Establishment Of Cyclophosphamide Immunosuppressive Model In 4 Weeks Old Juvenile Rat And Application

It is not identical in terms of drug metabolism and efficacy between young and adult individuals.Data from adult animal models are not suitable for assessing the safety and efficacy in child. The use of juvenile animal models is recognized and recommended by authorities and become a consensus. We expected to establish a model of cyclophosphamide (CYP) induced immunosuppression in 4-weeks-old juvenile rats for immunomodulatory drug development and evaluation.The study used hematology, flow cytometry, immunohistochemistry, histopathology, Western Blot and RT-PCR method. Firstly, screened modeling conditions according to the clinical, peripheral blood cell count and thymus index. Secondly, we analyzed the changes of body weight, peripheral blood cells,lymphocyte subsets, immune organs, the expression of CDKN1A protein and gene, intestinal mucosal immunity and comparison of Immunological Indexes with adult rats. Finally, verity the model with reference drug. The results as follows:First, the modeling conditions were screened. CYP 30 mg/kg for the best modeling conditions after oral administration twice a day. 4w juvenile rats showed significant immunosuppression and body weight loss, but no rat deaths and other serious side effects, and immunosuppressive levels can be gradually restored.When the model was established, the rats are normal in clinical observation. Compared with the control group, the number of peripheral blood cells, such as the total number of white blood cells.,neutrophils and lymphocytes decreased significantly; the lymphocytes Subtypes were strongly inhibited and B cells is more stronger; CDKN1A was up-regulated; thymus and bone marrow was significantly inhibited; intestinal mucosal immune index include intestinal sIgA secretion decreased, the number of PPs, IELs and goblet cells decreased in CYP group. Compared with 8-week-old adult rats, immune indicators of 4-week-old juvenile rats rapidly decreased and slowly recovery.The model was applied and validated using G-CSF and Pidotimod as reference drug. G-CSF administration group show significant granulocyte ascending effect, included neutrophils and mononuclear cells. The spleen organ coefficient was increased and the number of PPs were increased.NK cells were strongly increased; the CDKN1A down regulated; but the effect of lymphocyte promotion is limited. The Pidotimod group shows body weight higher than model group; the number of white blood cells and TBNK cells were increased; the expression of CDKN1A gene was decreased. The model can correctly reflect the immunoenhancement effects of G-CSF and Pidotimod which is consistent with the reference.In summary, it is first to use 4-week-old SD rats to establish CYP induced immunosuppressive animal model. CYP upregulation CDKNIA to affect cell cycle and subsequent immunosuppressive effects on innate immunity and acquired immunity is verified in juvenile rats. 4-week-old rats’immunosuppressive effects was similar to 8-weeks-old but stronger and the recovery ability was weak.The inhibition effects of CYP on intestinal mucosa were observed. The new established animal models provided more choice for mucosal immunomodulatory research and it is an appropriate animal model for developing immunomodulatory drugs for young individuals.