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OBJECTIVE: N-acetylcysteine (NAC) is an anti-oxidant and mucolytic effective against bacterial biofilms, making it useful in the treatment of chronically discharging ears that are unresponsive to traditional treatment methods. The objective of this study was to evaluate the otologic safety of intratympanic NAC combined with Ciprodex® in an animal model. METHODS: Baseline distortion product otoacoustic emissions (DPOAE) and auditory brainstem response (ABR) measurements were performed for both ears on thirteen guinea pigs from the animal care research facilities of the McGill University Health Center. This was followed by intratympanic administration of control solution (Ciprofloxacin 0.3%/Dexamethasone 0.1%) to the left ear and experimental solution (1.25% NAC/Ciprofloxacin 0.3%/Dexamethasone 0.1%) to the right ear. Three additional intratympanic injections were performed over the next fourteen days. DPOAE and ABR measurements were repeated 3-4 weeks after the initial procedure. Outcome measures included differences in DPOAE and ABR thresholds after intervention, clinical evidence of vestibular dysfunction and histological evidence of ototoxicity. RESULTS: There were no significant differences in the ABR thresholds and DPOAE results of the control and experimental ears at baseline and after intervention. There was neither clinical manifestation of vestibular dysfunction nor histological evidence of ototoxicity. CONCLUSION: Our study suggests that intratympanic 1.25% NAC with ciprofloxacin and dexamethasone is safe in guinea pigs and support its potential use in the treatment of chronically discharging ears. Further studies in humans are required to analyze its efficacy relative to conventional treatments. LEVEL OF EVIDENCE: Animal Research.
Assuntos
Acetilcisteína , Ototoxicidade , Humanos , Animais , Cobaias , Acetilcisteína/farmacologia , Modelos Animais , Ciprofloxacina , DexametasonaRESUMO
The role of acetylcholine (ACh) in visual processing in the mammalian retina has been the focus of research for many decades. Pioneering work on the localization of ACh discovered that the neurotransmitter is synthesized and stored in a distinct subpopulation of amacrine (starburst) cells. It has been shown that ACh release is regulated to a low resting "tonic" level, much like what is observed at the neuromuscular junction (NMJ). If there were a dysfunction in the tonic release of ACh, might post-synaptic changes render the targets of ACh [i.e., retinal ganglion cells (RGCs)] vulnerable to disease? During my time at Pharmacia & Upjohn (PNU), selective nicotinic ACh receptor (nAChR) agonists (e.g., PNU-282987) were developed as a possible therapy for central nervous system (CNS) diseases. As RGCs are the main targets of neurodegeneration in glaucoma, could the activation of this target provide neuroprotection? In response to this question, experiments to identify alpha7 nAChRs in the retina (i.e., target ID studies) followed by "proof-of-concept" experiments were conducted. Target ID studies included binding studies with retinal homogenates, [125I]-alpha-bungarotoxin (α-BTX) autoradiography, and fluorescently tagged α-BTX binding in retinal slices. Imaging studies of intracellular calcium dynamics in the retinal slice were conducted. Reverse transcription-polymerase chain reaction (RT-PCR) analysis with alpha7 nAChR knockout mice using the "laser-capture microdissection" technique, in situ hybridization studies, and RT-PCR analysis of the human retina were conducted. Collectively, these experiments confirmed the presence of alpha7 nAChRs on specific cells in the retina. "Proof-of-concept" neuroprotection studies demonstrated that PNU-282987 provided significant protection for RGCs. This protection was dose dependent and was blocked with selective antagonists. More recently, evidence for the generation of new RGCs has been reported with PNU-282987 in rodents. Interestingly, the appearance of new RGCs is more pronounced with eye-drop application than with intravitreal injection. One could postulate that this reflects the neurogenic activation of alpha7 receptors on the retinal pigment epithelium (RPE) (eye drops) vs. a neuroprotective effect on RGCs (injections). In conclusion, there does appear to be a cholinergic retinal "tone" associated with RGCs that could be utilized as a neuroprotective therapy. However, a distinct cholinergic neurogenic mechanism also appears to exist in the outer retina that could possibly be exploited to generate new RGCs lost through various disease processes.
RESUMO
PNU-282987, a selective alpha7 nicotinic acetylcholine receptor agonist, has previously been shown to have both neurogenic and broad regenerative effects in the adult murine retina. The objective of this study was to assay the molecular mechanism by which PNU-282987 promotes the production of Muller-derived progenitor cells through signaling via the resident retinal pigment epithelium. These Muller-derived progenitor cells generate a myriad of differentiated neurons throughout the retina that have previously been characterized by morphology. Herein, we demonstrate that topical application of PNU-282987 stimulates production of functional neurons as measured by electroretinograms. Further, we examine the mechanism of how this phenomenon occurs through activation of this atypical receptor using a transcriptomic approach isolated retinal pigment epithelium activated by PNU-282987 and in whole retina. We provide evidence that PNU-282987 causes a bi-modal signaling event in which early activation primes the retina with an inflammatory response and developmental signaling cues, followed by an inhibition of gliotic mechanisms and a decrease in the immune response, ending with upregulation of genes associated with specific retinal neuron generation. Taken together, these data provide evidence that PNU-282987 activates the retinal pigment epithelium to signal to Muller glia to produce Muller-derived progenitor cells, which can differentiate into new, functional neurons in adult mice. These data not only increase our understanding of how adult mammalian retinal regeneration can occur, but also provide therapeutic promise for treating functional vision loss.
Assuntos
Neurônios Retinianos , Epitélio Pigmentado da Retina , Animais , Benzamidas , Compostos Bicíclicos com Pontes , Mamíferos/genética , Camundongos , Neurogênese , Neuroglia , Retina , TranscriptomaRESUMO
The adult mammal lacks the ability to regenerate neurons lost to retinal damage or disease in a meaningful capacity. However, previous studies from this laboratory have demonstrated that PNU-282987, an α7 nicotinic acetylcholine receptor agonist, elicits a robust neurogenic response in the adult murine retina. With eye drop application of PNU-282987, Müller glia cells re-enter the cell cycle and produce progenitor-like cells that can differentiate into various types of retinal neurons. In this study, we analyzed the regenerative capability of PNU-282987 in two retinal disease models and identified the source of newly regenerated neurons. Wild-type mice and mice with a transgenic Müller-glia lineage tracer were manipulated to mimic loss of retinal cells associated with glaucoma or photoreceptor degeneration. Following treatment with PNU-282987, the regenerative response of retinal neurons was quantified and characterized. After onset of photoreceptor degeneration, PNU-282987 was able to successfully regenerate both rod and cone photoreceptors. Quantification of this response demonstrated significant regeneration, restoring photoreceptors to near wild-type density. In mice that had glaucoma-like conditions induced, PNU-282987 treatment led to a significant increase in retinal ganglion cells. Retrograde labeling of optic nerve axon fibers demonstrated that newly regenerated axons projected into the optic nerve. Lineage tracing analysis demonstrated that these new neurons were derived from Müller glia. These results demonstrate that PNU-282987 can induce retinal regeneration in adult mice following onset of retinal damage. The ability of PNU-282987 to regenerate retinal neurons in a robust manner offers a new direction for developing novel and potentially transformative treatments to combat neurodegenerative disease.
Assuntos
Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Modelos Animais de Doenças , Regeneração Nervosa/fisiologia , Degeneração Retiniana/tratamento farmacológico , Células Ganglionares da Retina/fisiologia , Neurônios Retinianos/fisiologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Ciclo Celular , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/metabolismo , Glaucoma/fisiopatologia , Pressão Intraocular/fisiologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Neurogênese , Agonistas Nicotínicos/farmacologia , Degeneração Retiniana/metabolismoRESUMO
Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 µl of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.
Assuntos
Técnicas de Cultura/métodos , Glaucoma/etiologia , Modelos Animais , Células Ganglionares da Retina/patologia , Animais , Glaucoma/patologia , Ratos , Células Ganglionares da Retina/efeitos dos fármacos , Cloreto de Sódio/administração & dosagem , Cloreto de Sódio/toxicidadeRESUMO
BACKGROUND: Glaucoma is characterized as a neuropathic disease that causes progressive degeneration of retinal ganglion cells (RGCs) in the retina, resulting in irreversible loss of vision. All conventional treatments for glaucoma are focused on reducing intraocular pressure (IOP) in the anterior chamber of the eye. However, these treatments alone are insufficient to halt the progression of the disease. As a result, neuroprotective strategies have been developed that prevent retinal neuron loss and disease progression. METHODS: The goal of this review is to summarize and discuss neuroprotective strategies in glaucoma at the level of the retina and the ganglion cell layer instead of treatments targeting IOP. Recent and past neuroprotective therapies used to prevent the loss of retinal ganglion cells, the loss of axons in the optic nerve and the loss of vision and function associated with glaucoma are presented. RESULTS: Pharmacological approaches have targeted specific receptors, signaling cascades and neurotrophic factors to induce neuroprotection in the retina, while others have focused on the mechanism of cellular loss associated with glaucoma, including excitotoxicity, oxidative stress and apoptotic processes. In addition to neuroprotective pharmacological treatments, stem cell, gene therapy and viral research have demonstrated neuroprotection against the loss of RGCs in glaucomatous conditions. CONCLUSION: It is likely that future development for glaucoma treatment will include a combination of these treatments to prevent the pathophysiology of glaucoma.
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Glaucoma/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Animais , Glaucoma/patologia , Humanos , Pressão Intraocular/efeitos dos fármacos , Fármacos Neuroprotetores/química , Retina/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologiaRESUMO
The α7nAChR agonist, PNU-282987, has previously been shown to have a neuroprotective effect against loss of retinal ganglion cells (RGCs) in an in vivo glaucoma model when the agent was injected into the vitreous chamber of adult Long Evans rat eyes. Here, we characterized the neuroprotective effect of PNU-282987 at the nerve fiber and retinal ganglion cell layer, determined that neuroprotection occurred when the agonist was applied as eye drops and verified detection of the agonist in the retina, using LC/MS/MS. To induce glaucoma-like conditions in adult Long Evans rats, hypertonic saline was injected into the episcleral veins to induce scar tissue and increase intraocular pressure. Within one month, this procedure produced significant loss of RGCs compared to untreated conditions. RGCs were quantified after immunostaining with an antibody against Thy 1.1 and imaged using a confocal microscope. In dose-response studies, concentrations of PNU-282987 were applied to the animal's right eye two times each day, while the left eye acted as an internal control. Eye drops of PNU-282987 resulted in neuroprotection against RGC loss in a dose-dependent manner using concentrations between 100 µM and 2 mM PNU-282987. LC/MS/MS results demonstrated that PNU-282987 was detected in the retina when applied as eye drops, relatively small amounts of PNU-282987 were measured in blood plasma and no PNU-282987 was detected in cardiac tissue. These results support the hypothesis that eye drop application of PNU-282987 can prevent loss of RGCs associated with glaucoma, which can lead to neuroprotective treatments for diseases that involve α7nAChRs.
Assuntos
Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Glaucoma/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Animais , Benzamidas/farmacocinética , Compostos Bicíclicos com Pontes/farmacocinética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Glaucoma/metabolismo , Glaucoma/patologia , Coração/efeitos dos fármacos , Masculino , Microscopia Confocal , Miocárdio/metabolismo , Fármacos Neuroprotetores/farmacocinética , Soluções Oftálmicas/farmacocinética , Soluções Oftálmicas/farmacologia , Ratos Long-Evans , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Solução Salina Hipertônica , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
OBJECTIVE: The aim was to study the prevalence of otolaryngologic surgeries in pediatric patients with eosinophilic esophagitis (EoE). METHODS: Retrospective cohort study at a tertiary care center. The type of otolaryngologic surgeries performed in patients with diagnosis of EoE was recorded during a 5-year period. RESULTS: Seventy-five percent of patients were male, with average age of EoE diagnosis at 7.5 years with an 83% incidence of atopy. Cohort analysis revealed that 33% (119/362) had a total of 275 otolaryngologic surgeries. Surgeries performed on 119 patients are as follows: 20% bilateral myringotomy with tubes, 14% tonsillectomy, 18.5% adenoidectomy, 1.4% sinus irrigation, 3.3% bronchoscopy, and 1.4% laryngotracheoplasty (LTP); 63% of patients underwent multiple procedures. Thirty percent of patients undergoing bilateral myringotomy with tube placement (BMT) needed additional tubes. Four of 5 LTP patients had successful operations. Twelve percent of patients had EoE diagnosis prior to an otolaryngologic surgery. CONCLUSION: Thirty-three percent of children with EoE required otolaryngologic surgical intervention and nearly one-third who underwent BMT required additional ear tubes. A large fraction of children with EoE will undergo an otolaryngologic surgery, only a minority with a preoperative EoE diagnosis. Until the nature of this relationship is clarified, the high coincidence with otolaryngologic surgeries dictates that otolaryngologists should be familiar with diagnosis of EoE in patients.
Assuntos
Esofagite Eosinofílica/cirurgia , Procedimentos Cirúrgicos Otorrinolaringológicos/estatística & dados numéricos , Criança , Esofagite Eosinofílica/diagnóstico , Esofagite Eosinofílica/epidemiologia , Esofagoscopia , Feminino , Seguimentos , Humanos , Masculino , Prevalência , Estudos Retrospectivos , Wisconsin/epidemiologiaRESUMO
OBJECTIVES: There are many studies that evaluate the role of surgery in the treatment of complications of pediatric acute sinusitis; however there are few studies, if any that report the incidence of surgery following recovery from acute complicated sinusitis. The goal of this study was to report the incidence and indications for surgical intervention after recovery from complications of pediatric acute sinusitis. METHODS: We reviewed the records of all children admitted to a tertiary care children's hospital between January 2005 and September 2010 with a diagnosis of sinusitis and an orbital or intracranial complication. Eighty-six patients met inclusion criteria. Charts were reviewed for type of complication, initial treatment (medical or surgical), type of procedure, secondary procedures, age, and comorbidities. Statistical analysis was completed using independent samples student t-tests and Mann-Whitney tests. RESULTS: A total of 86 patients with a mean age of 6.38 years (2 months to 18 years) were identified. Eighty patients had orbital complications while six presented with intracranial complications. Twenty-seven patients (31%) underwent sinus surgery during the acute phase of their illness whereas 59 patients (69%) were treated medically. After hospitalization and recovery for acute complicated sinusitis, surgery was performed on nine patients (mean age 4.86 years) within 1 month to 2 years post hospitalization. Of the nine patients who required secondary surgery following resolution of the initial complicated sinusitis, four patients were following initial surgical intervention and five patients had initially resolved their complication with medical therapy alone. Indications for subsequent surgery included failure of medical therapy for persistent rhinosinusitis (8 patients) and second complication (1 patient). CONCLUSIONS: This study suggests that following resolution of complicated pediatric rhinosinusitis, very few patients may need further surgical intervention. Subsequent intervention is best guided by clinical judgment, symptoms during outpatient clinic visits, and failure of medical therapy.
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Encefalopatias/complicações , Doenças Orbitárias/complicações , Sinusite/complicações , Sinusite/cirurgia , Doença Aguda , Adolescente , Encefalopatias/cirurgia , Criança , Pré-Escolar , Feminino , Hospitalização , Humanos , Incidência , Lactente , Masculino , Doenças Orbitárias/cirurgia , Estudos Retrospectivos , Resultado do Tratamento , WisconsinRESUMO
PURPOSE: The purpose of this study was to analyze the neuroprotective effect of an α7 nAChR agonist, PNU-282987, using an in vivo model of glaucoma in Long Evans rats. METHODS: One eye in each animal was surgically manipulated to induce glaucoma in control untreated animals and in animals that were treated with intravitreal injections of PNU-282987. To induce glaucoma-like conditions, 0.05 mL of 2 M NaCl was injected into the episcleral veins of right eyes in each rat to create scar tissue and increase intraocular pressure. The left eye in each rat acted as an internal control. One month following NaCl injection, rats were euthanized, retinas were removed, flatmounted, fixed, and nuclei were stained with cresyl violet or RGCs were immunostained with an antibody against Thy 1.1 or against Brn3a. Stained nuclei in the RGC layer and labeled RGCs in NaCl-injected retinas were counted and compared with cell counts from untreated retinas in the same animal. RESULTS: NaCl injections into the episcleral veins caused a significant loss of cells by an average of 27.35% (± 2.12 SEM) in the RGC layer within 1 month after NaCl injection, which corresponded to a significant loss of RGCs. This loss of RGCs was eliminated if 5 µL of 100 µM PNU-282987 was injected into the right eye an hour before NaCl injection. CONCLUSIONS: The results from this study support the hypothesis that the α7 agonist, PNU-282987, has a neuroprotective effect in the rat retina. PNU-282987 may be a viable candidate for future therapeutic treatments of glaucoma.
Assuntos
Benzamidas/uso terapêutico , Compostos Bicíclicos com Pontes/uso terapêutico , Glaucoma/prevenção & controle , Agonistas Nicotínicos/uso terapêutico , Doenças do Nervo Óptico/prevenção & controle , Degeneração Retiniana/prevenção & controle , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Contagem de Células , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Feminino , Glaucoma/induzido quimicamente , Glaucoma/patologia , Injeções Intravítreas , Masculino , Fármacos Neuroprotetores/uso terapêutico , Doenças do Nervo Óptico/induzido quimicamente , Doenças do Nervo Óptico/patologia , Ratos , Ratos Long-Evans , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/patologia , Células Ganglionares da Retina/patologia , Espectrometria de Massas em Tandem , Tonometria Ocular , Receptor Nicotínico de Acetilcolina alfa7/agonistasRESUMO
The objective of this study was to determine the neuroprotective role of tropisetron on retinal ganglion cells (RGCs) as well as to explore the possible mechanisms associated with alpha7 nAChR-induced neuroprotection. Adult pig RGCs were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured for 3 days under control untreated conditions, in the presence of 500 µM glutamate to induce excitotoxicity, and when tropisetron was applied before glutamate to induce neuroprotection. 500 µM glutamate decreased RGC survival by an average of 62% compared to control conditions. However, RGCs pretreated with 100 nM tropisetron before glutamate increased cell survival to an average of 105% compared to controls. Inhibition studies using the alpha7 nAChR antagonist, MLA (10 nM), support the hypothesis that tropisetron is an effective neuroprotective agent against glutamate-induced excitotoxicity; mediated by α7 nAChR activation. ELISA studies were performed to determine if signaling cascades normally associated with excitotoxicity and neuroprotection were up- or down-regulated after tropisetron treatment. Tropisetron had no discernible effects on pAkt levels but significantly decreased p38 MAPK levels associated with excitotoxicity from an average of 15 ng/ml to 6 ng/ml. Another mechanism shown to be associated with neuroprotection involves internalization of NMDA receptors. Double-labeled immunocytochemistry and electrophysiology studies provided further evidence that tropisetron caused internalization of NMDA receptor subunits. The findings of this study suggest that tropisetron could be an effective therapeutic agent for the treatment of degenerative disorders of the central nervous system that involves excitotoxicity.
Assuntos
Ácido Glutâmico/toxicidade , Indóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Agonistas Nicotínicos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Aconitina/análogos & derivados , Aconitina/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Indóis/antagonistas & inibidores , Antagonistas Nicotínicos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Tropizetrona , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Previous studies have reported that activation of nicotinic acetylcholine (ACh) receptors (nAChRs) on cultured pig retinal ganglion cells (RGCs) has a neuroprotective effect against glutamate-induced excitotoxicity. However, the mechanism linking nAChRs to neuroprotection is unknown. Here, we tested the hypothesis that signaling cascades involving p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) --> Akt are involved in linking activation of nAChRs to neuroprotection in isolated pig RGCs. In ELISA studies, regulation of phosphorylated p38 MAPK and Akt were analyzed after inducing excitotoxicity or neuroprotection in the presence and absence of specific inhibitors for p38 MAPK and PI3K. ELISA results demonstrated that ACh significantly increased phosphorylated Akt and decreased p38 MAPK. Glutamate increased phosphorylated p38 MAPK but had no significant effect on phosphorylated Akt. Other ELISA studies using p38 MAPK and PI3K inhibitors also supported the hypothesis that ACh up-regulated Bcl-2 levels downstream from PI3K and Akt, whereas glutamate down-regulated Bcl-2 levels downstream from p38 MAPK. RGC survival was subsequently assessed by culturing RGCs in conditions to induce excitotoxicity or neuroprotection in the presence or absence of specific inhibitors of p38 MAPK or PI3K. The p38 MAPK inhibitor significantly decreased the number of RGCs that died by glutamate-induced excitotoxicity but had no effect on the number of cells that survived because of ACh-induced neuroprotection. PI3K inhibitors significantly decreased cell survival caused by ACh-induced neuroprotection but had no effect on cell death caused by glutamate-induced excitotoxicity. These results demonstrate that glutamate mediates excitotoxicity through the p38 MAPK signaling pathway and that ACh provides neuroprotection by stimulating the PI3K --> Akt --> Bcl-2 signaling pathway and inhibiting the p38 MAPK --> Bcl-2 pathway.
Assuntos
Agonistas de Aminoácidos Excitatórios/toxicidade , Ácido Glutâmico/toxicidade , Receptores Colinérgicos/fisiologia , Células Ganglionares da Retina/fisiologia , Transdução de Sinais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Receptores Colinérgicos/química , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Transdução de Sinais/efeitos dos fármacos , SuínosRESUMO
PURPOSE: To determine which glutamate receptor (GluR) subtypes are responsible for glutamate-induced excitotoxicity in cultured adult pig retinal ganglion cells (RGCs) and to characterize the neuroprotective effect of acetylcholine (ACh) on pig RGCs. METHODS: Adult pig RGCs were isolated from other retinal tissue by a modified panning technique using Thy 1.1 antibody. Isolated RGCs were cultured in control media and media containing: glutamate, NMDA, or KA; glutamate and CNQX, MK-801, or AP-7; ACh, nicotine or muscarine; ACh and alpha-bungarotoxin (Bgt) or methyllycaconitine (MLA); and glutamate and choline or glutamate, choline, and MLA. To determine cell viability, cells were loaded with calcein and counted. RESULTS: Ninety-eight percent of isolated cells were immunolabeled with Thy 1.1 antibody. Chronic exposure to 500 microM glutamate decreased the number of surviving large and small RGCs, compared to control conditions. This glutamate-induced excitotoxicity was mediated through both NMDA and non-NMDA GluRs. In neuroprotective studies, ACh, nicotine, and choline significantly reduced glutamate-induced excitotoxicity in adult pig RGCs through alpha-Bgt-sensitive nicotinic ACh receptors (nAChRs). DISCUSSION: This was the first report of a modified panning technique to isolate adult pig RGCs. Cell viability was relatively high using this method, and both large and small RGCs grew extensive neurites in culture. The finding that both NMDA and non-NMDA GluRs were involved in glutamate-induced excitotoxicity suggests that isolated pig RGCs provide a good model for glaucoma. In addition, activation of AChRs may be useful in protecting RGC from excitotoxic insults occurring in neurodegenerative diseases such as glaucoma.
Assuntos
Acetilcolina/farmacologia , Ácido Glutâmico/toxicidade , Fármacos Neuroprotetores/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Bungarotoxinas/farmacologia , Separação Celular/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colina/farmacologia , Citoproteção , Agonistas de Aminoácidos Excitatórios/toxicidade , Antagonistas de Aminoácidos Excitatórios/farmacologia , Fluoresceínas/metabolismo , Nicotina/farmacologia , Receptores Nicotínicos/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , SuínosRESUMO
Chronic dehydration induces structural changes in the hypothalamic supraoptic nucleus (SON), including increased glutamate synapses and retraction of astroglial processes. We performed whole-cell recordings in acute hypothalamic slices to determine whether these changes increase tonic activation of presynaptic metabotropic glutamate receptors (mGluRs) by increasing ambient glutamate in the SON. Activation of presynaptic group III mGluRs caused a decrease in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in SON neurones that was significantly attenuated in slices from dehydrated rats (-27.8 %) compared with untreated rats (-41.7 %), suggesting a higher basal occupancy of mGluRs by ambient glutamate during dehydration. Blocking group III mGluRs caused an increase in the frequency of mEPSCs that was significantly higher in slices from dehydrated rats (+42.8 %) than untreated rats (+31.4 %), suggesting greater tonic activation of presynaptic mGluRs by ambient glutamate during dehydration. Increasing ambient glutamate levels by inhibiting astrocyte glutamate uptake resulted in a decrease in mEPSC frequency due to increased activation of presynaptic mGluRs. This was attenuated in slices from dehydrated rats (-35.4 %) compared with slices from untreated rats (-48.8 %), suggesting diminished astrocytic glutamate uptake during dehydration. Immunochemical analyses revealed a robust expression of the GLT-1 transporter protein in the SON, which was diminished in SON punches from dehydrated rats compared with untreated controls. Thus, dehydration leads to increased tonic activation of presynaptic mGluRs on glutamate terminals, consistent with a decrease in glutamate buffering capacity. The resulting reduction in glutamate release probability may compensate for the increase in glutamate release sites that occurs during dehydration.