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ABSTRACT: The aim of the current study was to examine the cartilage-specific binding property of polyarginine peptides (R4, 8, 12, and 16) and specifically to test octaarginine peptides for the optical imaging of articular cartilage in experimentally induced arthritis in mice.
Four rhodamine-labeled polyarginine peptides each with a different-length arginine chain (R4, 8, 12, or 16) were injected into the knee joints of C57BL/6J mice (n=20). The joints were excised 1h later and the fluorescent signal intensity in cartilage cryosections was compared for the four peptides. To examine the substrate of R8 in cartilage, femoral condyles obtained from another set of mice were treated with chondroitinase ABC (Ch'ase ABC), keratanase or heparitinase then immersed in R8-rhodamine. Fluorescent signals were examined by fluorescent microscopy. Next, R8-rhodamine was injected into the right knee joints of three control and three collagen antibody-induced arthritis (CAIA) mice, and fluorescent intensity in normal and degenerative cartilage was semi-quantitatively analysed on the histological sections using image software. Finally, femoral condyles from normal mice (n=2) and CAIA mice (n=2) were immersed in R8-rhodamine and calcein, then imaged using optical projection tomography (OPT).
Fluorescent signals were specifically detected in the cartilage pericellular matrix from the surface to the tide mark but were completely absent in the calcified layer or bone marrow. The number of arginine residues significantly influenced peptide accumulation in articular cartilage, with R8 accumulating the most. The fluorescent signal in the femoral condylar cartilage diminished when it was treated with Ch'ase ABC. R8 accumulation was significantly decreased in the degenerative cartilage of CAIA mice, and this was demonstrated both histologically and in three-dimensional (3D)-reconstruction image by OPT.
R8 may be a useful new experimental probe for optical imaging of normal and arthritic articular cartilage.
Osteoarthritis and Cartilage 04/2009; 17(9):1209-18. · 3.90 Impact Factor
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ABSTRACT: One hundred and twenty years ago, Camillo Golgi described reticular or fenestrated sheaths of extracellular matrix (ECM), enwrapping the cell bodies, axon initial segments and proximal dendrites of certain pyramidal and non-pyramidal neurons in the adult mammalian central nervous system. Such structures, currently termed perineuronal nets (PNs), are preferentially associated with GABAergic, parvalbumin-containing fast-spiking inhibitory types of cells, and mostly expressing the Kv3.1 subunit of voltage-gated potassium channels. Although some neuroanatomists validated the existence of the PNs on selected neurons in the adult brain, there was a lack of agreement concerning their origin, composition and function. Recent data suggest that PNs result from the visualization of ECM molecules that are confined to the space interposed between glial processes and the nerve cells that they outline. The substance confined to these spaces could be visualized selectively by antibodies directed to glycoproteins, proteoglycans, markers for hyaluronan, some plant lectins recognizing N-acetylgalactosamine or by monoclonal antibodies directed to epitopes on unknown molecules. The PNs appear only after birth, which they could serve as recognition molecules between certain neurons and their surrounding cells. Other putative roles include stabilization of synapses, maintenance of cellular relationships in the adult brain, concentration of growth factors around certain neurons, generation of a polyanionic ion-buffering microenvironment, as well as prevention of extracellular-space occlusion and the formation of certain link with the intracellular cytoskeleton.
Anantomia Histologia Embryologia 11/2005; 34(s1):45 - 45. · 0.90 Impact Factor
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ABSTRACT: The basal lamina of high endothelial venules (HEVs) in human palatine tonsils was intensely stained with cationic colloidal iron at pH 1.5 and with aldehyde fuchsin. This basal lamina exhibited a thick and double- or triple-layered structure forming small compartments, in which many lymphocytes were aligned. Digestion with hyaluronidase or collagenase eliminated both the colloidal iron and aldehyde fuchsin stainings of the basal lamina of HEVs. Treatment with chondroitinase ABC reduced colloidal iron staining, but did not interfere with the aldehyde fuchsin staining. Digestion with neuraminidase, keratanase, or heparitinase did not eliminate either the cationic colloidal or the aldehyde fuchsin staining. Digestion with neuraminidase reduced the colloidal iron staining on the luminal surface coat of the HEV. Electron microscopy of ultrathin sections revealed that cationic colloidal iron particles were deposited on the basal lamina of the HEV. The basal laminae of ordinary blood vessels were thin and single-layered, and stained only weakly with cationic colloidal iron. The present study suggests that negatively charged sites in the basal lamina of HEV derive mainly from a proteoglycan complex containing hyaluronic acid and chondroitin sulfate, which firmly binds collagen. This topochemical feature is suggested to be involved in the fascilitating migration of lymphocytes after passage through the endothelial layer.
Archives of Histology and Cytology 01/2002; 64(5):535-43. · 0.57 Impact Factor
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ABSTRACT: The three-dimensional distribution of the biliary tract in the rat was studied by scanning electron microscopy of biliary casts. The casts were prepared by a retrograde infusion of a low viscosity or monomeric methacrylate resin mixture into the common bile duct. No resin flow from the bile canaliculi to sinusoidal capillaries was ever noted. Bile canaliculi formed intricate meshworks and drained via the Hering's canals into the bile ductules. The bile canalicular meshworks of adjacent lobules intercommunicated with each other. The bile ductules formed a marked periportal plexus around the portal vein branch, and drained into the intrahepatic bile duct running along the portal vein branch. The junctional zone of the Hering's canal and bile ductule usually showed an ampullary dilation. When the Hering's canal directly drained into a thick bile ductule or into a periportal plexus of bile ductules, such an ampullary dilation at the origin of the bile ductule was never replicated. The extrahepatic bile duct protruded many crypt-like projections which presumably corresponded to parietal glands. It is suggested that the periportal plexus of bile ductules may store the bile as a substitute for the gallbladder.
Archives of Histology and Cytology 11/2001; 64(4):439-47. · 0.57 Impact Factor
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ABSTRACT: Light microscopic observations of healthy adult rat brain sections stained with anionic iron colloid indicated that 5-10% of neurons in the hippocampal subiculum and all neurons in the medial cerebellar nucleus possessed an intensely positively charged perineuronal net. This net was demonstrated to react to oxine, and therefore suggested to consist of guanidino compounds. It was further shown that the intensely positively charged perineuronal net, in accordance with the intensely negatively charged perineuronal net of proteoglycans, was digested by chondroitinase ABC, hyaluronidase, and collagenase, but not by endo-alphaN-acetylgalactosaminidase. This finding suggested that the former positively charged net might be linked to the latter negatively charged one.
Archives of Histology and Cytology 09/2001; 64(3):313-8. · 0.57 Impact Factor
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ABSTRACT: The Purkinje cells in the adult cat cerebellar cortex were found to possess perineuronal proteoglycans which could be stained with our fine cationic iron colloid and Fujita's highly concentrated aldehyde fuchsin, and digested by chondroitinase ABC/keratanase/ heparitinase and hyaluronidase. The Purkinje cells are surrounded by some collagenous elements which are stained with Gömöri's ammoniacal silver and digested by collagenase. The Purkinje cells also express nerve cell surface glycoproteins which are labeled with lectin Vicia villosa agglutinin and digested by a double treatment with collagenase and endo-alpha-N-acetylgalactosaminidase. Sole digestion by endo-alpha-N-acetylgalactosaminidase never erased the lectin labeling of the nerve cell surface glycoproteins. These findings suggest that the collagenous elements mediate the linkage of the perineuronal proteoglycans to the nerve cell surface glycoproteins. It is presumed that in mice and rats, the perineuronal nets of proteoglycans and nerve cell surface glycoproteins of the Purkinje cells are so thin or coarse that they can not be sufficiently visualized under the light microscope.
Archives of Histology and Cytology 06/2001; 64(2):203-9. · 0.57 Impact Factor
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ABSTRACT: Ferric chloride, when boiled with ammonium thiocyanate, ammonia and cacodylic acid, is converted into a fine anionic iron colloid which consists of 1.0-1.5 nm electron dense granules and gives a distinct Prussian blue reaction (OHTSUKA and MURAKAMI, 1986). Light microscopy of tissue sections stained with this fine anionic iron colloid at pH values of 6.0, 7.0 and 8.0 showed that the healthy adult rat brain contains a considerable number of neurons which possess an intensely positively charged perineuronal net. This net was most clearly demonstrable by staining with the anionic iron colloid at a pH value of 8.0, at which ionizations of almost all cationic sites of the tissue elements were obliterated. Transmission electron microscopy of ultrathin sections stained at a pH value of 8.0 showed that the anionic iron colloid was preferentially deposited in the perineuronal tissue spaces. These findings indicate that the intensely positively charged perineuronal net contains some strongly basic substances such as guanidino compounds, and occupies the perineuronal (perisynaptic) tissue space.
Archives of Histology and Cytology 03/2001; 64(1):45-50. · 0.57 Impact Factor
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ABSTRACT: The hippocampal subiculum in the adult rat brain contains many neurons with nerve cell surface glycoproteins which are linked by collagenous ligands to perineuronal proteoglycans. The nerve cell surface glycoproteins or their terminal N-acetylgalactosamines are digested by endo-alpha-N-acetylgalactosaminidase. The terminal N-acetylgalactosamines linked by the collagenous ligands are not digested by endo-alpha-N-acetylgalactosasminidase. The collagenous ligands associated with the terminal N-acetylgalactosamines were digested by collagenase. The newly exposed terminal N-acetylgalactosamines by this collagenase incubation were digested by endo-alpha-N-acetyl-galactosaminidase. These findings on the rat agree with those obtained in our previous studies of the adult mouse brain samples. Furthermore they emphasize our hypothetical model that the perineuronal proteoglycans are related--via collagen ligands--with the nerve cell surface glycoproteins.
Italian journal of anatomy and embryology = Archivio italiano di anatomia ed embriologia 02/2001; 106(2 Suppl 1):475-80.
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ABSTRACT: The free surface of the rat peritoneum was covered with a rich negative-charged substance which is distinctly stained with cationic colloidal iron (pH 1.5). Neuraminidase digestion erased this iron stain. Treatment with Limax flavus agglutinin (LFA), which has specific affinity to sialic acid, interferred with iron staining on the serosal surface. Transmission electron microscopy of the peritoneal samples stained with colloidal iron showed that colloidal particles were deposited on the free surface of the mesothelial cells. At pH 1.5, the colloidal particles aggregated in a dotted fashion; in those stained at pH 7.0, the particles arranged in fine strands (100-300 nm in length). This difference may occur as a structural transformation due to pH level changes. The string like structure seemed to correspond well to membrane associated sialomucin. The urinary surface of the rat glomerular podocytes possessed negatively charged sites detectable with cationic colloidal iron even at pH 1.5. Neuraminidase and LFA treatments erased iron staining. Substance containing sialic acid such as podocalyxin on the podocyte surface may be stained. This study shows that negatively charged sites of the substance covering the free surface of these regions repulse each other to maintain the serosal cavities or the podocyte end-feet slits.
Italian journal of anatomy and embryology = Archivio italiano di anatomia ed embriologia 02/2001; 106(2 Suppl 1):379-84.
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ABSTRACT: The accessory ascending cervical artery (Murakami et al., 1996), which arises from the subclavian artery and ascends between the scalenus anterior and medius muscles, was studied in 87 Japanese adult cadavers (174 sides), with special attention being given to its origin, distribution, and relationship to other arteries at the cervical or scalenus region. In 154 sides (88.5%), the accessory ascending cervical artery was found to originate from the subclavian artery behind the scalenus anterior muscle, and to branch out to the scalenus anterior and medius muscles as well as those entering the 5th and 6th intervertebral foramens along the 6th and 7th cervical nerves. This artery arose independently in 105 sides. The accessory ascending cervical artery issued off or formed a common trunk with the transverse cervical artery and/or costocervical trunk in 49 sides. In cases lacking the accessory ascending cervical artery, it was usually compensated for by the costocervial trunk and/or transverse cervical artery (18 sides). Common trunk formation with the vertebral, internal thoracic, or suprascapular arteries was not observed. The authors suggest that the accessory ascending cervical artery, the transverse cervical artery, and the costocervical trunk should be grouped into one arterial system, a system that may be a remnant of the precostal longitudinal anastomoses of intersegmental arteries of the dorsal aorta behind the scalenus anterior muscle.
Acta medica Okayama 01/2001; 54(6):243-52. · 0.84 Impact Factor
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ABSTRACT: The present study demonstrated the precise spatial relationship between meshes in the perineuronal proteoglycan network and the terminal boutons of synaptically associated axons. Sections from the rat cerebellum were stained with cationic colloidal iron (pH 1.0-1.5), and successively immunostained with anti-calbindin-D-28K monoclonal antibody. Cationic iron stained sulfated proteoglycans around the nerve cell of the medial cerebellar nucleus, whereas the anti-calbindin antibody labeled the Purkinje cells including their axons terminating on large neurons in the cerebellar nucleus. It was found that each synaptic bouton fits into a mesh of the perineuronal network. The individual meshes appeared to be divided by partitions faintly stained with the colloidal iron. Electron microscopy of cationic colloidal iron-stained ultrathin sections revealed that the synaptic boutons were separated from each other by the proteoglycan matrix and that each of them was further divided into two or more contact areas of presynaptic membrane by the same matrix. This suggests that individual synapses are protected against the effects of adjacent synaptic transmission, and that each of them may be subdivided by this manner of partitioning, like pads of a cat's paw.
Archives of Histology and Cytology 11/2000; 63(4):313-8. · 0.57 Impact Factor
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ABSTRACT: Histamine plays important roles in gastric acid secretion, inflammation, and allergic response. Histamine N-methyltransferase (HMT; EC 2.1.1.8) is crucial to the inactivation of histamine in tissues. In this study we investigated the immunohistochemical localization of this enzyme in guinea pig tissues using a rabbit polyclonal antibody against bovine HMT. The specificity of the antibody for guinea pig HMT was confirmed by Western blotting and the lack of any staining using antiserum preabsorbed with purified HMT. There was strong HMT-like immunoreactivity (HMT-LI) in the epithelial cells in the gastrointestinal tract, especially in the gastric body, duodenum, and jejunum. The columnar epithelium in the gallbladder was also strongly positive. Almost all the myenteric plexus from the stomach to the colon was stained whereas the submucous plexus was not. Other strongly immunoreactive cells included the ciliated cells in the trachea and the transitional epithelium of the bladder. Intermediately immunoreactive cells included islets of Langerhans, epidermal cells of the skin, alveolar cells in the lung, urinary tubules in the kidney, and epithelium of semiferous tubules. HMT-LI was present in specific structures in the guinea pig tissues. The widespread distribution of HMT-LI suggests that histamine has several roles in different tissues.
Journal of Histochemistry and Cytochemistry 08/2000; 48(7):943-54. · 2.72 Impact Factor
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ABSTRACT: The present study showed that many neurons in the adult rat brain possessed a perineuronal sulfated proteoglycan surface coat which reacted to cationic iron colloid and aldehyde fuchsin. This surface coat was stained supravitally with Ehrlich's methylene blue and doubly stained with Ehrlich's methylene blue and aldehyde fuchsin. The surface coat was also stained with Gömöri's ammoniacal silver and doubly stained with Gömöri's ammoniacal silver and cationic iron colloid. The surface coat was usually expressed together with a nerve cell surface glycoprotein net detectable with lectin Wisteria floribunda agglutinin. These findings indicate that the perineuronal proteoglycan surface coat is identical to Cajal's superficial reticulum and contains some collagenous elements. It was further demonstrated that collagenase digestion erased Gömöri's ammoniacal silver impregnation within the perineuronal proteoglycan surface coat.
Acta medica Okayama 07/2000; 54(3):111-8. · 0.84 Impact Factor
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ABSTRACT: The present study aimed for a clear visualization of faintly deposited colloidal iron in tissue sections for light microscopy. Paraffin blocks containing paraformaldehyde-fixed brain tissue from healthy adult mice were cut into sections 10-15 microm thick. After deparaffinization, the sections were stained with fine cationic iron colloid at a pH value of 1.0-1.5, and treated with a mixture of potassium ferrocyanide and hydrochloride for Prussian blue reaction. Some sections were further treated with Bodian's protein silver after the Prussian blue reaction. This sensitized development of Prussian blue reaction with Bodian's protein silver more clearly visualized the faintly deposited cationic colloidal irons than the demonstration by Prussian blue reaction alone, and allowed an enhanced visualization of the perineuronal nets of sulfated proteoglycans in the brain. Thus, such fine perineuronal sulfated proteoglycans as those in the CA3 field of the hippocampus, which are weakly stained with cationic iron colloid and usually overlooked by a demonstration with only a Prussian blue reaction, could be clearly visualized with striking contrast by the sensitized development with Bodian's protein silver after the Prussian blue reaction. Preliminary hyaluronidase digestion erased Bodian's protein silver development of perineuronal sulfated proteoglycans. Though some axonal fibers were also additionally stained with Bodian's protein silver itself, this sensitized development is useful to enhance such weak colloidal iron signals as are hardly detectable by only Prussian blue reaction.
Archives of Histology and Cytology 02/2000; 63(5):459-65. · 0.57 Impact Factor
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ABSTRACT: Electron microscopy of ultrathin sections stained with cationic iron colloid revealed that the rat pineal gland is provided with wide and intensely negative-charged pericapillary spaces. Light microscopically, the negative charging of the pericapillary spaces was completely eliminated by digestion with hyaluronidase and chondroitinase ABC. This pericapillary negative charging was also erased by digestion with collagenase. The results indicate that the negative charging is derived from sulfated proteoglycans which are bound to collagen molecules. These sulfated proteoglycans in the pericapillary spaces may retain numerous water molecules to form a tissue gel, and so act as a selective sieve regulating the passage of tissue molecules.
Archives of Histology and Cytology 02/2000; 63(5):485-94. · 0.57 Impact Factor
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ABSTRACT: The present study demonstrates light and electron microscopic changes in neurons in the myenteric plexus of the rat ileum following four-hour ischemia. Macroscopically, an intestinal constriction occurred at the damaged portion at three weeks after ischemia; the segment oral to the constriction markedly swelled at four weeks. In light microscopy, at three weeks after ischemia, the myenteric neurons appeared spongy or foamy, containing many vacuoles in their somatic cytoplasm. At four weeks, the neuronal cytoplasm and nerve fiber bundles had disintegrated to form vacant spaces in the myenteric plexus. The neuronal nucleus of the damaged plexus did not show positive nick-end labeling. In electron microscopy, neuronal cytoplasm revealed degenerative signs already at one week after ischemia: a distended endoplasmic reticulum and swollen mitochondria with fragmentary cristae. The nerve fibers also showed destruction of the mitochondria, and degenerative changes in the postsynaptic sites appeared earlier than the presynaptic terminals. The results suggest that intestinal ischemia causes delayed neuronal death, which differs from the apoptotic process previously demonstrated in the ischemia-damaged brain.
Archives of Histology and Cytology 11/1999; 62(4):383-92. · 0.57 Impact Factor
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ABSTRACT: As our previous studies have indicated, the cingulate cortex of the adult mouse brain contains many neurons with rich cell surface glycoproteins which are linked by collagenous ligands to perineuronal proteoglycans. The present study demonstrated that exclusive incubation with endo-alpha-N-acetylgalactosaminidase abolished the lectin Vicia villosa or Wisteria floribunda agglutinin (VVA or WFA) labeling of the nerve cell surface glycoproteins, while it neither interfered with the cationic iron colloid or aldehyde fuchsin stainings of the perineuronal proteoglycans nor abolished the Gömöri's ammoniacal silver impregnation of the collagenous ligands. Double incubations with endo-alpha-N-acetylgalactosaminidase and collagenase did not eliminate the lectin VVA or WFA labeling of the nerve cell surface glycoproteins, though they did eliminate the cationic iron colloid and aldehyde fuchsin stainings of the perineuronal proteoglycans as well as the Gömöri's ammoniacal silver impregnation of the collagenous ligands. Triple incubations with endo-alpha-N-acetylgalactosaminidase, collagenase, and endo-alpha-N-acetylgalactosaminidase abolished the lectin VVA or WFA labeling of the nerve cell surface glycoproteins, and also eliminated the cationic iron colloid and aldehyde fuchsin stainings of the perineuronal proteoglycans and the Gömöri's ammoniacal silver impregnation of the collagenous ligands. These findings indicate that: the nerve cell surface glycoproteins or their terminal N-acetylgalactosamines are digested by endo-alpha-N-acetylgalactosaminidase; these galactosamines associated with the collagenous ligands or perineuronal proteoglycans are not digested by endo-alpha-N-acetylgalactosaminidase; and the terminal N-acetylgalactosamines newly exposed by collagenase incubation are digested by this galactosaminidase. It was further demonstrated that hyaluronidase incubation neither digests the collagenous ligands nor revives the lectin VVA or WFA labeling of the nerve cell surface proteoglycans.
Archives of Histology and Cytology 09/1999; 62(3):273-81. · 0.57 Impact Factor
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ABSTRACT: As our previous study has indicated, perineuronal proteoglycans in the adult mouse brain are associated with some collagenous molecules which can be stained with Gömöri's ammoniacal silver and are resistant to hyaluronidase digestion. The present study demonstrated that these molecules are thoroughly digested with collagenase, and suggests that they represent a hyaluronic acid-binding domain of the ligand proteoglycans connecting the perineuronal proteoglycans and nerve cell surface glycoproteins.
Archives of Histology and Cytology 06/1999; 62(2):199-204. · 0.57 Impact Factor
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ABSTRACT: As our previous studies have indicated, many subsets of neurons in the vertebrate brain possess a sulfated proteoglycan surface coat which reacts to cationic iron colloid and aldehyde fuchsin. The present study demonstrated that this surface coat is supravitally stained with Ehrlich's methylene blue, and doubly with this blue and aldehyde fuchsin, a finding suggesting its being identical to Cajal's superficial reticulum (red superficial) and to Golgi's reticular coating (revetement reticulare). The perineuronal surface coat was further stained with Gömöri's ammoniacal silver, and doubly with this silver and cationic iron colloid. These neurons with such a proteoglycan surface coat usually expressed cell surface glycoproteins which were labeled with lectin Wisteria floribunda agglutinin. Hyaluronidase digestion did not interfere with this lectin labeling of the glycoproteins, methylene blue and Gömöri's ammoniacal silver staining of the surface coat, while it erased the cationic iron colloid and aldehyde fuchsin staining of the surface coat. These findings suggest that the perineuronal proteoglycan surface coat is associated with some additional molecules which are resistant to hyaluronidase digestion and stainable with methylene blue and Gömöri's ammoniacal silver. The possibility is suggested that these molecules might represent "ligand proteoglycans" connecting the perineuronal proteoglycans and cell surface glycoproteins.
Archives of Histology and Cytology 04/1999; 62(1):71-81. · 0.57 Impact Factor
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ABSTRACT: Some rare anomalies of the celiaco-mesenteric system were observed postmortem in a Japanese adult male: a) The left gastric, common hepatic, splenic and superior mesenteric arteries arose independently from the abdominal aorta. b) The anterior inferior pancreaticoduodenal artery of the superior mesenteric artery issued a hepatic artery which ascended along the anterior surface of the pancreas and gave off the right gastroepiploic, right gastric and cystic arteries. c) The common hepatic artery gave off an anastomosing branch to the superior mesenteric artery. d) The left gastric artery gave off the left accessory hepatic artery. e) The splenic artery issued the accessory middle colic artery. f) The left inferior phrenic artery gave off the esophageal branch. These anomalies are discussed in light of a typological system which we proposed in a previous paper for the celiaco-mesenteric system.
Acta medica Okayama 11/1998; 52(5):239-44. · 0.84 Impact Factor
