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Biomedical subjects

R H Funk

Publications and source records attributed to R H Funk.

At least 37 records · Page 2Linked to original sources
PubMed1999

Ultrastructural identification of caveolae and immunocytochemical as well as biochemical detection of caveolin in chondrocytes.

Using fluorescence immunocytochemistry, transmission electron microscopy and Western blotting, we have shown that caveolae and caveolin are abundant on chondrocytes of different cartilaginous structures of newborn and adult rat knee joints. Caveolin was detected in chondrocytes of the outer layer of articular cartilage, in the fibrocartilage of the menisci, and in fibrocartilage-like cells at tendon and ligament insertions. Electron microscopical studies revealed caveolae-like invaginations along the plasmalemmal membrane of articular chondrocytes and fibrocartilage cells. Immunoblot analysis demonstrated caveolin in detergent-insoluble and soluble complexes isolated from cultured rat chondrocytes.

Age Factors↗
PubMed1999

Microvasculature of the rat optic nerve head.

PURPOSE: To describe the arterial blood supply, capillary bed, and venous drainage of the rat optic nerve head. METHODS: Ocular microvascular castings from 6 Wistar rats were prepared by injection of epoxy resin through the common carotid arteries. After polymerization, tissues were digested with 6 M KOH, and the castings washed, dried, and coated for scanning electron microscopy. RESULTS: Immediately posterior to the globe, the ophthalmic artery trifurcates into the central retinal artery and two posterior ciliary arteries. The central retinal artery directly provides capillaries to the nerve fiber layer and only contributes to capillary beds in the neck of the nerve head. The remainder is supplied by branches of the posterior ciliary arteries that are analogous to the primate circle of Zinn-Haller. Arterioles arising from these branches supply the capillaries of the transitional, or laminar, region of the optic nerve head. These capillaries are continuous with those of the neck and retrobulbar optic nerve head. All optic nerve head capillaries drain into the central retinal vein and veins of the optic nerve sheath. A flat choroidal sinus communicates with the central retinal vein, the choriocapillaris, and with large veins of the optic nerve sheath. CONCLUSIONS: The microvasculature of the rat optic nerve head bears several similarities to that of the primate, with a centripetal blood supply from posterior ciliary arteries and drainage into the central retinal and optic nerve sheath veins. Association of nerve sheath veins with the choroid represents an important difference from the primate.

Animals↗
PubMed1999

Confocal laser scanning microscopy of leukocyte adhesion in the microcirculation of the inflamed rat knee joint capsule.

OBJECTIVE: The aim of the study was to develop a model of intravital microscopy of the microcirculation of the rat knee joint capsule in acute inflammation with the help of confocal laser scanning microscopy (CLSM). METHODS: The microvascular architecture of the joint capsule was investigated by the use of corrosion casting techniques. Knee joint capsule microcirculation of anesthetized rats could be visualized by dissection of the ligamentum infrapatellare rectum and application of a CLSM. Rhodamine 6G was used as a leukocyte marker. An acute arthritis was induced by intraarticular injection of 0.1 mL of 1% Carrageenan solution. Each experiment lasted for 3 h. Leukocyte adhesion (LA) was measured in 100 microns of vessel length. Leukocyte-rolling velocity (Vwbc), systemic leukocyte count (WBC), and differential blood count (DBC) were monitored in defined intervals. Finally, myeloperoxidase (MPO) activity in frozen-tissue homogenate served as a parameter of neutrophil content. RESULTS: Leukocyte adhesion in carrageenan-treated animals was 9.9 +/- 0.2 leukocytes/100 microns vessel (mean +/- SD; n = 6), while sham- treated animals had 2.8 +/- 0.2. Monoclonal antibodies (MAb) to alpha 4 integrin (0.2 mg/kg) reduced LA to 2.6 +/- 0.1 (p < 0.01). Vwbc/gamma quotients were 0.37 micron s/s in control animals and 0.53 micron s/s in anti-alpha 4-treated animals (p < 0.01). Changes in DBC were marked by lymphopenia and granulocytosis after 180 min. At this time point-control animals had 5.1 G/L LC and 2.6 G/I PMN. Animals treated with unspecific antibody had 4.7 G/L LC and 4.9 G/L PMN, while anti-alpha 4-treated animals had 10.6 G/L LC and 2.4 G/L PMN. Photometrically determined extinction of oxidized tetramethylbenzidine (TMB; measure for MPO content) was 1.058 +/- 0.555 in control animals and 0.245 +/- 0.093 in sham-treated animals. Tissue homogenate from unspecific IgG treated group had 0.776 +/- 0.140 and from anti-alpha 4-group 0.334 +/- 0.155 (p < 0.05). CONCLUSIONS: Confocal laser scanning microscopy is a tool for the study of the microcirculation in nontransparent organs. The microcirculation of the acutely inflamed rat knee joint capsule can serve as a model of integrin-mediated LA in vivo. Antiadhesive treatment of animals can reduce the tissue infiltration with inflammatory cells.

Animals↗
PubMed1998

In situ observation of living pericytes in rat retinal capillaries.

We observed the retinal capillary pericytes of the rat in situ. Whole retinae were mounted, immediately post vivo, in a special tissue chamber for electronic light microscopy at high magnifications. Under electronic light microscopy the pericytes could be clearly distinguished from the endothelial cells. In addition, the contractile apparatus of the pericytes was demonstrated by immunohistochemistry with alpha-smooth muscle actin. Administration of angiotensin II as well as endothelin into the observation chamber caused a significant decrease of the mean capillary diameter (13 and 16% reduction, respectively) within 90 s. Carbachol, bradykinin, and histamine significantly increased the capillary diameter within 90 s (13, 20, and 18% increase, respectively). This study demonstrates that our method allows the analysis of vasoactive effects on the retinal capillary in situ. We observed that this type of capillary can actively change its diameter.

Actins↗
PubMed1998

Hints of a functional connection between the neuropeptidergic innervation of arteriovenous anastomoses and the appearance of epithelioid cells in the rabbit ear.

Peripheral blood flow can be regulated by specialized vessel segments, the arteriovenous anastomoses. Their wall consists of a relatively thick layer of smooth muscle cells and so-called epithelioid cells. The epithelioid cell is a specialized myogenic cell phenotype expressing nitric oxide synthase. We studied the innervation of the different segments of arteriovenous anastomoses in the rabbit ear using antisera against neuropeptide Y, tyrosine hydroxylase, calcitonin gene-related peptide and substance P, as well as neuron-specific enolase, calbindin D and neurotubulin. The participation was especially examined of neuropeptidergic innervation and a possible morphological connection to the occurrence of epithelioid cells and a paracrine function. The NADPH diaphorase reaction and alpha-smooth muscle actin immunoelectron microscopy served to distinguish epithelioid cells from smooth muscle cells. Using conventional fluorescence microscopy and confocal laser scanning microscopy, we found the most dense innervation pattern of pan-neuronal markers (neurotubulin, neuron-specific enolase), tyrosine hydroxylase-immunoreactive nerve fibres and neuropeptidergic nerve fibres (neuropeptide Y, calcitonin gene-related peptide, substance P) around the intermediate segment in arteriovenous anastomoses, whereas the venous segment was barely marked. Single nerve fibres penetrated into the medial layer and reached the epithelioid cells. Using immunoelectron microscopy, we found intercellular contacts between epithelioid cells, but not the gap junction protein connexin 43. Here, we report for the first time a correlation of the innervation pattern with epithelioid cell type in arteriovenous anastomoses. Our findings suggest that epithelioid cells of the arteriovenous anastomoses are controlled by a dense network of neuropeptidergic nerve fibres in functional connection to their paracrine role as a nitric oxide producer.

Animals↗
PubMed1998

Electronic light microscopy combined with spectrophotometry allows real-time analysis of structures at the subcellular level.

A combination of microscopy (video-enhanced contrast microscopy) and spectrophotometry (which analyses online the spectra from the microscopic picture) is presented. This set-up allows the in vivo microscopic observation of cellular and subcellular structures as well as simultaneous online determination of cellular chromophores. To test our equipment we measured spectra of isolated red blood cells and NG 108-15 cells and correlated these spectra with the corresponding video-enhanced microscopic picture. By labelling specific cell organelles of astrocytes with in vivo fluorescent dyes, we visualized mitochondria and captured the corresponding spectra.

Animals↗
PubMed1998

Innervation pattern of different cartilaginous tissues in the rat.

BACKGROUND: The innervation of skeletal tissues by sensory nerves is poorly understood - especially of nerve fibres which reach into the bony and cartilaginous tissue. METHODS: Samples of rat cartilaginous tissues from different locations (knee joint, vertebral column, temporomandibular joint) were fixed by perfusion and decalcified. The distribution of protein gene product (PGP) 9.5-, calcitonin gene-related peptide (CGRP)- and tachykinin (TK)-immunoreactive axons was analysed using fluorescence immunohistochemistry. RESULTS: Nerve fibres were detected in the outer regions of the hyaline cartilage of the knee joint, in the hyaline cartilage of the vertebral body, in the fibrocartilage of the intervertebral disc and menisci, and in the articular disc of the temporomandibular joint. Predominantly, they were found to be CGRP-immunoreactive. CONCLUSION: The neuropeptidergic innervation of the hyaline cartilage in different locations and the presence of nerve fibres in the fibrocartilage might indicate that in addition to the classical neuronal afferent and efferent pathway these fibres may also mediate trophic actions like tissue adaptation and repair.

Animals↗
PubMed1998

Arteriovenous anastomoses of the episcleral vasculature in the rabbit and rat eye.

PURPOSE: To obtain more information about the functional significance of arteriovenous anastomoses as regulatory elements, the authors investigated the topography and innervation of episcleral anastomoses in the rat and rabbit. MATERIALS AND METHODS: The topography of arteriovenous anastomoses in the limbal and episcleral vasculature of the rat and rabbit eye was studied by scanning electron microscopy of vascular resin casts. The perivascular distribution of the following neuropeptides was investigated by fluorescence immunohistochemistry: neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), and calcitonin gene-related peptide (CGRP). RESULTS: The episcleral arteriovenous anastomoses connect arterioles directly with the episcleral venous plexus which also drains the aqueous humor. The nerve fiber plexus around the episcleral arteriovenous anastomoses is far more dense than around arteriovenous connections at the limbal arcades. NPY-, VIP-, SP-, and CGRP-immunoreactive fibers concentrate at the arteriolar segment of the episcleral arteriovenous anastomoses and are reduced at the venular segment. Still, numerous VIP-IR fibers are on the venous side of the rabbit episcleral anastomoses. CONCLUSION: We conclude that the elaborated innervation of the episcleral anastomoses is a prerequisite for a subtle modulation of the blood flow and possibly of the aqueous humor outflow dynamics.

Animals↗
PubMed1997

Microtopography of the autonomic nerves in the rat knee: a fluorescence microscopic study.

BACKGROUND: The autonomic innervation of the joint is involved in different functions, such as sensory inputs, modulation of the function of immune cells, and trophic actions. To have a basis for further studies of the arthritic knee joint we have investigated the topographical distribution of different neuropeptides in knees of newborn and adult rats and in adult rats after arthritis induction. METHODS: The distribution of the neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), substance P (SP), and neuropeptide Y (NPY) was analyzed using fluorescence immunohistochemistry. Samples were investigated after fixation by perfusion and decalcification by a special method which allows studies in bone tissue. Vascular structures were analyzed by scanning electron microscopy (SEM) of vascular resin casts. RESULTS: In all tissues of the joint (synovial membrane, vessels, fibrous structures, bone, and cartilagineous tissues) CGRP and NKA are the most frequent neuropeptides. They are localized in free or perivascular fibers predominantly around arteries and arterioles. The NPY-ergic perivascular fibers even enter the vessel wall. Generally, SP-ergic fibers occur rarely. Free NKA- and CGRP-ergic nerve fibers spread out in the synovial lining layer reaching the synovial cavity and the outer layers of the articular and metaphyseal cartilage. In the cartilagineous tissue these nerves contact the chondrocytes. The density of NKA- and CGRP-immunoreactive fibers is lower in newborn rats than in adult rats. Six hours after arthritis induction SP-, NKA-, and CGRP-immunoreactivity is enhanced especially in perivascular fibers. The related vessels are dilated substantially. CONCLUSIONS: The distribution pattern of the autonomic nerves found in this study might reflect the functions of these nerves: control of the microcirculation, sensory and even trophic functions. The new finding of CGRP- and NKA-ergic fibers in the outer layer of the cartilage can also have implications for the pathogenesis of rheumatoid arthritis.

Animals↗
PubMed1997

Cytoskeletal characterization of arteriovenous epithelioid cells.

Data on the cytoskeleton of epithelioid cells in arteriovenous anastomosis (AVA) are sparse, but there is evidence that the (myo)-epithelioid cells of the AVAs represent a specialized smooth muscle cell type with less contractile properties. We demonstrated the expression of alpha-smooth muscle actin, smooth muscle myosin, calponin, caldesmon, and caveolin in epithelioid cells of rabbit ear and in human toes, finger tips, and glomus tumors by means of indirect immunofluorescence techniques and immunoelectron microscopy. Epithelioid cells in rabbit ear did not express vimentin, but it was present in human toes, finger tips, and glomus tumors. Epithelioid cells in human toes, finger tips, and glomus tumors did not express desmin, but it was present in rabbit ear. Epithelioid cells did not express cytokeratins. The epithelioid cells examined showed only a weak expression of the protein smoothelin, which occurs exclusively in contractile smooth muscle cells. Immunoelectron microscopical demonstration of (alpha-smooth muscle actin revealed a striking difference in the arrangement of actin filaments in the epithelioid cells as compared to that in the smooth muscle cells of blood vessels. The epithelioid cells contained a loose array of actin filaments, whereas the smooth muscle cells contained tightly packed parallel actin bundles. In the present study we observed a correlation between the lack of contractile marker protein expression in epithelioid cells and the presence of only a few filaments, although the epithelioid cells are alpha-smooth muscle actin positive. The reduced number of contractile elements in the epithelioid cells of rabbit and human anastomoses suggests a lower contractility of epithelioid cells compared to that of the surrounding smooth muscle cells in anastomoses. A second interesting difference between both cell types is the high number of caveolae in epithelioid cells. Immunoelectron microscopy showed a compact distribution of caveolae at the epithelioid cell border, but a more dispersed distribution of caveolae in the cytoplasm of the blood vessel endothelium. The benign glomus tumor was characterized by an expression pattern of cytoskeletal proteins similar to that of epithelioid cells, confirming its description as a benign tumor.

Animals↗
PubMed1997

Immunohistochemical detection of human skin nerve fibers.

The autonomic nervous system is involved in different functions such as transduction of afferent sensory inputs, trophic actions, modulation of immunologic events and thermoregulation. In the present investigation, we studied the pattern of human autonomic skin innervation with special reference to its relation to blood vessels, hair follicles, sweat glands and sensory receptors. For the first time, two clinically important areas have been compared: the skin of the forearm and of the face. Using indirect immunohistochemistry, we analyzed the distribution of calretinin (CR), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP), neurokinin A (NKA), vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), tyrosine hydroxylase (TH), histamine, serotonin, enkephalin, and, enzyme histochemically, NADPH-diaphorase (NADPH-d). In the epidermis, we found nerve fibers containing SP, NKA and CGRP. In the dermis, SP-, CR-, VIP-, CGRP- and NKA-positive nerve fibers were detected. Particularly the large nerve fibers contained CR. VIP-positive fibers occurred especially around hair follicles and sweat glands. CGRP-positive nerve fibers were located close to the epidermal basal membrane, in the wall of blood vessels, and to a lesser extent around hair follicles. Immunoreactivity for SP and NKA in the dermis was observed predominantly in the papillary layer near the epidermal basal membrane. All neuropeptides tested in this study were also detected in the nerve fibers of the subcutis. Most of them were CGRP- and VIP-positive. They occurred in association with sweat glands and large arteries. NPY-positive nerve fibers are predominant in the wall of arteries, arterioles and veins. Nerve fibers containing NKA and SP were less common and identified only in the walls of large arteries in deeper dermal layers. In double-staining experiments, the NADPH-d reaction and reactivity to tubulin revealed a partial co-localization in nerve fibers, blood vessel walls, around glands and ganglionic cells. VIP-positive fibers were more common in the face skin than in the forearm. However, in forearm we detected more NPY-, CGRP-, NKA- and SP-positive nerve fibers than in face skin. These findings are important for future studies on skin disorders, such as sensory neuropathies, inflammatory reactions or allergic responses of human skin.

Blood Vessels↗
PubMed1997

Blood supply of the retina.

An overview of the functional anatomy of the retinal microcirculatory system and its regulation mechanisms is presented. The retinal microvasculature is characterized by thin capillaries which leave large vessel-free spaces compared to other microvascular beds. Despite high blood flow velocities, the blood flow volume within the capillaries is relatively low. This results in a high arteriovenous pO2 difference and a small capacity to tolerate periods of low perfusion. Furthermore, from the optic nerve head on there is no autonomic perivascular innervation to control the microvascular tone. A control via the bloodstream (mediators, e.g. O2, CO2, hormones), astrocytes, neurites and Müller cells (mediators, e.g. NO, prostaglandins, neuropeptides) takes over. Finally, the role of pericytes in the control of retinal hemodynamics is discussed.

Animals↗
PubMed1996

Scanning electron microscopic study of episcleral arteriovenous anastomoses in the owl and cynomolgus monkey.

In the present study we have analyzed the architecture of the episcleral microvasculature in the owl and cynomolgus monkey using scanning electron microscopy of resin casts. Due to the topical pretreatment with nitroprusside the complete vasculature including segments of the aqueous humor outflow channels could be visualized. We found that 1-3 mm posterior to the limbus corneae the episcleral vessels form numerous arteriovenous anastomoses (AVA) in the size of small arterioles and venules. These AVA are also located at the collector channels near Schlemm's canal and at the episcleral venous plexus which drains the collector channels. It is assumed that the AVA might influence not only the circulation in the episcleral venous plexus but also in the aqueous humor outflow routes.

Animals↗
PubMed1996

Short-term hemodynamic changes in episcleral arteriovenous anastomoses correlate with venous pressure and IOP changes in the albino rabbit.

In the present study we have determined the effects of acute diameter changes in the recently discovered episcleral arteriovenous anastomoses (AVA) on episcleral venous pressure and intraocular pressure (IOP) in the rabbit eye. The pressure was measured in episcleral arterioles, AVA and veins (vascular pressure) with a pressure chamber mounted on the tip of a microendoscope. After constriction of the AVA following topical administration (100 micrograms) of epinephrine we observed a decrease in the episcleral vascular pressure as well as in the IOP. Acute widening of the AVA after topical administration of 5mg nitroprusside led to a significant increase in the pressure of arterioles, AVA, veins and IOP which lasted several min. After 0.5mg topical nitroprusside the blood flow in the AVA increased. No significant effects were seen in the intravascular pressures; the IOP had a tendency to decrease. Experimental lowering of the IOP to 10mmHg caused an increase of vascular diameters and of blood flow in the AVA. It is assumed that-at least in drastic hemodynamic disorders-the degree of the AVA-perfusion might influence aqueous humor outflow and IOP due to changes of the episcleral venous pressure.

Administration, Topical↗