| BackgroundsCranial radiotherapy is the major treatment for intracranial and head and neck tumors,and it is also the most effective treatment means for childhood acute lymphoblastic leukemia.At present,the commonly segmentation modes of cranial radiotherapy include conventional fractionated radiation and hypo-fractionated radiation.The former is characterized by small single irradiation dose(generally 2 Gy),longer treatment time,tumor cure rate of about 18%,mild side effects on the surrounding normal tissues,and higher recurrence rate of advanced tumors.The latter is mainly manifested in large single irradiation dose(generally>2.5 Gy),shorter treatment time,higher treatment ratio,and severe side effects on surrounding normal tissues.With the development of medical imaging technique,hypo-fractionated radiation is becoming a promising new strategy for most malignant tumors,including glioma.Notably,cell and tissue injuries can occur in non-irradiated tissues other than the irradiated tumor sites over the course of radiotherapy in both segmentation modes,and such effects are called radiation-induced abscopal effects(RIAEs).It is worth noting that infertility affects an estimated 18%of couples in childbearing-age globally,and males are believed to contribute to 50%of cases.Clinical studies have shown that cranial radiotherapy for head and neck tumors or childhood leukemia could cause male pituitary secretion dysfunction and damage male fertility.So far,only two relevant laboratory studies were retrieved that reported abscopal effects of cranial irradiation damaged the reproductive system of male animals and no in-depth mechanism research has been carried out.Considering that the patients of head and neck tumors tend to be younger,and their survival rate improved significantly,the fertility injury of male in childbearing age after receiving cranial radiotherapy should be paid more attention.Previous studies have shown that RIAEs are dynamic processes mediated by multiple factors,pathways and mechanisms,which also have a profound impact on the process of radiation and the development of organisms.Moreover,the existing studies are mostly based on in vitro experiments at the cell or tissue level.So far,the regularity and characteristics of reproductive system injury caused by cranial RIAEs are not clear,the mechanism has not been clarified,and no research reports on its prevention.Repetitive transcranial magnetic stimulation(rTMS)is a safe and noninvasive method of neuromodulation,which has been applied to the treatment of neurological and psychiatric diseases in clinic.Previously,our group has found that rTMS has obvious preventive and therapeutic effects on brain injury caused by cranial irradiation.Therefore,whether rTMS has the similar effects on spermatogenesis disorder caused by cranial irradiation?Based on the above questions,adult male mice were used as study objects,and administrated X-ray cranial irradiation in two segmentation modes,namely conventional fractionated radiation and hypo-fractionated radiation.Then,histomorphology,molecular biology,proteomics and other experimental methods were applied to observe the effects of cranial irradiation on the tissue structure of peripheral main organs,study the regularity and characteristics of spermatogenesis disorder caused by cranial irradiation,and explore the relevant mechanisms.Finally,we evaluated the preventive and therapeutic effects of rTMS on spermatogenesis disorder caused by cranial RIAEs.The whole thesis includes four parts as followed:Part Ⅰ:The Abscopal Effects Induced by Cranial Irradiation on the Tissue Structure of Peripheral Main OrgansObjective:To evaluate the protection of lead shielding device and the sensitivity of peripheral main organs to the cranial RIAEs.Methods:Firstly,Monte Carlo simulation was used to calculate the absorbed dose of the main organs when mouse head was irradiated with 20 Gy X-ray,so as to evaluate the protective effects of the shielding device.Then,the awake mice were irradiated with X-ray cranial irradiation in the conventional fractionated mode(2 Gy×10 d)or hypo-fractionated mode(5 Gy×4 d),respectively.The total dose was 20 Gy and the dose rate of head was 2.33 Gy/min,and meanwhile,the dose rate of the testicular area under the shielding device was 0.01 Gy/min.Notably,we added a shielded irradiation group to observe the effects of scattering dose on peripheral main organs.At 4 W after cranial irradiation,the organ index of peripheral main organs was calculated,including testis,heart,liver,spleen,lung,kidney and thymus.The morphological structure of the above organs and intestine was observed by hematoxylin and eosin(HE)staining,and the testicular ultrastructure was estimated by transmission electron microscope(TEM).Finally,the sperm quality was evaluated in terms of sperm count,abnormality rate,survival rate and apoptosis rate.Results:The results of Monte Carlo simulation showed that the average absorbed dose of peripheral main organs was low during 20 Gy X-ray cranial irradiation,and the absorbed dose of testis was only 0.9‰of that of the head,which was basically consistent with the measured dose of testicular area.Compared with sham group,the testis index of mice decreased significantly in the two mode cranial irradiation groups,while the organ index of other peripheral organs did not change significantly;the morphological structure of testis and epididymis was obviously damaged,while that of other peripheral organs showed only slightly or no obvious damage;the ultrastructure of testicular seminiferous tubules was seriously damaged,especially in spermatogonia;the sperm quality decreased significantly,mainly in sperm count.Compared with 2 Gy×10 d group,the damage in 5Gy×4 d group was more obvious.Moreover,no obvious changes were found in the above indicators in the shielded irradiation group.Conclusion:Under the experimental conditions,the protection of the lead shielding device is effective and the cranial irradiation does not cause obvious scattering to the peripheral main organs.Compared with other peripheral main organs,testis is more sensitive to cranial RIAEs,mainly manifested as spermatogenesis disorder.Furthermore,the damage induced by hypo-fractionated radiation is more obvious.Part Ⅱ:The Regularity and Characteristics of Spermatogenesis Disorder Induced by Abscopal Effects of Cranial IrradiationObjective:To clarify the regularity and characteristics of spermatogenesis disorder induced by abscopal effects of cranial irradiation.Methods:Adult male mice were administered X-ray cranial irradiation(5 Gy×4 d).At 1W,2 W and 4 W after cranial irradiation,HE staining,immunofluorescence(IF)staining,immunohistochemical(IHC)staining,TUNEL staining,Evans blue(EB)fluorescence imaging,enzyme-linked immunosorbent assay(ELISA),western blotting(WB),quantitative real-time polymerase chain reaction(q RT-PCR)and other experimental methods were performed to detect the changes of testicular morphological structure,sperm quality,structure and secretory function of Sertoli cells,secretory function of Leydig cells,proliferation and differentiation ability of SSCs and testicular cell apoptosis.Results:Compared with sham group,the testicular morphological structure was significantly damaged and the sperm quality decreased significantly at 2 W after cranial irradiation,which mainly manifested in the decrease of sperm count and the increase of abnormality rate,and continuously aggravated to 4 W.EB exudation in seminiferous tubules increased and the ultrastructure of blood-testis barrier(BTB)was seriously damaged.The ability of Sertoli cells secreting SCF decreased at 1 W,then recovered slowly,and increased significantly at 4 W after cranial irradiation.The level of serum testosterone(T)decreased significantly at 1 W and 2 W,and recovered to normal level at 4W after cranial irradiation.The levels of PLZF protein and m RNA in testis decreased significantly at 1 W,and continuously decreased to 4 W after cranial irradiation.The level of C-kit protein in testis decreased significantly at 1 W,then recovered slowly,and increased continuously to 4 W after cranial irradiation.Moreover,the protein expression of C-kit increased mainly in SSCs.The ratio of Bcl 2/Bax in testis decreased significantly at 2 W,and constantly decreased to 4 W after cranial irradiation.The level of cleaved caspase 3 and TUNEL positive cells in testis at 4 W increased significantly,and the latter mostly located in the outermost layer of seminiferous tubules.Conclusion:These results suggested that cranial irradiation can damage the reproductive system of male mice,manifested as destructing of testis morphological structure and tissue function,increasing the permeability of BTB,causing the secretory function disorder of Sertoli cells and Leydig cells,inhibiting SSCs proliferation,disrupting SSCs differentiation and inducing SSCs apoptosis.Part Ⅲ:The Mechanisms of Spermatogenesis Disorder Induced by Abscopal Effects of Cranial IrradiationObjective:To reveal the mechanisms of spermatogenesis disorder induced by abscopal effects of cranial irradiation,and provide the experimental and theoretical basis for the prevention and treatment of male reproductive injury caused by cranial radiotherapy.Methods:Tandem mass tag(TMT)quantitative proteomics was used to screen the differentially expressed proteins(DEPs)of mouse testis after cranial irradiation,and bioinformatics analysis was used to analyze the key molecules and signal pathways related to spermatogenesis.Then,based on the above results,ELISA,colorimetry,IHC staining,IF staining,WB and q RT-PCR were used to observe the changes of oxidative stress and inflammatory response in testis,serum and brain,as well as the secretory function of the hypothalamic-pituitary-gonadal(HPG)axis to explore its underlying mechanisms.Results:Cranial irradiation significantly altered the protein expression profile of mouse testis.DEPs were mainly involved in biological processes such as spermatogenesis,cell differentiation,innate immune response,immune system,and apoptosis,etc.GO analysis showed spermatogenesis was closely related to oxidative stress and inflammatory response.The results of oxidative stress showed that compared with sham group,the activity of T-SOD obviously decreased in serum and it did not change significantly in testis and brain,and the concentration of MDA in testis,serum and brain increased significantly at 1 W,2 W and 4 W after cranial irradiation.The results of inflammatory factors showed that compared with sham group,the concentrations of IL-10,TNF-αand TGF-β1 in brain did not change significantly at 1 W,2 W and 4 W after cranial irradiation;the concentrations of TNF-αand TGF-β1 both in serum and testis both increased significantly at 1 W,and were still higher than the normal level at 4 W after cranial irradiation.Moreover,the number of F4/80~+cells in testis increased significantly at 4 W after cranial irradiation,and the number of F4/80~+/CD86~+cells was significantly higher than that of F4/80~+/CD206~+cells.The levels of neuron specific enolase(NSE)and S100calcium binding protein B(S100B)in serum increased significantly at 1 W,and were still higher than normal level at 4 W after cranial irradiation.The result of blood brain barrier(BBB)permeability showed that compared with sham group,EB exudation around brain micro vessels increased significantly at 2 W,and continuously increased at 4 W after cranial irradiation.The results of sex hormones showed that compared with sham group,the levels of Gn RH,FSH and LH in serum were disordered,the morphological structure of adenohypophysis was significantly damaged at 1 W,2 W and 4 W after cranial irradiation,and the number of Brd U~+cells in adenohypophysis decreased significantly at 4W after cranial irradiation.KEGG analysis showed DEPs were significantly enriched in PI3K/Akt pathway.The result of WB showed that compared with sham group,the levels of P-PI3K and P-Akt in testis increased significantly at 4 W after cranial irradiation.The result of IHC showed that P-Akt was mainly expressed in the cytoplasm of testicular interstitial tissue and Sertoli cells.Conclusion:Cranial irradiation can damage the testicular structure and spermatogenesis through brain-blood-testicular secondary oxidative stress and inflammatory response,as well as the secretory dysfunction of HPG axis.The PI3K/Akt pathway in testicular tissue was involved in spermatogenesis disorder induced by abscopal effects of cranial irradiation.Part Ⅳ:The Preventive and Therapeutic Effects of rTMS on Spermatogenesis Disorder Induced by Abscopal Effects of Cranial IrradiationObjective:To evaluate the preventive and therapeutic effects of rTMS on spermatogenesis disorder induced by abscopal effects of cranial irradiation,so as to provide technical support for fertility preservation of tumor patients in childbearing age.Methods:Mice in 5 Gy×4 d and sham groups were stimulated by rTMS using Magneuro60 TMS instrument with matched small animal magnetic stimulation coil.The frequency of rTMS was 10 Hz,the total number of pulses was 1000,5 d/W for continuously 4 W.The preventive and therapeutic effects of rTMS were evaluated from the aspects of sperm quality,testicular morphology and secretory function,proliferation and differentiation of SSCs,and its mechanism was discussed from oxidative stress,inflammatory response and the secretory function of HPG axis.Results:Compared with 5 Gy×4 d group,the general health status and sperm quality of mice in 5 Gy×4 d+rTMS group were significantly improved,which were mainly manifested in the increase of sperm count and the decrease of abnormality rate.Meanwhile,the damage of testicular tissue structure and the EB exudation in seminiferous tubules were significantly alleviated,the protein levels of PLZF and Brd U in testis increased significantly.The concentration of MDA in testis,serum and brain decreased significantly.In serum,the concentration of IL-10 increased,and the concentration of TNF-αand TGF-β1 decreased.The disturbance of Gn RH,FSH and LH in serum was partially relieved,and the structural damage of the adenohypophysis was alleviated.rTMS had no significant effect on sperm quality and testicular morphology of normal mice.Conclusion:rTMS can exert the preventive and therapeutic effects on the spermatogenesis disorder caused by abscopal effects of cranial irradiation,and its mechanisms may be related to reduce the subsequent cascade of oxidative stress and inflammatory response injury,and regulate the secretory dysfunction of HPG axis through preventive and therapeutic effects on radiation brain injury. |
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