Immunohistochemical expression of collagen types I, III, IV and alpha-actin in the uterine horns of nulliparous and multiparous beagles.

Collagen and smooth muscle cells play essential roles in the remodelling of uterine tissue during pregnancy and involution. To investigate the immunoreactivity and distribution pattern of collagen types I, III, IV and smooth muscle alpha-actin resulting from these processes, two homogenous groups of nulliparous and multiparous beagles were evaluated by immunohistochemistry. Immunostaining patterns of collagens I and III delineated the uterine connective tissue fibers and revealed their dual presence within fibers of both beagle groups. Collagen III staining, in particular, was more pronounced and especially evident in superficial fiber sections. The numerous, large arteries in the myometrial stratum vasculare of multiparous uteri exhibited a highly thickened intima, which distinctly expressed type I and III collagens. Intense collagen IV immunolabeling was discernable in the basement membranes of vascular endothelia and smooth muscle cells. Staining of the basement membranes of the luminal and glandular epithelia, conversely, was either absent or very weak. No difference in the immunoreactivity and distribution of the assessed collagens and actin could be detected between nulliparous and multiparous dogs. Overall, and with the exception of sclerotic arteries, immunohistochemical analysis revealed that the expression of uterine collagens and actin does not change in the uterus of multiparous beagles, even after seven elapsed pregnancies.

Influence of ovariectomy on the canine striated external urethral sphincter (M. urethralis): a stereological analysis of slow and fast twitch fibres.

Slow and fast twitch fibres were stereologically analysed in the morphologically defined and most strongly developed ventral and ventrolateral region of the external urethral sphincter (M. urethralis) using five sexually intact anestrous and five ovariectomized nulliparous beagles. The estimated mean total cross-sectional area of the investigated part of the muscle consisted of 4.2 mm2 (15.1%) type I fibre, 10.0 mm2 (32.1%) type II fibre, and 16.3 mm2 (52.8%) connective tissue in the control group. The corresponding absolute mean value of type I fibres (3.7 mm2/13.5%) was statistically lower in ovariectomized animals. No significant difference between groups was observed in the relative number of transverse profiles of type I and II fibres; type I fibres comprised 23.8% and type II 76.2% of all muscle fibres in the sexually intact group, but 21.8% and 78.2% in the ovariectomy group, respectively. The ovariectomized dogs exhibited a statistically significant lower type I and II fibre number and a concomitant slightly larger mean single profile area (diameter) of fibre type II compared with the control animals. The significantly reduced number and decreased total cross-sectional area of the fatigue-resistant type I fibres in ovariectomized dogs suggest a predominant weakening of the fibre type I portion of the M. urethralis as consequence of ovariectomy. The effect could be mediated by sex hormonal factors and may contribute to the development of postspaying urinary incontinence in female dogs.

Elastic fibre system of the female canine urethra. Histochemical identification of elastic, elaunin and oxytalan fibres.

The elastic system fibres of the female urethra were investigated in seven dogs of different breeds. The fibres were stained and differentiated with orcein, Verhoeff's iron hematoxylin, and pararosanilin-based aldehyde fuchsin, with and without previous oxidation. Orcein and aldehyde fuchsin revealed some subepithelial longitudinally orientated delicate fibres (elaunin fibres) and numerous coarse longitudinal fibres (elastic fibres) in the deeper subepithelial connective tissue containing the vascular plexus, as well as a network of fibres of different calibers in the periurethral connective tissue. Elastic system fibres were also found in association with the sinusoids of the vascular plexus and the urethral smooth musculature. Verhoeff's iron hematoxylin only reacted distinctly with coarse (elastic) fibres. When applied following oxidation, aldehyde fuchsin disclosed an extensive meshwork of additional delicate fibres (oxytalan fibres) in the subepithelial connective tissue adjacent to the basement membrane. Oxytalan fibres were also discernible in the sinusoidal adventitia of the vascular plexus, as well as between smooth and striated muscle fibres. Due to the predominantly longitudinal orientation of the elastic system fibres and the low urethral resistance to manual expression of urine from the bladder post mortally, when the elasticity of the urethra is still intact and the activity of other continence factors can be excluded, the elastic tissue is not judged to be a major contributory factor to urinary continence.

Stereological analysis of the urethra in sexually intact and spayed female dogs.

The perfusion-fixed urethrae of 5 sexually intact nulliparous and 5 ovariohysterectomized beagles were analyzed stereologically. Different urethral tissue components were estimated stereologically in each of the three proximal urethral quarters and compared between the two groups of dogs. Considerable individual variability within all groups was observed. In all the three urethral quarters considered, average smooth muscle and connective tissue volumes were generally lower in spayed than in intact animals, both in absolute and relative terms. However, only the difference between the mean relative volumes of urethral quarter I was statistically significant. In contrast, mean absolute and relative volumes of the vascular plexus were generally greater in the ovariohysterectomized dogs as compared to the animals of the other group--deviations in statistical relevance concerning the first urethral quarter were noted. Mean epithelial and striated muscle volumes did not vary essentially between the spayed and intact group of dogs. Despite the fact that all findings were not always statistically meaningful, our research nevertheless demonstrates that ovariohysterectomy decreases smooth muscle mass and connective tissue; it also elevates vascular plexus volumes in the urethra of spayed animals. Moreover, our results imply a general weakening of the urethral wall as a sequel to ovariohysterectomy, and this, in turn, could be seen to affect the closure mechanism of the urethra.

Morphological, histochemical and stereological analysis of the female canine M. urethralis.

The M. urethralis was morphologically investigated in ten medium-sized female dogs of different breeds and age, as well as histochemically and stereologically analysed in a homogeneous group of five female beagles. Macroscopically, the muscle was essentially confined to the distal third and most strongly developed in the fourth quarter of the urethra. Here, it surrounded the urethra transversely at the ventral and lateral aspects, passing with its caudal fibres dorsally onto the vagina. The muscle fibres were assembled in groups of different sizes and usually separated by thick connective tissue septae. Based on the myofibrillar actomyosin ATPase (mATPase) reaction, type I and two main subtype II fibres could be differentiated. Type II fibres were, however, indistinguishable by their metabolic enzyme activities since both subclasses displayed oxidative-glycolytic properties. The subtype II fibres containing the more acid-labile mATPase activity were classified as IIA, whereas the other main subtype was designated IIS (subtype) and considered as peculiar to the dog. In addition, the investigation revealed a rare fibre type exhibiting the histochemical profile of IIC fibres. There was no evidence of classical glycolytic IIB fibres. The M. urethralis was composed of 24% type I and 76% type II fibres with an average diameter of 34.9 and 28.5 microns, respectively. Connective tissue constituted 52.8% of the total muscle volume. Due to the predominance of fast twitch II fibres, the urethral muscle is not designed to maintain a high tone over long periods of time. Its primary role is to function rapidly and intermittently guaranteeing urinary continence during stress situations when other continence factors are overburdened.

Morphology and stereology of the female canine urethra correlated with the urethral pressure profile.

Perfusion-fixed urethrae of five sexually intact nulliparous female beagles in anestrus, between 2 and 3 years of age, were morphologically and morphometrically investigated. Absolute and relative volumes of different tissue components were stereologically estimated per urethral quarter. A discrete smooth muscle sphincter could neither be identified in the bladder neck nor in the urethra. However, the circularly arranged smooth muscle fibers in the proximal half of the urethra were regarded as being capable of sphincteric function. The connective tissue, composed of collagenous and longitudinally orientated numerous elastic fibers, accounted for the most predominant constituent. It comprised 77.3 to 78.2% of the total volume in the proximal three urethral quarters. The vascular plexus was most prominently developed in the proximal two urethral quarters, and its relative and absolute volumes significantly exceeded those of the third quarter. The discovered direct connections between small arteries and the sinusoids of the vascular plexus emphasize its importance as a major contributory component to urinary continence. The striated musculus urethralis was essentially confined to the distal half of the urethra and most strongly developed in the fourth quarter, where it surrounded the urethra transversely at its ventral and lateral aspects. The caudal muscle fibers continued dorsally to the vagina. A transitional epithelium bounded the urethral lumen. Its superficial cell layer progressively flattened towards the external urethral orifice, changing into a stratified squamous lining as also seen in the vestibulum. Recorded pressures, or pressure changes, could neither be correlated with volumes nor with morphology of particular tissue elements in the different urethral quarters.

In vitro growth characteristics of bovine herpesvirus 4 (BHV-4) as revealed by indirect immunofluorescence assay with monoclonal antibodies and polyvalent antisera.

The bovine herpesvirus 4 (BHV-4) isolate LVR-140 growing in MDBK cell cultures was investigated by indirect immunofluorescence (IF), using bovine antisera and 15 monoclonal antibodies (MAb). The cytopathogenic effect was discernible between 20 and 30 hours post inoculation (p.i.) and developed slowly as has been reported for other BHV-4 strains. According to the protein specificity as revealed by radioimmunoprecipitation and the intracellular localisation of reacting antigens as detected by IF, the MAb could be assigned to one of three groups. The initial antigen expression was first detected at 8 hours p.i. and was limited to a proportion of the infected cells only, despite the high multiplicity of infection used; infectious cell-associated progeny virus was first recognized at 12 hours p.i.