Direct confocal acquisition of fluorescence from X-gal staining on thick tissue sections

X-gal staining is a common procedure used in the histochemical monitoring of gene expression by light microscopy. However, this procedure does not permit the direct confocal acquisition of images, thus preventing the identification of labelled cells on the depth (Z) axis of tissue sections and leading sometimes to erroneous conclusions in co-localization and gene expression studies. Here we report a technique, based on X-gal fluorescence emission and mathematically-based optical correction, to obtain high quality fluorescence confocal images. This method, combined with immunofluorescence, makes it possible to unequivocally identify X-gal-labelled cells in tissue sections, emerging as a valuable tool in gene expression and cell tracing analysis

Carotid Body Transplants as a Therapy for Parkinson’s Disease

Affecting over 1.5 million people across the world, Parkinson's disease is a progressive neurological condition characterized, in part, by the loss of dopaminergic neurons in the substantia nigra pars compacta. It affects 1.5% of the global population over 65 years of age. As life expectancy is increasing, over the next few years the number of patients with Parkinson´s disease will grow exponentially. To date, there are no available treatments that are capable of curing Parkinson´s disease, and the current goal of therapy, dopamine replacement strategies, is to reduce symptoms. After several years of disease progression, treatment is complicated by the onset of motor fluctuations and dyskinesias. This information reveals the great importance and social need of finding an effective therapeutic intervention for Parkinson´s disease. This exemplary new book reviews some of the most outstanding examples of new drugs currently in pharmaceutical development or new targets currently undergoing the validation process to try to reach the Parkinson´s drug market in the next few years as potential disease modifying drugs. Providing up to date and comprehensive coverage, this book will be essential reading for researchers working in academia and industry in any aspect of medicinal chemistry or drug discovery

Selective Glial Cell Line-Derived Neurotrophic Factor Production in Adult Dopaminergic Carotid Body Cells In Situ and after Intrastriatal Transplantation

Glial cell line-derived neurotrophic factor (GDNF) exerts a notable protective effect on dopaminergic neurons in rodent and primate models of Parkinson’s disease (PD). The clinical applicability of this therapy is, however, hampered by the need of a durable and stable GDNF source allowing the safe and continuous delivery of the trophic factor into the brain parenchyma. Intrastriatal carotid body (CB) autografting is a neuroprotective therapy potentially useful in PD. It induces long-term recovery of parkinsonian animals through a trophic effect on nigrostriatal neurons and causes amelioration of symptoms insomePDpatients. Moreover, the adult rodentCBhas been shown to express GDNF. Here we show, using heterozygous GDNF/lacZ knock-out mice, that unexpectedly CB dopaminergic glomus, or type I, cells are the source of CB GDNF. Among the neural or paraneural cells tested, glomus cells are those that synthesize and release the highest amount ofGDNFin the adult rodent (as measured by standard and in situ ELISA). Furthermore,GDNFexpression by glomus cells is maintained after intrastriatal grafting and in CB of aged and parkinsonian 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated animals. Thus, glomus cells appear to be prototypical abundant sources of GDNF, ideally suited to be used as biological pumps for the endogenous delivery of trophic factors in PD and other neurodegenerative diseases

Combined treatment of graft versus host disease using donor regulatory T cells and ruxolitinib

Donor derived regulatory T lymphocytes and the JAK1/2 kinase inhibitor ruxolitinib are currently being evaluated as therapeutic options in the treatment of chronic graft versus host disease (cGvHD). In this work, we aimed to determine if the combined use of both agents can exert a synergistic efect in the treatment of GvHD. For this purpose, we studied the efect of this combination both in vitro and in a GvHD mouse model. Our results show that ruxolitinib favors the ratio of thymic regulatory T cells to conventional T cells in culture, without afecting the suppressive capacity of these Treg. The combination of ruxolitinib with Treg showed a higher efcacy as compared to each single treatment alone in our GvHD mouse model in terms of GvHD incidence, severity and survival without hampering graft versus leukemia efect. This benefcial efect correlated with the detection in the bone marrow of recipient mice of the infused donor allogeneic Treg after the adoptive transfer

Comparative analysis for the presence of IgG anti-aquaporin-1 in patients with NMO-Spectrum disorders

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected

GDNF y cuerpo carotídeo: implicaciones funcionales y uso en terapia celular

El proyecto que se desarrolla en esta Tesis Doctoral se ha centrado en el estudio de los mecanismos celulares y moleculares por los que el trasplante de cuerpo carotídeo (CC) produce recuperación en modelos experimentales de la enfermedad de Parkinson (EP), con especial interés en la expresión del factor neurotrófico GDNF (del inglés “glial cell line derived neurotrophic factor”; Lin y col., 1993) en dicho órgano. Este trabajo surge a partir de estudios previos realizados en el grupo de investigación, en los que se observó que el implante intraestrial de agregados celulares de CC produce una importante recuperación histológica y funcional en modelos animales de parkinsonismo (Espejo y col., 1998; Luquin y col., 1999; Toledo-Aral y col., 2003). A partir de estos resultados se ha propuesto el uso del tejido carotídeo en terapia celular en la EP (Arjona y col., 2003; Mínguez-Castellanos y col., 2007), así como en otras alteraciones del sistema nervioso central (Yu y col., 2005). El objetivo general de esta Tesis Doctoral fue profundizar en el conocimiento de la expresión de GDNF en el cuerpo carotídeo, así como en las consecuencias que dicha expresión pueda tener en el uso de este órgano en terapia celular. A continuación se detallas los principales objetivos específicos. 1. Estudiar la selectividad de la expresión de GDNF en el cuerpo carotídeo, en comparación con otros tejidos catecolaminérgicos usados en trasplantes realizados en enfermos de Parkinson. Determinar dentro del cuerpo carotídeo el tipo celular que expresa el GDNF. 2. Analizar la capacidad de las células del cuerpo carotideo para secretar GDNF y compararla con otros tipos celulares dopaminérgicos. 3. Examinar si la alta expresión de GDNG observada en el cuerpo carotídeo, se mantiene una vez trasplantado al estriado. Comprobar si el implante intraestriatal de cuerpo carotídeo induce la expresión de GDNF en el parénquima cerebral o en macrófagos activados. 4. Evaluar en el modelo experimental de ratón, si la capacidad del cuerpo carotídeo para producir GDNF se afecta por el envejecimiento o por el parkinsonismo inducido por MPTP. 5. Estudiar si el factor neurotrófico GDNF juega algún papel en el desarrollo embrionario del cuerpo carotídeo. 6. Comprobar cómo afecta la hipoxia crónica a los niveles de expresión de GDNF en el cuerpo carotídeo, tanto en individuos jóvenes como de edad avanzada. Analizar la participación del GDNF en la expansión del cuerpo carotídeo en hipoxia crónica

Long-term immunosuppression for CNS mouse xenotransplantation: Effects on nigrostriatal neurodegeneration and neuroprotective carotid body cell therapy

Abstract Background: The use of long-term immunosuppressive treatments on neural trans plantation has been controversial during the last decades. Although nowadays there is a consensus about the necessity of maintaining a permanent state of immunosup pression to preserve the survival of cerebral grafts, little is known about the effects that chronic immunosuppression produces both on the neurodegenerative process and on transplants function. Methods: Here, we establish a new immunosuppressive protocol, based on the dis continuous administration of CsA (15 mg/kg; s.c.) and prednisone (20 mg/kg; s.c.), to produce long-term immunosuppression in mice. Using this treatment, we analyse the effects that long-term immunosuppression induces in a chronic 1-methyl-4-phenyl-1, 2,3,6,-tetrahydropyridine (MPTP) model of parkinsonism and on the neuroprotective and neurorestorative anti-parkinsonian actions exerted by rat carotid body (CB) xenografts. Results: This protocol preserves the survival of rat CB xenotransplants maintaining the general wellness of the grafted mice. Although permanent immunosuppression does not prevent the MPTP-induced cell death of nigral neurons and the consequent degeneration of dopaminergic striatal innervation, allowing for its use as Parkinson’s disease (PD) model, it reduces the microglial activation and slightly declines the stri atal damage. Moreover, we reported that chronic administration of immunosuppres sant drugs does not alter the neuroprotective and restorative anti-parkinsonian actions of rat CB xenografts into parkinsonian mice. Conclusions: This new immunosuppressive protocol provides a new murine model to assay the long-term effects of cerebral xenografts and offer a pharmacological alternative to the commonly used genetic immunodeficient mice, allowing the use of genetically modified mice as hosts. In addition, it will permit the experimental analysis of the effects produced by human CB xenografts in the chronic PD murine model, with the final aim of using CB allografts as an option of cell therapy in PD patients

A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise.

The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sym pathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers un dergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic depen dency mediated through GDNF-family receptor-a2 (GFRa2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innerva tion for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-depen dent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innerva tion reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to mod erate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects

Immunization with α-synuclein/Grp94 reshapes peripheral immunity and suppresses microgliosis in a chronic parkinsonism model

Neuroinflammation mediated by chronically activated microglia, largely caused by abnormal accumulation of misfolded α-synuclein (αSyn) protein, is known to contribute to the pathophysiology of Parkinson’s disease (PD). In this work, based on the immunomodulatory activities displayed by particular heat-shock proteins (HSPs), we tested a novel vaccination strategy that used a combination of αSyn and Grp94 (HSPC4 or Gp96) chaperone and a murine PD model. We used two different procedures, first, the adoptive transfer of splenocytes from αSyn/Grp94-immunized mice to recipient animals, and second, direct immunization with αSyn/Grp94, to study the effects in a chronic mouse MPTP-model of parkinsonism. We found that both approaches promoted a distinct profile in the peripheral system -supported by humoral and cellular immunity- consisting of a Th1-shifted αSyn-specific response accompanied by an immune-regulatory/Th2-skewed general phenotype. Remarkably, this mixed profile sustained by αSyn/Grp94 immunization unexpectedly led to strong suppression of microglial activation in the substantia nigra and striatum, pointing to a newly described beneficial effect of anti-αSyn Th1-responses in the context of PD. This strategy is the first to target αSyn and report the suppression of PD-associated microgliosis. Overall, we show that the αSyn/Grp94 combination supports a distinct and long-lasting immune profile in the peripheral system, which has an impact at the CNS level by suppressing chronic microglial activation in an MPTP model of PD. Furthermore, our study demonstrates that reshaping peripheral immunity by vaccination with appropriate misfolding protein/HSP combinations could be highly beneficial as a treatment for neurodegenerative misfolding diseases