Melanopsin+RGCs Are fully Resistant to NMDA-Induced Excitotoxicity.

We studied short- and long-term effects of intravitreal injection of N-methyl-d-aspartate (NMDA) on melanopsin-containing (m+) and non-melanopsin-containing (Brn3a+) retinal ganglion cells (RGCs). In adult SD-rats, the left eye received a single intravitreal injection of 5µL of 100nM NMDA. At 3 and 15 months, retinal thickness was measured in vivo using Spectral Domain-Optical Coherence Tomography (SD-OCT). Ex vivo analyses were done at 3, 7, or 14 days or 15 months after damage. Whole-mounted retinas were immunolabelled for brain-specific homeobox/POU domain protein 3A (Brn3a) and melanopsin (m), the total number of Brn3a+RGCs and m+RGCs were quantified, and their topography represented. In control retinas, the mean total numbers of Brn3a+RGCs and m+RGCs were 78,903 ± 3572 and 2358 ± 144 (mean ± SD; n = 10), respectively. In the NMDA injected retinas, Brn3a+RGCs numbers diminished to 49%, 28%, 24%, and 19%, at 3, 7, 14 days, and 15 months, respectively. There was no further loss between 7 days and 15 months. The number of immunoidentified m+RGCs decreased significantly at 3 days, recovered between 3 and 7 days, and were back to normal thereafter. OCT measurements revealed a significant thinning of the left retinas at 3 and 15 months. Intravitreal injections of NMDA induced within a week a rapid loss of 72% of Brn3a+RGCs, a transient downregulation of melanopsin expression (but not m+RGC death), and a thinning of the inner retinal layers.

Decrease in retinal neuronal cells in streptozotocin-induced diabetic mice.

PURPOSE: Little is known about retinal neuronal loss in the retinas of diabetic mice. The purpose of this study was the quantitative assessment of retinal neural cell number in diabetic mice. METHODS: Five-week-old C57BL/6 mice were used as a diabetic model with streptozotocin. Mice were studied over the course of 6 and 12 weeks after the onset of diabetes. Intraocular pressure (IOP) was measured with a noninvasive TonoLab tonometer. The retinal ganglion cells (RGCs) were counted at two different time points after the induction of diabetes and examined using the immunofluorescence technique and quantitative analysis. RESULTS: The diabetic mice had significantly elevated IOP levels at 6 and 12 weeks after the onset of diabetes compared with the age-matched control mice (p<0.01 and p<0.001, respectively). The temporal course of Brn3a+ RGC and Neuronal Nuclei+RGC (NeuN+ RGC) loss induced by intraperitoneal injection of streptozotocin followed a similar trend. At 6 and 12 weeks after the onset of diabetes, the number of Brn3a+ RGCs (p<0.05 at 6 weeks; p<0.001 at 12 weeks) and NeuN+ RGCs (p<0.05 at 6 weeks; p<0.001 at 12 weeks) was significantly lower in diabetic mice than age-matched control mice. In the retinal flatmounts, the number of Brn3a+ RGCs (p<0.05 at 6 weeks, p<0.01 at 12 weeks) was also significantly lower in diabetic mice than control mice. The IOP in diabetic mice was negatively related with RGCs in cross sections. The cut-off value of IOP was 14.2 mmHg for diabetes. CONCLUSIONS: This is a specific quantitative study of neural cell loss in the retina during diabetes. These data suggest that retinal neural cell reduction occurs in diabetic mice. It indicates that RGC loss may be an important component of diabetic retinopathy.

Detection of cervical abnormalities in a developing country using measurement of Brn-3a in cervical smears.

OBJECTIVE: We have previously shown that Brn-3a is elevated in biopsies from women in the United Kingdom with high grade cervical neoplasia, and that it specifically trans-activates the HPV URR in vitro and in vivo. The aim of this study is to establish the relationship of Brn-3a, HPV E6 and pathological diagnosis in cervical smear from women in a developing country with a high prevalence of cervical disease. This is a follow-up of our previous work in which for the first time Brn-3a and E6 levels in cervical smears from women in United Kingdom were shown to correlate with the histological diagnosis of cervical neoplasia and were even better in predicting underlying pre-malignant disease than conventional procedures. METHOD: Cervical smears from 295 women with cervical abnormalities attending gynecological clinics in Brazil were used to make RNA. The mRNA levels of Brn-3a and HPV E6 were measured and the data obtained were used to establish the relationship between Brn-3a and the histological diagnosis. RESULTS: The cellular transcription factor Brn-3a was readily measured in cervical smears from the Brazilian population. Its presence was shown to be frequently associated with the expression of HPV E6. The measured level of Brn-3a parallels the severity of the cervical ailment and predicts the pathological categories. CONCLUSIONS: The ability of Brn-3a to predict for cervical ailments is independent to the geographical characteristics of the population, and hence it could be used routinely as an adjunct to colposcopy and pathological diagnosis in developing and developed countries.