Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background

The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells.

Methods

A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses.

Results

Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles.

Conclusion

We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.     

Influence of different supplements and sugarcane (Saccharum officinarum L.) cultivars on intake, digestible variables and methane production of dairy heifers under tropical conditions

The sulphur hexafluoride (SF(6)) gas tracer method was used to measure methane (CH(4)) production of crossbred (3/4 Holstein x Zebu) dairy heifers fed two types of sugarcane (Saccharum officinarum L.; cultivar IAC-862480 (CC1) or cultivar IAC-873184 (CC2)) and supplemented with urea or concentrate. The study was performed at Embrapa Southeast Cattle, S?o Carlos, SP, Brazil, using a completely randomised design. Differences between treatments were significant for digestibility of dry matter, organic matter and energy. When animals were supplemented with urea differences between sugarcane cultivars did occur for NDF consumption, but not for daily methane production. This suggest that variation in chemical composition of sugarcane did not affect bovine ruminal CH(4) emissions. Concentrate inclusion in animal diet increased digestible organic matter intake, improving the nutrient intake by animals, but did not reduce CH(4) production expressed as a percentage of gross energy intake.     

Leptospirosis as the most frequent infectious disease impairing productivity in small ruminants in Rio de Janeiro,Brazil

Despite the importance of small ruminants breeding in developing countries, milk/meat productivity remains unsatisfactory. Infectious diseases, such as leptospirosis, brucellosis, and small ruminant lentiviruses (SRLVs), contribute to this scenario. The objective of the present study was to determine the role of each of these diseases in the productivity of small ruminants breeding in Rio de Janeiro, Brazil. In goats, 343 samples were tested for leptospirosis, 560 for Brucella abortus, and 506 for caprine arthritis-encephalitis (CAE), whereas in sheep, 308 samples were tested for leptospirosis, 319 for B. abortus, 374 for Brucella ovis, and 278 for Maedi-Visna (MV). Regarding leptospirosis, 25.9% of goats and 47.4% sheep were seroreactive, with serovar Hardjo the most prevalent in both species. Anti-B. abortus agglutinins were found in 0.7% of all samples, exclusively in goats. In relation to SRLVs, 8.6% of goats and 3.2% of sheep samples were positive for CAE and MV, respectively. Leptospirosis was the major infectious problem in the small ruminants sampled and may contribute to impaired productivity of these animals.     

Update on the role of innate immune receptors during Brucella abortus infection

The innate immune system constitutes an efficient defense mechanism against invading microbial pathogens. Recent studies have revealed the intracellular signaling cascades involved in the TLR-initiated immune response to Brucella spp. infection. However, there is a piece of the puzzle missing that is the role of non-TLR receptors in innate immunity. The involvement of TLR receptors in brucellosis has been investigated by different research groups. It was demonstrated that TLR2 clearly does not play any role in controlling Brucella abortus infection in vivo, whereas TLR9 has been shown to be required for clearance of this bacterium in infected mice. The participation of adaptor molecules, such as MyD88 and TRIF has also been discussed. Recently, we and others have reported the critical role of MyD88- and not TRIF-mediated signaling in dendritic cell maturation and in vivo resistance during B. abortus infection. However, the relationship between specific Brucella molecules and non-TLR receptors and signal transduction pathways needs to be better understood. It is now clear that the interaction between TLRs and recently identified cytosolic innate immune sensors is crucial for mounting effective immune responses. Finally, this review discusses the mechanisms used by Brucella to escape detection by the host innate immune system.     

Learning by doing: a participatory methodology for systematization of experiments with agroforestry systems, with an example of its application

Participatory research methods have helped scientists to understand how farmers experiment and to seek partnerships with farmers in developing technologies with enhanced relevance and adoption. This paper reports on the development of a participatory methodology to systematize long-term experimentation with agroforestry systems carried out in a hotspot of biodiversity by non-governmental organizations and local farmers. A methodological guide for systematization and techniques used for Participatory Rural Appraisal formed the basis of our work. We propose an analytical framework that recognizes systems of reflexive and learning interactions, in order to make the learned lessons explicit. At the process level, the main lessons and recommendations are as follows. It is important to establish partnerships to conduct innovative and complex experimentation with agroforest. Participatory systematization allows us to improve the methodological aspects of design, implementation and management of on-farm participatory experimentation. It also serves to synthesize the main findings and to extract lessons from agroforestry systems experiments. It fosters the technical improvement of agroforestry systems. It creates possibilities for reflection on agroforestry systems by farmers, extensionists and researchers, as well as their learning with respect to management of such systems. The findings are placed in the context of current theory on participatory experimentation in agriculture. Extractive and interactive approaches help to produce rich insights of mutual interest through collaboration by identifying local, regional and global convergences, complementarities, and conflicts of interest; which affect the advance of new eco-friendly technologies, to both improve the livelihoods and to reverse biodiversity loss and environmental degradation.     

An experimental assessment of landscape configuration effects on frog and toad abundance and diversity in tropical agro-savannah landscapes of southeastern Brazil

Amphibians are an imperiled group of vertebrate animals that typically have biphasic life histories involving a shift from aquatic larval habitats to terrestrial adult habitats. Habitat loss is the greatest threat to amphibians and the importance of the spatial configuration of terrestrial and breeding habitats upon the landscape in determining amphibian persistence is poorly known. The information gap is particularly acute in tropical landscapes that simultaneously host the greatest and most imperiled amphibian fauna on Earth. We installed 125 artificial ponds at different distances from forest fragments embedded in an agricultural matrix in southeastern Brazil. Constructed ponds attracted 13 anuran species; ponds at the forest fragment-matrix transition hosted a greater abundance and higher species richness of frogs and toads than those installed either far from or well within forest fragments. Forest fragments larger than 70 ha in agricultural areas harbored more individuals than smaller fragments. Our results indicate that landscape configuration has an important influence on frog and toad distribution and abundance in tropical agricultural landscapes and we suggest guidelines for maintaining favorable configurations of aquatic and terrestrial habitats for conserving this rich and imperiled species suite.     

Slaframine toxicosis in Brazilian horses causing excessive salivation

Ingestion of food contaminated with slaframine, an alkaloid produced by Rhizoctonia leguminicola, causes a mycotoxicosis, characterised by excessive salivation. Twenty‐eight horses demonstrated this clinical sign after the consumption of alfalfa hay which on inspection showed dark patches on many of the stems. The presence of slaframine (1.5 ppm) in this hay was confirmed by gas chromatography and mass spectroscopy. This is the first equine slaframine toxicosis case reported in Brazil.     

Embryonic and fetal development in - pigmy rice rat - Oligoryzomys sp. (Rodentia, Sigmodontinae) and its significance for being a new experimental model

Oligoryzomys (Cricetidae, Sigmodontinae) is a common rodent genus from South America that includes a couple of very similar species. Related species have been used as experimental model for understanding several diseases for which these species are reservoirs. In order to provide a better understanding of the embryological aspects of this group, herein we showed data on the embryonic and fetal development in Oligoryzomys sp. Eight specimens of different stages of gestation were obtained from the Collection of the Zoology Museum of University of Sao Paulo, Brazil. Gestational ages were estimated by crown-rump-length according to Evans and Sack (1973). To address our analysis after examining the gross morphology, tissues from several organs were processed for light and scanning electron microscopy. Morphological data on the systems (nervous system, cardiorespiratory system, intestinal tract and urogenital system) were described in detail. Finally, the findings were compared with what is known about embryological aspects in other rodent species in order to establish similarities and differences during the organogenesis in different species.     

Gastroprotective potential of frutalin, a D-galactose binding lectin, against ethanol-induced gastric lesions

The present study was designed to verify whether frutalin (FTL) affords gastroprotection against the ethanol-induced gastric damage and to examine the underlying mechanism(s). Gastric damage was induced by intragastric administration of 0.2 ml of ethanol (96%). Mice in groups were pretreated with FTL (0.25, 0.5 and 1 mg/kg; i.p.), cimetidine (100 mg/kg; p.o.), or vehicle (0.9% of NaCl, 10 mL/kg; p.o.), 30 min before ethanol administration. They were sacrificed 30 min later, the stomachs excised, and the mucosal lesion area (mm2) measured by planimetry. Gastroprotection was assessed in relation to inhibition of gastric lesion area. To study the gastroprotective mechanism(s), its relations to capsaicin-sensitive fibers, endogenous prostaglandins, nitric oxide, sulphydryls, ATP-sensitive potassium channels, adrenoceptors, opioid receptors and calcium channels were analyzed. Treatments effects on ethanol-associated oxidative stress markers GSH and MDA were measured in gastric tissue. FTL afforded a dose-unrelated gastroprotection against the ethanol damage. However, it failed to prevent the ethanol-induced changes in the levels of GSH and MDA. It was observed that the gastroprotection by FTL was greatly reduced in animals pretreated with capsazepine, indomethacin, L-NAME or glibenclamide. Considering the results, it is suggested that the FTL could probably be a good therapeutic agent for the development of new medicine for the treatment of gastric ulcer.     

Comparison of the effects of epidural or intravenous methadone on the minimum alveolar concentration of isoflurane in dogs

The effects of epidural and intravenous (IV) methadone (0.5mg/kg) on the minimum alveolar concentration of isoflurane (ISO(MAC)) were compared in dogs. Six dogs (16.5 ± 2.5 kg bodyweight) received three treatments in random order during isoflurane anaesthesia, with a 7 day washout interval between each study. Methadone was injected via a lumbosacral epidural catheter introduced 10 cm cranially into the epidural canal and the electrical stimulation for ISO(MAC) determination was applied either to the thoracic (EP(T) treatment) or to the pelvic limb (EP(P) treatment) during separate study days. In the IV treatment, ISO(MAC) was determined via electrical stimulation of the pelvic limb. Variables were recorded before (baseline), 2.5 and 5h after drug injection. The ISO(MAC) decreased significantly (P<0.05) from baseline at 2.5 and 5h after methadone in all treatments. At 2.5h, the magnitude of ISO(MAC) reduction did not differ between treatments (mean decreases from baseline: 30-33%). The ISO(MAC) reduction lasted longer following epidural methadone in the thoracic limb (decreases from baseline: 30% at 5h in the EP(T) treatment vs. 19% and 16% in the EP(P) and IV treatments, respectively). Although the isoflurane sparing effect provided by epidural methadone was not significantly greater than IV methadone during the initial stage (2.5h), it was more prolonged than the IV route in specific dermatomes (5h in the thoracic limb) with the epidural technique employed. Methadone may therefore provide a greater isoflurane sparing effect when administered epidurally, compared to IV, when noxious stimulation occurs in specific dermatomes.