Daniel Rodrigo Marinowic

The dose-response effect of acute intravenous transplantation of human umbilical cord blood cells on brain damage and spatial memory deficits in neonatal hypoxia-ischemia

Despite the beneficial effects of cell-based therapies on brain repair shown in most studies, there has not been a consensus regarding the optimal dose of human umbilical cord blood cells (HUCBC) for neonatal hypoxia-ischemia (HI). In this study, we compared the long-term effects of intravenous administration of HUCBC at three different doses on spatial memory and brain morphological changes after HI in newborn Wistar rats. In addition, we tested whether the transplanted HUCBC migrate to the injured brain after transplantation. Seven-day-old animals underwent right carotid artery occlusion and were exposed to 8% O(2) inhalation for 2 h. After 24 h, randomly selected animals were assigned to four different experimental groups: HI rats administered with vehicle (HI+vehicle), HI rats treated with 1×10(6) (HI+low-dose), 1×10(7) (HI+medium-dose), and 1×10(8) (HI+high-dose) HUCBC into the jugular vein. A control group (sham-operated) was also included in this study. After 8 weeks of transplantation, spatial memory performance was assessed using the Morris water maze (MWM), and subsequently, the animals were euthanized for brain morphological analysis using stereological methods. In addition, we performed immunofluorescence and polymerase chain reaction (PCR) analyses to identify HUCBC in the rat brain 7 days after transplantation. The MWM test showed a significant spatial memory recovery at the highest HUCBC dose compared with HI+vehicle rats (P<0.05). Furthermore, the brain atrophy was also significantly lower in the HI+medium- and high-dose groups compared with the HI+vehicle animals (P<0.01; 0.001, respectively). In addition, HUCBC were demonstrated to be localized in host brains by immunohistochemistry and PCR analyses 7 days after intravenous administration. These results revealed that HUCBC transplantation has the dose-dependent potential to promote robust tissue repair and stable cognitive improvement after HI brain injury.

Bone marrow mononuclear cells reduce seizure frequency and improve cognitive outcome in chronic epileptic rats.

AIMS Epilepsy affects 0.5-1% of the worlds population, and approximately a third of these patients are refractory to current medication. Given their ability to proliferate, differentiate and regenerate tissues, stem cells could restore neural circuits lost during the course of the disease and reestablish the physiological excitability of neurons. This study verified the therapeutic potential of bone marrow mononuclear cells (BMMCs) on seizure control and cognitive impairment caused by experimentally induced epilepsy. MAIN METHODS Status epilepticus (SE) was induced by lithium-pilocarpine injection and controlled with diazepam 90 min after SE onset. Lithium-pilocarpine-treated rats were intravenously transplanted 22 days after SE with BMMCs obtained from enhanced green fluorescent protein (eGFP) transgenic C57BL/6 mice. Control epileptic animals were given an equivalent volume of saline or fibroblast injections. Animals were video-monitored for the presence of spontaneous recurrent seizures prior to and following the cell administration procedure. In addition, rats underwent cognitive evaluation using a Morris water maze. KEY FINDINGS Our data show that BMMCs reduced the frequency of seizures and improved the learning and long-term spatial memory impairments of epileptic rats. EGFP-positive cells were detected in the brains of transplanted animals by PCR analysis. SIGNIFICANCE The positive behavioral effects observed in our study indicate that BMMCs could represent a promising therapeutic option in the management of chronic temporal lobe epilepsy.

Cytotoxicity of esthetic, metallic, and nickel-free orthodontic brackets: Cellular behavior and viability

INTRODUCTION In this study, we evaluated the cellular viability of various esthetic, metallic, and nickel-free orthodontic brackets. METHODS The sample was divided into 11 groups (n = 8): cellular control, negative control, positive control, metallic, polycarbonate, 2 types of monocrystalline ceramic, 3 types of nickel free, and polycrystalline ceramic brackets. Cell culture (NIH/3T3-mice fibroblasts) was added to the plates of 96 wells containing the specimens and incubated in 5% carbon dioxide at 37°C for 24 hours. Cytotoxicity was analyzed qualitatively and quantitatively. Cell growth was analyzed with an inverted light microscope, photomicrographs were obtained, and the results were recorded as response rates based on modifications of the parameters of Stanford according to the size of diffusion halo of toxic substances. Cell viability was analyzed (MTT assay); a microplate reader recorded the cell viability through the mitochondrial activity in a length of 570 nm. The values were statistically analyzed. RESULTS All tested brackets had higher cytotoxicity values than did the negative control (P <0.05), with the exception Rematitan and Equilibrium (both, Dentaurum, Ispringen, Germany) (P >0.05), suggesting low toxicity effects. The values showed that only polycarbonate brackets were similar (P >0.05) to the positive control, suggesting high toxicity. CONCLUSIONS The brackets demonstrated different ranges of cytotoxicity; nickel-free brackets had better biocompatibility than the others. On the other hand, polycarbonate brackets were made of a highly cytotoxic material for the cells analyzed.

Transplantation of bone marrow mononuclear cells modulates hippocampal expression of growth factors in chronically epileptic animals.

In previous studies, transplantation of bone marrow mononuclear cells (BMMCs) in epileptic animals has been found to be neuroprotective. However, the mechanism by which the BMMCs act remains unclear. We hypothesize that BMMCs may provide neuroprotection to the epileptic brain through trophic support. To test our hypothesis, we studied the temporal expression of neurotrophins after BMMC transplantation in the epileptic rat hippocampus.

Two intrathecal transplants of bone marrow mononuclear cells produce motor improvement in an acute and severe model of spinal cord injury

OBJECTIVE: We studied transplants of bone marrow mononuclear cells (BMMC) by lumbar puncture (LP) in a severe model of spinal cord injury (SCI) using clip compression. METHODS: BMMCs or saline solution were transplanted by LP 48 hours and 9 days post injury. Motor function was evaluated by BBB scale, histological analysis by Nissl technique and the verification of cell migration by PCR analysis. RESULTS: The BBB had significantly improved in rats treated with BMMCs by LP compared with controls (p<0.001). The histological analysis did not showed difference in the lesional area between the groups. The PCR analysis was able to found BMMCs in the injury site. CONCLUSIONS: two BMMC transplants by LP improved motor function in a severe model of SCI and BMMC was found in the injury site.

Transplantation of bone marrow mononuclear cells prolongs survival, delays disease onset and progression and mitigates neuronal loss in pre-symptomatic, but not symptomatic ALS mice

Cell-based therapy provides a novel strategy to restore lost neurons or modulate the degenerating microenvironment in amyotrophic lateral sclerosis (ALS). This study verified the therapeutic potential of bone marrow mononuclear cells (BMMCs) in SOD1G93A mice. BMMCs were obtained from enhanced green fluorescent protein (EGFP) transgenic C57BL/6 mice (EGFPBMMCs) or from SOD1G93A transgenic mice (mSOD1BMMCs) and given to mice at the pre-symptomatic or late symptomatic stage. Survival, body weight and motor performance data were recorded. DNA integrity was evaluated using the alkaline comet assay. The spinal cords were collected to assess motoneuron preservation and cell migration. EGFPBMMCs and mSOD1BMMCs transplantation to pre-symptomatic SOD1G93A mice prolonged survival and delayed disease progression. The effects were more significant for the EGFPBMMC-transplanted mice. In late symptomatic mice, EGFPBMMCs promoted a discrete increase in survival, without other clinical improvements. DNA from EGFPBMMCs and mSOD1BMMCs was found in the spinal cords of transplanted animals. DNA damage was not modified by BMMCs in any of the studied groups. Despite positive behavioral effects observed in our study, the limited results we observed for late transplanted mice call for caution before clinical application of BMMCs in ALS.

Safety and Seizure Control in Patients with Mesial Temporal Lobe Epilepsy Treated with Regional Superselective Intra-arterial Injection of Autologous Bone Marrow Mononuclear Cells

Temporal lobe epilepsy (TLE) is a highly prevalent syndrome among people with epilepsy, and is usually refractory to drug treatment. Structural and physiological changes, such as hippocampal sclerosis, are often present in TLE patients. The objective of this study is to evaluate the feasibility and safety of intra‐arterial infusion of autologous bone marrow mononuclear cells (BMMC) in adults with medically refractory mesial TLE (MTLE) and unilateral hippocampal sclerosis (HS). We enrolled 20 patients who had been diagnosed with MTLE‐HS and were refractory to medical treatment. All patients underwent a neurological evaluation, magnetic resonance imaging with hippocampal volumetry, video‐electroencephalography (EEG) with ictal recording, and a neuropsychological test battery focusing on verbal and nonverbal memory domains. After bone marrow aspiration and subsequent cell preparation, the BMMC were infused by selective posterior cerebral artery catheterization. Patients were followed for 6 months. Safety of the procedure, seizure frequency, neuropsychological evaluation, EEG variables, routine brain magnetic resonance imaging and hippocampal volumetry were considered measurements of outcome. Any serious intercurrent clinical event or adverse effects related to the procedure were reported. No additional lesions and no significant hippocampal volumetric changes were observed. EEG recordings showed a decrease in theta activity and spike density. At 6 months, eight patients (40%) were seizure free. A significant increase in the memory scores over time was observed. The BMMC autologous transplant for the treatment of temporal lobe epilepsy is feasible and safe. The seizure control achieved in this novel study supports the therapeutic potential of stem cell transplants in MTLE‐HS patients. Copyright

Use of induced pluripotent stem cells (iPSCs) and cerebral organoids in modeling the congenital infection and neuropathogenesis induced by Zika virus: MAJOLO et al.

Infection with Zika virus (ZIKV) was recently demonstrated to be associated with damage to the central nervous system, especially microcephaly and the Guillain‐Barré syndrome. This finding had alarmed public health agencies and mobilized institutions around the world to search for more information about the virus, its effects, pathophysiological mechanisms, and potential immunizations and treatments. Given the increasing interest in using iPSCs and cerebral organoids to model the congenital infection and neuropathogenesis induced by ZIKV, the aim of this review was to present an up‐to‐date summary of the publications on the association of ZIKV with microcephaly, using iPSCs and organoids. According to our review, the number of studies has decreased concomitantly with a decrease in the number of cases. The presence of subclinical lesions at birth, which may eventually present cognitive or behavioral problems in the future, suggests that persistent research efforts on the virus should be undertaken by the global health community till the threat is completely wiped out.

Minimal Detectable Amounts of Exogenous DNA after Systemic Cell Engraftment

In cellular therapy models, the establishment of accurate methods to track the homing, cell fate and survival are fundamental to ensure safety and efficacy. The PCR is used to detect donor cells DNA in xenotransplant models. Five different concentrations of EGFP+ DNA from C57/BL6 mouse muscle tissue: 1,000 pg/μL, 500 pg/μL, 100 pg/μL, 10 pg/μL e 1 pg/μL were used to contaminate 10 ng/μL of DNA Wistar rat. Polymerase chain reaction was performed using 2 μL of each sample. EGFP+DNA were detected in samples containing 2,000 pg, 1,000 pg, 200 pg and 20 pg. Samples with 2 pg EGFP+DNA did not show signal. Here we demonstrate an alternative to proving the presence or confirm the absence, of exogenous DNA in organs and tissues. The concentration curve for cell migration detection by real time PCR will allow the use in systemically transplanted cells in animal models subjected to cell therapy.

Measurement of the Compound and Extracellular Action Potential of the Spinal Cord: Technical Report and Experimental Applications

Introduction Experimental studies of the spinal cord before and after injury caused by trauma are necessary to evaluate the new therapeutic options, such as stem cell therapy. In those experiments, it is mandatory to measure motor and sensitive function adequately to the bias and increase true results. The objective is to describe the technique used to obtain the compound and extracellular action potentials in the spinal cord. Materials and Methods To obtain the spinal cord compound action potentials (CAPs), the spinal cord of the rat spine was immediately placed in a Perspex chamber covered with Sylgard and superfused at (2 mL/min) with Krebs solution and glucose. The solution was maintained at pH 7.2 and 5% CO2/95% O2. The Ag/AgCl needle electrodes fixed the tissue in the chamber. The needle electrodes were connected to an operational amplifier and to a programmable signal conditioner model. The electrical stimulation was done by suction bipolar electrode placed in the inferior segment of the spinal cord. The electrode was connected to an analogical stimulator, which delivered stimulus at 01 Hz for a 0.2 milliseconds duration. The data always began to be recorded after 15 minutes of stimulation to ensure the stability of the preparation. The recordings of the CAPs were made during 30 minutes directly to an AxoScope software. The events were analyzed by the Clampfit software. To acquire extracellular action potential, the spinal cord was cooled in Krebs solution, previously gassed with a 95% O2 and 5% CO2 mixture to obtain a pH value of 7.2 to 7.4. A 400 µm thick coronal section was made. The spinal cord slice was recovered during at least 30 minutes at 32°C in Krebs solution and transferred to a submersion-type recording chamber with Krebs solution, 95% O2 and 5% CO2. Extracellular recordings were made using 5 to 10 MΩ resistance glass pipettes. An iridium stimulation electrode was connected to an analogical stimulator and positioned at the proximal side of the tissue to deliver electrical stimuli (0.1 Hz, 0.2 ms). The recording electrode was placed in the opposite or distal side. The recordings were performed using an amplifier connected to a programmable signal conditioner. The experiments were recorded during 1 hour. The data were retrieved by an AxoScope software and analyzed by the Clampfit software. Results The compound and extracellular action potential could be generated in both experiments indicating that the functional analysis of spinal cord neurons can be assessed by in vitro stimulation of the spinal cord tissue or its slice. The control values for the amplitude of CAPs was 4.4 ± 0.3 mV (n = 3). The rise time of the CAPs was 1.73 ± 03 ms; the decay time was 4.2 ± 2 ms, and the duration was 4.2 ± 0.8 ms. The analysis of the extracellular action potentials showed that the mean amplitude, rise time, decay time, and duration were 2.17 ± 0.3 mV, 0.4 ± 0.2, 4 ± 1, and 2.1 ± 0.2 ms, respectively. Conclusion The analysis of compound and extracellular action potential can be used as outcome measurement of spinal cord functionality. This can measure more precisely, for example, the neuronal repopulation and motor and sensitive recovery of spinal cord injury.