Canine cognitive dysfunction and Alzheimerʼs disease

Neurodegenerative diseases present a major and increasing burden in the societies worldwide. With aging populations, the prevalence of neurodegenerative diseases is increasing, yet there are no effective cures and very few treatment options are available. Alzheimer’s disease is one of the most prevalent neurodegenerative conditions and although the pathology is well studied, the pathogenesis of this debilitating illness is still poorly understood. This is, among other reasons, also due to the lack of good animal models as laboratory rodents do not develop spontaneous neurodegenerative diseases and human Alzheimer’s disease is only partially mimicked by transgenic rodent models. On the other hand, older dogs commonly develop canine cognitive dysfunction, a disease that is similar to Alzheimer’s disease in many aspects. Dogs show cognitive deficits that could be paralleled to human symptoms such as disorientation, memory loss, changes in behavior, and in their brains, beta amyloid plaques are commonly detected both in extracellular space as senile plaques and around the blood vessels. Dogs could be therefore potentially a very good model for studying pathological process and novel treatment options for Alzheimer’s disease. In the present article, we will review the current knowledge about the pathogenesis of canine cognitive dysfunction, its similarities and dissimilarities with Alzheimer’s disease, and developments of novel treatments for these two diseases with a focus on canine cognitive dysfunction

vzpostavitev in določitev značilnosti epitelne celične linije iz kozje mlečne žleze

Demanding transcriptomic studies in combination with challenging experiments in livestock animal species could be replaced by good in vitro models mimicking the function of ruminant mammary gland. The objective of our study was to establish epithelial cell line obtained from primary cell culture of lactating goat mammary gland. Mammary tissue from lactating Saanen goat (Caprahircus) was digested in collagenase and hyaluronidase solution and plated on plastic flasks. When growing on plastic, typical cobblestone morphology of epithelial cells and larger irregularly shaped cells, corresponding to myoepithelial cells were observed. When growth medium was supplemented with lactogenic hormones (insulin, hydrocortisone, and prolactin) and cells were cultured on plastic for extended period of time at high density, dome-like structures appeared as a result of cell to cell contact induced differentiation. Immunofluorescence staining using antibodies against smooth muscle [alfa]-actin, vimentin and various cytokeratins were used to distinguish between different cell types. Cell types of epithelial and myoepithelial cells were confirmed. Complete differentiation of cells was achieved when growing them on a commercial basal membrane matrix preparation which contains laminin, collagen IV, and various growth factors. Cells grown on basal membrane matrix in growth medium supplemented with lactogenic hormones differentiated morphologically and functionally. Spherical structures that resembled the alveoli of lactating mammary gland were observed. Reverse transcription PCR (RT-PCR) was performed on the total RNA extracted from the cultured cells in order to detect the potentially present milk protein mRNAs.Zahtevne transkriptomske študije, ki vključujejo poskuse na živih živalih, je mogoče nadomestiti z ustreznimi in vitro modeli. Cilj naše raziskave je bil vzpostaviti celično linijo epitelnih celic, pridobljenih iz primarne celične kulture kozje mlečne žleze v laktaciji. Žlezno tkivo kože (Capra hircus) sanske pasme smo razgradili v raztopini kolagenaze in hialuronidaze in nacepili v plastične posodice. Pri rasti na plastični podlagi so se pojavile značilne epitelne strukture v obliki tlakovcev in večje celice nepravilnih oblik, ki so po zunanjosti ustrezale mioepitelnim celicam. Ob dodatku laktogenih hormonov (inzulin, hidrokortizon, prolaktin) v medij in po daljšem obdobju rasti ter ob visoki gostoti celic na plastični podlagi so se oblikovale kupolaste strukture, ki so posledica diferenciacije zaradi medceličnih interakcij. Za karakterizacijo različnih celičnih tipov smo uporabili imunofluorescenčno barvanje ya [alfa]-aktin gladkih mišic, vimentin in različne citokeratine. Z barvanjem smo potrdili prisotnost epitelnih in mioepitelnih celic. Popolno diferenciacijo celic smo dosegli z gojenjem na komercialno pripravljenem matriksu, ki posnema bazalno membrano in vsebuje laminin, kolagen IV in različne rastne faktorje. Celice so se na podlagi iz ekstracelularnega matriksa in ob dodatku laktogenih hormonov morfološko in funkcionalno diferencirale. Nastale so sferične strukture, podobne alveolam mlečne žleze v laktaciji. Z reverzno verižno reakcijo s polimerazo (RT-PCR) smo na izolirani RNA preverili prisotnost mRNA za mlečne proteine

Markers and antibodies for characterization of goat mammary tissue and the derived primary epithelial cell cultures

A selection of commercially available antibodies, targeted against markers employed in studies of mammary gland biology, was tested to determine their reactivity in goat mammary tissue and the derived tissue cultures. Expression of the markers smooth muscle actin (SMA), selected keratins (KRT) 5, 14, 18, and 19, CD24 molecule (CD24), epithelial cell adhesion molecule (EPCAM), mucin 1 (MUC1), integrin subunit alpha 6 (ITGA6; CD49F), integrin subunit beta 1 (ITGB1; CD29), cyclin dependent kinase inhibitor 1A (CDKN1A; p21), membrane metalloendopeptidase (MME; CD10), progesterone receptor (PGR), estrogen receptor 1 (ESR1), and vimentin (VIM) was first assessed on mRNA level, using reverse transcription PCR (RT-PCR). The reactivity of the antibodies in the tissue sections and the derived tissue cultures was determined using immunofluorescence. The result of this study is a list of commercially available antibodies, raised mostly against human antigens, which also recognize orthologous goat antigens and are useful for characterization of different mammary cell types. Additionally, primers that are functional in detecting expression of mammary lineage markers in goat mammary mRNA isolates were validated. The suggested antibodies, PCR primers, and the described methods are of practical value for researchers interested in characterization and isolation of cell types comprising mammary tissue of goats and probably other ruminant

Identification of goat mammary stem/progenitor cells

Goat mammary gland epithelial cells have been used to establish primary and permanent cell lines but to date there is no data on mammary stem cells in this species. The detection and characterization of goat mammary stem cells (gMaSCs) is an important task for a better understanding of the cyclic character of the mammary gland development which will also offer a potential for manipulation of lactation yield and persistency. The objective of the present study was to demonstrate that a subpopulation of MaSCs resides in the goat mammary gland. Mammary tissue from lactating Saanen goats (Capra hircus) was dissociated and processed to a single cell suspension. Using an in vitro colony-forming assay we demonstrated that distinct colony types, which expressed specific lineage markers, arose from unipotent progenitors. Using two different growth media we showed that the frequencies of caprine clonogenic progenitors differed according to growth conditions. Goat epithelial cells were transplanted under the kidney capsule of non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice where they formed organized bilayered structures. Our results indicate the presence of MaSCs in the caprine mammary gland. These data represent the first description of the tissue hierarchy of the goat mammary gland and demonstrate the regenerative potential of goat adult mammary stem cells

Neural differentiation of canine mesenchymal stem cells/multipotent mesenchymal stromal cells

Background: The ability of adipose tissue-derived multipotent mesenchymal stromal cells/mesenchymal stem cells (ASCs) to differentiate in neural lineages promises progress in the field of regenerative medicine, especially for replacing neuronal tissue damaged by different neurological disorders. Reprogramming of ASCs can be induced by the growth medium with neurogenic inductors and specific growth factors. We investigated the neural differentiation potential of canine ASCs using several growth media (KEM, NIMa, NIMb, NIMc) containing various combinations of neurogenic inductors: B27 supplement, valproic acid, forskolin, N2-supplement, and retinoic acid. Cells were first preconditioned in the pre-differentiation neural induction medium (mitogenically stimulatedSTIM1), followed by the induction of neuronal differentiation. Results: After 3, 6, and 9 days of neural induction, elongated neural-like cells with bipolar elongations were observed, and some oval cells with light nuclei appeared. The expression of neuronal markers tubulin beta III (TUBB3), neurofilament H (NF-H), microtubule-associated protein-2 (MAP2), and glial fibrillary acidic protein (GFAP) was observed using immunocytochemistry, which confirmed the differentiation into neurons and glial cells. Flow cytometry analysis showed high GFAP expression (between 70 and 90% of all cells) after cells had been growing three days in the neural induction medium a (NIMa). Around 25% of all cells also expressed adult neuronal markers NF-H and MAP2. After nine days of ASCs differentiation, the expression of all neural markers was reduced. There were no differences between the neural differentiation of ASCs isolated from female or male dogs. Conclusions: The differentiation repertoire of canine ASCs extends beyond mesodermal lineages. Using a defined neural induction medium, the canine ASCs differentiated into neural lineages and expressed markers of neuronal and glial cells, and also displayed the typical neuronal morphology. Differentiated ASCs can thus be a source of neural cellular lineages for the regenerative therapy of nerve damage and could be useful in the future for therapy or the modelling of neurodegenerative diseases

Nitrosative stress in the frontal cortex from dogs with canine cognitive dysfunction

Canine cognitive dysfunction (CCD) is an age-related disorder similar to human Alzheimer\u27s disease (AD) that occurs in elderly dogs. Nitrosative stress has been implicated as one of the causes leading to neurodegenerative diseases, particularly AD. Its involvement in the development of CCD has not been studied so far. In the present study, immunohistochemical staining detected all three isoforms of nitric oxide synthases (nNOS, eNOS, and iNOS) and 3-nitrotyrosine (3-NT) in brains from CCD-affected dogs and non-demented control dogs in all layers of the canine frontal cortex. In CCD-affected and non-demented brains, nNOS was highly expressed in pyramidal-like neurons in the upper cortical layers. nNOS has also been observed in astrocytes in the CCD frontal cortex. The nNOS immunohistochemical staining was statistically significantly elevated in dogs with CCD in comparison to non-demented dogs. Blood vessel wall cells were positive for eNOS, which was also expressed in astrocytes and neurons. Intense 3-NT immunoreactivity was observed in the upper cortical layers, where amyloid-beta deposits spread in the last stage of CCD. Brain cells in the same area were highly immunoreactive for iNOS. This infers that neuroinflammation and nitrosative stress might exacerbate the neurodegenerative process in CCD-affected brains, ultimately leading to cognitive impairmen