Gene expression profile in experimental frozen-thawed ovarian grafts treated with scaffold-base delivery of adipose tissue-derived stem cells

Purpose: Gelfoam scaffold is a feasible and safe non-invasive technique for Adipose tissue-derived Stem Cell (ASC)-delivery in the treatment of frozen-thawed ovarian autografts. This study seeks to analyze the genes expression profile of rat frozen-thawed ovarian autografts treated with scaffold-based delivery of adipose tissue-derived stem cells. Methods: Eighteen adult Wistar rats were distributed into three groups: Control (frozen-thawed only); Group 1 (G1) and Group 2 (G2) (frozen-thawed ovaries treated with culture medium or ASC, respectively). Both treatments were performed immediately after autologous retroperitoneal transplant with scaffold-based delivery. The ovarian grafts were retrieved 30 days after transplantation. Quantitative gene expression (qPCR) for apoptosis, angiogenesis, and inflammatory cytokines (84 genes in each pathway) were evaluated by RT-PCR. Graft morphology (HE), apoptosis (cleaved-caspase-3), neoangiogenesis (VEGF), and cellular proliferation (Ki-67) were assessed. Results: In grafts treated with ASC, the apoptosis pathway showed the highest number of genes over-regulated – 49 genes – compared to inflammation cytokines and angiogenesis pathway – 36 and 23 genes respectively, compared to grafts treated with culture medium. Serpinb5 family was highlighted in the angiogenesis pathway and Cxcl6 in the inflammation cytokines pathway. In the apoptosis pathway, the most over-regulated gene was Capsase14. ASC treatment promoted the reduction of cleaved caspase-3 in the theca internal layer and increased cell proliferation by Ki-67 in the granulosa layer without altering VEGF. A mild inflammatory infiltrate was observed in both groups. Conclusion: ASC therapy in rat frozen-thawed ovarian autografts promoted an abundance of genes involved with apoptosis and inflammatory cytokines without compromising the ovary graft morphology and viability for short time. Further studies are necessary to evaluate the repercussion of apoptosis and inflammation on the graft in the long term

N-acetylcysteine improves morphologic and functional aspects of ovarian grafts in rats

PURPOSE: To evaluate morphological and functional aspects of the ovarian graft in transplanted rats treated with NAC.METHODS: Female Wistar rats, virgin, 3 to 4 months old, weighing 200-250 grams were used in experiments. The rats have been kept in proper sanitary conditions, receiving food and water ad libitum. Five groups (n=10, each) were constituted: 4 groups treated subcutaneously with NAC, at doses of 150, 300, 600 and 1200 mg/kg (NAC150, NAC300, NAC600 and NAC1200, respectively), one hour of before the ovarian transplantation and control group (GTx) - treated with physiological solution and submitted to ovarian transplantation. The rats were anesthetized and submitted to autologous left ovarian transplantation, without anastomosis in retroperitoneum, and contralateral oophorectomy. During follow-up of 4 or 15 days, the estrous cycle was evaluated by vaginal smears to determine cycle regularity. At the end of 4th or 15th days, rats were re-anesthetized and blood and graft were obtained to estradiol analysis and morphological assessment. Data were analysed by One Way Analysis of Variance (ANOVA) or ANOVA on ranks complemented by Student-Newman-Keuls test.RESULTS: At 4th day, viable follicles in the graft did not altered by NAC treatments. The NAC300 and NAC600 groups showed increasing in follicle atresia (p=0.012) compared to GTx and NAC1200 group. At 15th day, 50% of GTx, NAC150, and NAC300 rats showed regular oestrous cycle; 83% of NAC600 and 100% of NAC1200 rats returned to regular cycle. NAC1200 group showed increasing in primordial follicle compared to GTx, NAC150 or NAC300 (p=0.011). NAC did not interfere in estradiol levels after 4 or 15 days of transplantation.CONCLUSION: In autologous ovarian transplantation, high dose of NAC promotes graft viability with recovery of estrous cycle.Federal University do Vale do São FranciscoUNIFESPUniversity of São Paulo Medical School Obstetrics and Gynecology DepartmentFederal University of Ceara Faculty of MedicineUSP FM Department of Clinical MedicineUNIFESPSciEL

Estudo do esfregaço vaginal de ratas submetidas a transplante autólogo de ovário: impacto do precondicionamento isquêmico remoto

PURPOSE: To evaluate the remote ischemic preconditioning (R-IPC) impact on the quality of the ovarian graft by means of vaginal smear of transplanted rats. METHODS: Sixty rats were used divided in six groups: Control; Fresh transplant (TxF); Cryopreserved transplant (TxC); R-IPC; R-IPC + fresh transplant (TxF+R-IPC); R-IPC + cryopreserved transplant (TxC+R-IPC). R-IPC was performed in the common iliac artery. Autologous ovarian tissue was implanted integrally in the retro peritoneum. On the first PO day, vaginal smear collection was daily initiated. After 30 days, the PO day when the estrous cycle was re-initiated was considered for analysis as well as the estrous days and the number of estrous cycles. RESULTS: R-IPC showed a tendency to an early estrus re-initiation (p>0.05) as well as increase the number of cycles in the fresh transplanted group while in the cryopreserved transplant the number of cycles was similar, regardless of the stimulus R-IPC (p>0.05). The animals which had undergone fresh grafts had a longer estrous period than the ones which had undergone cryopreserved grafts, with or without R-IPC (p0,05), assim como aumento no número de cilcos estrais nos grupos com transplante fresco enquanto que no criopreservado o número de ciclos foi semelhante, independente do PCI-R (p>0,05). Os animais que receberam enxertos frescos apresentaram mais dias na fase estro do que os criopreservados, com ou sem PCI-R. CONCLUSÃO: O PCI-R promoveu retorno mais precoce da atividade ovariana no PO e maior freqüência de estro, sendo os resultados mais consistentes nos enxertos frescos do que nos criopreservados.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)UNIFESP Surgery and Research Postgraduate ProgramUniversidade Federal de São Paulo (UNIFESP) Department of PathologyUNIFESP Gynecological DivisionUniversidade Federal de São Paulo (UNIFESP) Department of Morphology Histology and Structural Biology DivisionUniversidade Federal de São Paulo (UNIFESP) Department of Surgery Operative Technique and Experimental Surgery DivisionUNIFESP, Surgery and Research Postgraduate ProgramUNIFESP, Department of PathologyUNIFESP, Gynecological DivisionUNIFESP, Department of Morphology Histology and Structural Biology DivisionUNIFESP, Department of Surgery Operative Technique and Experimental Surgery DivisionSciEL

Adipose tissue-derived stem cell therapy in rat cryopreserved ovarian grafts

Abstract\ud The preliminary results of ovarian transplantation in clinical practice are encouraging. However, the follicular depletion caused by ischemic injury is a main concern and is directly related to short-term graft survival. Cell therapy with adipose tissue-derived stem cells (ASCs) could be an alternative to induce early angiogenesis in the graft. This study aimed to evaluate ASCs therapy in rat cryopreserved ovarian grafts. A single dose of rat ASC (rASCs) or vehicle was injected into the bilateral cryopreserved ovaries of twelve adult female rats immediately after an autologous transplant. Daily vaginal smears were performed for estrous cycle evaluation until euthanasia on postoperative day 30. Follicle viability, graft morphology and apoptosis were assessed. No differences were found with respect to estrous cycle resumption and follicle viability (P > 0.05). However, compared with the vehicle-treated grafts, the morphology of the ASCs-treated grafts was impaired, with diffuse atrophy and increased apoptosis (P < 0.05). ASCs direct injected in the stroma of rat cryopreserved ovarian grafts impaired its morphology although may not interfere with the functional resumption on short-term. Further investigations are necessary to evaluated whether it could compromise their viability in the long-term.We thank: 1) São Paulo Research Foundation (FAPESP) for grant support\ud (Process numbers: 2010/17897-5 and 2012/09469-9) and Coordenação de\ud Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for post-doctoral\ud scholarship

Effect of hypovolemic shock in anastomose of small intestine of rats

Objective Study the effect of hypovolemic shock on small intestinal anastomose in adult rats. Method Ninety male rats were randomly divided into five groups: standard, hypovolemic shock, anastomose alone, hypovolemic shock + anastomose and hypovolemic shock + anastomose + blood reinfusion. Hypovolemic shock was achieved by bleeding 30% of the blood volume of the animal. A single layer extramucosal intestinal anastomose was performed. Following intestinal anastomose, the blood volume was restored in group shock, anastomose and blood reinfusion, using heparinized autologous. Results and Conclusion In the experimental model used, hypovolemic shock provoked histological lesions to the mucosa and increased colagen fiber deposition into the submucosa in the region of intestinal anastomose which were progressive with post-operative period. In addition, when the hypovolemic shock group were pressure tested there was a tendency towards a weakening of the intestinal wall at day 7 which became more evident at day 21 as compared to the control groups.Racional O choque hipovolêmico está incluído entre os fatores de risco para anastomoses intestinais, entretanto, sua ação sobre anastomoses do intestino delgado tem sido pouco estudada e, quando abordado, é avaliado como parâmetro de comparação com a cicatrização de cólons e não como alvo central das investigações. Objetivo - Estudar o efeito do choque hipovolêmico na anastomose do intestino delgado de ratos. Método - Foram utilizados 90 ratos machos adultos, distribuídos em cinco grupos: padrão, choque, anastomose, choque mais anastomose e choque mais anastomose mais reposição. O choque hipovolêmico foi obtido pela sangria de 30% da volemia do animal. Realizou-se anastomose término-terminal em plano único extra-mucoso, à média distância, entre o duodeno e a válvula ileocecal. A reposição volêmica no grupo choque, mais anastomose mais reposição foi realizada com o sangue autólogo. Resultados e Conclusão - Do ponto de vista histológico, o choque hipovolêmico provocou alterações progressivas na região da anastomose intestinal, quanto à lesão da túnica mucosa e o aumento na deposição de fibras colágenas na tela submucosa, com a evolução do período pós-operatório. Quanto à pressão de ruptura promoveu, em relação aos grupos controle, tendência a diminuição da resistência da parede intestinal e da anastomose intestinal com a evolução do período pós-operatório.UEPA Departamento de Saúde IntegradaUEPA Laboratório de Cirurgia ExperimentalUNIFESP-EPM Departamento de Gastroenterologia PediátricaUNIFESP, EPM, Depto. de Gastroenterologia PediátricaSciEL