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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 28, 2001 as doi:10.1096/fj.00-0666fje. |
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Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA
2Correspondence: Department of Ophthalmology and Visual Sciences, Box 8096, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO, 63110, USA. E-mail: Neufeld{at}vision.wustl.edu
SPECIFIC AIMS
We have used a rat model of ischemia/reperfusion injury of the retina to study various risk factors that are relevant to neural injury in the human central nervous system (CNS). We have determined whether age, diet, diabetes, the presence of pigment, or chronic moderately elevated intraocular pressure (IOP) influence the degeneration of retinal ganglion cells (RGCs).
PRINCIPAL FINDINGS
In all experiments, quantitation was by retrograde labeling of RGCs with Fluoro-Gold and counting of RGCs in retinal flat mounts. Ischemia/reperfusion was by anterior chamber cannulation and elevation of the IOP above systolic blood pressure.
1. Effect of aging on RGC survival
There is an inherent loss of RGCs with age. The density of
RGCs is less in the older animals at equivalent retinal positions. In
2-year-old animals, the number of RGCs in the central retina (781 +/-
35 cells/field; mean +/- SE) is 25% less than in
2-month-old animals (1037 +/- 75 cells/field; P < 0.05).
In the peripheral retina, 2-year-old animals had 40% fewer RGCs
(276+/-7 cells/field) than 2-month-old animals (459 +/- 49
cells/field; P < 0.05). Assuming a linear decrease over
time, the slopes indicating the rate of the loss of RGCs are about the
same in the central and peripheral retina. Thus, although a larger
percentage of RGCs are lost with age in the peripheral retina than in
the central retina, the number of RGCs actually lost in the peripheral
and central retina is similar because of the higher density in the
central region. Therefore, a relatively constant number of RGCs die
with age in different retinal locations.
The remaining RGCs in the retina of older rats are more susceptible to
ischemia/reperfusion damage than RGCs in young rats. The percentages of
RGCs lost 1 wk after ischemia in the 2-year-old animals were 38 +/-
1% in the peripheral region and in the central region, 28 +/- 3%.
These values were significantly different from 2-month-old animals,
which were 21 +/- 3% (P < 0.01) in the peripheral region
and 15.25 +/- 1% (P < 0.01) in the central region,
respectively (values for the controls in Fig. 1C
, D
).
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2. Effect of caloric restriction on RGC survival
As expected, the body weights of rats in the caloric restricted
groups were significantly lower than the body weights of the rats in
the control groups after 12 wk. In young animals, there was 25% less
body weight gain over this period in the caloric restricted group
compared with the control group. In old animals, which were no longer
gaining weight, caloric restriction caused a 16% loss of body weight
after 12 wk compared with controls (Fig. 1A
, B
).
Young animals on caloric restriction suffered less damage to their RGCs
in response to ischemia/reperfusion (Fig. 1C
, D
). In the
peripheral retina, the percentage RGCs lost in animals that were under
caloric restriction was decreased significantly, although there was no
statistical difference in the central retina. Similarly, in old animals
that were caloric restricted, the percent of RGCs lost in the
peripheral retina was decreased significantly (Fig. 2
). Thus, caloric restriction was neuroprotective against ischemia for
peripheral RGCs in young and old animals.
|
3. Effect of diabetes on RGC survival
There was 22% less body weight gain over 8 wk in the diabetic
group (intravenous streptozocin; blood glucose 400 mg/dl) compared
with the control group. Comparing normal eyes between the diabetes and
control groups, the number of RGCs in the diabetes group (819 +/- 141
cells/field) was not different from the control group (798 +/- 129
cells/field) in the central retina. In the peripheral retina, there was
also no significant difference between the diabetes group (396 +/- 66
cells/field) and the control group (322 +/- 72 cells/field). Thus, the
diabetic condition does not cause loss of RGCs.
When diabetic animals underwent retinal ischemia/reperfusion, there was
a small but significantly greater percentage RGC loss in the peripheral
and central retina of the diabetic group than in the nondiabetic
control group (Fig. 2)
. Thus, despite lower body weight, preexisting
diabetes causes increased susceptibility of RGCs to
ischemia/reperfusion damage.
4. Effect of glaucoma (chronic, moderately elevated IOP) on RGC
survival
The loss of RGCs due to ischemia/reperfusion in eyes that had
long-standing chronic, moderately elevated IOP was compared with eyes
that had normal IOP. The animals in both groups were the same age at
the time that retinal ischemia/reperfusion was performed, but eyes with
long-standing chronic, moderately elevated IOP had 21% fewer RGCs. The
percent loss of the remaining RGCs in the eyes with glaucoma after
ischemia/reperfusion (26 +/- 3%; mean +/- SE) was
significantly greater than in eyes with normal IOP (16 +/- 1%;
P < 0.05). Thus, RGCs in eyes with preexisting glaucoma
were more susceptible to ischemia/reperfusion damage.
CONCLUSIONS
Our experiments demonstrate that the loss of neurons due to ischemia/reperfusion damage can be influenced by many of the conditions that are considered risk factors in human neurodegenerative diseases. We have used as our model the loss of RGCs after retinal ischemia/reperfusion. This is a useful model because CNS neurons can be exposed to damaging conditions without violating the blood–brain barrier, all of the RGCs can be labeled retrogradely for specific identification, and the loss of RGCs can be quantified by comparing the experimental eye to the contralateral, control eye of the same animal. There are few, if any, other models of CNS degeneration that offer such specificity and quantitation. The results that we have obtained by studying RGCs with this model may generalize to other retinal and CNS neurons.
Our focus on RGCs is based on our interest in glaucoma. In this human disease, RGCs degenerate in response to the effects of elevated intraocular pressure. Elevated IOP is considered a primary risk factor. Other risk factors for developing glaucoma are older age, race, and diabetes. The definition of ‘risk factor’ in the context of this disease relates to the development of visual field abnormalities, for which the underlying changes are due to the degeneration of RGCs. Thus, in an experimental setting, such risk factors would be expected to influence the susceptibility of RGCs to a standardized, reproducible damaging stimulus.
From our studies, aging is the most significant risk factor for the loss of RGCs. As the animal ages, there is an inherent loss of RGCs similar to that reported for the cells of the outer nuclear layer and the inner nuclear layer of the retina. The loss of RGCs as the rat ages relative to its life expectancy appears similar to that reported for the human, suggesting that the rate of the inherent, age-related loss of RGCs is related to life span.
There are fewer RGCs in the retina of an aged rat. These remaining RGCs could have more, less, or the same susceptibility to damaging stressful conditions. Our data clearly indicate that when the remaining RGCs throughout the retina of an older eye are stressed by retinal ischemia/reperfusion, a greater percentage of these cells degenerate in old animals than in young animals.
The factors underlying the loss of RGCs with age and the greater loss of RGCs in older animals after ischemia/reperfusion damage are unknown. Undoubtedly factors that promote increased susceptibility must relate to changes in genes and/or gene products. Age-related changes in genetic expression in a tissue are only beginning to be studied with the availability of microarray technology. In our experiments, we could not identify which cells were contributing to the increased susceptibility. With age there may be changes in gene expression in the blood vessels in the tissue, in the glial cells and/or in the neurons themselves that contribute to increased damage after retinal ischemia/reperfusion.
Caloric restriction has been studied extensively and is the only regimen that extends both median and maximum life span in rats and other animals. In the CNS, caloric restriction reduces ischemia/reperfusion damage in the middle cerebral artery occlusion model of stroke. Our data demonstrate that caloric restriction is neuroprotective for peripheral RGCs against ischemia/reperfusion injury in both young and old animals. As discussed above for the increased susceptibility with age, the cellular and molecular changes in a caloric restricted animal that impart neuroprotection and which cells contribute to neuroprotection in these animals are unknown.
We also sought to determine whether a history of chronic, moderately elevated intraocular pressure would make the remaining RGCs more or less susceptible to ischemia/reperfusion damage. The eyes studied in this experiment had lost a considerable number of RGCs due to the chronic, moderately elevated IOP, had optic nerve cupping, and a loss of axons in the optic nerve due to the glaucomatous state. However, the remaining RGCs in these eyes were more susceptible to the ischemia/reperfusion challenge. This implies that the remaining RGCs were somehow compromised or had a lower threshold of susceptibility because of the glaucomatous state.
We have used a relatively straightforward approach to determine whether
clinically significant risk factors for retinal ischemia can be modeled
experimentally. Our results clearly demonstrate that older age and
previously existing disease states, like diabetes and glaucoma,
adversely affect the susceptibility of certain neurons in the retina to
the damage associated with ischemia/reperfusion (Fig. 3
). Our experiments demonstrate that risk factors modifying CNS injury
can be quantitatively modeled, setting the stage for investigating the
underlying cellular and molecular mechanisms that alter
susceptibility.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0666fje ; to cite this
article, use FASEB J. (March 28, 2001) 10.1096/fj.00-0666fje 
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IOP: elevated intraocular pressure.