RESUMO
The nee mouse model exhibits characteristic features of congenital glaucoma, a common cause of childhood blindness. The current study of nee mice had two components. First, the time course of neurodegeneration in nee retinal flat-mounts was studied over time using a retinal ganglion cell (RGC)-marker, BRN3A; a pan-nuclear marker, TO-PRO-3; and H&E staining. Based on segmentation of nuclei using ImageJ and RetFM-J, this analysis identified a rapid loss of BRN3A+ nuclei from 4 to 15 weeks of age, with the first statistically significant difference in average density compared to age-matched controls detected in 8-week-old cohorts (49% reduction in nee). Consistent with a model of glaucoma, no reductions in BRN3A- nuclei were detected, but the combined analysis indicated that some RGCs lost BRN3A marker expression prior to actual cell loss. These results have a practical application in the design of experiments using nee mice to study mechanisms or potential therapies for congenital glaucoma. The second component of the study pertains to a discovery-based analysis of the large amount of image data with 748,782 segmented retinal nuclei. Using the automatedly collected region of interest feature data captured by ImageJ, we tested whether RGC density of glaucomatous mice was significantly correlated to average nuclear area, perimeter, Feret diameter, or MinFeret diameter. These results pointed to two events influencing nuclear size. For variations in RGC density above approximately 3000 nuclei/mm2 apparent spreading was observed, in which BRN3A- nuclei-regardless of genotype-became slightly larger as RGC density decreased. This same spreading occurred in BRN3A+ nuclei of wild-type mice. For variation in RGC density below 3000 nuclei/mm2, which only occurred in glaucomatous nee mutants, BRN3A+ nuclei became smaller as disease was progressively severe. These observations have relevance to defining RGCs of relatively higher sensitivity to glaucomatous cell death and the nuclear dynamics occurring during their demise.
Assuntos
Núcleo Celular/patologia , Glaucoma/patologia , Células Ganglionares da Retina/metabolismo , Tomografia de Coerência Óptica/métodos , Animais , Contagem de Células , Modelos Animais de Doenças , Glaucoma/congênito , Glaucoma/metabolismo , Camundongos , Camundongos Mutantes , Células Ganglionares da Retina/patologiaRESUMO
Exfoliation syndrome is a leading cause of secondary glaucoma worldwide. Among the risk-factors for exfoliation syndrome and exfoliation glaucoma that have been investigated, a genetic association with 15q24.1 is among the most striking. The leading candidates for the causal gene at this locus are LOXL1 and/or LOXL1-AS1, but studies have not yet coalesced in establishing, or ruling out, either candidate. Here, we contribute to studies of the 15q24.1 locus by making a partially humanized mouse model in which 166 kb of human genomic DNA from the 15q24.1 locus was introduced into the mouse genome via BAC transgenesis (B6-Tg(RP11-71M11)Andm). Transgenic expression of human genes in the BAC was only detectable for LOXL1-AS1. One cohort of 34 mice (21 experimental hemizygotes and 13 non-carrier control littermates) was assessed by slit-lamp exams and SD-OCT imaging at early (1-2 months) and mid (4-5 months) time points; fundus exams were performed at 5 months of age. A second smaller cohort (3 hemizygotes) were aged extensively (>12 months) to screen for overt abnormalities. Across all genotypes and ages, 136 slit-lamp exams, 128 SD-OCT exams, and 42 fundus exams detected no overt indices of exfoliation syndrome. Quantitatively, small, but statistically significant, age-related declines in ganglion cell complex thickness and total retinal thickness were detected in the hemizygotes at 4 months of age. Overall, this study demonstrates complexity in gene regulation from the 15q24.1 locus and suggests that LOXL1-AS1 is unlikely to be a monogenic cause of exfoliation syndrome but may contribute to glaucomatous retinal damage.
RESUMO
Purpose: Despite popularity of optical coherence tomography (OCT) in glaucoma studies, it's unclear how well OCT-derived metrics compare to traditional measures of retinal ganglion cell (RGC) abundance. Here, Diversity Outbred (J:DO) mice are used to directly compare ganglion cell complex (GCC) thickness measured by OCT to metrics of retinal anatomy measured ex vivo with retinal wholemounts and optic nerve histology. Methods: J:DO mice (n = 48) underwent fundoscopic and OCT examinations, with automated segmentation of GCC thickness. RGC axons were quantified from para-phenylenediamine-stained optic nerve cross-sections and somas from BRN3A-immunolabeled retinal wholemounts, with total inner retinal cellularity assessed by TO-PRO and subsequent hematoxylin staining. Results: J:DO tissues lacked overt disease. GCC thickness, RGC abundance, and total cell abundance varied broadly across individuals. GCC thickness correlated significantly to RGC somal density (r = 0.58) and axon number (r = 0.44), but not total cell density. Retinal area and nerve cross-sectional area varied widely. No metrics were significantly influenced by sex. In bilateral comparisons, GCC thickness (r = 0.95), axon (r = 0.72), and total cell density (r = 0.47) correlated significantly within individuals. Conclusions: Amongst outbred mice, OCT-derived measurements of GCC thickness correlate significantly to RGC somal and axon abundance. Factors limiting correlation are likely both biological and methodological, including differences in retinal area that distort sampling-based estimates of RGC abundance. Translational Relevance: There are significant-but imperfect-correlations between GCC thickness and RGC abundance across genetic contexts in mice, highlighting valid uses and ongoing challenges for meaningful use of OCT-derived metrics.
Assuntos
Glaucoma , Doenças do Nervo Óptico , Animais , Glaucoma/diagnóstico , Hematoxilina , Camundongos , Doenças do Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Tomografia de Coerência Óptica/métodosRESUMO
Chédiak-Higashi syndrome (CHS) is a lethal disorder caused by mutations in the LYST gene that involves progressive neurologic dysfunction. Lyst-mutant mice exhibit neurologic phenotypes that are sensitive to genetic background. On the DBA/2J-, but not on the C57BL/6J-background, Lyst-mutant mice exhibit overt tremor phenotypes associated with loss of cerebellar Purkinje cells. Here, we tested whether assays for ataxia could measure this observed strain-dependency, and if so, establish parameters for empowering phenotype- and candidate-driven approaches to identify genetic modifier(s). A composite phenotypic scoring system distinguished phenotypes in Lyst-mutants and uncovered a previously unrecognized background difference between wild-type C57BL/6J and DBA/2J mice. Accelerating rotarod performance also distinguished phenotypes in Lyst-mutants, but at more advanced ages. These results establish that genetic background, Lyst genotype, and age significantly influence the severity of CHS-associated neurologic deficits. Purkinje cell quantifications likewise distinguished phenotypes of Lyst-mutant mice, as well as background differences between wild-type C57BL/6J and DBA/2J mice. To aid identification of potential genetic modifier genes causing these effects, we searched public datasets for cerebellar-expressed genes that are differentially expressed and/or contain potentially detrimental genetic variants. From these approaches, Nos1, Prdx2, Cbln3, Gnb1, Pttg1 were confirmed to be differentially expressed and leading candidates.
Assuntos
Ataxia Cerebelar/patologia , Síndrome de Chediak-Higashi/complicações , Mutação , Doenças do Sistema Nervoso/patologia , Animais , Ataxia Cerebelar/etiologia , Modelos Animais de Doenças , Feminino , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Doenças do Sistema Nervoso/etiologia , FenótipoRESUMO
Pain stimulates some behaviors (e.g., withdrawal responses) and depresses other behaviors (e.g., feeding and locomotion). We are developing methods for testing candidate analgesics using measurements of pain-depressed behaviors. Such assays may model important aspects of clinical pain and complement traditional procedures that measure pain-stimulated behaviors. The present study characterized the effects of a chronic pain manipulation (monosodium iodoacetate (MIA)-induced osteoarthritis) on wheel running in rats. Rats had 24 h voluntary access to running wheels. Duration of running wheel acquisition was manipulated such that rats had either 21 or 7 days of running wheel access prior to MIA administration. Wheel running was monitored for an additional 21 days following MIA administration. MIA produced concentration- and acquisition length-dependent decreases in wheel running. Parallel experiments demonstrated that MIA produced concentration-dependent tactile allodynia and shifts in hind limb weight bearing. MIA was differentially potent across assays with a potency rank: weight-bearing≥von Frey>running wheel. MIA produced greater depression of wheel running in rats with relatively high baseline running rates compared to rats with relatively low baseline running rates. The differential potency of MIA across assays and apparent rate-dependent effects in running wheels may impact our traditional interpretations of preclinical nociceptive and antinociceptive testing.
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Iodoacetatos/toxicidade , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Osteoartrite/induzido quimicamente , Osteoartrite/fisiopatologia , Dor/fisiopatologia , Analgésicos/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Doença Crônica , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Membro Posterior , Masculino , Dor/tratamento farmacológico , Medição da Dor/métodos , Ratos , Ratos Sprague-Dawley , Suporte de CargaRESUMO
AIMS: The current studies were designed to compare the in vivo potencies of the opioid antagonists 6beta-naltrexol and naltrexone in blocking the effects of the opioid agonist hydrocodone following intravenous (i.v.) or oral (p.o.) administration. MAIN METHODS: Adult male CD-1 mice were used for all experiments. The 55 degrees C tail-flick assay was used to assess the CNS antinociceptive activity of hydrocodone, and a charcoal meal gastrointestinal transit assay was used to assess the peripheral effects of hydrocodone. Graded antagonist dose-response curves for i.v. and p.o. 6beta-naltrexol and naltrexone were generated to determine ID(50) antagonist potency estimates against fixed doses of hydrocodone. KEY FINDINGS: Both antagonists produced dose-related blockade of hydrocodone-induced antinociception and inhibition of gastrointestinal transit. Naltrexone was between 5- and 13-fold more potent than 6beta-naltrexol in blocking a CNS effect of hydrocodone, whereas the drugs were nearly equipotent in blocking inhibition of gastrointestinal transit. Co-administration studies indicated an approximate 10-fold greater potency of 6beta-naltrexol for antagonism of hydrocodone-induced inhibition of gastrointestinal transit versus antinociception, whereas naltrexone blocked both effects with near equal potency. 6beta-naltrexol produced a longer duration of antagonist blockade and had a slower time to peak effect compared to naltrexone. SIGNIFICANCE: The pharmacology of 6beta-naltrexol differentiates it from currently available opioid antagonists. This includes an intermediate selectivity for peripheral versus central opioid receptors, a long duration of action, and neutral antagonist qualities in opioid exposed systems. These properties render it a drug candidate for a co-formulation product with opioid analgesics to reduce peripheral opioid side effects and limit abuse potential.
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Analgésicos Opioides/antagonistas & inibidores , Analgésicos Opioides/farmacologia , Trânsito Gastrointestinal/efeitos dos fármacos , Hidrocodona/antagonistas & inibidores , Hidrocodona/farmacologia , Naltrexona/análogos & derivados , Antagonistas de Entorpecentes , Administração Oral , Animais , Relação Dose-Resposta a Droga , Injeções Intravenosas , Masculino , Camundongos , Naltrexona/farmacologia , Medição da Dor/efeitos dos fármacos , Tempo de Reação/efeitos dos fármacosRESUMO
AIMS: Pain depresses expression of many behaviors, and one goal of analgesic treatment is to restore pain-depressed behaviors. Assays that focus on pain-depressed behaviors may contribute to preclinical assessment of candidate analgesics. MAIN METHODS: This study compared effects of the mu opioid receptor agonist morphine (an acknowledged analgesic), the dopamine receptor antagonist haloperidol (a non-analgesic sedative), the adenosine receptor antagonist caffeine (a non-analgesic stimulant) and the neurokinin-1 receptor antagonist CJ 11,974-01 (a candidate analgesic) on acetic acid-induced writhing (a traditional pain-stimulated behavior) and acetic acid-induced suppression of locomotor activity (a pain-depressed behavior) in male ICR mice. Drug effects on non-depressed (baseline) locomotor activity were also examined. KEY FINDINGS: I.P. administration of acetic acid (0.18-1%) was equipotent in stimulating writhing and depressing locomotor activity. Morphine blocked both acid-induced stimulation of writhing and depression of locomotion, although it was 56-fold less potent in the assay of acid-depressed locomotion. Haloperidol and CJ 11,974-01 decreased acid-stimulated writhing, but failed to block acid-induced depression of locomotion. Caffeine had no effect on acid-stimulated writhing or acid-depressed locomotor activity, although it did increase non-depressed locomotion. Thus, morphine was the only drug to block both acid-stimulated writhing and acid-depressed locomotion. SIGNIFICANCE: Complementary assays of pain-stimulated and pain-depressed behaviors may improve the predictive validity of preclinical studies that assess candidate analgesic drugs. The low potency of morphine to block acid-induced depression of locomotion suggests that locomotor activity may be a relatively insensitive measure for studies of pain-depressed behavior.