Ординатура / Офтальмология / Английские материалы / Ocular Neuroprotection_Levin, Polo _2003

The prior light-rearing conditions of experimental animals, age, and diet are additional factors that influence light damage. Irrespective of the type of light exposure used, dark-reared rats are about twoto threefold more susceptible to light damage than comparable cyclic light reared animals. Dark-reared rats also incur RPE cell damage, whereas in cyclic light–reared rats the RPE is less involved. Young cyclic light–reared rats ( 25 days) are generally quite resistant to light damage compared with adult animals [38]. Among adult rats we found a twofold increase in visual cell loss at 12 months versus 2-month-old animals after 24 h green light exposures. Ironically, dark rearing resulted in no age-related increase in retinal light damage, but those animals were still more susceptible to damage than cyclic light reared animals [39]. In rats fed vitamin A–deficient diets, or in those fed essential fatty acid–deficient diets, retinal light damage is also decreased [40,41]. Recently we tested the start time of light exposure as a variable. Our findings indicate that light exposure beginning at 1 a.m. causes oneto twofold more damage than light exposure at 9 a.m. [42]. With the exception of mydriatics, anesthetics such as halothane also reduce light damage susceptibility in rodents by preventing rhodopsin regeneration [43]. These factors have been summarized in Table 2.

C.Outcomes of Retinal Light Damage

Depending on the types of information sought and the measurements used, conclusions regarding the extent and nature of light-induced cell loss can vary. Among the typical techniques used—histology, electrophysiology, and biochem- istry—each has its limitations, and correlative measurements are recommended. In this section we point out some of these limitations and other problems associated with interpretations based on the time course of retinal degeneration. Our

Table 2 Some Experimental Factors Which Alter Retinal

Light Damage in Rats

approach will be a quantitative assessment of visual cell loss arising from the synchronous involvement of photoreceptors during intense light exposure.

1. Measurements of Visual Cell Loss

Much of our understanding of lights’ pathological effects on the retina has come from histology. It is the most widely used approach, providing information on the types of cells involved and their locations within quadrants of the retina. No other approach is as good in describing organelle damage within cells during or after light exposure. From such studies we know that the entire visual cell becomes involved during damage, including the ROS, inner segment mitochondrial rich region, nucleus, and even the synaptic end plate. Depending on the type of light and the animal model used RPE damage also occurs, either simultaneously or shortly after visual cell damage [6,38]. The major drawback is that histology is descriptive and is often presented as a “snapshot” that is interpreted as representative damage in the entire tissue. While this may be true, the examples chosen frequently are those that depict the greatest morphological changes. Accordingly, histological measurements that assess visual cell loss by morphometric analysis over the entire eye, including ONL thickness, ROS length, and/or rod cell nuclear counts, are required. Such approaches require serial sectioning and proper cell alignment for accurate conclusions regarding ONL volume and ROS length. Obviously this is painstaking and tedious work, requiring considerable expertise. One of the first examples of this approach was published by Rapp and Williams [26] in their detailed analysis of the superior and inferior hemispheres of lightdamaged rat eyes.

Electroretinography is one of the best examples of unbiased functional analysis of photoreceptor survival and loss. Its strength lies in its repetitive use for following ERG changes in the same animals. By comparing scotopic A- and B- wave amplitudes, latencies, and thresholds, which reflect the summed electrical signals generated primarily by intact photoreceptors and Muller cells, much can be learned about the extent of visual cell damage and its time course. Photopic analysis, to determine cone cell responses, provides valuable functional measurements of histologically identifiable cell types that survive light damage. This is particularly useful in cone-dominant animal models because purified cone pigments have never been isolated from the retina. Unfortunately, simple ERG analysis and morphometry of visual cell loss do not always correlate as well as expected. This may be related to RPE cell damage and the subsequent shunting of current generated in response to light stimuli [3]. Despite this limitation, electroretinography, using Ganzfeld illumination, provides an average summated response from the entire population of synaptically intact photoreceptors remaining after light damage. In one of the few comprehensive studies comparing ERG analysis and morphometry in light-damaged rat retinas Sugawara, Sieving, and

Bush [44] report a high degree of correlation between the two techniques and discuss potential drawbacks in electroretinography. The interested reader is referred to this thorough study.

Classical biochemistry offers many opportunities to assess retinal damage and cell loss because of the variety of measurements that can be made with the same tissue. As with electroretinography and morphometry, the two eyes of the same animal can also provide correlative assessments of light damage. This is based on many studies showing that both eyes have identical photoreceptor cell numbers and components, and that when intense light exposures are properly conducted the extent of visual cell loss is the same. Typically, biochemical measurements are “average responses” determining changes in the entire tissue that remains following damage or cell loss. Because acute light-induced retinal degeneration induces synchronous cell involvement, much has been learned about the mechanism(s) of light damage and cell death from these techniques. The relative ease by which biochemical measurements can be made is another advantage, but the choice of measurement and the time course of damage can lead to erroneous conclusions.

Because rhodopsin is essentially contained within the ROS of rod photoreceptors and damaged cellular material is removed by the RPE, it can be used as an endpoint determination of rod cell survival following light damage. When compared with the rhodopsin level in similarly dark adapted, but unexposed, littermate controls, the extent of visual cell loss can reasonably be quantified. Although this technique works well in normal rats, in RCS rats its use alone is unsatisfactory. Because phagocytosis is deficient in these animals, rhodopsin accumulates in the partially degraded photoreceptor debris, and estimates of visual cell loss by DNA measurements differ by as much as 50% from simple rhodopsin determinations [30].

Through the activation of endonucleases, DNA is degraded in dying photoreceptors, even in the absence of phagocytosis by RPE. However, DNA is also present in cells of the inner nuclear layers. To quantitate photoreceptor cell DNA losses, therefore, it is necessary to subtract the DNA content of the inner retina layers. Based on this approach and using old RCS rat retinas, which have lost their photoreceptor cells, we determined that about 70% of retinal DNA is contained within the ONL. By using this subtraction technique, reasonable conclusions about visual cell loss and DNA fragmentation in photoreceptors can be achieved from retinal DNA measurements following light damage.

Similarly, about 50% of retinal protein and lipid is contained within the photoreceptor cell layer of the adult rat retina. Because extensive light-induced visual cell loss can result in the loss of up to one half of the retinal protein, this needs to be controlled for in typical enzyme measurements. For example, the activity of trans retinol dehydrogenase, which is present in photoreceptors, is decreased by as much as 25% following light exposure, but the Muller cell en-

zyme glutamine synthetase (GS) is unaffected by light [45]. These measurements need to be reported per retina, because GS activity per milligram of protein would show about a 50% increase; clearly a different and erroneous conclusion. Therefore, despite the relative ease and advantage of biochemical measurements, knowledge of the distribution of retinal proteins, enzymes, DNA, or lipid within the normal retina is essential for quantitative assessments of visual cell loss in degenerating tissues.

2. Time Course of Damage

The relative order of events leading to retinal damage and degeneration is important, not only from a mechanistic standpoint but also for possible therapeutic interventions. Time course measurements beginning with the light-mediated bleach of rhodopsin (the trigger for light damage) and proceeding through the final common pathway of cell death can also lead to reliable conclusions regarding endpoint determinations of retinal cell loss. As cellular apoptosis appears to be a primary cause of retinal degenerations in animal models and in human disease we can also learn much from its measurement. There are, however, potential problems associated with time course measurements of retinal light damage and cell loss.

The decision to use one type of measurement or another in light damage studies rests on the important variable of when the determination is made. In rats a somewhat normal ERG is recordable for several hours after intense light treatment and rhodopsin regenerates in darkness to about 60% of control over a period of 6 h to 1 day even though photoreceptors are severely damaged [25,63]. Subsequently, the ERG declines and the level of rhodopsin decreases as photoreceptors die and degenerate over the next 4–6 days. Ten to 14 days after intense light, most of the degenerated photoreceptors have been removed and repair in the surviving cells is nearly complete. At this time morphometry, ERG, and biochemical measurements more accurately reflect total visual cell loss.

Over the same 2-week “recovery period,” retinal DNA levels decline from about 90% of control immediately after light exposure to a final level commensurate with the extent of visual cell loss and the percent of rhodopsin loss. Thus, although light leads to DNA degradation, often there is little measurable loss during a typical light exposure period. Conclusions regarding the nature of DNA degradation also depend on the timing of the measurements and the technique used. An example of this is shown in Figure 5, which depicts neutral and alkaline gel electrophoretic analysis of DNA from rat retinas.

Using neutral gels, a pattern of DNA ladders is present in cyclic light– reared rats immediately after 24 h of light exposure. This pattern is not as apparent in dark-reared rat retinas until 2 days after a 3 h light treatment. In both cases DNA ladders are not present 4 to 16 days after light treatment. Alkaline gel

Figure 5 Gel electrophoresis of rat retinal DNA. (A,B) neutral agarose gel electrophoresis (C,D) alkaline agarose gels. Each lane was loaded with a total of 2 g DNA from three different rat retinas. (Adapted from Ref. 46.)

electrophoresis, which detects single strand breaks in DNA and larger fragments, shows a pattern of DNA breakdown that coincides with the apoptotic pattern in cyclic light rats, but that precedes the same pattern in dark reared rat retinas by at least 12 h. What does this mean? Simply put, if DNA electrophoresis is conducted 4–16 days after light exposure, no evidence of the nature of DNA degradation is detectable. Furthermore, by neutral gel analysis, apoptosis could be detected in one group of rats after 12 h in darkness but is barely detectable in the other group of animals. While this example is for DNA [46], similar time-depen- dent differences in retinal gene expression and protein or enzyme analysis can be found. The bottom line is that conclusions based on single time-point determinations of the extent or nature of pathological changes in the retina can vary considerably depending on when those measurements are made.

D.Prevention of Retinal Light Damage

Having discussed some variables associated with light-induced retinal degeneration, the goal of this section is experimental design related to its prevention. The literature is replete with claims that one treatment or another reduces the extent of visual cell loss from light, but upon further analysis these findings are often

viewed as marginal. That is not to say that extending visual cell life by, say, 5 to 10% is not important. As almost any clinician can attest, there are patient quality-of-life benefits from preserving vision in age-related or genetically based conditions. Because the mechanisms of retinal cell loss in human disorders and experimental retinal degenerations bear a striking resemblance, well-designed interventions in animal models of light damage offer hope for future clinical trials. With an aim toward improving the efficacy of drug, antioxidant, or dietary treatments, we discuss some of the problems associated with these in animal models.

1. Endogenous or Adaptive Processes

The relative susceptibility or resistance of the retina to light damage depends on a variety of conditions already discussed (see Table 2). These include rearing conditions and age, both of which contribute to changes in retinal gene expression. Because these changes precede intense light treatment, they are endogenous in nature and can greatly affect experimental outcomes. Simple differences in rhodopsin levels, or other visual cell proteins, which are altered by rearing conditions or age, indicate that control measurements should be made before light damage experiments are done. The same is true for transgenics or retinal gene knockout models as these can alter in unexpected ways other proteins or processes in the retina. For example, transgenic rd mice overexpressing Bcl-2 were found to be resistant to retinal light damage, but also to have lower rhodopsin levels than controls; a possible reason for the finding [47]. C-fos knockout mice also have reduced rhodopsin levels and fewer photoreceptor cells than c-fos / animals [48], although the basic conclusion regarding the importance of this transcription factor in light-induced apoptotic cell death is unchanged.

In addition to providing mechanistic insights into light damage, the use of genetically modified animal models has implications regarding the progression of inherited or age-related retinal disease. Early work with RCS rats, which have a recessive mutation in phagocytosis of ROS tips by RPE, showed that both lightrearing conditions [49] and intense light exposure [50] accelerate the rate of retinal degeneration. In albino mice, LaVail et al. [23] found light damage susceptible and resistant strains, a finding that was confirmed in F1 heterozygotes [24]. In some cases of these “spontaneous mutations,” retinal light sensitivity led to studies into the underlying gene defect. However, with prescribed transgene expression or knockout models, questions about specific proteins and the role of light environment can be addressed more directly. Using transgenic mice expressing a mutated SOD gene, Mittag et al. [51] found that they were more susceptible to retinal light damage than nontransgenic animals. In mice [27] and in rats [28] with rhodopsin mutations, light rearing and/or intense exposures were found to influence the rates of retinal degeneration. Arrestin knockout mice exhibit re-