Evaluation of equine (Equus cabbalus) corneal endothelium stored in EUSOL-C® preservation medium / Avaliação do endotélio da córnea de equinos (Equus cabbalus) acondicionado em meio de preservação de córnea Eusol-C®

The objective of this study was evaluate the maintenance of the corneal endothelium of horses in cold EUSOL-C® preservation medium over different periods (seven and 14 days) using scanning electron microscopy. A total of 20 pairs of eyes from horses were analysed. The corneas were divided into four groups of 10 corneas each (G1, G2, G3 and G4): G1 - the samples were kept in the preservation medium for seven days; G3 - the samples were kept in the preservation medium for for 14 days; G2 and G4 were formed by the control corneal buttons of G1 and G3, respectively. The average cell loss observed in G1 was 7.62%, in G2 it was 7.04%, in G3 9.12% and in G4 7.16%. No statistically significant differences were observed between the four groups. It was concluded that the Eusol-C® hypothermic preservation medium provided satisfactory preservation of the corneal endothelium in equine species for up to 14 days.

Objetivou-se avaliar a manutenção do endotélio da córnea de equinos em meio de preservação a frio EUSOL-C® em diferentes períodos de acondicionamento (sete e 14 dias) utilizando microscopia eletrônica de varredura. Foram avaliados 40 bulbos oculares de 20 equinos. Os bulbos oculares foram divididos em quatro grupos (G1, G2, G3 e G4), nos quais: G1 foi composto por 10 botões corneais acondicionados em meio de preservação para córnea Eusol-C® durante sete dias; o G3 foi formado por 10 botões corneais acondicionados em meio de preservação para córnea Eusol-C® durante 14 dias. O G2 e G4 foram formados pelas córneas controle armazenadas imediatamente em glutaraldeído. A média da perda celular observada no G1 foi de 7,62%, no G2 foi de 7,04 %, no G3 foi de 9,12% e o no G4 foi de 7,16%. Não foram observadas diferenças estatisticamente significativas entre os quatro grupos. Foi possível concluir que o meio de preservação hipotérmico Eusol-C® proporcionou de forma satisfatória a preservação do endotélio da córnea na espécie equina durante o período de até 14 dias.

Evaluation of equine (Equus cabbalus) corneal endothelium stored in EUSOL-C® preservation medium / Avaliação do endotélio da córnea de equinos (Equus cabbalus) acondicionado em meio de preservação de córnea Eusol-C®

The objective of this study was evaluate the maintenance of the corneal endothelium of horses in cold EUSOL-C® preservation medium over different periods (seven and 14 days) using scanning electron microscopy. A total of 20 pairs of eyes from horses were analysed. The corneas were divided into four groups of 10 corneas each (G1, G2, G3 and G4): G1 - the samples were kept in the preservation medium for seven days; G3 - the samples were kept in the preservation medium for for 14 days; G2 and G4 were formed by the control corneal buttons of G1 and G3, respectively. The average cell loss observed in G1 was 7.62%, in G2 it was 7.04%, in G3 9.12% and in G4 7.16%. No statistically significant differences were observed between the four groups. It was concluded that the Eusol-C® hypothermic preservation medium provided satisfactory preservation of the corneal endothelium in equine species for up to 14 days.(AU)

Objetivou-se avaliar a manutenção do endotélio da córnea de equinos em meio de preservação a frio EUSOL-C® em diferentes períodos de acondicionamento (sete e 14 dias) utilizando microscopia eletrônica de varredura. Foram avaliados 40 bulbos oculares de 20 equinos. Os bulbos oculares foram divididos em quatro grupos (G1, G2, G3 e G4), nos quais: G1 foi composto por 10 botões corneais acondicionados em meio de preservação para córnea Eusol-C® durante sete dias; o G3 foi formado por 10 botões corneais acondicionados em meio de preservação para córnea Eusol-C® durante 14 dias. O G2 e G4 foram formados pelas córneas controle armazenadas imediatamente em glutaraldeído. A média da perda celular observada no G1 foi de 7,62%, no G2 foi de 7,04 %, no G3 foi de 9,12% e o no G4 foi de 7,16%. Não foram observadas diferenças estatisticamente significativas entre os quatro grupos. Foi possível concluir que o meio de preservação hipotérmico Eusol-C® proporcionou de forma satisfatória a preservação do endotélio da córnea na espécie equina durante o período de até 14 dias.(AU)

Propriedades tensiométricas do peritônio da paca (Cuniculus paca) a fresco e conservado em glicerina 98 por cento / Tensiometer peritoneum properties of the paca (Cuniculus paca) fresh and preserved in glycerin 98 por cento

In constant searching for alternative biological material to perform implants and new options of experimental animal models, the objective of this investigation was to describe the mechanical properties of the peritoneum paca (Cuniculus paca Linnaeus, 1766) fresh and preserved in 98% glycerin. Samples of fresh and preserved in glycerin for periods of 30, 60 and 90 days were subjected to mechanical tests. Four adult animals, male or female, with mean body weight of eight kilograms, were used for collecting samples of the peritoneum. All tissues preserved in glycerin 98% showed a decrease in stiffness and increase in ductility and toughness. Considering the maximum force applied to the peritoneum, significant increase was observed in values (p<0.01) of samples stored for 60 and 90 days when compared to fresh material. In relation to the stretch variable, an increase was observed in all storage time of glycerin samples, verifying significant difference (p<0,01) when compared with the fresh samples. The variable area also showed significance (p <0.01) between the values of the fresh samples (5.40 mm²) and preserved in the glycerin by periods of 30 days (4.50 mm²), 60 days (9.00 mm²) and 90 days (7.20 mm²), thus indicating that the area of this membrane increased by 0.033 mm² per day. Generally, it was concluded that the 98% glycerin is a substance effective for the preservation of the peritoneum of the agouti paca, therefore improves its mechanical properties allowing the support membranes greater deformation forces. Thus, the results obtained in mechanical tests of the peritoneum of paca suggest its use as an alternative biological material.(AU)

Na busca constante, tanto de material biológico alternativo para a realização de implantes, quanto de novas opções de modelos de experimentação animal, o objetivo desta investigação foi descrever o comportamento mecânico do peritônio da paca (Cuniculus paca Linnaeus, 1766) a fresco e conservado em glicerina a 98%. Amostras frescas e conservadas em glicerina por períodos de 30, 60 e 90 dias foram submetidas a testes mecânicos de tração. Quatro animais adultos, três machos e uma fêmeas, com peso corporal médio de oito quilogramas, foram utilizados para colheita das amostras de peritônio. Todos os tecidos conservados em glicerina a 98% apresentaram diminuição na rigidez e aumento na ductibilidade e tenacidade. Considerando-se a força máxima aplicada ao peritônio, evidenciou-se aumento significativo nos valores (p<0,01) das amostras conservadas por 60 e 90 dias, quando comparado ao material a fresco. Com relação a variável alongamento, notou-se aumento nos valores relativos aos materiais em glicerina em todos os tempos de conservação, verificando-se diferença significativa (p<0,01) entre os valores das amostras a fresco. A variável área também se apresentou significativa (p<0,01) entre os valores das amostras a fresco (5,40 mm²) e os preservados em glicerina pelos períodos de 30 dias (4,50 mm²), 60 dias (9,00 mm²) e 90 dias (7,20 mm²), indicando assim, que a área desta membrana aumentou em 0,033 mm² por dia. Mediante os resultados observados, concluiu-se que a glicerina 98% é uma substância eficiente para a conservação do peritônio da paca, pois melhorou suas propriedades mecânicas permitindo que as membranas suportem maiores forças de deformação. Assim, os resultados obtidos nos ensaios mecânicos do peritônio da paca sugerem sua utilização como mais uma opção de material biológico.(AU)

Efeitos de imunossupressores sobre o sistema imunológico em transplantes de células germinativas em trutas arco-íris

Resumo YOSHINAGA, T.T. Efeitos de imunossupressores sobre o sistema imunológico em transplantes de células germinativas em trutas arco-íris Os transplantes de células germinativas e enxertos de testículo podem ser aplicados na reprodução de espécies comerciais e na conservação de espécies ameaçadas de extinção. No entanto, a maior limitação dos transplantes está na sua limitada eficiência, devido à baixa porcentagem de hospedeiros capazes de gerar gametas derivados do doador e da rejeição de enxertos de testículo alogênicos após poucas semanas de sua implantação. O sistema imunológico dos hospedeiros podem estar envolvidos na baixa de eficiência dos transplantes e na rejeição dos enxertos. Desta forma, este trabalho buscou verificar se a administração de drogas imunossupressoras como o tacrolimus e ciclosporina em emulsão poderiam ser utilizados para prolongar a viabilidade de enxertos de testículo em trutas arco-íris. Na primeira parte, experimentos in vitro com leucócitos do sangue periférico demonstraram que ambos o tacrolimus e a ciclosporina impedem a proliferação celular mesmo sob estímulo proliferativo da concanavalina. Nos ensaios in vivo, ambas as doses de tacrolimus (0,5 e 1,5 mg/kg) e a mais baixa de ciclosporina (20 mg/kg) inibiram significativamente a expressão de il2 no rim cefálico três dias após a injeção. Já a dose de 40 mg/kg de ciclosporina inibiu a expressão de il2 por até sete dias após a injeção. Na segunda parte, enxertos alogênicos de testículo foram realizados em animais tratados semanalmente com emulsões de tacrolimus. As análises histológicas (coloração H.E.) e as de RT-PCR (vasa e txdnc6) demonstraram a presença de espermatogônias na primeira semana e indicaram a presença de espermátides/espermatócitos na quinta semana, respectivamente, em alguns animais do grupo tratado com a dose de 0,5 mg/kg de tracrolimus. No grupo tratado com a dose mais alta de tacrolimus (1,5 mg/kg) e no grupo controle (sem imunossupressor), células germinativas ou marcadores destas não foram detectados. Estes resultados, portanto, fazem do tacrolimus um imunossupressor promissor para utilização em enxertos alogênicos e também em transplantes de células germinativas de truta arco-íris. A administração concomitante de um outro imunossupressor conjugado ao tacrolimus (na baixa dosagem) para inibir duas ou mais vias de ativação do sistema imunológico, conforme utilizado em transplantes de órgãos em humanos, pode ser uma alternativa para otimizar os efeitos imunossupressivos nos animais receptores. Palavras-Chave Reprodutores substitutos; imunossupressão;transplante de células germinativas;

Abstract YOSHINAGA, T.T Effects of immunosuppressants on immune system in germ cell transplantation in rainbow trout Germ cell transplantation and testis graft can be applied for the reproduction of commercial or endangered species. However, mechanisms of rejection from the host immune system might limit the production of surrogate gametes and progeny after transplantation and induce rejection of testis allografts within few weeks. In this work, we aimed to administer immunosuppressants-containing emulsions in order to verify whether they are capable to prevent immune rejection and promote testis allograft survival in rainbow trout. In the first part of this study, it was demonstrated in vitro that tacrolimus and cyclosporine were able to inhibit leucocyte proliferation even under concanavalin-induced mitotic stimulation. In in vivo experiments, both dosage of tacrolimus (0,5 and 1,5 mg/kg) and a lower of cyclosporine (20 mg/kg) inhibited significantly the expression of il2 in head kidney at three days post-injection. A higher dosage of cyclosporine (40 mg/kg) was able to inhibit il2 expression for up to seven days post-injection. In the second part, testis allografts were conducted in fish treated weekly with tacrolimus-containing emulsions. Histological (H.E. staining) and RT-PCR (vasa and txdnc6) analysis demonstrated the presence of spermatogonias in the first week and indicated the presence of spermatids/spermatocytes in the fifth week, respectively, in some animals treated with 0,5 mg/kg of tacrolimus. In the group treated with the highest tacrolimus dose (1,5 mg/kg) and in the control group (without immunosupressant), no germ cells or their respective markers were detected. These results suggest that tacrolimus comprise a promising immunosuppressant, with applications to testis allografts or germ cell transplantation in rainbow trout. Co-administration combining tacrolimus (at lower dose) with other immunosuppressive drugs for inhibiting more than two activation pathways of the immune system, as done in human organ transplantation, can be an alternative to optimize the immunossupressive effects in host organisms. Keywords Surrogate broodstock; immunosuppression; germ cell transplantation;

Experimental ovarian transplantation on stomach for bone repair in ovariohysterectomized rabbits

PURPOSE: To evaluate the bone repair process in ovariohysterectomized rabbit submitted to an ovarian transplant to stomach that may supplying some quantity of estrogen occurs to improve bone healing. METHODS: In 20 female rabbits three holes of 1, 2 and 3mm diameter in tibial shaft were made and after that all animals received OHE through a ventral incision and they were randomly divided into two groups of ten rabbits each. In group one, animals received one of their self-ovaries that transplanted on serosal layer of stomach and group two did not receive treatment. Animals were kept during bone healing for a period of 45 days and radiological, biochemical, biomechanical and histopathological evaluation. RESULTS: The tibial defects in group one healed completely after 45 days and had more callous than second group. There is significant difference between two groups after operation in 21, 28 and 35 days about estrogen, progesterone and phosphatase Alkaline. The maximum forces in group one, were significantly higher than that for the group two. CONCLUSION:Ovarian transplantation prevents the effects of ovariohysterectomized on bone healing of rabbit tibia, suggesting that unilateral transplanted ovaries can substitute for the action of ovaries on the skeleton in ovariohysterectomized animals.(AU)

SOROEPIDEMIOLOGIA DE Toxoplasma gondii EM DOADORES DE CÓRNEAS DO ESTADO DO RIO DE JANEIRO, BRASIL

A toxoplasmose é causada por um protozoário parasito intracelular obrigatório, Toxoplasma gondii. A maioria dessas infecções é assintomática ou benigna, mas pode causar consequências graves ou fatais em pacientes imunodeficientes, transplantados e no feto. A transmissão pode ocorrer pela ingestão de carne crua, vegetais contaminados, transfusão de sangue, transplantes de órgãos e através da placenta da mãe para o feto. Em muitos países, as taxas de prevalência variam de 15% a 85% na população humana. Em crianças, a prevalência é relativamente baixa, aumentando com a idade e com mais exposição a fatores de riscos durante o curso da vida. No Brasil, a soroprevalência da infecção por T. gondii é alta, em torno de 20 e 97% da população adulta. No presente estudo, o objetivo foi determinar a frequência de anticorpos IgG e IgM anti- T. gondii no soro de doadores de córneas do Banco de Tecido Ocular Pedro Sélmo Thiesen, Banco de Olhos de Volta Redonda, estado Rio de Janeiro, Brasil. Para tanto, foram coletadas 426 amostras de soros a serem testadas por meio do ensaio imunoenzimático (ELISA) e da reação de imunofluorescência indireta (RIFI) para detecção de anticorpos IgG e IgM específicos. Do total dos doadores, 262 (61,5%) eram do gênero masculino e 164 (38,5%) do feminino. A média da idade dos doadores foi 52.65 ± 16.96 anos. Sendo a idade mínima de 10 anos e a máxima de 80 anos. A faixa etária que obteve maior número de doadores foi a 51-60 anos (n=106/24,88%) seguida de 61- 70 anos (n= 99/23,23%), da faixa etária 41-50 anos (n= 65/ 15,26%) e da faixa etária 71 80 anos (n=61/14,32%). Os doadores eram oriundos de sete das oito regiões do estado do Rio de Janeiro Sendo a região Metropolitana (n=302/ 70,90%) e do Médio Paraíba (n=101/ 23,71%) com a maior concentração de doadores. O município que obteve maior número de doadores foi o de Duque de Caxias, com 87 (20,42%), seguido do município do Rio de Janeiro com 86 (20,19%) e Volta Redonda com 80 (18,78%) doadores. A principal causa de mortes dos 426 doadores de córneas foi o acidente vascular encefálico (AVC ou AVE), com 147 (34,50%), seguido de traumatismo crânio encefálico (TCE), com 89 (20%) e câncer com 25 (5,87%) doadores. Estimando-se o índice de concordância Kappa entre as duas técnicas sorológicas empregadas, encontrou-se = 0.69 para ELISA e/ou RIFI IgG e =0.14 para ELISA e/ou RIFI IgM. De acordo com os critérios de seleção os valores de Kappa estavam com concordância substancial para ELISA e/ou RIFI IgG e com ligeira concordância para o ELISA e/ou RIFI IgM. Considerando-se os resultados dos testes RIFI e/ou ELISA, a prevalência da infecção pelo T. gondii foi confirmada pela positividade de anticorpos IgG e IgM anti- T. gondii, sendo de 79,34% e 3,99%, respectivamente. Foi observada no gênero masculino uma positividade de 4,58% e 80,92% para anticorpos anti- T. gondii classe IgM e classe IgG, respectivamente. Enquanto os percentuais de sororreagentes no gênero feminino foram 3,05% e 76,83% para anticorpos anti- T. gondii classe IgM e classe IgG, respectivamente. Na faixa etária dos 71- 80 anos foram observados os maiores percentuais de doadores IgM e IgG soropositivos sendo 9(14,76%) e 55(90,16%) para anticorpos anti- T. gondii classe IgM e IgG, respectivamente. A região do Médio Paraíba obteve 4,95% e metropolitana 3,98% dos percentuais de doadores reagentes aos anticorpos IgM anti- T. gondii. Em relação à causa de óbitos, 12% dos doadores reagentes aos anticorpos IgM anti- T. gondii faleceram em decorrência de neoplasias. E 91,18% dos doadores foram IgG soropositivos e vieram a óbito devido a transtorno no aparelho respiratório. Estes dados demonstram a importância de inquéritos soroepidemiológicos regional e nacional contínuos para definir as estratégias de saúde pública v que possam reverter e reduzir a elevada prevalência da infecção pelo T. gondii na população geral e consequentemente na população doadora de córneas.

Toxoplasmosis is caused by an obligate intracellular protozoan parasite, Toxoplasma gondii. Most of these infections are asymptomatic or benign, but may cause severe or fatal consequences in immunodeficient patients, transplant recipients and in the fetus. Transmission may occur by eating uncooked meat, contaminated vegetables, blood transfusion, and organ transplantation and across the placenta from the mother to the fetus. In many countries, seroprevalence rates ranging from 15% to 85% have been reported of the human population. In children, the prevalence is relatively low, increasing with age and with more exposure to risk factors during the course of life. In Brazil, the seroprevalence of T. gondii infection is high, around 50 and 80% of the adult population. In the present study, our aim was to determine the prevalence of T. gondii IgG and IgM antibodies in eye donors of Rio de Janeiro, Brazil. Four hundred twenty-six serum samples collected from eye donors, in the city of Volta Redonda, State of Rio de Janeiro, Brazil. The samples were tested by enzyme immunoassay (ELISA) and the Indirect Fluorescent Antibody Test (IFAT) for IgG and IgM antibodies. From the 426 donors, 262 were male and 164 female donors. The average age of donors was 52.65 ± 16.96 years. It is the minimum age of 10 years and maximum 80 years. The age group that obtained the highest number of donors was 51-60 years (n = 106/24.88%) followed by the age group 61- 70 years (n = 99/23.23%), the age group 41-50 years (n = 65/15.26%) and the age group 71-80 years (n = 61/14.32%). Donors were from seven of the eight regions of the state of Rio de Janeiro Being the metropolitan region (n = 302/70.90%) and the Middle Paraíba (n = 101/23.71%) with the highest concentration of donors. The city that obtained the highest number of donors was the Duque de Caxias, with 87 (20.42%) donors, followed by the city of Rio de Janeiro with 86 (20.19%) donors and Volta Redonda with 80 (18.78 %) donors. The main cause of death of the 426 corneal donors was the stroke, with 147 (34.50%) donors, followed by cranial trauma, 89 (20%) donors, and cancer with 25 (5.87%) donors. The Kappa index between the two serological techniques employed is estimated, = 0.69 for ELISA and/or IFAT IgG and = 0.14 to ELISA and / or IgM IFAT. According to the selection criteria, the Kappa values were in substantial agreement for ELISA and / or IFAT IgG and slight agreement for ELISA and/or IFAT IgM. The prevalence of toxoplasmosis was confirmed by positive IgG and IgM anti-T. gondii, being 79.34% and 3.99%, respectively. In males positivity of 4.58% and 80.92% for anti-T. gondii IgM and IgG, respectively, was observed. While the percentage of seropositive in females were 3.05% and 76.83% for T. gondii antibodies anti-IgM and IgG, respectively. The age group of 71- 80 years obtained the highest percentages of IgM and IgG seropositive donors, 9(14.76%) and 55(90.16%) for anti- T. gondii IgM and IgG, respectively. The middle Paraíba region obtained 4.95% and the metropolitan 3.98% of soropositives donors of IgM anti T. gondii. Regarding the cause of death, 12% of reagents donors IgM antibodies anti- T. gondii died due to cancer. In addition, 91.18% of donors were IgG seropositive and they had died due to disorder of the respiratory system. These data demonstrate the importance of regional and national seroepidemiologic continuous surveys to define public health strategies to reverse and reduce high serologic prevalence in the general population and consequently in the cornea donors population.

Obtenção de células mononucleares da medula óssea pela punção do tubérculo umeral de coelhos / Acquirement of bone marrow mononuclear cells by puncture of the humerus tubercle of rabbits

As células-tronco mesenquimais têm sido isoladas da medula óssea e utilizadas na reparação e regeneração de defeitos osteocondrais. O objetivo desse estudo foi avaliar eficiência da técnica de colheita da medula óssea em 12 coelhos adultos, machos, da raça Nova Zelândia, com peso entre 3,0 kg a 4,5 kg. Após flexão da articulação escapulo-umeral, foi inserida no tubérculo umeral uma agulha de biópsia de Rosenthal heparinizada, sendo aspirados 2,0 mL de medula óssea. No isolamento pelo gradiente de densidade Ficoll-paque, obteve-se um rendimento médio de 6,25 x 10° células mononucleares/mL. A técnica de colheita de medula óssea por punção do tubérculo umeral demonstrou ser eficaz e de fácil execução, devido ao alto rendimento de células mononucleares isoladas das amostras.

Bone marrow derived-mesenchymal stem cells have been isolated and used for osteochondral defects regeneration and repair. The aim of this study was evaluate the efficiency of bone marrow harvest technique using twelve adult male New Zealand rabbits, with weight between 3.0 kg to 4.5 kg. After shoulder joint flexion, a heparinized Rosenthal biopsy needle was inserted through the humeral tubercle and further aspiration of 2.0 mL bone marrow. In the isolation with Ficoll-paque gradient density, was achieved a mean yield of 6.25 x 10° mononuclear cells per milliliter. The bone marrow harvest technique by puncture of the humerus tubercle was easily accomplished and effective, due to high yield of isolated mononuclear cells of samples.

Negative effect of vitrification of whole rat ovarian tissue and its subsequent autotransplantation into omentum

This study was designed to examine the viability of follicles in whole rat ovary after vitrification-warming and transplantation into omentum. Sixteen 6-8 week old female Wistar rats were randomly divided into four groups, with four rats in each group. Freshly isolated ovaries saved as a control (group 1; fresh ovaries) in formalin-fixed or vitrified immediately after dissection (group 2; vitrified ovaries). In group 3 (fresh grafts), ovarian tissues were dissected out and implanted into the omentum. In group 4 (vitrified grafts), ovarian tissues were dissected out and used for autologous transplantation into omentum after vitrification and warming. Ovaries in the vitrified groups were exposed sequentially to two vitrification solutions: 10% ethylene glycol and 10% DMSO in holding medium [HM; human tubal fluid (HTF) medium with 20% FBS] for 15 min and then 20% ethylene glycol, 20% DMSO and 0.5 M sucrose in HM for 2 min before plunging into liquid nitrogen. After warming at 37°C, cryoprotectants were diluted with 1.0 M sucrose in HTF for 10 min. All of the collected ovaries underwent hematoxylin and eosin-stained paraffin serial sections. Follicles at each developmental stage were counted and divided according to the morphological changes. Among the morphologically changed follicles after vitrification, developing and antral follicles significantly accounted for high proportions compared to the fresh control group. A significant decrease in the percentage of healthy follicles was observed in the vitrified ovarian tissues implanted into the omentum in comparison with the fresh omental graft (8.3% vs. 46.3%, P < 0.05). Cryopreservation of whole rat ovarian tissue by the vitrification method used had detrimental effect on follicles. Subsequent autotransplantation of the ovaries into the omentum was not able to support viability of follicles and also insufficient blood supply associated ischemia caused more damage and significant decrease in the number of follicles.(AU)

Negative effect of vitrification of whole rat ovarian tissue and its subsequent autotransplantation into omentum

This study was designed to examine the viability of follicles in whole rat ovary after vitrification-warming and transplantation into omentum. Sixteen 6-8 week old female Wistar rats were randomly divided into four groups, with four rats in each group. Freshly isolated ovaries saved as a control (group 1; fresh ovaries) in formalin-fixed or vitrified immediately after dissection (group 2; vitrified ovaries). In group 3 (fresh grafts), ovarian tissues were dissected out and implanted into the omentum. In group 4 (vitrified grafts), ovarian tissues were dissected out and used for autologous transplantation into omentum after vitrification and warming. Ovaries in the vitrified groups were exposed sequentially to two vitrification solutions: 10% ethylene glycol and 10% DMSO in holding medium [HM; human tubal fluid (HTF) medium with 20% FBS] for 15 min and then 20% ethylene glycol, 20% DMSO and 0.5 M sucrose in HM for 2 min before plunging into liquid nitrogen. After warming at 37°C, cryoprotectants were diluted with 1.0 M sucrose in HTF for 10 min. All of the collected ovaries underwent hematoxylin and eosin-stained paraffin serial sections. Follicles at each developmental stage were counted and divided according to the morphological changes. Among the morphologically changed follicles after vitrification, developing and antral follicles significantly accounted for high proportions compared to the fresh control group. A significant decrease in the percentage of healthy follicles was observed in the vitrified ovarian tissues implanted into the omentum in comparison with the fresh omental graft (8.3% vs. 46.3%, P < 0.05). Cryopreservation of whole rat ovarian tissue by the vitrification method used had detrimental effect on follicles. Subsequent autotransplantation of the ovaries into the omentum was not able to support viability of follicles and also insufficient blood supply associated ischemia caused more damage and significant decrease in the number of follicles.

Transplante renal em bloco de doadores-cadáveres infantis: experiência do hcfmrp-usp

The high demand for organs for transplantation has made it necessary to consider using the youngest of potential donors in order to increase the organ supply. Previous reports of decreased graft survival and increased complication rates have made surgeons wary of using such kidneys. This is a chart review with the objective to analyse the results achieved with transplantation of children kidneys ( 2 years) in the HCFMRP-USP from 1998 to 2000. A total of 5 pacients received en bloc renal transplants from donors aged 9 months to 2 years old, weighthing between 8 and 14 kg. Results: Only one patient lost the allograft within 8 days of transplantation due to allograft trombosis. Two patients had ureteral complications, one with ureteral necrosis and urinary fistula and the other with total ureterovesical anastomosis disrupture. Both were successfully repaired. Intra operatory complication was experienced in one pacient, with arterial anastomosis rupture. In loco reperfusion and repair was made. Another patient had inferior renal pole rupture, treated with polar nefrectomy at the 4º PO. Despite such problems, mean follow up of 20,7 months showed serum creatinine ranging from 0,9 to 2 mg/100ml, with no serious complications. Conclusion: Our data support the use of en bloc child cadaveric kidneys, even from infant donors less than two year of age, when the organ necessity supplant the higher rates of complications.

A grande procura de órgãos para transplante tornou necessária a aceitação de potenciais doadores pediátricos a fim de aumentar a oferta. Trabalhos anteriores relatando menor sobrevida do enxerto e taxas maiores de complicação fizeram com que cirurgiões considerassem o assunto com cautela. Esse é um estudo retrospectivo com o objetivo de analisar os resultados de transplantes renais em bloco de doadores infantis ( 2 anos) no HCFMRP-USP entre 1998 e 2000. Um total de cinco pacientes foi submetido a transplante renal em bloco de doadores com idades entre 9 meses e 2 anos de vida, pesando entre 8 e 14 kg. Apenas um paciente perdeu o enxerto no oitavo dia após transplante devido à trombose. Dois pacientes tiveram complicações ureterais, um com necrose do ureter e fístula urinária e outro com ruptura completa da anastomose ureterovesical. Ambos foram reparados com sucesso. Complicação intra-operatória foi observada em um paciente, com ruptura da anastomose arterial. Corrigiu-se com reperfusão in loco e reanastomose. Outro paciente apresentou ruptura do pólo renal inferior, tratada com nefrectomia polar no quarto dia pós-operatório. Seguimento médio de 20,7 meses mostrou, apesar de tais problemas, creatinina sérica variando entre 0,9 e 2 mg/100 ml, sem complicações sérias. Nossos achados sustentam o uso de rins infantis, em bloco, mesmo de doadores com menos de 2 anos de idade, desde que a necessidade do órgão seja maior que o risco oferecido pelo alto índice de complicações.

TRANSPLANTE RENAL EM BLOCO DE DOADORES-CADÁVERES INFANTIS: EXPERIÊNCIA DO HCFMRP-USP

The high demand for organs for transplantation has made it necessary to consider using the youngest of potential donors in order to increase the organ supply. Previous reports of decreased graft survival and increased complication rates have made surgeons wary of using such kidneys. This is a chart review with the objective to analyse the results achieved with transplantation of children kidneys ( 2 years) in the HCFMRP-USP from 1998 to 2000. A total of 5 pacients received en bloc renal transplants from donors aged 9 months to 2 years old, weighthing between 8 and 14 kg. Results: Only one patient lost the allograft within 8 days of transplantation due to allograft trombosis. Two patients had ureteral complications, one with ureteral necrosis and urinary fistula and the other with total ureterovesical anastomosis disrupture. Both were successfully repaired. Intra operatory complication was experienced in one pacient, with arterial anastomosis rupture. In loco reperfusion and repair was made. Another patient had inferior renal pole rupture, treated with polar nefrectomy at the 4º PO. Despite such problems, mean follow up of 20,7 months showed serum creatinine ranging from 0,9 to 2 mg/100ml, with no serious complications. Conclusion: Our data support the use of en bloc child cadaveric kidneys, even from infant donors less than two year of age, when the organ necessity supplant the higher rates of complications.

A grande procura de órgãos para transplante tornou necessária a aceitação de potenciais doadores pediátricos a fim de aumentar a oferta. Trabalhos anteriores relatando menor sobrevida do enxerto e taxas maiores de complicação fizeram com que cirurgiões considerassem o assunto com cautela. Esse é um estudo retrospectivo com o objetivo de analisar os resultados de transplantes renais em bloco de doadores infantis ( 2 anos) no HCFMRP-USP entre 1998 e 2000. Um total de cinco pacientes foi submetido a transplante renal em bloco de doadores com idades entre 9 meses e 2 anos de vida, pesando entre 8 e 14 kg. Apenas um paciente perdeu o enxerto no oitavo dia após transplante devido à trombose. Dois pacientes tiveram complicações ureterais, um com necrose do ureter e fístula urinária e outro com ruptura completa da anastomose ureterovesical. Ambos foram reparados com sucesso. Complicação intra-operatória foi observada em um paciente, com ruptura da anastomose arterial. Corrigiu-se com reperfusão in loco e reanastomose. Outro paciente apresentou ruptura do pólo renal inferior, tratada com nefrectomia polar no quarto dia pós-operatório. Seguimento médio de 20,7 meses mostrou, apesar de tais problemas, creatinina sérica variando entre 0,9 e 2 mg/100 ml, sem complicações sérias. Nossos achados sustentam o uso de rins infantis, em bloco, mesmo de doadores com menos de 2 anos de idade, desde que a necessidade do órgão seja maior que o risco oferecido pelo alto índice de complicações.

Inhibition of mouse and rat lymphoproliferation by gangliosides

Our previous study have demonstrated that Glycosphingolipids (GSLs) have an immunosuppressive effect on murine lymphoproliferation and IL-2 production. In the present study we examined the effect of a pool of Gangliosides (Gang) on spleen lymphocyte proliferation from either isogeneic strains of Wistar rats or BALB/c mice. Two hundred-fifty grams adult female isogeneic Wistar rats and 8-week-old BALB/c mice were used. The animals were sacrificed and the spleen harvested aseptically for cellular assays. Spleen cells suspensions were obtained by homogenization in RPMI 1640 with a loose tissue grinder. After washing, the cells were suspended in RPMI 1640 supplemented. Cell viability was measured by Trypan blue exclusion. Cells were cultured in triplicate using increasing concentrations of Gang (1; 2; 5; 10; 15; 20 mug/well) and in the presence of Concanavalin A. The cells were incubated for 48 hours and were pulsed with [³H] thymidine 18 hours prior to harvesting on glass fiber paper for counting in a beta-counter. Data were presented as rate of inhibition, as previously described. At concentrations 1 and 2 mug/well, Gang stimulated lymphoproliferation (30% and 50%, rats and mice respectively), while at concentration from 5 to 20 mug/well an increasing inhibition was observed for spleen cells from both mouse and rat (from 40% up to 80%). In preliminary studies we observed inhibition of mixed lymphocyte reaction on spleen cells from rats treated with Gang for 10 days (data not shown). Our data suggest that Gang may be investigated as a immunosuppressive drug in organ transplantation.

Inhibition of mouse and rat lymphoproliferation by gangliosides

Our previous study have demonstrated that Glycosphingolipids (GSLs) have an immunosuppressive effect on murine lymphoproliferation and IL-2 production. In the present study we examined the effect of a pool of Gangliosides (Gang) on spleen lymphocyte proliferation from either isogeneic strains of Wistar rats or BALB/c mice. Two hundred-fifty grams adult female isogeneic Wistar rats and 8-week-old BALB/c mice were used. The animals were sacrificed and the spleen harvested aseptically for cellular assays. Spleen cells suspensions were obtained by homogenization in RPMI 1640 with a loose tissue grinder. After washing, the cells were suspended in RPMI 1640 supplemented. Cell viability was measured by Trypan blue exclusion. Cells were cultured in triplicate using increasing concentrations of Gang (1; 2; 5; 10; 15; 20 mug/well) and in the presence of Concanavalin A. The cells were incubated for 48 hours and were pulsed with [³H] thymidine 18 hours prior to harvesting on glass fiber paper for counting in a beta-counter. Data were presented as rate of inhibition, as previously described. At concentrations 1 and 2 mug/well, Gang stimulated lymphoproliferation (30% and 50%, rats and mice respectively), while at concentration from 5 to 20 mug/well an increasing inhibition was observed for spleen cells from both mouse and rat (from 40% up to 80%). In preliminary studies we observed inhibition of mixed lymphocyte reaction on spleen cells from rats treated with Gang for 10 days (data not shown). Our data suggest that Gang may be investigated as a immunosuppressive drug in organ transplantation.