C. Van Ginneken

Canine Mammary Tumours, an Overview

Canine mammary tumours (CMTs) are the most common neoplasms in intact female dogs. Although the prevalence of these tumours decreases in regions where preventive ovari(ohyster)ectomy is performed, it remains an important disease entity in veterinary medicine. Moreover, treatment options are limited in comparison with human breast cancer. Nevertheless, recent human treatment protocols might have potential in bitches suffering from CMTs.

NITRIC OXIDE SYNTHASE EXPRESSION IN ENTERIC NEURONS DURING DEVELOPMENT IN THE PIG DUODENUM

Abstract The expression of the constitutive neural isoform of nitric oxide synthase (bNOS) is dynamic and thus forms an ideal parameter to evaluate whether development and region affect the enteric nervous system. By applying NADPH-diaphorase histochemistry on whole-mount preparations of the myenteric and submucosal plexuses and by using the ’unbiased counting frame’, a qualitative and quantitative description of bNOS-expression in enteric neurons in the pig duodenum in various developmental stage and region was obtained. Examinations were carried out on the oral and aboral duodenum of fetal pigs from the second half of gestation, of 1–2-day-old pigs and of 6–8-week-old pigs. In the pig duodenum, three enteric plexuses were readily distinguished: the inner submucous, the outer submucous and the myenteric plexuses. All three plexuses already harboured, to different degrees, bNOS-expressing neurons at midgestation. Although the enteric nervous system was present at midgestation, the enteric neurons had not yet reached their adult phenotype and morphology. During gestation, the number of inner submucous bNOS-expressing neurons increased approximately 50-fold, whereas after birth that number fell to about 10% of the prenatal value. During further postnatal development it returned to prenatal values. In addition, the number of bNOS-expressing myenteric neurons doubled postnatally. These changes favour a role for NO in mediating the development of enteric neurons and point to a greater necessity for inhibitory innervation in the adult pig as compared with the fetal pig. Furthermore, the number of bNOS-expressing outer submucosal and myenteric neurons was significantly higher in the oral duodenal segment compared with the aboral duodenal segment. This regional difference suggests that the oral duodenal segment is more prominently involved in the regulation of NO-mediated gastrointestinal processes than the aboral one. The developmentally and regionally dependent bNOS-expression can be explained by shifts and differences in the balanced system of hormones, presynaptic input and target-derived signals that affects neurotransmitter expression.

Stereologic Characteristics of Pig Small Intestine During Normal Development

Stereologic methods were used to study the behavior of the pigs intestinal wall during periods that are characterized by a high incidence of gastrointestinal disorders. For this purpose conventionally stained transverse and vertical paraffin sections were made of the small intestine (duodenum, jejunum, and ileum) of fetal, neonatal, and weaned pigs. The volumes of the intestinal walls were estimated using Cavalieris method. Subsequently, the surface density (Sv) of the tunica mucosa and the volume densities (Vv) of the different small intestinal elements were estimated. Finally, the surface and volumes per serosal surface area (Ss and Vs) were calculated. The decrease of Sv can be attributed to the finding that the mucosal surface increases to a lesser extent compared with the volume of the intestinal wall. The Vs of the various layers increased postnatally, illustrating that the intestinal wall thickens. Despite an increasing total mucosal surface, this postnatal thickening causes Ss to decline. Each of these changes is temporally related to dietary changes, an increased antigen load, and an increased need for protection. Additionally, the regional differences of the various parameters match the qualitative descriptions of the small intestine of the pig and relate to region-specific functions.

Nutritional interventions to prevent and rear low-birthweight piglets

Selection for hyperprolific sows, as a means of increasing litter size and profit, has resulted in an increased number of low-birthweight (LBW) piglets. These LBW piglets might suffer from increased morbidity and mortality during the early neonatal period. In addition, they show reduced growth performance, meat and carcass quality, which leads to an important economic loss for the farmer in the post-natal period. Therefore, nutritional interventions can be undertaken to prevent and rear LBW piglets. In the first part of this review, the preventive strategies at the sow level will be discussed. Approaches in preventing LBW piglets are to optimize the intrauterine environment via supplementing the sow during gestation. In the second part of this review, the interventions at the piglet level will be described. To increase the survival and growth rates of LBW piglets, one must focus on ensuring adequate colostrum and milk intake. Interventions include supplementing piglets, split nursing, split weaning and cross-fostering. Additional interventions increasing the probability of optimal post-natal food intake will be discussed.

Postnatal and diet-dependent increases in enteric glial cells and VIP-containing neurones in preterm pigs

Abstract  A mature enteric nervous system (ENS) is required to ensure a normal pattern of intestinal motility in order to regulate digestion after birth. We hypothesized that neuronal and glial components of the ENS would mature during the first postnatal days in preterm pigs that are a sensitive animal model of food intolerance and necrotizing enterocolitis (NEC). Stereological volume densities of the general neuronal population [assessed by βIII‐tubulin immunoreactivity (IR)] and subsets of neuronal (VIP‐IR and nitrergic IR) and glial cells (GFAP‐IR and S100‐IR) were determined in the small intestine of newborn preterm piglets (93% gestation), after 3 days of receiving total parenteral nutrition (TPN) and after 3 days of TPN plus 2 days of enteral feeding with sow’s colostrum or milk formula. Following TPN, VIP in the myenteric and inner submucous plexus and GFAP in the inner submucous plexus increased, while the relative volume of the total neuronal population remained constant. Introduction of enteral food induced variable degrees of food intolerance and NEC, especially after formula feeding, a diet that gave rise to a higher myenteric VIP and GFAP content in the inner submucous plexus than colostrum feeding. However, the ENS seemed unaffected by the presence of NEC‐like intestinal lesions. Nevertheless, this study shows that the ENS is highly plastic during the first days after premature birth and adapts in an age‐ and diet‐dependent manner. The observed postnatal adaptation in enteric VIP and GFAP may help to maintain intestinal homeostasis during suboptimal feeding regimens in preterm neonates.

Evaluation of immunohistochemical markers of lymphatic and blood vessels in canine mammary tumours.

Canine mammary tumours (CMTs) are the most common neoplasms in intact female dogs. Bitches with spontaneously arising CMTs represent a promising animal model for human breast cancer research. The aim of the present study was to develop an immunohistochemical protocol for the identification of blood and lymphatic vessels in CMTs. Antibodies specific for human lymphatic vessels (prox-1, lyve-1, podoplanin and D2-40) and blood vessels (von Willebrand factor [vWf], CD31 and CD34) were utilized. Serial sections of 18 samples (eight samples of normal canine mammary tissue, five benign and five malignant CMTs) were examined. Antibodies specific for podoplanin, D2-40 and CD34 showed no immunoreactivity with canine tissue. Prox-1 and CD31 were determined to be the most suitable markers for lymphatic and blood vessels, respectively.

Age-related differences in mucosal barrier function and morphology of the small intestine in low and normal birth weight piglets.

To test the hypothesis that the mucosal maturation of the small intestine is altered in low birth weight piglets, pairs of naturally suckled low birth weight (LBW, n = 20) and normal birth weight (NBW, n = 20) littermate piglets were selected and sampled after 0, 3, 10, and 28 d of suckling. In vivo intestinal permeability was evaluated via a lactulose-mannitol absorption test. Other indirect measurements for mucosal barrier functioning included sampling for histology and immunohistochemistry (intestinal trefoil factor [ITF]), measuring intestinal alkaline phosphatase (IAP) activity, and immunoblotting for occludin, caspase-3, and proliferating cell nuclear antigen (PCNA). The lactulose-mannitol ratio did not differ between NBW and LBW piglets, but a significant increase in this ratio was observed in 28-d-old piglets (P = 0.001). Small intestinal villus height did not differ with age (P = 0.02) or birth weight (P = 0.20). In contrast, villus width (P = 0.02) and crypt depth (P < 0.05) increased gradually with age, but no birth-weight-related differences were observed. LBW piglets had significantly (P = 0.03) more ITF immunoreactive positive cells per villus area compared to NBW piglets, whereas no age (P = 0.82) or region-related (P = 0.13) differences could be observed. The activity of IAP in the small intestine was higher in newborn piglets compared to the older piglets. No significant differences in cell proliferation in the small intestine was observed (P = 0.47) between NBW and LBW piglets; the highest proliferation was seen in piglets of 28 d of age (P = 0.01). Newborn piglets had significantly fewer apoptotic cells, whereas more apoptotic cells were seen in piglets of 10 d of age (P < 0.01). In conclusion, birth weight did not affect the parameters related to intestinal barrier function investigated in this study, suggesting that the mucosal barrier function is not altered in LBW piglets. Nevertheless, these results confirm that the mucosal barrier function in the small intestine of piglets alters with age.

Supplementing formula-fed piglets with a low molecular weight fraction of bovine colostrum whey results in an improved intestinal barrier

To test the hypothesis that a low molecular weight fraction of colostral whey could affect the morphology and barrier function of the small intestine, 30 3-d-old piglets (normal or low birth weight) were suckled (n = 5), artificially fed with milk formula (n = 5), or artificially fed with milk formula with a low molecular weight fraction of colostral whey (n = 5) until 10 d of age. The small intestine was sampled for histology (haematoxylin and eosin stain; anti-KI67 immunohistochemistry) and enzyme activities (aminopeptidase A, aminopeptidase N, dipeptidylpeptidase IV, lactase, maltase, and sucrase). In addition, intestinal permeability was evaluated via a dual sugar absorption test and via the measurement of occludin abundance. Artificially feeding of piglets reduced final BW (P < 0.001), villus height (P < 0.001), lactase (P < 0.001), and dipeptidylpeptidase IV activities (P < 0.07), whereas crypt depth (P < 0.001) was increased. No difference was observed with regard to the permeability measurements when comparing artificially fed with naturally suckling piglets. Supplementing piglets with the colostral whey fraction did not affect BW, enzyme activities, or the outcome of the dual sugar absorption test. On the contrary, the small intestines of supplemented piglets had even shorter villi (P = 0.001) than unsupplemented piglets and contained more occludin (P = 0.002). In conclusion, at 10 d of age, no differences regarding intestinal morphology and permeability measurements were observed between the 2 BW categories. In both weight categories, the colostral whey fraction affected the morphology of the small intestine but did not improve the growth performances or the in vivo permeability. These findings should be acknowledged when developing formulated milk for neonatal animals with the aim of improving the performance of low birth weight piglets.

A stereological evaluation of secretin and gastric inhibitory peptide-containing mucosal cells of the perinatal small intestine of the pig

Stereological methods were used to quantify secretin and gastric inhibitory peptide (GIP)‐immunoreactivity (GIP‐IR) in paraffin sections of the duodenum, jejunum and ileum of fetal and neonatal piglets. In addition, sections were processed for GLP‐1‐immunohistochemistry. The volume density of the tunica mucosa increased after birth, giving rise to a decreased volume density of the tela submucosa and tunica muscularis. Generally known region‐specific morphological distinctions were reflected in differing volume densities of the various layers. The highest volume density of GIP‐IR epithelial cells was observed in the jejunum of the neonate. In contrast, the volume density of secretin‐IR epithelial cells was highest in the duodenum of both fetal and neonatal piglets. The volume occupied by GIP‐IR and secretin‐IR epithelial cells increased in the jejunum after birth. Additionally, ileal secretin‐IR epithelial cells were more numerous in the neonatal piglet. In conclusion, the quantitative and qualitative presence of GIP‐IR and secretin‐IR epithelial cells agree with earlier reports of their presence and co‐localization between GIP‐IR and GLP‐1‐IR, in the pig small intestine. Furthermore, the differences suggest that age‐ and region‐related functional demands are temporally and probably causally related with the morphological diversification of the intestine and its endocrine cells.

Intrinsic Innervation of the Stomach of the Fetal Pig: an Immunohistochemical Study of VIP‐Immunoreactive Nerve Fibres and Cell Bodies

Using an immunohistochemical technique, the presence and distribution of vasoactive intestinal polypeptide (VIP) was investigated in cryostat sections, both tangential and transverse, of the fetal pigs stomach. In all fetuses and in all gastric segments investigated, VIP‐like immunoreactive (IR) nerve‐cell bodies were seen in all intramural ganglia, and VIP‐IR nerve fibres were found in all layers of the gastric wall except the tunica serosa. Consequently, VIP‐IR nerve fibres were found to form a periglandular network, to accompany arterioles, to interconnect the intramural ganglia, to encircle both VIP‐IR‐negative and ‐positive neurons, and were found in all muscle layers. Despite the fact that VIP‐IR seems to be restricted to the intramural nervous elements, some nonspecific‐reacting VIP‐IR glandular cells were noticed in the basal parts of the fundic, antral and pyloric gastric glands.