Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and the highly reactive free radical species, NO. Two major constitutive isoforms, neuronal and endothelial NOS, have been ented in the retina. These constitutive isoforms produce small amounts of NO while the inducible isoform, iNOS, produces high levels of NO. Excess production of NO by iNOS has beem implicated in the complications of diabetes. Since NO is a highly labile molecule, its potential localization is best achieved by immunocytochemical studies of NOS.

Semiquantitative colloidal gold labeled immunocytochemical studies of endothelial NOS (eNOS) and iNOS were done on sections of neural retina in eyes of obese, noninsulin dependent diabetic BBZ/Wor rats with diabetes of five months to twelve months duration. Age matched, nondiabetic control retinas were from eyes from BBDR/Wor rats.

Levels of iNOS were elevated in diabetic (33.9 ± 10.0 gold particles) as compared to age matched nondiabetic control (3.5 ± 2.8 gold particles) retinas.

Cephalad pulmonary mucociliary clearance driven by cilia of the ciliated airway epithelium provides probably the most important line of defense against inhaled toxins and particulate material for organs of the thoracic cavity. We demonstrate the reorganization of the cytoskeleton and the endoplasmic reticulum of airway epithelial cells upon cigarette smoke inhalation by employing DIC, LSCM, TEM and widefield fluorescence microscopy and have correlated this reorganization to changes in ciliary beat frequency (CBF) via FFT analysis. Neonatal pigs were used to provided healthy tracheal epithelial tissue. Exposure to cigarette smoke causes rapid ultrastructural changes including: ciliary distortion and detachment, ciliary abscission and severe alteration in endoplasmic reticulum structure suggesting profound disruption of essential membrane-cytoskeletal linkages.

To examine changes in CBF we describe a simple approach, using a laser scanning confocal microscope (LSCM) and an analog-to-digital computer converter/analyzer for the acquisition of data from biological systems that undergo rapid periodic movement, such as ciliary motion.

There have rarely been reported on radiation induced hematopoietic cells apoptosis and the recovery ,the expression and significance of oncogenes, tumor suppresser genes, hematopoietic grouth factors and their receptors in bone marrow.

By meams of routine pathology, specific stainning, electron microscope, immunocytochemistry, image analysis, in situ hybridization, in situ terminal end labelling, in situ PCR and DNA electrophoresis, The machanisms of hematopoietic cells apoptosis and then recovery in mouse bone marrow were studies through LAC A mouse total body irradiation with 60Co γ -rays.

The results showed: The bone marrow in each radiated group appeared obvious injury and then recovery, and the lessions were the most typical in 5.5Gy irradiation group, the pathologic changes were divided into four phases: namely the phase of hematopoietic cells apoptosis,bone marrow at 6hours after radiation. Furthermore, the higher radiation dose, the more the apoptotic cells were(table l).

Ultrastructural Pathology of capillary endothelial cells in autoimmune inflammatory myopathies has been intensively investigated in the last two decades, but much less is known about the alterations of capillary pericytes. In other diseases as diabetes and arterial hypertension perycites abnormalities have been reported. In this work we report the pericyte ultrastructural pathology in autoimmune inflammatory myopathies.

Patients admitted to the study were attending the rheumatology clinics at the Caracas University Hospital. Diagnoses were systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, and muscle paraneoplastic phenomenon.

Needle muscle biopsies were processed with routine techniques for transmission electron microscopy and observed in a Hitachi H-500 electron microscope.

Two different processes were observed, pericyte hypertrophy and cell degeneration. In the first one cytoplasmic proliferation is evident (Fig. 1). Some capillaries embraced by the pericyte were found (Fig. 2). In the second one cytoplasmic vacuolation and capillary necrosis (Fig. 3) were evident. Usually, pericyte and endothelial degeneration were concomitant.

Modern diagnosis of biopsied muscle requires a multidisciplinary approach, including routine histology and immunohistochemistry, cryosectioning for histochemistry, and (in selected cases) electron microscopy or biochemical evaluation. Routine paraffin embedding offers excellent histological detail. Cryosections provide fresh tissue for histochemical reactions that delineate fiber types and reveal special architectural alterations. Electron microscopy is applied in selected instances to provide diagnostic detail not otherwise apparent. Biochemical analysis of frozen tissue is used to define certain inborn errors of metabolism. This integrated approach allows diagnostic precision far beyond the limited information available from routine paraffin sections. We present examples of diagnoses made possible using these special techniques.

Chronic, slowly progressive neuropathies show grouping of fiber types and target fiber formation, apparent in special histochemical preparations. Routine histological preparations may be normal or show only nonspecific changes. Central core disease shows areas of pallor evident in the central areas of many myofibers in histochemical preparations.

The CD34 antigen is a single chain, heavily glycosylated transmembrane protein originally identified in hematopoietic stem cells. Subsequently it was found to be expressed in cells of normal endothelium and a variety of soft tissue tumors, including: epithelioid sarcoma, solitary fibrous tumor,’ gastrointestinal stromal tumor and dermatofibrosarcoma protuberans. Using paraffin immunohistochemistry, membranous and cytoplasmic staining for CD34 can be seen in these lesions, with most showing a combination of both. The findings of cytoplasmic staining is unexpected, as CD34 is a transmembrane cell surface protein. We analyzed the ultrastructural localization of CD34 on two soft tissue tumors with membranous and combined membranous and cytoplasmic staining.

Biopsy specimens were fixed with a mixture of 4% paraformaldehyde and 1% glutaraldehyde in 0.1M sodium cacodylate buffer, pH = 7.4 for 1-2 hours, and embedded in Poly/Bed 812 with osmium tetroxide post-fixation, or in LR White without osmication. The immunogold method was performed on ultrathin sections placed on Nickel grids,

Pheochromocytoma is a rare tumor found in approximately 2 individuals per million of the population. In 90% of cases, it arises from the chromaffin cells of the adrenal medulla. These cells secrete catecholamines: epinephrine and norepinephrine which have a sympathomimetic action. The classical clinical presentation is paroxysms of extreme elevation of systemic blood pressure. Although pheochromocytoma accounts for only 0.1 % of cases of systemic hypertension, it is important to make the diagnosis, as the sudden release of large quantities of catecholamines into the circulation may prove fatal, as happened in our patient.

We present a case of a 25 year old woman, who collapsed suddenly, while she was feeding her 8 month old baby. She went into ventricular fibrillation and was defibrillated 3 times. There was no response to vigorous resuscitative measures.

Past Medical History: No definitive findings. Vague history of palpitations and an “abnormal heart beat”, which had been considered to be benign.

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.