Jared R. Jackson and Dr. Robert Seegmiller, Physiology and Developmental Biology
Introduction
Stickler syndrome, caused by a mutation of the Collagen 2A1 gene, is the most common cause of inherited retinal detachment in children. A mutation of the same gene in mice causes Disproportionate Micromelia (Dmm). “In cases where mouse mutations occur in genes that are also responsible for human disorders, the mouse strains provide essential model systems for studies of pathogenetic mechanisms.” The purpose of this study is to find and document evidence of retinal detachment that is hypothesized in heterozygous (Dmm/+) mutant mice.
Retinal Detachment
In mice, histological identification of retinal detachment is distinguished from retinal detachment caused by human error during tissue preparation (also known as artifact) by photoreceptor degeneration and the presence of subretinal fluid (SRF). Because of their dependence on the choriocapillaries, photoreceptors degenerate after only hours of separation from the pigment epithelium of the eye. Proteinaceous fluid frequently accumulates beneath the retina and is strongly eosinophilic. When the detachment has persisted for a few weeks, macrophages or mobilized retinal pigment epithelial cells accumulate in the subretinal space and in the SRF.
Methods
Mice heterozygous for the COL2A1 mutation (Dmm/+) age 6 and 10 months were compared with control mice of the same age. Mice were euthanized via carbon dioxide asphyxiation followed by cervical dislocation, then both eyes from each animal were taken for study. Each eye was entirely removed by grasping the tissue deep to the globe of the eye with curved forceps and gently pulling outward; the tissue were fixed in 4% paraformaldehyde in TBS solution to stop the natural deterioration of the tissue. The tissue was then processed and then embedded in paraffin. The tissues were oriented such that they produced 6 μM thick sagittal sections when cut on a microtome. Sections were placed on slides and stained with Hematoxylin and Eosin (H&E).
Tissues from 6 month old animals were processed in the Seegmiller lab, then cut and stained with H&E by lab technicians at the Utah Valley Regional Medical Center (UVRMC) histology lab. Tissues from 10 month old animals were fixed at the Seegmiller lab, then processed and cut at the UVRMC lab. After analysis of the 6 month old tissues, we decided to have some of the 10 month old tissue sections stained H&E and some with a trichrome stain in an attempt to better visualize what we think are macrophages in the photoreceptor layers of the specimens.
Analysis
Prepared slides of the heterozygous mutants were analyzed and compared against those of the control mice in an attempt to identify differences. Control mice should show no signs of retinal detachment. Retinal detachment in the mutant with the presence of SRF, the presence of a large number of macrophages, or both will suggest that the Col2A1 mutation is causing the detachment. Retinal detachment without the presence of SRF or a large number of macrophages indicates a preparatory artifact.
Months
These specimens showed the same signs of retinal detachment due to preparatory artifact that the control specimen did. I then looked for the presence of SRF and macrophages to confirm that the mutation was part of the cause. Some of the sections showed what may be SRF. There was also an abundance of small clear cells located in the photoreceptor layers of the detached retina. The hypothesis is that these are macrophages which have accumulated to dispose of degenerating photoreceptors following retinal detachment while the specimen was still alive. This would suggest that the mutation does actually cause retinal detachment in mice.
10 Months
These specimens also showed signs of retinal detachment due to preparatory artifact, especially cracking and tearing of the retina. There was an increased incidence of macrophages compared to the control, but not as significant a number as the 6 month sample. There was also no definitive evidence of SRF. Interestingly, there was an increased incidence of macrophages. This may be because this layer has also begun deteriorating due to the mutation.
Conclusion
This study gives preliminary if not definitive evidence that there is a difference in the eyes of mutant mice and control mice. Though the number of macrophages in the 10 month sample was fewer than expected, their abundance in the 6 month old sample shows promising evidence that with increased sample size this model can be verified as one for the study of human eye defects.
I was disappointed that I was unable to avoid the preparatory artifacts in this study—my recommendation for the continuation of the study is to embed the tissue in plastic as it is more easily cut without shattering and tearing than paraffin-embedded tissue. It appears that paraffin has trouble infiltrating the entire eye, leaving hollow pockets in the tissue which cause much of the artifact I encountered. Plastic resin should infiltrate more completely and fix this problem.
A continuation of the study is currently underway using the recommended method, and a few samples of tissue have been embedded in plastic and been cut much more cleanly than those embedded in paraffin. I am optimistic that this method will rid the tissue of artifacts so prevalent in paraffin-embedded tissue. Eliminating the retinal detachment due to preparatory artifact would make the analysis of tissues much simpler, and defects would be easier to detect.
References
- Stickler Syndrome. The Iris Fund, http://www.berf.org.uk/pdf/stickler_syndrome.pdf
- Disproportionate micromelia (Dmm) in mice caused by a mutation in the c-propeptide coding
region of Col2a1, Pace, J. M., et al, Developmental Dynamics, 208:25-33, 1997. - Systematic Evaluation of the Mouse Eye: Anatomy, Pathology, and Biomethods, pp. 216-217,
262-266, Smith, R. S. et al, 2002. - Histochemical Immunoflourescence and Ultrastructural Differences in Fetal Cartilage Among
Three Genetically Distinct Chonrodystrophic Mice, Seegmiller, R. E. et al, Teratology, 38 (6): 579-
592, 1988 - Euthanasia, dehydration, and staining procedures are found in the Seegmiller research lab SOP
manual.