Effects Of Chemotherapeutic Drug Cyclophosphamide On Cerebellar Development And Function

Objective: To explore the effects of chemotherapeutic drug cyclophosphamide on the proliferation and apoptosis of granule neuronal progenitors andoligodendrocytes in the cerebellum during development,the effects on the behavioral function in adulthood and to further explore the cellular and molecular mechanism in the process.We aim to provide valuable information for clinical use of the drug cyclophosphamide in children.Methods: We specifically gave a single intraperitoneal injection(50?g/g)of cyclophosphamide or the same volume of PBS as a control to C57BL/6 strain mice at postnatal day 6(P6).We observed the effects at 1 hour,48 hours,96 hours and 8 weeks after the injection.H&E staining was performed to analyze histological effects of cyclophosphamide in the developing cerebellum.Staining for EdU and TUNEL was used to estimate the cell proliferation and apoptosis.Immunofluorescent staining was used to identify the changes in different types of cells in the cerebellum.Rortarod test and Hanging Wire test were used to evaluate the motor coordination and balance.The differentiation markers and genes related to Sonic Hedgehog(SHH)signaling were measured via quantitative real-time PCR(RT-PCR)or immunoblotting.Results: We found that the thickness of external granule layer(EGL)of the experimental group cerebellum decreased sharply at P8,48 hours after the intraperitoneal injection of cyclophosphamide(P<0.001)and almost disappeared.At the same time,the number of Math1 positive cells in the EGL also decreased dramatically(P < 0.001).By staining for EdU and TUNEL,we found that the proliferation of cells in the EGL was significantly reduced(P < 0.001),while the number of apoptotic cells increased(P < 0.001)in the experimental group at P8.In the internal granule layer(IGL)of the cerebellum of experimental group,the proliferation of Olig2 positive glial cells was decreased(P<0.01),and their apoptosis was not affected at P8.Proliferation and apoptosis of Purkinje cells below the cerebellar molecular layer(ML)were unaffected.When the mice of both groups grew to adulthood,8 weeks after the treatment,we found that there were no differences in the ability of coordination and balance between the two groups.After the mice were sacrificed and the cerebellum were dissected,we found that the cerebellum of the experimental group was smaller than the control one(P<0.001),while the multilayer laminar structure of cerebella was largely restored.These inspired us to explore whether the severely damaged cerebellum has undergone a replenishing process to restore the ability of coordination and balance in adulthood.In fact,we found that at P10,96 hours after the treatment,there was a significant increase in the proliferation of cells in the EGL and IGL in the experimental group.Through the staining for specific markers,we found that the cells compensating in the EGL including Math1 positive cells and Sox2 positive cells.The Olig2 positive glial cells in the IGL also increased.At this stage,the expression level of the markers for neuronal differentiation in the experimental cerebellum was relatively lower.At P10,96 hours after the cyclophosphamide treatment,we found that the cerebellum replenished the injury.Furthermore,we found that the Sonic Hedgehog(SHH)signaling,which regulates the development of the cerebellum was highly activated in the experimental group,and thus we speculated that the SHH signaling,enhanced the capability of proliferation quickly after cyclophosphamide administration was stopped,which allowed the developing cerebellum to catch up and to gradually replenish the injury.Conclusion: The chemotherapeutic agent cyclophosphamide induces an immediate damage to the developing cerebellum,but the cerebellar multilayer laminar structure and motor function can be largely restored if the agent is stopped shortly after use.