The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.
Part 1: Pre-processing of tissue
Part 2: Removal of the eyeball from the orbital cavity
Part 3: Dissect the retina from the eyecup
Part 4: Sampling
Part 5: Representative Results:
In our laboratory, we routinely perform immunohistochemistry on cryosectioned retinae (Figure 2). In this case we are interested in the isodensity of cannabinoid receptors (CB1) in the primate retina. We also examine the effects of prenatal ethanol exposure on the primate visual system. To this end, we are interested in cell density and layer thickness in the fovea and in the peripheral retina. To accomplish this, we embed pieces of retina in Epon, slice at 700nm on an ultramicrotome, and stain with 1% toluidine blue (Figure 3).
Figure 1 Flatmount Retina. Radial cuts are used to flatten the retina.
Figure 2 Immunostaining. Cryosection of peripheral retina immunostained for CB1 where the retinal ganglion cells are heavily labeled with some labeling in the inner and outer nuclear layers. The thickness of this section is 14 m and stained on the slide. RG – retinal ganglion layer; IP – inner plexiform layer; IN – inner nuclear layer; OP – outer plexiform layer; ON – outer nuclear layer.
Figure 3 Fovea. Section through the fovea that was embedded in Epon and sliced on an ultramicrotrome at 700nm. The photoreceptor layer (PR) was used to align the sections. Notice that the entire extent of the photoreceptors can be identified indicating an appropriate angle in the coronal plane. Density measurements were taken at 300 m, 500 m, and 800 m from the center of the foveal pit using the Bioquant Imaging system.
RG – retinal ganglion layer; IP – inner plexiform layer; IN – inner nuclear layer; OP – outer plexiform layer; ON – outer nuclear layer; scale bar = 50 m
The preparation of the retina as a wholemount allows for the analysis of the topography and spatial distribution of either the ganglion cell layer or the endothelial cells of the retinal blood vessels3. Quantification of cell density in the periphery of primate retina is readily accomplished. however, in perifoveal and foveal regions, the stacking of multiple layers in the ganglion cell layer obstructs quantification. To circumvent this potential bias, the fovea and perifoveal region can be dissected from the wholemount preparation, embedded in Epon, and serially sectioned using an ultramicrotome to obtain semi-thin sections in the coronal plane2,4. There are a number of other disadvantages to the wholemount preparation, which can be overcome with alternative sampling paradigms.
Taking isometric samples from the retina and sectioning in the coronal plane on either a cryostat or vibratome allows for specific examination of the different layers, which cannot be readily performed on a wholemount preparation. Sectioning in this manner also allows for the application of multiple immnohistochemistry protocols5. These sections can then be removed from the slide, embedded in Epon and sliced on an ultramicrotome. With an ultramicrotome minor changes in the cutting angle can be made to ensure a standard coronal plane through the photoreceptors. Once a standard plane has been obtained, layer thickness can be measured and compared between retinal regions and subjects. Furthermore, Epon embedded tissue can be used in electron microscopy studies to reveal ultrastructural characteristics of the retina6.
The authors would like to thank Ikiel Ptito for his technical support. We are grateful to Frank Ervin, Roberta Palmour and the staff of Behavioural Sciences Foundation Laboratories located in St Kitts, West Indies, for their continued support of our primate work.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Scalpel | Fine Science Tools | 10003-12 | ||
Scalpel blades | Fine Science Tools | 10011-00 | ||
Spring scissors | Fine Science Tools | 15020-15 | ||
Scissors | Fine Science Tools | 14090-11 | Any surgical scissors are sufficient | |
Rongeurs | Fine Science Tools | 16121-14 | ||
Forceps | Fine Science Tools | 11027-12 | ||
Filter paper | Fisher | 09-924-150 | ||
Camel or Sable Hair paintbrush | Art supply store |