Two juvenile macaque monkeys (Macaca mulatta) 6.3 and 5.5 kg, served as subjects. All experimental protocols were approved by the University Committee of Animal Resources at the University of Rochester Medical Center, complied with the Public Health Service policy on Humane Care and Use of Laboratory Animals, and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Axial lengths of the imaged eyes were measured with an IOLMaster (Carl Zeiss Meditec, Jena, Germany) (5 determinations for each eye, standard deviation approximately 3% of mean) in the anesthetized monkey, and used to calculate transverse and axial retinal distances. Each monkey was fitted with a rigid, gas permeable contact lens to preserve the corneal epithelium and reduce astigmatism and spherical refractive error. Standard ophthalmic trial lenses were used to reduce residual astigmatism and spherical error to values that could be corrected with the AO deformable mirror.
The fluorescence adaptive optics scanning laser ophthalmoscope (FAO-SLO) instrument, monkey imaging methods, and the post-processing methods have been previously described [10
]. For in-vivo
imaging, the monkeys were anesthetized with isoflurane at a dosage (typically 2%) sufficient to minimize large ocular movements and eliminate microsaccades. Vital signs and body temperature were continuously monitored. The monkey retinas were imaged in-vivo
with the AO system using reflected light at 794 nm ± 17 nm to generate a high-resolution fundus photograph of the peripapillary region. Two regions of interest were selected (along the temporal arcuate NFL, see Figure ), and 1000 frames were captured at each focal plane in a series of focus depths. We used a deformable mirror to focus through the retina at 0.1 D steps from the inner retinal surface, above the nerve fiber layer, to the pigmented epithelium. The Elmsley model eye was used to convert diopters of focus (F) to axial distance using the following formula: microns of focus = 4/3 [1/[power(D) +focus(D)]-1/power(D)]×106
, where P is the power of the eye in diopters, calculated by linearly scaling eye size by the measured axial length assuming an emmetropic eye. For the monkeys used in this study, 0.1D corresponded to approximately 26 μm.
Figure 1 Map of the location in the retina of monkey 1 where in-vivo images (heavy border squares) and RPC network thickness measures (dots) were made. The superior retina was prepared as a wholemount, but because this preparation compressed the tissue no measurements (more ...)
In each imaging session we used two 0.5 mL intravenous injections of sodium fluorescein (scaled to be equivalent to the standard human dose in ml/body weight) to enhance the contrast of the vasculature at the locations of interest. We obtained a reflectance video with 794 nm light and simultaneously used 488 nm laser excitation and recorded the emitted fluorescein fluorescence at 520 nm ± 35 nm. Each region of interest (ROI) was imaged at depths ranging from the NFL to the RPE in 0.1D steps. The detector gain was continually optimized to minimize the number of saturated pixels in the image. Because of the weak fluorescence signal from the retina, 1000 raw video frames were dual-registered and averaged using methods described previously [10
]. (Although high signal/noise was present in single frames obtained in the first few seconds after fluorescein injection, registration and averaging allowed us to obtain high quality images up to 30 minutes after injections, despite the faint fluorescence present at this time). Single frame and registered/averaged frame results are similar in human subjects.
The monkeys were euthanized for histological analysis. Under deep anesthesia, both monkeys were perfused, initially with 2 liters of saline to flush blood from the vascular system and then with two liters of 4% paraformaldehyde, infused over a 1 hour period. The eyes were fixed further in the eyecup for an additional 40 minutes. The retinas were cut in half along the horizontal raphe, and relief cuts were made in each section. The two in-vivo imaging areas, located in the superior retina were preserved in one tissue block. The superior retinal ROI was simultaneously incubated with three primary antibodies, one which binds to neurofilaments (rabbit polyclonal neurofilament 200, Chemicon, Temecula, CA) and the other to blood vessel endothelium (mouse monoclonal CD31, and mouse monoclonal von Willebrandts factor, Lab Vision, Fremont, CA). Dye-coupled secondary antibodies were applied to label the neurofilaments with Alexa 555 (goat anti-rabbit (H&L)) and the vessels with Alexa 488 (goat anti-mouse (H&L)) (Invitrogen, Carlsbad, CA). Then the superior retinal tissue was removed from solution, placed as a flat mount on a slide, covered in Vectashield (Vector, Burlingame, CA) to minimize fading of fluorescence, and cover slipped. The inferior retinal tissue was embedded in agar and sectioned (transverse sections parallel to the horizontal raphe) at 60 μm thickness on a vibratome (Microm, Walldorf, Germany). Every third section was reacted with the same antibodies as above, followed by dye-coupled secondary antibodies to label the neurofilaments with Alexa 543 and the vessels with Alexa 488, as well as 4', 6-diamino-2-phenylindole, dihydrochloride (DAPI) (Invitrogen, Carlsbad, CA) to identify nuclei of retinal neurons. The sections were covered in Vectashield and coverslipped.
Ex-vivo wholemount and transverse sections were imaged on a Zeiss 510 Meta confocal microscope using a 10× air objective with an NA of 0.3, an NA similar to that used to perform in-vivo FAO SLO. Six to ten 10× imaging fields were needed to visualize all areas of each section. The images were digitally combined to create one image per tissue section. The retinal location of the digitally assembled sections were registered against a color fundus photograph using vascular landmarks.
In-vivo and ex-vivo measurements
Figure illustrates the method that was used to measure the thickness of the nerve fiber and vascular layers in the retina at the locations indicated by large dots in Figure . Vessel caliber was also measured on these sections. Pairs of sections were dual-labeled for NF 200 and CD-31/Von Willenbrandt's factor. Because the fluorescence tags of the secondary antibodies (Alexa 543 and Alexa 488, respectively) differ in emission peak, we simultaneously recorded total fluorescence on a confocal microscope and separated fluorescence channels in post processing with ImageJ. Thus the images remain in registration throughout the analysis. Section A (Figure ) portrays the neurofilament label, the intensely labeled NFL and other retinal layers with light labelling. Section B (Figure ) portrays vessel labelling, showing the luminal border of retinal vascular endothelial cells.
Figure 2 Epi-fluorescence micrographs of adjacent retinal sections from the inferior retina of monkey 1 (see Figure 1 for location) labeled for (A) axons-neurofilament - N 200 and (B) vasculature -CD31/vWF. The RPC and nerve fiber layer thickness shown in the (more ...)
a. RPC thickness-
in-vivo - Thickness of the RPC bed was measured fromadaptive optics images, some of which are illustrated in Figure . The inner border of the RPC bed was taken as the first image which showed RPCs clearly and the outer border as the first image which showed the vascular pattern typical of the ganglion cell layer. Uncertainty in this measurement reflects the depth sampling interval (approximately 26 μm) as well as the estimate of which vessels were near the center of focus. We also measured maximal RPC thickness in four quandrants of each image and averaged the four measures.
Figure 3 Through-focus series of in-vivo adaptive optics images of RPCs from two retinal locations in monkey 1 and one retinal location in monkey 2. One location in both monkeys is near the optic disc; monkey 1 (a, d, g, j) and monkey 2 (c, f, i, l) and the second (more ...)
ex-vivo - RPC thickness was determined by measurements on transverse sections, as illustrated in Figure , of the distance from the most superficial (vitread) NFL vessels to the outermost RPC boundary, which is determined as the inflection in pattern between the extremely long capillaries with thin walls, little variation in caliber and parallel-linked structure oriented parallel to the NFL axons, and the polygonal pattern typical of the ganglion layer and retinal vasculature. The outer (sclerad) border of the RPC bed was also correlated with the location of the most superficial ganglion cell dendrites, which were highly visible due to neurofilament labelleing. These patterns were easily distinguishable within in-vivo images as well as histologic transverse sections. This measurement is illustrated in Figure as the distance between the two black arrows on a section close to the optic nerve head whose location is shown in Figure . Each ex-vivo result represents the average of five measurements over a 50 micron region centered at each data point.
b. NFL thickness - NFL thickness was determined by measuring neurofilament label in transverse sections as illustrated in Figure . Each result represents the average of five measurements over a 50 micron region centered at each data point. NFL thickness could not be measured from in-vivo imaging data.
c. vessel pattern - the vessel pattern within versus beneath the RPC bed was determined by appearance (RPCs, figure ,, and ) and (ganglion cell vasculature, figure . and ).
d. vessel diameter - was measured at 20 randomly chosen locations on each of the 6 in-vivo AOSLO RPC images illustrated in Figure , and using the open-source NIH software package, ImageJ. Twenty (20) random x, y locations were generated on each image and a measurement was taken of the caliber of the nearest RPC vessel by drawing a line segment across the vessel, whose length was registered by the software. Similar measures were made at 20 randomly chosen locations on each digital image from 6 sections from the lower retina of monkey 1, 3 sections at each of the two locations that correspond to the in-vivo imaging of the upper retina.