Differences between collagen morphologies, properties and distribution in diabetic and normal biobreeding and Sprague-Dawley rat sciatic nerves.

Both structural and functional differences between normal and diabetic nerve have been observed, in human patients and animal models. We hypothesize that these structural differences are quantifiable, morphologically and mechanically, with the ultimate aim of understanding the contribution of these differences to permanent nerve damage. The outer collagenous epineurial and perineurial tissues of mammalian peripheral nerves mechanically and chemically shield the conducting axons. We have quantified differences in these collagens, using whole-nerve uniaxial testing, and immunohistochemistry of collagens type I, III, and IV in diabetic and normal nerves. We present results of two studies, on normal and diabetic BioBreeding (BB), and normal, diabetic and weight-controlled Sprague-Dawley (SD) rats, respectively. Overall, we measured slightly higher uniaxial moduli (e.g. 5.9 MPa vs. 3.5 MPa, BB; 10.7 MPa vs. 10.0 MPa, SD at 40% strain) in whole nerves as well as higher peak stresses (e.g. 0.99 MPa vs. 0.74 MPa, BB; 2.16 MPa vs. 1.94 MPa, SD at 40% strain) in the diabetics of both animal models. We measured increased concentrations of types III and IV collagens in the diabetics of both models and mixed upregulation results were observed in type I protein levels. We detected small differences in mechanical properties at the tissue scale, though we found significant structural and morphometric differences at the fibril scale. These findings suggest that whole-tissue mechanical testing is not a sufficient assay for collagen glycation, and that fibrillar and molecular scale assays are needed to detect the earliest stages of diabetic protein glycation.

Impact of gastroenterology consultation on the outcomes of patients admitted to the hospital with decompensated cirrhosis.

Managed care has strongly discouraged generalists from referring patients to specialists in an effort to reduce the costs of health care. The aim of this study was to compare patient outcomes when generalists work together with gastroenterologists or alone in the management of patients admitted to the hospital with decompensated cirrhosis. Consecutive patients admitted to the hospital with decompensated cirrhosis over a 1-year period were identified. We compared the length of stay, cost of hospitalization, incidence of hospital readmission, and mortality for patients who did and those who did not have a gastroenterology (GI) consultation. A GI consultation was requested for 107 of the 197 patients (54.3%). Patients who had a GI consultation had a significantly shorter length of stay (5.6 +/- 3.5 vs. 10.1 +/- 5.8 days, P <.001) and a lower cost of hospitalization ($6,004 +/- $4,994 vs. $10,006 +/- $6,183, P <.001) than those patients who were managed by generalists alone. The 30-day incidence of readmission (13.3% vs. 27.8%, P =.01) and mortality (7.5% vs. 16.7%, P =.045) were significantly lower in the GI consultation group. During a median follow-up of 618 days (range, 2-970), patients who had a GI consultation during hospitalization had a significantly longer time to hospital readmission (P <.001) and improved survival (P =.02) compared with those who were managed by generalists alone. In conclusion, for patients admitted to the hospital with decompensated cirrhosis, individuals who were managed by generalists in conjunction with gastroenterologists had better outcomes than those who were managed by generalists alone.

Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein.

An hPepT1-GFP fusion construct was made to study drug inhibition of dipeptide uptake and apical, basolateral, or subcellular hPepT1 localization. The hPepT1 stop codon was mutated by polymerase chain reaction and was subsequently cloned into the pEGFP-N1 vector. The hPepT1-GFP fusion construct was then transfected into Caco-2 and HeLa cells, and drug inhibition was studied by inhibiting 3H-Gly-Sar uptake. Western blot analysis was used to determine hPepT1-GFP expression levels and confocal microscopy was used to examine the localization. Both anti-hPepT1 antibody and anti-GFP antibody recognized a 120-kd hPepT1-GFP fusion protein in the transfected cells. The 3H-Gly-Sar uptake in transfected HeLa cells was enhanced more than 20 times compared with the control. Valacyclovir (5 mmol/L) was able to completely inhibit 3H-Gly-Sar uptake in these transfected cells. Confocal microscopy showed that the hPepT1-GFP mainly localized in the Caco-2 cell apical membrane, but was present throughout the entire HeLa cell membranes. The hPepT1-GFP fusion protein was not found in either early endosome or lysosome of Caco-2 cells under normal conditions; however, it was detected in some subsets of lysosomes and early endosomes in phorbol 12-myristate 13-acetate (PMA)-treated Caco-2 cells.

Retroviral-mediated transduction of endothelial cells with the lac Z gene impairs cellular proliferation in vitro and graft endothelialization in vivo.

PURPOSE: An endothelialized lumen within a synthetic graft that expresses recombinant proteins with anticoagulant or antiproliferative activity has the potential to improve graft function. However, preliminary data suggest that genetic modification of endothelial cells (ECs) impairs their proliferation. The purpose of this investigation was to test the hypothesis that retroviral transduction of cultured ECs with the lac Z gene encoding for beta-galactosidase would decrease EC proliferation in vitro and graft endothelialization in vivo. METHODS: In vitro studies compared canine EC proliferation over a 14-day period among early-passage ECs (two and three) and late-passage ECs (six and nine) transduced with the BAG vector (containing the lac Z gene and the neomycin resistance gene), ECs transduced with the neomycin resistance gene only, the nontransduced ECs. In vivo canine studies assessed endothelialization of expanded polytetrafluoroethylene thoracoabdominal grafts seeded with autologous lac Z-transduced ECs (n = 7) or nontransduced ECs (n = 3) compared with that of nonseeded grafts (n = 3). Histochemical staining and DNA polymerase chain reaction was used 6 weeks after implantation to detect the presence of the lac Z gene in the grafts' cellular linings and perigraft tissues. Endothelialization was assessed by light microscopy and electron microscopy. RESULTS: Proliferation of late-passage lac Z-transduced ECs in vitro was significantly decreased compared with neomycin resistance-transduced ECs or nontransduced ECs. Among early-passage ECs smaller but significant decreases in proliferation were noted among lac Z-transduced cells compared with nontransduced cells. Six of seven expanded polytetrafluoroethylene grafts seeded with transduced ECs showed lac Z gene expression. Lac Z gene expression was not found on grafts seeded with nontransduced ECs or nonseeded grafts. The endothelialized luminal surface area was significantly less in grafts seeded with lac Z-transduced ECs compared with grafts seeded with nontransduced ECs. CONCLUSIONS: Retroviral-mediated transduction of canine ECs with the lac Z gene encoding for beta-galactosidase impairs EC proliferation in vitro and the ability of transduced ECs to form a confluent EC monolayer on the luminal surface of synthetic grafts in vivo.

Formation of the peripheral nervous system during tail regeneration in urodele amphibians: ultrastructural and immunohistochemical studies of the origin of the cells.

In the regenerating newt tail, epimorphic regeneration--which recapitulates morphologically normal embryonic development--proceeds along a rostrocaudal differentiation gradient. Innervation of the new myomeres results from the spinal roots of segments rostral to the amputation plane and from ventral roots emerging from the lateroventral region of the regenerating spinal cord, in which motor neurons are differentiating. Electron microscopy and an indirect immunofluorescence study with anti-glial fibrillary acid protein (GFAP) confirm that the ventrolateral part of the regenerated ependymal tube gives rise to cells of the ventral root sheath and the spinal ganglia. Anti-GFAP and anti-neurofilament antibodies showed that ependymoglial cells and Schwann cells may play a role in neuronal pathfinding by helping guide and stabilize pioneering axons as they extend toward the myomeres. The carbohydrate epitope NC-1 is expressed in the spinal cord, in sheath cells of the spinal ganglia and in the non-myelin-forming Schwann cells of the peripheral nervous system. L1, a Ca++ independent neural cell adhesion molecule, was detected in the axonal compartments of the regenerating spinal cord, on immature and/or non-myelin-forming Schwann cells within the peripheral nervous system (PNS), and on nerve fibers within the regenerate. These immunohistochemical observations collectively support the hypothesis that Schwann cells already present in the blastema could be involved in organizing neural pathways.

Extraocular muscle regeneration in primates. Local anesthetic-induced lesions.

Retrobulbar administration of several local anesthetics (0.75% bupivacaine, 2.0% mepivacaine or 2.0% lidocaine plus 1:100,000 epinephrine) in monkeys resulted in a low incidence of muscle fiber lesions in the extraocular muscles closest to the site of injection. Most lesions resulted in the degeneration and regeneration of muscle fibers on the surface of the muscles, but occasionally a massive internal lesion was seen. In contrast, large lesions were common in rectus muscles that received direct injections of local anesthetics in both monkeys and humans. The morphology and temporal sequence of muscle fiber degeneration and regeneration was similar to that seen in primate thumb muscles injured by anesthetic agents.

Early innervation of skeletal muscle during tail regeneration in urodele amphibians.

The innervation pattern of skeletal muscles was studied in the normal and regenerating tail of Notophthalmus viridescens. Silver staining for nerve endings and histochemical localization of acetylcholinesterase (AChE) were used for light microscopy. In In normal musculature, AChE positive reactions were localized at the ends of the muscle fibers where they are anchored on connective tissue septa by myotendinous junctions. At this level, silver staining shows nerve terminals forming endplates. During regeneration, positive reactions for AChE appear de novo as dense plates localized at the ends of the newly formed myotubes. The mechanisms involved in the localization of AChE on this surface seem to operate before previous local contacts by nerve terminals. From the ultrastructural data and immunohistochemical results with anti-laminin antibody, these observations suggest that regenerating muscle fibers determine a region of post-synaptic specialization in close relation with the organization of myotendinous regions and basement membrane formation. Nerve-muscle contacts appear at these levels at stage IV (15-20 days after amputation) in the stump and in the rostral part of the regenerate (transition zone). These nerve terminals are provided by the disorganized peripheral nervous system of the injured segment. In the regenerate a similar pattern of AChE reaction can be seen in every myotube, differentiating according to a rostro-caudal gradient. Innervation at the ends of the muscle fibers is in spatiotemporal relation with the exists of the ventral roots from the regenerating nerve cord as the regenerate continues to grow in length.

A histological study of local anesthetic-induced muscle degeneration and regeneration in the monkey.

Small amounts (1-2 ml) of local anesthetics (bupivacaine, lidocaine, and mepivacaine) were injected into the abductor pollicis brevis muscles of 35 monkeys. Control muscles were injected with saline. The muscles were preserved for histology from 4.5 h to 48 days after the injection. Histological damage to muscle fibers was evident from the time of the first sampling. Invasion of damaged muscle fibers by phagocytic cells was prominent by 2-3 days postinjection. At 4 to 5 days, areas of muscle fiber damage were characterized by dense concentrations of phagocytes and mononuclear myoblastic cells. At 6 days, fields of early myotubes were evident. Maturation of myotubes into immature cross-striated muscle fibers occurred over the next week. Occasional myotubes or immature regenerating muscle fibers were seen as late as 28 days. The topographical pattern of muscle fiber degeneration and regeneration showed a concentration along the surfaces of muscle fascicles or, if intrafascicular, around the presumed site of injection.

An electron microscopic study of local anesthetic-induced skeletal muscle fiber degeneration and regeneration in the monkey.

An electron microscopic study was done on abductor pollicis brevis muscles of 18 Rhesus monkeys after intramuscular injections of 0.75% bupivacaine, 2% mepivacaine, or 2% lidocaine + epinephrine. The muscles were examined for from 2 h to 28 days. Severe muscle fiber damage, consisting of breakdown of sarcolemma and myofibrils, was seen as early as 2 h. Phagocyte mediated fragmentation of the degenerating muscle fibers was at its peak during the third and fourth days. Myoblasts were abundant during the fourth day. Early myotubes appeared on the fifth and sixth days, and they matured during the second week. Satellite cells appeared alongside mature myotubes. Overall, the local anesthetic-induced breakdown and regeneration of skeletal muscle fibers in the monkey followed a course quite similar to that seen in the rat.

Free radical injury to skeletal muscles of young, adult, and old mice.

We tested the hypotheses that 1) muscles of old mice are more susceptible to injury than muscles of young and adult mice, and 2) secondary or delayed onset injury results from free radical damage. Extensor digitorum longus muscles were injured in situ by lengthening contractions. Injury was assessed by measurement of maximum isometric tetanic force (Po) expressed as a percentage of the control value and by morphological damage. Mice were treated with a free radical scavenger, polyethylene glycol-superoxide dismutase (PEG-SOD). Three days postinjury, the Po of 44% for muscles of nontreated old mice was significantly lower than the Po of 58 and 61% for those of young and adult mice. In each group, the secondary injury at 3 days was alleviated by treatment with PEG-SOD. For treated muscles of young, adult, and old mice, values for Po were 88, 80, and 70%, respectively. We conclude that muscles of old mice are more susceptible to injury than muscles of young or adult mice and that free radicals contribute to the secondary or delayed onset injury.

Ultrastructure of mepivacaine-induced damage and regeneration in rat extraocular muscle.

Adult rats were given a single retrobulbar injection of 50 microliters of 2.0% mepivacaine and the lateral rectus muscles were examined ultrastructurally at intervals from 15 min to 30 days post-injection. There were three purposes of the study: (1) to determine the extent of muscle fiber damage caused by the anesthetic; (2) to document the subsequent course of muscle fiber regeneration; and (3) to relate these findings to clinical data on possible adverse effects of local anesthetics on human extraocular muscle function. The lateral rectus muscle was massively damaged by exposure to the anesthetic, with membrane lesions seen as early as 15 min after the injection. Intracellular damage was followed by the phagocytic removal of the remnants of the damaged muscle fibers. The activation of satellite cells to myoblasts began during the phase of phagocytosis, and between 3 and 4 days after injection multinucleated myotubes actively forming sarcomeres appeared. Even during later stages of muscle fiber regeneration, evidence of damage was seen in muscle fibers that were not destroyed during the first 2 days post-injection. The results of this experiment show (1) that the vast majority of lateral rectus muscle fibers are rapidly broken down by the anesthetic, but that the destroyed muscle fibers are replaced by regenerating ones; and (2) that the ultrastructure of regeneration of extraocular muscle fibers differs little from the regeneration of somatic muscle fibers. The myotoxic effects of retrobulbarly applied local anesthetics in rats seem to be much greater than they are in primates.

Macular suprastructure, stereociliary bonding and kinociliary/stereociliary coupling in rat utricular macula.

This report considers rat utricular macular suprastructure after chemical treatment with the sodium salt of N,N-Naphthaloylhydroxylamine (NHA), used alone or in combination with tannic acid (TA). NHA and TA preserve calcium and complex carbohydrate-protein molecules, respectively. Macromolecules of supramacular substance appear comparable morphologically to material of the external lamina of glycocalyx. Similar material crosslinks stereocilia with approximately equal to 58 nm periodicity and couples parts of kinocilia with specific stereocilia. Particles which occur within kinocilia at certain attachment sites connect to dynein arms of the kinocilia. Interstereociliary connections are aligned with internal linkers to actin. Thus, a basis for dynamic communication between kinocilia and stereociliary actin has been shown. The mechanism would appear to be calcium dependent. The findings support the concept that kinocilia are motile and lead to the stereociliary tuft in vestibular hair cells.

The suprastructure of the saccular macula.

Inclusion of tannic acid in the primary fixative fortuitously preserved the macular suprastructure, from the macular surface to the otoconia, in rat. Results demonstrate that fluid-filled channels around the stereociliary tufts and a slit-like space immediately above the macula are artefacts of tissue preparation. Filaments fill the area between, and show linkage to, the macular surface and the more peripheral, closely knit layer identified here as the true otoconial membrane. The material also passes through the otoconial membrane at various sites to surround the otoconia and extends well beyond the maculas in all directions. Although it may prove later to be endolymph, it is referred to here as supramacular substance. Kinocilia and the tallest stereocilia as well as the otoconia are tethered to the otoconial membrane. Findings were confirmed in decalcified material. Results are discussed in terms of functional and biochemical implications, and by analogy to man-made acceleration sensing devices.

The distribution of serum albumin in the rat testis, studied by electron microscope immunocytochemistry on ultrathin frozen sections.

The distribution of serum albumin is of interest in the rat testis because this protein is the principal carrier for testosterone in the plasma and interstitial fluid of this species. We have localized extravascular serum albumin in the rat testis at the electron microscope level, using gold particle immunocytochemistry on ultrathin frozen sections of tissue fixed lightly by perfusion. The same localization was obtained with three different antisera. Preabsorption and normal rabbit serum controls were negative, and Western blots of testis extracts showed major activity only at the molecular weight of albumin. Serum albumin occurred in substantial concentration throughout extracellular space in the interstitial tissue, as well as in the space between the boundary layer and the base of the seminiferous epithelium. Immunoreactivity extended between Sertoli cells, as well as around spermatogonia and early primary spermatocytes (to stage 11), but did not traverse the Sertoli-Sertoli junctions that comprise the blood-testis barrier. Macrophages in the interstitial tissue showed some endocytic activity. If perfusion fixation was carried out in a manner that flushed most of the albumin from the interstitial space, then a layer of albumin remained on the surface of Leydig cells and many macrophages but was minimal or absent on the surface of other cell types that are normally in contact with albumin, such as Sertoli cells, spermatogonia, myoid cells, lymphatic endothelium, fibroblasts, or cells of blood vessels.