Pituitary pathology and gene expression in acromegalic cats

© 2019 Endocrine Society. The prevalence of GH-secreting pituitary tumors in domestic cats (Felis catus) is 10-fold greater than in humans. The predominant inhibitory receptors of GH-secreting pituitary tumors are somatostatin receptors (SSTRs) and D2 dopamine receptor (DRD2). The expression of these receptors is associated with the response to somatostatin analog and dopamine agonist treatment in human patients with acromegaly. The aim of this study was to describe pathological features of pituitaries from domestic cats with acromegaly, pituitary receptor expression, and investigate correlates with clinical data, including pituitary volume, time since diagnosis of diabetes, insulin requirement, and serum IGF1 concentration. Loss of reticulin structure was identified in 15 of 21 pituitaries, of which 10 of 15 exhibited acinar hyperplasia. SSTR1, SSTR2, SSTR5, and DRD2 mRNA were identified in the feline pituitary whereas SSTR3 and SSTR4 were not. Expression of SSTR1, SSTR2, and SSTR5 was greater in acromegalic cats compared with controls. A negative correlation was identified between DRD2 mRNA expression and pituitary volume. The loss of DRD2 expression should be investigated as a mechanism allowing the development of larger pituitary tumors

Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadate

<p>Abstract</p> <p>Background</p> <p>Fish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (<it>Sparus aurata</it>), a marine teleost with acellular bone, has been successfully used in recent years to study the function and regulation of bone and cartilage related genes during development and in adult animals. Tools recently developed for gilthead seabream, <it>e.g. </it>mineralogenic cell lines and a 4 × 44K Agilent oligo-array, were used to identify molecular determinants of <it>in vitro </it>mineralization and genes involved in anti-mineralogenic action of vanadate.</p> <p>Results</p> <p>Global analysis of gene expression identified 4,223 and 4,147 genes differentially expressed (fold change - FC > 1.5) during <it>in vitro </it>mineralization of VSa13 (pre-chondrocyte) and VSa16 (pre-osteoblast) cells, respectively. Comparative analysis indicated that nearly 45% of these genes are common to both cell lines and gene ontology (GO) classification is also similar for both cell types. Up-regulated genes (FC > 10) were mainly associated with transport, matrix/membrane, metabolism and signaling, while down-regulated genes were mainly associated with metabolism, calcium binding, transport and signaling. Analysis of gene expression in proliferative and mineralizing cells exposed to vanadate revealed 1,779 and 1,136 differentially expressed genes, respectively. Of these genes, 67 exhibited reverse patterns of expression upon vanadate treatment during proliferation or mineralization.</p> <p>Conclusions</p> <p>Comparative analysis of expression data from fish and data available in the literature for mammalian cell systems (bone-derived cells undergoing differentiation) indicate that the same type of genes, and in some cases the same orthologs, are involved in mechanisms of <it>in vitro </it>mineralization, suggesting their conservation throughout vertebrate evolution and across cell types. Array technology also allowed identification of genes differentially expressed upon exposure of fish cell lines to vanadate and likely involved in its anti-mineralogenic activity. Many were found to be unknown or they were never associated to bone homeostasis previously, thus providing a set of potential candidates whose study will likely bring insights into the complex mechanisms of tissue mineralization and bone formation.</p

Three-Dimensional Neurophenotyping of Adult Zebrafish Behavior

The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior

Positional Cloning of “Lisch-like”, a Candidate Modifier of Susceptibility to Type 2 Diabetes in Mice

In 404 Lepob/ob F2 progeny of a C57BL/6J (B6) x DBA/2J (DBA) intercross, we mapped a DBA-related quantitative trait locus (QTL) to distal Chr1 at 169.6 Mb, centered about D1Mit110, for diabetes-related phenotypes that included blood glucose, HbA1c, and pancreatic islet histology. The interval was refined to 1.8 Mb in a series of B6.DBA congenic/subcongenic lines also segregating for Lepob. The phenotypes of B6.DBA congenic mice include reduced β-cell replication rates accompanied by reduced β-cell mass, reduced insulin/glucose ratio in blood, reduced glucose tolerance, and persistent mild hypoinsulinemic hyperglycemia. Nucleotide sequence and expression analysis of 14 genes in this interval identified a predicted gene that we have designated “Lisch-like” (Ll) as the most likely candidate. The gene spans 62.7 kb on Chr1qH2.3, encoding a 10-exon, 646–amino acid polypeptide, homologous to Lsr on Chr7qB1 and to Ildr1 on Chr16qB3. The largest isoform of Ll is predicted to be a transmembrane molecule with an immunoglobulin-like extracellular domain and a serine/threonine-rich intracellular domain that contains a 14-3-3 binding domain. Morpholino knockdown of the zebrafish paralog of Ll resulted in a generalized delay in endodermal development in the gut region and dispersion of insulin-positive cells. Mice segregating for an ENU-induced null allele of Ll have phenotypes comparable to the B.D congenic lines. The human ortholog, C1orf32, is in the middle of a 30-Mb region of Chr1q23-25 that has been repeatedly associated with type 2 diabetes

Questionnaire-based behaviour analysis of Cavalier King Charles spaniels with neuropathic pain due to Chiari-like malformation and syringomyelia.

Chiari-like malformation (CM)/syringomyelia (SM) is a disease complex recognised in Cavalier King Charles spaniels (CKCSs) that can lead to neuropathic pain (NeP). In humans, NeP is associated with anxiety, depression and reduced quality of life (QoL). In this study, databases of three specialist veterinary centres were searched and CKCS breed societies and health forums were contacted to identify CKCS with an imaging diagnosis of CM/SM. Owners completed questionnaires on behaviour, signalment, general health status, NeP and QoL. Data were analysed from 122 dogs out of 564 questionnaires completed, after incomplete questionnaires and data from dogs that had other potentially debilitating disease processes were excluded. NeP severity score was significantly and positively correlated with 'stranger-directed' fear (r(S)=0.28), non-social fear (r(S)=0.34), 'separation-related' behaviour (r(S)=0.38), attachment behaviour (r(S)=0.24), excitability (r(S)=0.21) and proxy for pain sensation (r(S)=0.29). Increased NeP was also significantly associated with decreased QoL (r(S)=0.47), ability to settle (r(S)=0.26) and willingness to exercise (r(S)=0.50). Severity of NeP was positively associated with certain fear-associated behaviour and with decreased owner-perceived QoL. Thus, neurobehavioural changes should be considered in the management of NeP in CKCS with CM/SM

Long-term outcome of Cavalier King Charles spaniel dogs with clinical signs associated with Chiari-like malformation and syringomyelia.

The disease complex Chiari-like malformation (CM) and syringomyelia (SM) has been associated with the development of neuropathic pain (NeP), and commonly affects Cavalier King Charles spaniels (CKCS). This prospective cohort study followed 48 CKCSs with CM and/or SM and clinical signs suggestive of NeP for a period of 39 (±14.3) months from diagnosis. At the end of the study, 36 dogs were still alive; five dogs died of an unrelated or unknown cause, and seven were euthanased due to severe clinical signs suggestive of NeP. During the follow-up period, the clinical signs of scratching, facial rubbing behaviour, vocalisation and exercise ability were evaluated. Nine out of 48 dogs stopped scratching (P<0.001), but there was no statistically significant change in the number of dogs exhibiting exercise intolerance, vocalisation or facial rubbing behaviour. The overall severity of clinical signs based on a visual analogue scale (VAS) (0 mm: no clinical signs 100 mm: severe clinical signs) increased (from median 75 mm (interquartile ranges (IQR) 68-84) to 84 mm (IQR 71.5-91), P<0.001). A quarter of the dogs were static or improved. In general, the majority of the owners felt that the quality of life of their dogs was acceptable. Medical treatments received were gabapentin or pregabalin and/or intermittently, carprofen. The owner's perception of their animal's progress, and progress based on VAS, had strong positive correlation (Spearman's rank correlation (s(r)) 0.74, P<0.001). Overall, this study suggests that clinical signs suggestive of NeP progress in three-quarters of CKCSs with CM and/or SM

Establishment of primary cell cultures from fish calcified tissues

Fishes have been recently recognized as a suitable model organism to study vertebrate physiological processes, in particular skeletal development and tissue mineralization. However, there is a lack of well characterized in vitro cell systems derived from fish calcified tissues. We describe here a protocol that was successfully used to develop the first calcified tissue-derived cell cultures of fish origin. Vertebra and branchial arches collected from young gilthead seabreams were fragmented then submitted to the combined action of collagenase and trypsin to efficiently release cells embedded in the collagenous extracellular matrix. Primary cultures were maintained under standard conditions and spontaneously transformed to form continuous cell lines suitable for studying mechanisms of tissue mineralization in seabream. This simple and inexpensive protocol is also applicable to other calcified tissues and species by adjusting parameters to each particular case