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.     

Breeding objectives of Brangus cattle in Brazil

Our goal was to define a breeding objective for Brangus beef cattle in Brazil. Bioeconomic models were produced and used to estimate economic values (EVs). The scenarios simulated were typical full-cycle beef production systems that are used in tropical and subtropical regions. The breeding objective contained pregnancy rate (PR), warm carcass weight (WCW), mature cow weight (MCW), number of nematode eggs per gram of faeces (EPG) and tick count (TICK). Two models were used in series to estimate the EV. A deterministic model was used to simulate effects of PR, WCW and MCW on profitability with a constant parasite load. Subsequently, stochastic models were used to estimate economic values for TICK and EPG as consequences of their environmental effects on weight gains, mortality and health costs. The EV of PR, WCW, MCW, EPG and TICK, was US$1.59, US$2.11, −US$0.24, −US$5.35 and −US$20.88, respectively. Results indicate positive emphasis should be placed on PR (12.49%) and WCW (65.07%) with negative emphasis on MCW (13.92%), EPG (2.77%) and TICK (5.75%). In comparison with the indexes usually used, these results suggest a reformulation in the selection indexes of the beef production system in tropical and subtropical regions in order to obtain greater profitability.     

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Thirty‐five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Paraná, Brazil. Twenty‐two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff’s method and processed for acid‐fast bacilli staining, culture in Stonebrink and Lowenstein‐Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR‐positive results (54.5%) was similar to that of culture‐positive results (51.5%, P > 0.05). The percentage of PCR‐positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR‐negative were reanalysed using 2.5 μl DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.     

Detection of a novel chloramphenicol resistance plasmid from "equine" Staphylococcus sciuri

A small chloramphenicol resistance (Cm) plasmid of 4.65 kB could be detected in an "equine" Staphylococcus sciuri-culture. This plasmid, designated as pSC3, was identified by interspecific protoplast transformation. On the basis of restriction endonuclease analyses a detailed restriction map of pSC3 could be constructed. This allowed structural comparisons of pSC3 with Cm-plasmids of other staphylococcal species from infections of humans and animals and identification of pSC3 as a member of the pC 221-family of staphylococcal Cm-plasmids. The pSC3-plasmid encoded an inducible chloramphenicol acetyltransferase as confirmed by enzymatic assays. This enzyme could be demonstrated in cell-free lysates of Cm-induced pSC3-transformants.     

Computed Tomography of the Lungs of the Dog by a Six-generation CT Scanner, Intravenous Contrast Medium and Different Windows

Computed tomography (CT) is a modern technique of image diagnosis particularly recommended in human medicine to evaluate the existence of pulmonary pathological changes such as neoplasms, metastasis, interstitial infiltrates, etc. In veterinary medicine, however, few anatomical and clinical CT studies in the dog have used apparatus of the latest generation, including injection of intravenous contrast and application of regional specific CT windows with different window width (WW) and window level (WL) to evaluate the lungs, the pulmonary vessels and the bronchial structures. This methodology allows the obtaining of clear CT images with high capacity of tissue discrimination and different shades of attenuation. In this work we have planned a tomographic study of the lungs of the dog by using a six-generation spiral CT scanner (Toshiba Ex Vision), belonging to the private Medical Institute of Radiology 'Irion' of Porto Alegre, Brazil. Four mixed-breed mature dogs (4–6 years, 15–20 kg) were used, two males and two females. The dogs were anaesthetized and kept in a maximum inspiration when obtaining the images. Dogs were placed in a stretcher in a ventral or sternal recumbency. Previously, the contrast urografin® was injected in the cephalic vein. Different CT windows were applied in order to increase the quality of the images: pulmonary window (WW 928; WL -680), high-resolution pulmonary window (WW 1085; WL -750), and soft tissue window (WW 652; WL -34). The use of intravenous contrast, different CT windows and a modern CT apparatus produced excellent images of the pulmonary parenchyma, the pleural cavity, the pulmonary veins, the lobular rami of the pulmonary artery and the lobular bronchi.     

Gross Anatomy of the Brachial Plexus in the Giant Anteater (Myrmecophaga tridactyla)

Ten forelimbs of five Myrmecophaga tridactyla were examined to study the anatomy of the brachial plexus. The brachial plexuses of the M. tridactyla observed in the present study were formed by the ventral rami of the last four cervical spinal nerves, C5 through C8, and the first thoracic spinal nerve, T1. These primary roots joined to form two trunks: a cranial trunk comprising ventral rami from C5‐C7 and a caudal trunk receiving ventral rami from C8‐T1. The nerves originated from these trunks and their most constant arrangement were as follows: suprascapular (C5‐C7), subscapular (C5‐C7), cranial pectoral (C5‐C8), caudal pectoral (C8‐T1), axillary (C5‐C7), musculocutaneous (C5‐C7), radial (C5‐T1), median (C5‐T1), ulnar (C5‐T1), thoracodorsal (C5‐C8), lateral thoracic (C7‐T1) and long thoracic (C6‐C7). In general, the brachial plexus in the M. tridactyla is similar to the plexuses in mammals, but the number of rami contributing to the formation of each nerve in the M. tridactyla was found to be larger than those of most mammals. This feature may be related to the very distinctive anatomical specializations of the forelimb of the anteaters.     

Reproductive seasonality in the mare: neuroendocrine basis and pharmacologic control

Reproductive seasonality in the mare is characterized by a marked decline in adenohypophyseal synthesis and secretion of LH beginning near the autumnal equinox. Thus, ovarian cycles have ceased in most mares by the time of the winter solstice. Endogenous reproductive rhythms in seasonal species are entrained or synchronized as a result of periodic environmental cues. In the horse, this cue is primarily day length. Hence, supplemental lighting schemes have been used managerially for decades to modify the annual timing of reproduction in the mare. Although a full characterization of the cellular and molecular bases of seasonal rhythms has not been realized in any species, many of their synaptic and humoral signaling pathways have been defined. In the mare, neuroendocrine-related studies have focused primarily on the roles of GnRH and interneuronal signaling pathways that subserve the GnRH system in the regulatory cascade. Recent studies have considered the role of a newly discovered neuropeptide, RF-related peptide 3 that could function to inhibit GnRH secretion or gonadotrope responsiveness. Although results that used native peptide sequences have been negative in the mare and mixed in all mammalian females, new studies that used an RFRP3 antagonist (RF9) in sheep are encouraging. Importantly, despite continuing deficits in some fundamental areas, the knowledge required to control seasonal anovulation pharmacologically has been available for >20 yr. Specifically, the continuous infusion of native GnRH is both reliable and efficient for accelerating reproductive transition and is uniquely applicable to the horse. However, its practical exploitation continues to await the development of a commercially acceptable delivery vehicle.     

A three‐dimensional cell culture system as an in vitro canine mammary carcinoma model for the expression of connective tissue modulators

In this study, derived complex carcinoma (CC) and simple carcinoma (SC) cell lines were established and cultured under two‐dimensional (2D) and three‐dimensional (3D) conditions. The 3D was performed in six‐well AlgiMatrix? (LifeTechnologies®, Carlsbad, CA, USA) scaffolds, resulting in spheroids sized 50–125 µm for CC and 175–200 µm for SC. Cell viability was demonstrated up to 14 days for both models. Epidermal growth factor receptor (EGFR) was expressed in CC and SC in both systems. However, higher mRNA and protein levels were observed in SC 2D and 3D systems when compared with CC (P < 0.005). The connective tissue modulators, metalloproteinases‐1, ‐2, ‐9 and ‐13 (MMPs), relaxin receptors 1 and 2 (RXR1 and RXR2) and E‐cadherin (CDH1) were quantitated. All were upregulated similarly when canine mammary tumour (CMT)‐derived cell lines were cultured under 3D AlgiMatrix, except CDH1 that was downregulated (P < 0.005). These results are promising towards the used of 3D system to increase a high throughput in vitro canine tumour model.