Influence of macronutrient composition of commercial diets on circulating leptin and adiponectin concentrations in overweight dogs.

Leptin and adiponectin play important roles in obesity-related inflammation and comorbidities. Previous research suggests that alterations in dietary macronutrient composition can modify circulating leptin and adiponectin concentrations in people, but limited research on this subject has been performed in dogs. This study investigated the effects of commercial high protein (HP), high fat (HF) and high carbohydrate medium protein (HCMP) diets on baseline (T-1 ) concentrations, post-prandial peak concentrations and total release in a ten-hour time span of leptin and adiponectin in dogs, when compared to a maintenance high carbohydrate low protein (HCLP) diet. Thirty-six overweight dogs were fed the HCLP diet in a one-week control period, after which the animals were assigned to one of three groups. In three four-week periods, each group was fed all test diets in a different sequence. At the last day of each period, blood was sampled at one hour before feeding (T-1 ) and at three (T3 ), six (T6 ) and nine (T9 ) hours after feeding. Feeding caused peak leptin concentrations at T6 and T9 (p < .001). No significant post-prandial change in adiponectin concentrations was found (p = .056). The HP diet resulted in lower leptin peak concentrations (p = .004) and AUCT-1-T9 (p = .01), but none of the diets influenced baseline leptin concentrations (p = .273). Baseline adiponectin concentrations were lower for the HF diet (p = .018) and HCMP (p < .001), and the HP, HF and HCMP AUCT-1-T9 (p < .001) were lower compared with the HCLP diet. Female dogs had lower adiponectin baseline concentrations (p = .041) and AUCT-1-T9 (p = .023) than male dogs. In conclusion, the HP diet was associated with the lowest post-prandial peak leptin concentration and the least decrease in adiponectin release, suggesting that a HP diet may improve immune-metabolic health and post-prandial satiety in overweight dogs.

Inflammatory profiles in canine intervertebral disc degeneration.

BACKGROUND: Intervertebral disc (IVD) disease is a common spinal disorder in dogs and degeneration and inflammation are significant components of the pathological cascade. Only limited studies have studied the cytokine and chemokine profiles in IVD degeneration in dogs, and mainly focused on gene expression. A better understanding is needed in order to develop biological therapies that address both pain and degeneration in IVD disease. Therefore, in this study, we determined the levels of prostaglandin E2 (PGE2), cytokines, chemokines, and matrix components in IVDs from chondrodystrophic (CD) and non-chondrodystrophic (NCD) dogs with and without clinical signs of IVD disease, and correlated these to degeneration grade (according to Pfirrmann), or herniation type (according to Hansen). In addition, we investigated cyclooxygenase 2 (COX-2) expression and signs of inflammation in histological IVD samples of CD and NCD dogs. RESULTS: PGE2 levels were significantly higher in the nucleus pulposus (NP) of degenerated IVDs compared with non-degenerated IVDs, and in herniated IVDs from NCD dogs compared with non-herniated IVDs of NCD dogs. COX-2 expression in the NP and annulus fibrosus (AF), and proliferation of fibroblasts and numbers of macrophages in the AF significantly increased with increased degeneration grade. GAG content did not significantly change with degeneration grade or herniation type. Cytokines interleukin (IL)-2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, immune protein (IP)-10, tumor necrosis factor (TNF)-α, and granulocyte macrophage colony-stimulating factor (GM-CSF) were not detectable in the samples. Chemokine (C-C) motif ligand (CCL)2 levels in the NP from extruded samples were significantly higher compared with the AF of these samples and the NP from protrusion samples. CONCLUSIONS: PGE2 levels and CCL2 levels in degenerated and herniated IVDs were significantly higher compared with non-degenerated and non-herniated IVDs. COX-2 expression in the NP and AF and reactive changes in the AF increased with advancing degeneration stages. Although macrophages invaded the AF as degeneration progressed, the production of inflammatory mediators seemed most pronounced in degenerated NP tissue. Future studies are needed to investigate if inhibition of PGE2 levels in degenerated IVDs provides effective analgesia and exerts a protective role in the process of IVD degeneration and the development of IVD disease.

Long-Term Survival of Transplanted Autologous Canine Liver Organoids in a COMMD1-Deficient Dog Model of Metabolic Liver Disease.

The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression. Organoid-derived adult liver stem cells can be cultured indefinitely to create sufficient cell numbers for transplantation, and they are amenable to gene correction. This study provides preclinical proof of concept of the potential of cell transplantation in a large animal model of inherited copper toxicosis, such as Wilson's disease, a Mendelian disorder that causes toxic copper accumulation in the liver. Hepatic progenitors from five COMMD1-deficient dogs were isolated and cultured using the 3D organoid culture system. After genetic restoration of COMMD1 expression, the organoid-derived hepatocyte-like cells were safely delivered as repeated autologous transplantations via the portal vein. Although engraftment and repopulation percentages were low, the cells survived in the liver for up to two years post-transplantation. The low engraftment was in line with a lack of functional recovery regarding copper excretion. This preclinical study confirms the survival of genetically corrected autologous organoid-derived hepatocyte-like cells in vivo and warrants further optimization of organoid engraftment and functional recovery in a large animal model of human liver disease.

Intradiscal application of rhBMP-7 does not induce regeneration in a canine model of spontaneous intervertebral disc degeneration.

INTRODUCTION: Strategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain. METHODS: Canine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 µg, 25 µg, and 250 µg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue. RESULTS: In vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 µg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 µg and 250 µg of rhBMP-7. CONCLUSIONS: An intradiscal bolus injection of 2.5 µg, 25 µg, and 250 µg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 µg rhBMP-7, and to a lesser extent 25 µg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients.

Mammary steroid metabolizing enzymes in relation to hyperplasia and tumorigenesis in the dog.

Progesterone and estradiol play a crucial role in the control of mammary gland proliferation and tumour formation in the dog. However, little is known whether steroid metabolizing enzymes are present within the canine mammary gland that may play a modulating role in the bioavailability of progesterone and estrogen. In this study we investigated the expression of the steroid metabolizing enzymes 5alpha-reductase (type I and type II) and aromatase in relation to hyperplasia or tumorigenesis in the canine mammary tissue. The relative mRNA concentrations were examined by a semi-quantitative reverse-transcriptase PCR analysis (RT-PCR). In addition the affinity of dihydroprogesterone (5alpha-reduced metabolite of progesterone) for canine progesterone receptors was investigated. Quantification of the RT-PCR products revealed that in mammary tumours a significantly higher expression of aromatase is present in comparison to normal mammary tissue. Furthermore, significant decrease in expression of both aromatase and 5alpha-reductase type II enzymes was found in hyperplasic mammary tissue compared to tumours. The changes in expression of type II 5alpha-reductase and aromatase were highly correlated. 5alpha-Reduction of progesterone to dihydroprogesterone resulted in a six-fold less affinity for the canine progesterone receptor. It is concluded that hyperplasia is associated with a decreased expression of type II 5alpha-reductase and aromatase enzymes, whereas in tumours the opposite situation is found.

Expression stability of reference genes for quantitative RT-PCR of healthy and diseased pituitary tissue samples varies between humans, mice, and dogs.

Pituitary surgery generates pituitary tissue for histology, immunohistochemistry, and molecular biological research. In the last decade, the pathogenesis of pituitary adenomas has been extensively studied in humans, and to a lesser degree in dogs, and tumor oncogenesis has been studied in knock-out mice, often by means of quantitative reversed-transcriptase PCR (RT-qPCR). A precondition of such analyses is that so-called reference genes are stably expressed regardless of changes in disease status or treatment. In this study, the expression of six frequently used reference genes, namely, tata box binding protein (tbp), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (ywhaz), hydroxymethylbilane synthase (hmbs), beta-2-microglobulin (b2m), succinate dehydrogenase complex subunit A (sdha), and glyceraldehyde 3 phosphate dehydrogenase 1 (gapdh), was studied in pituitary tissue (normal and adenoma) from three species (humans, mice, and dogs). The stability of expression of these reference genes differed between species and between healthy and diseased tissue within one species. Quantitative analysis based on a single reference gene that is assumed to be stably expressed might lead to wrong conclusions. This cross-species analysis clearly emphasizes the need to evaluate the expression stability of reference genes as a standard and integral aspect of study design and data analysis, in order to improve the validity of the conclusions drawn on the basis of quantitative molecular analyses.