Specific Biomarkers in Spinocerebellar Ataxia Type 3: A Systematic Review of Their Potential Uses in Disease Staging and Treatment Assessment.

Spinocerebellar ataxia type 3 (SCA3) is the most common type of disease related to poly-glutamine (polyQ) repeats. Its hallmark pathology is related to the abnormal accumulation of ataxin 3 with a longer polyQ tract (polyQ-ATXN3). However, there are other mechanisms related to SCA3 progression that require identifying trait and state biomarkers for a more accurate diagnosis and prognosis. Moreover, the identification of potential pharmacodynamic targets and assessment of therapeutic efficacy necessitates valid biomarker profiles. The aim of this review was to identify potential trait and state biomarkers and their potential value in clinical trials. Our results show that, in SCA3, there are different fluid biomarkers involved in neurodegeneration, oxidative stress, metabolism, miRNA and novel genes. However, neurofilament light chain NfL and polyQ-ATXN3 stand out as the most prevalent in body fluids and SCA3 stages. A heterogeneity analysis of NfL revealed that it may be a valuable state biomarker, particularly when measured in plasma. Nonetheless, since it could be a more beneficial approach to tracking SCA3 progression and clinical trial efficacy, it is more convenient to perform a biomarker profile evaluation than to rely on only one.

Neurofilament light chain as a biomarker for acute hepatic porphyrias.

Background: Acute hepatic porphyrias (AHP) represent a rare group of inherited metabolic disorders of heme biosynthesis pathway. This study aims to determine the diagnostic and prognostic value of serum neurofilament light chain (NfL) as potential biomarker for AHP. Methods: We conducted a cross-sectional observational study to evaluate NfL levels in patients with AHP. They were divided in different groups: normal health individuals; patients with definitive diagnosis of AHP during acute episodes; patients with AHP and infrequent attacks; patients with AHP and recurrent attacks; asymptomatic individuals with positive genetic testing and urinary delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) levels elevated 4 or more times ("high excretors"); asymptomatic individuals with exclusive positive genetic test; control group with Hereditary Amyloidosis related to Transthyretin with Polyneuropathy (ATTRv-PN). Results: During acute attacks, serum NfL levels were 68 times higher compared to normal controls and disclosed a strong correlation with ALA and PBG levels; also exhibited elevated levels in patients with chronic symptoms regardless of the number of disease attacks compared to healthy controls, and at similar levels to patients with ATTRv-PN, which is a model of progressive neuropathy. Conclusion: This study represents the first to establish NfL as a biomarker for AHP, disclosing NfL as a sensitive biomarker for axonal damage and chronic symptom occurrence. This study not only underscores that neurological damage associated with the disease in any patient, irrespective of the number of attacks, but also reinforces the progressive and profoundly debilitating nature of acute and chronic symptoms observed in individuals with AHP.

Systematic review and evidence gap mapping of biomarkers associated with neurological manifestations in patients with COVID-19.

OBJECTIVE: This study aimed to synthesize the existing evidence on biomarkers related to coronavirus disease 2019 (COVID-19) patients who presented neurological events. METHODS: A systematic review of observational studies (any design) following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines and the Cochrane Collaboration recommendations was performed (PROSPERO: CRD42021266995). Searches were conducted in PubMed and Scopus (updated April 2023). The methodological quality of nonrandomized studies was assessed using the Newcastle‒Ottawa Scale (NOS). An evidence gap map was built considering the reported biomarkers and NOS results. RESULTS: Nine specific markers of glial activation and neuronal injury were mapped from 35 studies published between 2020 and 2023. A total of 2,237 adult patients were evaluated in the included studies, especially during the acute phase of COVID-19. Neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) biomarkers were the most frequently assessed (n = 27 studies, 77%, and n = 14 studies, 40%, respectively). Although these biomarkers were found to be correlated with disease severity and worse outcomes in the acute phase in several studies (p < 0.05), they were not necessarily associated with neurological events. Overall, 12 studies (34%) were judged as having low methodological quality, 9 (26%) had moderate quality, and 9 (26%) had high quality. CONCLUSIONS: Different neurological biomarkers in neurosymptomatic COVID-19 patients were identified in observational studies. Although the evidence is still scarce and conflicting for some biomarkers, well-designed longitudinal studies should further explore the pathophysiological role of NfL, GFAP, and tau protein and their potential use for COVID-19 diagnosis and management.

High Levels of NfL, GFAP, TAU, and UCH-L1 as Potential Predictor Biomarkers of Severity and Lethality in Acute COVID-19.

Few studies showed that neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), total tubulin-associated unit (TAU), and ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) may be related to neurological manifestations and severity during and after SARS-CoV-2 infection. The objective of this work was to investigate the relationship among nervous system biomarkers (NfL, TAU, GFAP, and UCH-L1), biochemical parameters, and viral loads with heterogeneous outcomes in a cohort of severe COVID-19 patients admitted in Intensive Care Unit (ICU) of a university hospital. For that, 108 subjects were recruited within the first 5 days at ICU. In parallel, 16 mild COVID-19 patients were enrolled. Severe COVID-19 group was divided between "deceased" and "survivor." All subjects were positive for SARS-CoV-2 detection. NfL, total TAU, GFAP, and UCH-L1 quantification in plasma was performed using SIMOA SR-X platform. Of 108 severe patients, 36 (33.33%) presented neurological manifestation and 41 (37.96%) died. All four biomarkers - GFAP, NfL, TAU, and UCH-L1 - were significantly higher among deceased patients in comparison to survivors (p < 0.05). Analyzing biochemical biomarkers, higher Peak Serum Ferritin, D-Dimer Peak, Gamma-glutamyltransferase, and C-Reactive Protein levels were related to death (p < 0.0001). In multivariate analysis, GFAP, NfL, TAU, UCH-L1, and Peak Serum Ferritin levels were correlated to death. Regarding SARS-CoV-2 viral load, no statistical difference was observed for any group. Thus, Ferritin, NFL, GFAP, TAU, and UCH-L1 are early biomarkers of severity and lethality of SARS-COV-2 infection and may be important tools for therapeutic decision-making in the acute phase of disease.

Immunohistochemical characterization of stem cell, vascular, neural, and differentiation markers in the apical papilla and dental pulp of human teeth at various stages of root development.

This study aimed to evaluate the expression of several differentiation markers in the apical papilla (AP) and dental pulp (DP) of human permanent teeth. Twenty young human teeth were extracted and classified according to three Moorrees tooth development stages: initial root formation (Ri), root length ½ (R1/2), and root length complete (Rc). Immunohistochemical assays were performed using STRO-1, VEGF Receptor-2, Neurofilament heavy (NFH), and Nestin antibodies and analyzed under light microscopy. Decalcified, formalin fixed paraffin embedded tooth sections stained with hematoxylin and eosin showed an apical cell rich zone between the DP and AP. The AP revealed fewer vascular and cellular components than the DP. STRO-1 was expressed on vascular and neuronal elements beneath the odontoblast (OB) and in the sub-odontoblastic (SOB) zone, and VEGFR-2 positive cells were observed in the endothelium, arterioles, and blood vessels. Neuroepithelial stem cell protein (Nestin) was highly expressed in differentiated odontoblasts in the predentin odontotoblast and odontoblast cell processes. Neurofilament heavy (NFH) was expressed in mature axons throughout the DP. STRO-1 and VEGFR-2 microvascular expression was higher at the stages Ri and R1/2 while STRO-1 and NFH expression showed strong spatial distribution of Rc neuronal elements as compared to Ri and R1/2. Differentiated OB and SOB cells showed Nestin expression, indicating a reservoir of newly differentiated odontoblast-like cells.

Following the Clues: Usefulness of Biomarkers of Neuroinflammation and Neurodegeneration in the Investigation of HTLV-1-Associated Myelopathy Progression.

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease due to axonal damage of the corticospinal secondary to an inflammatory response against infected T-cells. In the present work, we aimed to evaluate biomarkers of neurodegeneration and neuroinflammation in the definition of HAM/TSP prognosis. Neurofilament light (NfL) and phosphorylated heavy (pNfH) chains, total Tau protein, cellular prion protein (PrPc), inflammatory chemokines, and neopterin were quantified in paired cerebrospinal fluid (CSF) and serum samples from HAM/TSP patients (n=21), HTLV-1 asymptomatic carriers (AC) (n=13), and HTLV-1 seronegative individuals with non-inflammatory non-degenerative neurological disease (normal-pressure hydrocephalus) (n=9) as a control group. HTLV-1 proviral load in peripheral blood mononuclear cells and the expression of chemokine receptors CCR4, CCR5, and CXCR3 in infected CD4+ T-cells (HTLV-1 Tax+ cells) were also assessed. CSF levels of Tau, NfL, and pNfH were similar between groups, but PrPc and neopterin were elevated in HAM/TSP patients. Most individuals in the control group and all HTLV-1 AC had CSF/serum neopterin ratio < 1.0, and two-thirds of HAM/TSP patients had ratio values > 1.0, which positively correlated with the speed of disease progression and pNfH levels, indicating active neuroinflammation. HAM/TSP patients showed high serum levels of CXCR3-binding chemokines (CXCL9, CXCL10, and CXCL11) and elevated CSF levels of CCL2, CCL3, CCL4, CCL17, CXCL5, CXCL10, and CXCL11. Indeed, CXCL10 concentration in CSF of HAM/TSP patients was 5.8-fold and 8.7-fold higher in than in HTLV-1 AC and controls, respectively, and correlated with CSF cell counts. HAM/TSP patients with typical/rapid disease progression had CSF/serum CXCL10 ratio > 1.0 and a higher frequency of CXCR3+Tax+CD4+ T-cells in blood, which indicated a positive gradient for the migration of infected cells and infiltration into the central nervous system. In conclusion, the slow progression of HAM/TSP abrogates the usefulness of biomarkers of neuronal injury for the disease prognosis. Thus, markers of inflammation provide stronger evidence for HAM/TSP progression, particularly the CSF/serum neopterin ratio, which may contribute to overcome differences between laboratory assays.

Decreased Neurofilament L Chain Levels in Cerebrospinal Fluid and Tolerogenic Plasmacytoid Dendritic Cells in Natalizumab-Treated Multiple Sclerosis Patients - Brief Research Report.

BACKGROUND: Neurofilament Light (NfL) chain levels in both cerebrospinal fluid (CSF) and serum have been correlated with the reduction of axonal damage in multiple sclerosis (MS) patients treated with Natalizumab (NTZ). However, little is known about the function of plasmacytoid cells in NTZ-treated MS patients. OBJECTIVE: To evaluate CSF NfL, serum levels of soluble-HLA-G (sHLA-G), and eventual tolerogenic behavior of plasmacytoid dendritic cells (pDCs) in MS patients during NTZ treatment. METHODS: CSF NfL and serum sHLA-G levels were measured using an ELISA assay, while pDCs (BDCA-2+) were accessed through flow cytometry analyses. RESULTS: CSF levels of NfL were significantly reduced during NTZ treatment, while the serum levels of sHLA-G were increased. Moreover, NTZ treatment enhanced tolerogenic (HLA-G+, CD274+, and HLA-DR+) molecules and migratory (CCR7+) functions of pDCs in the peripheral blood. CONCLUSION: These findings suggest that NTZ stimulates the production of molecules with immunoregulatory function such as HLA-G and CD274 programmed death-ligand 1 (PD-L1) which may contribute to the reduction of axonal damage represented by the decrease of NfL levels in patients with MS.

Brain Proteomic Analysis on the Effects of the Antidepressant Fluoxetine.

Psychiatric disorders such as major depression are linked to early mortality, and patients affected by these conditions are at an increased risk of developing other diseases that are characteristic of the old and very old. Antidepressants are prescribed in the treatment of depression, although the mechanism of how they exert their therapeutic effects is only partly understood. To shed further light on their mode of action, this chapter presents a protocol using two-dimensional differential in-gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for identifying a proteomic signature in guinea pig brains after treatment with the antidepressant, fluoxetine. As this signature pointed toward changes in synaptic structure, we also present a protocol for Western blot analysis targeting selected proteins identified by the combined 2D-DIGE-MALDI-TOF mass spectrometry procedure. Such validation experiments are critical for the translation of putative biomarkers into preclinical and clinical studies.

Relationship of Neurofilament Light (NfL) and Cognitive Performance in a Sample of Mexican Americans with Normal Cognition, Mild Cognitive Impairment and Dementia.

INTRODUCTION: This study characterized the relationship between plasma NfL and cognition in a community-based sample of older Mexican Americans. METHODS: 544 participants completed a battery of neuropsychological tests and were diagnosed using clinical criteria. NfL was assayed using Simoa. NfL levels across groups and tests were analyzed. RESULTS: Difference in NfL was found between normal and impaired groups and was related to global cognition, processing speed, executive functions and a list of learning tasks with a significant negative effect for all diagnostic groups. NfL had a negative impact on processing speed, attention, executive functions and delayed and recognition memory for both normal and MCI groups. CONCLUSION: The research supports plasma NfL as a marker of cognitive impairment related to neurodegenerative processes in Mexican Americans and may be a marker of early changes in cognition in those with normal cognition and at risk for developing MCI.

A Pilot Cross-Sectional Study to Investigate the Biomarker Potential of Phosphorylated Neurofilament-H and Immune Mediators of Disability in Patients With 5 Year Relapsing-Remitting Multiple Sclerosis.

Objective: To test the feasibility of conducting a full-scale project evaluating the potential value of the phosphorylated neurofilament H (pNF-H) and several cytokines as disability markers in relapsing-remitting multiple sclerosis (RRMS). Methods: Twenty-four patients with 5-year RRMS evolution and eleven healthy control subjects entered the study. None of the participants had an inflammatory systemic or metabolic disease. Disability progression was evaluated using the Expanded Disability Status Scale. Serum level of pNF-H, the anti-inflammatory cytokine transforming growth factor-ß 1 (TGF-ß1), and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-17A (IL-17A), monocyte chemotactic protein-1 (MCP-1), and soluble intercellular cell-adhesion molecule 1 (sICAM-1) were quantified using enzyme-linked immunosorbent assay (ELISA). Results: The patients had higher serum level of TGF-ß1, IL-6, sICAM-1, and pNF-H. Based on these findings, a sample of at least 49 controls and 89 recent-onset RRMS patients is required to find an at least 1-point between-group difference in pNF-H with a power of 80% and an α error = 0.05. The progression of the disease was correlated with the level of pNF-H (Spearman rho = 0.624, p = 0.006), but not with the cytokines'. Conclusions: The serum level of pNF-H, EDSS score-correlated, might stand for a potential biomarker of disability in RRMS reflecting progressive axonal damage and cumulative neurological deterioration. The novelty of these results warrants conducting a larger confirmatory trial.

Serum Neurofilament Light, Glial Fibrillary Acidic Protein and Tau Are Possible Serum Biomarkers for Activity of Brain Metastases and Gliomas.

BACKGROUND: Primary central nervous system (CNS) tumors and brain metastases (BMs) are major causes of morbidity and mortality, accompanied by low survival rates. Efforts to early discovery of CNS malignancies are critical. However, to date, there are no biomarkers approved for detection of cancer activity in the brain. Blood levels of neurofilament light (NfL) and tau, as well as glial fibrillary acidic protein (GFAp), show promise as biomarkers for brain injury in previous studies. Therefore, we performed a cross-sectional study to investigate correlations of those biomarkers with CNS activity of gliomas and BMs. METHODS: Serum samples of 36 participants of a single centered institution were tested for NfL, GFAp and tau with Simoa immunoassay, and correlated with clinical and radiological data. RESULTS: NfL and GFAp levels were significantly associated with the state of intracranial disease (analysis of variance (ANOVA), PsNfL = 0.03; ANOVA, PGFAp = 0.03). Although statistically significant (P = 0.04), differences in concentrations were not clinically meaningful for tau levels. Serum NfL (sNfL) and GFAp concentrations were higher in the group of patients with CNS tumors with disease in progression versus CNS with stable disease (P = 0.03 and P = 0.01, respectively). In addition, sNfL were higher in patients with metastatic solid tumors with known BMs than in those with metastatic tumors with no BM (P = 0.0004). CONCLUSION: sNfL and GFAp both apparently vary closely with presence and activity of gliomas and BMs. Further studies in larger populations are needed to expand these findings.

Neurofilament light chain in the assessment of patients with multiple sclerosis / Neurofilamento de cadeia leve na avaliação de pacientes com esclerose múltipla

ABSTRACT Multiple sclerosis (MS) is an autoimmune, inflammatory, and degenerative disease of the central nervous system. Axonal degeneration is triggered by inflammation and is the pathological substrate of progressive disability in patients with MS. Therapeutic interventions can reduce inflammatory activity, thus delaying neurodegeneration and the progression of disability. Disease activity and neurodegeneration are assessed mainly through clinical evaluation and magnetic resonance imaging. These measures lack sensitivity and accuracy, so new biomarkers are necessary. Several markers have been studied and to date the most promising is neurofilament light (NfL), a component of the axonal cytoskeleton, which is released into cerebrospinal fluid (CSF) following axonal damage. In the present study, we review the current knowledge about CSF NfL determination in MS, clinically isolated syndrome, and radiologically isolated syndrome, and critically discuss how CSF NfL measurement may contribute to therapeutic decision-making in these patients.

RESUMO A esclerose múltipla (EM) é uma doença autoimune, inflamatória e degenerativa do sistema nervoso central. A degeneração axonal é deflagrada pelo processo inflamatório e é o substrato patológico da incapacidade na EM. As intervenções terapêuticas reduzem a inflamação retardando a neurodegeneração e a progressão da incapacidade. A neurodegeneração é avaliada pelo quadro clínico e pela ressonância magnética. Estas mensurações não suficientemente acuradas, havendo necessidade de novos biomarcadores. Diversos biomarcadores têm sido estudados e, até o presente, o mais promissor é o neurofilamento de cadeia leve (NfL). O mesmo é um componente do citoesqueleto que é liberado no líquido cefalorraquidiano após injúria axonal. No presente estudo nós revisamos o conhecimento atual acerca do NfL na EM, síndrome clinica isolada e síndrome radiológica isolada, discutindo criticamente como a determinação deste biomarcador pode contribuir na tomada de decisões clínicas.

Phospholipase D1 downregulation by α-synuclein: Implications for neurodegeneration in Parkinson's disease.

We have previously shown that phospholipase D (PLD) pathways have a role in neuronal degeneration; in particular, we found that PLD activation is associated with synaptic injury induced by oxidative stress. In the present study, we investigated the effect of α-synuclein (α-syn) overexpression on PLD signaling. Wild Type (WT) α-syn was found to trigger the inhibition of PLD1 expression as well as a decrease in ERK1/2 phosphorylation and expression levels. Moreover, ERK1/2 subcellular localization was shown to be modulated by WT α-syn in a PLD1-dependent manner. Indeed, PLD1 inhibition was found to alter the neurofilament network and F-actin distribution regardless of the presence of WT α-syn. In line with this, neuroblastoma cells expressing WT α-syn exhibited a degenerative-like phenotype characterized by a marked reduction in neurofilament light subunit (NFL) expression and the rearrangement of the F-actin organization, compared with either the untransfected or the empty vector-transfected cells. The gain of function of PLD1 through the overexpression of its active form had the effect of restoring NFL expression in WT α-syn neurons. Taken together, our findings reveal an unforeseen role for α-syn in PLD regulation: PLD1 downregulation may constitute an early mechanism in the initial stages of WT α-syn-triggered neurodegeneration.

The cerebrospinal fluid in multiple sclerosis: far beyond the bands / O líquido cefalorraquidiano na esclerose múltipla: muito além das bandas

ABSTRACT The cerebrospinal fluid analysis has been employed for supporting multiple sclerosis diagnosis and ruling out the differential diagnoses. The most classical findings reflect the inflammatory nature of the disease, including mild pleocytosis, mild protein increase, intrathecal synthesis of immunoglobulin G, and, most typically, the presence of oligoclonal bands. In recent years, new biomarkers have emerged in the context of multiple sclerosis. The search for new biomarkers reflect the need of a better evaluation of disease activity, disease progression, and treatment efficiency. A more refined evaluation of disease and therapy status can contribute to better therapeutic choices, particularly in escalation of therapies. This is very relevant taking into account the availability of a greater number of drugs for multiple sclerosis treatment in recent years. In this review, we critically evaluate the current literature regarding the most important cerebrospinal fluid biomarkers in multiple sclerosis. The determination of biomarkers levels, such as chemokine ligand 13, fetuin A, and mainly light neurofilament has shown promising results in the evaluation of this disease, providing information that along with clinical and neuroimaging data may contribute to better therapeutic decisions.

RESUMO A análise do líquido cefalorraquidiano tem sido empregada para avaliação diagnóstica da esclerose múltipla e a exclusão dos diagnósticos diferenciais. Os achados clássicos refletem a natureza inflamatória da doença, incluindo discreta pleocitose, leve hiperproteinorraquia, aumento da síntese intratecal de imunoglobulina G e, mais tipicamente, a presença de bandas oligoclonais. Nos últimos anos, surgiram novos biomarcadores para esclerose múltipla, e esta busca por marcadores reflete a necessidade de melhor avaliar a atividade e a progressão da doença, bem como a eficácia terapêutica. Uma avaliação mais refinada da atividade da doença e da resposta aos medicamentos pode contribuir para melhores decisões terapêuticas, particularmente no que se refere à troca de medicação. Isto é muito importante nos dias de hoje, quando surgem novas opções medicamentosas. Neste artigo de revisão, avaliamos criticamente a literatura atual referente aos novos marcadores liquóricos na esclerose múltipla. A mensuração destes marcadores, como a quimiocina CXCL13, fetuína A e, principalmente, o neurofilamento de cadeia leve, demonstrou resultados promissores na avaliação da doença, provendo informações que, em conjunto com dados clínicos e de neuroimagem, podem contribuir para melhores decisões terapêuticas.

High concentration of trichlorfon (1mM) disrupts axonal cytoskeleton and decreases the expression of plasticity-related proteins in SH-SY5Y cells.

Some organophosphorus compounds (OPs) induce a neurodegenerative disorder known as organophosphate-induced delayed neuropathy (OPIDN), which is related to irreversible inhibition of neuropathy target esterase (NTE) and impairment of neurite outgrowth. The present study addresses the effects of trichlorfon, mipafox (neuropathic model) and paraoxon (non-neuropathic model) on neurite outgrowth and neuroplasticity-related proteins in retinoic-acid-stimulated SH-SY5Y cells, a cellular model widely used to study the neurotoxicity of OPs. Mipafox (20µM) decreased cellular differentiation and the expression of neurofilament 200 (NF-200), growth associated- (GAP-43) and synaptic proteins (synapsin I and synaptophysin); whereas paraoxon (300µM) induced no effect on cellular differentiation, but significant decrease of NF-200, GAP-43, synapsin I and synaptophysin as compared to controls. However, the effects of paraoxon on these proteins were significantly lower than the effects of mipafox. In conclusion, axonal cytoskeletal proteins, as well as axonal plasticity-related proteins are more effectively affected by neuropathic (mipafox) than by non-neuropathic (paraoxon) OPs, suggesting that they might play a role in the mechanism of OPIDN. At high concentration (1mM), trichlorfon induced effects similar to those of the neuropathic OP, mipafox (20µM), but also caused high inhibition of AChE. Therefore, these effects are unlikely to occur in humans at non-lethal doses of trichlorfon.

Quinolinic acid induces disrupts cytoskeletal homeostasis in striatal neurons. Protective role of astrocyte-neuron interaction.

Quinolinic acid (QUIN) is an endogenous metabolite of the kynurenine pathway involved in several neurological disorders. Among the several mechanisms involved in QUIN-mediated toxicity, disruption of the cytoskeleton has been demonstrated in striatally injected rats and in striatal slices. The present work searched for the actions of QUIN in primary striatal neurons. Neurons exposed to 10 µM QUIN presented hyperphosphorylated neurofilament (NF) subunits (NFL, NFM, and NFH). Hyperphosphorylation was abrogated in the presence of protein kinase A and protein kinase C inhibitors H89 (20 µM) and staurosporine (10 nM), respectively, as well as by specific antagonists to N-methyl-D-aspartate (50 µM DL-AP5) and metabotropic glutamate receptor 1 (100 µM MPEP). Also, intra- and extracellular Ca(2+) chelators (10 µM BAPTA-AM and 1 mM EGTA, respectively) and Ca(2+) influx through L-type voltage-dependent Ca(2+) channel (10 µM verapamil) are implicated in QUIN-mediated effects. Cells immunostained for the neuronal markers ßIII-tubulin and microtubule-associated protein 2 showed altered neurite/neuron ratios and neurite outgrowth. NF hyperphosphorylation and morphological alterations were totally prevented by conditioned medium from QUIN-treated astrocytes. Cocultured astrocytes and neurons interacted with one another reciprocally, protecting them against QUIN injury. Cocultured cells preserved their cytoskeletal organization and cell morphology together with unaltered activity of the phosphorylating system associated with the cytoskeleton. This article describes cytoskeletal disruption as one of the most relevant actions of QUIN toxicity in striatal neurons in culture with soluble factors secreted by astrocytes, with neuron-astrocyte interaction playing a role in neuroprotection.

Characterization of a murine neuron-enriched model of primary trigeminal ganglia cultures to study the interferon-ß antiviral effect against Herpes Simplex Virus type 1 / Caracterización de un modelo murino de cultivos primarios de ganglio trigeminal enriquecido en neuronas para el estudio del efecto antiviral del interferón-ß frente al virus Herpes simple tipo 1

Objective: To characterize a neuron-enriched primary TG culture and evaluate interferon- β expression and activity after HSV-1 infection. Materials and methods: The percentage of neurons present in cultures was assessed by neurofilament immunocytochemistry. Cultures were treated with interferon- β and infected with HSV-1, then viral antigen positive cells were counted and interferon- βexpression was assessed by quantitative PCR. Results: The culture contained 15% neurons and 85% non-neuronal cells. A cytopathic effect was observed, associated with high viral spread (72.9% neurons and 48.3% non-neuronal cells were positive for viral antigen). Interferon- β treatment impaired the cytopathic effect and decreased the infected neurons to 16.7% and infected non-neuronal cells to 7.8%. Viral infection at 6 h postinfection significantly increased the interferon- β transcripts by 18.2 fold, while at 18 h postinfection Interferon pre-treatment in infected cultures increased interferon- β transcription by 3.7 fold. Discussion: This culture model contained 15% neurons, which is 10 times higher compared to other reported cultures, and non-neuronal cells comprised 85% of cells in this culture. All types of cells were found to be infected, which is similar to that reported during acute infections in vivo . Additionally, interferon- βdecreased the infected cells, avoiding the cytopathic effect, which is similar to that reported in swine TG cultures. Conclusions: A neuron-enriched primary TG model was characterized. Interferon- β treatment protected cells from cytopathic effects and viral spread, while viral infection up-regulated interferon- β expression. This result means that interferon- β exerts an important antiviral effect against HSV-1 in these cultures.

Objetivo: Caracterizar un cultivo primario de ganglio trigeminal (GT) enriquecido en neuronas y evaluar la expresión de interferón- y su actividad frente a la infección con Herpes simple tipo 1 (HSV-1). Materiales y métodos: El porcentaje de neuronas fue determinado por inmunocitoquímica para neurofilamento. Los cultivos fueron tratados con interferón- β e infectados con HSV-1, y se cuantificaron las células positivas para antígeno viral por inmunocitoquímica y la expresión de interferón- β por PCR cuantitativa. Resultados: El cultivo presentó un 15% de neuronas y 85% de células no neuronales. Se encontró efecto citopático, asociado a una alta diseminación de la infección (72,9% neuronas y 48,3% de células no neuronales positivas para antígeno viral). El interferón- β evitó la aparición de efecto citopático y disminuyó las células infectadas a 16,7% en neuronas y a 7,8% las células no neuronales. La infección viral incrementó la expresión de transcritos de interferón- β 18,2 veces a las 6 h de infección, mientras que a las 18 h post infección el tratamiento con interferón incrementó esta expresión 3,7 veces. Discusión: Los cultivos presentaron un 15% de neuronas, lo cual es 10 veces más que en otros cultivos reportados. Las células no neuronales representan el 85% de las células del cultivo, y se evidenció que todos los tipos de células se infectaron; similar a lo que ha sido reportado durante infecciones agudas in vivo . Adicionalmente, el interferón- β disminuyó el porcentaje de células infectadas y evitó la aparición de efecto citopático, similar a lo que ha sido reportado en cultivos de GT porcino. Conclusiones: Se caracterizó un modelo de cultivo primario de GT enriquecido en neuronas. Interferón- β protegió las células del efecto citopático y la diseminación viral mientras que la infección viral incrementó la expresión de interferón- β. Por lo tanto, el interferón- β ejerció un papel antiviral importante frente al HSV-1 en estos cultivos.

Inmunohistoquímica de la inervación del conducto deferente humano / Immunohistochemistry in the innervations of the human vas deferens

El conducto deferente humano presenta una pared muscular gruesa, donde el componente muscular liso ocupa la parte media y más prominente. Esta composición histológica le permite al órgano desarrollar las potentes contracciones durante el proceso de la eyaculación y emisión del semen. Objetivos: Analizar la presencia y distribución de la positividad inmunohistoquímica a Neurofilamentos (NF) en las paredes del conducto deferente humano. Pacientes, Materiales y Método: De tres pacientes sometidos a orquiectomía radical por diagnóstico de Seminoma, se obtuvieron los conductos deferentes fijados en formol tamponado (pH 7,2). Mediante procedimientos histológicos de rutina, se obtuvieron secciones de 5 um de espesor en portaobjetos silanizados. Se procedió al desarrollo del protocolo de inmunohistoquímica usando anticuerpos específicos contra Neurofilamentos (NF); las imágenes se obtuvieron con cámara fotográfica digital CCD Micrometrics, en microscopio óptico Olympus CX31. Resultados: En los subcompartimientos de las secciones transversales de la pared de los conductos deferentes humanos, se observa la reacción inmunohistoquímica positiva a NF. Sin embargo, los fascículos nerviosos se concentran en la adventicia, mientras que en la mucosa y pared muscular son en extremo escasos y finos. Conclusión: En el conducto deferente existe una inervación preferencial dispuesta en la adventicia del órgano, siendo posible que la potencia de la contracción de la pared en base a la actividad muscular, requiera factores adicionales de estimulación.

The human vas deferens has a thick muscular wall, where the smooth muscle component occupies the middle and most prominent. This composition allows the organ histologic develop powerful contractions during ejaculation process and the semen. Objectives. To analyze the presence and distribution of immunologically positlve for Neurofilament (NF) on the walls of human vas deferens. Patients, Materiall and Methods.' Three patients undergoing radical orchiectomy for seminoma diagnosis were obtained vas deferens fixed in buffered formain (pH 12). By routine histological procedures, sections were obtained 5 um thick on silylated slides. We proceeded to the development of immunohistochemical protocol using specific antibodies against Neurofilament (NF); the digitized images were obtained with CCO Micrometrics digital camera, in the light microscope Olympus CX31. Results: In the subcompartments of the cross sections of the wall of human vas deferens, there is a positive immunohistochemical reaction to NF However, nerve bundles are concentrated in the adventitia, whereas in the mucosa and muscle wall are extremely rare and fine. Conclusion: In the vas deferens there willing preferential innervation in the adventitia of the court, it being possible for the power of contraction of the wall of the body based on muscle activity, stimulation requires additional factors.