Background
Degenerative myelopathy (DM) is a progressive neurodegenerative disease frequently found in Pembroke Welsh Corgis (PWCs). Most DM-affected PWCs are homozygous for the mutant superoxide dismutase 1 (
SOD1
) allele; however, the genetic examination for the SOD1 mutation does not exclusively detect symptomatic dogs. In order to identify novel biomarkers, the plasma microRNA (miRNA) profiles of PWCs with DM were investigated.
Results
Quantification of the plasma levels of 277 miRNAs by an RT-qPCR array identified 11 up-regulated miRNAs and 7 down-regulated miRNAs in DM-affected PWCs from those in wild-type SOD1 PWCs. A pathway analysis identified 3 miRNAs: miR-26b, miR-181a, and miR-196a, which potentially regulate several genes associated with SOD1. In order to validate the diagnostic accuracy of the candidate miRNAs in the aged PWC population, candidate miRNAs in plasma were measured by RT-qPCR and a receiver operating characteristic (ROC) curve analysis was performed. miR-26b had the largest area under the ROC curve for distinguishing DM PWCs from healthy PWCs (sensitivity, 66.7%; specificity, 87.0%). The plasma level of miR-26b was significantly higher in the DM group than in the healthy control group. A positive correlation was observed between increases in the plasma level of miR-26b and disease progression.
Conclusions
These results suggest that plasma miR-26b is a potential novel diagnostic biomarker of DM.
Electronic supplementary material
The online version of this article (10.1186/s12917-019-1944-3) contains supplementary material, which is available to authorized users.
Epithelial-mesenchymal transition (EMT) plays a crucial role in metastasis of epithelial tumors; however, it is challenging to detect EMT by cytology. In the present study, EMT was visualized by fluorescenceimmunocytochemistry (FICC). Air-dried smears from epithelial tumors of dogs (n = 22) and cats (n = 9) were stained using mouse monoclonal anti-E-cadherin and rabbit monoclonal anti-vimentin antibodies.Enzymatic immunohistochemistry (IHC) revealed that 51.6 % (8/22 in dogs, 8/9 in cats) of the cases showed EMT. In dogs, FICC could detect EMT in 62.5 % (5/8) of those cases. In cats, FICC could detect EMT in 100 % (8/8) of the cases. In conclusion, the present FICC method could successfully detect EMT using conventional air-dried cytology smear slides.
The argyrophilic nucleolar organizer regions (AgNORs) are cellular proliferation markers, crucial for predicting the clinical course and aggressiveness of tumors. The purpose of this study was to establish an easy and practical AgNOR staining method in the cytology of dogs and cats. Air-dried cytological slides were prepared from dogs (n = 14) and cats (n = 12). Ethanol, acetone, and formalin were tested as fixatives for AgNOR staining. Subsequently, various methods of Romanowsky-based counterstains were tested before and after AgNOR staining. Clear and strong AgNOR spots were observed with all fixatives, and post-May-Grünwald staining was the best counterstaining method. The established method showed clear AgNOR spots even in the long-term storage samples and Romanowsky-stained ones.
Canine degenerative myelopathy (DM) is an adult-onset, chronic, progressive neurodegenerative disease reported in multiple canine breeds, including the German Shepherd Dog (GSD). Clinical signs include progressive motor neuron paralysis, which begins in the pelvic limbs and eventually leads to respiratory distress, which may necessitate euthanasia. A common DM-associated mutation is a single nucleotide substitution that causes an amino acid substitution (c.118G>A, p.E40K) in the canine SOD1 gene. This SOD1 mutation and the clinical progression rate of A/A risk genotype in the Japanese GSD population have not been analyzed before. Therefore, the aim of this study was to determine the frequency of the mutated allele and analyze the clinical progression rate in the Japanese GSD population. We studied 541 GSDs registered with the Japanese German Shepherd Dog Registration Society between 2000 and 2019. Genotyping was performed using real-time PCR with DNA extracted from the hair roots of each dog. The study revealed 330 G/G dogs (61%), 184 G/A dogs (34%), and 27 A/A dogs (5%), indicating a frequency of the mutant allele of 0.220, which are in Hardy–Weinberg equilibrium. We analyzed the clinical signs in A/A dogs with an age limit of 10 years based on information obtained from the dogs’ owners. Of the seven A/A dogs older than 10 years, owners reported DM-related clinical signs, indicating a clinical progression rate of 100%. These results, further genotyping, and thorough clinical examinations of SOD1 A/A risk genotype will help control and prevent DM in the Japanese GSD population.
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