Endogenous Peroxidatic Activity Research Articles

Electron-dense tracer evidence for a blood-brain barrier in the cuttlefish Sepia officinalis.

Electron-dense tracers were used to study the permeability of the blood-brain interface in a cephalopod mollusc, the cuttlefish Sepia officinalis. Gel filtration established that horseradish peroxidase is a suitable tracer for in vivo injection, but microperoxidase is not, being subject to binding by plasma proteins. Perfusion-fixed brain vertical and optic lobes showed no endogenous peroxidatic activity. Horseradish peroxidase was injected intravenously, and allowed to circulate for 10-35 min before tissue fixation by immersion or perfusion. Horseradish peroxidase reaction product was undetectable in the bulk of the brain parenchyma. In microvessels, venous vessels and at the brain surface, horseradish peroxidase penetrated the layers of endothelial and pericyte cells, being stopped by the layer of perivascular glia. In arterial vessels, tracer restriction occurred at the level of the pericytes. In the region of tracer blockade, a gradient of tracer could be traced in the intercellular cleft, from high at the luminal end to undetectable at the tissue end. The clefts of the restricting zone were generally wide (15-20 nm), with faint periodicities or linking structures spanning the cleft, and contained a fibrillar extracellular material. Perfusion of lanthanum chloride in saline for 15 min, followed by precipitation of lanthanum phosphate during fixation, resulted in lanthanum tracer distribution similar to that of horseradish peroxidase. Horseradish peroxidase was seen filling extracellular spaces within the neuropile when the blood-brain barrier was breached by a stab wound, indicating that the interstitium itself does not restrict tracer diffusion. It is concluded that Sepia has a blood-brain barrier tight to horseradish peroxidase and ionic lanthanum. The restricting junction is not a typical zonula occludens or septate junction, but appears to reduce tracer penetration by a filtering mechanism within the extracellular cleft. The barrier is formed by perivascular glial cell processes in the microvessels and venous vessels, but by pericytes in arterial vessels. This organization suggests that a glial blood-brain barrier may be the primitive condition, and a barrier associated with vascular elements (endothelium/pericyte) a later development.

Endogenous peroxidatic activity in astrocytes after spinal cord injury

After spinal cord injury, endogenous peroxidatic-like activity develops along the axis of the cord. At 2 weeks postinjury, this activity appears in cells whose processes are intimately associated with microvessels. The objectives of this study were to further characterize this response and to identify the cellular localization of endogenous peroxidatic-like activity. After traumatic injury to the rat spinal cord, adjacent sections of spinal cord were processed in medium to visualize antiglial fibrillary acidic protein, endogenous peroxidatic activity, or cytochrome oxidase activity. In addition, certain sections, stained for endogenous peroxidatic-like activity, were prepared for electron microscopy. To identify the nature of the activity, some sections were exposed to an incubation medium that included inhibitors of either catalase or heme protein activity. The distribution of prominent glial fibrillary acidic protein immunoreactivity in the dorsal columns corresponded to that of marked staining for endogenous peroxidatic-like activity and cytochrome oxidase. At the ultrastructural level, endogenous peroxidatic-like activity was identified in the cytoplasmic compartment of the astrocyte. This activity was abolished when potassium cyanide (an inhibitor of heme protein) was added to the incubation medium. Spinal cord injury elicited a pronounced cellular response along the axis of the cord that was characterized by enhanced staining for antiglial fibrillary acidic protein, cytochrome oxidase activity, and endogenous peroxidatic-like activity. It is not clear whether pronounced cytochrome oxidase activity corresponded to astrocytes that also expressed prominent endogenous peroxidatic-like activity. However, according to both light and ultrastructural findings, endogenous peroxidatic-like activity was prominently associated with the astrocytic cytoplasm. The biochemical nature of the peroxidatic activity is unknown, but these results suggest that it is related to a heme-containing protein.

In human macrophages the development of the endogenous peroxidatic activity pattern coincides with the expression of the differentiation antigen RFD7: An ultrastructural immunocytochemical study

In human macrophages the development of the endogenous peroxidatic activity pattern coincides with the expression of the differentiation antigen RFD7: An ultrastructural immunocytochemical study

An improved method for the inhibition of endogenous peroxidase non-deleterious to lymphocyte surface markers. Application to immunoperoxidase studies on eosinophil-rich tissue preparations.

The inhibitory effect of phenylhydrazine and azide combined with either pre-formed or nascent hydrogen peroxide H2O2 upon endogenous peroxidatic activity, expressed by tissue eosinophils in different disease states, was investigated. It was found that whilst endogenous peroxidatic activity due to eosinophils in a Hodgkin's disease and a histiocytosis X case were adequately inhibited by phenylhydrazine combines with pre-formed or nascent H2O2, the eosinophils in the Onchocerca volvulus nodule were either not at all or only partly inhibited by the two regimens. On the other hand, a combination of azide with nascent H2O2 proved consistently effective against this resistant form of endogenous peroxidatic activity. Using human tonsil sections this protocol was shown to be non-deleterious to T4('CD4'), T6('CD1') and T8('CD8') lymphocyte surface antigens as evidenced by the application of a standard indirect immunoperoxidase technique and the relevant monoclonal antibodies.

The development of the resident pattern of endogenous peroxidatic activity in mouse peritoneal macrophages coincides with the expression of the differentiation antigen F4/80. A combined method for immunoperoxidase labeling and endogenous peroxidase cytochemistry.

In the mouse the maturation of mononuclear phagocytes was followed by comparing the ultrastructural pattern of endogenous peroxidatic activity (PA) at different time points during an acute peritonitis induced with newborn calf serum (NCS). Exudate macrophages demonstrate PA only in lysosomes, whereas resident macrophages have reaction product in the nuclear envelope (NE) and rough endoplasmic reticulum (RER). Transitional cells called "exudate-resident" macrophages have PA in the NE, RER, and some virginal lysosomes. In addition, peroxidase-negative macrophages were also present. A monoclonal antibody, F4/80, that specifically recognizes a mouse macrophage differentiation antigen (Austyn and Gordon, 1981) was used in this study. To compare the indirect immunoperoxidase labeling of this antigen and the endogenous peroxidase cytochemistry on the cellular level, a combined method was developed. Finally, the method was applied to the peritoneal cells at different time points after intraperitoneal injection of NCS in mice. The relative numbers of cells demonstrating the different patterns of endogenous PA and the proportions of each subpopulation expressing F4/80 antigen were estimated. It appeared that the expression of the antigen F4/80 coincides with the development of the resident pattern of PA. It is therefore concluded that the macrophages with the resident pattern of endogenous peroxidase are derived from monocyte-like exudate macrophages. In addition, the results indicate that both exudate-resident macrophages and at least a part of the peroxidase-negative macrophages are transitional forms.

The accumulation of [35S]methimazole by monocytes and macrophages.

We have used an automatic cell harvester and micro culture techniques to examine the accumulation of [35S]methimazole by monocytes, macrophages and lymphocytes. Significant temperature-dependent accumulation of the drug was found in resting monocytes and macrophages; this was increased up to 4-fold by phagocytosis. Lymphocytes accumulated little or no drug and myeloma and leukaemic cell lines accumulated none. These results show that two interrelated cells with endogenous peroxidatic activity take up the antithyroid drug methimazole providing further support for the concept that immunosuppression by this drug in Graves' disease is mediated via an action on antigen-presenting cells.

Peroxidase-positive endothelial cells in rat liver.

Rat liver fixed by perfusion with low glutaraldehyde concentrations was incubated in diaminobenzidine-containing medium to stain for peroxidase. Endogenous peroxidatic activity was found not only in Kupffer cells but also in the endothelial cells lining the sinusoids and central veins. The reaction product was localized in the nuclear envelope and endoplasmic reticulum. The peroxidatic activity in endothelial cells showed a concentration-dependent sensitivity to glutaraldehyde: in liver samples fixed with 0.25% glutaraldehyde, approx. 23% of the sinusoidal endothelial cells and 65% of central vein endothelium were peroxidase-positive; with 0.5% glutaraldehyde, only approx. 8% of the sinusoidal endothelial cells contained detectable amounts of the reaction product; with 1.5% glutaraldehyde all endothelial cells were consistently peroxidase-negative. No peroxidatic activity could be found in liver endothelial cells following isolation by centrifugal elutriation. Endothelial cell peroxidase may possibly be involved in defense responses of liver and/or, as a part of prostaglandin synthase system, in prostanoid production.

Non-lymphoid cells from rat intestinal lymph.

Peripheral lymph of all mammalian species examined contains a subpopulation of nonlymphoid cells (NLC) absent from central lymph (1). These cells have often been considered to be mononuclear phagocytes on the basis of their strikingly irregular surface morphology. In the past, investigation of the properties of these cells has been restricted to large experimental animals in which direct cannulation of peripheral lymphatics is feasible (2,3). The lack of inbred strains of these animals has hampered attempts to analyze functional properties of NLC. However, it has been found that following mesenteric lymphadenectomy in the rat NLC appear in thoracic duct lymph. These NLC show marked heterogeneity with regard to their surface morphology as visualized by scanning electron microscopy, some being veiled while others have fine or blunt pseudopodia. The cells also vary in the amount of nonspecific esterase activity and endogenous peroxidatic activity they display (4). These cells are derived locally from intestinal lymph draining both Peyer’s patch and non-Peyer’s patch areas (4). In this paper we show that they are of bone marrow origin and turnover rapidly.KeywordsStimulatory CapacityMixed Leukocyte ReactionIntestinal LymphThoracic Duct LymphPeripheral White Blood Cell CountThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Ultrastructural demonstration of endogenous peroxidatic activity in mammalian arterial wall

Endogenous peroxidatic activity has been demonstrated at the ultrastructural level in large arteries of rabbit and rat using diaminobenzidine. The reaction was positive in endothelial cells of both species and also in the smooth muscle cells of rat arteries. The reaction product was localized in the nuclear envelope and endoplasmic reticulum of the reactive cells. Since the enzymatic activity was extremely sensitive to fixation, best visualization was obtained in unfixed, directly incubated tissues in which additional mitochondrial staining occurred due to the activity of endogenous cytochrome c/cytochrome oxidase system. The peroxidatic activity was partially sensitive to cyanide and could be completely abolished by azide and aminotriazole. It has been suggested that the observed endogenous peroxidatic activity of the arterial wall components reflects the activity of prostaglandin endoperoxide synthetase and, indirectly, production of prostacyclin (PGI2).

Endogenous peroxidatic activity in the cerebral and cerebellar cortex of normal adult rats.

There are at least three sources of endogenous peroxidatic activity that are present in both the cerebral and the cerebellar cortex. All three sources, being neuronal, astrocytic or hemoglobin-dependent, may obscure the interpretation of studies using exogenous horseradish peroxidase. The activities are to a variable extent influenced by fixation. From the present results DAB-incubation of cryostate sectioned brain appears to be an acceptable method for histochemical demonstration of the mitochondrial cytochrome chain enzymes.

Difficulties involved in diaminobenzidine histochemistry of endogenous peroxidase

Basically peroxidase (PO) histochemistry is used in different areas: haematologic physiopathology, estrogen-dependent genito-mammary physiopathology and immunoperoxidase histochemistry, both in electron microscopic cytochemistry and at the light microscopic level. In immunochemistry, endogenous peroxidatic activity is inhibited by means of various procedures. On the contrary, in haematology and in reproduction biology stainability of endogenous PO is mandatory; controls are achieved using different inhibitors. Literature data on the most appropriated technical conditions concerning fixation, processing and incubation, for PO staining are reviewed.

The distribution of myelin basic protein in central nervous system lesions of multiple sclerosis and acute experimental allergic encephalomyelitis.

To obtain more information about early events in central nervous system myelin injury in multiple sclerosis (MS), a comparative study was conducted of the distribution of myelin basic protein (BP) in tissue lesions of both MS and experimental allergic encephalomyelitis (EAE). Sixteen postmortem specimens containing lesions from 4 persons with MS and the brains of 14 guinea pigs with acute EAE induced with BP were studied. Cryostat sections of quick-frozen material were fixed, treated with rabbit antibody to BP, and processed by immunoperoxidase techniques. The reaction of antibody to BP was reduced or absent in MS lesions but normal in uninvolved surrounding tissue. Alterations in BP generally paralleled changes in staining of myelin by histological methods. Marked diminution of reactivity with anti-BP occurred in early lesions of MS and extended far beyond any identifiable inflammatory elements. In both parenchyma and perivascular areas, lipid-laden macrophages in MS plaques frequently contained BP material in addition to a variable amount of endogenous peroxidatic activity. Compared with MS, BP was relatively well preserved in brains of guinea pigs with EAE. At the light microscopy level, normal-appearing patterns of BP existed adjacent to the perivascular cellular infiltrates, and macrophages containing BP material were rare. The results of this study suggest differences between MS and acute EAE in both removal of BP from the central nervous system and its subsequent disposal.

Endogenous peroxidatic activity in brain stem neurons as demonstrated by their staining with diaminobenzidine in normal squirrel monkeys

In order to test for the presence or absence of endogenous peroxidatic activities within the brains of normal squirrel monkeys, sections from the brain stems were incubated in buffered media of diaminobenzidine (DAB) and hydrogen peroxide, according to the methods of Graham and Karnovsky 20 or Novikooff and Goldfischer 39. It was found that neurons within certain regions were consistently stained both media. These reactive neurons belong to the extrapyramidal motor system (globus pallidus, substantia nigra (pars diffusa), red nucleus, pontine and mesencephalic reticular formation), to certain cranial nerve nuclei (oculomotor, trochlear, abducens, mesencephalic trigeminal, vestibular) and to relay nuclei of the auditory pathway (superior olivary nucleus and nucleus of the trapezoid body). Neurons within other regions of the brain stem, such as thalamus, hypothalamus and corpus striatum were not stained. On the other hand, the neuropil, excluding the neurons, in other motor areas (such as the subthalamic nucleus, inferior olivary nucleus and corpus striatum) and other auditory relay nuclei (inferior colliculus, nucleus of lateral lemniscus) were also significantly stained. Processes around the Purkinje cells of the cerebellum were particularly reactive. Under the electron microscope, reaction products were localized prominently along the cristae and limiting membranes of the mitochondria and within peroxisome-like bodies of the staiined neurons. Non-reactive neurons have little or no reaction products in their organelles. The staining was absent when DAB was withheld, and it was weak or absent if H 2O 2 was omitted from the media. The addition of inhibitors of catalase, 3-amino-1, 2,4-triazole and 2,6-dichlorophenolindophenol did not affect the staining intensity, whereas the addition of potassium cyanide, a strong inhibitor of metalloenzymes, especially heme-enzymes, almost completely abolished the reactions. In addition, a 3-day ‘bleaching treatment’ with 30% H 2O 2 did not affect the reactions. These results suggest that the reactivity in the above regions was due not to neuromelanin or other pigments, but rather to heme-enzymes and/or other hemoproteins. It was postulated that a higher concentration of the reactive agents was characteristic of certain neuronal types in the brain stem of the squirrel monkeys, and could be related to the cells' functional needs, energy requirements and perhaps to the metabolism of their specific neurotransmitters.

Effects of allylisopropylacetamide (AIA) on the fine structure and peroxidase activity of microbodies in rat hepatic cells.

Multiple intraperitoneal injections of AIA in rats cause a decrease in matrix material and loss of endogenous peroxidatic activity in hepatic cell microbodies. These changes are detectable as early as 48 hours after commencing injections and are prominent by 5 days. On cessation of injections, recovery of normal microbody morphology is rapid but peroxidatic activity of microbodies returns more slowly. There is no evidence of increased destruction of microbodies during the recovery period. These observations support the concept that microbody constituents may be able to exchange independently of turnover of the entire organelle.