A simplified cuff technique for abdominal aortic transplantation in mice.

BACKGROUND: Allograft arteriopathy is still a leading cause of late organ failure. The aortic allograft model in mice has been used to study chronic rejection and has given useful information in the development of graft arteriosclerosis. However, the technical difficulties of small vessel anastomoses still continue to limit its widespread use. We introduce a new simple method for aortic transplantation in mice. METHODS: The descending aorta or infrarenal aorta from the donor mouse was anastomosed to the infrarenal aorta using a cuff technique. Aortic transplantation was performed in 30 mice, 10 isografts and 20 allografts. No immunosuppression was administered, and the recipients were sacrificed at day 28. The grafts were histologically analyzed. RESULTS: Implantation of grafts could be completed in an average of 23 min. There was no technical failure in all 60 anastomoses. The overall survival rate was 93.3%. Histology of aortas revealed typical aspects of chronic rejection in the allografts at day 28. No significant lesion was observed in isografts. CONCLUSIONS: We have developed an innovative, stable, and simple aortic transplantation model in mice, which is useful for vascular research in transplantation and beyond.

Postsurgical food and water consumption, fecal corticosterone metabolites, and behavior assessment as noninvasive measures of pain in vasectomized BALB/c mice.

Recognition of pain and stress is a common challenge when working with laboratory mice. The aim of the current study was to identify noninvasive parameters to assess the severity and duration of possible pain and stress after vasectomy in BALB/c mice. Mice underwent isoflurane anesthesia with or without vasectomy. Body weight, food and water intake, and fecal corticosterone metabolites (FCM) were measured 3 d before and 3 d after the procedure. Behavior was recorded 1, 2, 4, and 8 h after the procedure. Food and water consumption and defecation were reduced postoperatively in the vasectomized group compared with mice given anesthesia only. FCM were elevated the first day after anesthesia in the control mice but not in the vasectomized group. Vasectomy resulted in behavioral changes that were not seen in the group that was anesthetized only. In conclusion, food and water consumption and pain-related behaviors, but not FCM, may be useful as noninvasive parameters to assess postoperative pain and stress in vasectomized mice.

Long-term culture and transplantation of spermatogonial stem cells from BALB/c mice.

Development of a culture system that supports self-renewal and proliferation of spermatogonial stem cells (SSCs) is enormously valuable for experimental research and potential treatment for male infertility. Although several research groups had reported their successes in SSC isolation and culture, the two current accepted culture systems are different in cell enrichment methods, serum and growth factors. Previous researches also indicated SSCs from different mouse strains required different culture conditions. Here we report for the first time that SSCs from BALB/c mice could be cultured in an improved culture system for 3 months. The modified culture system consisted of an improved enzymatic procedure, the enrichment of undifferentiated spermatogonia by differential adherence selection of isolated SSCs, mouse embryonic fibroblast feeder cells, StemPro-34 SFM medium supplemented with glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor and GDNF-family receptor alpha1 (GFRalpha1). The improved digestion method increased the viability and enrichment efficiency of isolated testis cells. Furthermore, basal culture medium with 10% fetal bovine serum as selected medium could increase the number of germ cell colonies in the initiation stage of culture. Cultured SSCs were characterized morphologically and formed typical colonies. Immunocytochemical staining and RT-PCR showed that cultured SSCs expressed Oct-4, GFRalpha1, Sox2 and several other special genes resembling undifferentiated spermatogonia. Spermatogonia transplantation further confirmed that cultured SSCs were functionally normal and could restore complete spermatogenesis. The culture methods described here could serve as a paradigm to establish conditions for the culture of SSCs from other species, allowing identification of universal factors necessary for proliferation of SSCs.

Microscopic wire guide-based orotracheal mouse intubation: description, evaluation and comparison with transillumination.

Airway access is needed for a number of experimental animal models, and the majority of animal research is based on mouse models. Anatomical conditions in mice are small, and the narrow glottic opening allows intubation only with a subtle technique. We therefore developed a microscopic endotracheal intubation method with a wire guide technique in mice anaesthetized with halothane in oxygen. The mouse is hung perpendicularly with its incisors on a thread fixed on a vertical plate. The tongue is placed with a pair of forceps between the left hand's thumb and forefinger and slightly pulled, while the neck and thorax are positioned using the third and fourth fingers. By doing so, the neck can be slightly stretched, which allows optimal visualization of the larynx and the vocal cords. To ensure a safe intubation, a fine wire guide is placed under vision between the vocal cords and advanced about 5 mm into the trachea. An intravenous 22G x 1 in. plastic or Teflon catheter is guided over this wire. In a series of 41 mice, between 21 and 38 g, the success rate for the first intubation attempt was >95%. Certainty of the judgement procedure was 100% and success rate was higher using the described method when compared with a transillumination method in a further series. The technique is safe, less invasive than tracheostomy and suitable for controlled ventilation and pulmonary substance application.

Actividad de superóxido dismutasa y las concentraciones de glutation y malonaldehído en los tejidos pulmonar y cardiaco post (perfusión, preservación y reperfusión) del bloque cardiopulmonar / Superoxide dismutase activity and glutathione and malonaldehyde concentration in pulmonary and cardiac tissue post (perfusion, preservation and reperfusion) of cardiopulmonary block

Introducción. Los periodos de isquemia y de reperfusión involucra la producción de radicales libres (RL), un grupo de especies oxidantes derivadas del matabolismo de oxígeno molecular. Durante la reperfusión, se generan (RL) al restablecer la circulación sanguínea dado el aporte de oxígeno, además de que durante la isquemia, el metabolismo aeróbico del bloque cardiopulmonar continúa por el oxígeno remanente en los alvéolos. La degradación de los RL es a través de receptores específicos como la enzima superóxido dismutasa (SOD) y el glutatión (GLU). Si estos mecanismos receptores no funcionan adecuadamente, los RL pueden dar inicio a la peroxidación lipídica. El manoaldehído (MAL) es un producto final de la peroxidación lipídica y su cuantificación refleja el daño celular por RL. Objetivo. Utilizando un modelo experimental murino de perfusión, preservación y reperfusión cardiopulmonar a través de la arteria pulmonar con solución de Krebs-Henseleit a 4ºC y con un flujo de 0.2 mL/min, el objetivo de este trabajo fue: determinar y evaluar la actividad de la enzima SOD y las concentraciones de GLU y MAL en homogeneizados tisulares de corazón y de pulmón como respuesta al daño ocasionado por efecto de la perfusión, preservación y reperfusión cardiopulmonar. Material y métodos. Utilizamos 14 bloques cardiopulmonares de murinos Balb-C divididos en dos grupos de estudio. Los bloques fueron: Grupo 1 (n=7): perfundidos en forma inmediata y continua únicamente durante el tiempo necesario para exaguinarlos y Grupo 2 (n=7): perfundidos en forma inmediata y continua para exanguinarlos, preservados durante 24 horas a 4ºC y reperfundidos durante 30' bajo las mismas condiciones dadas para la perfusión. Concluidas las perfusiones y la reperfusiones, preparamos homogeneizados del corazón y de los pulmones para determinar la actividad SOD y las concentraciones de GLU y MAL mediante espectrofotometría. Resultados. La actividad de SOD antes y después de la preservación fue mayor en el tejido pulmonar que en el tejido cardiaco. En ambos tejidos, la actividad de SOD y la concentración de GLU obtenidas previa preservación prolongada. La concentración de MAL fue similar en ambos homogeneizados tisulares tanto antes como después de la preservación del bloque cardiopulmonar y no se modificó por efecto de la reperfusión

Reproduçäo de um modelo experimental de enxerto de coraçäo em orelha de camundongo / Reproduction of an experimental model of heart allograft in the mouse ear

A transplantaçäo de órgäos é uma modalidade terapêutica bem estabelecida no tratamento da falência de órgäos. Os avanços neste campo deve-se muitas vezes, aos estudos de imunobiologia de transplante e da açäo dos vários imunossupressores em modelos experimentais de transplante em animais. Objetivo: Desenvolver um modelo experimental de transplante de órgäo sólido (coraçäo) a ser utilizado nos estudos de imunobiologia, para análise da reaçäo de rejeiçäo para testar novos imunossupressores. Material e métodos: Oito animais recém-nascidos submetidos à toracotomia, tiveram os coraçöes extraídos e, posteriormente, implantados em lobos auriculares de quatro camundongos adultos isogênicos. A avaliaçäo da "pega" dos enxertos foi definida pela sensaçäo tátil dos batimentos dos coraçöes implantados e pela análise histopatológica dos mesmos. Resultados: Quatro dias após os transplantes, constatou-se movimentos contráteis em 06 dos 08 enxertos, o que näo foi percebido nos outros dois. Em 06 enxertos, a macroscopia e a microscopia foram sugestivas de "pega" dos transplantes, enquanto a análise de 2 enxertos, em um mesmo receptor, evidenciou processo compatível com rejeiçäo aguda dos mesmos. Discussäo: A explicaçäo para a rejeiçäo encontrada em dois dos enxertos, seria a histoimcompatibilidade Hy entre os doadores e o receptor. Conclusäo: O modelo experimental descrito mostrou-se viável para o estudo de imunobiologia de transplante, podendo ser utilizado com reprodutividade na avaliaçäo da açäo "in vivo" dos imunossupressores.

Xenografts of rat islets into diabetic mice. An evaluation of new smaller capsules.

Healthy rat islets were encapsulated in alginate-polylysine-alginate capsules measuring 0.25-0.35 mm in diameter using a modified encapsulation technique. The encapsulated islets were transplanted intraperitoneally in nonimmunosuppressed streptozotocin-induced diabetic BALB/c mice. The diabetic condition of the experimental animals was reversed within two days following the transplantation and the animals remained normoglycemic for up to 308 days, with a mean xenograft survival of 219.8 +/- 46.2 days. Four and six months posttransplant the capsules were removed from two recipients. This resulted in regression to a hyperglycemic state. After a second transplant of encapsulated islets, the animals returned to normoglycemia. In control mice that received free unencapsulated islets, the xenografts remained functional for no more than 12 days. Our study clearly demonstrates that the encapsulation of islets in the new smaller capsules can effectively prolong xenograft survival without immunosuppression.