Efficacy Of Allogeneic Adipose Tissue-derived Mesenchymal Stem Cells For Treatment Of Dogs With Experimental Cystitis

Objective:Patients with cystitis generally experience complex symptoms known as lower urinary tract symptoms(LUTS),which are characterized by stranguria or dysuria,urinary urgency,urination in inappropriate locations and microscopic or macroscopic hematuria.Due to their short urethra,female animals of all species are more prone to ascending infection of the urinary tract.However,the available methods for cystitis management do not result in long-term control of symptoms and do not provide-pain relief to patients due to recurrence and drug resistance.The treatment of cystitis is still a challenge for veterinarians.The study aimed to the effect of administrating ADSCs on chemical cystitis which was induced by administering an ethanol solution.Bladder injury was evaluated during the treatment period.The bladder wall was evaluated by ultrasonography.Study Ⅰ Isolation,Characterization and Differentiation of Canine ADSCsSubcutaneous fat tissues from canine abdomens were obtained via germ-free procedures,mechanically shredded and digested with 0.1%collagenase.Subsequently,ADSCs were cultured in DMEM medium containing 10%FBS.Then,cell morphology was observed under microscope and cell surface antigen expression was analyzed by flow cytometry,respectively.Finally,osteogenic,adipogenic and chondrogenic differentiation were determined.Results:ADSCs was successfully isolated from fresh fat tissues of canine using enzyme digestion method.High expression of CD29(100%)and low expression of CD34(1.5%),CD45(5.6%)were observed in this cell by flow cytometry analysis.The ADSCs could be differentiated into adipocytes,osteoblasts and chondrocytes in vitro.The results indicated that ADSCs can be isolated from the subcutaneous fat tissue of canine and have multi-directional differentiation ability.Study Ⅱ Establishment of a Canine Experimental Cystitis Model12 healthy adult dogs(7 ± 2)kg were divided into the control group(A),saline group(B)and model group(C).Group B recieved bladder instillation with 0.9%saline.Canine cystitis was induced by bladder instillation with 0.6 mL/kg 75%alcohol in modell group.Results:There was no significant difference between the control group and the saline group.Anesthesia and catheterization would not lead to bladder injury.The results showed that inflammation was induced in bladder after infusing with alcohol.The histopatholigical section showed that the bladder mucosal swelling and white blood cells accumulated in the submucosa layers.A large number of red blood cells,white blood cell and tissue fragments appeared in the urine.The alcohol could obviously enhance the inflammatory response as indicated by the increased expression of IL-8 and enhanced the injury of the bladder mucosa by the increased expression of EGF.In summary,the canine cystitis model was successfully established.Study Ⅲ Efficacy of Allogeneic Adipose Tissue-Derived Mesenchymal Stem Cells For The Treatment of Dogs with Experimental CystitisThe present study examined the therapeutic effect of adipose-derived stem cells(ADSCs)on inflammatory cells and the severity of inflammation in an experimentally induced cystitis dog model.A total of 30 healthy adult beagles were divided into 3 groups:the ethanol group(A),the ADSC treatment group(B,C).ADSCs were prepared from the floating mature adipocyte fraction and the stromal vascular fraction of collagenase-digested canine omental adipose tissue.Chemical cystitis was induced by administering an ethanol solution(75%,0.6 mL/kg)in all groups.Dogs in group B and C received a single ADSC infusion(2×106 cells/kg bodyweight)and ADSC infusion(4X 106 cells/kg bodyweight)respectively.Bladder injury was evaluated during the treatment period.The bladder wall was evaluated by ultrasonography.Statistical significance was accepted as P<0.05.The ethanol solution significantly increased visceral hyperalgesia,bladder edema,hemorrhage,vascular permeability,and urinary cytokine(IL-8)levels(P<0.05).Conversely,ADSCs significantly attenuated these parameters compared to those in ethanol-instilled dogs(P<0.05).Additionally,ADSCs increased the level of urinary EGF compared to that in the ET group(P<0.05).Accordingly,ADSC treatment reduced inflammatory parameters and bladder injury compared to those in group A(P<0.05).Ultrasonographic images of the bladder lumen showed that the treated groups had a less thickened bladder wall.In conclusion,a single Ⅳ infusion of ADSCs improved bladder lesions,as assessed macroscopically,and slightly reduced bladder inflammation,as evaluated by histopathology,in dogs with cystitis.