RECRUITMENT OF BONE MARROW MONONUCLEAR CELL TO THE CENTRAL NERVOUS SYSTEM AFTER ALLOGENEIC CELL TRANSPLANTATION IN DOGS WITH CANINE DISTEMPER SEQUELS

Abstract

<h3>Background</h3> Canine distemper is an infectious disease that causes demyelinating encephalitis in dogs. Neurological signs of distemper are variable and may be permanent. There is no specific therapy for dogs with distemper sequels and a new alternative treatment can be used. Stem cell therapy has been studied as treatment option for several injuries, including brain injury. Bone marrow mononuclear cells (BMMNC) can be easily isolated and broadly applied as a therapeutic strategy in preclinical and clinical studies. However, there are some challenges associated with this therapeutic approach. One of them is the lack of studies on the characterization of canine BMMNC, and the other is the route of administration for efficient cell delivery. The aim of this study was to evaluate cell migration after allogeneic BMMNC transplantation in 10 animals with neurological complications of canine distemper. <h3>Methods</h3> Bone marrow was harvested from eight donors and BMMNC were isolated, characterized by flow cytometry, labeled with PKH26 dye and intravenously (IV) transplanted in the patients. The cell migration was assessed in the cerebrospinal fluid (CSF) of patients at different periods of time by fluorescence microscopy and by flow cytometry to quantitatively evaluate the percentage of labeled cells with PKH 26 dye. <h3>Results</h3> The mean volume of harvested bone marrow was 28.38 mL (±6.46 mL) and the average of mononuclear cells obtained was 8.84 × 10⁶ (±2.56 × 10⁶) cells/mL. BMMNC express CD45 (94.33%, ±4.72%), CD29 (96.10%, ±1.97%), CD44 (89.60%, ±3.63%), CD9 (79.48%, ±8.90%), CD90 (1.12%, ±0.63%), CD34 (1.15%, ±0.49%), CD14 (24.08%, ±15.35%) and CD8a (10.91%, ±8.93%). In flow cytometry analyses, it was observed an average of 93.0% (±9.9%) PKH26 labeled BMMNC and the average cell viability was 87.6% (±12.8%). Labelling with PKH26 had no negative effect on cell viability. It was showed the presence of labeled BMMNC recruited by central nervous system on CSF. Quantitative analysis by flow cytometry, demonstrate a wide variation in the percentage of labeled cells in different CSF collection times. Also, there was an increase on the percentage of PKH26-labeled cells in the CSF, with highest percentage between 4:00 and 5:30 hours after infusion with subsequent decrease. <h3>Conclusion</h3> This study shows that BMMNC can be efficiently labeled with PKH26 dye and these cells can be monitored after IV transplantation in animals with neurological complications of canine distemper.