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Central regulation of food intake during aging in the teleost fish Nothobranchius furzeri
Abstracts / Annals of Anatomy 207 (2016) 118–126
144.8 ± 12 (HP3), and 218.5 ± 23 (HP30) (CTR vs HP3 and HP30 P < 0.05; HP-3 vs HP30 P < 0.001); G␣gust -IR cells were 126.9 ± 26 (CTR), 158.4 ± 24 (HP3), 214 ± 26 (HP30) (CTR vs HP30 and HP3 vs HP30 P < 0.05). In the pyloric mucosa, G␣tran /5-HT-IR cells were 107.8 ± 28 (CTR), 141.3 ± 14 (HP3) and 207.5 ± 22 (HP-30) (HP30 vs CTR and HP3 P < 0.01), similarly the G␣gust /5-HT-IR cells were 127.3 ± 44 (CTR), 161 ± 13 (HP3) and 203.3 ± 24 (HP30) (HP30 vs CTR and HP3 P < 0.05). In pyloric mucosa, G␣tran /GHR-IR cells were 76 ± 22 (CTR), 106 ± 24 (HP3) and 191.5 ± 15 (HP30) (HP30 vs CTR and HP3 P < 0.01) likewise G␣gust /GHR-IR cells were 64.5 ± 23, 111.8 ± 33, 189.5 ± 34 in CTR, HP3 and HP30, respectively (HP30 vs CTR and HP3 P < 0.05). We demonstrated that HP (30d > 3d) evoked an increased density of G␣gust /G␣tran in 5-HT- and GHR -IR cells lending support to TR-mediated effects in metabolic homeostasis and satiety mechanisms. http://dx.doi.org/10.1016/j.aanat.2016.04.017 A canine serum free skin culture model for basic research studies and testing therapeutics V. Miragliotta 1,∗ , A. Pirone 1 , C. Lenzi 1 , M.P. Ricciardi 1 , M.F. della Valle 2 , F. Abramo 1 1 Department of Veterinary Sciences, University of Pisa, Pisa, Italy 2 Science Information and Documentation Centre (CeDIS), Innovet Italia SRL, Milano, Italy
Canine skin full thickness culture may offer unique opportunities both for overcoming the concerns on the use of animals for biomedical research and to study drug mechanism of action in a biologically relevant environment. Normal skin was obtained from donor dogs referred for mastectomy. Biopsy samples were cultured in triplicate in Williams’ E medium supplemented with penicillin/streptomycin, insulin, hydrocortisone and glutamine. General morphological features of epidermis, dermis and adnexa, morphometric assessment of epidermal thickness and keratinocyte proliferation (via Ki67 immunostaining) were assessed at day 1, 4 and 7. The effect of Epidermal Growth Factor (EGF) was also tested. Palmitoylethanolamide (PEA) was used to counteract mast cell degranulation induced by compound 48/80. General morphological features of skin anatomical structures were well maintained up to day 7, except adnexa which showed early sign of vacuolization mainly in the proliferative compartment of the bulb; scattered pyknotic nuclei were visible in the epidermis at day 7. Epidermal thickness decreased from day 0 to day 7. Keratinocyte proliferation decreased from day 1 to day 4 excluding peripheral areas of the cultured biopsies; no Ki67 positive nuclei were visible at day 7. Treatment with EGF induced the formation of “epithelial tongues” at the periphery of cultured biopsies. Mast cell degranulation induced by compound 48/80 was inhibited by PEA. The main concern of this model appeared to be interexperimental variability. Once thoroughly standardized this method may help in dissecting canine skin morpho-physiology and has the potential of characterizing the biological effects of dermatologic therapeutics. http://dx.doi.org/10.1016/j.aanat.2016.04.018
123
Investigation on skeletal development in small-sized breed newborn dogs: Anatomic and radiographic findings obtained by spontaneously died animals S.C. Modina 1,∗ , M.C. Veronesi 1 , M.E. Andreis 2 , V. Lodde 1 , B. Bolis 1 , M. di Giancamillo 2 1 Dipartimento di Scienze veterinarie e sanità pubblica, Università degli Studi di Milano, Milano, Italy 2 Dipartimento di Scienze veterinarie per la salute, la produzione animale e la sicurezza alimentare, Università degli Studi Milano, Milano, Italy
Species-specific measurements and ranges for normal skeletal growth are reliable reference data for various studies, including those related to age determination and disorders of growth and nutrition. In dog, limited information is available about normal skeletal growth characteristics and when present, they refer mostly to Beagles as an animal model. Because of the great number of pure breed dogs the main limitation of these studies relies on the need of homogeneous and sizeable populations; moreover each subject must be submitted to serial and multiple investigations that, although weakly invasive, are inadvisable in puppies, according to animal welfare rules. Finally, some analyses are not possible on living individuals. In this research we considered the possibility of using 27 remains of spontaneously died puppies (0–28 days) as a consistent population to study the skeletal development of small-sized breed dogs during the first month of life. These puppies belonged to different pure breeds and were categorized according to the standard breed adult body weight <7 kg (Brianza, 2006). Anatomic and radiographic measurements of limb bones length and of skull diameters were positively correlated with weight and age of the subjects and weight was positively correlated with radius bone mineral density (Spearman bivariate test, P < 0.01). Histological evidences confirmed the presence of the limb secondary ossification centres observed by X-rays and detailed the onset of their formation. These data suggest that cadavers of young animals may represent a useful tool to study skeletal development and possible disorders in dog. http://dx.doi.org/10.1016/j.aanat.2016.04.019 Central regulation of food intake during aging in the teleost fish Nothobranchius furzeri A. Montesano 1,2,∗ , N. Arcamone 1 , T. Genade 2 , P. de Girolamo 1 1
Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy 2 Biology Faculty, North Western College, Iowa, USA The mechanisms deputed to energetic control have been selected by ancestral diets resulting from the nutrient disposal during the evolution. They are regulated by a network of molecules controlling metabolic needs and influence caloric intake. Also the course of aging adds significant modifications to energy homeostasis and fuel metabolism in. An emerging model for aging research, a teleost fish Nothobranchius furzeri, the vertebrate with the shortest lifespan described in laboratory, is employed to investigate the regulation of Neuropeptide Y (NPY) and Orexin A and B in the hypothalamus. The study is carried out on specimens at different age stages and fed with different diets.
124
Abstracts / Annals of Anatomy 207 (2016) 118–126
Immunohistochemical analyses revealed the localization and co-labeling of NPY, Orexins A and B in some hypothalamic areas, as the dorsal hypothalamus (Hd) and diffuse inferior lobe (DIL). The co-labeled neurons were identified as serotoninergic. In addition, the median lifespan is prolonged of more than one week in subjects treated with hypocaloric diet. In the latter group, western blot analyses revealed high expression of NPY in the brain. These observations suggest that the role of NPY in food intake regulation is modulated during aging and hypocaloric regime, and is linked to the action of Orexin A. Furthermore, the co-presence of NPY and Orexin A in serotoninergic neurons could indicate the involvement of the two peptides also in the circadian rhythm regulation. http://dx.doi.org/10.1016/j.aanat.2016.04.020 Epithelial-like stem cells isolated from equine epidermis show regenerative capacities in vitro and in vivo M. Patruno 1,∗ , S. Broeckx 2 , T. Martinello 1 , C. Gomiero 1 , L. Maccatrozzo 1 , J. Spaas 2 1
Department of Comparative Biomedicine and Food Science, University of Padua, Padua, Italy 2 Global Stem Cell Technology-ANACURA group, Evergem, Belgium Stem cells have been found in hair follicles and dermis of mammals although also the epidermis contains a subpopulation of undifferentiated progenitor cells. However, only limited information concerning epidermis-derived epithelial-like stem/progenitor cells (EpSCs) is available, especially in a large animal model. In this research the purified cells from equine epidermis were characterized as EpSCs by means of positive expression for CD29, CD44, CD49f, CD90, Casein Kinase 2b, p63, and Ki67, low expression for cytokeratin (CK)14, negative expression for CD105, CK18, Wide CK, and Pan CK; in vitro, cells were differentiated toward keratinocytes and adipocytes fates. Moreover, to evaluate the regenerative capacities of EpSCs in vivo, six full-thickness skin wounds were performed in a 5-years old gelded horse: three were treated with allogeneic EpSCs and autologous platelet-richplasma (EpSC/PRP-treated) while three were treated with carrier fluid alone (PRP-treated). After 30 days the skin of EpSC/PRPtreated wounds was significantly thinner and exhibited increases in vascularization, elastin content, follicle-like structures and more restricted granulation tissue than the PRP-treated wounds, confirming that the stem cell treatment improved tissue repair after the clinical application. Recent data concern the comparison of autologous versus allogeneic EpSCs for treating the skin of our equine wound model (six French trotter mares). http://dx.doi.org/10.1016/j.aanat.2016.04.021 Effect of a mixture of short and medium chain fatty acids esterified to glycerol in milk replacer on weaning calves rumen papillae development L. Ragionieri ∗ , A. Cacchioli, F. Ravanetti, M. Botti, A. Ivanovska, F. Righi, A. Quarantelli, R. Panu, F. Gazza Department of Veterinary Sciences, University of Parma, Parma, Italy The supplementation of sodium butyrate in milk replacers (MR), commonly used to anticipate weaning, affects both small intestine and rumen development, determining earlier solid feed intake
which leads to better performances in cattle. The present study aims to determine the effects of a mixture of short and medium chain fatty acids esterified to glycerol (SMCFA) added to MR on rumen papillae development of weaning calves offered starter diet at libitum. Eight bull calves (10-days-old) were divided in two balanced groups: Control (C) and Treated (T). The latter received 0.2% SMCFA in MR besides starter diet. Animals were slaughtered at the age of 72 days. Histomorphometric analysis performed on samples from the cranio-dorsal sack of the rumen did not indicate differences in the mean number of papillae or in their total surface/cm2 of mucosa but rather in their shape. T group had more wide, short tonguelike or irregular shaped papillae and many interposed papillae compared to C group whose narrow, tongue-to-finger-like papillae, often showed secondary papillae or irregular branches. In T group, enhanced proliferation of the epithelial cells of the stratum basale, spinosum and granulosum was observed in the interpapillar region, while C group showed more cellular layers in the stratum corneum at the base of the papillae and in the interpapillar region. SEM analysis confirmed the different morphology of the papillae in the two groups and, at high magnification, revealed the presence of more dense cytoplasmic protrusions covering horn cell surfaces in T group. http://dx.doi.org/10.1016/j.aanat.2016.04.022 Calcitonin Gene-Related Peptide (CGRP) expression in the spinal cord and spinal ganglia of the Bottlenose Dolphin (Tursiops truncatus) A. Rambaldi 1,∗ , A. Grandis 1 , M. Mazzoni 1 , C. Tagliavia 1 , P. Clavenzani 1 , B. Cozzi 2 , C. Bombardi 1 1 Department of Veterinary Medical Science, University of Bologna, Bologna, Italy 2 Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro, Italy
CGRP is a neuropeptide especially involved in pain transmission. The localization of CGRP in the nervous system of different species has been extensively investigated, but no data are available on its distribution in the spinal cord and spinal ganglia of Cetaceans. Samples of spinal cord and spinal ganglia from three bottlenose dolphins (Tursiops truncatus) were used for this study. The tissue was stained with histological and immunohistochemical methods. Morphometric analysis of motor neurons and primary afferent neurons (PANs) of spinal ganglia was performed. In addition, the percentage of IR-neurons in both spinal cord and ganglia has been calculated. In the spinal cord the CGRP immunoreactivity was localized in small interneurons of laminae I-II and in large motoneurons of lamina IX. The immunostaining in motoneurons was mainly localized in the somata and appeared as a specific granular pattern. In the spinal cord intensely labelled fibers were observed in laminae I, II and X. In spinal ganglia the majority of CGRP-IR PANs showed a small or medium-sized somata and represented the 40% of the total neuronal population. The abundance of IR-fibers in laminae I and II is in agreement with the role played by this neuropeptide in the transmission of nociceptive stimuli. IR-fibers have also been observed in lamina X, which receives visceral afferent fibers; thus CGRP could be also involved in modulation of visceral sensibility. The presence of the CGRP in motoneurons suggests an interaction between these cells and skeletal muscle mediated by this neuropeptide. http://dx.doi.org/10.1016/j.aanat.2016.04.023
144.8 ± 12 (HP3), and 218.5 ± 23 (HP30) (CTR vs HP3 and HP30 P < 0.05; HP-3 vs HP30 P < 0.001); G␣gust -IR cells were 126.9 ± 26 (CTR), 158.4 ± 24 (HP3), 214 ± 26 (HP30) (CTR vs HP30 and HP3 vs HP30 P < 0.05). In the pyloric mucosa, G␣tran /5-HT-IR cells were 107.8 ± 28 (CTR), 141.3 ± 14 (HP3) and 207.5 ± 22 (HP-30) (HP30 vs CTR and HP3 P < 0.01), similarly the G␣gust /5-HT-IR cells were 127.3 ± 44 (CTR), 161 ± 13 (HP3) and 203.3 ± 24 (HP30) (HP30 vs CTR and HP3 P < 0.05). In pyloric mucosa, G␣tran /GHR-IR cells were 76 ± 22 (CTR), 106 ± 24 (HP3) and 191.5 ± 15 (HP30) (HP30 vs CTR and HP3 P < 0.01) likewise G␣gust /GHR-IR cells were 64.5 ± 23, 111.8 ± 33, 189.5 ± 34 in CTR, HP3 and HP30, respectively (HP30 vs CTR and HP3 P < 0.05). We demonstrated that HP (30d > 3d) evoked an increased density of G␣gust /G␣tran in 5-HT- and GHR -IR cells lending support to TR-mediated effects in metabolic homeostasis and satiety mechanisms. http://dx.doi.org/10.1016/j.aanat.2016.04.017 A canine serum free skin culture model for basic research studies and testing therapeutics V. Miragliotta 1,∗ , A. Pirone 1 , C. Lenzi 1 , M.P. Ricciardi 1 , M.F. della Valle 2 , F. Abramo 1 1 Department of Veterinary Sciences, University of Pisa, Pisa, Italy 2 Science Information and Documentation Centre (CeDIS), Innovet Italia SRL, Milano, Italy
Canine skin full thickness culture may offer unique opportunities both for overcoming the concerns on the use of animals for biomedical research and to study drug mechanism of action in a biologically relevant environment. Normal skin was obtained from donor dogs referred for mastectomy. Biopsy samples were cultured in triplicate in Williams’ E medium supplemented with penicillin/streptomycin, insulin, hydrocortisone and glutamine. General morphological features of epidermis, dermis and adnexa, morphometric assessment of epidermal thickness and keratinocyte proliferation (via Ki67 immunostaining) were assessed at day 1, 4 and 7. The effect of Epidermal Growth Factor (EGF) was also tested. Palmitoylethanolamide (PEA) was used to counteract mast cell degranulation induced by compound 48/80. General morphological features of skin anatomical structures were well maintained up to day 7, except adnexa which showed early sign of vacuolization mainly in the proliferative compartment of the bulb; scattered pyknotic nuclei were visible in the epidermis at day 7. Epidermal thickness decreased from day 0 to day 7. Keratinocyte proliferation decreased from day 1 to day 4 excluding peripheral areas of the cultured biopsies; no Ki67 positive nuclei were visible at day 7. Treatment with EGF induced the formation of “epithelial tongues” at the periphery of cultured biopsies. Mast cell degranulation induced by compound 48/80 was inhibited by PEA. The main concern of this model appeared to be interexperimental variability. Once thoroughly standardized this method may help in dissecting canine skin morpho-physiology and has the potential of characterizing the biological effects of dermatologic therapeutics. http://dx.doi.org/10.1016/j.aanat.2016.04.018
123
Investigation on skeletal development in small-sized breed newborn dogs: Anatomic and radiographic findings obtained by spontaneously died animals S.C. Modina 1,∗ , M.C. Veronesi 1 , M.E. Andreis 2 , V. Lodde 1 , B. Bolis 1 , M. di Giancamillo 2 1 Dipartimento di Scienze veterinarie e sanità pubblica, Università degli Studi di Milano, Milano, Italy 2 Dipartimento di Scienze veterinarie per la salute, la produzione animale e la sicurezza alimentare, Università degli Studi Milano, Milano, Italy
Species-specific measurements and ranges for normal skeletal growth are reliable reference data for various studies, including those related to age determination and disorders of growth and nutrition. In dog, limited information is available about normal skeletal growth characteristics and when present, they refer mostly to Beagles as an animal model. Because of the great number of pure breed dogs the main limitation of these studies relies on the need of homogeneous and sizeable populations; moreover each subject must be submitted to serial and multiple investigations that, although weakly invasive, are inadvisable in puppies, according to animal welfare rules. Finally, some analyses are not possible on living individuals. In this research we considered the possibility of using 27 remains of spontaneously died puppies (0–28 days) as a consistent population to study the skeletal development of small-sized breed dogs during the first month of life. These puppies belonged to different pure breeds and were categorized according to the standard breed adult body weight <7 kg (Brianza, 2006). Anatomic and radiographic measurements of limb bones length and of skull diameters were positively correlated with weight and age of the subjects and weight was positively correlated with radius bone mineral density (Spearman bivariate test, P < 0.01). Histological evidences confirmed the presence of the limb secondary ossification centres observed by X-rays and detailed the onset of their formation. These data suggest that cadavers of young animals may represent a useful tool to study skeletal development and possible disorders in dog. http://dx.doi.org/10.1016/j.aanat.2016.04.019 Central regulation of food intake during aging in the teleost fish Nothobranchius furzeri A. Montesano 1,2,∗ , N. Arcamone 1 , T. Genade 2 , P. de Girolamo 1 1
Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy 2 Biology Faculty, North Western College, Iowa, USA The mechanisms deputed to energetic control have been selected by ancestral diets resulting from the nutrient disposal during the evolution. They are regulated by a network of molecules controlling metabolic needs and influence caloric intake. Also the course of aging adds significant modifications to energy homeostasis and fuel metabolism in. An emerging model for aging research, a teleost fish Nothobranchius furzeri, the vertebrate with the shortest lifespan described in laboratory, is employed to investigate the regulation of Neuropeptide Y (NPY) and Orexin A and B in the hypothalamus. The study is carried out on specimens at different age stages and fed with different diets.
124
Abstracts / Annals of Anatomy 207 (2016) 118–126
Immunohistochemical analyses revealed the localization and co-labeling of NPY, Orexins A and B in some hypothalamic areas, as the dorsal hypothalamus (Hd) and diffuse inferior lobe (DIL). The co-labeled neurons were identified as serotoninergic. In addition, the median lifespan is prolonged of more than one week in subjects treated with hypocaloric diet. In the latter group, western blot analyses revealed high expression of NPY in the brain. These observations suggest that the role of NPY in food intake regulation is modulated during aging and hypocaloric regime, and is linked to the action of Orexin A. Furthermore, the co-presence of NPY and Orexin A in serotoninergic neurons could indicate the involvement of the two peptides also in the circadian rhythm regulation. http://dx.doi.org/10.1016/j.aanat.2016.04.020 Epithelial-like stem cells isolated from equine epidermis show regenerative capacities in vitro and in vivo M. Patruno 1,∗ , S. Broeckx 2 , T. Martinello 1 , C. Gomiero 1 , L. Maccatrozzo 1 , J. Spaas 2 1
Department of Comparative Biomedicine and Food Science, University of Padua, Padua, Italy 2 Global Stem Cell Technology-ANACURA group, Evergem, Belgium Stem cells have been found in hair follicles and dermis of mammals although also the epidermis contains a subpopulation of undifferentiated progenitor cells. However, only limited information concerning epidermis-derived epithelial-like stem/progenitor cells (EpSCs) is available, especially in a large animal model. In this research the purified cells from equine epidermis were characterized as EpSCs by means of positive expression for CD29, CD44, CD49f, CD90, Casein Kinase 2b, p63, and Ki67, low expression for cytokeratin (CK)14, negative expression for CD105, CK18, Wide CK, and Pan CK; in vitro, cells were differentiated toward keratinocytes and adipocytes fates. Moreover, to evaluate the regenerative capacities of EpSCs in vivo, six full-thickness skin wounds were performed in a 5-years old gelded horse: three were treated with allogeneic EpSCs and autologous platelet-richplasma (EpSC/PRP-treated) while three were treated with carrier fluid alone (PRP-treated). After 30 days the skin of EpSC/PRPtreated wounds was significantly thinner and exhibited increases in vascularization, elastin content, follicle-like structures and more restricted granulation tissue than the PRP-treated wounds, confirming that the stem cell treatment improved tissue repair after the clinical application. Recent data concern the comparison of autologous versus allogeneic EpSCs for treating the skin of our equine wound model (six French trotter mares). http://dx.doi.org/10.1016/j.aanat.2016.04.021 Effect of a mixture of short and medium chain fatty acids esterified to glycerol in milk replacer on weaning calves rumen papillae development L. Ragionieri ∗ , A. Cacchioli, F. Ravanetti, M. Botti, A. Ivanovska, F. Righi, A. Quarantelli, R. Panu, F. Gazza Department of Veterinary Sciences, University of Parma, Parma, Italy The supplementation of sodium butyrate in milk replacers (MR), commonly used to anticipate weaning, affects both small intestine and rumen development, determining earlier solid feed intake
which leads to better performances in cattle. The present study aims to determine the effects of a mixture of short and medium chain fatty acids esterified to glycerol (SMCFA) added to MR on rumen papillae development of weaning calves offered starter diet at libitum. Eight bull calves (10-days-old) were divided in two balanced groups: Control (C) and Treated (T). The latter received 0.2% SMCFA in MR besides starter diet. Animals were slaughtered at the age of 72 days. Histomorphometric analysis performed on samples from the cranio-dorsal sack of the rumen did not indicate differences in the mean number of papillae or in their total surface/cm2 of mucosa but rather in their shape. T group had more wide, short tonguelike or irregular shaped papillae and many interposed papillae compared to C group whose narrow, tongue-to-finger-like papillae, often showed secondary papillae or irregular branches. In T group, enhanced proliferation of the epithelial cells of the stratum basale, spinosum and granulosum was observed in the interpapillar region, while C group showed more cellular layers in the stratum corneum at the base of the papillae and in the interpapillar region. SEM analysis confirmed the different morphology of the papillae in the two groups and, at high magnification, revealed the presence of more dense cytoplasmic protrusions covering horn cell surfaces in T group. http://dx.doi.org/10.1016/j.aanat.2016.04.022 Calcitonin Gene-Related Peptide (CGRP) expression in the spinal cord and spinal ganglia of the Bottlenose Dolphin (Tursiops truncatus) A. Rambaldi 1,∗ , A. Grandis 1 , M. Mazzoni 1 , C. Tagliavia 1 , P. Clavenzani 1 , B. Cozzi 2 , C. Bombardi 1 1 Department of Veterinary Medical Science, University of Bologna, Bologna, Italy 2 Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro, Italy
CGRP is a neuropeptide especially involved in pain transmission. The localization of CGRP in the nervous system of different species has been extensively investigated, but no data are available on its distribution in the spinal cord and spinal ganglia of Cetaceans. Samples of spinal cord and spinal ganglia from three bottlenose dolphins (Tursiops truncatus) were used for this study. The tissue was stained with histological and immunohistochemical methods. Morphometric analysis of motor neurons and primary afferent neurons (PANs) of spinal ganglia was performed. In addition, the percentage of IR-neurons in both spinal cord and ganglia has been calculated. In the spinal cord the CGRP immunoreactivity was localized in small interneurons of laminae I-II and in large motoneurons of lamina IX. The immunostaining in motoneurons was mainly localized in the somata and appeared as a specific granular pattern. In the spinal cord intensely labelled fibers were observed in laminae I, II and X. In spinal ganglia the majority of CGRP-IR PANs showed a small or medium-sized somata and represented the 40% of the total neuronal population. The abundance of IR-fibers in laminae I and II is in agreement with the role played by this neuropeptide in the transmission of nociceptive stimuli. IR-fibers have also been observed in lamina X, which receives visceral afferent fibers; thus CGRP could be also involved in modulation of visceral sensibility. The presence of the CGRP in motoneurons suggests an interaction between these cells and skeletal muscle mediated by this neuropeptide. http://dx.doi.org/10.1016/j.aanat.2016.04.023