Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB).

A new method for demonstrating cytochrome oxidase activity, based upon the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic reaction product, has improved the localization of this enzyme over methods based upon the Nadi reaction, in both the light and electron microscopes. The reaction product occurs in nondroplet form, which more accurately delineates the localization of cytochrome oxidase in mitochondria of heart, liver, and kidney. In electron microscopic preparations the excess reaction product is found to overflow into the intracristate spaces and into the outer compartment between inner and outer limiting mitochondrial membranes. This finding suggests that the enzymatic activity of cytochrome c is located on the inner surface of the intracristate space which is the outer surface of the inner mitochondrial membrane. Succinic dehydrogenase activity has also been located at this site by using an osmiophilic ditetrazolium salt, TC-NBT. Considered together, the sites of reactivity of both parts of the respiratory chain have implications for the chemiosomotic hypothesis of Mitchell who suggests a mechanism of energy conservation during electron transport in the respiratory chain of the mitochondrion.

Ultrastructural demonstration of cytochrome oxidase activity by the Nadi reaction with osmiophilic reagents.

A new method for the subcellular and cytochemical demonstration of cytochrome oxidase has been developed with the introduction of N-benzyl-p-phenylenediamine (BPDA) and the discovery that indoanilines are osmiophilic. These indoanilines produced upon oxidation of BPDA in the presence of naphthols are highly colored compounds that yield electron-opaque coordination polymers of osmium (osmium black) that are amorphous, insoluble in water, and in organic solvents. The best methods for preparing rat tissue were in decreasing order: fixation in formaldehyde solution, fresh tissue slices, and frozen sections of fresh or fixed tissue. Ultrathin sections were counterstained by bridging with the thiocarbohydrazide-osmium tetroxide (T-O) procedure for enhancing underlying membranous structures. Cytochrome oxidase activity was noted primarily in mitochondria and occasionally in sarcotubules of heart, in mitochondria and occasionally in infoldings of the plasma membrane of renal tubular cells, and in mitochondria and, to a great extent, in endoplasmic reticulum of hepatic cells. Cytochrome oxidase activity produced deposits in droplet form, whereas dehydrogenase activity resulted in uniform staining of mitochondrial cristae, as recently demonstrated with an osmiophilic tetrazolium salt. Even more recently we have succeeded in demonstrating cytochrome oxidase activity in nondroplet staining on mitochondrial cristae with an osmiophilic benzidine-type reagent that apparently polymerizes upon oxidation (to be published later).

Biomaterials and biomedical devices.

This review discusses the factors important in the incorporation or integration of biomaterials and devices by tissue. Methods for surface modification and surface-sensitive techniques for analysis are cited. In vitro methods to evaluate the biocompatibility or efficacy of certain biomaterials and devices are presented. Present and future directions in neural prostheses, cardiovascular materials, blood or bone substitutes, controlled drug delivery, orthopedic prostheses, dental materials, artificial organs, plasma- and cytapheresis, and dialysis are discussed.

Neutrophil pseudoplatelets: their discrimination by myeloperoxidase demonstration.

Neutrophils, especially in acute infection or the myeloid leukemias, may shed platelet-sized particles that can readily be distinguished from true platelets because they contain neutrophil myeloperoxidase. This enzyme, unlike platelet peroxidase, is not inhibited by glutaraldehyde. The myeloperoxidase and acid hydrolase levels and continuous plasma membranes of these cell-like particles suggest that they are functional cellular entities. They further differ from platelets in that they contain nuclear remnants, occur in bacteria-laden pus and inflammatory exudates, are ingested by macrophages, and do not adhere to each other or aggregate. They could be involved in the immune response to pathogens or contribute to trauma and healing by facilitating deployment of neutrophil acid hydrolase, neutral protease, and myeloperoxidase.

Phi bodies: peroxidatic particles that produce crystalloidal cellular inclusions.

Unique, spindle-shaped particles (phi bodies) and rods with peroxidatic activity are found in certain epithelial cells of normal mice, clofibrate-fed rats, and in leukemic leukocytes. The ellipsoidal shape of phi bodies apparently results from the deformation of spherical granules by extrusion of axial crystalloid that subsequently fragments into rods.

Osmiophilic polymer generation: catalysis by transition metal compounds in ultrastructural cytochemistry.

A transition metal compound that is bound in tissues by any appropriate cytochemical reaction may catalyze the generation of an insoluble osmiophilic polymer from organic monomers such as 3,3'-diaminobenzidine. When the polymers are treated with osmium tetroxide, electron-opaque, insoluble osmium blacks (coordination polymers of osmium) are formed at the sites of the particular macromolecule or enzyme permitting its light, and electron, microscopic localization. This approach represents a distinct advantage over earlier cytochemical methods because the shorter incubation time needed here results in less artifactual deposition of metal ions, and less tendency to crystallize the reaction product. In addition, the shorter incubation times permit longer fixation of tissues and hence less artifact due to enzyme diffusion.

Coordination polymers of osmium: the nature of osmium black.

The design of cytochemical reagents that yield osmiophilic products from which an osmium black may be derived on exposure to osmium tetroxide has resulted in new methods described previously for the ultrastructural demonstration of enzyme activity and functional groups of macromolecules with the electron microscope. Attempts to determine the nature of the osmium black end products have been frustrated by their insolubility. The preparation of watersoluble analogs and their characterization as polymers suggest that the insoluble osmium blacks are coordination polymers. This is consonant with the unusually favorable properties of osmium black in electron microscopy. Although polymers of osmium have frequently been postulated as the end products of reaction of osmium tetroxide with tissue conistituents or with other organic compounds, this is the first example of their characterization.

Carcinogens 3,4-benzpyrene and 3-methylcholanthrene: induction of mitochondrial oxidative enzymes.

Sections of liver from rats injected with 3,4-benzpyrene and 3-methylcholanthrene, when incubated in mediums specific for the histochemical demonstration of mitochondrial oxidative enzymes, show greater activity of several of these enzymes than do sections from control rats. This observation was confirmed by comparison of the staining of mitochondria isolated from the control and from "induced" rats. The fact that an inhibitor of protein synthesis, actinomycin D, effectively diminished the stimulation provided evidence that the stimulation of activity is due to an increase in enzyme synthesis, generally called induction.

Staining tissue for light and electron microscopy by bridging metals with multidentate ligands.

"Osmium black," a pigment very useful for cytological staining in both light and electron microscopy, may be deposited selectively at the tissue-binding sites of other metal ions by bridging OsO(4) to the tissue bound metal ion through a multidentate ligand.

Facilitated light microscopic cytochemical diagnosis of acute myelogenous leukemia.

Hydroperoxidase-positive Phi bodies and rods are much more prominent and prevalent than rods visualized with a Romanovsky-type stain (Auer rods) in immature leukocytes of patients with active acute myelogenous leukemia (AML). They are readily observed with the light microscope in peripheral blood or marrow films of AML patients stained to show their peroxidatic activity. In many of these patients, Auer rods, which apparently constitute only a small subpopulation of the hydroperoxidase-positive Phi bodies and rods, were detected with difficulty, if at all. The hydroperoxidase-positive Phi bodies and rods were observed in 92% of 36 patients with active disease. They were never observed in leukocytes of patients with other hematopoietic disorders or of normal individuals. Thus, they facilitated the distinction of AML from acute lymphocytic leukemia and chronic granulocytic leukemia in blast crisis. They were absent in full clinical remission after chemotherapy and were greatly diminished in partial remission. They were present in disease relapse and reappeared in five patients who had been in full remission. These results suggest that these hydroperoxidase-positive enlarged particles are pathognomonic of AML and that monitoring them with the light microscope may aid in guiding its clinical management.

Osmiophilic reagents in electronmicroscopic histocytochemistry.

Direct histocytochemical staining methods on undisrupted tissues, stabilized by chemical fixation, potentially offer perhaps the most reliable approach to the study of the enzymes of the cell with relation to its ultrastructure. The atoms which, for the most part, comprise the biomacromolecules and enzymes of cells and tissues contribute little to their inherent electron opacity or ability to scatter electrons differentially. The latter property of a substance is responsible for its observation with the electron microscope. Since the introduction of osmiophilic reagents into cytochemistry (HANKER et al. 1964), the selective deposition of relatively large amounts of polymeric osmium black reaction products at the subcellular sites of insoluble or immobilized enzymes or biomacromolecules has facilitated their demonstration with the light and electron microscopes. Perhaps the most widely employed osmiophilic reagent in histocytochemistry has been DAB which was introduced by GRAHAM and KARNOVSKY (1966a, b). Although it receives its widest use for demonstrating the sites to which the exogenous ultrastructural tracer horseradish peroxidase (HRP) is transported in vertebrate tissues, it is also widely employed for the demonstration of catalase in peroxisomes with the media of FAHIMI (1969) or of NOVIKOFF and GOLDFISCHER (1969), and for the demonstration of cytochrome oxidase with the medium of SELIGMAN et al. (1968a). The importance of this reagent lies in its ability to undergo oxidative polymerization forming an insoluble osmiophilic melanin-like product (HANKER et al. 1972a) which comforms well to ultrastructure, at the sites of enzymic or nonenzyme proteins which catalyze its oxidation. In the past few years, studies in our laboratory have shown that a rational approach to the histocytochemical demonstration of enzymes could be devised. It is based on the selective deposition of transition metal compounds at the sites of enzymes that resemble hemoproteins in their ability to catalyze the oxidative polymerization of DAB. The most useful of these compounds, cupric ferrocyanide (Hatchett's brown) was also introduced into cytochemistry by Karnovsky's laboratory (KARNOVSKY 1964; KARNOVSKY and ROOTS 1974). By the use of natural substrates, when available, or synthetic substrates which liberate or form a reducing agent at the sites of the enzymatic activity, many diverse types of enzymes have been demonstrated by methods depending on this principle known as catalytic osmiophilic polymer generation. DAB has probably been the most useful histocytochemical reagent of the past decade. Yet its borderline carcinogenicity and the frequent interruption of a supply of good quality DAB have encouraged research into a substitute reagent. A new substitute for DAB has resulted from the study of artificial melanins in our laboratory for several years. It consists of a mixture of p-phenylenediamine and pyrocatechol and is much better than DAB for the demonstration of HRP used as a cytochemical tracer...

Experimental studies on the ultrastructural localization of acetylcholinesterase in the mediobasal hypothalamus of the rat.

Acetylcholinesterase (AChE) activity has been demonstrated ultrastructurally in neurons of the arcuate nucleus and associated with fibers in the arcuate nucleus neuropil and the median eminence (ME) of the rat. In addition, the effects of neonatal monosodium glutamate (MSG) treatment and Halász deafferentation on the AChE staining and localization have been studied. Neonatal MSG-treatment resulted in loss of the majority of AChE-positive neurons in the arcuate nucleus while leaving neuropil staining intact. Halász deafferentation caused a loss of arcuate neuropil activity while leaving the neuronal staining unaltered. These observations are consistent with previous biochemical results suggesting the existence of a cholinergic tuberoinfundibular system with nerve cells in the arcuate nucleus and terminals in the median eminence. In addition, the deafferentation experiments indicated that extra-hypothalamic cholinergic fibers may innervate the arcuate nucleus. Supporting evidence from other biochemical studies and the curious paucity of histochemical and biochemical AChE activity in the ME are also discussed.

Evaluation in cats of a new material for cranioplasty: a composite of plaster of Paris and hydroxylapatite.

The materials ordinarily used to reconstruct bone defects in the calvaria and facial bones either are difficult to shape, are partially resorbed by the body, or are likely to become infected if used near a contaminated area such as the frontal sinus. Calcium sulfate hemihydrate (plaster of Paris) has been known for years to have excellent reparative qualities in bone defects, but ordinarily it is quickly resorbed. Consequently, a new material, a composite of a dense form of plaster of Paris and hydroxylapatite, was devised to provide nonabsorbable hydroxylapatite particles for bone to form around and within during the phase of plaster absorption. Two types of this material were evaluated in cranial defects in cats. Each of the plaster of Paris/hydroxylapatite mixtures was placed into a surgically unroofed frontal sinus and into a contralateral parietal trephine hole in a group of 32 cats. Two cats in each group succumbed to anesthesia, leaving two sets of 30 cats. During the entire follow-up period there was only one other death, with no evidence of wound infection, wound dehiscence, implant rejection, or cerebral dysfunction among the survivors. The cats in each group were sacrificed at 1, 2, 3, 5, 7, 8, 9, 10, or 12 months after operation. Following sacrifice, both the frontal and parietal defects were exposed and examined visually, histologically, and with histomorphometric analysis for new bone formation. New bone formation was present as early as 1 month after operation and continued to increase during the 12 months of the study. Based upon these osteogenic qualities, the ease of shaping the composite, and the lack of infection in the frontal sinus region, it is concluded that this substance could be a valuable new material for human cranioplasty.

Silver methenamine staining for scanning electron microscopy of bone sections containing biomaterials.

Sections of tissue containing orthopedic materials are currently used to study the compatibility of those materials and to perform electron probe microanalysis at the material-tissue interface. Identification of the cells in contact with the material by Scanning electron microscopy (SEM) is of interest. We have developed a method for staining cells and tissue structures embedded in polymethyl methacrylate with silver methenamine once the sections have been obtained. Sections were prepared by grinding, and the silver methenamine was applied after oxidation with periodic acid. The procedure was carried out in a microwave oven. Backscatter SEM showed staining of the cell nucleus membrane, chromatin, the nuclear organizers, and the chromosomes of dividing cells. The cytoplasm and the cytoplasmic membrane were also stained. Collagen fibers of the extracellular matrix and the mineralized matrix of bone were labeled. Material particles in the macrophages were easily recognizable and Energy-Dispersive Spectrometer were not impaired by the presence of silver in the preparation.

[Coloration of cytologic thick sections containing biomaterials with silver methenamine. Usefulness for scanning electron microscopy]. / Coloration de coupes histologiques épaisses contenant des biomatériaux par la silver methenamine. Utilité pour l'examen en microscopie électronique à balayage.

UNLABELLED: Prostheses implanted in hard tissues cannot be processed for electron microscopic examination or microanalysis in the same way as those in other tissues. For these reasons, we have developed a method allowing light and electron microscopic studies as well as microanalysis of the interface between bone and biomaterials after the sections glycoproteins have been stained with silver methenamine. Silver can be evidenced by SEM in back scattered mode. MATERIALS AND METHODS: Tranverse sections of a human femur containing an HA-coated prostheses were obtained with a diamond saw and ground to a thickness of 50-100 microns. The sections were stained in a microwave oven using a 1% silver methenamine solution. They have been examined by back-scatter SEM operated at 25 KV. EDS has been performed on cellular inclusions and extracellular bone matrix. RESULTS: Type I and III collagen fibers, and reticulin fibers were stained. The mineralized matrix was heavily colored. At the cell level, the nuclear and cytoplasm membranes, the chromatin and ribosomes were shown. The characteristic peaks of the Ag spectrum are distinct from those of the elements used in orthopaedic biomaterials and did not impair their identification.