Effects of melatonin on both testicular regeneration and recovery of spermatogenesis in busulfan-treated rats

Abstract Testicular damage is one of the most hazardous effects of chemotherapy as it is frequently associated with oligozoospermia and azoospermia. This study aimed at evaluating the protective effect of melatonin in a rat model of busulfan-induced testicular injury. Rats were divided into four groups: control, melatonin, busulfan, busulfan plus melatonin. After 15 days, the semen was collected from the epididymis and testes were assessed. Sperm removed from cauda epididymis and analyzed for sperm count and viability. Testis tissues were also removed, fixed in formalin and were embedded in paraffin. Sections of testis tissue were stained with hematoxylin-eosin for histological examination and prepared for TUNEL (Terminal deoxynucleotide transferase dUTP Nick End Labeling) assay to detect apoptosis and PCNA (proliferating cell nuclear antigenassay) to detect proliferation cells. Serum and testes supernatants were separated to detect testosteron level and oxidative stress parameters. In histological examination, degenerative changes in seminiferous tubules were observed in the experimental groups. In biochemical examination, the total oxidant status (TOS) levels in Busulfan group were significantly higher than in the control group while the total antioxidant status (TAS) levels of all the groups were similar. In conclusion, the beneficial properties of melatonin treatment by its potent anti-oxidants may reduce adverse effects of chemotherapy in the reproductive system in a rodent system.

Establishing a nonlethal and efficient mouse model of male gonadotoxicity by intraperitoneal busulfan injection / 亚洲男科学杂志(英文版)

An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%-87% of mice die when administered an intraperitoneal injection of 40 mg kg-1. In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg-1) on day 36 or a dose of 40 mg kg-1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg-1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg-1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.

Establishing a nonlethal and efficient mouse model of male gonadotoxicity by intraperitoneal busulfan injection / 亚洲男科学杂志(英文版)

An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%-87% of mice die when administered an intraperitoneal injection of 40 mg kg-1. In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg-1) on day 36 or a dose of 40 mg kg-1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg-1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg-1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.

Damaged male germ cells induce epididymitis in mice / 亚洲男科学杂志(英文版)

Epididymitis can be caused by infectious and noninfectious etiological factors. While microbial infections are responsible for infectious epididymitis, the etiological factors contributing to noninfectious epididymitis remain to be defined. The present study demonstrated that damaged male germ cells (DMGCs) induce epididymitis in mice. Intraperitoneal injection of the alkylating agent busulfan damaged murine male germ cells. Epididymitis was observed in mice 4 weeks after the injection of busulfan and was characterized by massive macrophage infiltration. Epididymitis was coincident with an accumulation of DMGCs in the epididymis. In contrast, busulfan injection into mice lacking male germ cells did not induce epididymitis. DMGCs induced innate immune responses in epididymal epithelial cells (EECs), thereby upregulating the pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), as well as the chemokines such as monocyte chemotactic protein-1 (MCP-1), monocyte chemotactic protein-5 (MCP-5), and chemokine ligand-10 (CXCL10). These results suggest that male germ cell damage may induce noninfectious epididymitis through the induction of innate immune responses in EECs. These findings provide novel insights into the mechanisms underlying noninfectious epididymitis, which might aid in the diagnosis and treatment of the disease.

Hematopoietic stem cell injury in mice induced by busulfan / 中国比较医学杂志

Objective To study the hematopoietic stem cell injury(HSC)induced by busulfan. Methods C57BL/6 mice were treated with i.p. injection of 20 mg/kg or 40 mg/kg busulfan. All mice were euthanized at 15 days and 30 days after busulfan treatment for analysis of the peripheral blood cell counts, bone marrow cellularity and HPC (LKS-, lineage-sca1 -c-kit+), HSC(LSK, lineage-sca1 +c-kit+)and long-term HSC(CD34 - LSK, CD34 - lineage-sca1 +c-kit+)frequency. The colony-forming unit-granulocyte and macrophage(CFU-GM)ability of HPC was measured by colony-forming cell(CFC)assay,and the HSC self-renewal capacity was analyzed by single-cell colony-forming assay. Results The busulfan administration decreased the WBC,RBC and PLT compared with control mice. The HPC function (CFU-GM)was impaired(P < 0.05), and the HSC colony forming ability was decreased at 15 days after busulfan treatment(P < 0.05), whereas the body weight of the mice didn't change significantly after busulfan treatment. Conclusions Our findings suggest that busulfan can induce hematopoietic stem cell injury,and provide a support for the study of hematopoietic stem cell injury mechanism.

Establishment of Effective Mouse Model of Premature Ovarian Failure Considering Treatment Duration of Anticancer Drugs and Natural Recovery Time

OBJECTIVES: This study was aimed to establish the most effective premature ovarian failure (POF) mouse model using Cyclophosphamide (CTX), busulfan (Bu), and cisplatin considering treatment duration of anticancer drugs and natural recovery time. METHODS: POF was induced by intraperitoneally injecting CTX (120 mg/kg)/Bu (12 mg/kg) for 1 to 4 weeks or cisplatin (2 mg/kg) for 3 to 14 days to C57BL/6 female mice aged 6 to 8 weeks. Controls were injected with equal volume of saline for the same periods. Body weight was measured every week, and ovarian and uterine weights were measured after the last injection of anticancer drug. To assess ovarian function, POF-induced mice were superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin, and then mated with male. After 18 hours, zygotes were retrieved and cultured for 4 days. Finally, the mice were left untreated for a period of times after the final injection of anticancer drug, and the time for natural recovery of ovarian function was evaluated. RESULTS: After 2 weeks of CTX/Bu injection, ovarian and uterine weights, and ovarian function were decreased sharply. Cisplatin treatment for 10 days resulted in a significant decrease in ovarian and uterine weight, and ovarian function. When POF was induced for at least 2 weeks for CTX/Bu and for at least 10 days for cisplatin, ovarian function did not recover naturally for 2 weeks and 1 week, respectively. CONCLUSIONS: These results suggest that CTX/Bu should be treated for at least 2 weeks and cisplatin for at least 10 days to establish the most effective primary ovarian insufficiency mouse model.

Outcome of Hematopoietic Stem Cell Transplantation in Wiskott-Aldrich Syndrome / 임상소아혈액종양

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a very rare disease and patients who do not receive timely treatment suffer from bleeding, infection, and malignancy. Hematopoietic stem cell transplantation (HSCT) has been recognized as an effective treatment, but the standard transplantation protocol has not been established. We report the outcomes of WAS patients who underwent HSCT in our institution. METHODS: We retrospectively studied patients who underwent HSCT at Seoul National University Children's Hospital from 2005 to 2018. Busulfan-based myeloablative conditioning regimen was used, and an intensive daily therapeutic drug monitoring (TDM) for busulfan dosing was started for effective myeloablation and to reduce toxicity since 2008. We collected and analyzed data regarding symptoms, engraftment, transplantation-related toxicities, and survival. RESULTS: Six WAS patients who received HSCT were evaluated. The median age of the patients at diagnosis was 5 years (range, 1–11). There were 2 matched unrelated donor bone marrow transplantations, 3 matched unrelated peripheral blood stem cell transplantations (PBSCT), and 1 haploidentical PBSCT. No patient experienced engraftment failure. Three patients developed grades II to IV acute graft-versus-host disease (GVHD). Two patients had veno-occlusive disease (VOD). Two patients died (due to VOD and acute GVHD). The 5-year overall survival was 66.7% with 8 years of median follow-up. Particularly, a patient who underwent haploidentical PBSCT using targeted busulfan is alive with a follow-up duration of 3 years after HSCT. CONCLUSION: In conclusion, WAS patients may be cured with HSCT with targeted busulfan-based myeloablative conditioning. But, long-term and multi-center studies are needed.

Busulfan plus melphalan versus high-dose melphalan as conditioning regimens in autologous stem cell transplantation for newly diagnosed multiple myeloma

BACKGROUND: High-dose melphalan (HDMEL) represents the standard conditioning regimen before autologous stem cell transplant (ASCT) in multiple myeloma (MM), but recent updates have suggested combination of melphalan with bulsulfan (BUMEL) is also associated with favorable outcomes. We performed the current study to address the lack of comparative studies between the two conditioning regimens in Asian populations. METHODS: Using the Korean National Health Insurance and Korean Health Insurance Review and Assessment Service databases, 1,304 patients newly diagnosed with MM undergoing ASCT between January 2010 and December 2014 were identified. Patients were divided according to conditioning regimen (HDMEL vs. BUMEL), and after case matching, 428 patients undergoing HDMEL conditioning were compared to 107 patients undergoing BUMEL conditioning with respect to clinical course and treatment outcomes. RESULTS: The 3-year progression-free survival (PFS) was 52.5% for the HDMEL conditioning group versus 70.3% for the BUMEL conditioning group (P=0.043). The 3-year overall survival (OS) was 82.0% versus 83.5% (P=0.525), respectively. Although not statistically significant, BUMEL conditioning was associated with more platelet transfusion, while HDMEL was associated with more granulocyte colony stimulating factor support. In multivariate analysis, BUMEL conditioning was not inferior to HDMEL conditioning in regard to both PFS and OS. CONCLUSION: Our study confirmed that BUMEL is an effective and well-tolerated alternative to HDMEL conditioning, with better PFS.

Anti-CD3 Antibody Ameliorates Transfusion-Associated Graft-Versus-Host Disease in a Chemotherapy-Based Mouse Model With Busulfan and Fludarabine

ABSTRACT To establish a transfusion-associated graft-versus-host disease (TA-GVHD) mouse model with busulfan and fludarabine for effective treatment evaluation. BALB/c (H-2d) mice were injected with busulfan (15 mg/kg) and fludarabine (30 mg/kg) twice a day for 4 days. The mice were transfused with 106 T cell-depleted bone marrow (TCD-BM )and cells in different groups 3 days after chemotherapy: syngeneic BALB/c, MHC minor mismatch DBA/2 (H-2d), or MHC major mismatch C57BL/6(H2-b). Recipient BALB/c mice were injected with either blood only or blood+splenocyte. TA-GVHD was monitored in terms of body weight loss, clinical scores, and survival. Dexamethasone (50 mg/kg), cyclophosphamide (50 mg/kg), cyclosporine A (30 mg/kg), and anti-CD3 (1 mg/kg) were injected to each group to examine the treatments. Blood transfusion alone is insufficient to induce TA-GVHD in a chemotherapy-based mouse model. A MHC-mismatched TA-GVHD model can be induced by splenocyte and blood transfusion. This MHC-mismatched TA-GVHD model was resistant to dexamethasone treatment. Treatment based on anti-CD3 monoclonal antibody slightly ameliorated TA-GVHD. Treatment effectiveness was associated with T-cell depletion following activation by anti-CD3. Busulfan and fludarabine chemotherapy regimen can be used to establish a TA-GVHD mouse model. Anti-CD3 monoclonal antibody is a potential alternative to treat TA-GVHD.

Dose-dependent effects of busulfan on dog testes in preparation for spermatogonial stem cell transplantation / 한국실험동물학회지

Successful male germ cell transplantation requires depletion of the host germ cells to allow efficient colonization of the donor spermatogonial stem cells. Although a sterilizing drug, busulfan, is commonly used for the preparation of recipient models before transplantation, the optimal dose of this drug has not yet been defined in dogs. In this study, 1-year-old mongrel dogs were intravenously injected with three different concentrations of busulfan (10, 15, or 17.5 mg/kg). Four weeks after busulfan treatment, no fully matured spermatozoa were detected in any of the busulfan-treated groups. However, small numbers of PGP9.5-positive spermatogonia were detected in all treatment groups, although no synaptonemal complex protein-3-positive spermatocytes were detected. Of note, acrosin-positive spermatids were not detected in the dogs treated with 15 or 17.5 mg/kg busulfan, but were detected in the other group. Eight weeks after busulfan treatment, the dogs treated with 10 mg/kg busulfan fully recovered, but those in the other groups did not. PGP9.5-positive spermatogonia were detected in the 10 mg/kg group, and at a similar level as in the control group, but these cells were rarely detected in the 15 and 17.5 mg/kg groups. These results suggest that a dose of 15-17.5 mg/kg is optimal for ablative treatment with busulfan to prepare the recipient dogs for male germ cell transplantation. At least eight weeks should be allowed for recovery. The results of this study might facilitate the production of recipient dogs for male germ cell transplantation and can also contribute to studies on chemotherapy.

Application of stereological methods for unbiased estimation of sperm morphology in the mice induced by busulfan / 대한해부학회지

Busulfan is an anticancer drug, which causes the apoptosis germ cells and azoospermia in humans and animals. Abnormal morphology of spermatozoa related to the male infertility. The sperm morphology is evaluation of sperm size, shape and appearance characteristics should be assessed by carefully observing a stained sperm sample under the microscope. Evaluation of sperm morphology has been considered as one of the most important factors for a successful fertilization and determining sperm quality. The mice were assigned to tow experimental groups: control and busulfan. Each group included six mice that were housed under standard conditions. The volume was estimated using the nucleator method. The sperm's flegellum and mid-piece length was estimated by counting the number of intersections between the tails and Merz grid test line in an unbiased counting frame, superimposed on live images of sperms. Our results demonstrated a significant different in the volume and surface of the sperm's head and the length of the sperm's flagellum in the control and busulfan groups. Busulfan can effect on the volume of the sperm's head and the length of the sperm's flagellum in rat.

The effect of chemotherapeutic drugs on CD19-CAR-T cells in vitro / 实用医学杂志

Objective The research about the effect of different chemotherapeutic drugs on CD19?CAR?T cells with CCK8 test to provide the theoretical support about the development of chemotherapy for clinical support. Methods Extract T cells from a normal adult peripheral blood and synthesize CD19?CAR?T cell. CD19?CAR?T cells were treated with different doses of chemotherapeutic drugs for 24,48,72 h and(or)96 h,and inhibition rate was calculated. Results First,we observed that the inhibition rates of fludarabine and Mafosfamide for CD19?CAR?T cells were increasing with the time and concentration (P 0.05). Finally ,Cyclophosphamide had no effect in CD19?CAR?T cells in vitro (P > 0.05). Conclusion Mafosfamide and Fludarabine can inhibit the CD19?CAR?T cells. Cyclophosphamide have no activity in vitro.

Population pharmacokinetics of intravenous infusing busulfan in patients undergoing hematopoietic stem cell transplantation / 中国药学杂志

OBJECTIVE: To establish a population pharmacokinetic model of intravenous infusing busulfan in HSCT patients, and to explore physiological and pathological factors which may influence the pharmacokinetic parameters. METHODS: We have collected clinical history information of 35 patients undergoing HSCT surgery and taking busulfan intravenous infusion for treatment. These information such as physiological and pathological factors and busulfan concentration data were used to perform the population pharmacokinetic analysis by applying the method of nonlinear mixed effects modeling(NONMEM). RESULTS: A statistical model of busulfan is established, including variables such as body weight, sex, serum creatinine clearance. The success of 973 out of 1 000 times resampling trials (by bootstrap) shows that the newly parameters value are very close to the estimate value calculated from the final model by NONMEM, which demonstrates the established population pharmacokinetic model of busulfanis stable, effective and predictable. CONCLUSION: The population pharmacokinetic model is established, which is capable of depicting the pharmacokinetic characteristics of busulfan. It is found that patients' weight, gender and creatinine clearance influence pharmacokinetic parameters, which can be useful and valuable for the clinical individualized dosing regimens.

Prospective validation of a novel dosing scheme for intravenous busulfan in adult patients undergoing hematopoietic stem cell transplantation

The objective of this study was to externally validate a new dosing scheme for busulfan. Thirty-seven adult patients who received busulfan as conditioning therapy for hematopoietic stem cell transplantation (HCT) participated in this prospective study. Patients were randomized to receive intravenous busulfan, either as the conventional dosage (3.2 mg/kg daily) or according to the new dosing scheme based on their actual body weight (ABW) (23×ABW(0.5) mg daily) targeting an area under the concentration-time curve (AUC) of 5924 µM·min. Pharmacokinetic profiles were collected using a limited sampling strategy by randomly selecting 2 time points at 3.5, 5, 6, 7 or 22 hours after starting busulfan administration. Using an established population pharmacokinetic model with NONMEM software, busulfan concentrations at the available blood sampling times were predicted from dosage history and demographic data. The predicted and measured concentrations were compared by a visual predictive check (VPC). Maximum a posteriori Bayesian estimators were estimated to calculate the predicted AUC (AUC(PRED)). The accuracy and precision of the AUC(PRED) values were assessed by calculating the mean prediction error (MPE) and root mean squared prediction error (RMSE), and compared with the target AUC of 5924 µM·min. VPC showed that most data fell within the 95% prediction interval. MPE and RMSE of AUCPRED were -5.8% and 20.6%, respectively, in the conventional dosing group and −2.1% and 14.0%, respectively, in the new dosing scheme group. These fi ndings demonstrated the validity of a new dosing scheme for daily intravenous busulfan used as conditioning therapy for HCT.

Targeted busulfan and fludarabine-based conditioning for bone marrow transplantation in chronic granulomatous disease / 소아과

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by impaired phagocytic function. Hematopoietic stem cell transplantation (HSCT) is a definitive cure for CGD; however, the use of HSCT is limited because of associated problems, including transplantation-related mortality and engraftment failure. We report a case of a patient with CGD who underwent successful HSCT following a targeted busulfan and fludarabine reduced-toxicity myeloablative conditioning. Intravenous busulfan was administered once daily for 4 consecutive days (days –8 to –5), and the target area under the curve was 75,000 µg·hr/L. Fludarabine (40 mg/m2) was administered once daily for 6 consecutive days from days –8 to –3. Antithymocyte globulin (2.5 mg/kg/day) was administered from days –4 to –2. The patient underwent successful engraftment and did not have any severe toxicity related to the transplantation. Conditioning with a targeted busulfan and fludarabine regimen could provide a better outcome for HSCT in CGD, with close regulation of the busulfan dose.

Humanizing NOD/SCID/IL-2Rγnull (NSG) mice using busulfan and retro-orbital injection of umbilical cord blood-derived CD34+ cells

BACKGROUND: Humanized mouse models are still under development, and various protocols exist to improve human cell engraftment and function. METHODS: Fourteen NOD/SCID/IL-2Rγnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34+ hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM), spleen, and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation. RESULTS: Most of the NSG mice tolerated the regimen well. The percentage of hCD45+ and CD19+ cells rose significantly in a time-dependent manner. The median percentage of hCD45+cells in the BM was 55.5% at week 8, and 67.2% at week 12. The median percentage of hCD45+ cells in the spleen at weeks 8 and 12 was 42% and 51%, respectively. The median percentage of hCD19+ cells in BM at weeks 8 and 12 was 21.5% and 39%, respectively (P=0.04). Similarly, the median percentage of hCD19+ cells in the spleen at weeks 8 and 12 was 10% and 24%, respectively (P=0.04). The percentage of hCD19+ B cells in PB was 23% at week 12. At week 8, hCD3+ T cells were barely detectable, while hCD7+ was detected in the BM and spleen. The percentage of hCD3+ T cells was 2‒3% at week 12 in the BM, spleen, and PB of humanized NSG mice. CONCLUSION: We adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45+ cells than earlier studies without any significant toxicity. And human CD45+ cell engraftment at week 8 was comparable to that of week 12.

Clinical study on treatment of high-risk acute leukemia with unrelated cord blood transplantation / 器官移植

Objective To investigate the effect of unrelated cord blood transplantation (UCBT)on the treatment of high-risk childhood and adult acute leukemia.Methods Ten patients with high-risk acute leukemia underwent UCBT.Among the 1 0 patients,3 were children and 7 were adults with the median age of 29 years old (1 1 -41 years old).Six patients underwent one-unit cord blood transplantation and four patients underwent two-unit cord blood transplantation.The myeloablative conditioning regimen without antithymocyte globulin (ATG)was adopted.Cytarabine (Ara-C),fludarabine (Flu)or total body irradiation (TBI)was added on the basis of busulfan (Bu)and cyclophosphamide (Cy).Ciclosporin and mycophenolate mofetil were used to prevent graft-versus-host disease (GVHD).Results The transplantation was successful in 8 (80%) patients.The median implant-time of leukocytes was 1 9 d(1 4-25 d)and that of platelets was 40 d(33-60 d).Three patients developed acute GVHD and no patient developed chronic GVHD.The median follow-up time was 24 months (1 -29 months).Seven patients remained in disease-free survival.Both the 2-year overall survival and disease-free survival rates were 66.7%.Conclusions UCBT is feasible in the treatment of high-risk acute leukemia.UCBT is the preferred option for the high-risk patients without HLA-identical sibling donors,which is characterized by low incidence of GVHD and low recurrence rate.It may make patients with acute leukemia remain long-term survival.

Limited sampling strategies for clinical plasma concentration monitoring of busulfan in hematopoietic stem cell transplantation patients / 中国药学杂志

OBJECTIVE: To establish limited sampling strategy to estimate area under the curve (AUC) of HSCT patients who had received busulfan (Bu) intravenous infusion. METHODS: Plasma samples were collected at certain time points from 22 HSCT patients who had received busulfan intravenous infusion. The LC-MS/MS method was used to measure blood concentration of busulfan. The classical method was used to calculate pharmacokinetic parameters. To estimate AUC0-6, measured blood concentration data was used to build a multiple linear regression model which was subsequently validated. Consistency between results produced by the traditional method and the proposed LSS respectively was assessed by the intraclass correlation coefficient (ICC) and Bland-Altman (BA) analysis. RESULTS: It is shown that model based on concentrations on two sampling time points (2 and 5 h) is able to predict AUC0-6 accurately (adjusted r2=0.917. MPE=0.2%, RMSE=4.48%). The 95% confidence interval of ICC is 0.914-0.984 while the limit of agreement in the BA is -0.9-8.8. Results produced by limited sampling strategy are nearly consistent with AUC0-6 produced by the classical method. CONCLUSION: The proposed limited sampling strategy to estimate AUC0-6 based on C2 and C5 can be used to busulfan blood concentration monitoring in clinical practice.

Induction of Spermatogenesis by Bone Marrow-derived Mesenchymal Stem Cells in Busulfan-induced Azoospermia in Hamster

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) have potential of differentiation and they secrete anti-inflammatory cytokines and growth factors which make them appropriate for cell therapy. AIM OF THE WORK: Were to evaluate the healing effect of BM-MSCs transplantation on germinal cells of busulfan-induced azoospermic hamsters. MATERIAL AND METHODS: In the present experimental case control study, BM-MSCs were isolated from bone marrow of donor albino hamsters. Five mature male recipient hamsters received two doses of 10 mg/kg of busulfan with 21 days interval to stop endogenous spermatogenesis. After induction of azoospermia, right testis of hamsters was injected with 106 BM-MSCs via efferent duct and the left one remained as azoospermia control testis. Five normal mature hamsters were selected as normal intact control. After 35 days, testes and epididymis of three groups were removed for histological evaluation. RESULTS: Histomorphological analyses of BM-MSCs treated testes and epididymis showed the epithelial tissue of seminiferous tubules had normal morphology and spermatozoa were present in epididymis tubes. Spermatogenesis was observed in most cell-treated seminiferous tubules. The untreated seminiferous tubules were empty. CONCLUSION: Transplanted BM-MSCs could successfully induce spermatogenesis in seminiferous tubules of azoospermic hamster. Therefore, BM-MSCs can be an attractive candidate in cell transplantation of azoospermia.

Exploration of the dynamic responses of Sertoli cells to depletion of spermatogonial stem cells in mouse testis / 中国实验动物学报

Objective To explore the dynamic responses of Sertoli cells to depletion of spermatogonial stem cells by busulfan.Methods After intraperitoneal injection of 15, 30 or 44 mg/kg busulfan to mice, the spermatogenesis and the expression of GDNF, PLZF, Nanog and GFRɑ1 mRNA were assessed by real-time quantitative PCR at 5 and 28 days after the busulfan treatment.Results Glial cell line-derived neutrophic factor ( GDNF ) was significantly increased and showed a dose-dependent trend at 5 days after busulfan treatment, but no significant difference was seen in the expression of promyelocytic leukemia zinc finger(PLZF) and GDNF family receptorα-1(GFRα1).The testicular histology also appeared no significant difference at 5 days after busulfan treatment.At 28 days after busulfan treatment, the relative expression lev-els of GDNF, PLZF, Nanog and GFRɑ1 mRNA were drastically increased.Morphological observation showed that spermat-ogenesis damages became even more severe as the busulfan dose increased.Conclusions Sertoli cell response to the de-pletion of spermatogonia occurs as early as the fifth day after busulfan treatment.Production of GDNF in Sertoli cells shows a compensatory increase, which may stimulate spermatogonial stem cells to accelerate their self-renewal, reflected by the enhancing expression of Nanog and PLZF, and ultimately promote the restoration of spermatogenesis.