Chapter 2 Methodology and technique development
2.5. Light-dark preferential test (LDPT)
2.5.4. Statistical analysis and strategy
On the advice o f statisticians from the Department o f Psychology, University of Sheffield, ANOVA (analysis o f variance) was the chosen statistical method for data analysis. ANOVA is a powerful parametric statistical method that applies to
categorical independent variables associated with continuous dependent variables. It is a natural and logical extension o f the t-test because it compares means between groups. It has greater application than the t-test because comparison between two or more groups can be made. ANOVA uses the F probability distribution and, like the t- test or chi-square test, degrees o f freedom must be taken into account. Here both one
way and two-way ANOVA analyses are required and multiple factors influences are also taken into account. Here it is used to test the null hypothesis o f no difference between different groups.
An analysis o f variance (ANOVA) was performed on the data using a commercial statistical package (StatView, SAS Institute Inc, USA). In the cross-sectional study, a 3-factor ANOVA with one repeated measure (Group [normal versus dystrophic] x Age [6 weeks, 3 months, 6 months, 12 months] x Test [RUN 1, RUN 2]) was used to analyse the data. Test was the repeated measure. For the longitudinal study, a 2-factor ANOVA with one repeated measure (Group [normal versus dystrophic] x Age [6 weeks, 3 months, 6 months, 12 months]) was used to analyse the data. Age was the repeated measure. The aim was to examine whether normal sighted mice would spend significantly more time in the dark compartment compared to visually impaired dystrophic rd mice. The hypothesis was there was no difference in the time they spent in the dark compartment.
The transplant animals were separated into two groups, a histology-positive group and a histology-negative group, based on the presence or absence o f graft post transplantation. Using these criteria, a 2-factor ANOVA with one repeated measure (Group [histology-positive, histology-negative. Sham] x transplantation [pre transplantation, post-transplantation]) was used to analyse the data. Pre- and post transplantation measurements were the repeated measure. The hypothesis was that there was no difference in DCT between the histology-positive group and the others.
2.6, Retinal threshold measurement
Dr Y. Sauvé performed the retinal threshold measurements in our electrophysiology laboratory at the Institute o f Ophthalmology. My involvement was in preparing the animals and in helping to collect the data and assisting Dr Sauvé with the
experiments. Animals were anaesthetised with urethane (1.25 g/kg, intraperitoneally), the head held by a nose bar and the test eye stabilised with three equidistant
subconjunctival sutures attached to the fixation frame. This provided maximal eye stability without substantially obstructing the animal's field o f vision. In order to eliminate the potential effects o f variations in resting pupil size, full dilation was obtained with topical Tropicamide (1.0%; Alcan Laboratories Ltd, UK). A non correcting contact lens protected the cornea. Body temperature was monitored and maintained at 37°C. A translucent hemisphere (Ganzfield bowl, 110 cm in diameter) was centred on the optic disc projection o f the tested eye as visualised by an
ophthalmoscope (Figure 6 A). A one-hour period o f adaptation at 0.02 cd/m^ was implemented prior to recording to ensure rod activation. Single- and multi-unit recordings were made from the superficial layers o f the SC to a depth o f 100 - 300 pm using glass-coated tungsten electrodes (custom made in Dr Sauvé’s laboratory; resistance: 0.5 MQ); care was taken to record sufficiently superficially to avoid the larger field cells o f the deep stratum griseum superficiale and stratum opticum. Preliminary studies in which the SC was exposed by aspiration o f the overlying cortex indicated that recording sites (localised by stereotaxic positioning) covered the full extent o f the SC with the exception o f its medial bank, which was located just under the superior sagittal sinus. No cortical ablations were made in this study in order to avoid possible alterations o f SC light-evoked responses. The microelectrode penetrations were made along a rectilinear grid o f 100 pm steps. At sites where a
response to whole-eye illumination was found, an attempt was made to define a discrete receptive field (RF) for that unit. The visual field was systematically searched by moving spots o f light (38mm diameter with luminance o f up to 4.5 log cd/m^ higher than background) across the surface o f the translucent hemisphere (background luminance kept at 0.02 cd/m^). Once a response could be elicited in this manner, the RF was located and its centre defined. The RF was then illuminated with a flashing light centred on the RF (spot o f 38 mm in diameter with 1 second duration at 3 seconds intervals). Neutral density filters were added progressively (minimal steps o f 0.1 log filters; unfiltered stimulus was 4.5 log illumination above
background) to establish the minimal illumination required to elicit a response.
Threshold values for a total o f about 76 points (usually separated by 200-pm recording steps in the SC) were presented as maps with topological reference to the retina (Figure 6B). In area where there was no response even with the highest setting, symbol “n” is designated or an auditory response when there was no response with light, symbol “a” is designated in the results.
To further characterise the electrical signal recorded, thus responsiveness o f the RF illumination, a post-stimulus time histograms (PSTH) can be assembled over 30 consecutive stimuli (1-second duration, 3-second intervals). To maximise the response a stimulus of 4.5 log unit above background is used. PSTH reveals the latency o f the ON-response (seconds), the activity o f the response in number of spikes o f electrical signal and the duration o f the ON-response.
Baseline study
A total o f 3 normal mice (one 6-week-old, two 3-month-old) and 9 dystrophic mice (five 8-week-old, one 9-week-old, three 3 -month-old) was used for the baseline study. A 76-point retinotopic map was plotted across the SC as described above. For operational reasons, all the baseline recordings were performed on the right eye. As the electrophysio logical test was a terminal experiment (no recovery from anaesthesia was allowed according to Home Office regulations), animals were sacrificed after each experiment and the eyes were harvested for histology as described above.
Transplanted group
A total o f 18 dystrophic rd mice that received “good” retinal transplants, as defined by good intra-operative subretinal graft appearance observed through the operating microscope, were included in the study. All the threshold testing was performed 2 to 3 weeks after transplantation when the mice were between 8 to 11 weeks o f age (Table 7). All the animals received bilateral transplants and only the side with the “good” transplant was tested, therefore a total o f 18 retinotopic threshold maps were obtained (Figure 11).
Table 7. Retinal threshold measurement time points for transplanted animals. (Subject codes relate to animal from Figure 11)
Subjects Side Age at time of tests (weeks) Weeks after transplantation
XIII R 10 3 XIV L 10 3 XV L 10 3 XVI R 11 3 XVII R 11 3 XVIII R 11 3 XIX R 8 2 XX R 8 2 XXI R 8 2 XXII L 9 3 XXIII L 8 2 XXIV L 8 2 XXV L 11 3 XXVI R 10 2 XXVII L 9 3 XXVIII R 9 3 XXIX L 9 3 XXX R 9 3
Figure 6
Retinal threshold measurement apparatus. (A) The retinal threshold measurement apparatus is seen. The light source is mounted on guide wires behind the semi
transparent hemisphere (the ganzfield bowl). The subject (ARROW) is mounted with a nose bar and positioned at the centre o f the hemisphere. (B) Schematic diagram showing the electrode penetrating into the superficial layer o f the superior colliculus for threshold measurement. When a sufficient light stimulus is presented and the retinal threshold is reached at a particular point o f the retina, an electrical signal o f on- and off- responses can be recorded. Threshold is measured by inserting different neutral density filters in front of the tested eye until no recording can be made. A 76- points retinotopic map is eventually obtained