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(Investigative Ophthalmology and Visual Science. 2001;42:2074-2084.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Neuroprotective Effects of {alpha}2-Selective Adrenergic Agonists against Ischemia-Induced Retinal Ganglion Cell Death

María P. Lafuente, María Paz Villegas-Pérez, Paloma Sobrado-Calvo, Antonio García-Avilés, Jaime Miralles de Imperial and Manuel Vidal-Sanz

From the Laboratorio de Oftalmología Experimental, Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, Spain.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate in adult rats the effects of two {alpha}2-selective adrenergic agonists ({alpha}2-SAs; AGN 191103 and AGN 190342) on retinal ganglion cell (RGC) survival after transient retinal ischemia.

METHODS. RGCs were labeled with a Fluorogold (FG) tracer applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 60 or 90 minutes. In one group, a single dose of saline or one {alpha}2-SA was administered intraperitoneally (IP) or topically 1 hour before ischemia. In another group, a single dose of AGN 190342 was administered IP, 1, 2, 4, 24, or 72 hours after ischemia. Rats were processed 7, 14, or 21 days later. Densities of surviving RGCs were estimated by counting FG-labeled cells in 12 standard retinal areas.

RESULTS. Seven days after 60 or 90 minutes of retinal ischemia, death had occurred in 36% or 47%, respectively, of the RGC population, and by 21 days the loss of RGCs amounted to 42% or 62%, respectively. Systemic pretreatment with an {alpha}2-SA resulted in enhanced survival of ischemic-injured RGCs. Topical pretreatment with an {alpha}2-SA prevented up to 100% of the ischemia-induced RGC loss. Pretreatment with an {alpha}2-SA abolished the secondary slow RGC loss that occurred between days 7 and 21 after ischemia. When administered shortly after ischemia (up to 2 hours) AGN 190342 rescued substantial proportions of RGCs destined to die and diminished slow RGC death.

CONCLUSIONS. Pretreatment and early posttreatment with an {alpha}2-SA induces marked long-lasting neuroprotective in vivo protection against ischemia-induced cell death in RGCs.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The retinal ganglion cell (RGC) population of the rodent has been used to investigate regenerative1 2 3 4 5 6 7 and degenerative8 9 responses of mature central nervous system (CNS) neurons to axonal injury. Soon after axotomy, large proportions of RGCs die progressively.9 RGCs are capable of overcoming axotomy-induced death, at least temporarily, when different substances, such as neurotrophins,10 11 trophic factors,12 13 14 or compounds that may directly or indirectly halt the apoptotic cell death process15 16 17 are administered exogenously.

Recently, we have investigated the pattern of RGC loss that follows transient ischemia of the retina induced by elevation of intraocular pressure18 or by selective ligature of the ophthalmic vessels (SLOV).19 In both situations, ischemia-induced RGC death is a progressive event that takes place in at least two phases: an early rapid and a later more protracted period of cell loss. The amount and duration of these periods of cell loss are determined by the duration of the period of ischemia.18 19

The {alpha}2-adrenergic receptors are G protein–coupled receptors localized in the retina within the RGC layer, inner nuclear layer, and inner segments of the photoreceptors.20 21 22 Recent evidence indicates that retinal {alpha}2-adrenergic receptors mediate neuroprotective responses in the retina. {alpha}2-Selective adrenergic agonists ({alpha}2-SAs) mediate the expression of bFGF mRNA in the inner segments of photoreceptors, and this, in turn, may be responsible for neuroprotection against light-induced photoreceptor damage.22 In addition, {alpha}2-SAs are neuroprotective for adult rat RGCs against partial crush injury of the optic nerve (ON).23 Furthermore, preliminary reports suggest that {alpha}2-SAs are neuroprotective against pressure-induced retinal ischemia24 and chronic elevation of intraocular pressure.25

We report the effects of two potent {alpha}2-SAs (AGN 191103 and AGN 190342) on RGC death induced by SLOV. We present evidence indicating that {alpha}2-SAs, when administered systemically or topically before retinal ischemia, resulted in the rescue of up to 100% of the RGC population. Furthermore, the rescuing effects persisted during the 21-day period of study, and this resulted in inhibition of the protracted slow phase of RGC death that follows retinal ischemia. Early posttreatment with an {alpha}2-SA also diminished the early rapid and secondary slow loss of RGCs. Overall, these in vivo studies indicate that {alpha}2-adrenergic receptors mediate a potent neuroprotective effect in the retina and prevent the devastating consequences of ischemia on the RGC population.

Parts of this work have been presented in preliminary report and abstract form.26 27


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Adult Sprague-Dawley rats (200–250 g) were obtained from the breeding colony of Murcia University and from Harlan Interfauna Ibérica (Barcelona, Spain). Animal care and experimental procedures were performed according to institutional guidelines, European Union regulations, and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All surgical manipulations were performed with rats under general anesthesia. During recovery from anesthesia, the animals were placed in their cages, and a steroid-antibiotic ointment containing neomycin and dexamethasone (Fludronef; Iquinosa, Madrid, Spain) was applied over the ocular surface to prevent corneal desiccation. The rats had free access to food and water and were maintained in cages in temperature-controlled rooms with a 12-hour light–dark cycle (light period from 8 AM to 8 PM). Light intensity in the cages ranged from 8 to 24 lux.

{alpha}2-Adrenergic–Selective Agonists
The {alpha}2-SAs imidazolidin-zylidene-(5-methyl-quinoxalin-6-yl)-amine (AGN 191103) and 5-bromo-6-[2-imidazolin-zyl-amino] quinoxaline (AGN 190342) also known as brimonidine tartrate (BMD) or UK-1434 were obtained from Allergan, Inc. (Irvine, CA). Yohimbine, a selective {alpha}2 adrenoceptor antagonist, was purchased from Sigma Chemical Co. (Madrid, Spain). Both {alpha}2-SAs were dissolved in saline and either administered intraperitoneally (IP) or instilled topically on the eye. Yohimbine was dissolved in phosphate-buffered saline and administered IP.

Surgical Manipulations
Retrograde Labeling of Retinal Ganglion Cells.
RGCs were labeled retrogradely with a Fluorogold (FG) tracer (Fluorogold; Fluorochrome, Inc., Engelwood, CO) applied to both superior colliculi (SCi), which are the main RGC targets in the brain,28 according to described methods.11 18 19 Rats were anesthetized with an IP injection of a mixture of ketamine (75 mg/kg; Ketolar; Parke-Davis, SL, Barcelona, Spain) and xylazine (10 mg/kg; Rompún; Bayer, SA, Barcelona, Spain) in sterile saline. The midbrain was exposed, the pia mater overlying the SCi was removed, and a small pledget of gelatin sponge (Espongostan Film; Ferrosan A/S, Soeborg, Denmark) soaked in a solution of 3% FG in saline containing 10% dimethyl sulfoxide was applied over the surface of both SCi to label the RGCs retrogradely. Previous observations from this laboratory11 18 have documented that 7 days after FG application in the brain, the densities of FG-labeled RGCs are similar to those obtained when other retrogradely transported fluorescent and nonfluorescent tracers are applied to the main retinorecipient target regions in the brain.8 9 29 Moreover, FG persists within the somata of the retrogradely labeled neurons for up to 4 weeks after tracer application to the target, without apparent leakage or fading,18 30 thus enabling identification of the RGC population for up to 4 weeks after application.

Induction of Transient Periods of Retinal Ischemia.
Previous studies from this laboratory have shown that 60 and 90 minutes of transient ischemia induced by SLOV produce consistent and predictable patterns of RGC loss that have been recently characterized.19 Thus, for the present experiments, periods of 60 or 90 minutes of transient retinal ischemia were induced in the left eye 7 days after FG application. Transient ischemia in the left retina was induced by SLOV, according to described methods.19 31 In brief, the left ON head was exposed in the orbit,1 the superior aspect of its dural sheath was opened longitudinally, and a 10-0 nylon suture was introduced between the dural sheath and the ON and tied around the sheath, to interrupt blood flow through the ophthalmic vessels, which run in an inferior and nasal aspect within the sheath.32 33 34 Care was taken not to damage the ON.

Interruption of retinal blood flow during ischemia was assessed by direct ophthalmoscopy of the eye fundus through the surgical microscope. The animals that did not show a complete interruption of retinal blood flow during the ischemic period were excluded. At the end of the ischemic period, the ligature was released, and retinal reperfusion was assessed through the surgical microscope. The animals that did not show a complete recovery of retinal blood flow within the first few minutes after the ligature was released were also excluded. Eye fundus inspection was facilitated, because most eyes appeared mydriatic after the induction of transient ischemia. When necessary, a drop of 1% tropicamide (Colirio de Tropicamida; Alcon-Cusi Laboratories, Barcelona, Spain) was applied topically to induce mydriasis.

Because xylazine can activate {alpha}2-adrenergic receptors,22 and ketamine may also have a neuroprotective effect against ischemia,35 for the induction of retinal ischemia, the animals were anesthetized with an IP injection of 7% chloral hydrate in saline (0.42 mg/g body weight).

Animals treated with an {alpha}2-SA showed prolonged periods of recovery from anesthesia. {alpha}2-adrenergic agonists are used in clinical anesthesiology before surgery for their hypnotic and anesthetic properties and after surgery to prolong sedation.36 Thus, this prolonged recovery from anesthesia was probably related to the effects of the {alpha}2-SA on the central nervous system (CNS). A dose-dependent hypnotic response in rats36 37 mediated through the {alpha}2-adrenergic receptor subtype in the locus ceruleus38 has been observed with dexmedetomidine, another {alpha}2-SA.

A proportion of the animals treated with an {alpha}2-SA also showed a transient partial opalescence of the lens, which reverted within the first few minutes after onset of reperfusion. This partial opalescence of the lens did not preclude assessment of retinal blood flow through the microscope, and at present we have no clear explanation for it.

Intraorbital Sectioning of the ON.
In one group of animals, 7 days after FG application the left ON was sectioned at its exit from the eye, according to previously described methods.1 8 9 29 In brief, the animals were anesthetized as for the other surgical manipulations, the ON head was accessed in the orbit, its dural sheath opened longitudinally, and the ON was sectioned close to its origin in the eye with scissors. The eye fundus was examined before and after the procedure, and the animals that showed any disturbance in retinal blood flow were excluded.

Drug Administration and Groups of Animals
RGC Survival after SLOV.
Effects of Pretreatment with an {alpha}2-SA.
In one group, animals subjected to 60- or 90-minute periods of transient retinal ischemia were pretreated 1 hour before with an {alpha}2-SA. These animals were divided into two subgroups, depending on the route of administration of the {alpha}2-SA. One received a single IP injection of AGN 191103 (0.1 or 0.01 mg/kg body weight) or BMD (0.1 or 1 mg/kg body weight), and the other received two 5-µl drops of AGN 191103 (0.1% or 0.05% in saline) or BMD (0.5% in saline) topically in the left eye. These doses were selected based on preliminary experiments (data not shown) in which we examined after 7 days the effects of different doses of BMD (0.0001%, 0.001%, 0.01%, and 0.1% in saline) administered topically 1 hour before ischemia. As a control, similar groups of animals were treated with saline administered IP or instilled topically. These animals were processed 7, 14, or 21 days after ischemia.

To investigate whether the effects observed with an {alpha}2-SA were mediated through {alpha}2-adrenergic receptors, in a group of animals subjected to 90 minutes of retinal ischemia, we administered a single IP injection of yohimbine (5 mg/kg body weight), a selective {alpha}2 adrenoceptor antagonist, 80 minutes before the induction of retinal ischemia and 20 minutes before IP administration of a single dose of BMD (1 mg/kg body weight). The effects of yohimbine alone were also tested in a similar group of rats in which BMD was not administered. These groups were analyzed 7 days later.

RGC Survival after SLOV.
Effects of Posttreatment with an {alpha}2-SA.
An additional group of animals subjected to 90 minutes of retinal ischemia were treated with an IP injection of BMD (1 mg/kg body weight) at various times (1, 2, 4, 24, or 72 hours) after the onset of reperfusion. These animals were processed 7, 14, or 21 days later.

RGC Survival after ON Sectioning.
Effects of the {alpha}2-SA.
The animals with the left ON transected intraorbitally were treated 1 hour before with a single IP injection of saline, BMD (1 mg/kg body weight), or AGN 191103 (0.1 mg/kg body weight) dissolved in saline. These animals were processed 7 days later.

Tissue Processing
The animals were anesthetized with an overdose of 7% chloral hydrate and were perfused transcardially through the ascending aorta, first with saline and then with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Both eyes were removed and the retinas were dissected as flattened wholemounts by making four radial cuts (the deepest one in the superior pole), postfixed for an additional hour in the same fixative, rinsed in 0.1 M phosphate buffer, and mounted vitreal side up on subbed slides and covered with a mounting medium containing 50% glycerol in 0.1 M sodium carbonate buffer (pH 9) containing 0.04% p-phenylenediamine.39

Estimation of RGC Survival.
The retinas were examined and photographed through a fluorescence microscope (Axiophot; Carl Zeiss, Oberkochen, Germany) equipped with an ultraviolet (BP365/12, LP397) filter that allows the observation of the white-gold fluorescence of the tracer.

Mean densities of FG-labeled RGCs in the left experimental retinas, as well as in the right control nonischemic retinas, were estimated according to a protocol already described.8 9 11 18 19 In brief, labeled RGCs were counted in photographs taken from 12 standard rectangular areas (0.36 x 0.24 mm) of each retina situated, three in every retinal quadrant at 0.875, 1.925, and 2.975 mm from the optic disc. The number of labeled cells in the photographs was divided by the area of the region to obtain mean densities of labeled cells per square millimeter, and the densities obtained in the 12 areas were pooled to calculate a mean RGC density per retina. Cell counts were performed by the same investigator in a masked fashion. The identity of the retinas that led to the micrographs was unknown until cell counts from different groups were completed.

Results are reported as mean RGC densities per retina and mean RGC densities (±SEM) per group of animals. Statistical analysis of the differences between groups of retinas or groups of animals was performed by nonparametric ANOVA (Statistix ver. 1.0 for Windows 95; Analytical Software, Tallahassee, FL). The Kruskal-Wallis test was used to compare more than two groups and the Mann-Whitney test was used when comparing two groups only. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Using quantitative neuroanatomic techniques, the current studies were conducted to investigate the effects of {alpha}2-SAs on RGC survival after 60 or 90 minutes of retinal ischemia induced by SLOV and different reperfusion intervals. The main findings of this study can be summarized as follows: Seven days after transient ischemia of 60 or 90 minutes duration, there was an early loss of approximately 36% or 47%, respectively, of the RGC population, followed by a slower loss of 6% or 15%, respectively, of the RGC population, between days 7 and 21 after ischemia. Systemic or topical pretreatment with a single dose of an {alpha}2-SA protected RGCs from ischemia-induced cell death. The neuroprotection may affect up to 100% of the RGC population at 7 days, when {alpha}2-SAs are administered topically. Furthermore, pretreatment with an {alpha}2-SAs abolished the slow loss of RGCs that takes place between 7 and 21 days after insult. Systemic treatment with a single IP dose of BMD 1 or 2 hours after the onset of reperfusion reduced the initial as well as the slow loss of RGCs that followed ischemia. Posttreatment with BMD at 4, 24, or 72 hours after the onset of reperfusion had no effect on RGC survival after transient ischemia.

FG-Labeled RGCs in Control and Experimental Retinas
In the nonischemic (right) retinas of the experimental animals, the only retinal cells that appeared labeled with FG were RGCs. These had the typical punctate and diffuse gold fluorescence delineating their somas and occasionally the initial segments of their primary dendrites (Fig. 1) . Mean densities of FG-labeled RGCs were rather consistent for all the control groups analyzed. For example, the mean FG-labeled RGC densities found in the right control retinas of the groups treated with vehicle or AGN 191103 and analyzed 7, 1, or 21 days later, ;T1-T4>showed comparable results (Tables 1 2 3 and 4 ; Fig. 2A ). However, there were slight variations in mean densities of FG-labeled RGCs in some of the control right retinas of the groups treated with BMD (Table 5 ; Fig. 4A ). These variations can be attributed to slight differences in the efficiency of tracer application or in the batches of the tracer used and have also been observed in previous studies in this laboratory.11 18 Nevertheless, the overall densities were comparable to those found in previous studies in which FG was applied to the superficial layers of the SCi and RGCs were estimated for similar regions of the retina.11 18 The mean density of labeled RGCs in the right (nonischemic) retinas of each subgroup of rats was considered 100% survival for their contralateral, experimental left retinas.



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  		Figure 1. Fluorescence micrographs from representative regions of flatmounted retinas in animals treated 1 hour before retinal ischemia with a single IP injection of saline or saline containing AGN 191103 (0.1 mg/kg body weight). Retinal ganglion cells were labeled with FG applied to both SCi 7 days before retinal ischemia. The left retinas were subjected to 90 minutes of transient ischemia by SLOV, and the right nonischemic retinas were used as a control. Seven days after 90 minutes of ischemia, the left retinas of animals treated with vehicle (A) showed smaller numbers of FG-labeled RGCs than those treated with AGN 191103 (B). Twenty-one days after ischemia, the difference was even greater between (C) vehicle- and (D) AGN 191103-treated retinas. In addition to FG-labeled RGCs, there were also cell debris and microglial cells (C, arrows) labeled with FG. A nonischemic control right retina is shown in (E). Scale bar, 50 µm.

 

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  		Table 1. Densities of FG-Labeled RGCs after 60 Minutes of Retinal Ischemia

 

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  		Table 2. Densities of FG-Labeled RGCs 7 Days after 60 Minutes of Retinal Ischemia

 

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  		Table 3. Densities of FG-Labeled RGCs 7 Days after 90 Minutes of Retinal Ischemia

 

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  		Table 4. Densities of FG-Labeled RGCs 7 Days after Intraorbital Optic Nerve Transection

 


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  		Figure 2. (A) Mean densities of FG-labeled retinal ganglion cells at different survival intervals (7, 14, or 21 days) after 90 minutes of SLOV. Rats were treated 1 hour before ischemia with a single IP injection of saline (0.9% NaCl) or saline containing AGN 191103 (0.1 or 0.01 mg/kg body weight) or BMD (0.1 or 1 mg/kg body weight). All probabilities are by Mann-Whitney test. *Significantly greater than vehicle-treated retinas (P = 0.0011); {dagger}significantly different (P = 0.007); {ddagger}not different (P = 0.26); §not different (P = 0.21); ¶significantly different (P = 0.02). (B) Neuroprotection by {alpha}2-adrenergic agonist AGN 191103 from ischemia-induced RGC death at 7 days and prevention of further slow loss of RGCs that follows between 7 and 21 days. FG-labeled RGCs (expressed as mean ± SEM percentages of their right nonischemic retinas) in the left experimental retinas of groups of rats 7, 14, and 21 days after 90 minutes of transient ischemia induced by left SLOV. Pretreatment with an IP injection of saline alone (vehicle) resulted in the progressive loss of 47%, 52%, and 62%, respectively, of the RGC population. Systemic pretreatment with IP injections of saline containing AGN 191103 (0.1 or 0.01 mg/kg body weight) 1 hour before ischemia resulted 7 and 14 days later in the rescue of 25% and 27%, 28% and 39%, and 31% and 49%, respectively, of the RGC population. Furthermore, AGN 191103 protected against the slow RGC loss that occurred between day 7 and day 21 after ischemia (n = 6 for all groups).

 

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  		Table 5. Densities of FG-Labeled RGCs after 90 Minutes of Retinal Ischemia

 


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  		Figure 4. (A) Mean densities of FG-labeled RGCs at different survival intervals (7, 14, or 21 days) after 90-minute left eye SLOV. Rats were treated with a single IP injection of saline containing BMD (1 mg/kg body weight) 1, 2, 4, 24, or 48 hours after the onset of reperfusion. *Values from vehicle-treated retinas shown in the first two columns of Figure 2A . {dagger}Not different from vehicle-treated retinas at similar survival intervals (P > 0.05); {ddagger}significantly greater than vehicle-treated retinas (P < 0.05); §significantly different (P = 0.007); ¶not different (P = 0.202); #significantly different (P = 0.0152). (B) Posttreatment with BMD (AGN 190342) reduced initial and delayed ischemia-induced RGC death. Data represent mean densities of FG-labeled RGCs (expressed as percentages ± SEM of the right nonischemic retinas) in the experimental left retinas of groups of rats 7, 14, or 21 days after 90-minute SLOV for. Rats were treated with a single IP injection of saline (vehicle) 1 hour before ischemia or saline containing BMD (AGN 190342; 1 mg/kg body weight) 1 or 2 hours after the onset of reperfusion. Vehicle treatment resulted in the progressive loss of 47%, 52%, and 62%, respectively, of the RGC population. Posttreatment with AGN 190342 1 hour after ischemia resulted in significantly greater densities of FG-labeled RGCs when compared with corresponding retinas in vehicle-treated groups. Furthermore, the delayed loss of RGCs between 14 and 21 days observed in the vehicle-treated groups was not observed in the BMD-treated groups. Vehicle data from the first two columns of Figure 2A . For most groups, n = 5–6. Posttreatment with BMD at 21 days, n = 11.

 
In the experimental left retinas, RGCs were also identified by their typical FG fluorescence. The densities of FG-labeled RGCs in these groups varied according to the different treatments of the study. When densities were less than those found in the contralateral right retinas, other cells with small somas and fine tortuous processes that were intensely labeled with FG also appeared (Fig. 1) . These cells had the typical morphology of microglia.11 18 40 Retinal microglial cells phagocytose the debris of degenerating RGCs41 and become FG labeled.42

Pretreatment with an {alpha}2-SAs
Systemic Administration.
Effects of 60 Minutes of Ischemia.
We initially investigated the effects of systemic pretreatment with an {alpha}2-SAs on RGC death induced by SLOV for 60 minutes. By day 7, mean densities of FG-labeled RGCs in the left retinas had diminished to 64% of those found in the right retinas in the vehicle-treated group (Table 1) . RGC densities in the left retinas of rats pretreated with a single IP dose of an {alpha}2-SA 1 hour before ischemia were significantly greater than those found in the left retinas of the vehicle-treated animals and corresponded to 97% and 93% of the densities found in their right retinas, in the groups treated with 0.01 and 0.1 mg/kg AGN 191103, respectively, and to 90% and 93%, in the animals treated with 0.1 and 1 mg/kg BMD, respectively. Thus, at the doses administered in these experiments, the two {alpha}2-SAs greatly improved RGC survival and rescued between 26% and 33% of the RGC population by 7 days after ischemia (Table 1) .

To determine whether the rescue effect observed with the {alpha}2-SAs was a transitory phenomenon, two additional groups of animals pretreated with an IP injection of vehicle or AGN 191103 (0.01 and 0.1 mg/kg) and subjected to 60 minutes of ischemia were analyzed 14 and 21 days after injury. The densities of FG-labeled RGCs in the groups treated with AGN 191103 were still greater than those obtained in the vehicle-treated groups 14 and 21 days after ischemia. Moreover, RGC densities in the left retinas of the groups treated with AGN 191103 did not decrease between 7 and 21 days after ischemia (Table 1) , showing that in this group there was no further RGC loss and that the neuroprotective effect induced by the {alpha}2-SAs was not transitory but persisted for the 21-day period of study.

Effects of 90 Minutes of Ischemia.
We then investigated the effects of systemic pretreatment with an {alpha}2-SA after 90 minutes of transient ischemia, which is a more severe insult to the retina. In the vehicle-treated group, RGC densities in the left retinas decreased to approximately 53% by day 7 after ischemia (Fig. 2) . At this time point, however, RGC densities in the left eyes of the animals pretreated with an {alpha}2-SA were significantly greater than those obtained in the vehicle-treated animals (Fig. 1) . Expressed as proportions of their fellow right eyes, these densities corresponded to 81% and 88% in the animals treated with 0.01 and 0.1 mg/kg of AGN 191103, respectively, and to 91% in the animals treated with BMD (Fig. 2) . Thus, at the doses used in these experiments, the two {alpha}2-SAs rescued between 28% and 38% of the RGC population by 7 days after ischemia (Fig. 2) .

To determine whether the rescuing effects observed with the {alpha}2-SAs were short lived, additional groups of animals pre-treated with vehicle or with AGN 191103 were analyzed 14 and 21 days after ischemia. The densities of FG-labeled RGCs in the left retinas of the vehicle-treated group decreased significantly from 53% to 38% between 7 and 21 days after ischemia (Figs. 1 2) . RGC densities in the left retinas of the AGN 191103-treated group were significantly greater than those obtained in the left retinas of the vehicle-treated group at 14 and 21 days after ischemia. Also, these densities did not decrease significantly between days 7 and 21 after ischemia, showing that the rescuing effects were sustained and that the treatment completely prevented RGC death between these two periods after ischemia (Figs. 1 2) . Thus, AGN 191103 not only protected against the early loss that is apparent by 7 days after ischemia, but also prevented the slow loss of RGCs observed between days 7 and 21 (Fig. 2) .

Topical Administration.
Effects of 60 Minutes of Ischemia.
In animals subjected to 60 minutes of ischemia and pretreated topically with vehicle 1 hour before, the densities of FG-labeled RGCs in the left retinas had decreased by 7 days to approximately 67% of the densities found in their right contralateral intact retinas (Table 2) . Seven days after ischemia, when the animals were pretreated topically 1 hour before ischemia with two 5-µl drops of saline containing BMD (0.1% or 0.5%), the mean densities of FG-labeled RGCs in the left retinas were not different from the densities obtained in the right control retinas. These results show that BMD had protected the whole RGC population from ischemia-induced death (Table 2) .

Effects of 90 Minutes of Ischemia.
The mean RGC densities obtained seven days after injury in the left retinas of the animals subjected to 90 minutes of ischemia and pretreated topically with vehicle represented approximately 54% of the densities found in their right contralateral intact retinas (Table 5) . In contrast, the groups of animals treated 1 hour before ischemia with two 5-µl drops of saline containing BMD (0.1% or 0.5%) or AGN 191103 (0.05%) showed mean densities of FG-labeled RGCs at 7 days that were similar to those found in the control right eyes (Fig. 3) . This shows that topical instillation resulted again in maximal neuroprotection (Table 5) . It is possible that topical instillation of the drug resulted in higher retinal concentrations,43 and this would explain the higher results obtained with {alpha}2-SAs administered through this route.



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  		Figure 3. Micrographs from wholemounted left and right retinas of an experimental animal 1 week after 90 minutes of transient ischemia in the left eye. Fluorescent gold (FG) tracer was applied to both SCi 7 days before ischemia. The left eye received, two 5-µl drops of BMD (0.5% in saline) 1 hour before ischemia. FG-labeled RGCs were evenly distributed throughout the retinal quadrants in both the experimental left (A) and contralateral nonischemic (B) retinas. Micrographs were 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, ver. 4.1; Media Cybernetics, Silver Spring, MD). When FG-labeled RGCs were counted in 12 standard areas, the densities were similar in both retinas. The superior aspect of the retinas is between the 1- and 2-o’clock orientations. Scale bar, 1 mm.

 
Further groups were analyzed after longer reperfusion intervals to determine whether the neuroprotective effects of single topical instillation of BMD (0.5%) were also maintained for longer periods. Fourteen days after 90 minutes of transient ischemia of the retina and topical instillation of vehicle, the mean densities of FG-labeled RGCs in the left eyes had significantly diminished to 48% of the densities found in the right control eyes (Table 5) . At this same survival interval, the mean densities of FG-labeled RGCs in the left retinas pretreated topically with BMD were not different from the mean densities found in the control right eyes. Thus, topical pretreatment with BMD protected the whole RGC population up to day 14 after ischemia (Table 5) . Twenty-one days after 90 minutes of retinal ischemia and topical instillation of vehicle, the mean densities of FG-labeled RGCs in the left retinas had further diminished to 29% of those found in the control right eyes. At this same time point, the densities of FG-labeled RGCs in the retinas pretreated topically with BMD represented 88% of the densities found in the control right retinas and were not different from the densities obtained in the group treated with BMD analyzed at day 14 (Table 5) . Thus, topical instillation of BMD before 90 minutes of transient ischemia rescued approximately 83% of the population of RGCs destined to die at 21 days (Table 5) .

Effect of {alpha}2-Adrenoceptor Inhibitors.
To investigate whether {alpha}2-SAs’ neuroprotective effects are specifically mediated through {alpha}2-adrenoreceptors, we assessed RGC survival under a combined treatment of yohimbine and BMD. Yohimbine, a selective inhibitor of {alpha}2-adrenoceptors, was administered shortly before BMD. Seven days after 90 minutes of ischemia, the densities of labeled RGCs in the left retinas of animals pretreated with yohimbine and BMD were similar to those found in animals pretreated with yohimbine alone (Table 3) . Furthermore, these densities were similar to the densities found in the left retinas of the animals that received an IP injection of vehicle (Table 3) . Thus, yohimbine blocked the neuroprotective effects of the {alpha}2-SA on RGC survival 7 days after 90 minutes of retinal ischemia, and treatment with yohimbine alone had no effect on RGC survival. Overall, these results indicate that the neuroprotective effect of BMD after ischemia was specifically mediated through {alpha}2-adrenoreceptors.

Effect of {alpha}2-SAs on RGC Survival after ON Section.
To determine whether {alpha}2-SAs were also effective in preventing axotomy-induced RGC death, additional groups of rats were pretreated with a single IP injection of saline, AGN 191103 (0.1 mg/kg), or BMD (1 mg/kg) 1 hour before sectioning of the intraorbital left ON. These animals were analyzed 7 days later. Concordant with previous studies,10 11 our results show that 7 days after intraorbital ON transection, approximately 45% of the RGCs were lost in the group of animals treated with saline. Comparable results were observed in the groups pretreated with AGN 191103 or BMD (Table 4) , documenting that these two {alpha}2-SAs did not protect RGCs against the effects of ON section.

Posttreatment with an {alpha}2-SA
To investigate whether the administration of an {alpha}2-SA after 90 minutes of ischemia had neuroprotective effects on RGCs, in an additional groups of animals a single IP dose of BMD (1 mg/kg body weight) was administered 1, 2, 4, 24, or 72 hours after the onset of reperfusion, and the animals were analyzed 7, 14, or 21 days later. These results were compared with those obtained in corresponding groups of animals pretreated IP with saline.

In the group of animals treated 1 hour after ischemia and analyzed 7, 14, or 21 days later, the densities of FG-labeled RGCs in the left retinas were significantly greater than those obtained in the left retinas of the vehicle-treated control animals (Fig. 4) . These results show that BMD administered 1 hour after the onset of reperfusion increased RGC survival after ischemia. Furthermore, because the densities of FG-labeled RGCs in the left retinas of the animals treated with BMD did not decrease significantly between 14 and 21 days after ischemia and the densities of RGCs in the left retinas of the vehicle-treated rats decreased significantly between these periods, it is possible that BMD not only rescued a proportion of RGCs from early death but also halted the slow secondary loss of RGCs observed between 14 and 21 days (Fig. 4) . The group of animals treated with BMD 2 hours after ischemia also showed significantly greater densities of FG-labeled RGCs in the left retinas than the vehicle-treated group, at both 7 and 21 days after ischemia (Fig. 4) . However, the densities of FG-labeled RGCs in the left retinas of the animals posttreated with BMD 4, 24, or 72 hours after ischemia and analyzed 7 or 21 days later were similar to those found in the vehicle-treated group (Fig. 4A) . Taken together, these results indicate that there is a time window of approximately 2 hours after onset of reperfusion for effective posttreatment with an {alpha}2-SA after retinal ischemia.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The experimental strategies used in this study allowed quantitative estimations of RGC loss after retinal ischemia and provided evidence for the in vivo neuroprotective effects of {alpha}2-SAs on ischemia-induced RGC death. We show that SLOV for 60 or 90 minutes induced the loss at 7 days of approximately 36% or 47%, respectively, of the RGC population, and between 7 and 21 days there was an additional loss of 6% or 15%, respectively. The major finding we report is that pretreatment with an {alpha}2-SA can protect the entire RGC population against ischemia-induced cell death, and this effect persisted for the 21-day period of study. Furthermore, when administered up to 2 hours after the onset of reperfusion, {alpha}2-SAs increased RGC survival and diminished delayed RGC death.

Early Neuroprotective Effects of Pretreatment with an {alpha}2-SA
Seven days after 60 or 90 minutes of ischemia, the percentages of surviving RGCs diminished to 64% or 53%, respectively, of their control values. This is in agreement with studies from this18 19 and other44 45 46 47 48 laboratories showing an abrupt RGC loss after transient retinal ischemia. A single IP injection of AGN 191103 or BMD, 1 hour before 60 or 90 minutes of ischemia resulted 7 days later in the rescue of 26% or 38%, respectively, of the RGC population. Moreover, topical instillation of AGN 191103 or BMD, 1 hour before 60 or 90 minutes of ischemia, resulted 7 days later in densities of surviving RGCs similar to those found in nonischemic fellow retinas (Tables 2 5) , showing that the {alpha}2-SA protected the entire RGC population from ischemia-induced RGC death. Previous studies have used antiapoptotic drugs,17 49 N-methyl-D-aspartate (NMDA) antagonists,50 51 52 nitric oxide synthase inhibitors,53 neurotrophic factors,17 54 55 56 or gangliosides57 to protect RGCs after ischemia. These studies show increased RGC survival after ischemia, but none has reported protection of the entire RGC population. Because the neuroprotective effects of {alpha}2-SAs were abolished when yohimbine was administered (Table 3) , it is likely that the neuroprotective effects of {alpha}2-SAs are mediated through activation of {alpha}2-adrenoceptors.

How {alpha}2-SAs may prevent the early loss of RGCs after retinal ischemia is not clear.58 It is known that {alpha}2-SAs upregulate retinal levels of endogenous bFGF and diminish intraocular levels of glutamate and aspartate after retinal ischemia. Systemic administration of an {alpha}2-SA upregulates the expression of bFGF mRNA in the retina,22 and exogenous administration of bFGF increases RGC survival after transient pressure-induced retinal ischemia.54 55 Ischemia-induced neuronal death is associated with excessive release of excitatory amino acids and stimulation of the NMDA subtype of ionotropic glutamate receptors.59 60 The administration of dextromethorphan, a glutamate receptor antagonist50 and selective NMDA antagonists48 diminish ischemia-induced RGC loss. Systemic administration of an {alpha}2-SA diminishes intraocular levels of glutamate and aspartate after pressure-induced retinal ischemia,61 and increases RGC survival after partial crush injury to the ON, a lesion that also produces excessive release of glutamate into the vitreous and mimics excitotoxicity.23

Sustained Neuroprotective Effects of Pretreatment with an {alpha}2-SA
After the early loss of RGCs that occurred in the first 7 days after ischemia, RGC death progressed within the next 2 weeks. This is in agreement with a recent study indicating that transient ischemia induces a rapid loss of RGCs followed by a slower rate of cell death that progresses during the following weeks.19 The progressive loss of RGCs observed in the present study was more evident in the group of animals analyzed after 90 minutes of ischemia. Between 7 and 21 days there was a significant loss of 15% of the RGC population in the vehicle-treated group. To determine whether the in vivo protective effects of {alpha}2-SAs were transitory, we examined RGC survival 14 and 21 days after ischemia and systemic or topical administration of AGN 191103 (Fig. 2 ; Table 5 ). Twenty-one days after 90 minutes of transient ischemia, RGC densities in the group pretreated systemically with AGN 191103 resulted in densities of FG-labeled RGCs that were comparable to those obtained at 7 days, suggesting that the rescuing effects persisted for the 21-day period of study (Fig. 2) . Similarly, topical pretreatment with AGN 190342 resulted by 7 and 14 days in densities of surviving RGCs that were similar to those in their control contralateral retinas (Table 5) . Moreover, in this group of animals, at 21 days the densities of surviving RGCs did not differ from those obtained at 14 days.

Previous studies on ischemia-induced RGC death have shown increased RGC survival after ischemia with the administration of exogenous substances (described earlier), but in most of these studies RGC survival was investigated only during the first 7 to 14 days after ischemia. Our results indicate that both the early abrupt and the secondary protracted phase of cell loss that follows retinal ischemia19 may be halted with a single dose of an {alpha}2-SA administered before ischemia.

Ongoing studies from this laboratory indicate that SLOV also induces death of other non-RGC neurons,62 and systemic administration of an {alpha}2-SA preserves inner and outer nuclear retinal cells, as well as the cytoarchitecture of retinal layers.62 Thus, it is conceivable that the sustained effects observed in this study are related not only to the initial rescue of RGCs but also to the preservation of other non-RGC retinal neurons. Other studies have indicated that ameliorating the early RGC loss does not ensure prevention of delayed RGC loss.48

Posttreatment Neuroprotective Effects
We further investigated whether {alpha}2-SAs would be neuroprotective when administered after retinal ischemia. When administered 1 hour after the onset of reperfusion, BMD was also effective in preventing the death of approximately 19%, 12%, and 22% of the RGC population by 7, 14, or 21 days, respectively (Fig. 4) . Moreover, the densities of FG-labeled RGCs did not decrease further between 14 and 21 days in the BMD-treated groups, but there were significant reductions in the vehicle-treated groups. Taken together, these results show that, when administered 1 hour after ischemia, BMD partially prevented RGC death and diminished the slow loss of RGCs observed between 7 and 21 days. In the group of animals treated with BMD 2 hours after ischemia there were also significant neuroprotective effects. However, the neuroprotective effect was lost when BMD was injected 4, 24, or 72 hours after onset of reperfusion, presumably because it were administered at a time when a large proportion of RGCs were already committed to die.48

RGC death after transient ischemia of the retina may result in both acute excitotoxic necrotic cell death and delayed apoptotic cell death.44 45 46 47 48 Because posttreatment with an {alpha}2-SA resulted in increased RGC survival after ischemia and diminished the secondary slow loss of RGCs, it is possible that {alpha}2-SAs have antiapoptotic effects. Recent studies, in neuronally differentiated PC12 cells deprived of serum and nerve growth factor (NGF) and in cerebellar granule neurons placed in low-K+ medium, indicate that {alpha}2-SAs may halt apoptotic degradation by maintaining the mitochondrial permeability and avoiding release of degradation-signaling factors, suggesting that {alpha}2-SAs act early on the cascade of events leading to apoptosis.63 The absence of neuroprotection of {alpha}2-SAs against axotomy-induced RGC death observed in this study (Table 4) may be interpreted as an indication that the pathogenic pathways activated by transient ischemia or axotomy differ,17 although leading to a common cell death mechanism, apoptosis. An inhibitor of a late event in apoptosis, the irreversible wide-range CPP32-like caspase inhibitor Z-DEVD-cmk, was found to be neuroprotective against both axotomy- and ischemia-induced RGC death.17 An effect on the early events that lead to ischemia-induced apoptosis could explain why {alpha}2-SAs were effective against ischemia- but not axotomy-induced RGC death.

Finally, it is possible that the neuroprotective effects of {alpha}2-SAs are due to specific upregulation of survival pathways in the retina through activation of Müller cells.64 Systemic administration of an {alpha}2-SA induces activation in Müller cells of extracellular signal-regulated kinases (ERKs),64 which are classic members of the mammalian mitogen-activated protein kinases (MAPKs). The ERK signaling pathway appears to mediate survival effects.65 For example, brain-derived neurotrophic factor (BDNF)–mediated neuroprotection in ischemic–hypoxic brain injury in vivo is known to be mediated through activation of ERKs.66 In addition to ERKs, two other types of MAPK, known as stress-activated protein kinases, are thought to be involved in the signaling pathways to apoptosis induced by various stress and injury stimulus, including glutamate excitotoxicity17 67 and cerebral ischemia.68 The observation that {alpha}2-SAs selectively activate MAPKs in Müller cells may be taken as an indication that these cells, which maintain integrity and normal function of the retina, may play an important role in protecting retinal neurons.64


   Acknowledgements
 
The authors thank Larry Wheeler for helpful discussions during the course of this work, Constantino Sotelo for critical comments on the manuscript, Marcelino Avilés for advice, and Sergio Mayor-Torroglosa, Diego Martínez-Pérez, and M. Elena Aguilera-Meseguer for technical support.


   Footnotes
 
Supported by Fundación Séneca Grant PB/18/FS/97 from the Regional Government of Murcia, Grants FIS 98/0341 and 99/0190 from the Spanish Ministry of Health, and an unrestricted grant from Allergan, Inc.

Submitted for publication January 3, 2001; revised April 2, 2001; accepted May 15, 2001.

Commercial relationships policy: F.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Manuel Vidal-Sanz, Departamento de Oftalmología, Universidad de Murcia, E-30.100 Espinardo, Murcia, Spain. ofmmv01{at}fcu.um.es


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 Introduction
 Methods
 Results
 Discussion
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