Time-related effects of general functional training in spinal cord-injured rats

OBJECTIVES: This prospective, randomized, experimental study with rats aimed to investigate the influence of general treatment strategies on the motor recovery of Wistar rats with moderate contusive spinal cord injury. METHODS: A total of 51 Wistar rats were randomized into five groups: control, maze, ramp, runway, and sham (laminectomy only). The rats underwent spinal cord injury at the T9-T10 levels using the NYU-Impactor. Each group was trained for 12 minutes twice a week for two weeks before and five weeks after the spinal cord injury, except for the control group. Functional motor recovery was assessed with the Basso, Beattie, and Bresnahan Scale on the first postoperative day and then once a week for five weeks. The animals were euthanized, and the spinal cords were collected for histological analysis. RESULTS: Ramp and maze groups showed an earlier and greater functional improvement effect than the control and runway groups. However, over time, unexpectedly, all of the groups showed similar effects as the control group, with spontaneous recovery. There were no histological differences in the injured area between the trained and control groups. CONCLUSION: Short-term benefits can be associated with a specific training regime; however, the same training was ineffective at maintaining superior long-term recovery. These results might support new considerations before hospital discharge of patients with spinal cord injuries.

Experimental investigations of heat transfer coefficients of cutting fluids in metal cutting processes: analysis of workpiece phenomena in a given case study

This paper reports an experimental method to estimate the convective heat transfer of cutting fluids in a laminar flow regime applied on a thin steel plate. The heat source provided by the metal cutting was simulated by electrical heating of the plate. Three different cooling conditions were evaluated: a dry cooling system, a flooded cooling system and a minimum quantity of lubrication cooling system, as well as two different cutting fluids for the last two systems. The results showed considerable enhancement of convective heat transfer using the flooded system. For the dry and minimum quantity of lubrication systems, the heat conduction inside the body was much faster than the heat convection away from its surface. In addition, using the Biot number, the possible models were analyzed for conduction heat problems for each experimental condition tested.

IL-12p40 Deficiency Leads to Uncontrolled Trypanosoma cruzi Dissemination in the Spinal Cord Resulting in Neuronal Death and Motor Dysfunction

Chagas' disease is a protozoosis caused by Trypanosoma cruzi that frequently shows severe chronic clinical complications of the heart or digestive system. Neurological disorders due to T. cruzi infection are also described in children and immunosuppressed hosts. We have previously reported that IL-12p40 knockout (KO) mice infected with the T. cruzi strain Sylvio X10/4 develop spinal cord neurodegenerative disease. Here, we further characterized neuropathology, parasite burden and inflammatory component associated to the fatal neurological disorder occurring in this mouse model. Forelimb paralysis in infected IL-12p40KO mice was associated with 60% (p<0.05) decrease in spinal cord neuronal density, glutamate accumulation (153%, p<0.05) and strong demyelization in lesion areas, mostly in those showing heavy protein nitrosylation, all denoting a neurotoxic degenerative profile. Quantification of T. cruzi 18S rRNA showed that parasite burden was controlled in the spinal cord of WT mice, decreasing from the fifth week after infection, but progressive parasite dissemination was observed in IL-12p40KO cords concurrent with significant accumulation of the astrocytic marker GFAP (317.0%, p<0.01) and 8-fold increase in macrophages/microglia (p<0.01), 36.3% (p<0.01) of which were infected. Similarly, mRNA levels for CD3, TNF-alpha, IFN-gamma, iNOS, IL-10 and arginase I declined in WT spinal cords about the fourth or fifth week after infection, but kept increasing in IL-12p40KO mice. Interestingly, compared to WT tissue, lower mRNA levels for IFN-gamma were observed in the IL-12p40KO spinal cords up to the fourth week of infection. Together the data suggest that impairments of parasite clearance mechanisms in IL-12p40KO mice elicit prolonged spinal cord inflammation that in turn leads to irreversible neurodegenerative lesions.

Multiple Intravenous Administrations of Human Umbilical Cord Blood Cells Benefit in a Mouse Model of ALS

Background: A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) is the use of cell-based therapies that can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (25x10(6) cells) of mononuclear cells from human umbilical cord blood (MNC hUCB) administered intravenously to pre-symptomatic G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages. Methodology/Principal Findings: Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice received 10(6) or 2.5x10(6) cells from 13 weeks of age. A third, pre-symptomatic, group received 10(6) cells from 9 weeks of age. Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in mouse spinal cords. Results showed that mice receiving 10(6) cells pre-symptomatically or 2.5x10(6) cells symptomatically significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice. Astrocytes and microglia were significantly reduced in all cell-treated groups. Conclusions/Significance: These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This multiple cell infusion approach may promote future clinical studies.

Impaired blood-brain/spinal cord barrier in ALS patients

Vascular pathology, including blood-brain/spinal cord barrier (BBB/BSCB) alterations, has recently been recognized as a key factor possibly aggravating motor neuron damage, identifying a neurovascular disease signature for ALS. However, BBB/BSCB competence in sporadic ALS (SALS) is still undetermined. In this study, BBB/BSCB integrity in postmortem gray and white matter of medulla and spinal cord tissue from SALS patients and controls was investigated. Major findings include (1) endothelial cell damage and pericyte degeneration, (2) severe intra- and extracellular edema, (3) reduced CD31 and CD105 expressions in endothelium, (4) significant accumulation of perivascular collagen IV, and fibrin deposits (5) significantly increased microvascular density in lumbar spinal cord, (6) IgG microvascular leakage, (7) reduced tight junction and adhesion protein expressions. Microvascular barrier abnormalities determined in gray and white matter of the medulla, cervical, and lumbar spinal cord of SALS patients are novel findings. Pervasive barrier damage discovered in ALS may have implications for disease pathogenesis and progression, as well as for uncovering novel therapeutic targets. (C) 2012 Elsevier B.V. All rights reserved.

Generating unconstrained two-dimensional non-guillotine cutting patterns by a Recursive Partitioning Algorithm

In this study, a dynamic programming approach to deal with the unconstrained two-dimensional non-guillotine cutting problem is presented. The method extends the recently introduced recursive partitioning approach for the manufacturer's pallet loading problem. The approach involves two phases and uses bounds based on unconstrained two-staged and non-staged guillotine cutting. The method is able to find the optimal cutting pattern of a large number of pro blem instances of moderate sizes known in the literature and a counterexample for which the approach fails to find known optimal solutions was not found. For the instances that the required computer runtime is excessive, the approach is combined with simple heuristics to reduce its running time. Detailed numerical experiments show the reliability of the method. Journal of the Operational Research Society (2012) 63, 183-200. doi: 10.1057/jors.2011.6 Published online 17 August 2011

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells. Microsc. Res. Tech. 75:766770, 2012. (C) 2011 Wiley Periodicals, Inc.

Proteome analysis of spinal cord during the clinical course of monophasic experimental autoimmune encephalomyelitis

The induction of autoimmune encephalomyelitis (EAE) in Lewis rats results in a period of exacerbation followed by complete recovery. Therefore, this model is widely used for studying the evolution of multiple sclerosis. In the present investigation, differentially expressed proteins in the spinal cord of Lewis rats during the evolution of EAE were assessed using the combination of 2DE and MALDI-TOF MS. The majority of the differentially expressed proteins were identified during the acute phase of EAE, in relation to naive control animals. On the other hand, recovered rats presented a similar protein expression pattern in comparison with the naive ones. This observation can be explained, at least in part, by the intense catabolism existent in acute phase due to nervous tissue damage. In recovered rats, we have described the upregulation of proteins that are apparently involved in the recovery of damaged tissue, such as light and medium neurofilaments, glial fibrillary acidic protein, tubulins subunits, and quaking protein. These proteins are involved mainly in cell growth, myelination, and remyelination as well as in astrocyte and oligodendrocyte maturation. The present study has demonstrated that the inflammatory response, characterized by an increase of the proliferative response and infiltration of autoreactive T lymphocytes in the central nervous system, occurs simultaneously with neurodegeneration.

Diaphragmatic pacing stimulation in spinal cord injury: anesthetic and perioperative management

OBJECTIVE: The standard therapy for patients with high-level spinal cord injury is long-term mechanical ventilation through a tracheostomy. However, in some cases, this approach results in death or disability. The aim of this study is to highlight the anesthetics and perioperative aspects of patients undergoing insertion of a diaphragmatic pacemaker. METHODS: Five patients with quadriplegia following high cervical traumatic spinal cord injury and ventilator-dependent chronic respiratory failure were implanted with a laparoscopic diaphragmatic pacemaker after preoperative assessments of their phrenic nerve function and diaphragm contractility through transcutaneous nerve stimulation. ClinicalTrials.gov:NCT01385384. RESULTS: The diaphragmatic pacemaker placement was successful in all of the patients. Two patients presented with capnothorax during the perioperative period, which resolved without consequences. After six months, three patients achieved continuous use of the diaphragm pacing system, and one patient could be removed from mechanical ventilation for more than 4 hours per day. CONCLUSIONS: The implantation of a diaphragmatic phrenic system is a new and safe technique with potential to improve the quality of life of patients who are dependent on mechanical ventilation because of spinal cord injuries. Appropriate indication and adequate perioperative care are fundamental to achieving better results.

Temporal modifications in bone following spinal cord injury in rats

Introduction: The aim of this study was to investigate the temporal modifications in bone mass, bone biomechanical properties and bone morphology in spinal cord injured rats 2, 4 and 6 weeks after a transection. Material and methods: Control animals were randomly distributed into four groups (n = 10 each group): control group (CG) - control animals sacrificed immediately after surgery; spinal cord-injured 2 weeks (2W) - spinal cord-injured animals sacrificed 2 weeks after surgery; spinal cord-injured 4 weeks (4W) - spinal cord-injured animals sacrificed 4 weeks after surgery; spinal cord-injured 6 weeks (6W) - spinal cord-injured animals sacrificed 6 weeks after surgery. Results: Biomechanical properties of the right tibia were determined by a threepoint bending test and injured animals showed a statistically significant decrease in maximal load compared to control animals. The right femur was used for densitometric analysis and bone mineral content of the animals sacrificed 4 and 6 weeks after surgery was significantly higher compared to the control animals and animals sacrificed 2 weeks after surgery. Histopathological and morphological analysis of tibiae revealed intense resorptive areas in the group 2 weeks after injury only. Conclusions: The results of this study show that this rat model is a valuable tool to investigate bone remodeling processes specifically associated with SCI. Taken together, our results suggest that spinal cord injury induced bone loss within 2 weeks after injury in rats.

Cutting Edge: Brazilian Pemphigus Foliaceus Anti-Desmoglein 1 Autoantibodies Cross-React with Sand Fly Salivary LJM11 Antigen

The environmental factors that contribute to the development of autoimmune diseases are largely unknown. Endemic pemphigus foliaceus in humans, known as Fogo Selvagem (FS) in Brazil, is mediated by pathogenic IgG4 autoantibodies against desmoglein 1 (Dsg1). Clusters of FS overlap with those of leishmaniasis, a disease transmitted by sand fly (Lutzomyia longipalpis) bites. In this study, we show that salivary Ags from the sand fly, and specifically the LJM11 salivary protein, are recognized by FS Abs. Anti-Dsg1 monoclonal autoantibodies derived from FS patients also cross-react with LJM11. Mice immunized with LJM11 generate anti-Dsg1 Abs. Thus, insect bites may deliver salivary Ags that initiate a cross-reactive IgG4 Ab response in genetically susceptible individuals and lead to subsequent FS. Our findings establish a clear relationship between an environmental, noninfectious Ag and the development of potentially pathogenic autoantibodies in an autoimmune disease. The Journal of Immunology, 2012, 189: 1535-1539.

Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation

Isolation of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) from full-term deliveries is a laborious, time-consuming process that results in a low yield of cells. In this study we identified parameters that can be helpful for a successful isolation of UCB-MSCs. According to our findings, chances for a well succeeded isolation of these cells are higher when MSCs were isolated from UCB collected from normal full-term pregnancies that did not last over 37 weeks. Besides the duration of pregnancy, blood volume and storage period of the UCB should also be considered for a successful isolation of these cells. Here, we found that the ideal blood volume collected should be above 80 mL and the period of storage should not exceed 6 h. We characterized UCB-MSCs by morphologic, immunophenotypic, protein/gene expression and by adipogenic differentiation potential. Isolated UCB-MSCs showed fibroblast-like morphology and the capacity of differentiating into adipocyte-like cells. Looking for markers of the undifferentiated status of UCB-MSCs, we analyzed the UCB-MSCs' protein expression profile along different time periods of the differentiation process into adipocyte-like cells. Our results showed that there is a decrease in the expression of the markers CD73, CD90, and CD105 that correlates to the degree of differentiation of UCB-MSCs We suggest that CD90 can be used as a mark to follow the differentiation commitment degree of MSCs. Microarray results showed an up-regulation of genes related to the adipogenesis process and to redox metabolism in the adipocyte-like differentiated MSCs. Our study provides information on a group of parameters that may help with successful isolation and consequently with characterization of the differentiated/undifferentiated status of UCB-MSCs, which will be useful to monitor the differentiation commitment of UCB-MSC and further facilitate the application of those cells in stem-cell therapy.

The effects of particulate ambient air pollution on the murine umbilical cord and its vessels: A quantitative morphological and immunohistochemical study

Previous studies have shown that particulate matter (PM) compromise birth weight and placental morphology. We hypothesized that exposing mice to ambient PM would affect umbilical cord (UC) morphology. To test this, mice were kept in paired open-top exposure chambers at the same location and ambient conditions but, in one chamber, the air was filtered (F) and, in the other, it was not (NF). UCs were analysed stereologically and by immunohistochemistry to localize isoprostane and endothelin receptors. The cords of mice from NF chambers were smaller in volume due to loss of mucoid connective tissue and decrease in volume of collagen. These structural changes and in umbilical vessels were associated with greater volumes of regions immunostained for isoprostane, ETAR and ETBR. Findings indicate that the adverse effects of PM on birth weight may be mediated in part by alterations in UC structure or imbalances in the endogenous regulators of vascular tone and oxidative stress. (C) 2012 Elsevier Inc. All rights reserved.

Considerations for Assessing Maximum Critical Temperatures in Small Ectothermic Animals: Insights from Leaf-Cutting Ants

The thermal limits of individual animals were originally proposed as a link between animal physiology and thermal ecology. Although this link is valid in theory, the evaluation of physiological tolerances involves some problems that are the focus of this study. One rationale was that heating rates shall influence upper critical limits, so that ecological thermal limits need to consider experimental heating rates. In addition, if thermal limits are not surpassed in experiments, subsequent tests of the same individual should yield similar results or produce evidence of hardening. Finally, several non-controlled variables such as time under experimental conditions and procedures may affect results. To analyze these issues we conducted an integrative study of upper critical temperatures in a single species, the ant Atta sexdens rubropiosa, an animal model providing large numbers of individuals of diverse sizes but similar genetic makeup. Our specific aims were to test the 1) influence of heating rates in the experimental evaluation of upper critical temperature, 2) assumptions of absence of physical damage and reproducibility, and 3) sources of variance often overlooked in the thermal-limits literature; and 4) to introduce some experimental approaches that may help researchers to separate physiological and methodological issues. The upper thermal limits were influenced by both heating rates and body mass. In the latter case, the effect was physiological rather than methodological. The critical temperature decreased during subsequent tests performed on the same individual ants, even one week after the initial test. Accordingly, upper thermal limits may have been overestimated by our (and typical) protocols. Heating rates, body mass, procedures independent of temperature and other variables may affect the estimation of upper critical temperatures. Therefore, based on our data, we offer suggestions to enhance the quality of measurements, and offer recommendations to authors aiming to compile and analyze databases from the literature.

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Background aims. Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues. Methods. MSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (alpha-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive (R) system. After a mean of 18 +/- 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays. Results. Post-thaw cell recovery: 114 +/- 2.90% (mean +/- SEM). Recovery of viable cells: 93.46 +/- 4.41%, 90.17 +/- 4.55% and 81.03 +/- 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 +/- 1.56%, 72.71 +/- 2.12%, 70.20 +/- 2.39% and 63.02 +/- 2.33% (P<0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 +/- 0.17 X, with a doubling time of 38 +/- 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure. Conclusions. A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.