RESUMEN
PURPOSE: To study the population of intrinsically photosensitive retinal ganglion cells (melanopsin-expressing RGCs, m+RGCs) in P23H-1 rats, a rat model of inherited photoreceptor degeneration. METHODS: At postnatal (P) times P30, P365, and P540, retinas from P23H dystrophic rats (line 1, rapid degeneration; and line 3, slow degeneration) and Sprague Dawley (SD) rats (control) were dissected as whole-mounts and immunodetected for melanopsin and/or Brn3a. The dendritic arborization of m+RGCs and the numbers of Brn3a+RGCs and m+RGCs were quantified and their retinal distribution and coexpression analyzed. RESULTS: In SD rats, aging did not affect the population of Brn3a+RGCs or m+RGCs or the percentage that showed coexpression (0.27%). Young P23H-1 rats had a significantly lower number of Brn3a+RGCs and showed a further decline with age. The population of m+RGCs in young P23H-1 rats was similar to that found in SD rats and decreased by 22.6% and 28.2% at P365 and P540, respectively, similarly to the decrease of the Brn3a+RGCs. At these ages the m+RGCs showed a decrease of their dendritic arborization parameters, which was similar in both the P23H-1 and P23H-3 lines. The percentage of coexpression of Brn3a was, however, already significantly higher at P30 (3.31%) and increased significantly with age (10.65% at P540). CONCLUSIONS: Inherited photoreceptor degeneration was followed by secondary loss of Brn3a+RGCs and m+RGCs. Surviving m+RGCs showed decreased dendritic arborization parameters and increased coexpression of Brn3a and melanopsin, phenotypic and molecular changes that may represent an effort to resist degeneration and/or preferential survival of m+RGCs capable of synthesizing Brn3a.
Asunto(s)
Regulación de la Expresión Génica , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/genética , Células Ganglionares de la Retina/patología , Factor de Transcripción Brn-3A/genética , Animales , Muerte Celular , Modelos Animales de Enfermedad , Técnicas para Inmunoenzimas , Células Fotorreceptoras de Vertebrados/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Ganglionares de la Retina/metabolismo , Opsinas de Bastones , Factor de Transcripción Brn-3A/biosíntesis , Factor de Transcripción Brn-3A/inmunologíaRESUMEN
To investigate the long-term effects of laser-photocoagulation (LP)-induced ocular hypertension (OHT) in the innermost and outermost (outer-nuclear and outer segment)-retinal layers (ORL). OHT was induced in the left eye of adult rats. To investigate the ganglion cell layer (GCL) wholemounts were examined at 1, 3 or 6 months using Brn3a-immunodetection to identify retinal ganglion cells (RGCs) and DAPI-staining to detect all nuclei in this layer. To study the effects of LP on the ORL up to 6 months, retinas were: i) fresh extracted to quantify the levels of rod-, S- and L-opsin; ii) cut in cross-sections for morphometric analysis, or; iii) prepared as wholemounts to quantify and study retinal distributions of entire populations of RGCs (retrogradely labeled with fluorogold, FG), S- and L-cones (immunolabeled). OHT resulted in wedge-like sectors with their apex on the optic disc devoid of Brn3a(+)RGCs but with large numbers of DAPI(+)nuclei. The levels of all opsins diminished by 2 weeks and further decreased to 20% of basal-levels by 3 months. Cross-sections revealed focal areas of ORL degeneration. RGC survival at 15 days represented approximately 28% and did not change with time, whereas the S- and L-cone populations diminished to 65% and 80%, or to 20 and 35% at 1 or 6 months, respectively. In conclusion, LP induces in the GCL selective RGCs loss that does not progress after 1 month, and S- and L-cone loss that progresses for up to 6 months. Thus, OHT results in severe damage to both the innermost and the ORL.
Asunto(s)
Coagulación con Láser/efectos adversos , Hipertensión Ocular/patología , Retina/patología , Animales , Western Blotting , Recuento de Células , Modelos Animales de Enfermedad , Femenino , Hipertensión Ocular/etiología , Opsinas/metabolismo , Ratas , Ratas Sprague-Dawley , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Conos/efectos de la radiación , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/efectos de la radiaciónRESUMEN
We have investigated the effects of light-emitting diode (LED)-induced phototoxicity (LIP) on cone-photoreceptors and their protection with brimonidine (BMD), brain-derived neurotrophic factor (BDNF), pigment epithelium-derived factor (PEDF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF). In anesthetized, dark adapted, adult albino rats a blue (400 nm) LED was placed perpendicular to the cornea (10 sec, 200 lux) and the effects were investigated using Spectral Domain Optical Coherence Tomography (SD-OCT) and/or analysing the retina in oriented cross-sections or wholemounts immune-labelled for L- and S-opsin and counterstained with the nuclear stain DAPI. The effects of topical BMD (1%) or, intravitreally injected BDNF (5 µg), PEDF (2 µg), CNTF (0.4 µg) or bFGF (1 µg) after LIP were examined on wholemounts at 7 days. SD-OCT showed damage in a circular region of the superotemporal retina, whose diameter varied from 1,842.4±84.5 µm (at 24 hours) to 1,407.7±52.8 µm (at 7 days). This region had a progressive thickness diminution from 183.4±5 µm (at 12 h) to 114.6±6 µm (at 7 d). Oriented cross-sections showed within the light-damaged region of the retina massive loss of rods and cone-photoreceptors. Wholemounts documented a circular region containing lower numbers of L- and S-cones. Within a circular area (1 mm or 1.3 mm radius, respectively) in the left and in its corresponding region of the contralateral-fellow-retina, total L- or S-cones were 7,118±842 or 661±125 for the LED exposed retinas (nâ=â7) and 14,040±1,860 or 2,255±193 for the fellow retinas (nâ=â7), respectively. BMD, BDNF, PEDF and bFGF but not CNTF showed significant neuroprotective effects on L- or S-cones. We conclude that LIP results in rod and cone-photoreceptor loss, and is a reliable, quantifiable model to study cone-photoreceptor degeneration. Intravitreal BDNF, PEDF or bFGF, or topical BMD afford significant cone neuroprotection in this model.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Electrónica , Proteínas del Ojo/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Luz/efectos adversos , Factores de Crecimiento Nervioso/farmacología , Fármacos Neuroprotectores/farmacología , Quinoxalinas/farmacología , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Conos/efectos de la radiación , Serpinas/farmacología , Animales , Tartrato de Brimonidina , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Modelos Animales de Enfermedad , Femenino , Ratas Sprague-Dawley , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Factores de Tiempo , Tomografía de Coherencia ÓpticaRESUMEN
AIMS: To investigate the cause of retinal ganglion cell (RGC) loss in dystrophic aged Royal College of Surgeons (RCS) rats. METHODS: RCS-p+ (dystrophic) female rats of postnatal times (P365, P450 and P540) and age-matched RCS-p1 rdy+ (non-dystrophic) rats were used. In whole-mounted retinas, RGCs were doubly labelled with Fluorogold (FG) retrogradely transported from the superior colliculi and Brn3a immunohistochemistry. RGC axons were labelled with anti-neurofilament antibodies. Automatic image analysis techniques allowed quantification of the total population of RGCs per retina and construction of isodensity maps to investigate RGC topology. RESULTS: Dystrophic retinas showed at all times studied wedge-shaped sectors devoid of FG(+) and Brn3a(+) RGCs. These sectors were also devoid of neurofilament-labelled axons. The total number of FG(+)RGC and Brn3a(+)RGC per retina was significantly smaller in dystrophic rats at P540, revealing RGC death at this age. The total number of FG(+)RGCs was smaller than those of Brn3a(+)RGCs at P540, indicating a disturbance of the retrograde axonal transport at this age. CONCLUSIONS: RGC double labelling documents that sectorial RGC loss in aged dystrophic RCS rats is mainly due to RGC death, although a deficit of the retrograde axonal transport exists also at the more advanced ages.