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

Abstract 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 (Gl) 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 Cap-sasel4. 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. HIGHLIGHTS The scaffold-based delivery therapy with adipose tissue-derived stem cells in the rat ovarian autografts seems to be the best option when compared to direct injection or systemic route. Ovarian grafts treated with adipose tissue-derived stem cells showed the highest number of genes over-regulated in the apoptosis pathway, compared to inflammation cytokines and angiogenesis pathway. Capsase14 was the most over-regulated gene in the apoptosis pathway. The treatment with adipose tissue-derived stem cells in ovarian grafts treated didn't compromise the ovary graft morphology and viability for short time.

Does a bilateral polypropylene mesh alter the duct deferens morphology, testicular size and testosterone levels? Experimental study in rats

Abstract Purpose To evaluate the effect of a PP mesh on duct deferens morphology, testicular size and testosterone levels. Methods Forty adult male rats were distributed into groups: 1) no surgery; 2) inguinotomy; 3) mesh placed on the duct deferens; and 4) mesh placed on the spermatic funiculus. After 90 postoperative days, the inguinal region was resected, and blood samples were collected for the measurement of serum testosterone (pg/dl). The ducts deferens were sectioned in three axial sections according to the relationship with the mesh — cranial, medial and caudal. The wall thickness and duct deferens lumen area were measured. Results The morphology of the duct deferens was preserved in all groups. The mesh placement did not alter this morphology in any of the analyzed segments. Surgery, with or without mesh placement, did not alter the morphology, wall thickness or lumen area (p>0.05). In all operated groups, serum testosterone levels were similar (p>0.05) but there was a decrease in testicle size (p<0.05). Conclusion Surgery, with or without mesh placement, did not alter the morphology of the duct deferens and, although this treatment resulted in testicular size reduction, it did not affect serum testosterone levels.

Polypropylene and polypropylene/polyglecaprone (Ultrapro(r)) meshes in the repair of incisional hernia in rats

PURPOSE: To compare the inflammatory response of three different meshes on abdominal hernia repair in an experimental model of incisional hernia. METHODS: Median fascial incision and skin synthesis was performed on 30 Wistar rats. After 21 days, abdominal hernia developed was corrected as follows: 1) No mesh; 2) Polypropylene mesh; and, 3) Ultrapro(r) mesh. After 21 days, the mesh and surrounding tissue were submitted to macroscopic (presence of adhesions, mesh retraction), microscopic analysis to identify and quantify the inflammatory and fibrotic response using a score based on a predefined scale of 0-3 degrees, evaluating infiltration of macrophages, giant cells, neutrophils and lymphocytes. RESULTS: No significant difference was seen among groups in adherences, fibrosis, giant cells, macrophages, neutrophils or lymphocytes (p>0.05). Mesh shrinkage was observed in all groups, but also no difference was observed between polypropylene and Ultrapro mesh (7.0±9.9 vs. 7.4±10.1, respectively, p=0.967). Post-operatory complications included fistula, abscess, dehiscence, serohematic collection and reherniation, but with no difference among groups (p=0.363). CONCLUSION: There is no difference between polypropylene (high-density) and Ultrapro(r) (low-density) meshes at 21 days after surgery in extraperitoneal use in rats, comparing inflammatory response, mesh shortening, adhesions or complications. .

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. .

Study on the vaginal smear of rats submitted to autologous ovarian transplant: impact of remote ischemic preconditioning / 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 (p<0.05). CONCLUSION: R-IPC promoted earlier re-initiation of ovarian activity in the PO and greater estrous frequency, with more consistent results in the fresh grafts than in the cryopreserved ones.

OBJETIVO: Avaliar o impacto do precondicionamento isquêmico remoto (PCI-R) na qualidade do enxerto ovariano através dos esfregaços vaginais dos animais transplantados. MÉTODOS: Foram utilizadas 60 ratas, distribuídas em seis grupos de estudo: Controle; Transplante fresco; Transplante criopreservado; PCI-R; PCI-R + Transplante fresco; PCI-R + Transplante criopreservado. O PCI-R foi realizado na artéria ilíaca comum. O tecido ovariano foi implantado íntegro no retroperiônio. No 1º dia de pós-operatório (PO) foram coletados esfregaços vaginais diariamente. Após 30 dias foram considerados para análise o dia de PO de retorno do estro, assim como o número de dias em estro e de ciclos estrais. RESULTADOS: PCI-R mostrou tendência em reinício mais precoce do cilo estral (p>0,05), 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.

Indirect evaluation of estrogenic activity post heterotopic ovarian autograft in rats / Avaliação indireta da atividade estrogênica após transplante heterotópico de ovário em ratas

PURPOSE: To morphologically evaluate the estrogenic effect on the uterus and vagina of rats submitted to ovarian autografts. METHODS: Twenty Wistar EPM-1 adult rats were bilaterally ovariectomized, followed by ovarian transplants in retroperitoneal regions. The animals were divided in four groups of five animals, according to the day of euthanasia: G4, G7, G14 and G21, corresponding to the 4th, 7th, 14th and 21st day after surgery, respectively. Vaginal smears were collected from the first day of surgery until euthanasia day. After that, the vagina and uterus were removed, fixed in 10 percent formaldehyde and submitted to histological analysis and stained with hematoxiline and eosine. RESULTS: All animals showed estrous cycle changes during the experiment. In 4th day, the uterus showed low action of estrogen with small number of mitosis and eosinophils as well as poor development. On the 7th day, the endometrium was atrophic without mitotic signals and presented a small number of eosinophils. On the 14th and 21th days the histological findings were similar, with the presence of mitosis in the endometrial glands and intense leucocyte infiltration with a large number of eosinophils. Morphometric results showed that the endometrial and myometrial thickness as well as the number of eosinophils presented the highest values during the 14th and 21th days of the evaluation. The 7th day group also presented the lowest eosinophil numbers. Vaginal epithelium features were: 4th and 7th day groups presented non-keratinized stratified epithelium with 5 and 2 cell layers, respectively. The 14th and 21st day groups presented non-keratinized stratified epithelium with 14 and 15 cell layers. CONCLUSION: Experimental ovarian autografts in the evaluated organs presented maximum estrogen activity after the 21st day of surgery, according to morphological and morphometric data.

OBJETIVO: Avaliar morfologicamente a atividade estrogênica no útero e vagina de ratas submetidas a transplante autólogo de ovário. MÉTODOS: Foram utilizadas 20 ratas adultas, Wistar EPM-1, submetidas à ooforectomia bilateral seguida de transplante de ovário em retroperitônio, distribuídas em grupos, conforme a data pré-estabelecida para eutanásia, com cinco animais cada: G4, G7, G14 e G21 - eutanásia no 4º, 7º, 14º e 21º dia de pós-operatório, respectivamente. No 1º dia após a cirurgia foi iniciada coleta de esfregaço vaginal diariamente até o dia pré-estabelecido para eutanásia. Após a eutanásia foi realizada exérese em bloco da vagina e útero, que foram fixados em formol 10 por cento para processamento histológico e coloração pela Hematoxilina-Eosina. RESULTADOS: Todos os animais mostraram alterações do ciclo estral no decorrer dos dias do experimento. No 4º dia, o útero mostrava sinais de baixa atividade estrogênica, o endométrio apresentava poucas figuras de mitose, pouco desenvolvimento e pequena quantidade de eosinófilos. Já no 7º dia, o endométrio encontrava-se atrofiado, sem atividade mitótica e com raros eosinófilos. No 14º e 21º dias os achados foram semelhantes, com presença de mitose nas glândulas endometriais e intenso infiltrado leucocitário, com predomínio de eosinófilos. Os resultados da morfometria mostraram que tanto as espessuras endometrial e miometrial quanto o número de eosinófilos foram maiores no 14º e 21º dias de avaliação, com menores valores no 7º dia. As características do epitélio vaginal foram: epitélio pavimentoso não queratinizado no 4º e 7º (com cerca de cinco e duas camadas celulares, respectivamente) e pavimentoso queratinizado em no 14º e 21º dias (com cerca de 14 e 15 camadas celulares, respectivamente) CONCLUSÃO: O transplante experimental autólogo de ovário manteve a atividade estrogênica nos órgãos avaliados, com atividade estrogênica máxima ao final de 21 dias, conforme parâmetros morfológicos...