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Additional file 1: View of the white matter (arrow) exposed in the ventricle of a hyh mouse to be dissected out.

Additional file 5: Percentages respect to total number of cells (labelled with DAPI) of mature oligodendrocytes (Olig2-positive and NG2-negative) relative to OPCs (Olig2-positive and NG2-positive). This has been represented for the white matter of wt (n = 6) and hyh (n = 6) mice. * p

Additional file 3: Overexpressed proteins as indicators of astrocyte reaction detected by UHPLC–HRMS. Abundance ratios (Sample hyh) / (Control wt) for GFAP (Gfap, glial fibrillary acidic protein) and vimentin (vim) (wt, n = 4; hyh, n = 5). The sum PEP score corresponds to the score calculated based on the posterior error probability (PEP) values of the peptide spectrum matches (PSM). The PEP indicates the probability that an observed PSM is a random event. Sum PEP score is calculated as the negative logarithms of the PEP values of the connected PSM.

Additional file 5. : Mass spectrometry analysis of the BM-MSC secretome under TNFα stimulation. Sum PEP Score corresponds to the score calculated on the basis of the posterior error probability (PEP) values of the peptide spectrum matches (PSM). Sum PEP Score indicates the probability that an observed PSM is incorrect. Molecular weights (MW) of the proteins are shown. Number of peptides and percentage of the identified proteins are indicated.

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)

El Trabajo Fin de Grado (TFG), asignatura obligatoria que el estudiante debe realizar autónoma e individualmente, es un trabajo original que será presentado y defendido ante un Tribunal de Evaluación. Su implantación ha manifestado debilidades y carencias (comunicación oral y escrita) del alumnado. Se proponen actividades para mejorar estas capacidades. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Universidad de Málaga (PIE17-150).

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).

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).

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 FEDER

Comunicación oral. El resumen no se publica por deseo de los autores NG2 cells are considered oligodendrocyte precursor cells (OPC). In pathological conditions, NG2 cells contribute to generate oligodendrocytes and reactive astrocytes. This study has been designed to uncover the role of NG2 cells in congenital hydrocephalus using the hyh mouse model. Materials and methods Brain sections and whole mount preparations were obtained from embryos and postnatal hyh and control mice. NG2 positive (NG2+) cells were co-labelled with different IHC markers to deepest identification. Additionally, trying to understand reproducibility of our results in different neurodegenerative conditions, same NG2 identification approach was performed using ventricular walls explants from control mice after mechanical induction of astrocyte reaction. Results A higher number of NG2+ cells were found in the hyh mice compared to the control mice. In addition, NG2+ cells in the hyh mice showed a higher NG2 antigen content compared to the control mice. In the hyh mouse, colocalization results showed that most of NG2+ cells were identified as OPC cells and pericytes, but never as reactive astrocytes o microglial cells. However, in the same approach performed in neurodegenerative conditions, NG2+ cells were mostly identified as reactive astrocytes. Conclusions NG2 progenitors appear to be affected in hyh mutant mice giving rise to a different NG2+population which role in hydrocephalus is still unkown. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Background Bone marrow-derived mesenchymal stem cells (BM-MSC) are considered as a potential therapeutic tool for neurodegenerative diseases due to their ability for migrating into damaged tissue. Additionally, these cells have been proven to produce neuroprotective factors when they are transplanted into damaged tissue. In this research we uncover a neuroprotective role of BM-MSC on congenital hydrocephalus and we study the molecular mechanisms behind it. Materials and methods Fluorescent BM-MSC were analyzed by flow-cytometry and multilineage cell differentiation. They were brain-ventricle injected into hyh hydrocephalic mice. Wild-type and saline-injected hyh mice were used as controls. Inmunohistochemical analyses were performed in fixed brain sections. Inflammatory reaction and neuroprotective factors were studied using quantitative RT-PCR. Metabolites and osmolytes related to brain damage were studied by High Resolution Magic Angle Spinning spectroscopy (HRMAS). RT-PCR and HRMAS were performed in fresh tissue. Results Integrated BM-MSC were identified inside the periventricular astrocyte reaction. IL-1alpha/beta and IL6 levels indicated the absence of inflammatory response in the transplanted tissue after BM-MSC integration. BM-MSC were found producing neuroprotective factors, including GDNF, NGF, BDNF and VEGF. Reduction of osmolytes and neural/glial-function related metabolites levels were detected in hyh mice after BM-MSC injection. These levels mimicked the wild-type situation and indicate partial recovery. Conclusions The BM-MSC can play a neuroprotective effect on congenital hydrocephalus in the hyh mouse by the production of neuroprotective factors and the reduction of osmolytes and metabolites related to tissue damage. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

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.

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.

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 Tech