Complicaciones de la laringectomía total: experiencia de 7 años en un hospital universitario de Paraguay / Complications of total laryngectomy: a 7-year experience in a university hospital in Paraguay

Introducción: El cáncer de laringe es la neoplasia maligna más común de las vías aerodigestivas superiores. La laringectomía total es el tratamiento de elección en casos avanzados, pero se asocia a una alta tasa de complicaciones. Objetivos: Conocer la prevalencia de las complicaciones posquirúrgicas de la laringectomía total y los factores asociados en pacientes con cáncer de laringe. Materiales y métodos: Estudio observacional, descriptivo, retrospectivo, de corte transversal, revisando el comportamiento de la laringectomía total y sus principales complicaciones en la Cátedra y Servicio de Otorrinolaringología del Hospital de Clínicas de la Facultad de Ciencias Médicas de la Universidad Nacional de Asunción, Paraguay, de 2015 a 2022. Se incluyeron pacientes mayores de 18 años, de ambos sexos, postoperados de laringectomía total, con diagnóstico anatomopatológico de neoplasia de laringe. Se excluyeron pacientes no operados, con fichas incompletas o que abandonaron el tratamiento. Se analizaron variables demográficas, clínicas, quirúrgicas y anatomopatológicas. Resultados: Se incluyeron 10 pacientes, todos varones, con edad media de 56,3 ± 10,2 años. El 90% presentaba hábitos tóxicos. La complicación más frecuente fue la fístula faringocutánea (70%), seguida por infección del sitio quirúrgico (10%) y sangrado posoperatorio (10%). El 71,4% de las fístulas se resolvieron con medidas conservadoras. El 30% tenía afectación supraglótica y el 57,1% de los que presentaron complicaciones recibieron radioterapia previa. Conclusión: Las complicaciones de la laringectomía total son frecuentes, principalmente la fístula faringocutánea. La afectación supraglótica y la radioterapia previa se asociaron a mayor tasa de complicaciones. Se requieren estudios prospectivos con muestras más grandes para confirmar estos hallazgos.

Introduction: Laryngeal cancer is the most common malignant neoplasm of the upper aerodigestive tract. Total laryngectomy is the treatment of choice in advanced cases, but it is associated with a high rate of complications. Objectives: To determine the prevalence of postoperative complications of total laryngectomy and associated factors in patients with laryngeal cancer. Materials and methods: Observational, descriptive, retrospective, cross-sectional study, reviewing the behavior of total laryngectomy and its main complications in the Department of Otorhinolaryngology of the Hospital de Clínicas, Faculty of Medical Sciences, National University of Asunción, Paraguay, from 2015 to 2022. Patients over 18 years of age, of both sexes, who underwent total laryngectomy, with anatomopathological diagnosis of laryngeal neoplasm were included. Non-operated patients, those with incomplete records or who abandoned treatment were excluded. Demographic, clinical, surgical and anatomopathological variables were analyzed. Results: Ten patients were included, all male, with a mean age of 56.3 ± 10.2 years. Ninety percent had toxic habits. The most frequent complication was pharyngocutaneous fistula (70%), followed by surgical site infection (10%) and postoperative bleeding (10%). Conservative measures resolved 71.4% of the fistulas. Thirty percent had supraglottic involvement and 57.1% of those who presented complications received previous radiotherapy. Conclusion: Complications of total laryngectomy are frequent, mainly pharyngocutaneous fistula. Supraglottic involvement and previous radiotherapy were associated with a higher rate of complications. Prospective studies with larger samples are required to confirm these findings.

Infection of Mouse Neural Progenitor Cells by Toxoplasma gondii Reduces Proliferation, Migration, and Neuronal Differentiation in Vitro.

Congenital toxoplasmosis constitutes a major cause of pre- and postnatal complications. Fetal infection with Toxoplasma gondii influences development and can lead to microcephaly, encephalitis, and neurologic abnormalities. Systematic studies concerning the effects of neural progenitor cell infection with T. gondii are unavailable. Cortical intermediate progenitor cells cultivated as neurospheres obtained from E16.5 Swiss Webster mice were infected with T. gondii (ME49 strain) tachyzoites to mimic the developing mouse cerebral cortex in vitro. Infection was associated with decreased cell proliferation, detected by Ki-67 staining at 48 and 72 hours after infection in floating neurospheres, and reduced cellularity at 96 hours. Transient decreases in the expression of the neurogenesis-related transcription factors T-box brain protein 1, mouse atonal homolog protein 1, and hairy and enhancer of split protein 1 were found in infected cultures, while the level of transcription factor SOX-2 remained unaltered. Neurogenic potential, assessed in plated neurospheres, was impaired in infected cultures, as indicated by decreased late neuronal marker neurofilament heavy chain immunoreactivity. Infected cultures exhibited decreased overall migration rates at 48 and 120 hours. These findings indicate that T. gondii infection of neural progenitor cells may lead to reduced neurogenesis due to an imbalance in cell proliferation alongside an altered migratory profile. If translated to the in vivo situation, these data could explain, in part, cortical malformations in congenitally infected individuals.

Mucocele fronto-etmoidal gigante / Giant frontoethmoidal mucocele

Se presenta el caso de un paciente varón de 18 años, con un gran mucocele frontoetmoidal derecho, postoperado en dos oportunidades anteriores, que acudió a nuestro servicio por un empeoramiento de la diplopía. Al examen físico se visualizaba un desplazamiento del globo ocular hacia abajo y afuera. Se le realizó estudios de imágenes, una tomografía computarizada y una resonancia magnética nuclear que sugerían un mucocele frontoetmoidal derecho. Se le realizó una sinusotomía tipo Draf III para drenaje de la lesión, con mejoría de los síntomas.

We present the case of an 18-year-old male patient with a large right frontoethmoidal mucocele, postoperatively on two previous occasions, who came to our department due to worsening diplopia. Physical examination revealed a downward and outward displacement of the eyeball. Imaging studies, computed tomography, and magnetic resonance imaging were performed that suggested a right frontoethmoidal mucocele. A type Draf III sinusotomy was performed to drain the lesion, with improvement of the symptoms.

P2X7 Receptor Triggers Lysosomal Leakage Through Calcium Mobilization in a Mechanism Dependent on Pannexin-1 Hemichannels.

The P2X7 receptor is a critical purinergic receptor in immune cells. Its activation was associated with cathepsin release into macrophage cytosol, suggesting its involvement in lysosomal membrane permeabilization (LMP) and leakage. Nevertheless, the mechanisms by which P2X7 receptor activation induces LMP and leakage are unclear. This study investigated cellular mechanisms associated with endosomal and lysosomal leakage triggered by P2X7 receptor activation. We found that ATP at 500 µM and 5 mM (but not 50 µM) induced LMP in non-stimulated peritoneal macrophages. This effect was not observed in P2X7-deficient or A740003-pretreated macrophages. We found that the P2X7 receptor and pannexin-1 channels mediate calcium influx that might be important for activating specific ion channels (TRPM2 and two-pore channels) on the membranes of late endosomes and lysosomes leading to LMP leakage and consequent cathepsin release. These findings suggest the critical role of the P2X7 receptor in inflammatory and infectious diseases via lysosomal dysfunction.

GD3 synthase deletion alters retinal structure and impairs visual function in mice.

Gangliosides are glycosphingolipids abundantly expressed in the vertebrate nervous system, and are classified into a-, b-, or c-series according to the number of sialic acid residues. The enzyme GD3 synthase converts GM3 (an a-series ganglioside) into GD3, a b-series ganglioside highly expressed in the developing and adult retina. The present study evaluated the visual system of GD3 synthase knockout mice (GD3s-/- ), morphologically and functionally. The absence of b- series gangliosides in the retinas of knockout animals was confirmed by mass spectrometry imaging, which also indicated an accumulation of a-series gangliosides, such as GM3. Retinal ganglion cell (RGC) density was significantly reduced in GD3s-/- mice, with a similar reduction in the number of axons in the optic nerve. Knockout animals also showed a 15% reduction in the number of photoreceptor nuclei, but no difference in the bipolar cells. The area occupied by GFAP-positive glial cells was smaller in GD3s-/- retinas, but the number of microglial cells/macrophages did not change. In addition to the morphological alterations, a 30% reduction in light responsiveness was detected through quantification of pS6-expressing RGC, an indicator of neural activity. Furthermore, electroretinography (ERG) indicated a significant reduction in RGC and photoreceptor electrical activity in GD3s-/- mice, as indicated by scotopic ERG and pattern ERG (PERG) amplitudes. Finally, evaluation of the optomotor response demonstrated that GD3s-/- mice have reduced visual acuity and contrast sensitivity. These results suggest that b-series gangliosides play a critical role in regulating the structure and function of the mouse visual system.

Human mesenchymal stem cell therapy promotes retinal ganglion cell survival and target reconnection after optic nerve crush in adult rats.

BACKGROUND: Optic-nerve injury results in impaired transmission of visual signals to central targets and leads to the death of retinal ganglion cells (RGCs) and irreversible vision loss. Therapies with mesenchymal stem cells (MSCs) from different sources have been used experimentally to increase survival and regeneration of RGCs. METHODS: We investigated the efficacy of human umbilical Wharton's jelly-derived MSCs (hWJ-MSCs) and their extracellular vesicles (EVs) in a rat model of optic nerve crush. RESULTS: hWJ-MSCs had a sustained neuroprotective effect on RGCs for 14, 60, and 120 days after optic nerve crush. The same effect was obtained using serum-deprived hWJ-MSCs, whereas transplantation of EVs obtained from those cells was ineffective. Treatment with hWJ-MSCs also promoted axonal regeneration along the optic nerve and reinnervation of visual targets 120 days after crush. CONCLUSIONS: The observations showed that this treatment with human-derived MSCs promoted sustained neuroprotection and regeneration of RGCs after optic nerve injury. These findings highlight the possibility to use cell therapy to preserve neurons and to promote axon regeneration, using a reliable source of human MSCs.

Paracrine signaling of human mesenchymal stem cell modulates retinal microglia population number and phenotype in vitro.

PURPOSE: Cellular therapy with mesenchymal stem cells (MSC) is emerging as an effective option to treat optic neuropathies. In models of retinal degeneration, MSC injected in the vitreous body protects injured retinal ganglion cells and stimulate their regeneration, however the mechanism is still unknown. Considering the immunomodulating proprieties of MSC and the controversial role of microglial contribution on retinal regeneration, we developed an in vitro co-culture model to analyze the effect of MSC on retinal microglia population. METHODS: We used whole adult rat retinal explants in co-culture with human Wharton's jelly mesenchymal stem cells (hMSC) separated by a transwell membrane and analyzed hMSC effect on both retinal ganglion cells (RGCs) and retinal microglia. RESULTS: hMSC in co-culture protected RGCs after 3 days in vitro by paracrine signaling. In addition, hMSC reduced microglia population and inhibited the pro-inflammatory phenotype of the remaining microglia. CONCLUSIONS: Using a co-culture model, we demonstrated the paracrine effect of hMSC on RGC survival after injury concomitant with a reduction of microglial population. Paracrine signaling of hMSC also changed microglia phenotype and the expression of antiinflammatory factors in the retina. Our results are consistent with a detrimental effect of microglia on RGC survival and regeneration after injury.

Erratum to "Preconditioning of Rat Bone Marrow-Derived Mesenchymal Stromal Cells with Toll-Like Receptor Agonists".

[This corrects the article DOI: 10.1155/2019/7692973.].

Neuroprotection from optic nerve injury and modulation of oxidative metabolism by transplantation of active mitochondria to the retina.

Mitochondrial dysfunctions are linked to a series of neurodegenerative human conditions, including Parkinson's disease, schizophrenia, optic neuropathies, and glaucoma. Recently, a series of studies have pointed mitotherapy - exogenous mitochondria transplant - as a promising way to attenuate the progression of neurologic disorders; however, the neuroprotective and pro-regenerative potentials of isolated mitochondria in vivo have not yet been elucidated. In this present work, we tested the effects of transplants of active (as well-coupled organelles were named), liver-isolated mitochondria on the survival of retinal ganglion cells and axonal outgrowth after optic nerve crush. Our data show that intravitreally transplanted, full active mitochondria incorporate into the retina, improve its oxidative metabolism and electrophysiological activity at 1 day after transplantation. Moreover, mitotherapy increases cell survival in the ganglion cell layer at 14 days, and leads to a higher number of axons extending beyond the injury site at 28 days; effects that are dependent on the organelles' structural integrity. Together, our findings support mitotherapy as a promising approach for future therapeutic interventions upon central nervous system damage.

Effects of a combinatorial treatment with gene and cell therapy on retinal ganglion cell survival and axonal outgrowth after optic nerve injury.

After an injury, axons in the central nervous system do not regenerate over large distances and permanently lose their connections to the brain. Two promising approaches to correct this condition are cell and gene therapies. In the present work, we evaluated the neuroprotective and neuroregenerative potential of pigment epithelium-derived factor (PEDF) gene therapy alone and combined with human mesenchymal stem cell (hMSC) therapy after optic nerve injury by analysis of retinal ganglion cell survival and axonal outgrowth. Overexpression of PEDF by intravitreal delivery of AAV2 vector significantly increased Tuj1-positive cells survival and modulated FGF-2, IL-1ß, Iba-1, and GFAP immunostaining in the ganglion cell layer (GCL) at 4 weeks after optic nerve crush, although it could not promote axonal outgrowth. The combination of AAV2.PEDF and hMSC therapy showed a higher number of Tuj1-positive cells and a pronounced axonal outgrowth than unimodal therapy after optic nerve crush. In summary, our results highlight a synergistic effect of combined gene and cell therapy relevant for future therapeutic interventions regarding optic nerve injury.

Preconditioning of Rat Bone Marrow-Derived Mesenchymal Stromal Cells with Toll-Like Receptor Agonists.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are dynamic cells that can sense the environment, adapting their regulatory functions to different conditions. Accordingly, the therapeutic potential of BM-MSCs can be modulated by preconditioning strategies aimed at modifying their paracrine action. Although rat BM-MSCs (rBM-MSCs) have been widely tested in preclinical research, most preconditioning studies have employed human and mouse BM-MSCs. Herein, we investigated whether rBM-MSCs modify their phenotype and paracrine functions in response to Toll-like receptor (TLR) agonists. The data showed that rBM-MSCs expressed TLR3, TLR4, and MDA5 mRNA and were able to internalize polyinosinic-polycytidylic acid (Poly(I:C)), a TLR3/MDA5 agonist. rBM-MSCs were then stimulated with Poly(I:C) or with lipopolysaccharide (LPS, a TLR4 agonist) for 1 h and were grown under normal culture conditions. LPS or Poly(I:C) stimulation did not affect the viability or the morphology of rBM-MSCs and did not modify the expression pattern of key cell surface markers. Poly(I:C) did not induce statistically significant changes in the release of several inflammatory mediators and VEGF by rBM-MSCs, although it tended to increase IL-6 and MCP-1 secretion, whereas LPS increased the release of IL-6, MCP-1, and VEGF, three factors that were constitutively secreted by unstimulated cells. The neurotrophic activity of the conditioned medium from unstimulated and LPS-preconditioned rBM-MSCs was investigated using dorsal root ganglion explants, showing that soluble factors produced by unstimulated and LPS-preconditioned rBM-MSCs can stimulate neurite outgrowth similarly, in a VEGF-dependent manner. LPS-preconditioned cells, however, were slightly more efficient in increasing the number of regrowing axons in a model of sciatic nerve transection in rats. In conclusion, LPS preconditioning boosted the production of constitutively secreted factors by rBM-MSCs, without changing their mesenchymal identity, an effect that requires further investigation in exploratory preclinical studies.

Nivel de satisfacción sobre la seguridad y complicaciones a cuatro años de la introducción de la técnica Microelectrólisis Percutánea (MEP®) Sport como práctica fisioterapéutica / Vigilância em segurança e complicações quatro anos após a introdução da técnica Microeletrólise Percutânea (MEP®) Sport como prática fisioterapêutica / Surveillance on safety and complications four years after the introduction of Percutaneous Microelectrolisis (MEP®) Sport technique as a physical therapy practice

RESUMEN El objetivo de este trabajo es investigar la experiencia de los fisioterapeutas formados en microeletrólisis percutánea sport y conocer la cantidad de aplicaciones realizadas semanalmente, los efectos adversos presentados y el nivel de satisfacción de los terapeutas con sus pacientes. Se realizó una encuesta que evaluó la opinión y la experiencia de profesionales certificados en microelectrolisis percutánea sport. Los datos fueron obtenidos a través de la plataforma virtual SurveyMonkey, enviando por correo electrónico una invitación a 1.096 fisioterapeutas de América Latina. Respondieron el cuestionario 315 profesionales, entre los cuales 165 (56,51%) atienden de uno a cinco pacientes por semana. Las respuestas sobre efectos adversos fueron: nunca he tenido complicaciones (56,79% - 159 respuestas); choque hipotensivo (19,64% - 55 respuestas.); alergia al metal (5,36% - 15 respuestas). Los sitios/patologías a que más se aplican la microelectrolisis percutánea sport son: tendón rotuliano (10,77% - 198 respuestas.); tendón de aquiles (9,58% - 176 respuestas); tendón supraespino (9,36% - 172 respuestas.); fascitis plantar/espolón calcáneo (8,05% - 148 respuestas.); y puntos-gatillo (7,18% - 132 respuestas.). La satisfacción de los profesionales fue: satisfecho (51,87% - 152 respuestas) y muy satisfecho (40,96% - 120 respuestas). Las respuestas de los pacientes fueron: satisfecho (61,90% - 182 respuestas) y muy satisfecho (29,93% - 88 respuestas). La técnica MEP se aplica principalmente en tendinopatías y produce resultados satisfactorios y muy satisfactorios tanto para los pacientes como para los terapeutas, con baja presencia de efectos adversos.

RESUMO O objetivo do trabalho é pesquisar sobre a experiência dos fisioterapeutas formados em MEP Sport, conhecer a quantidade de aplicações realizadas semanalmente, os efeitos adversos que tenham sido apresentados e o nível de satisfação dos terapeutas com seus pacientes. Realizou-se uma enquete de perguntas mistas que avaliam a opinião e experiência de profissionais certificados em MEP Sport. Os dados foram obtidos por meio da plataforma virtual SurveyMonkey, enviando por correio eletrônico um convite a 1.096 fisioterapeutas da América Latina. Responderam 315 profissionais, destes, 165 (56,51%) atendem de um a cinco pacientes por semana. As respostas sobre efeitos adversos foram: nunca tive complicações (56,79% - 159 respostas); choque hipotensivo (19,64% - 55 respostas.); alergia ao metal (5,36% - 15 respostas). Os locais/patologias em que mais se aplicam MEP são: T. rotuliano (10,77% - 198 respostas.); T. Aquiles (9,58% - 176 respostas.); T. supraespinhoso (9,36% - 172 respostas.); fascite plantar/esporão calcâneo (8,05% - 148 respostas.); e pontos gatilhos (7,18% - 132 respostas.). A satisfação dos profissionais foi: satisfeito (51,87%, 152 respostas.) e muito satisfeito (40,96%, 120 respostas). As respostas dos pacientes foram: satisfeito (61,90%, 182 respostas.) e muito satisfeito (29,93%, 88 respostas). A técnica MEP é aplicada principalmente em tendinopatías e produz resultados satisfatórios e muito satisfatórios tanto para os pacientes quanto para os terapeutas, com baixa presença de efeitos adversos.

ABSTRACT This work aims to recollect information about the experience of physical therapists trained in MEP Sport, to know how many treatments they did per week, the adverse effects that might have appeared and the patients and therapists' satisfaction. A mixed multiple choice survey with the option of choosing one or more alternatives to assess the opinion and experience of physical therapists trained in MEP Sport was carried out. SurveyMonkey was used for data collection. The invitations were sent by email to 1.096 physical therapists of Latin America. The survey was answered by 315 professionals, of whom 165 (56,51%) treat 1 to 5 patients per week. The answers about adverse effects were: I've never had adverse effects: 159 answers (56,79%), Hypotensive shock: 55 answers (19,64%), Allergy to metal 15 answers (5,36%). The most common areas/conditions where the MEP is applied are: Patellar tendon (10,77% - 198 answ.), Achilles tendon, (9,58% - 176 answ.), Supraspinatus tendon (9,36% - 172 answ.), Plantar fasciitis/Calcaneal spurs (8,05% - 148 answ.), Trigger points (7,18% - 132 answ.). The professionals' satisfaction was: Satisfied (51,87%, 152 answ.) and Very Satisfied (40,96%, 120 answ.). Patients' satisfaction was: Satisfied (61,90%, 182 answ.) and Very satisfied (29,93%, 88 answ.). MEP is applied mainly in tendinopathies and produces satisfactory and very satisfactory results, both for patients and professionals, with low incidence of adverse effects.

Long-term neuronal survival, regeneration, and transient target reconnection after optic nerve crush and mesenchymal stem cell transplantation.

BACKGROUND: Retina and/or optic nerve injury may cause irreversible blindness, due to degeneration of retinal ganglion cells. We and others have previously shown that the intravitreal injection of mesenchymal stem cells (MSCs) protects injured retinal ganglion cells and stimulates their regeneration after optic nerve injury, but the long-term effects of this therapy are still unknown. METHODS: We injected rat MSC (rMSC) intravitreally in adult (3-5 months) Lister Hooded rats of either sex after optic nerve crush. Retinal ganglion cell survival, axonal regeneration, and reconnection were analyzed 60 and 240 days after crush by immunohistochemistry for Tuj1, anterograde labeling with cholera-toxin B and by immunohistochemistry for nerve growth factor-induced gene A (NGFI-A, driven by light stimulation) in the superior colliculus after a cycle of light deprivation-stimulation. Visual behaviors (optokinetic reflex, looming response, and preference for dark) were analyzed 70 days after crush. RESULTS: rMSC treatment doubled the number of surviving retinal ganglion cells, preferentially of a larger subtype, and of axons regenerating up to 0.5 mm. Some axons regenerated to the lateral geniculate nucleus and superior colliculus. NGFI-A+ cells were doubled in rMSC-treated animals 60 days after crush, but equivalent to vehicle-injected animals 240 days after crush, suggesting that newly formed synapses degenerated. Animals did not recover visual behaviors. CONCLUSIONS: We conclude that rMSC-induced neuroprotection is sustained at longer time points. Although rMSCs promoted long-term neuroprotection and long-distance axon regeneration, the reconnection of retinal ganglion cells with their targets was transitory, indicating that they need additional stimuli to make stable reconnections.

Bone-marrow mononuclear cell therapy in a mouse model of amyotrophic lateral sclerosis: Functional outcomes from different administration routes.

Amyotrophic lateral sclerosis (ALS) is a chronic degenerative disease that mainly affects motor neurons, leading to progressive paralysis and death. Recently, cell therapy has emerged as a therapeutic alternative for several neurological diseases, including ALS, and bone-marrow cells are one of the major cell sources. Considering the importance of pre-clinical trials to determine the best therapeutic protocol and the hope of translating this protocol to the clinical setting, we tested bone-marrow mononuclear cell (BMMC) therapy administered by different routes in the SOD1G93A model of ALS. BMMCs were isolated from non-transgenic, age matched animals and administered intravenously (IV), intramuscularly (IM), and intravenously and intramuscular concomitantly (IV + IM). BMMC therapy had no significant beneficial effects when injected IV or IM, but delayed disease progression when these two routes were used concomitantly. BMMC IV + IM treatment reduced the number of microglia cells in the spinal cord and partially protected of neuromuscular-junction innervation, but had no effect in preventing motor-neuron loss. This study showed that injection of BMMC IV + IM had better results when compared to each route in isolation, highlighting the importance of targeting multiple anatomical regions in the treatment of ALS.

Nerve Growth Factor Role on Retinal Ganglion Cell Survival and Axon Regrowth: Effects of Ocular Administration in Experimental Model of Optic Nerve Injury.

Retinal ganglion cell (RGC) degeneration occurs within 2 weeks following optic nerve crush (ONC) as a consequence of reduced retro-transport of growth factors including nerve growth factor (NGF). The hypothesis that intravitreal (ivt) and eye drop (ed) administration of recombinant human NGF (rhNGF) might counteract ONC in adult rats is explored in this study. We found that both ivt- and ed-rhNGF reduced RGC loss and stimulated axonal regrowth. Chiefly, survival and regenerative effects of rhNGF were associated with a reduction of cells co-expressing Nogo-A/p75NTR at crush site borders, which contribute to glia scar formation following nerve injury, and induce further degeneration. We also found that ocular application of rhNGF reduced p75NTR and proNGF and enhanced phosphorylation of TrkA and its intracellular signals at retina level. Nogo-R and Rock2 expression was also normalized by ed-rhNGF treatment in both ONC and contralateral retina. Our findings that ocular applied NGF reaches and exerts biological actions on posterior segment of the eye give a further insight into the neurotrophin diffusion/transport through eye structures and/or their trafficking in optic nerve. In addition, the use of a highly purified NGF form in injury condition in which proNGF/p75NTR binding is favored indicates that increased availability of mature NGF restores the balance between TrkA and p75NGF, thus resulting in RGC survival and axonal growth. In conclusion, ocular applied NGF is confirmed as a good experimental paradigm to study mechanisms of neurodegeneration and regeneration, disclose biomarkers, and time windows for efficacy treatment following cell or nerve injury.

Vaccination With Recombinant Filamentous fd Phages Against Parasite Infection Requires TLR9 Expression.

Recombinant filamentous fd bacteriophages (rfd) expressing antigenic peptides were shown to induce cell-mediated immune responses in the absence of added adjuvant, being a promising delivery system for vaccination. Here, we tested the capacity of rfd phages to protect against infection with the human protozoan Trypanosoma cruzi, the etiologic agent of Chagas Disease. For this, C57BL/6 (B6) and Tlr9-/- mice were vaccinated with rfd phages expressing the OVA257-264 peptide or the T. cruzi-immunodominant peptides PA8 and TSKB20 and challenged with either the T. cruzi Y-OVA or Y-strain, respectively. We found that vaccination with rfd phages induces anti-PA8 and anti-TSKB20 IgG production, expansion of Ag-specific IFN-γ, TNF-α, and Granzyme B-producing CD8+ T cells, as well as in vivo Ag-specific cytotoxic responses. Moreover, the fd-TSKB20 vaccine was able to protect against mortality induced by a high-dose inoculum of the parasite. Although vaccination with rfd phages successfully reduced both parasitemia and parasite load in the myocardium of WT B6 mice, Tlr9-/- animals were not protected against infection. Thus, our data extend previous studies, demonstrating that rfd phages induce Ag-specific IgG and CD8+ T cell-mediated responses and confer protection against an important human parasite infection, through a TLR9-dependent mechanism.

CD60b: Enriching Neural Stem/Progenitor Cells from Rat Development into Adulthood.

CD60b antigens are highly expressed during development in the rat nervous system, while in the adult their expression is restricted to a few regions, including the subventricular zone (SVZ) around the lateral ventricles-a neurogenic niche in the adult brain. For this reason, we investigated whether the expression of C60b is associated with neural stem/progenitor cells in the SVZ, from development into adulthood. We performed in vitro and in vivo analyses of CD60b expression at different stages and identified the presence of these antigens in neural stem/progenitor cells. We also observed that CD60b could be used to purify and enrich a population of neurosphere-forming cells from the developing and adult brain. We showed that CD60b antigens (mainly corresponding to ganglioside 9-O-acetyl GD3, a well-known molecule expressed during central nervous system development and mainly associated with neuronal migration) are also present in less mature cells and could be used to identify and isolate neural stem/progenitor cells during development and in the adult brain. A better understanding of molecules associated with neurogenesis may contribute not only to improve the knowledge about the physiology of the mammalian central nervous system, but also to find new treatments for regenerating tissue after disease or brain injury.

Time-Dependent Nerve Growth Factor Signaling Changes in the Rat Retina During Optic Nerve Crush-Induced Degeneration of Retinal Ganglion Cells.

Nerve growth factor (NGF) is suggested to be neuroprotective after nerve injury; however, retinal ganglion cells (RGC) degenerate following optic-nerve crush (ONC), even in the presence of increased levels of endogenous NGF. To further investigate this apparently paradoxical condition, a time-course study was performed to evaluate the effects of unilateral ONC on NGF expression and signaling in the adult retina. Visually evoked potential and immunofluorescence staining were used to assess axonal damage and RGC loss. The levels of NGF, proNGF, p75NTR, TrkA and GFAP and the activation of several intracellular pathways were analyzed at 1, 3, 7 and 14 days after crush (dac) by ELISA/Western Blot and PathScan intracellular signaling array. The progressive RGC loss and nerve impairment featured an early and sustained activation of apoptotic pathways; and GFAP and p75NTR enhancement. In contrast, ONC-induced reduction of TrkA, and increased proNGF were observed only at 7 and 14 dac. We propose that proNGF and p75NTR contribute to exacerbate retinal degeneration by further stimulating apoptosis during the second week after injury, and thus hamper the neuroprotective effect of the endogenous NGF. These findings might aid in identifying effective treatment windows for NGF-based strategies to counteract retinal and/or optic-nerve degeneration.

Intraspinal bone-marrow cell therapy at pre- and symptomatic phases in a mouse model of amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that selectively affects the motor neurons. The details of the mechanisms of selective motor-neuron death remain unknown and no effective therapy has been developed. We investigated the therapy with bone-marrow mononuclear cells (BMMC) in a mouse model of ALS (SOD1(G93A) mice). METHODS: We injected 10(6) BMMC into the lumbar portion of the spinal cord of SOD1(G93A) mice in presymptomatic (9 weeks old) and symptomatic (14 weeks old) phases. In each condition, we analyzed the progression of disease and the lifespan of the animals. RESULTS: We observed a mild transitory delay in the disease progression in the animals injected with BMMC in the presymptomatic phase. However, we observed no increase in the lifespan. When we injected BMMC in the symptomatic phase, we observed no difference in the animals' lifespan or in the disease progression. Immunohistochemistry for NeuN showed a decrease in the number of motor neurons during the course of the disease, and this decrease was not affected by either treatment. Using different strategies to track the BMMC, we noted that few cells remained in the spinal cord after transplantation. This observation could explain why the BMMC therapy had only a transitory effect. CONCLUSION: This is the first report of intraspinal BMMC therapy in a mouse model of ALS. We conclude this cellular therapy has only a mild transitory effect when performed in the presymptomatic phase of the disease.