Tempol improves neuroinflammation and delays motor dysfunction in a mouse model (SOD1G93A) of ALS

The development of new therapeutic strategies to treat amyotrophic lateral sclerosis (ALS) is of utmost importance. The use of cyclic nitroxides such as tempol may provide neuroprotection and improve lifespan. We investigated whether tempol (50 mg/kg) presents therapeutic potential in SOD1G93A transgenic mice. Tempol treatment began at the asymptomatic phase of the disease (10th week) and was administered every other day until week 14, after which it was administered twice a week until the final stage of the disease. The animals were sacrificed at week 14 (initial stage of symptoms—ISS) and at the end stage (ES) of the disease. The lumbar spinal cord of the animals was dissected and processed for use in the following techniques: Nissl staining to evaluate neuronal survival; immunohistochemistry to evaluate astrogliosis and microgliosis (ISS and ES); qRT-PCR to evaluate the expression of neurotrophic factors and pro-inflammatory cytokines (ISS); and transmission electron microscopy to evaluate the alpha-motoneurons (ES). Behavioral analyses considering the survival of animals, bodyweight loss, and Rotarod motor performance test started on week 10 and were performed every 3 days until the end-stage of the disease. The results revealed that treatment with tempol promoted greater neuronal survival (23%) at ISS compared to untreated animals, which was maintained until ES. The intense reactivity of astrocytes and microglia observed in vehicle animals was reduced in the lumbar spinal cords of the animals treated with tempol. In addition, the groups treated with tempol showed reduced expression of proinflammatory cytokines (IL1β and TNFα) and a three-fold decrease in the expression of TGFβ1 at ISS compared with the group treated with vehicle. Altogether, our results indicate that treatment with tempol has beneficial effects, delaying the onset of the disease by enhancing neuronal survival and decreasing glial cell reactivity during ALS progression in SOD1G93A mice161CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP300553/2013-9; 303085/2017-72013/16168-8; 2014/06892-3; 2017/02895-6; 2018/05006-

Chemically-Induced RAT Mesenchymal Stem Cells Adopt Molecular Properties of Neuronal-Like Cells but Do Not Have Basic Neuronal Functional Properties

Induction of adult rat bone marrow mesenchymal stem cells (MSC) by means of chemical compounds (β-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanizole) has been proposed to lead to neuronal transdifferentiation, and this protocol has been broadly used by several laboratories worldwide. Only a few hours of MSC chemical induction using this protocol is sufficient for the acquisition of neuronal-like morphology and neuronal protein expression. However, given that cell death is abundant, we hypothesize that, rather than true neuronal differentiation, this particular protocol leads to cellular toxic effects. We confirm that the induced cells with neuronal-like morphology positively stained for NF-200, S100, β-tubulin III, NSE and MAP-2 proteins. However, the morphological and molecular changes after chemical induction are also associated with an increase in the apoptosis of over 50% of the plated cells after 24 h. Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na+ or K+ currents and do not fire action potentials. Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death. Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro

Analyses of metabolic and morphological myocardium state using microPET technique.

<p>Infected animals with <i>T. cruzi</i> for 1 month were treated with PBS or MSC and analyzed 15–30 days after transplantation. The cardiac assessment was obtained by regional uptake of the glucose analogue ¹⁸F-FDG. (<b>A</b>) Representative image of the whole body, horizontal plane, assessed by microPET technique of a control animal. Note the high glucose activity in the heart. (<b>B–D</b>) Transversal plane images of the heart in high magnification showing the right and left ventricles. It is possible to observe right ventricle dilation in the untreated chagasic animal in image (C). (<b>E–F</b>) Graphs showing the glucose uptake in (E) left and (F) right ventricles. (<b>G</b>) Graph demonstrating the dilation in the right ventricle of infected mice and the recovery by MSC therapy. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001.</p

Tracking of X-Sight-labeled MSCs in control and chagasic mice 2 or 15 days after cell transplantation.

<p>The cells were intravenously injected (tail vein) in control and chagasic animals 1 month after infection for tracking by IVIS. (<b>A</b>) Representative images showing the distribution of transplanted MSCs viewed from in the ventral surface of the body. (<b>B</b>) Images of <i>ex vivo</i> organs showing the distribution of labeled MSCs. Note the majority of the signal is localized in the lung, liver and spleen of the animals. (<b>C–H</b>) Quantification of fluorescence intensity of <i>ex vivo</i> organs shown in images (B). The evaluated organs were (<b>C</b>) heart, (<b>D</b>) bladder, (<b>E</b>) lung, (<b>F</b>) liver, (<b>G</b>) spleen and (<b>H</b>) kidney. <i>P</i><0.05.</p

Detection of X-Sight761 nanoparticles into MSCs <i>in vitro</i>.

<p>X-Sight incorporation was observed by IVIS and confocal microscopy. (<b>A-A″</b>) Fluorescence microscopy images showing the high efficiency of X-Sight to label MSCs after 4 hours of incubation. (<b>A</b>) Staining wih the nuclear stain DAPI. (<b>A′</b>) X-Sight nanoparticles. (<b>A″</b>) Merge of images showing that apparently all MSCs incorporated nanoparticles. (<b>B</b>) Confocal microscope image showing, at high magnification, that X-Sight nanoparticles were incorporated into cell cytoplasm. (<b>C–E</b>) Detection of labeled MSCs by IVIS technique in 96-well plate. (<b>C</b>) Analysis of retention time of X-Sight for up to 4 weekes using different cell plating densities. (<b>D</b>) Representative IVIS image showing labeled cells at different concentrations in a 96-well plate, 2 days after intial exposure of 4 hours. (<b>E</b>) Graph showing a linear correlation between the number of cells and fluorescence mean intensity of the same cells represented in image (D). Scale bar in image (A″) and (B) = 50 µm.</p

Tracking of labeled MSCs with X-Sight in the hearts of control and chagasic mice 2 or 15 days after cells transplantation.

<p>Although the majority of the signal is localized in the lung, liver and spleen of the animals it was possible to observe labeled cells in the heart. (<b>A</b>) Same <i>ex vivo</i> heart images shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001971#pntd-0001971-g002" target="_blank">Figure 2B</a> represented in high magnification showing a greater number of cells migrated to chagasic hearts when compared with control hearts. (<b>B</b>) Statistical analysis of heart fluorescent intensity represented in (A). We observed a stronger signal in the chagasic hearts 2 days after transplantation when compared to the others groups, suggesting that there is a preferential migration to the damage tissue. (<b>C-C″</b>) Representative confocal microscopy images showing X-Sight-labeled cells (arrows) in heart slice of chagasic animals. Only a small number of labeled cells was found in the slices. (<b>C</b>) DAPI. (<b>C′</b>) X-Sight nanoparticles. (<b>C″</b>) Merge of images. Scale bar = 20 µm. <i>P</i><0.05.</p

Mesenchymal Stem Cell Therapy in Diabetic Cardiomyopathy

The incidence and prevalence of diabetes mellitus (DM) are increasing worldwide, and the resulting cardiac complications are the leading cause of death. Among these complications is diabetes-induced cardiomyopathy (DCM), which is the consequence of a pro-inflammatory condition, oxidative stress and fibrosis caused by hyperglycemia. Cardiac remodeling will lead to an imbalance in cell survival and death, which can promote cardiac dysfunction. Since the conventional treatment of DM generally does not address the prevention of cardiac remodeling, it is important to develop new alternatives for the treatment of cardiovascular complications induced by DM. Thus, therapy with mesenchymal stem cells has been shown to be a promising approach for the prevention of DCM because of their anti-apoptotic, anti-fibrotic and anti-inflammatory effects, which could improve cardiac function in patients with DM

Segurança do transplante autólogo, intra-arterial, de células mononucleares da medula óssea na fase aguda do acidente vascular cerebral isquêmico Intra-arterial autologous bone marrow mononuclear cell transplantation for acute ischemic stroke

O acidente vascular cerebral (AVC) é a terceira causa de óbito e a principal causa de incapacidade em indivíduos adultos. Embora a mortalidade do AVC esteja diminuindo em alguns países, a morbidade tem aumentado em razão do envelhecimento da população e do aumento da sobrevida dos pacientes¹. O tratamento com ativador do plasminogênio tissular recombinante (rt-PA) é eficaz quando instituído em até 3 horas após o início dos sintomas², porém seu uso está limitado a cerca de 5% dos pacientes na fase aguda do AVC isquêmico. Além disso, nenhum agente para neuroproteção teve sua eficácia comprovada em estudos clínicos em humanos. Portanto, outras estratégias terapêuticas precisam ser desenvolvidas. Em modelos animais, o uso de células-tronco correlacionou-se com melhora funcional após o AVC³. Publicações recentes têm demonstrado a segurança do tratamento com células mononucleares da medula óssea (CMMO) injetadas via intracoronária em pacientes portadores de cardiopatia isquêmica aguda ou crônica4,5. Baseado nesses dados iniciais, há crescente interesse no estudo do transplante com CMMO na fase aguda do AVC. Relatamos o primeiro caso de transplante autólogo de CMMO via intra-arterial na fase aguda do AVC isquêmico.<br>Stroke is the third cause of death and the leading cause of disability in adult subjects. Although stroke mortality has been declining in some countries, stroke morbidity has been increasing due to the aging of population and patients improved survival.¹ Treatment with recombinant tissue plasminogen activator (rtPA) is successful provided it is administered within 3 hours of symptoms onset,² but its use is limited to about 5% of the patients with acute ischemic stroke. Furthermore, no neuroprotective agent has yet been proven effective in human clinical trials. The development of other therapeutic strategies is, therefore, warranted. The use of stem cells in animal models has led to functional improvement following stroke.³ Recent publications have shown that bone marrow mononuclear cells (BM-MNC) therapy through intracoronary injection is a safe procedure in patients with acute or chronic ischemic heart disease.4,5 Based on these preliminary data, there has been growing interest in the study of BM-MNC transplantation for acute ischemic stroke. We report the first case of intra-arterial autologous BM-MNC transplantation for acute ischemic stroke

Therapeutic Benefit of the Association of Lodenafil with Mesenchymal Stem Cells on Hypoxia-induced Pulmonary Hypertension in Rats

Pulmonary arterial hypertension (PAH) is characterized by the remodeling of pulmonary arteries, with an increased pulmonary arterial pressure and right ventricle (RV) overload. This work investigated the benefit of the association of human umbilical cord mesenchymal stem cells (hMSCs) with lodenafil, a phosphodiesterase-5 inhibitor, in an animal model of PAH. Male Wistar rats were exposed to hypoxia (10% O2) for three weeks plus a weekly i.p. injection of a vascular endothelial growth factor receptor inhibitor (SU5416, 20 mg/kg, SuHx). After confirmation of PAH, animals received intravenous injection of 5.105 hMSCs or vehicle, followed by oral treatment with lodenafil carbonate (10 mg/kg/day) for 14 days. The ratio between pulmonary artery acceleration time and RV ejection time reduced from 0.42 &plusmn; 0.01 (control) to 0.24 &plusmn; 0.01 in the SuHx group, which was not altered by lodenafil alone but was recovered to 0.31 &plusmn; 0.01 when administered in association with hMSCs. RV afterload was confirmed in the SuHx group with an increased RV systolic pressure (mmHg) of 52.1 &plusmn; 8.8 normalized to 29.6 &plusmn; 2.2 after treatment with the association. Treatment with hMSCs + lodenafil reversed RV hypertrophy, fibrosis and interstitial cell infiltration in the SuHx group. Combined therapy of lodenafil and hMSCs may be a strategy for PAH treatment