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

from different groups were finished. For statistical analysis, RGC densities were compared using two non-parametric tests: the one-way ANOVA Kruskal-Wallis and Mann-Whitney tests, with P values of 0.05 considered statistically significant.

3.General Appearance of Retinas Labeled with FG or Di-ASP

In control retinas, only the retinal cells labeled with FG have the typical characteristics of RGCs. These are observed in the RGC layer, but there is a small proportion of displaced RGCs (Dogiel corpuscles) that may be observed in the inner plexiform or inner nuclear layers. FG-labeled RGCs have the typical punctate and diffuse FG fluorescence, which delineates their soma and occasionally initial segments of their primary dendrites (Fig. 4a, b). In a recent study, the densities of FG-labeled RGCs in the control retinas of large series of rats were estimated following the above-described methods [29,30]. Although there were small variations in the mean densities of FG-labeled RGCs in some control retinas, overall these were rather consistent [29,30] (Fig. 5). These variations can be attributed to slight differences in tracer application or in the efficiency of the batches of tracer employed, and also have been observed in previous studies from this laboratory [18,19]. Nevertheless, in the experimental groups, the densities obtained for the right intact retinas may be used as 100% survival for their contralateral, left operated retinas.

In the experimental left retinas subjected to various periods of ischemia, in addition to RGCs, microglial cells also appear intensely labeled with FG. These cells, which can be easily distinguished from RGCs on the basis of their morphology (cells with bright fluorescence in their small soma and multiple fine tortuous processes), phagocytose the debris of degenerating FG-labeled RGCs and thus become FG-labeled [24]. FG-labeled microglial cells have been found in a number of studies in which FG was applied to the SCI before retinal injury in the rat [18,19,36] and goldfish [40].

In control and experimental retinas in which we used DiAsp as retrograde tracer, the only cells that appeared DiAsp-labeled were RGCs. These showed typical punctate and diffuse DiAsp fluorescence delineating the soma, axon, and, occasionally, primary and secondary RGC dendrites (Figs. 6, 7). The mean densities of RGCs labeled with di-ASP applied to intraorbital ON stump in a control group of nine rats (22) was somewhat smaller than the densities of labeled RGCs when other nonlipophilic tracers were applied to the SC or the ON (17–19). This could be explained by the limited solubility of the dye, which resulted in lower labeling efficiency. Because DiAsp application involves transection of the ON, the low numbers of DiAsp-labeled RGCs found in our experiments could also be explained by the axotomy-induced retrograde degeneration of RGCs. However, this is not likely because axotomy-induced RGC death first appears between

Figure 5 Micrograph of the whole-mounted right retina in an adult PVG rat 1 week after FluoroGold application to both superior colliculi. FG-labeled RGCs are evenly distributed throughout the retinal quadrants. The micrograph was prepared with the aid of a motorized stage on a photomicroscope with a high-resolution camera connected to an image analysis system with an automatic frame grabber device (Image-Pro Plus, V4.1; Media Cybernetics, Silver Spring, MD, USA). The superior aspect of the retinas is between 1 and 2 o’clock orientation. (Scale bar1 mm.)

4 and 5 days after ON section close to the eye [18,26], and we estimated RGC densities 3 days after dye application [22]. Moreover, in experiments in which DiAsp was applied to the SC to retrogradely label RGCs, Thanos [24] reported densities of DiAsp-labeled RGCs similar to those obtained when we applied DiAsp to the cut ON, indicating that DiAsp has lower labeling efficiency than other neuronal tracers. Finally, the absence of DiAsp-labeled microglial cells in our experiments further supports that there is no RGC death in the first 3 days after ON section.

III. DISCUSSION

Several requirements need to be met for the above-mentioned protocols to be useful. These include, among others (1) reproducibility of the same insult given to the retina; (2) consistency of the results produced with a given type of insult; and (3) reliable estimates of the effects of the insult in the survival of the RGC population and/or other retinal neurons.

A.Requirements Needed for the Protocols to Be Useful

1. Reproducible Insult

For an insult to be reproducible, it should be performed equally every time and should also lead to similar results. Thus, an identical procedure should be performed every time that transient ischemia is given to the retina, whether this is induced by elevated IOP or by ligature of the ophthalmic vessels. A number of investigators monitor the level of the IOP reached to induce blood arrest with the use of intraocular cannulas connected to a manometer system [41–46]. In our experiments the eye fundus was constantly inspected to ensure flow arrest, but accurate measurements of the IOP were not done. Nevertheless, studies in which a number of animals were treated with similar periods of ischemia showed consistent numbers of surviving RGCs [19], suggesting that small fluctuations in the intraocular pressure reached to arrest blood supply did not affect RGC survival substantially.

Ligature of the ophthalmic vessels seems to be a rather consistent method to induce blood arrest into the retina [30]. The reproducibility of this method relies on the ability of the investigator to perform in identical fashion the dissection and ligature of the ophthalmic vessels that run within the ON sheath. Another variable to be taken into account when performing these studies is the difference in the period of time (usually a few minutes) from ligature release to full retinal reperfusion.

2. Consistency of Injury-Induced Cell Loss

The results of a large series of experiments in which transient ischemia of the rat retina was induced by elevated IOP were highly consistent for the different groups analyzed after different survival (reperfusion) intervals [19]. Similarly, consistent results in the amount of RGC loss were observed in another series of experiments in which transient ischemia of the rat retina was induced by ligature of the ophthalmic vessels and RGCs were labeled with DiAsp [31] or FG [29,30,32]. Altogether, these studies indicate that RGC survival after different periods of transient ischemia and survival intervals is a highly consistent and predictable finding.

3. Reliable Estimates of Surviving Cells

The studies mentioned above were based on counts of cells retrogradely labeled with dyes applied to their cut axons or their targets. The efficiency of the tracer employed—that is, the proportion of retinal ganglion cells that becomes retrogradely labeled—should be well established. Another important issue is to be able to reproduce consistently similar proportions of labeled retinal ganglion cells every time the dye is applied.

The techniques employed in these experiments sampled similar regions of the retinas located within the central retina, where higher densities of RGCs are observed [13], but overlook RGC densities within more peripheral areas of the retina. While this is a reliable and consistent method to obtain RGC densities, a more accurate estimate should include counting all the FG-labeled RGCs within each retina, but this is major job to be done manually. RGC counts with an automatic image analysis system of a digital image of the retina has been precluded by technical difficulties in identifying, single FG-labeled neurons, FGlabeled microglial cells, and FG-labeled cells that are located close together as it happens for the central regions of the retina (see Fig. 4a,b). Recent advances in image-analysis may permit in the future more detailed analysis off the RGC population [23,39], including the identification of different types of RGCs, as defined by their morphological properties [47].

Screening studies on the effects of neuroprotectants with the above-men- tioned techniques rely on the interpretation of labeled RGCs as alive, rescued neurons. In some instances, neuronal cells were labeled prior to injury with tracers applied to their main targets in the brain (e.g., Refs. 34,30). In other instances these cells were labeled after injury with tracers applied to their targets [23], or their cut axons [31]. While this assumption is generally accepted for many of these studies [18,19,23,29–32,34,35], we cannot be certain that rescued RGCs retain all their normal physiological properties. Other studies have shown that retinal cells may undergo functional and metabolic deficits before they actually die [16,48,49]. For example, chronic ligature of both carotid arteries did not induce evident loss of photoreceptors until 9 months later, but functional correlates indicated that both the a and b waves of the electroretinogram were clearly pathological, as early as 90 days after bilateral carotid occlusion [50]. This highlights the need for further studies to ascertain functional viability of retinal neurons surviving ischemic injury [51].

B.Advantages and Disadvantages of These Methods

1. Increasing IOP Above Systolic Levels

Increasing the IOP above systolic levels with two sutures pulling from both sides of the eye has the advantage of being a noninvasive method. No puncture is given

to the eye and/or the retina, and therefore nonspecific neuroprotective effects [35] that may originate as a consequence of lens injury [36] are discarded. However, the main disadvantage of this method is that it is tedious and time consuming; constant observation of the eye fundus of the retina to ascertain blood arrest is required to adjust tension on the sutures when necessary.

The duration of the period of transient ischemia influences both the amount and pattern of RGC loss [19]. This fact should be taken into account when designing experiments aimed at investigating possible neuroprotective effects. Furthermore, we ignore whether in addition to blood flow arrest, increased intraocular pressure also induces compression or axotomy-like damage to retinal axons. If this were to be the case, an additional axotomy-like insult is given to the retina.

2. Ligature of the Ophthalmic Vessels

Transient ischemia of the retina may be induced with methods that do not increase the intraocular pressure. Transient ligature of the carotid [48,52] and vertebral arteries [53] induces retinal ischemia but may also affect the brain. Clamping or ligating the ophthalmic vessels together with the ON have been used to induce retinal ischemia [35,54–57]. However, these were short-term experiments and, in addition to retinal ischemia, these procedures also induced ON axotomy. For the purpose of our studies, aimed at investigating the shortand long-term effects of retinal ischemia, we have preferred to induce retinal ischemia by the selective ligature of the ophthalmic vessels, avoiding direct mechanical damage to ON fibers.

Selective ligature of the ophthalmic vessels was first used in monkeys by Hamasaki and Kroll [58] and later applied to rats [28–32,51]. Selective ligature of the ophthalmic vessels induces in the retina pathological findings that are similar to those found after ischemia induced by elevated IOP, including the loss of RGCs and the thinning of the IPL, INL, and OPL [59]. This method does not increase the IOP and, thus, there is no intraocular compression of retinal axons, avoiding axotomy-like injury to RGC fibers. The effects of ligature of the ophthalmic vessels on the ON head were not studied, but it is likely that transient ligature of the ophthalmic vessels also induces transient ischemia of the ON head. The main source of blood flow to the ON head in the rat comes from branches of the posterior ciliary arteries as well as from arterioles arising from the central retinal artery [6], and thus it is likely that ligature of the ophthalmic vessels affects blood flow within the ON head.

C.Further Implications of These Models

Retinal ischemia induced by any of the above-discussed methods induces an early loss of RGCs [19,31]. The loss of RGCs that appears shortly after retinal ischemia may be prevented, at least in part, with the use of different neuroprotective agents.

A recent article reviews several studies on RGC neuroprotection after retinal ischemia [27]. The variety of drugs employed in these experiments may be taken as an index of the many steps involved in RGC death that may be halted, and further points to the multiple factors involved in the process of ischemia-induced RGC death (see Table 7, Ref. 27). The techniques described here do not address questions related to the specific mechanisms of action of putative neuroprotectants. An efficient neuroprotective drug should diminish injury-induced RGC death, and this may be accomplished either through a direct effect on injured RGCs or indirectly by activating other non-RGC neurons or nonneuronal cells of the retina. Thus, whether the neuroprotective effect of a given drug is directly mediated through the RGCs themselves, or indirectly mediated through other cells in the retina, should be tackled with additional methods.

In addition to the early loss of RGCs, retinal ischemia also induces a protracted loss of RGCs [19,31]. The mechanism by which the initial insult triggers such a long-lasting and prolonged process of RGC death is unknown. Whether injury triggers the immediate death of a subset of RGCs, and this in turn leads to secondary RGC death is currently unknown [31]. In this context, it is worth noticing that ideal neuroprotection should prevent both the early as well as the secondary slow loss of RGCs that were not primed to die in the first instance but that disappear with time [29–32].

Therapeutic interventions after CNS ischemia are at present mainly directed toward the so-called penumbra zone, a region of incomplete ischemia that surrounds the core of the infarcted region, which is most susceptible to reperfusion damage. The above-discussed models involve complete ischemia of the retina, and there may be differences in susceptibility of different retinal neurons depending on their location within the retina [55,60]. However, the concept of penumbra zone after ischemia may not be fully reproducible in these models of retinal ischemia. Moreover, a few minutes of ischemia induce irreversible damage in the CNS [61–63], whereas in the retinal models examined in these studies (increase of the IOP and SLOV), the transient period of ischemia required to produce RGC death is substantially longer. For example, 5 min of global cerebral ischemia induced by bilateral carotid artery occlusion are enough to produce neuronal death of nearly all the CA1 pyramidal neurons within the gerbil hippocampus after 5 min global cerebral ischemia [61,62]. In contrast, periods of 45–60 min of pressure-induced retinal ischemia are required to induce RGC death [19].

Transient ischemia of the retina has been used as a basic research model for glaucoma because ischemia may be involved in the pathogenic mechanism of this disease. In addition, both ischemia and glaucoma involve progressive loss of RGCs [19]. However, both the above-discussed methods to induce retinal ischemia elicit a rapid and massive RGC loss, and this is clearly not the case for chronic neurodegenerative conditions such as glaucoma, where RGC death appears to be a progressive but much slower event. Moreover, retinal ischemia also

induces the loss of other non-RGC neurons [59] and this may not be the case for glaucoma [64].

Ligature of the ophthalmic vessels does not fully resemble the clinical situation of acute interruption of the blood flow either by central artery or vein occlusion. In these diseases, the outer retina is usually not affected, while ligature of the ophthalmic vessels interrupts blood supply to the retina and choroid, thus leading to different pathological findings. Preliminary results indicate that after ligature of the ophthalmic vessels, not only the RGC layer but the other retinal layers were severely affected by the initial period of transient ischemia [65].

The ligature of the ophthalmic vessels and the pressure-increased models to induce retinal ischemia do not fully reproduce human conditions. Nevertheless, these are useful models in neurobiology research to induce progressive neuronal degeneration of the RGC population, and are suitable models to examine the consequences of such insults in the different retinal layers after retinal ischemia. The fact that other retinal layers also degenerate with time after these insults may provide an opportunity to explore the progressive loss of other non-RGC neurons of the retina, including the inner and outer nuclear layer neurons. Moreover, these models may be used as powerful in vivo tools to screen for compounds that may have relevant neuroprotective effects against ischemia-induced neuronal cell death [29–32,65].

IV. CONCLUSIONS

The above described methods to induce retinal ischemia cannot be extrapolated to clinical conditions in the human eye. Nevertheless, they provide a powerful tool to examine the fate of retinal neurons after ischemia and to further explore future therapeutic interventions to lessen the effects of transient ischemia.

ACKNOWLEDGMENTS

This work was supported by research grants from the Regional Government of Murcia (Fundacio´n Se´neca, PI-92/00540/FS/01), the Spanish Ministry of Science and Technology (BFI2002-03742), The Spanish Ministry of Health (03/13; FIS PI020407), and the European Union (QLK6-CT-2000-00569 and QLK6-CT- 2001-00385). The authors thank A. Avile´s, M. E. Aguilera, and J. M. Bernal for technical support.

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