Análisis Vertical

stimulus

Time-domain analysis of ERG OPs revealed no early (two month) delays of initial OP latency in hq disease mice when compared to the wild-type mice (Figure 3.2A). However, by three months of age hq mice demonstrate a significant delay (p = 0.005, Figure 3.2A) of initial OP latency by 24.6%. This is indicative of neural retinal feedback delay between the photoreceptors, INL and GCL of the retina. The trend of OP delayed response continues at four months and is significantly delayed at five to seven months (p = 0.0115, Figure 3.2A). Summed OP amplitudes demonstrated no significant changes at two or three months of age in the hq mice indicating normal INL and GCL function

Figure 3.1.8PCR amplification confirmed Aif genotype. Aif genotyping PCR

amplicons were electrophoresed through a 1.5% agarose gel and stained with SYBR®

Safe DNA Gel Stain (Invitrogen, Burlington, ON). A 100 bp DNA ladder (FroggiaBio Inc, Toronto, ON) was used as a size standard (STD) to estimate amplicon size. A single 537 bp amplicon indicated a wild-type Aif allele (XY) while a single 725 bp amplicon indicated the presence of an Aif proviral insertion.93 Heterozygote, hq carriers

demonstrated the presence of both 537 bp and 725 bp amplicons. A no DNA control (NC) was created with water prior to amplification and added to the PCR mastermix to eliminate the possibility of a false positive.

(Figure 3.2B). However by four months of age, a significant deterioration of summed OP amplitudes (p = 0.0222) by 26.5% occurred in hq mice resulting in reduced INL and GCL function (Figure 3.2B). The largest effect on summed OP amplitudes occurred at five to seven months with a significant reduction (p = 0.003, Figure 3.2B) by 54.4%. No statistical conclusions can be made about initial OP latency or summed OP amplitudes at eight to ten months of age as the hq cohort only contains two mice, although general trends appear to continue (Figure 3.2A,B).

3.3 Frequency-domain analysis of ERG OPs show early frequency changes in hqY disease mice followed by later changes in OP power and energy (10 cd•s/m2 -

stimulus)

hqY OP frequency demonstrated early, two-month changes with a significant reduction by nearly 14% in comparison to the wild type. Significant reductions were also present at three (p = 0.0015, 26.6%) and four months (p = 0.0248, 25.9%) of age (Figure 3.3A). This is indicative of delayed periodicity and reduced neural feedback mechanisms. hqY overall OP power showed no early significant changes at two, three, or four months in comparison to the wild type (Figure 3.3B). This indicated adequate uniform retinal response of the INL and GCL of the hq mice. However by five to seven months of age, overall OP power demonstrated a significant reduction (p = 0.0028) by nearly 54.5% in comparison to the wild type (Figure 3.3B). Total OP waveform energy, indicative of total retinal INL, GCL and retinal response across the entire retina, demonstrated no early changes at two or three months of age in hqY mice (Figure 3.3C). A significant decline

Figure 3.2.9Electroretinography demonstrates delayed retinal responses and

decreases in initial OP latency followed by subsequent reductions in summed OP amplitudes in the hq retina following a 10 cd•s/m2 stimulus. (A) Initial latency of OP2

indicates the functional response of the INL and GCL cells to photoreceptor hyperpolarization. Delayed initial latency of OP2 is evident at three months in hq mice with a significant delay of nearly 24.6% in comparison to the wild type. The delay in hq initial OP response is also evident at five to seven months with an overall delay by 30.8%. (B) Summed amplitude is indicative of the overall retinal response and continuity of neural feedback of the INL and GCL. Significant reductions in hq OP summed amplitude are first apparent at four months of age resulting in an overall reduction by nearly 26.5%. In five-to-seven-month-old hq mice, the disease reduces summed OP amplitudes by 54.5%. The effect of age on genotype was also examined for all measured variables (data not shown, Appendix B, Table B.6A) Sample size per cohort can be found in Figure 2.1B. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.005) and error bars represent ± 1 standard error of the mean.

Figure 3.3.10hqY mice have changes in OP waveform frequency as early as two

months of age, followed by subsequent reductions of OP power and energy following stimulus of 10 cd•s/m2

. (A) Significant OP frequency reductions were evident immediately at two months of age with a 14% decline in comparison to the wild type. OP frequency continued to decline in hqY mice at 3 (26.6% reduction) and four months (25.9% reduction) of age in comparison to the wild type. This was indicative of a prolonged INL and GCL periodicity. (B) OP power is a measure of uniform retinal response of the INL and GCL. OP power showed a significant reduction of 54.5% by five to seven months in hq mice and continued to decline until eight to ten months. (C) OP energy is the summation of total OP retinal energy and was not significantly different at two or three months of age in hq mice. However, by four months of age hq mice show a significant reduction of OP energy by nearly 28.3% which progresses to a nearly 52.3% reduction by five to seven months of age. This indicates progressive disease in the hq retina particularly in the INL and GCL response. The effect of age on genotype was also examined for all measured variables (data not shown, Appendix B, Table B.6B) Sample size per cohort can be found in Figure 2.1B. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.005) and error bars represent ± 1 standard error of the mean.

(p = 0.029) is evident at four months of age resulting in a 28.3% reduction of OP energy (Figure 3.3C). The decline in OP energy continues as the hq disease progresses and by five to seven months an overall significant reduction (p = 0.0006) by 52.3% is apparent (Figure 3.3C). No statistical conclusions can be made about OP frequency, power or total energy at eight to ten months of age as the hq cohort only contains two mice, although general trends appear to continue (Figure 3.3A, B,C).

3.4 Time-domain analysis of OPs indicate decreased summed amplitude in hq