Brigitte Decker

Localization of Carbonic Anhydrase IV in Rat and Human Heart Muscle

We investigated carbonic anhydrase IV (CA IV) in rat and human heart with immunohistochemical methods by both light and electron microscopy. In cryosections that were incubated with anti-CA IV/FITC, the capillaries showed a strong reaction for CA IV. In paraffin and semithin sections treated with anti-CA IV/ABC (avidin-biotin-peroxidase complex) blood vessels, capillaries, and sarcolemma (SL) were positively stained. By staining ultrathin sections with anti-CA IV/immunogold, CA IV could also be demonstrated at the latter two locations, including the specialized sarcolemmal structures intercalated discs, and T-tubules. In addition, by this method CA IV was seen to be associated with the sarcoplasmic reticulum (SR). The absence of immunostaining in SR and/or SL with some techniques probably indicates a problem of accessibility of the antigenic sites. In line with the immunohistochemical results, CA IV mRNA expression was visualized in both endothelial and muscle cells by in situ hybridization histochemistry.

Collagen Fibril Organization in the Patellar Tendon Autograft After Posterior Cruciate Ligament Reconstruction: A Quantitative Evaluation in a Sheep Model

We replaced the posterior cruciate ligament in 30 skel etally mature sheep with a patellar tendon autograft using the central third of the ipsilateral patellar tendon. The healing autograft was compared with the contralat eral posterior cruciate ligament and the patellar tendons and posterior cruciate ligaments of nonoperated ani mals. The collagen fibril diameters were analyzed using transmission electron photomicrographs of fibril cross sections taken at six periods during the 2 years after surgery. The patellar tendon and posterior cruciate liga ment were characterized by a broad, nongaussian dis tribution of collagen fibril diameters. The autografts shifted to a unimodal distribution by an increase of small-diameter collagen fibrils. The frequency of small- diameter fibrils measuring up to 100 nm was 99% after 2 years. At that time, these small-diameter fibrils rep resented 91.6% of the area covered by collagen fibrils. The mean diameter of the collagen fibrils in the au tografts significantly decreased to 45% of the controls at Week 26 and remained at this level until the end of this study. The percentage of area covered by collagen fibrils per 1 μm2 was 78% of the controls 2 years post operatively. This study suggests that the patellar tendon autograft could not reproduce the collagen fibril orga nization of the posterior cruciate ligament. This may be a biologic factor responsible for inconsistent results in posterior cruciate ligament replacement.

The relationship of mechanical properties to morphology in patellar tendon autografts after posterior cruciate ligament replacement in sheep

In a sheep model the posterior cruciate ligament (PCL) was replaced by a patellar tendon autograft (PTAG) using the central one-third of the ipsilateral patellar tendon (PT). The sheep were sacrificed at 16, 26, 52 and 104 weeks postoperation. The PTAG, and, as controls, the contralateral PCL and PT were harvested. These were examined using biomechanical testing as well as light and transmission electron microscopy, including immunohistological techniques. The material properties (maximum stress, elastic modulus) were compared to the morphological features. The cellular distribution, the distribution of glycosaminoglycans (GAGs), the collagen fibril diameter and the occurrence of Type III collagen were studied. Prior to transplantation, the PTAG was shown to be superior in maximum stress (57.2 +/- 5.5 MPa vs 41.3 +/- 1.9 MPa) and elastic modulus (368.8 +/- 49.3 MPa vs 172.3 +/- 14.6 MPa) to the PCL. The early decline in material properties of the PTAG (maximum stress 22% and elastic modulus 42% of the control) after free grafting paralleled a cell- and capillary-rich PTAG tissue with remnants of necrosis and a poorly organized extracellular matrix. Two years after implantation, with progressive alignment of the tissue matrix, maximum stress and elastic modulus acquired approximately 60 and 70% of the control, respectively. However, there was also an evidence of degenerative changes characterized by acellular areas, loss of the normal bundling pattern of collagen fibers and abnormal accumulation of GAGs. Ultrastructurally, there was a predominant shift to thin collagen fibrils in the PTAG compared to PCL and PT, both consisting of thick and thin collagen fibrils. Thin fibrils were demonstrated to be, in part, split thick fibrils as well as newly formed fibrils. Most of these thin fibrils revealed a positive reaction with antibodies to Type III collagen.

Membrane-associated carbonic anhydrase IV in skeletal muscle: subcellular localization

Carbonic anhydrase IV (CA IV) was examined by light microscopy and electron microscopy in rat soleus muscle. Semithin sections of aldehyde-fixed Epon-embedded muscle were stained with rabbit anti-rat lung CA IV and the avidin-biotin-peroxidase complex. With this technique, capillaries and sarcolemma showed positive CA IV staining. For electron microscopy, rat soleus specimens were aldehyde-fixed, with or without subsequent osmication, and embedded in Epon. Ultrathin sections were immunostained with anti-rat lung CA IV/immunogold. Omitting osmium allowed ample antigen-antibody reactions but could not prevent the release of glycosylphosphatidylinositol-anchored CA IV from the membranes, which led to apparent background staining. Postosmication significantly reduced tissue antigenicity but kept the antigen bound to the membranes and thus allowed a very precise localization of CA IV. By electron microscopy, membrane-bound CA IV is found to be associated with capillary endothelium, sarcolemma, and sarcoplasmic reticulum (SR). Conceivably, the presence of SR staining in ultrathin sections and its absence in semithin sections reflect a problem of accessibility of the antigenic sites.

Biological aspects of long-term failure of autografts after cruciate ligament replacement

SummaryThe alterations of the ultrastructure of the posterior cruciate ligament autograft of patellar tendon origin were examined in a sheep model 1 year after surgery. The ultrastructure was also compared with that of the normal contralateral posterior cruciate ligament and patellar tendon. The most striking finding was the unimodal distribution of the collagen fibrils, with a predominance of loosely packed thin fibrils in the central portion of the autograft. The results suggested that the remodeled autograft tissue became highly organized but never exhibited the ultrastructural features of a ligament. This could be responsible for the decreased biomechanical properties and the long-term failure of a patellar tendon autograft.ZusammenfassungDie ultrastrukturellen Veränderungen eines freien Patellarsehnentransplantates beim hinteren Kreuzbandersatz wurden in einem Schafsmodell 1 Jahr nach Transplantation untersucht. Die Feinstruktur der normalen kontralateralen Patellarsehne und des hinteren Kreuzbandes wurden mit der des Transplantates verglichen. Die unimodale Verteilung der Fibrillendurchmesser zugunsten dünner Fibrillen im zentralen Bereich des Transplantates werden als Ursache für die verminderten biomechanischen Eigenschaften und die langfristige Transplantatinsuffizienz diskutiert.

Alterations of glycosaminoglycans during patellar tendon autograft healing after posterior cruciate ligament replacement a biochemical study in a sheep model

In each of 30 skeletally mature sheep, the posterior cruciate ligament was replaced in one knee by a free patellar tendon autograft using the central third of the ipsilateral patellar tendon. The healing autograft was compared with the contralateral posterior cruciate ligament and the patellar tendons and posterior cruciate ligaments of nonoperated animals. The content of glycosaminoglycans, chondroitin sulfate disaccharides, and dermatan sulfate disaccharides was assessed bio-chemically at six periods during the 2 years after surgery. The total glycosaminoglycans and chondroitin sulfate disaccharides in the native posterior cruciate ligament was threefold that in the native patellar tendon. In contrast, the amount of dermatan sulfate disaccharides was similar in both the native tendon and native ligament. In the autograft, glycosaminoglycans and chondroitin sulfate disaccharides increased significantly to about 144% and 172%, respectively, of the contralateral posterior cruciate ligament at Week 104. The dermatan sulfate disaccharides in the autograft also showed a significant increase up to Week 26, followed by a remarkable but not significant decrease until the end of the study. In the contralateral posterior cruciate ligament, the dermatan sulfate disaccharides increased significantly between Weeks 52 and 104. Thus, the amount of dermatan sulfate disaccharides was similar in both the autograft and the contralateral posterior cruciate ligament after 2 years. This study suggests that the patellar tendon autograft did not completely assume the biochemical properties of the posterior cruciate ligament.

Filtration Characteristics of the Single Isolated Perfused Glomerulus of Myxine glutinosa

Using an in vitro microperfusion technique, the filtration characteristics of single isolated glomeruli of the Atlantic hagfish (Myxine glutinosa) were investigated. The suitability of the method as a model to study glomerular function was evaluated. Experiments with a protein-free perfusate led to a filtration coefficient Kf (0.189 +/- 0.181 nl x s-1 x mm Hg-1) that was in the same order of magnitude as determined in vitro for other vertebrates. Morphometric analysis on serial thin sections (1 microns) revealed a glomerular capillary surface (A) of 1.84 +/- 0.72 mm2. A linear relationship between glomerular diameter and A allowed the calculation of the hydraulic conductivity Lp (0.618 +/- 0.384 microliter x s-1 x mmHg-1 x cm-2). The data indicate that the isolated perfused glomerulus of M. glutinosa has filtration characteristics similar to higher vertebrates. The glomeruli of M. glutinosa, which in contrast to glomeruli of other animals are easy to dissect, are a suitable model for the study of glomerular filtration and permeability characteristics of vertebrates. An example of a possible application of the model is a study into the effects of adrenaline on glomerular filtration characteristics. Adrenaline led to increases in ultrafiltration pressure (PUF), single nephron glomerular filtration rate (SNGFR) and filtration fraction (FF); Kf remained unaffected.

Histochemical aspects of the proteoglycans of patellar tendon autografts used to replace the posterior cruciate ligament

In the female German black-faced sheep the posterior cruciate ligament was replaced by a free patellar tendon autograft and after 2, 6, 16, 26 and 52 weeks tissue samples of the grafts center (axial region far from bones) were removed for histochemistry and electron microscopy. To localize the proteoglycans Alcian Blue and 0.3 M MgCl2 were added to the fixative solution. The distribution of the proteoglycans in the graft was compared to that of a normal patellar tendon and of a normal posterior cruciate ligament. In the patellar tendon spindle-shaped cells predominated and proteoglycans appeared as short filaments at regular intervals between the collagen fibrils. In the posterior cruciate ligament chondroid cells and long filaments in a net-work-like arrangement were seen. In the patellar tendon autografts short interfibrillar filaments prevailed after 2, 6 and 16 weeks. After 26 weeks and particularly after 52 weeks long filaments also appeared. Digestion with Chondroitinase ABC, AC and Hyaluronidase suggested that the short filaments were PGs containing dermatan sulfate. In grafts, in the early phases the fibroblasts predominated, while in the late phases mainly chondroid cells were observed. The grafts showed aspects of the normal posterior cruciate ligament. However, differences remained, for example the thin collagen fibrils, which could represent one of the reasons for a secondary graft failure.

Changes in the extracellular matrix of the autogenous patellar tendon graft after posterior cruciate ligament reconstruction: a biochemical study in sheep

The left knee joints of female German sheep were operated, replacing the posterior cruciate ligament by the central third of the patellar tendon. After 2, 6, 16, 26 and 52 weeks the graft of the operated leg as well as the contralateral central third of the patellar tendon and the posterior cruciate ligament were dissected and used for biochemical analysis. The total glycosaminoglycan content in grafts increases within the first year up to 5 fold and is higher than that of the patellar tendon, but is lower than that of the cruciate ligament. The distribution pattern of glycosaminoglycans differ in the posterior cruciate ligament and the patellar tendon. In cruciate ligament the main components are chondroitin sulfate (76%) and hyaluronan (15%). In the patellar tendon a higher portion of dermatan sulfate (approx. 16%) was found, next to 52% chondroitin sulfate and 22% hyaluronan. During the examination time the grafts show changes in the concentration and the distribution pattern of glycosaminoglycans. The chondroitin sulfate content increases during the experimental period from 1.4 +/- 1.2 mumol/g dry weight (d.w.) to 8.7 +/- 2.9 mumol/g d.w. After 1 year the chondroitin sulfate content in the graft does not differ significantly from that of the cruciate ligament. In the grafts the concentration of chondroitin (non sulfated) increases after 6 weeks up to 1.3 +/- 0.6 mumol/g d.w. in comparison to the value after 2 weeks (0.2 +/- 0.1 mumol/g d.w.) and also in the other groups (16, 26 and 52 weeks) it remains significantly increased. After 1 year the dermatan sulfate content in the graft has increased up to the fifth-fold compared to the value after 2 weeks and is higher than in the patellar tendon and in the cruciate ligament. In graft the hyaluronan content (1.0 +/- 0.4 mumol/g d.w. does not differ significantly in the groups 2, 6, 16, 26 and 52 weeks after operation, but in all five groups it is lower than in the cruciate ligament (2.4 +/- 1.0 mumol/g d.w.). Dermatan and heparan sulfate are not or only little detected in all three tissues. The distribution pattern of glycosaminoglycans in graft shows chondroitin sulfate and dermatan sulfate being the major parts of glycosaminoglycans with a slight increase of these components in the different groups during the experimental period. Hyaluronan makes up to 24 +/- 5% of the whole content of glycosaminoglycans after 2 weeks and decrease to 8 +/- 1% after 52 weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

Primary skeletal muscle cells cultured on gelatin bead microcarriers develop structural and biochemical features characteristic of adult skeletal muscle

A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T‐tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regarding the expression of isoforms of myosin heavy chains (MHC), we could demonstrate that from day 16 on, no non‐adult MHC isoform mRNAs are present. Instead, on day 28 the myotubes express predominantly adult fast MHCIId/x mRNA and protein. This MHC pattern resembles that of fast muscles of adult rabbits. In contrast, primary myotubes grown on matrigel‐covered culture dishes express substantial amounts of non‐adult MHC protein even on day 21. To conclude, primary myotubes grown on microcarriers in their later stages exhibit many features of adult skeletal muscle and characteristics of fast type II fibers. Thus, the culture represents an excellent model of adult fast skeletal muscle, for example, when investigating molecular mechanisms of fast‐to‐slow fiber‐type transformation.