G. Klima

Anatomy and innervation of the rhabdosphincter of the male urethra.

The striated sphincter of the male urethra and its innervation are still a subject of controversy. Essentially, two concepts of its anatomy can be found in the literature. Some authors describe the rhabdosphincter as part of the urogenital diaphragm caudal to the prostate, others as a striated muscle which extends from the base of the bladder to the “urogenital diaphragm.”

A comparative study of three different biomaterials in the engineering of skeletal muscle using a rat animal model

Defects caused by traumatic or postsurgical loss of muscle mass may result in severe impairments of the functionality of skeletal muscle. Tissue engineering represents a possible approach to replace the lost or defective muscle. The aim of this study was to compare the suitability of three different biomaterials as scaffolds for rat myoblasts, using a new animal model. PKH26-fluorescent-stained cultured rat myoblasts were either seeded onto polyglycolic acid meshes or, alternatively, suspended in alginate or in hyaluronic acid-hydrogels. In each of the eight Fisher CDF-344 rats, four capsule pouches were induced by subcutaneous implantation of four silicone sheets. After two weeks the silicone sheets were removed and myoblast-biomaterial-constructs were implanted in the preformed capsules. Specimens were harvested after four weeks and examined histologically by H&E-staining and fluorescence microscopy. All capsules were well-vascularized. Implanted myoblasts fused by forming multinucleated myotubes. This study demonstrates that myoblasts seeded onto different biomaterials can be successfully transplanted into preformed highly vascularized capsule pouches. Our animal model has paved the way for studies of myoblast-biomaterial transplantations into an ectopic non-muscular environment.

Histologic Pattern of Small Bowel Allograft Rejection in the Rat

Accessory small bowel transplantation was performed in a rat model, and the histologic sequence of acute rejection with and without immunosuppression with cyclosporine defined. The first rejection episodes occurred on the fifth postoperative day, defined as phase I. The mucosa and submucosa of the grafts were infiltrated by mononuclear and polynuclear leukocytes, and lymphocytes were scattered along the myenteric ganglia. Three to five days later (phase II) the infiltrate intensified and was associated with villous flattening and sloughing of epithelial cells. The muscular layer was invaded by numerous lymphocytes and neutrophils. At the end of the rejection episode complete destruction of the mucosa and the muscular layer developed (phase III). As no lesion other than lymphocytic infiltration could be detected in the mucosa in phases I and II of rejection, which may be considered reversible, and this change is known to be unspecific, biopsies of the mucosa alone are inadequate for a purely histologic diagnosis of small bowel allograft rejection.

Reconstruction of the lower urinary tract using autologous muscle transfer and cell seeding: current status and future perspectives

Abstract Functioning free-muscle transfer has established itself for the treatment of skeletal muscle deficiency over the last two decades. The capability of skeletal muscle to empty a spherical reservoir has been shown to be lessened if the muscle has contracted due to dissection. Currently there is no established curative treatment for bladder acontractility. Experimental data and preliminary clinical results have shown that innervated free latissimus dorsi muscle (LD) may serve as a substitute for a dysfunctional detrusor. In a clinical protocol, latissimus dorsi detrusor myoplasty (LDDM) was applied in 11 patients (age 9–68 years) with bladder acontractility due to spinal cord injury (seven patients), congenital malformations (two patients), detrusor myopathy (one patient), and idiopathic causes (one patient) who had required catheterization for bladder emptying for a minimum of 2 years. In all, 10 of 11 patients were capable of voiding volitionally, with eight of them no longer requiring catheterization, throughout the follow-up period of 12–46 months. Transplanted LD activity was confirmed by ultrasonography and flow-mode computerized tomography. It can thus be concluded that emptying of the bladder can be induced through the contractility of reinnervated free LD that has been wrapped around the bladder. An innervated free-LD flap does not undergo the severe muscle fibrosis, contracture, and atrophy that occur after transfer of completely or partially denervated pedicled muscle. Apart from the restoration of deficient detrusor function, microneurovascular transfer of a free-LD flap may be combined with tissue engineering to serve as a basis for bladder augmentation and substitution.

Rejection of kidney and pancreas after pancreas-kidney transplantation.

On the basis of 26 combined pancreas-kidney transplants we questioned whether both organs undergo rejection simultaneously. Reliable diagnosis of pancreas-graft rejection was made possible by monitoring exocrine graft function, including quantitative measurements of pancreatic juice, its amylase content, and pancreatic juice cytology. In addition, diagnosis of pancreas rejection was based on regular flow studies, daily urinary neopterin excretion, and a retrospective analysis of the clinical course. Clinical symptoms, blood chemistry, and, primarily, histology were used to assess rejection of the kidney allograft. In 18 cases the kidney and pancreas were rejected together; in 8 cases the kidney or the pancreas was rejected. Although both organs were rejected at the same time in most cases, either organ can be rejected alone. Thus, the kidney cannot be used to monitor the pancreas allograft in every case.

Preservation of Small Bowel Grafts – A Comparison with Two Standard Solutions

This study investigates the use of EuroCollins (EC) and University of Wisconsin (UW) solution, two major preservation fluids, for small bowel preservation. After in situ flushing, grafts were cold-stored at 4 degrees C with either EC for 30 min (group 1a), 6 h (group 1b) and 12 h (group 1c) or with UW for 30 min (group 2a), 6 h (group 2b) and 12 h (group 2c). Using UW, cold ischemia was extended to up to 18 h (group 2d). As a control, small intestines were flushed and stored for the same time periods in cold saline (group 3a-c). Survival in group 1b was 66% versus 100% in group 2b. After 12 h 80% survived in group 2c, but there were not survivors in group 1c. After 18 h of cold storage, survival was only observed in group 2d (25%). Saline was ineffective after 6 h of preservation. Histology at the end of preservation revealed characteristic changes for EC (intracellular vacuoles) and UW (amorphic granules). We conclude that with UW small bowel can be preserved for up to 12 h.

Regeneration of Sympathetic Activities in Small Bowel Transplants

In order to study the activity of noradrenergic nerve fibres along mesenteric arteries in small-intestinal grafts, the entire jejunoileum was transplanted heterotopically in an isogeneic rat model (group I, n = 12). To assess their influence on graft motility, 9 cm of jejunum were transplanted in an orthotopic position and three bipolar electrodes sutured to the seromuscular layer of the graft (group II, n = 10). Fasting motility was recorded up to postoperative day 42. Animals of group I were sacrificed from day 7 on at weekly intervals and mesenteries were analysed histochemically by fluorescence microscopy. After the 1st and 2nd week, grafts were found to be completely depleted of noradrenaline. At the end of the 3rd week, fluorescence became detectable along graft mesenteric arteries and showed normal intensity from the end of week 4. Migrating myoelectric complexes (MMCs) were present in all animals of group II. The variability of the MMC period (mean 12.6 min; SD 6.2 min) expressed as variation coefficient (median 36.5; 14.6-74) did not change during the observation period. From these findings it is concluded that there are no extrinsic noradrenergic activities in rat small-intestinal transplants for the first 3 postoperative weeks but they do recover there after. Their influence on graft function remains unclear: MMC periodicity, however, was no influenced.

Myoelectric activity during small bowel allograft rejection

The effect of rejection on myoelectric activity of an orthotopically transplanted small intestinal segment (group I,N=14) was studied. Electrodes were placed on grafts and recipient small bowel. Isografts (group II,N=5) and native bowel (group III,N=5) served as controls. The first morphological signs of rejection were seen on day 6 and steadily progressed until day 11, when the cellular infiltrate involved all layers of the bowel wall. Slow-wave frequencies remained unchanged throughout the observation period. No difference was detectable between grafts (group I: 31.9±1.65; group II: 31.36±0.7) and native bowel after transection (group I: 32.16±1.78; group II: 31.50±1.01), which was different (P=0.0001) from intact bowel of group III animals (38.4±0.81). Irregular MMCs were detectable in grafts from day 5 on and replaced after food intake by random spiking activities. At day 8, spiking activities disappeared in allografts, which showed a still preserved mucosal architecture, while slow-wave activities continued. These findings demonstrate that intestinal allografts during rejection develop paralysis before mucosal destruction is established, which might be of clinical relevance.

Increase in Intraepithelial Lymphocytes as an Early Marker of Rejection in a Fully Allogeneic Rat Small Bowel Transplantation Model

In a model of fully allogeneic heterotopic rat small bowel transplantation, the changes in intraepithelial lymphocyte (IEL) number and subpopulations were analysed. During early phases of rejection (5th postoperative day) a 4-fold increase in the number of IEL was observed when compared with native small bowel (4.05 vs. 15.84 IEL/100 epithelial cells). When cyclosporine was given, counts were still as high as 11.4 and 12.58 on the 5th and 10th postoperative days, respectively. The percentage of CD8+ IEL, constituting a major population (84%) in the untreated small bowel, was significantly decreased (46.4%) during early phases of rejection. At that time, the majority of intraepithelial mononuclear cells were both CD8- and CD4-. In cyclosporine-treated animals, this was not observed until the 10th postoperative day. Some 23% of IEL in untreated animals expressed MHC class I antigens of the host; 17.2% (5th postoperative day) and 19.8% (10th postoperative day) did so in the cyclosporine-treated animals. Transmission electron microscopy revealed lymphocytes that bore cytoplasmic buds and pseudopods protruding between the enterocytes. There was no morphological difference between the IEL of rejected allografts and native small bowel. Due to the unspecific histologic changes associated with rejection, interpretation of histopathologic findings in mucosa biopsies of the allograft can be rather troublesome. An increase in the number of IEL is therefore a welcome additional marker of rejection.

Altered distribution of MHC class II antigens on enterocytes during acute small bowel allograft rejection in rats

Class II major histocompatibility complex (MHC) antigen induction was investigated on enterocytes of heterotopic rat small bowel allografts in the Lewis-Brown Norway strain combination and on isografts in the Lewis-Lewis strain combination. I a antigens were detected with monoclonal antibodies using an immunoperoxidase technique. Generally, MHC class II antigens were not exhibited in the isografted group, with the exception of two long-term isografts that presented the same pattern as normal small bowel. In these cases, I a was expressed in a patchy distribution predominantly in the villi, and only very few enterocytes stained positive in Lieberkühns crypts. Allografted rats showed a typical pattern of I a expression on the enterocytes during the rejection course. The initial expression was confined to the crypts, indicating a very early stage of rejection when compared to histological findings. More advanced stages of rejection were accompanied by increasing I a biosynthesis in the crypts and I a expression by the epithelium lining the villi. Cyclosporin (CyA) was not able to fully inhibit MHC class II antigen expression; however, the appearance of I a was delayed.