Quantification of protein A-gold staining for peroxisomal enzymes by confocal laser scanning microscopy.

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific beta-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.(J Histochem Cytochem 47:1343-1349, 1999)

Peroxisomes in permanent and provisional kidneys. Phylogenic and ontogenic considerations.

Peroxisomes in three forms of vertebrate kidney (pronephros, mesonephros, and metanephros), as permanent or provisional kidney, are summarized concerning their ultrastructure and developmental changes. Because the peroxisome is known to be diverse in mammalian metanephros, and species difference is its distinctive feature among cell organelles, information should be obtained on each kidney of each species. The ultrastructural and biochemical features of peroxisomes have at least been partly delineated in the metanephros and mesonephros, but nothing is known about the pronephros. Ultrastructural studies of the metanephric peroxisomes are present in mammals, birds, and reptiles, but information on their development is restricted to mammals and birds. As for the mesonephric peroxisomes, both ultrastructural and developmental data have been accumulating on mammals and amphibians, and ultrastructural information is present on fishes, but not on birds and reptiles. At present, studies on peroxisomes of provisional kidney have been restricted to mammalian mesonephros. The common features of renal peroxisomes previously examined are that they are spherical cell organelles with a single limiting membrane in ultrastructure, and are positive for catalase. Information on the ultrastructure and enzymes is not sufficient at present for comparing the ontogenesis of renal peroxisomes with their phylogenesis.

Early active motion and weightbearing after cross-stitch achilles tendon repair.

Twenty-two closed Achilles tendon ruptures caused by sports injuries in 22 patients (average age, 37.6 years) were repaired with Kirschmayer core suture and cross-stitch epitenon suture, and early active ankle motion with weightbearing was implemented after surgery. This study was undertaken to evaluate the effectiveness of the repair technique and rehabilitation protocol by assessing clinical results and magnetic resonance imaging findings. The follow-up period averaged 24.6 months. Twenty of the tendons (91%) healed without rerupture, and two tendons (9%) suffered a partial rerupture at 23 and 56 days, respectively. Active ankle extension reached from the minus range to 0 degree in an average of 9.7 days, and ankle motion recovered to normal in an average of 6.0 weeks. Full weightbearing without heel raising became possible in an average of 16.4 days, and heel raising with both legs became possible in an average of 7.3 weeks. The patients returned to full sports activity in 13.1 weeks. The interval until the area of high-intensity signal at the tendon repair site on T2-weighted magnetic resonance imaging scans became intermediate-intensity signal averaged 6.9 weeks, and the tendon repair site became low-intensity signal in an average of 12.6 weeks, demonstrating excellent tendon healing. Treatment employing Kirschmayer core suture and cross-stitch epitenon suture may help athletes return to sports activity in a shorter period than that allowed by previous methods of repair for Achilles tendon ruptures.

Expression of adhesion molecules and fibronectin of activated peritoneal surface with lipopolysaccharide (LPS) analyzed with immuno SEM.

To disclose cell-to-cell interaction associated with the defensive mechanism of the peritoneum, the peritoneum was stimulated with lipopolysaccharide (LPS) and analyzed three-dimensionally, ultrastructurally, and immunohistochemically with immunoSEM (scanning electron microscopy). The activated hepatic peritoneal surface demonstrated numerous microvilli with the adhesion molecules ICAM-1 and VCAM-1. They were restricted to villi and peaked at 1.5 microg/g body weight of LPS. Delicate strands appeared moderately and were interwoven among microvilli with increasing LPS. These strands did not express ICAM-1 or VCAM-1, but fibronectin. Leukocytes began to adhere to the peritoneal surface above die value of LPS (2.5 microg). These results suggest that the peritoneal surface gives a defensive sheet for cell-to-cell interaction through adhesion molecules and fibronectin.

[Motoneuronal death following prevention of reinnervation during postnatal development].

The hypothesis that survival of motoneurons depends on targets postnatally was tested in Wistar rats of known age. Under ether anesthesia, the cut distal end of a medial gastrocnemius (MG) nerve was anchored to the lateral gastrocnemius (LG) muscle to prevent reinnervation. The compound action potential (CAP) in response to stimulation of the MG nerve was recorded from L4 and L5 ventral roots to evaluate the degree of motoneuronal death in the nerve. CAPs of MG nerves were greatly reduced 1 to 8 months after the operation in the rats operated on at 6 or 9 days of age, which indicates that the majority of MG motoneurons died after the operation. In contrast, the CAPs in the animals which were operated on at older than 12 days were similar to normal animals. However, the number of motor units that remained in the implanted MG nerve was 1.3 on the average in the rats operated on at 12-days-old. In normal rats, it was 87. Thus, the results indicate that in the rats operated on at 12 days of age, a majority of MG motoneurons survived without making functional synapses. We conclude that there is a sharp critical period, between 9 and 12 days after birth, during which motoneuronal target dependency changes.

Ender nailing for unstable surgical neck fractures of the humerus in elderly patients.

The purpose of this study was to assess the effects of stabilization of displaced and unstable surgical neck fractures of the humerus by Ender nailing followed by protected passive range of motion exercise of the shoulder. Thirty-four patients (29 women and 5 men) with such fractures were treated. The average age of patients was 69.5 years (range, 48-86 years), and the average followup period was 9.9 months (range, 6-22 months). Protected passive range of motion of the shoulder was started in 2 weeks after surgery for 25 patients and in 3 to 4 weeks for 9 patients. All patients experienced pain relief immediately after surgery. Bone union occurred at 5.9 weeks (range, 4-10 weeks) in all but 1 patient. The average range of motion of the shoulder at followup was 129.7 degrees elevation and 43.2 degrees external rotation. The average limitation of elbow extension was 4.3 degrees. The most important points of this procedure are to obtain immediate relief of pain, to stabilize the unstable fragment, and to begin protected passive range of motion of the shoulder before extensive adhesions develop.

Spatial distribution of cell adhesion molecules on the peritoneal surface in the cecal perforation-induced peritonitis.

For understanding the immunological functions of the peritoneum, spatial localization of integrins and their ligands was studied by immuno-SEM on the peritoneal surface of mice with cecal perforation-induced peritonitis. The cecal peritoneum 24 hr after perforation was stained with specific antibodies against LFA-1, Mac-1, VLA-4, ICAM-1, VCAM-1, and fibronectin diluted with cold University of Wisconsin (UW) solution in conjunction with immuno-gold labeling. The spatial localization of those cell adhesion molecules was detected by backscatter electron (BSE) imaging with field emission scanning electron microscope (FESEM). Numerous leukocytes with diverse surface ultrastructure were observed on the peritoneal surface by FESEM. Some leukocytes were in contact with mesothelial cells, and others adhered to the exposed underlying connective tissue. The BSE imaging showed the ubiquitous distribution of Mac-1 on all membrane domains of leukocytes, i.e., cell body, ruffles, and microvilli. In contrast, predominant expressions of LFA-1 and VLA-4 were discernible on ruffles/microvilli of some leukocytes. The mesothelial cells remaining in the inflamed area expressed both ICAM-1 and VCAM-1 on their microvilli. The fibronectin was detected on presumable collagen fibers and/or fibrin over the exposed smooth muscle layer as well as on fibrin extending between leukocyte aggregation. The spatial microlocalization of integrins was clarified on the leukocytes emigrated in peritonitis, and their ligands were detected on the inflamed peritoneum.

Morphological analysis of leucocyte transmigration in the pleural cavity.

The role that pleural mesothelial cells play in leucocyte transmigration into the pleural cavity was investigated in lipopolysaccharide-stimulated mice. Changes in mesothelial cell morphology and changes in expression of adhesion molecules on mesothelial cells and leucocytes were analysed by light microscopy, immunohistochemistry, transmission electron microscopy (TEM) and immuno-scanning electron microscopy (immuno-SEM). After stimulation, the mesothelial cells separated completely from one another before leucocyte penetration across the mesothelial layer occurred. These changes occurred primarily in the immediate vicinity of ribs, where a large number of leucocytes accumulated. Immuno-SEM showed that the expression of intercellular adhesion molecule-1 (ICAM-1) on the parietal pleural mesothelial cells was significantly up-regulated by lipopolysaccharide stimulation, and that of vascular cell adhesion molecule-1 (VCAM-1) was induced. Both were restricted to the microvilli of the mesothelial cells. By contrast, expression of intercellular adhesion molecule-2 (ICAM-2), platelet/endothelial cell adhesion molecule-1 (PECAM-1), mucosal addressin cell adhesion molecule-1 (MAdCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1), peripheral node addressin (PNAd) and fibronectin were not detected. Lymphocyte function associated antigen-1 (LFA-1), macrophage-1 molecule (Mac-1) and very late appearing antigen-4 (VLA-4), all ligands of ICAM-1 and VCAM-1, were present on the transmigrated neutrophils and macrophages. These findings demonstrate that the immediate vicinity of ribs is a source of leucocyte migration into the pleural space.

Three-dimensional morphological analysis of antigen-antibody reaction in hepatic sinusoids preserved in hypothermic UW solution.

ICAM-1 antigen-antibody reaction was visualized by three-dimensional immunoscanning electron microscopy of hepatic sinusoids in rat liver treated with hypothermic University of Wisconsin (UW) organ preservation solution. The results were compared with similar antigen-antibody reactions carried out with immunoliposomes injected in vivo. Morphologically, the hepatic sinusoids were preserved well during the hypothermic procedure. Endothelial cells had a large number of fenestrations, which partly aggregated and formed sieve plates. ICAM-1 expression was induced by injection of LPS and detected by monoclonal antibody in the UW solution followed by gold-labeled secondary antibody. ICAM-1 was restricted mostly to the unique areas of sieve plates with immature, small fenestrations. A similar distribution of ICAM-1 was present when detected by in vivo injection of immunoliposomes containing the monoclonal ICAM-1 antibody. The results showed that antigen-antibody reactions can take place in livers preserved in hypothermic UW solution. Further, the reaction is similar to that which could occur in vivo during transplantation. This suggests that it may be possible to block potentially harmful antigen-antibody reactions by addition of appropriate antibodies to hypothermic UW solution prior to transplantation.