monocyte/macrophage lineage.1 Macrophage depletion impairs wound healing, while overexpression of macrophage products is associated with chronic inflammatory states. If light-activatable pharmacologic agents could be selectively targeted to macrophages, clinical conditions resulting from overexpression such as postsurgical adhesions, hypertrophic scarring, and the formation of intimal hyperplasia could be suppressed. Similarly, adjusting light dose parameters to provide a stimulatory effect could selectively enhance wound repair processes.

To test these hypothesis, photoactivatable cytotoxins (photosensitizers, PS) coupled to specific receptor ligands have been evaluated for localization in cells found in cutaneous tissue using a non-invasive imaging method, two-photon microscopy.

The two-photon microscope (TPM) has been found to be a useful tool for imaging cells with submicron resolution thus allowing for the determination of photosensitizer localization within cells. Additional uses of the TPM will include imaging into epithelial tissue grown in culture and tracking cells in wounds.

The purpose of this study was to evaluate the biological response of a bone substitute material placed around Ti dental implants surgically positioned in tooth extraction sites of canine mandible. Under general anesthesia six adult mongrel canine had bilateral extractions of the fourth premolarbicuspid. Each extraction site was modeled into a 4 wall defect with box-like dimensions (1.0 mm vertically x 1.2 mm horizontally x 0.75 mm laterally). Utilizing the Branemark technique, screwtype implants with healing screws (10 mm x 4 mm), were placed in the prepared sites. A synthetic hydroxyapatite bone cement (HAC) [BoneSource®, Howmedica Laboratories, Inc.] was prepared and filled in around each implant. As controls, three canine had HAC placed in prepared sites without implants. Prior to periodontal flap suture closure a n-butyl-2-cyanoacrylate biodegradable tissue adhesive [Histoacryl®], was placed over implants and HAC. One canine each was euthanized at one month (m), two m, and three m postop.

Assessment of peroxisome proliferation is important in drug safety studies, since peroxisome proliferation often precedes tumor development in rodents. Proliferation can be measured morphometrically on sections by immunostaining for the peroxisomal enzyme catalase. Previous studies have used tissue embedded in acrylic resins such as LR White, which requires tissue sizes of 1-2 mm. Glycol methacrylate (GMA) is an acrylic resin that preserves antigenicity in much larger pieces of tissue. In this study we applied immunogold-silver staining (IGSS) to GMA sections for the quantitation of peroxisome proliferation in rat livers.

Sprague-Dawley rats were given clofibrate, a known peroxisome proliferator, for 14 days. Livers were removed, fixed in neutral buffered formalin for 4 hours, and embedded in GMA. Sections (3 μm thick) were incubated with antibody to catalase followed by protein A-gold (PAG), and the gold label was amplified using a commercial silver enhancement kit. Tissue sections were counterstained with propidium iodide (PI).

The mixed-lineage leukemia (MLL) gene is associated with more than 25 translocations of chromosome band 1 lq23 in the human leukemias. In certain unusually aggressive leukemias, the N-terminal portion of MLL fuses in frame to a portion of the transcription factor AF4. As a part of our investigation of the role of the MLL-AF4 fusion oncoprotein in Leukemogenesis, we studied the cellular sites of distribution of MLL-AF4 in the Hela carcinoma cells and U937 myeloid cells by immunofluorscene, confocal imaging and immuno-electron microscopy. Hela and U937 cells were stably transfected with a FLAG epitope-tagged MLL-AF4 (F-MLL-AF4) under the control of a tetracycline-responsive promoter and the expression of F-MLL-AF4 detected by indirect immunofluorescence (IF) with an anti-FLAG monoclonal antibody. These studies revealed that in both cell types the antibodies labeled two nuclear components, the nucleoli in an intense manner and the nucleoplasm in a punctate (100 to 200 dots) pattern. No antibody staining was observed in control (in the presence of tetracycline) cells.

An elevated low-density lipoprotein (LDL) in the plasma is the most recognized risk factor in the pathogenesis of atherosclerosis. On the other hand, the concentration of high density lipoprotein (HDL), is inversely correlated with atherosclerotic coronary artery disease. The transport pathways of LDL across the arterial wall has been well established. However, little is known about the transport mechanism(s) of HDL across the arterial wall. In this study, HDL was conjugated to colloidal gold for investigation of the ultrastructural aspects of binding and uptake of HDL by aortic endothelium.

High density lipoproteins were isolated from fresh rat plasma by sequential ultracentrifugation. HDL-colloidal gold conjugates were prepared according to the modified method of Kao et al. Sprague-Dawley rats were anesthetized and the vasculature was cleared of blood by perfusion with oxygenated PBS supplemented with 1 mM CaCl2 and 14 mM glucose at 37 °C through the abdominal aorta using the vena cava as an outlet.

Basic fibroblast growth factor (bFGF) is a potent angiogenic polypeptide. It promotes angiogenesis in vivo and in vitro by stimulating migration, proliferation and proteolytic activity of endothelial cells. Whereas several effects of exogenous bFGF on endothelial cells have been described, it has remained unclear how endogenous bFGF produced by vascular endothelial cells regulate angiogenesis.

To further investigate functional implications of the distribution of bFGF, we undertook the present study. Our aims were (i) to identify the specific location of bFGF in endothelial cells using electron microscopy immunogold labeling technique (ii) to determine the distribution of bFGF in capillaries of different types of tissues.

Tissue samples from sciatic nerve, hippocampus, adrenal gland and kidney of normal adult rats were fixed in 4% paraformaldehyde/1 to 5% glutaraldehyde and embedded in Spurr's resin. Ultrathin sections were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-ZymoGenetics) specific for bFGF using a two-step immunogold labeling method.

Basic Fibroblast Growth Factor (bFGF) is a multifunctional polypeptide which has been shown to play a pivotal role in the survival and differentiation of nerve cells. Several trophic and non-trophic functions of this protein have been suggested in peripheral nerves. In spite of ample information about the distribution and effects of bFGF in central nervous system, few data are available concerning the localization of this protein in peripheral nerves. In view of the role of bFGF in regulation of trophic and non-trophic functions, we particularly focused on the presence and precise location of bFGF in peripheral nerves at the electron microscope level.

Spurr's resin embedded ultrathin sections from adult rats’ sural nerves were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-Zymogenetics) specific for bFGF using two-step immunogold labeling method. Control samples were treated with either an equivalent volume of blocking solution (omitting the primary antibody) or an irrelevant antibody (Factor VIII, VGF, anti-histamine, anti-fibroblast 5B5).

C57BL/6 mice, along with several other mouse genotypes, have served as models for human presbycusis (age-related hearing losses). C57BL/6 mice and their genetic substrain C57/M6 show progressively severe hearing losses, starting as early as 30 days postnatally. The hearing losses result from sweeping degeneration of sensory (hair) cells and neurons that begins in the basal end of the cochlea and advances apically. Although the underlying mechanisms orchestrating sensory and neural degeneration are not known, it is possible to correlate degenerative events with the cytoplasmic levels and distribution patterns of a marker molecule, such as acid phosphatase (AP). AP, a representative lysosomal enzyme, plays a role in both normal cellular metabolism and degenerative changes (trauma and senescence). AP activity is visualized histochemically at the light and electron microscopic levels by the presence of dense deposits within lysosomes.

The mouse strain C57BL/6 suffers from the early onset of deafness. The deafness is attributable to sweeping degeneration of hair cells and the auditory nerve. Degeneration advances with age from the base to the apex of the cochlea. In the present study, neuronal condition was determined by linking relative levels of neuron specific enolase (NSE) in spiral ganglion neurons to their cytological appearances. NSE is an enolase isoform that is expressed only in nervous tissue and neuroendocrine glands.

Mice ranged in age from 3 days to 10 months. Inner ears were fixed with 2.5% paraformaldehyde in 0.1 M cacodylate buffer, decalcified for 24 hrs with EDTA, and then embedded in either paraffin or polyester wax. The latter, which melts at a lower temperature, caused much less neuronal shrinkage during routine processing. Deparaffinized sections were exposed first to rabbit anti-bovine NSE antibodies, then to biotinylated swine anti-rabbit antibodies, and finally to peroxidase-labelled avidin.

The purpose of this scientific investigation was to determine the presence and specific cellular localization of left-handed Z-RNA, within germinative zone (GZ) epithelium of the lens (Fig. 1), using anti-Z-RNA IgG polyclonal antibodies. Right-handed B-DNA has the ability to adopt the Z-DNA conformation in vitro (Sinden, 1994). Right-handed A-RNA can be transformed into Z-RNA under specific conditions (Hall et al., 1984), and Z-RNA has been identified in cultured cells (Zarling et al., 1990). Strong evidence supports the idea of Z-DNA in vivo (Sinden, 1994). Removal of proteins by fixatives can induce supercoiling which stabilizes Z-DNA (Sinden, 1994).

Anti-Z-RNA antibodies were produced in rabbits immunized with injections of Z-RNA: brominated-poly[ribosomal(G-C)]. For light microscopy, immunohistochemical studies (ABC method), normal dog lens tissues (1 yr old) were fixed in Carnoy's, embedded in paraffin and sectioned 2 μm thick. For electron microscopy (immunogold staining), pieces of epithelium from the GZ of normal dog lens (1 yr old) were fixed with 5% glutaraldehyde in 0.05 M phosphate buffer solution, pH 7.3.

Catalase is one of the several important antioxidant enzymes that detoxifies hydrogen peroxide (H2O2) to nontoxic H2O+O2. While previous studies prove that catalase plays an important role in the mechanism of enzymatic antioxidant defenses, the basal level of catalase is inadequate to deal widi excessive generation of H2O2 during ischemia and inflammation of me central nervous system. Although administered exogenous catalase can significantly reduce free oxygen radical damage in inflammatory response and demyelination disorders, it does not eliminate H2O2. Moreover, catalase has to be administered daily to increase its level. Recently, viral mediated gene delivery has been successfully demonstrated in many mammalian tissues, including in vivo gene transfer into ocular tissue. Gene expression was observed in retinal photoreceptor, RPE, Corneal endothelial and trabecular meshwork. Although it has already been reported that in vitro human endothelial cells express a 2-4 fold increase in catalase level within 24 hours after administration of the viral-catalase cDNA complex, there is no information available regarding in vivo human catalase gene transfer.

Axonemal dynein is a high molecular weight Mg2+-ATPase which occurs across the eukaryotic phylogenetic spectrum and interacts with the microtubular doublets of ciliary axonemes to effect ciliary beating. Unlike cytoplasmic dyneins which serve intracellular functions and are relatively ubiquitous in all cells, axonemal dynein is expressed exclusively in mature and nascent ciliated cells. Ciliated cells figure prominently in mucociliary clearance, a primary defense mechanism of the mammalian respiratory airways. Acting in concert with mucus secreting cells, the mucociliary escalator maintains virtual sterility in the airways and lung. Although ciliated cells are the predominant cell type in the airways and serve a critical function in respiratory health, they also are particularly vulnerable to injury. The strategic functional importance of the ciliated cell as well as its susceptibility to injury by infectious agents and irritants has led to development of epithelial cell culture systems which facilitate epithelium-specific studies of injury.

Microwave energy (MWE) as a method of rapid tissue processing is gaining increasing support as an alternative to routine chemical processing in diagnostic laboratory environments. Chemical fixatives and conventional fixation times used for standardized preservation of tissues can result in serious alterations in morphology as a consequence of solubilization and conformational changes of proteins and lipids. These untoward changes typically result in compromised antigenicity of many tissue proteins. With MWE processing, tissue antigens can be distinctly better preserved, antigen retrieval made more replicate and histochemical and immunochemical reactions made ultrafast. The present study had two objectives: to compare MWE processed skin to conventionally processed skin for ultrastructural integrity; to compare MWE expression of antigenicity of selected skin proteins which are usually sensitive to conventional chemical processing. Skin proteins selected were bullous pemphigoid antigen (BPA) and laminin.

The average sperm nucleus is only 8 micrometers long, but contains 1 meter of DNA. We have previously described how the sperm cell is able to package this DNA into the small volume of the sperm head. The only DNA structure that exists in both somatic cells and sperm nuclei is the organization of DNA loop domains that are attached at their bases to the nuclear skeleton, the nuclear matrix. We compared the structure of one gene in sperm nuclei to that of spermatogenic and adult cells.

Individual loop domains of the 5S rRNA gene cluster were visualized by fluorescent in situ hybridization (FISH). Nuclei were isolated from hamster spermatozoa, spermatogonia, pachytene spermatocytes, round spermatids, and adult liver cells. They were then extracted to remove the protamines or histones, fixed, hybridized to a biotinylated 5S rDNA probe, then viewed by epifluorescence microscopy.

Myosin is composed of two high-molecular weight heavy chains and four low-molecular weight hght chains. In both vertebrate and invertebrate skeletal muscle, each myosin heavy chain is associated with two myosin light chains. In skeletal muscle myosins studied by X-ray diffraction, each myosin heavy chain binds one of each of two distinct classes of hght chains. Thus, while isoform distributions may vary within and between fibers, the spatial distribution of each class of light chain should be uniform within the A band and between sarcomeres and fibers. Since no such study exists for crustacean myosin, we investigated the spatial distribution of the hght chains within the superficial flexor muscle (SFM) of the lobster, Homarus americanus, using immunoelectron microscopy. The SFM contains two classes of myosin hght chains, termed “alpha” (Mr = 21,000 to 23,500) and “beta” (Mr = 18,000 to 18,500). Immunocytochemical electron microscopic results suggest that the alpha light chains are not uniformly distributed at the subsarcomere level.

Cisplatin, a commonly used broad spectrum chemotherapeutic drug, has been proven effective in the treatments of bladder, lung, ovarian, head and neck, testicular and breast cancers. One of the major side effects of this drug is its gastrointestinal toxicity, which includes severe nausea, vomiting and in case of rats bloating of the stomach and gastric ulceration. In case of rats, it manifests as bloating of the stomach and gastric ulceration since the rats don't have vomiting reflex. This has been attributed to hypocalcemia induced by cisplatin treatment which results in the suppression of acetylcholine and constitutive nitric oxide release inducing a bloating of the stomach. This has further been shown to induce an increase in the acid content of the stomach. There are no studies available on any changes of gastric protection after cisplatin treatment Gastrin has been shown to have gastroprotective effects against injury by maintaining the gastric mucosal integrity and gastric mucosal blood flow.

Poly-[(trans-1,2-diaminocyclohexane) platinumj-carboxyamylose (“poly-plat“) is a second generation analog of cisplatin which enhances the immune system with greater efficacy in vitro and in vivo “Poly-plat” contains 1/5 the platinum of CDDP and demonstrates less toxicity. In order to understand the mechanism of action of this compound an in vitro and in vivo study was performed. Swiss Webster mice and isolated murine peritoneal macrophages were treated with “poly-plat” (10 mg/kg). The Swiss Webster mice were given bolus injections and sacrificed at 2 and 12 days. Peritoneal macrophages were then isolated and allowed to incubate in culture for 24 h. Peritoneal macrophages were also isolated from normal mice and treated with the drugs for 2 h. After treatments the macrophages were placed in fresh media and allowed to incubate 24 h. Supematants were isolated at various times during culture for immunocytochemical analysis.

Both in vitro and in vivo studies showed enhanced immunostimulation after their respective treatments.

Oocyte meiotic maturation occurs without the aid of centrioles in the mouse; instead the division spindles are organized by a number of microtubule organizing centers (MTOC). Immunofluorescence microscopy using anti-γ tubulin shows that multiple MTOCs migrate to surround the central nucleus (germinal vesicle, GV) during early maturation.. However, at the EM level, recognizable MTOCs have never been seen prior to the nucleation of microtubules (MT) which occurs near the time of nuclear envelope breakdown. Recently, I described multivesicular aggregates (MVAs) as the organelles most likely to represent immature MTOCs prior to MT nucleation4. These are first seen at time zero by EM as two large aggregates in the cortex, with sizes averaging 10um. MVA subsequently fragment into smaller units as they migrate centrally.

Materials and Methods: Female ICR mice were injected IP with 100 iu of chorionic gonadotropin (Sigma) to stimulate follicle growth, and ovaries were removed 48 hours later.

Confocal laser scanning microscopy is an excellent method to study nerve cell morphology and the three-dimensional distribution and interrelationship of dentrites and axons in the central nervous system. The cerebellum has been taking as a model of a gray center.

The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum,5 mm thick, were incubated in 10,30 and 100 μm solutions of FM4-64 in sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Mossy and climbing fibers were traced in the cerebellar white and gray substances .They exhibited high fluorescence signal at the level of myelin sheath.

Mossy fibers were identified in the granular layer by their typical rosette formation and dichotomous bifurcation pattern. Climbing fibers bundles were observed crossing the granular layer and giving collateral branches around Golgi cell bodies.