Bruno Diaz Paredes

Bone Marrow Multipotent Mesenchymal Stromal Cells Do Not Reduce Fibrosis or Improve Function in a Rat Model of Severe Chronic Liver Injury

The objective of our study was to evaluate the therapeutic potential of bone marrow mesenchymal stromal cells (MSC) in a rat model of severe chronic liver injury. Fourteen female Wistar rats were fed exclusively an alcoholic liquid diet and received intraperitoneal injections of carbon tetrachloride every other day during 15 weeks. After this period, eight animals (MSC group) had 1 × 107 cells injected into the portal vein while six animals (placebo group) received vehicle. Blood analysis was performed to evaluate alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin before cell therapy and 1 and 2 months after cell or placebo infusion. Fibrosis was evaluated before and 1 month after cell or placebo injection by liver biopsies. Two months after cell delivery, animals were sacrificed and histological analysis of the livers was performed. Fibrosis was quantified by histomorphometry. Biopsies obtained before cell infusion showed intense collagen deposition and septa interconnecting regenerative nodules. One month after cell injection, this result was unaltered and differences in fibrosis quantification were not found between MSC and placebo groups. ALT and AST returned to normal values 2 weeks after cell or placebo infusion, without significant differences between experimental groups. Two months after cell or placebo injection, albumin had also returned to normal values and histological results were maintained, again without differences between MSC and placebo groups. Therefore, under our experimental conditions, MSC were unable to reduce fibrosis or improve liver function in a rat model of severe chronic liver injury.

Therapeutic window for treatment of cortical ischemia with bone marrow-derived cells in rats

The beneficial effect of treatment with bone marrow mononuclear cells (BMMCs) was evaluated in different therapeutic windows in a rat model of focal ischemia induced by thermocoagulation of the blood vessels in the left motor, somestesic, and sensorimotor cortices. We also compared the therapeutic benefits between BMMCs and bone marrow-derived mesenchymal stem cells (MSCs). BMMCs and MSCs were obtained from donor rats and injected into the jugular vein after ischemia. BMMCs-treated animals received approximately 3x10(7) cells at post-ischemic days (PIDs) 1, 7, 14, or 30. MSCs-treated animals received approximately 3x10(6) cells at PIDs 1 and 30. Control animals received only the vehicle. The animals were then evaluated for functional sensorimotor recovery weekly with behavioral tests (cylinder test and adhesive test). Significant recovery of sensorimotor function was only observed in the cylinder test in animals treated with BMMCs at PIDs 1 and 7. Similar effects were also observed in the animals treated with MSCs 1 day after ischemia, but not in animals treated with MSCs 30 days after ischemia. Significant decrease in glial scarring did not seem to be a mechanism of action of BMMCs, since treatment with BMMCs did not change the level of expression of GFAP, indicating no significant change in the astrocytic scar in the periphery of the ischemic lesion. These results suggest that BMMCs might be an efficient treatment protocol for stroke only in the acute/subacute phase of the disease, and its efficiency in inducing functional recovery is similar to that of MSCs.

Zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells

Zika virus (ZIKV) infection has been associated with severe complications both in the developing and adult nervous system. To investigate the deleterious effects of ZIKV infection, we used human neural progenitor cells (NPC), derived from induced pluripotent stem cells (iPSC). We found that NPC are highly susceptible to ZIKV and the infection results in cell death. ZIKV infection led to a marked reduction in cell proliferation, ultrastructural alterations and induction of autophagy. Induction of apoptosis of Sox2+ cells was demonstrated by activation of caspases 3/7, 8 and 9, and by ultrastructural and flow cytometry analyses. ZIKV-induced death of Sox2+ cells was prevented by incubation with the pan-caspase inhibitor, Z-VAD-FMK. By confocal microscopy analysis we found an increased number of cells with supernumerary centrosomes. Live imaging showed a significant increase in mitosis abnormalities, including multipolar spindle, chromosome laggards, micronuclei and death of progeny after cell division. FISH analysis for chromosomes 12 and 17 showed increased frequency of aneuploidy, such as monosomy, trisomy and polyploidy. Our study reinforces the link between ZIKV and abnormalities in the developing human brain, including microcephaly.

Distribution of Mesenchymal Stem Cells and Effects on Neuronal Survival and Axon Regeneration after Optic Nerve Crush and Cell Therapy

Bone marrow-derived cells have been used in different animal models of neurological diseases. We investigated the therapeutic potential of mesenchymal stem cells (MSC) injected into the vitreous body in a model of optic nerve injury. Adult (3–5 months old) Lister Hooded rats underwent unilateral optic nerve crush followed by injection of MSC or the vehicle into the vitreous body. Before they were injected, MSC were labeled with a fluorescent dye or with superparamagnetic iron oxide nanoparticles, which allowed us to track the cells in vivo by magnetic resonance imaging. Sixteen and 28 days after injury, the survival of retinal ganglion cells was evaluated by assessing the number of Tuj1- or Brn3a-positive cells in flat-mounted retinas, and optic nerve regeneration was investigated after anterograde labeling of the optic axons with cholera toxin B conjugated to Alexa 488. Transplanted MSC remained in the vitreous body and were found in the eye for several weeks. Cell therapy significantly increased the number of Tuj1- and Brn3a-positive cells in the retina and the number of axons distal to the crush site at 16 and 28 days after optic nerve crush, although the RGC number decreased over time. MSC therapy was associated with an increase in the FGF-2 expression in the retinal ganglion cells layer, suggesting a beneficial outcome mediated by trophic factors. Interleukin-1β expression was also increased by MSC transplantation. In summary, MSC protected RGC and stimulated axon regeneration after optic nerve crush. The long period when the transplanted cells remained in the eye may account for the effect observed. However, further studies are needed to overcome eventually undesirable consequences of MSC transplantation and to potentiate the beneficial ones in order to sustain the neuroprotective effect overtime.

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

BackgroundDomestic dogs and cats are very well known to develop chronic hepatic diseases, including hepatic lipidosis and cirrhosis. Ultrasonographic examination is extensively used to detect them. However, there are still few reports on the use of the ultrasound B-mode scan in correlation with histological findings to evaluate diffuse hepatic changes in rodents, which represent the most important animal group used in experimental models of liver diseases. The purpose of this study was to determine the reliability of ultrasound findings in the assessment of fatty liver disease and cirrhosis when compared to histological results in Wistar rats by following up a murine model of chronic hepatic disease.ResultsForty Wistar rats (30 treated, 10 controls) were included. Liver injury was induced by dual exposure to CCl4 and ethanol for 4, 8 and 15 weeks. Liver echogenicity, its correlation to the right renal cortex echogenicity, measurement of portal vein diameter (PVD) and the presence of ascites were evaluated and compared to histological findings of hepatic steatosis and cirrhosis. Liver echogenicity correlated to hepatic steatosis when it was greater or equal to the right renal cortex echogenicity, with a sensitivity of 90%, specificity of 100%, positive and negative predictive values of 100% and 76.9% respectively, and accuracy of 92.5%. Findings of heterogeneous liver echogenicity and irregular surface correlated to liver cirrhosis with a sensitivity of 70.6%, specificity of 100%, positive and negative predictive values of 100% and 82.1% respectively, and accuracy of 87.5%. PVD was significantly increased in both steatotic and cirrhotic rats; however, the later had greater diameters. PVD cut-off point separating steatosis from cirrhosis was 2.1 mm (sensitivity of 100% and specificity of 90.5%). One third of cirrhotic rats presented with ascites.ConclusionThe use of ultrasound imaging in the follow-up of murine diffuse liver disease models is feasible and efficient, especially when the studied parameters are used in combination. The potential implication of this study is to provide a non-invasive method that allows follow-up studies of fatty liver disease and cirrhosis of individual rats for pre-clinical drug or cell based therapies.

Early Double-Negative Thymocyte Export in Trypanosoma cruzi Infection Is Restricted by Sphingosine Receptors and Associated with Human Chagas Disease

The protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironmental and lymphoid compartments. Acute infection results in severe atrophy of the organ and early release of immature thymocytes into the periphery. To date, the pathophysiological effects of thymic changes promoted by parasite-inducing premature release of thymocytes to the periphery has remained elusive. Herein, we show that sphingosine-1-phosphate (S1P), a potent mediator of T cell chemotaxis, plays a role in the exit of immature double-negative thymocytes in experimental Chagas disease. In thymuses from T. cruzi-infected mice we detected reduced transcription of the S1P kinase 1 and 2 genes related to S1P biosynthesis, together with increased transcription of the SGPL1 sphingosine-1-lyase gene, whose product inactivates S1P. These changes were associated with reduced intrathymic levels of S1P kinase activity. Interestingly, double-negative thymocytes from infected animals expressed high levels of the S1P receptor during infection, and migrated to lower levels of S1P. Moreover, during T. cruzi infection, this thymocyte subset expresses high levels of IL-17 and TNF-α cytokines upon polyclonal stimulation. In vivo treatment with the S1P receptor antagonist FTY720 resulted in recovery the numbers of double-negative thymocytes in infected thymuses to physiological levels. Finally, we showed increased numbers of double-negative T cells in the peripheral blood in severe cardiac forms of human Chagas disease.

Effects of bone marrow-derived mononuclear cells from healthy or acute respiratory distress syndrome donors on recipient lung-injured mice.

Objective:The advantage of using autologous bone marrow–derived mononuclear cells to treat acute respiratory distress syndrome patients is to prevent immunological rejection. However, bone marrow–derived mononuclear cells may be altered by different acute respiratory distress syndrome etiologies, resulting in questionable efficacy and thus limited clinical application. We aimed to investigate the effects of bone marrow–derived mononuclear cells obtained from healthy and acute respiratory distress syndrome donors on pulmonary and extrapulmonary acute respiratory distress syndrome. Design:Prospective, randomized, controlled experimental study. Setting:University research laboratory. Subjects:Two hundred and twenty-five C57BL/6 mice. Interventions:Acute respiratory distress syndrome was induced by Escherichia coli lipopolysaccharide intratracheally (ARDSp) or intraperitoneally (ARDSexp). Control mice (Healthy) received saline solution intratracheally (Cp) or intraperitoneally (Cexp). After 24 hours, whole bone marrow cells were analyzed in vitro: 1) colony-forming unit–fibroblasts and 2) hematopoietic stem cells, neutrophils, T helper lymphocytes, B lymphocytes, and nonhematopoietic precursors. After cell characterization, all groups received saline or bone marrow–derived mononuclear cells (2 × 106), obtained from Cp, Cexp, ARDSp, and ARDSexp donor mice, IV, on day 1. Measurements and Main Results:On day 1, in ARDSp, different patterns of colony formation were found, with nonstromal cells (mainly neutrophils) predominating over fibroblastoid colonies. In ARDSexp, irregular colony-forming unit–fibroblasts morphology with dispersed proliferating colonies and a greater number of hematopoietic stem cells were observed. In ARDSp, colony-forming unit–fibroblasts count was higher but not measurable in ARDSexp. In ARDSp, monocytes and T lymphocytes were increased and hematopoietic precursor cells reduced, with no significant changes in ARDSexp. On day 7, bone marrow–derived mononuclear cells improved survival and attenuated changes in lung mechanics, alveolar collapse, inflammation, pulmonary fibrosis, and apoptosis in the lung and distal organs, regardless of donor type. Conclusions:Bone marrow–derived mononuclear cells from ARDSp and ARDSexp donors showed different characteristics but were as effective as cells obtained from healthy donors in reducing inflammation and remodeling, suggesting the utility of autologous transplant of bone marrow–derived mononuclear cells in the clinical setting.

Sustained effect of bone marrow mononuclear cell therapy in axonal regeneration in a model of optic nerve crush.

In adult mammals, the regeneration of the optic nerve is very limited and at the moment there are several groups trying different approaches to increase retinal ganglion cell (RGC) survival and axonal outgrowth. One promising approach is cell therapy. In previous work, we performed intravitreal transplantation of bone-marrow mononuclear cells (BMMCs) after optic nerve crush in adult rats and we demonstrated an increase in RGC survival and axon outgrowth 14 days after injury. In the present work, we investigated if these results could be sustained for a longer period of time. Optic nerve crush was performed in Lister-hooded adult rats and BMMC or saline injections were performed shortly after injury. Neuronal survival and regeneration were evaluated in rats׳ retina and optic nerve after 28 days. We demonstrated an increase of 5.2 fold in the axon outgrowth 28 days after lesion, but the BMMCs had no effect on RGC survival. In an attempt to prolong RGC survival, we established a new protocol with two BMMC injections, the second one 7 days after the injury. Untreated animals received two injections of saline. We observed that although the axonal outgrowth was still increased after the second BMMC injection, the RGC survival was not significantly different from untreated animals. These results demonstrate that BMMCs transplantation promotes neuroregeneration at least until 28 days after injury. However, the effects on RGC survival previously observed by us at 14 days were not sustained at 28 days and could not be prolonged with a second dose of BMMC.

Bone marrow-derived mononuclear cells promote improvement in glomerular function in rats with early diabetic nephropathy.

Background/Aims: Diabetic nephropathy is one of the main causes of end-stage renal disease. The present study investigated the effect of mononuclear cell (MC) therapy in rats subjected to diabetic nephropathy. Methods: Male Wistar rats were divided into control (CTRL), diabetic (DM), CTRL+MC and DM+MC groups. Diabetes was induced by a single injection of streptozotocin (45 mg/kg, i.p.) and, 4 weeks later, 2×107 MCs were injected via the jugular vein. Results: The rats in the DM and DM+MC groups showed increased glycemia, glomerular filtration rate and glomerular tuff area versus control groups. The glomerular filtration rate and glomerular tuff area were normalized in the DM+MC group. No alterations were observed in the fractional excretion of electrolytes and proteinuria between the DM and DM+MC groups. TGF-β1 protein levels in the DM group were significantly increased versus control animals and normalized in the DM+MC group. An increase in ED1+/arginase I+ macrophages and IL-10 renal expression was observed in the DM+MC group versus DM group. Conclusions: Bone marrow-derived MC therapy was able to prevent glomerular alterations and TGF-β1 protein overexpression and modulated glomerular arginase I+ macrophage infiltration in rats subjected to early diabetic nephropathy.

An ultrasound and histomorphological analysis of experimental liver cirrhosis in rats.

We investigated whether liver injury by dual exposure to ethanol and carbon tetrachloride (EtOH + CCl4) for 15 weeks would persist after hepatotoxic agents were removed (EtOH + CCl4/8wR). After 15 weeks of hepatic injury with ethanol (5.5%, m/v) and carbon tetrachloride (0.05, mL/kg, ip), 5 of 11 female Wistar rats were sacrificed. The other 6 rats were maintained for an additional 8 weeks without hepatotoxic agents. Ultrasonography showed increased liver echogenicity and dilation of portal vein caliber in both groups (EtOH + CCl4: 0.22 +/- 0.01 cm, P < 0.001; EtOH + CCl4/8wR: 0.21 +/- 0.02 cm, P < 0.01) vs control (0.16 +/- 0.02 cm). Histopathology showed regenerative nodules in both experimental groups. Histomorphometry revealed increased fibrosis content in both groups (EtOH + CCl4: 12.6 +/- 2.64%, P < 0.001; EtOH + CCl4/8wR: 10.4 +/- 1.36%, P < 0.05) vs control (2.2 +/- 1.21%). Collagen types I and III were increased in groups EtOH + CCl4 (collagen I: 2.5 +/- 1.3%, P < 0.01; collagen III: 1.3 +/- 0.2%, P < 0.05) and EtOH + CCl4/8wR (collagen I: 1.8 +/- 0.06%, P < 0.05; collagen III: 1.5 +/- 0.8%, P < 0.01) vs control (collagen I: 0.38 +/- 0.11%; collagen III: 0.25 +/- 0.06%). Tissue transglutaminase increased in both groups (EtOH + CCl4: 66.4 +/- 8%, P < 0.01; EtOH + CCl4/8wR: 58.8 +/- 21%, P < 0.01) vs control (7.9 +/- 0.8%). Cirrhosis caused by the association of CCl4-EtOH remained for at least 8 weeks after removal of these hepatotoxic agents. Ultrasound images can be a useful tool to evaluate advanced hepatic alterations.