Non-Image-Forming Light Driven Functions Are Preserved in a Mouse Model of Autosomal Dominant Optic Atrophy

Autosomal dominant optic atrophy (ADOA) is a slowly progressive optic neuropathy that has been associated with mutations of the OPA1 gene. In patients, the disease primarily affects the retinal ganglion cells (RGCs) and causes optic nerve atrophy and visual loss. A subset of RGCs are intrinsically photosensitive, express the photopigment melanopsin and drive non-image-forming (NIF) visual functions including light driven circadian and sleep behaviours and the pupil light reflex. Given the RGC pathology in ADOA, disruption of NIF functions might be predicted. Interestingly in ADOA patients the pupil light reflex was preserved, although NIF behavioural outputs were not examined. The B6; C3-Opa1Q285STOP mouse model of ADOA displays optic nerve abnormalities, RGC dendropathy and functional visual disruption. We performed a comprehensive assessment of light driven NIF functions in this mouse model using wheel running activity monitoring, videotracking and pupillometry. Opa1 mutant mice entrained their activity rhythm to the external light/dark cycle, suppressed their activity in response to acute light exposure at night, generated circadian phase shift responses to 480 nm and 525 nm pulses, demonstrated immobility-defined sleep induction following exposure to a brief light pulse at night and exhibited an intensity dependent pupil light reflex. There were no significant differences in any parameter tested relative to wildtype littermate controls. Furthermore, there was no significant difference in the number of melanopsin-expressing RGCs, cell morphology or melanopsin transcript levels between genotypes. Taken together, these findings suggest the preservation of NIF functions in Opa1 mutants. The results provide support to growing evidence that the melanopsin-expressing RGCs are protected in mitochondrial optic neuropathies

Probing the molecular basis of melanopsin induced light sensitivity

It has been demonstrated that retinal photoreception among mammals extends beyond rods and cones to include a small number of intrinsically photosensitive retinal ganglion cells (pRGCs), which are capable of responding to light due to expression of the melanopsin (OPN4) photopigment. OPN4 may have therapeutic potential if ectopically expressed in the degenerate retina in cases where photoreceptors are lost, but the other molecules involved in this light induced transduction cascade are less well characterized. Therefore I sought to probe further the mechanism of OPN4 mediated light sensitivity by siRNA mediated knock down of specific molecules in two mice models in which complete loss of rods and cones renders them almost exclusively dependent on the OPN4 pathway for light sensitivity. I generated siRNA probes against the long transcript variant of murine Opn4 mRNA and first tested these probes on the murine Neuro2A (N2a) cell line, before assessing effects in C3H/HeN rd and rodless/coneless rd/rd cl mice. siRNA was injected intravitreally into one eye and pupillometry was assessed, combined with molecular analyses at various timepoints. Reverse transcription polymerase chain reaction (RT-PCR) analysis in N2a cells confirmed Opn4 knockdown and immunolabelling techniques identified >85% silencing with siRNA. Pupil responses in the rd and rd/rd cl mice were inhibited by the siRNA injections in vivo which confirmed the functional effect of Opn4 silencing detected by molecular analysis. I therefore present a novel reproducible in vivo model in which siRNA induced silencing of the melanopsin pathway can be assessed by pupillometry and compared to levels of mRNA and protein at specific timepoints. Probes against other putative candidate genes, such as TRPC3, may unravel the molecular interactions of this pathway. This may help in future to induce light sensitivity in other retinal neurons in patients who are completely blind from photoreceptor loss.This thesis is not currently available in ORA

Quality of Life in Oral Cancer Patients in Greek Clinical Practice: A Cohort Study

Cancer of the oral cavity is one of the most common cancers all over the world. Oral cancer and its treatment impacts on patients’ Quality of Life (QOL). The purpose of the present study was to assess oral cancer patients’ QOL after the completion of surgical therapy, and to investigate factors affecting it. This was a prospective cohort study, conducted at the Department of Oral & Maxillofacial Surgery, of a large general public hospital in Northern Greece. The sample consisted of 135 consecutive eligible cancer patients. Three distinct questionnaires were used. The first one included questions regarding the participants’ demographic characteristics and relevant clinical information. The second comprised the European Organization for Research and Treatment core module (EORTC QLQ-C30) and its head and neck module EORTC QLQ-H&N35. The third was the Functional Assessment of Cancer Therapy–General (FACT-G) assessment of quality of life. We also included the physician-completed Karnofsky scale to assess the functional status of the participants. We found that location of the tumor affects QOL and specifically social contact (H = 17.89, p = 0.001), on the first assessment, and nutritional supplements (H = 22.49, p = 0.000), on the fourth assessment. QOL in patients deteriorates immediately after treatment but significantly improves over time. Health care professionals should take into account these results and arrange care plans in order to find ways to increase patients’ QOL

Pupil light reflex in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> mice.

<p>The average minimum pupil area expressed as a percentage of maximum dilation following illumination with various intensities of white light for <i>Opa1^+/+</i> (n = 5) and <i>Opa1^+/−</i> (n = 5) mice. All data are fitted with four term sigmoidal functions (solid lines) of the form y = y0+a/(1+exp(-(x-x0)/b)) (goodness of fit of fitted curve to actual data (R2): <i>Opa1^+/+</i> = 0.993 and <i>Opa1^+/−</i> = 0.995). A 2-way ANOVA using intensity and genotype as factors showed a significant effect of light intensity (p<0.0001) but no significant effect of genotype (p = 0.51) and no significant interaction between genotype and intensity (p = 0.99).</p

Melanopsin expression in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> retinae.

<p>Overall distribution of melanopsin-positive RGCs in a flatmount retina from (A) <i>Opa1</i>^+/+ and (B) <i>Opa1</i>^+/<i>−</i> mice. The total number of melanopsin expressing cells was not significantly different between genotypes (<i>Opa1^+/+</i>: n = 3; <i>Opa1^+/</i>: n = 3). (C) Quantification of melanopsin (<i>Opn4</i>) and <i>Opa1</i> gene expression by real time quantitative PCR. Expression levels in <i>Opa1^+/−</i> animals are plotted relative to wildtype data. No significant difference in expression was detected for <i>Opn4</i> between genotypes. A significant reduction in <i>Opa1</i> expression was observed in <i>Opa1^+/−</i> mice relative to wildtype controls (student's t-test. * = p<0.005). (D) Representative confocal images of melanopsin cells in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> retinae. A projected image of a confocal stack (from the inner plexiform layer to the ganglion cell layer) is shown for each genotype. An image at the plane of the outermost region of sublamina a and an image at the plane of the innermost region of sublamina b from the same image stacks is also shown.</p

Masking response in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> mice.

<p>(A) The average wheel running revolutions on the night (gray background) of the 3 h light pulse (white background) are plotted relative to the baseline levels (the night before the pulse) for <i>Opa1^+/+</i> (n = 6) and <i>Opa1^+/−</i> (n = 7) mice. The masking pulse completely suppressed activity in both genotypes immediately. ANOVA analysis found no significant effect of genotype on the baseline corrected activity levels (p = 0.468) (B) Hourly breakdown of activity during the masking pulse. A 2-way ANOVA using activity in each hour of light pulse and genotype as factors found a significant effect of hour of light pulse (p<0.005) but no significant effect of genotype (p = 0.143) and no interaction between genotype and light pulse hour (p = 0.359). All data are presented as mean ± SEM.</p

Phase shift behaviour in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> mice.

<p>Representative actograms from (A) <i>Opa1</i>^+/+ and (B) <i>Opa1</i>^+/<i>−</i> mice in constant dark (DD) conditions. Animals were exposed to 15 min light pulses every ∼15 days. Photon matched pulses at 480 nm (black arrow) or 525 nm (white arrow; 1×10¹¹ photons/s/cm²) were applied at CT16. Animals were also exposed to a dark sham pulse condition (grey arrow). (C) The size of the phase shift response are plotted for the 525 nm, 480 nm and sham conditions for <i>Opa1^+/+</i> (n = 6) and <i>Opa1^+/−</i> (n = 7) mice. A two-way ANOVA with genotype and wavelength as factors was performed. There was no significant effect of wavelength (<i>p</i> = 0.66) or genotype (<i>p</i> = 0.17) and the interaction of genotype and wavelength was not significant (<i>p</i> = 0.91).</p

Circadian behaviour in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> mice.

<p>Representative actograms from (A) <i>Opa1 </i>^+/+ and (B) <i>Opa1 </i>^+/− mice entrained to a 12/12 LD cycle and subsequently released into constant darkness (DD). Each horizontal line corresponds to one day and the data has been double plotted. The black vertical bars represent activity (i.e. wheel revolutions). The shaded region represents lights ON. (C) Table showing average period (τ), total activity levels and length of the active phase in LD and in DD for <i>Opa1^+/+</i> (n = 6) and <i>Opa1^+/−</i> (n = 7) mice. There were no significant differences between genotypes (unpaired students t-test; p values are shown). All data are presented as mean ± SEM.</p