Samuel Greggio

Hemispheric brain injury and behavioral deficits induced by severe neonatal hypoxia-ischemia in rats are not attenuated by intravenous administration of human umbilical cord blood cells.

Neonatal hypoxia-ischemia (HI) is an important cause of mortality and morbidity in infants. Human umbilical cord blood (HUCB) is a potential source of cellular therapy in perinatology. We investigated the effects of HUCB cells on spatial memory, motor performance, and brain morphologic changes in neonate rats submitted to HI. Seven-day-old rats underwent right carotid artery occlusion followed by exposure to 8% O2 inhalation for 2 h. Twenty-four hours after HI, rats received either saline solution or HUCB cells i.v. After 3 wk, rats were assessed using a Morris Water Maze and four motor tests. Subsequently, rats were killed for histologic, immunohistochemical, and polymerase chain reaction (PCR) analyses. HI rats showed significant spatial memory deficits and a volumetric decrease in the hemisphere ipsilateral to arterial occlusion. These deficits and decreases were not significantly attenuated by the injection of HUCB cells. Moreover, immunofluorescence and PCR analysis revealed few HUCB cells located in rat brain. Intravenous administration of HUCB cells requires optimization to achieve improved therapeutic outcomes in neonatal hypoxic-ischemic injury.

DNA damage in organs of mice treated acutely with patulin, a known mycotoxin

Patulin, a known mycotoxin, is considered a significant contaminant in apples, apple-derived products and feeds. This study investigated the genotoxic effects of patulin in multiple organs (brain, kidney, liver and urinary bladder) of mice using an in vivo comet assay. We assessed the mechanism underlying this genotoxicity by measuring the GSH content and the thiobarbituric acid-reactive species (TBARS) level. Male CF-1 mice were given 1.0-3.75 mg/kg patulin intraperitoneally. The effect of patulin was dose-dependent and the highest patulin dose induced DNA strand breaks in the brain (damage index, DI, in hippocampus increased from 36.2 in control animals to 127.5), liver (44.3-138.4) and kidneys (31.5-99); decreased levels of GSH (hippocampus--from 46.9 to 18.4 nmol/mg protein); and an increase in lipid peroxidation (hippocampus--from 5.8 to 20.3 MDA equivalents/mg protein). This finding establishes an interrelationship between the pro-oxidant and genotoxic effects of patulin. Pre-treatment administration of N-acetyl-cysteine reduced patulin-induced DNA damage (hippocampus--DI from 127.5 to 39.8) and lipid peroxidation (hippocampus--20.3 to 12.8 MDA equivalents/mg protein) by restoring cellular GSH levels, reinforcing the positive relationship between patulin-induced GSH depletion and DNA damage caused by systemic administration of this mycotoxin.

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.

Intra-arterial transplantation of human umbilical cord blood mononuclear cells in neonatal hypoxic-ischemic rats.

UNLABELLED Based on preclinical findings, cellular therapy has become a promising therapeutic approach for neonatal hypoxia-ischemia (HI). However, before translation into the clinical setting, new and effective routes of cell delivery must be determined. Intra-arterial (IA) delivery is an attractive route of cellular administration but has never been used in neonatal HI rats. AIMS In this study, we investigated the feasibility of IA transplantation of human umbilical cord blood (HUCB) mononuclear cells for the treatment of long-term behavior dysfunction and brain lesion after neonatal HI. MAIN METHODS Seven-day-old rats were subjected to a HI model and the animals received HUCB mononuclear cells into the left common carotid artery 24 h after HI insult. KEY FINDINGS At 9 weeks post-HI, intra-arterially transplanted HUCB mononuclear cells significantly improved learning and long-term spatial memory impairments when evaluated by the Morris water maze paradigm. There was no effect of neonatal HI insult or IA procedure on body weight and on motor coordination and balance when evaluated by the accelerating rotarod test. Cellular transplantation by the IA route did not restore neonatal HI-induced brain damage according to stereological volume assessment. Furthermore, HUCB mononuclear cells were tracked in the injured brain and peripheral organs of HI transplanted-rats by nested polymerase chain reaction analysis at different time points. SIGNIFICANCE Our findings contribute to the translational knowledge of cell based-therapy in neonatal HI and demonstrate for the first time that IA transplantation into rat pups is a feasible route for cellular delivery and prevents long-term cognitive deficits induced by experimental neonatal HI.

A novel preclinical rodent model of collagenase-induced germinal matrix/intraventricular hemorrhage.

Germinal matrix/intraventricular hemorrhage (GMH/IVH) is a complication that arises in premature infants associated with neurological sequelae. Greater understanding of GMH/IVH is needed to develop therapies, a goal that depends on the existence of appropriate animal models. Towards this goal, we aimed to develop a rodent model of GMH/IVH based on collagenase-induced hemorrhage that exhibits histological and neurological consequences similar to that seen in patients. Male 6-day-old rats were placed on a warming pad and anesthetized with halothane/nitrous oxide delivered by face mask. Uni- or bilateral periventricular injections of 2-μl collagenase (2.0 U) were performed freehand with a needle inserted percutaneously. Sham rats were infused with saline. Early neonatal development, long-term motor and cognitive performances and alterations in brain volume were assessed. Collagenase-based GMH/IVH negatively affected ambulation, surface righting and negative geotaxis outcomes more evidently in bilaterally infused rats, which also presented an early decrease in brain volume, as assessed by the Cavalieri method. In adult animals, a unilateral collagenase infusion produced no significant alteration on forepaw preference. Only bilaterally infused rats presented an impairment of object recognition memory and locomotor deficit. Nevertheless, histological evaluation also demonstrated a persistent brain volume reduction in bilaterally infused rats. Our study provides a pioneering animal model of collagenase-based GMH/IVH, which can be used to evaluate preventive strategies and potential therapeutic interventions for this disorder.

NAP prevents acute cerebral oxidative stress and protects against long-term brain injury and cognitive impairment in a model of neonatal hypoxia–ischemia

Hypoxia-ischemia (HI) is a common cause of neonatal brain damage with lifelong morbidities in which current therapies are limited. In this study, we investigated the effect of neuropeptide NAP (NAPVSIPQ) on early cerebral oxidative stress, long-term neurological function and brain injury after neonatal HI. Seven-day-old rat pups were subjected to an HI model by applying a unilateral carotid artery occlusion and systemic hypoxia. The animals were randomly assigned to groups receiving an intraperitoneal injection of NAP (3 μg/g) or vehicle immediately (0 h) and 24 h after HI. Brain DNA damage, lipid peroxidation and reduced glutathione (GSH) content were determined 24 h after the last NAP injection. Cognitive impairment was assessed on postnatal day 60 using the spatial version of the Morris water maze learning task. Next, the animals were euthanized to assess the cerebral hemispheric volume using the Cavalieri principle associated with the counting point method. We observed that NAP prevented the acute HI-induced DNA and lipid membrane damage and also recovered the GSH levels in the injured hemisphere of the HI rat pups. Further, NAP was able to prevent impairments in learning and long-term spatial memory and to significantly reduce brain damage up to 7 weeks following the neonatal HI injury. Our findings demonstrate that NAP confers potent neuroprotection from acute brain oxidative stress, long-term cognitive impairment and brain lesions induced by neonatal HI through, at least in part, the modulation of the glutathione-mediated antioxidant system.

NAP prevents hippocampal oxidative damage in neonatal rats subjected to hypoxia-induced seizures

Neonatal seizures in which hypoxic-ischemic encephalopathy is the main triggering etiology have a challenging diagnosis and limited efficacy of treatment. NAP (NAPVSIPQ) has shown extensive neuroprotective and antioxidant capacity in vitro and in vivo. To evaluate its neuroprotective role in the context of seizures associated with perinatal hypoxia, we assessed the integrity of DNA and lipid membranes as well as the redox status in the hippocampus of 10-day-old rats exposed to hypoxia-induced seizures (HS) with and without NAP treatment. Rats were exposed to transient global hypoxia (12 min exposure to 5-7% O2 was able to induce electrographic seizures) or room air with subsequent intraperitoneal NAP (0.03, 0.3 or 3 microg/g) or vehicle administration. Results showed elevated DNA damage immediately after the insult until 72 h post-HS, while oxidized bases were only detected 3, 6 and 24 h later. In addition, thiobarbituric acid reactive species peaked at 6 h in parallel with decreased levels of reduced glutathione between 3 and 72 h post-HS insult. Our findings expand on the knowledge about the time course of HS-induced oxidative damage and demonstrate for the first time that a single NAP injection dose-dependently prevents HS-induced oxidative damage to DNA and lipid membranes, in correlation with modulation of the glutathione system. Hence, NAP may represent a promising therapeutic strategy for avoiding HS-induced oxidative damage.

O uso de células-tronco na asfixia perinatal: do laboratório à prática clínica

OBJECTIVES: To present recent scientific evidence on the effects of stem cell transplantation in animal models of neonatal hypoxic-ischemic brain injury and address the translational relevance of cell therapy for clinical application in this context. SOURCES: The PubMed and Scopus databases were used to select articles. The selection criterion was the specificity of articles regarding the subject studied, preferably articles published from 2000 onward. We also reviewed classic articles from previous years that were applicable to this review. SUMMARY OF THE FINDINGS: Stem cells from different exogenous sources may exhibit neuroprotective properties in experimental models of neonatal hypoxia-ischemia. In most animal experiments, the morphological and functional benefits observed were independent of neural differentiation, suggesting associated mechanisms of action, such as the release of trophic factors and inflammatory modulation. CONCLUSIONS: Based on the experimental studies analyzed, cell therapy may become a promising therapeutic approach in the treatment of children with hypoxic-ischemic encephalopathy. However, further studies are warranted to elucidate potential mechanisms of action of these cells and to define safe and effective clinical strategies.

Use of stem cells in perinatal asphyxia: from bench to bedside

OBJECTIVES To present recent scientific evidence on the effects of stem cell transplantation in animal models of neonatal hypoxic-ischemic brain injury and address the translational relevance of cell therapy for clinical application in this context. SOURCES The PubMed and Scopus databases were used to select articles. The selection criterion was the specificity of articles regarding the subject studied, preferably articles published from 2000 onward. We also reviewed classic articles from previous years that were applicable to this review. SUMMARY OF THE FINDINGS Stem cells from different exogenous sources may exhibit neuroprotective properties in experimental models of neonatal hypoxia-ischemia. In most animal experiments, the morphological and functional benefits observed were independent of neural differentiation, suggesting associated mechanisms of action, such as the release of trophic factors and inflammatory modulation. CONCLUSIONS Based on the experimental studies analyzed, cell therapy may become a promising therapeutic approach in the treatment of children with hypoxic-ischemic encephalopathy. However, further studies are warranted to elucidate potential mechanisms of action of these cells and to define safe and effective clinical strategies.