Stereology and computer assisted three-dimensional reconstruction as tools to study probiotic effects of Aeromonas hydrophila on the digestive tract of germ-free Artemia franciscana nauplii.

AIMS: Validation of stereology and three-dimensional reconstruction for monitoring the probiotic effect of Aeromonas hydrophila on the gut development of germ-free Artemia franciscana nauplii. METHODS AND RESULTS: Germ-free Artemia nauplii were cultured using Baker's yeast and dead Aer. hydrophila. Live Aer. hydrophila were added on the first day to the treatment group. The gut length and volume were monitored on days two and four using stereology and three-dimensional reconstruction. Both methods showed comparable results. Stereology was least labour intensive to estimate volumes, while three-dimensional reconstructions rendered architectural and topographical data of the gut. Moreover, a positive effect of probiotic bacterium, Aer. hydrophila is likely. CONCLUSION: Slight increment in the growth of the digestive tract of A. franciscana nauplii exerted by probiotic bacteria could be detected using stereology and three-dimensional reconstruction. SIGNIFICANCE AND IMPACT OF THE STUDY: The gnotobiotic Artemia rearing system is unique to investigate the effects of micro-organisms on the development of nauplii. However, in the base of this model system, only survival counts and length measurements exist as monitoring tools. Therefore, additional tools such as stereology and three-dimensional reconstruction are prerequisite to obtain more powerful analysis.

Locations of gut-associated lymphoid tissue in the 3-month-old chicken: a review.

The lymphoid tissue that is associated with the intestinal tract, the so-called gut-associated lymphoid tissue (GALT), is well developed in the chicken. Depending on the location, it is present as aggregations of lymphoid cells, or organized in lymphoid follicles and tonsils. From proximal to distal, the intestinal tract contains a pharyngeal tonsil, diffuse lymphoid tissue and lymphoid follicles in the cervical and thoracic parts of the oesophagus, an oesophageal tonsil, diffuse lymphoid tissue in the proventriculus, a pyloric tonsil, Peyer's patches, Meckel's diverticulum, two caecal tonsils, diffuse lymphoid tissue in the rectum, the bursa of Fabricius, and diffuse lymphoid tissue in the wall of the proctodeum. The lymphoid tissues are frequently covered by a lympho-epithelium that is infiltrated by lymphoid cells. Such an epithelium often contains M or microfold cells, which are specialized in antigen sampling and transport antigens to the underlying lymphoid tissue. A solid knowledge of the avian GALT could contribute to the development of vaccines to be administered orally. Additionally, immune stimulation via pre- and probiotics is based on the presence of a well-developed intestinal immune system.

Absence of arterial phenotype in mice with homozygous slc2A10 missense substitutions.

Arterial tortuosity syndrome (ATS, MIM# 208050) is a rare autosomal recessive connective tissue disease, mainly characterized by widespread arterial involvement with elongation, tortuosity, and aneurysms of the large and middle-sized arteries (Callewaert et al., 2008, Hum Mutat 29:150-158). Recently, mutations were identified in the SLC2A10 gene encoding the facilitative glucose transporter GLUT10 (Coucke et al., 2006, Nat Genet 38:452-457). It was hypothesized that loss-of-function of the transporter results in upregulation of the transforming growth factor beta (TGFbeta) signaling pathway (Coucke et al., 2006, Nat Genet 38:452-457). We anticipated that a mouse model would help to gain more insight in the complex pathophysiological mechanism of human ATS. Here, we report that two mouse models, homozygous respectively for G128E and S150F missense substitutions in glut10 do not present any of the vascular, anatomical, or immunohistological abnormalities as encountered in human ATS patients. We conclude that these mouse strains do not phenocopy human ATS and cannot help the further elucidation of pathogenetic mechanisms underlying this disease.

Anatomy of the lower respiratory tract in domestic birds, with emphasis on respiration.

This manuscript describes the anatomy of the lower respiratory tract in domestic bird species including the chicken and pigeon. The here described anatomical structures play a major role avian respiration, which is fundamentally different from respiration in mammals. During inspiration and expiration, a continuous caudocranial airflow is present within the tertiary bronchi of the Paleopulmo, while the Neopulmo, which is only present in phylogenetically recent species, is characterized by tidal respiration. Various anatomical structures and aerodynamic mechanisms have been described in an attempt to explain the proposed mechanism of respiration. The air sac system that is essential for avian respiration usually comprises an unpaired clavicular air sac and paired cervical, cranial and caudal thoracic, and abdominal air sacs. The latter are by far the larger and are interwoven with the abdominal organs.

Anatomy of the upper respiratory tract in domestic birds, with emphasis on vocalization.

This work reviews the anatomy of the upper respiratory tract in domestic birds including the chicken and pigeon. Non-exhaustive additional information on other bird species, illustrating the extraordinary diversity in the biological class Aves, can be found in several footnotes. The described anatomical structures are functionally considered in view of avian sound production. In particular, the Syrinx is invaluable. Its most important structures are the Labia and the lateral and medial tympaniform membranes in non-songbirds and songbirds, respectively. These structures produce sound by vibrating during expiration and eventually inspiration.

Neurons expressing serotonin-1B receptor in the basolateral nuclear group of the amygdala in normally behaving and aggressive dogs.

The present study aimed to quantify neurons expressing the serotonin-1B receptor and evaluate numerical differences in normally behaving and pathologically aggressive dogs in order to assess whether the serotonin-1B receptor is involved in pathological canine aggression. Because previous studies have reported structural alterations in the basolateral nuclear group (BNG) of the amygdaloid body of aggressive dogs, this structure was selected as region of interest in the present study. Indirect immunohistochemistry was applied to visualise the serotonin-1B-receptor-positive neurons. Immunoreactivity was located predominantly within the neuronal cell bodies and adjacent neuronal processes. In the aggressive dogs the BNGs contained a significantly higher number of serotonin-1B-receptor-positive neurons compared to the normally behaving dogs. This number was strongly correlated with the total number of neurons per BNG, which was also significantly increased in aggressive dogs compared to normal dogs. The percentage of neurons expressing the serotonin-1B receptor did not differ significantly between both groups. No significant asymmetries were observed for the number and percentage of serotonin-1B-receptor-positive neurons. Potential relationships between the present findings and the etiology of aggressive behaviour, the neuroprotective role of the serotonin-1B receptor and receptor dysfunction are discussed.

Cell-specific localization of progesterone receptors in the bovine ovary at different stages of the oestrous cycle.

This immunohistochemical study describes the localization of progesterone receptors (PR) in the bovine ovary of 23 cows at different stages of the oestrous cycle. In primordial, primary and secondary follicles the score for PR in the follicle cells increased progressively with the maturation of the follicle. In vital tertiary follicles and cystic atretic follicles a moderate score for PR was found, while in obliterative atretic follicles the score was much lower. Scores were high in corpora hemorrhagica, low in corpora lutea and still lower in corpora albicantia. Low PR scores were also found in the tunica albuginea and surface epithelium. Cyclic variations of PR immunoreactivity were manifest in most ovarian tissues. Follicular scores for PR were high in oestrus and decreased during the following stages, whereas scores in corpora lutea cells varied according to a characteristic pattern with high levels during oestrus and metoestrus. The variations in the scores for PR in the different ovarian cell types suggest a cell-specific and cycle-dependent influence of progesterone. A negative correlation was found between the PR scores and the plasma progesterone concentration.

Anatomical Description of the Presence and Variability of the Digital Manica Flexoria in the Equine Digital Flexor Tendon Sheath.

During endoscopy (tenoscopy) of the distal aspect of the equine digital flexor tendon sheath (DFTS), the digital manica flexoria can be visualized connecting the distal branches of the superficial digital flexor tendon. However, this structure has been inconsistently described and variably named in the veterinary literature. The objectives of this study were to describe the presence, configuration and variability of the digital manica flexoria in the equine distal limb. Dissection of 144 equine cadaveric limbs revealed the presence of this structure in all the feet, although different types and conformations were identified. In the forelimbs, a membranous digital manica flexoria predominated (94%; P < 0.001), in particular a synovial bridge type (83%; P < 0.001). In the hindlimbs, a tendinous digital manica flexoria predominated (93%; P < 0.001), in particular the oblique-crossing of tendinous bundles (61%; P < 0.001). Passage dorsal to the digital manica flexoria towards the distal DFTS was only possible in 22 of the 144 limbs, all forelimbs. Clinicians should be aware of the intra- and inter-individual anatomical variations of the digital manica flexoria to avoid misinterpretation during ultrasonographic and tenoscopic examinations of the DFTS.

Increased volume and neuronal number of the basolateral nuclear group of the amygdaloid body in aggressive dogs.

Involvement of the basolateral nuclear group (BNG) in pathological canine aggression was assessed by stereological determination of the volume of the BNG and quantification of the numerical density and total number of BNG neurons in normally behaving and aggressive dogs. A bilateral BNG enlargement of 40% was observed in the aggressive group. This enlargement appeared to be caused by a significantly increased number of BNG neurons. Other alterations such as an increased vessel density, oedema and scar tissue were not observed in any of the examined BNGs. The potential role of neurotrophins and stress hormones in the increased number of BNG neurons is discussed.

Neurokinin-1 receptor in the basolateral nuclear group of the canine amygdala--comparative study in normal and aggressive dogs.

Substance P and its NK-1 receptor are involved in the modulation of aggressive behavior. Because of the role of the basolateral nuclear group (BNG) of the amygdala in canine aggression, neurokinin-1 receptor (NK-1) immunoreactivity in this brain region was assessed stereologically in 7 normally behaving and 6 pathologically aggressive dogs. The first aim of this study was to obtain information about the absolute number of neurons expressing the NK-1 receptor in the canine BNG because absolute numbers of neurons expressing the NK-1 receptor are not documented in literature. Additionally, an exploratory comparison was made between NK-1 expressing neurons in the BNGs of normally behaving and aggressive dogs. Results showed a very low amount (1-2%) of BNG neurons containing the NK-1 receptor in both groups. Aggressive dogs had significantly more NK-1-receptor-positive BNG neurons than normal dogs, but the numerical densities and fractions of receptor-positive neurons did not differ significantly between both groups. Combined with the fact that aggressive dogs have 27% more neurons in their BNGs than normal dogs, as reported in a previous study, these findings suggest a limited role for the NK-1-receptor-positive neurons within the BNG in the modulation of canine aggression. The present report of absolute numbers of neurons expressing the NK-1 receptor in the canine BNG could however be useful for further quantitative studies.

Cell-specific localisation of apoptosis in the bovine ovary at different stages of the oestrous cycle.

Apoptosis was localized in all ovarian cell types of 23 cows in various stages of the oestrous cycle, using the detection of active caspase-3, in situ end labelling (TUNEL) and DNA fluorescent staining (DAPI). Very few apoptotic cells were found in primordial, primary, secondary and vital tertiary follicles. In contrast, apoptosis in atretic tertiary follicles was much more frequent, and high apoptotic scores were recorded when using the TUNEL technique and lower scores with the caspase-3 assay. Cystic atretic follicles showed in general a higher apoptotic score than obliterative atretic follicles, with intermediate to high scores in granulosa cells and lower scores in theca cells. In corpora lutea, large and small lutein cells had intermediate to high scores using the caspase-3 assay, and intermediate to low scores using the TUNEL assay. Irrespective of the detection method, the scores were higher in lutein cells than in the capsular stroma cells. In all ovarian structures examined, variations in apoptotic scores were seen in the different cycle stages, suggesting a cycle-dependent influence on apoptosis, although correlations with plasma progesterone concentrations were low.

Morphology of the Canine Omentum Part 2: The Omental Bursa and its Compartments Materialized and Explored by a Novel Technique.

The canine omental bursa is a virtual cavity enclosed by the greater and lesser omentum. While previous representations of this bursa were always purely schematic, a novel casting technique was developed to depict the three-dimensional organization of the omental bursa more consistently. A self-expanding polyurethane-based foam was injected into the omental bursa through the omental foramen in six dogs. After curing and the subsequent maceration of the surrounded tissues, the obtained three-dimensional casts could clearly and in a reproducible way reveal the omental vestibule, its caudal recess and the three compartments of the splenic recess. The cast proved to be an invaluable study tool to identify the landmarks that define the enveloping omentum. In addition, the polyurethane material can easily be discerned on computed tomographic images. When the casting technique is preceded by vascular injections, the blood vessels that supply the omentum can be outlined as well.

Morphology of the Canine Omentum Part 1: Arterial Landmarks that Define the Omentum.

Although the omentum remains an enigmatic organ, research during the last decades has revealed its fascinating functions including fat storage, fluid drainage, immune activity, angiogenesis and adhesion. While clinicians both in human and veterinary medicine are continuously exploring new potential omental applications, detailed anatomical data on the canine omentum are currently lacking, and information is often retrieved from human medicine. In this study, the topographic anatomy of the canine greater and lesser omentum is explored in depth. Current nomenclature is challenged, and a more detailed terminology is proposed. Consistent arteries that are contained within folds of the superficial omental wall are documented, described and named, as they can provide the anatomical landmarks that are necessary for unambiguous scientific communication on the canine omentum. In an included dissection video, the conclusions and in situ findings described in this study are demonstrated.

Topography and ultrasonographic identification of the equine pulmonary vein draining pattern.

Information on ultrasound examination of equine pulmonary veins is scarce due to a lack of in-depth anatomical information. Each pulmonary vein drains a specific lung lobe region, after which those veins merge into a collecting antrum, before opening into the left atrium through their respective ostia. The aim of this study was, by using anatomical dissection and silicone casting of equine cardiopulmonary sets, to study the venous drainage of both lungs and the position of the ostia and to investigate whether the ostia can be identified and differentiated using ultrasound. Three out of the four ostia could be observed echocardiographically in the standing horse. The ostium draining the most caudal aspects of both lungs showed little variability, while the ostium draining the rest of the right lung could be used as an easily recognisable landmark, since it was located adjacent to the interatrial septum. The identification of the equine pulmonary vein ostia using ultrasound might allow for the determination of size and flow patterns in the assessment of cardiovascular disease.

Quantification of neurons expressing androgen receptor and volume estimation of the basolateral nuclear group of the canine amygdaloid body.

A protocol was developed for the stereological quantification of neurons expressing androgen receptor (AR) in the basolateral nuclear group (BNG) of the canine amygdaloid body. The Cavalieri method was used to estimate the BNG volume and the physical disector technique was applied for assessing the numerical densities and total numbers of both ordinary and AR-positive BNG neurons. The overall number of BNG neurons and the BNG volume were assessed on Nissl-stained sections, while AR was visualised using indirect immunohistochemistry. The morphological differentiation between neurons, astrocytes, and oligodendrocytes in these immunohistochemical sections was hampered by the cytoplasmic localisation of AR in these cells. Therefore, an additional criterion was developed based on the nuclear diameters of these cells. With the cutoff value of 7.4 microm, a sensitivity of 97.7% and specificity of 97.6% were obtained. A negative correlation was found between the BNG volume and the numerical density of its neurons, implicating that a large BNG will not necessarily have a higher number of neurons. Therefore, the numerical density or BNG volume should always be assessed in addition to the total number of neurons, justifying the use of the physical disector instead of the fractionator technique in the present study. However, higher coefficients of error were obtained for the total number of neurons with the physical disector method because of the indirect measurement of cell numbers. Therefore, the precision of the estimates must be high enough when using the disector method to compensate the precision loss caused by this indirect calculation of the total cell number.

Anatomical localisation and histology of the ovine tonsils.

The topography and histologic structure of the various tonsils were studied anatomically and microscopically in 15 sheep aged between 9 and 15 months. The palatine, pharyngeal and paraepiglottic tonsils were readily visible macroscopically. They consisted mainly of secondary lymph nodules and were encapsulated in dense connective tissues. The epithelium covering the tonsils and their crypts was frequently infiltrated heavily by lymphocytes. The tubal tonsil and the tonsil of the soft palate were macroscopically visible after fixation in 2% acetic acid. These tonsils consisted of scattered lymph nodules, aggregations of lymphocytes and diffuse lymphoid tissue. They were not encapsulated, and therefore the borders of these tonsils could not be clearly delineated. The lingual tonsil was not macroscopically visible in sheep and consisted of scattered small aggregations of lymphocytes.

Morphology of caprine skin glands involved in buck odour production.

The distribution and morphology of the cornual, sub-caudal, mental and preputial glands were studied macro- and microscopically in four Toggenburg and eight miniature male goats. Although the cornual and sub-caudal glands could be readily located macroscopically, the mental glands in the inter-mandibular region and the preputial glands at the preputial orifice were not visible macroscopically. On histological section, all glands were found to be composed of lobulated sebaceous tissue combining both normal and modified holocrine secretory units. Over a period of 18 months, five consecutive glandular swabs for scent tests were taken to assess the influence of age and season on buck odour production. Buck odour was most apparent in the cornual gland area, less distinct at the mental gland region, and faint or absent in the other glandular areas. Surgical removal of the cornual glands caused a decrease in buck odour and persisting scent was ascribed to smaller skin glands dispersed in the cranial body half. Complete absence of buck odour was only observed in castrated bucks.

Variability in the eruption of the permanent incisor teeth in sheep.

The incisor teeth of 176 sheep of six breeds were inspected every two to three months for a year to record the shedding of the deciduous teeth and the eruption of the permanent teeth. In all the breeds the permanent central incisors erupted at between 12 and 18 months of age. In 96 per cent of the sheep the permanent middle incisors erupted at between 18 and 26 months; and in 92 per cent the permanent lateral incisors erupted at between 24 and 36 months of age. The permanent corner teeth erupted at between 32 and 44 months in 96 per cent of the sheep. The gingival redness and swelling accompanying the eruption of a permanent tooth disappeared within two months. In 14 cases two pairs of incisors erupted during the year, in 18 cases the incisors erupted asymmetrically, and in 22 cases no incisors erupted. Rotation of one incisor was observed in five sheep and was combined with dental deviation in one.

The pulmonary veins of the pig as an anatomical model for the development of a new treatment for atrial fibrillation.

The layout of the porcine atriopulmonary junction and immediately adjacent structures was investigated by gross anatomical and vascular corrosion casting studies to meet the need for more in-depth anatomical insights when using the pig as an animal model in the development of innovative approaches for surgical cardiac ablation in man. The veins from the right cranial and middle lung lobes drain through a common ostium in the left atrium, whereas a second ostium receives the blood returning from all other lung lobes, although limited variation to this pattern was observed. Surrounding anatomical structures that are most vulnerable to ablation damage as reported in man are located at a safer distance from the pulmonary veins in pigs, yet a certain locations, comparable risks are to be considered. Additionally, it was histologically confirmed that myocardial sleeves extend to over a centimetre in the wall of the pulmonary veins.

The ligaments of the canine hip joint revisited.

Numerous conventional anatomical textbooks describe the canine hip joint, but many contradictions, in particular regarding the ligament of the femoral head, are present. This paper presents a brief overview of the different literature descriptions. These are compared with own observations that have resulted in a revised description of the anatomy of the ligament of the femoral head in the dog. To this purpose, the hip joints of 41 dogs, euthanized for reasons not related to this study and devoid of lesions related to hip joint pathology, were examined. It was observed that the ligament of the femoral head is not a single structure that attaches only to the acetabular fossa, as generally accepted, but it also connects to the transverse acetabular ligament and is complemented by a strong accessory ligament that courses in caudal direction to attach in the elongation of the acetabular notch that extends on the cranioventral surface of the body of the ischium. The description of this accessory ligament in conventional anatomical handbooks is incomplete. This description of the accessory ligament of the femoral head could support the research unravelling the etiopathogenesis of hip instability.