in this work, we develop a robust, extensible tool to automatically and accurately count retinal ganglion cell axons in optic nerve (on) tissue images from various animal models of glaucoma. We adapted deep learning to regress pixelwise axon count density estimates, which were then integrated over the image area to determine axon counts. the tool, termed Axonet, was trained and evaluated using a dataset containing images of on regions randomly selected from whole cross sections of both control and damaged rat ons and manually annotated for axon count and location. this rat-trained network was then applied to a separate dataset of non-human primate (nHp) on images. Axonet was compared to two existing automated axon counting tools, AxonMaster and AxonJ, using both datasets. Axonet outperformed the existing tools on both the rat and nHp on datasets as judged by mean absolute error, R 2 values when regressing automated vs. manual counts, and Bland-Altman analysis. Axonet does not rely on hand-crafted image features for axon recognition and is robust to variations in the extent of on tissue damage, image quality, and species of mammal. therefore, Axonet is not speciesspecific and can be extended to quantify additional ON characteristics in glaucoma and potentially other neurodegenerative diseases. Glaucoma is the leading cause of irreversible blindness worldwide 1,2 , and thus is a significant research focus. This optic neuropathy is characterized by degeneration and loss of retinal ganglion cells (RGCs), which carry visual signals from the retina to the brain. Therefore, an important outcome measure in studying glaucomatous optic neuropathy, particularly in animal models of the disease, is the number and appearance of RGC axons comprising the optic nerve 3,4 , usually evaluated from images of optic nerve cross sections. Using images obtained by light microscopy is known to result in an axon count underestimation of around 30% relative to counts from images obtained by transmission electron microscopy 5,6. However, light microscopy is widely used to count optic nerve axons because of its lower cost and favorable time requirements for tissue preparation. Therefore, in this work we focus on axon counting in optic nerve images generated by light microscopy. Manual counting is the gold standard approach to quantify RGC axons, but it is extremely labor-intensive, since RGC axon numbers in healthy nerves range from the tens of thousands in mice to more than a million in humans 7. Further complicating axon quantification is the fact that axon appearance can be highly variable. For example, in the healthy nerve, most axons are characterized by a clear central axoplasmic core and a darker myelin sheath; following previous work 5,8 , we will refer to such an appearance as "normal". However, in damaged nerves (and even occasionally in ostensibly heathy nerves), other axon appearances occur, such as an incomplete myelin sheath and/or a darker axoplasmic region. Such variability further increases the time needed for axon counting,...
Purpose Genipin has been proposed as a possible neuroprotective therapy in myopia and glaucoma. Here, we aim to determine the effects of prolonged genipin-induced scleral stiffening on visual function. Methods Eyes from Brown Norway rats were treated in vivo with either a single 15 mM genipin retrobulbar injection or sham retrobulbar injection and were compared to naïve eyes. Intraocular pressure, optomotor response, and electroretinograms were repeatedly measured over 4 weeks following retrobulbar injections to determine visual and retinal function. At 4 weeks, we quantified retinal ganglion cell axon counts. Finally, molecular changes in gene and protein expression were analyzed via real-time polymerase chain reaction (RT-PCR) and proteomics. Results Retrobulbar injection of genipin did not affect intraocular pressure (IOP) or retinal function, nor have a sustained impact on visual function. Although genipin-treated eyes had a small decrease in retinal ganglion cell axon counts compared to contralateral sham-treated eyes (−8,558 ± 18,646; mean ± SD), this was not statistically significant ( P = 0.206, n = 9). Last, we did not observe any changes in gene or protein expression due to genipin treatment. Conclusions Posterior scleral stiffening with a single retrobulbar injection of 15 mM genipin causes no sustained deficits in visual or retinal function or at the molecular level in the retina and sclera. Retinal ganglion cell axon morphology appeared normal. Translational Significance These results support future in vivo studies to determine the efficacy of genipin-induced posterior scleral stiffening to help treat ocular diseases, like myopia and glaucoma.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.