Neuropathological events in an animal model resembling human fetal post-hemorrhagic hydrocephalus

Introduction: In premature newborns, intraventricular hemorrhages (IVH) probably trigger the disruption of the neurogenic ventricular zone. Most of the cases with severe IVH develop post-hemorrhagic hydrocephalus (PHH). A mouse model with IVH has been developed to research into the common neuropathological events present in PHH and into possible therapies. Methods: In two-day-old mice, the blood serum from littermates was injected into the ganglionic eminence of one hemisphere or both hemispheres. Fourteen days later, a histopathological analysis was carried out. In the case of injection in one hemisphere, the effects were compared with the contralateral non-injected hemisphere. Results: Mice with IVH developed the following neuropathological effects. The ependyma was found denuded and replaced by reactive astrocytes. A reaction of astrocytes over-expressing aquaporin-4 and of NG2 cells was also found developed in the white matter. Alterations in the neurogenesis were also common in the ventricular zone and in the white matter. Conclusions: The animal model of IVH developed shows similar neuropathological events to other forms of congenital hydrocephalus and can be used to research into therapies for PHH.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. PI15/0619 (ISCIII/FEDER) to AJJ

IPSC differentiation into ependymal progenitors to treat ventricular damage during hydrocephalus

Introduction: During both obstructive congenital hydrocephalus and post-hemorrhagic hydrocephalus additional pathological events are intimately associated with their ethiology: a) a detrimental inflammatory response; b) severe damage of the underlying periventricular nervous tissue, including white matter, and c). Therapeutic approaches have been directed to overcome a) and b), however recovery of damaged neuroepithelium/ependyma is, in our present, an important therapeutic gap. Methods: Human and mouse induced pluripotent stem cells (iPSC) have been artificially differented into ependymal progenitors. Intracerebroventricular (ICV) injections of iPCS are performed ex vivo and in vivo in the damaged ventricular wall. Their integration and differentiation has been studied by immunohistochemistry and histopathological analysis. Results: Mice and human ependymal progenitors are able to integrate and differentiate into ependyma in damaged ventricular wall. Stage of ependymal differentiation by the time of the injection defined different degrees of integration. Conclusions: IPSC appear to be a good ependymal progenitor source with no ethical controversy associated.RyC 2014-16980 Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Long-time effects of an experimental therapy with mesenchymal stem cells in congenital hydrocephalus

Introduction: Bone marrow-derived mesenchymal stem cells (BM-MSC) are a potential therapeutic tool due to their ability for migrating and producing neuroprotector factors when they are transplanted in other neurodegenerative diseases. Moreover, some investigations have shown that BM-MSC are able to modulate astrocyte activation and neuroprotector factor production. The aim of this study was to evaluate the long-time effects of a BM-MSC experimental therapy in the hyh mouse model of congenital hydrocephalus. Methods: BM-MSC were characterized in vitro and then transplanted into the ventricles of young hydrocephalic hyh mice, before they develop the severe hydrocephalus. Non-hydrocephalic normal mice (wt) and hydrocephalic hyh mice sham-injected (sterile saline serum) were used as controls. Samples were studied by analyzing and comparing mRNA, protein level expressions and immunoreaction related with the progression and severity of hydrocephalus. Results: Fourteen days after transplantation, hydrocephalic hyh mice with BM-MSC showed lower ventriculomegaly. In these animals, BM-MSC were found undifferentiated and spread into the periventricular astrocyte reaction. There, BM-MSC were detected producing several neuroprotector factors (BDNF, GDNF, NGF, VEGF), in the same way as reactive astrocytes. Total neocortical levels of NGF, TGF-β and VEGF were found increased in hydrocephalic hyh mice transplanted with BM-MSC. Furthermore, astrocytes showed increased expressions of aquaporin-4 (water channel protein) and Slit-2 (neuroprotective and anti-inflammatory molecule). Conclusions: BM-MSC seem to lead to recovery of the severe neurodegenerative conditions associated to congenital hydrocephalus mediated by reactive astrocytes.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. PI15/0619 (ISCIII/FEDER

Brain tissue recovery in obstructive congenital hydrocephalus after intraventricular transplantation of mesenchymal stem cells

Introduction: Bone marrow-derived mesenchymal stem cells (BM-MSC) are a potential therapeutic tool due to their ability for migrating and producing neuroprotector factors when transplanted. The aim of this study was to evaluate the short-time effects of a BM-MSC experimental therapy in the hyh mouse model with severe obstructive hydrocephalus. Methods: BM-MSC were characterized in vitro and then injected into the ventricles of hyh mice. Wild-type and saline-injected hyh mice were used as controls. Samples were studied by analyzing and comparing mRNA, protein and metabolites level expression in control and damaged tissue. Results: Undifferentiated BM-MSC were found to: i) spread into the periventricular astrocyte reaction region after four days post-injection, and, ii) be producing neuroprotector factors (GDNF and VEGF). Astrocytes located in periventricular edematous region increased their aquaporin-4 expression, as well as Slit2 expression (neuroprotective and anti-inflammatory molecule). There was also a significant reduction of osmolytes such as taurine and neuroexcytotoxic glutamate. Halved apoptotic cell death was detected in the periventricular walls. Conclusions: BM-MSC lead to recovery of the severe neurodegenerative conditions associated to congenital hydrocephalus mediated by reactive astrocytes.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Supported by Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech, and PI15/0619 (ISCIII/FEDER)

Bone marrow-derived mesenchymal stem cells characterization and transplantation in an animal model of congenital hydrocephalus

Congenital hydrocephalus is a disorder presenting a degeneration of the periventricular cerebral parenchyma and the white matter, which causes significant mortality and life-long neurological complications. There are currently no effective therapies for congenital hydrocephalus. Bone marrow-derived mesenchymal stem cells (BM-MSC) are considered as a potential therapeutic tool in neurodegenerative diseases, due to their ability to migrate to degenerated tissues and the production of growth factors. In the present study, using an animal model of congenital hydrocephalus, the hyh mouse, it has been studied the capacity of the BM-MSC to reach the degenerated regions exhibiting glial reactions and their probable neuroprotector effects. The BM-MSC were isolated from two different sources: a) transgenic mice expressing the monomeric red fluorescent protein (mRFP1); b) wild type mice. In the second case, the BM-MSC were labelled in vitro using bromodeoxyuridine, a fluorescent cell tracker and the lipophilic DiR. Before application, the cells were analysed using flow cytometry and immunofluorescence. The BM-MSC were injected into the retro-orbital sinus or into the lateral ventricle of hyh mice. After 24/96 hours of administration, the BM-MSC were detected under light, confocal and electron microscopes. The injected BM-MSC reached the degenerated periventricular regions and the disrupted neurogenic niches. They were detected in the periventricular parenchyma, around periventricular blood vessels and in the ventral meninges. Most of the applied BM-MSC expressed the glial cell-derived neurotrophic factor (GDNF), in the same way as the periventricular reactive astrocytes, suggesting a possible neuroprotector effect.FIS (Instituto de Salud Carlos III)-FEDER a AJJ. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Characterization and administration of bone marrow-derived mesenchymal stem cells in an animal model of congenital hydrocephalus

Bone marrow-derived mesenchymal stem cells (BM-MSC) are considered as a potential therapeutic tool in neurodegenerative diseases, due to their ability to migrate to degenerated tissues and the production of growth factors. Congenital hydrocephalus is a disorder characterized by a degeneration of the periventricular cerebral parenchyma and the white matter. In the present study, using an animal model of congenital hydrocephalus, the hyh mouse, it has been studied the capacity of the BM-MSC to reach the degenerated regions exhibiting glial reactions and their probable neuroprotector effects. The BM-MSC were isolated from two sources: a) transgenic mice expressing the monomeric red fluorescent protein (mRFP1); b) wild type mice. In the second case, the BM-MSC were labelled in vitro using bromodeoxyuridine, a fluorescent cell tracker and the lipophilic DiR. Before application, the cells were analysed using flow cytometry and immunofluorescence. The BM-MSC were injected into the retro-orbital sinus or into the lateral ventricle of hyh mice. After 24/96 hours of administration, they were detected under light, confocal and electron microscopes. The injected BM-MSC reached the degenerated periventricular regions and the disrupted neurogenic niches. They were detected in the periventricular parenchyma, around periventricular blood vessels and in the ventral meninges. Most of the applied BM-MSC expressed the glial cell-derived neurotrophic factor (GDNF), in the same way as the periventricular reactive astrocytes, suggesting a possible neuroprotector effect.Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tech. Instituto de Salud Carlos III, PI12/0631 con cofinanciación FEDER

Bone marrow-derived mesenchymal stem cells transplantation produces a tissue recovery in hydrocephalic mice

In congenital hydrocephalus, cerebrospinal fluid accumulation is associated to ischemia/hypoxia, metabolic impairment, neuronal damage and astrocytic reaction, which cause significant mortality and life-long neurological complications. Currently, there are no effective therapies for congenital hydrocephalus. Bone marrow-derived mesenchymal stem cells (BM-MSC) are considered as a potential therapeutic tool for neurodegenerative diseases due to their ability for migrating and producing neuroprotector factors when they are transplanted. The aim of this research was to study the ability of BM-MSC to reach the degenerated regions and to detect their neuroprotector effects, using an animal model of congenital hydrocephalus, the hyh mouse. Fluorescent BM-MSC were analyzed by flow-cytometry and multilineage cell differentiation. BM-MSC were brain-ventricle injected into hyh mice. Wild-type and saline-injected hyh mice were used as controls. Inmunohistochemical, RT-PCR and High Resolution Magic Angle Spinning spectroscopy (HRMAS) analyses were carried out. After administration, integrated BM-MSC were identified inside the periventricular astrocyte reaction. They were detected producing glial-derived neuroprotector factor (GDNF), neural growth factor (NGF), and brain-derived neuroprotector factor (BDNF). Tissue recovery was detected with a reduction of apoptotic cells in the periventricular walls and of the levels of glutamate, glutamine, taurine, and creatine, all of them markers of tissue damage in hydrocephalus.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. ISCIII PI15/00619 y FEDE

A metabolite profile reveals the presence of neurodegenerative conditions according to severity of hydrocephalus

Introduction: In obstructive congenital hydrocephalus, cerebrospinal fluid accumulation is associated with high intracranial pressure (ICP), ischemia/hypoxia, metabolic impairment, neuronal damage and astrocytic reaction. The hyh mutant mice exhibit two different forms of hydrocephalus evolution: severe and moderate. A study was carried out in hyh mice to detect a metabolite profile that define the tissue response in each hydrocephalus form. Methods: Metabolites levels in brain cortex were analyzed with 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance (1H HR-MAS) spectroscopy. The study was complemented with ICP recording and histopathological analysis. Results: Mice with severe hydrocephalus were found to have higher ICP and stronger astrocytic reaction. Several metabolites including glutamate and glutamine were found to correlate with the severity of hydrocephalus. The whole metabolite profile may be explained based in differential astrocyte reactions, neurodegenerative and ischemic conditions. The glutamate transporter EAAT2 and the metabolite taurine were found as key histopathological markers for the damaged parenchyma. Conclusions: Spectroscopy allowed the detection of a metabolite profile related to intracranial pressure and hydrocephalus severity, and therefore can be useful to monitor the efficacy of experimental therapies. Supported by Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech, and PI15/0619 (ISCIII/FEDER).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Supported by Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech, and PI15/0619 (ISCIII/FEDER)

Potential protective role of reactive astrocytes in the periventricular parenchyma in congenital hydrocephalus

Background Cerebrospinal fluid accumulation in hydrocephalus produces an elevation of intraventricular pressure with pathological consequences on the periventricular brain parenchyma including ischemia, oedema, oxidative stress, and accumulation of metabolic waste products. Here we studied in the hyh mouse, an animal model of congenital hydrocephalus, the role of reactive astrocytes in this clinical degenerative condition. Materials and Methods Wild type and hydrocephalic hyh mice at 30 days of postnatal age were used. Three metabolites related to the oxidative and neurotoxic conditions were analysed in ex vivo samples (glutathione, glutamine and taurine) using High Resolution Magic Angle Spinning (HR-MAS). Glutathione synthetase and peroxidase, glutamine synthetase, kidney-type glutaminase (KGA), and taurine/taurine transporter were immunolocated in brain sections. Results Levels of the metabolites were remarkably higher in hydrocephalic conditions. Glutathione peroxidase and synthetase were both detected in the periventricular reactive astrocytes and neurons. Taurine was mostly found free in the periventricular parenchyma and in the reactive astrocytes, and the taurine transporter was mainly present in the neurons located in such regions. Glutamine synthetase was found in reactive astrocytes. Glutaminase was also detected in the reactive astrocytes and in periventricular neurons. These results suggest a possible protective response of reactive astrocytes against oxidative stress and neurotoxic conditions. Conclusions Astrocyte reaction seems to trigger an anti-oxidative and anti-neurotoxic response in order to ameliorate pathological damage in periventricular areas of the hydrocephalic mice.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. PI15-00619 to AJJ

Aplicación de estrategias de aprendizaje para la adquisición de competencias profesionales y su evaluación mediante una rúbrica integradora en diversos títulos de grado de la Universidad de Málaga

El Espacio Europeo de Educación Superior ha supuesto importantes cambios en el proceso de enseñanza-aprendizaje para la adquisición de competencias transversales y específicas. La calidad es un factor estratégico, por lo que es necesario, por una parte, el diseño de estrategias por parte del docente para que el alumnado adquiera competencias profesionales, y por otra, la evaluación de la eficacia e incidencia de las distintas estrategias en el rendimiento académico del alumnado. En este trabajo se han desarrollado rúbricas que permiten evaluar el efecto de nuevas estrategias de aprendizaje para la adquisición de las siguientes competencias: razonamiento crítico y autocrítico, adquisición de conocimientos básicos y capacidad de aplicar los conocimientos adquiridos a casos prácticos, transmisión de información oral/escrita, y capacidad de trabajo en equipo. El estudio se ha desarrollado en ocho asignaturas de la Universidad de Málaga, de grados experimentales (varias ingenierías, biología, bioquímica, fisioterapia y psicología), y una asignatura de una disciplina más abstracta como son las matemáticas. Finalmente, el alumnado ha evaluado el alcance de estas estrategias en la adquisición de las competencias a través de una rúbrica (eRúbrica) común a todas las asignaturas, datos que han sido cotejados con el resultado final del examen.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech