Quantitative and Qualitative Evaluation of Photoreceptor Synapses in Developing, Degenerating and Regenerating Retinas

Quantitative and qualitative evaluation of synapses is crucial to understand neural connectivity. This is particularly relevant now, in view of the recent advances in regenerative biology and medicine. There is an urgent need to evaluate synapses to access the extent and functionality of reconstructed neural network. Most of the currently used synapse evaluation methods provide only all-or-none assessments. However, very often synapses appear in a wide spectrum of transient states such as during synaptogenesis or neural degeneration. Robust evaluation of synapse quantity and quality is therefore highly sought after. In this paper we introduce QUANTOS, a new method that can evaluate the number, likelihood, and maturity of photoreceptor ribbon synapses based on graphical properties of immunohistochemistry images. QUANTOS is composed of ImageJ Fiji macros, and R scripts which are both open-source and free software. We used QUANTOS to evaluate synaptogenesis in developing and degenerating retinas, as well as de novo synaptogenesis of mouse iPSC-retinas after transplantation to a retinal degeneration mouse model. Our analysis shows that while mouse iPSC-retinas are largely incapable of forming synapses in vitro, they can form extensive synapses following transplantation. The de novo synapses detected after transplantation seem to be in an intermediate state between mature and immature compared to wildtype retina. Furthermore, using QUANTOS we tested whether environmental light can affect photoreceptor synaptogenesis. We found that the onset of synaptogenesis was earlier under cyclic light (LD) condition when compared to constant dark (DD), resulting in more synapses at earlier developmental stages. The effect of light was also supported by micro electroretinography showing larger responses under LD condition. The number of synapses was also increased after transplantation of mouse iPSC-retinas to rd1 mice under LD condition. Our new probabilistic assessment of synapses may prove to be a valuable tool to gain critical insights into neural-network reconstruction and help develop treatments for neurodegenerative disorders

Retinal Morphology and Sensitivity Are Primarily Impaired in Eyes with Neuromyelitis Optica Spectrum Disorder (NMOSD)

<div><p>Background</p><p>Previous studies of neuromyelitis optica spectrum disorder (NMOSD) using spectral domain optical coherence tomography (SD-OCT) showed that the outer nuclear layer (ONL) in eyes without a history of optic neuritis (ON) was thinner than that of healthy controls. It remains unclear whether the ONL thinning is caused by a direct attack on the retina by an autoantibody or a retrograde degeneration.</p><p>Objective</p><p>To determine the mechanisms involved in the retinal damage in eyes with NMOSD without ON.</p><p>Methods</p><p>SD-OCT was used to determine the thicknesses of the different retinal layers of 21 eyes of 12 NMOSD patients without prior ON and 19 eyes of 10 healthy controls. Eyes with peripapillary retinal nerve fiber layer (RNFL) thinning were excluded to eliminate the confounding effects of retrograde degeneration. Microperimetry was used to determine the central retinal sensitivity. The data of the two groups were compared using generalized estimated equation models to account for inter-eye dependencies.</p><p>Results</p><p>The ganglion cell plus inner plexiform layer and the inner nuclear layer plus outer plexiform layer thicknesses of the NMOSD eyes were not significantly different from that of the control eyes (<i>P</i> = 0.28, <i>P</i> = 0.78). However, the ONL and average macular thickness (AMT) in the NMOSD eyes were significantly thinner than that of the control eyes (<i>P</i> = 0.022, <i>P</i> = 0.036). The retinal sensitivity in the central 10°, 10° to 2°, and 2° sectors were significantly lower in the NMOSD eyes than in the control eyes (<i>P</i> = 0.013, <i>P</i> = 0.022, <i>P</i> = 0.002).</p><p>Conclusions</p><p>The ONL thinning, AMT thinning, and reduced retinal sensitivity in eyes with NMOSD without significant peripapillary RNFL thinning are most likely due to direct retinal pathology.</p></div

Evaluation of retinal sensitivity.

<p>The retinal sensitivities were obtained from 37 points within the central 10 degrees of the foveal center. The average retinal sensitivity of the central 10°, central 10° to 2°, and central 2° were calculated. The macular integrity assessment (MAIA) was used to determine the retinal sensitivity. (A) Retinal sensitivity of a healthy control eye. Retinal sensitivities obtained from each point are shown on the left. The average sensitivity of central 10° and the histogram of threshold frequencies compared with built-in database of normal population are shown on the right. (B) Retinal sensitivity of an eye with neuromyelitis optica spectrum disorder (NMOSD). The patient was a 66-year-old woman who had history of longitudinally extensive transverse myelitis and was positive for serum autoantibody against aquaporin-4. The average retinal sensitivity of the central 10° was 27.0 dB whereas the average sensitivity of healthy control eyes was 29.39 dB.</p

Clinical characteristics of patients with NMOSD.

<p>Clinical characteristics of patients with NMOSD.</p

Hypothesis of different mechanisms between retrograde degeneration and direct retinal pathology.

<p>(A) In retrograde degeneration of neuromyelitis optica spectrum disorder (NMOSD), involvement of anterior visual pathway results in a thinning of the inner retinal layers including the retinal nerve fiber layer(RNFL) and ganglion cell-inner plexiform layer (GCIP). (B) In direct retinal pathology of NMOSD, a direct attack of Müller cells by anti-aquaporin 4 (AQP4) antibody results in a secondary loss of retinal neurons including ONL thinning.</p

Representative optical coherence tomographic (OCT) images and retinal segmentation of the images.

<p>(A) Orange segmentation lines define the ganglion cell layer and inner plexiform layer (GCIP), the inner nuclear layer and outer plexiform layer (INL+OPL), and the outer nuclear layer (ONL). (B) Disc circle mode of OCT was used to evaluate the peripapillary retinal nerve fiber layer thickness around the optic disc. (C) Macula map mode was used to evaluate the thickness of the different retinal layers including the GCIP, INL+OPL, and ONL within a 9 mm x 9 mm area centered on the fovea.</p

Result of optical coherence tomography and visual function tests.

<p>Result of optical coherence tomography and visual function tests.</p