Neuroprotection with alpha-2 agonists

Manuel Vidal-Sanz

Over the last few years, our laboratory has used several animal models to study injury-induced cell death in the retina, including axotomy-induced retinal injury, transient ischemia-induced reti-nal injury, and ocular hypertension-induced retinal injury. At the same time, we have also explored the possibility of preventing some of the injury-induced effects.

Retinal ganglion cells

In several recent studies, we induced transient ischemia of the retina by selective ligature of the ophthalmic vessels (LOV) in adult rats and mice.^1-3 We used the tracer fluorogold (FG) to prelabel the entire retinal ganglion cell (RCG) population.^4-6 Retinas were prepared as whole-mounts and examined and photographed under a fluorescence microscope equipped with a digital camera and a motorized stage, connected to an image analysis system. Images were processed using an automatic counter and data was obtained on the area of the retina, the total number of FG-labelled retinal ganglion cells for the retina, and mean density. We found that initial cell loss progressed over time. After 90 minutes of transient ischemia of the retina, approximately 40% of the retinal ganglion cell popula-tion was lost by five days, and the loss continued to progress for up one to two months. We examined the effects of a number of neuroprotectants against this injury and found that brimonidine, an alpha-2 agonist, was effective in preventing ischemia. To determine whether topically applied brimonidine reached the retina, we carried out a group of experiments^7,8 in which two drops of 0.5% brimonidine or saline were applied topically to the left eye of intact rats, and the retinas were collected at different sur-vival intervals to analyze mRNA levels. We found that a single instillation of brimonidine reached the retina and upregulated the expression of several neurotrophic factors.

In subsequent studies, we found that the neuroprotective effects of brimonidine were dose-dependent, with the 0.1% concentration achieving optimal neuroprotection against the early loss of RGCs.⁹ We also found that neuroprotection lasted for up to three weeks after the injury when the rat eye was treated one hour before ischemia with two drops of brimonidine. Neuroprotection also occurred when brimonidine was administered one or two hours after ischemia, but not when given four hours after the injury.¹⁰ Other studies documented that not all of the RGCs that survive ischemia retain their capacity for retro-grade and orthograde axonal transport.^11,12 However, RGCs rescued from ischemia-induced cell death with brimonidine retain this capacity.

Inner and outer retina

In a recent study, we investigated the long-term effects of transient LOV on the inner and outer retina in the rat model.¹³ We assessed these effects using electroretinogram (ERG) recordings of a- and b-wave amplitudes and a morphological analysis of layer thick-ness in paraffin sections. The ERG results showed significant reductions in the mean b-wave amplitudes of the ischemic eyes at eight and 12 weeks after LOV in the vehicle-treated group of animals, but not in the group pretreated with brimonidine.¹³ In the morphological analysis, the thickness of the inner nuclear layer and the inner plexiform layer had decreased by about one-third in the vehicle-treated animals. However, in those pretreated with brimonidine, there was no significant loss (see Fig. 1).

Fig. 1. Retinal cross sections 3 months after a 90-minute LOV. (Adapted from: Mayor-Torroglosa S, et al. IOVS 2005; 46: 3825-3835.)

Retino-tectal projection

In the same long-term study described above, we also examined the effects of ischemia on the retinotectal projection three months after transient ischemia of the retina, and assessed the neuroprotective effects of brimonidine. The retinotectal projection was orthogradely labeled with cholera toxin subunit B (CTB) injected in the eye and measured in serial coronal sections of the superior colliculus. In the unlesioned (control) rat, very densely CTB-labeled retinal axons filled the superficial layers of the superior colliculus. In contrast, CTB-labeled profiles were reduced in the vehicle-treated rat. In the brimonidine group, the densities of CTB-labeled profiles were significantly greater than those in the vehicle-treated group, indicating a prevention of the ischemia-induced effect on the visual projection. The results of a mathematical analysis of the CTB-labeled profiles indicated that approximately half of the entire retinotectal projection volume was lost three months after ischemia in animals not treated with brimonidine. Pretreatment with brimonidine significantly protected against this retinal damage and degeneration of retinal projection (see Fig. 2).

Fig. 2. Average volumes of the retinotectal projection. Shown is the mean (± SD) volume occupied by densely CTB-labeled retinal afferents in the two most superficial layers of the right SC in control and experimental groups of animals three months after 90 minutes of retinal ischemia to the left eye. (Adapted from: Mayor-Torroglosa S, et al. IOVS 2005; 46: 3825-3835.)

References

1. Vidal-Sanz M, Lafuente MP, Mayor S, de Imperial JM, Villegas-Perez MP. Retinal ganglion cell death induced by retinal ischemia. neuroprotective effects of two alpha-2 agonists. Surv Ophthalmol 2001; 45 Suppl 3: S261-267; discussion S273-266.
2. Vidal-Sanz M, Lafuente MP, Mayor-Torroglosa S, Aguilera ME, Miralles de Imperial J, Villegas-Perez MP. Brimonidine’s neuroprotective effects against transient ischaemia-induced retinal ganglion cell death. Eur J Ophthalmol 2001; 11 Suppl 2: S36-40.
3. Vidal-Sanz M, Lafuente MP, Sobrado-Calvo P, et al. Neuroprotection of retinal ganglion cells after different types of injury. Neurotoxicity Res 2000; 2: 215-227.
4. Salinas–Navarro M, Mayor–Torroglosa S, Holmes T, et al. Automatic Quantitative Analysis of Retinal Ganglion Cells That Project to the Superior Colliculi in Adult Sprague–Dawley Rats. IOVS 2005; 46: E-Abstract 271.
5. Villegas–Perez M, Aguilera M, Salinas–Navarro M, et al. Retinal Ganglion Cells In Adult Albino And Pigmented Rats: Spatial Distribu-tion And Quantitative Analysis. IOVS 2006; 47: E-Abstract 3318.
6. Vidal-Sanz M, Salinas-Navarro M, Avilés-Trigueros M, et al. Spatial Distribution and Quantitative Analysis of Retinal Ganglion Cells in Adult Albino Rodents. IOVS 2007; 48: E-Abstract 134.
7. Lonngren U, Napankangas U, Lafuente M, et al. The growth factor response in ischemic rat retina and superior colliculus after brimonidine pre-treatment. Brain Res Bull 2006; 71: 208-218.
8. Napankangas U, Lonngren U, Lafuente M, et al. The Expression of Growth Factors and Their Receptors After Transient Retinal Ischemia – Modulation by Alpha–2–Receptor Stimulation. IOVS 2006; 47: E-Abstract 4837.
9. Lafuente MP, Villegas-Perez MP, Mayor S, Aguilera ME, Miralles de Imperial J, Vidal-Sanz M. Neuroprotective effects of brimonidine against transient ischemia-induced retinal ganglion cell death: a dose response in vivo study. Exp Eye Res 2002; 74: 181-189.
10. Lafuente MP, Villegas-Perez MP, Sobrado-Calvo P, Garcia-Aviles A, Miralles de Imperial J, Vidal-Sanz M. Neuroprotective effects of alpha(2)-selective adrenergic agonists against ischemia-induced retinal ganglion cell death. IOVS 2001; 42: 2074-2084.
11. Lafuente Lopez-Herrera MP, Mayor-Torroglosa S, Miralles de Imperial J, Villegas-Perez MP, Vidal-Sanz M. Transient ischemia of the retina results in altered retrograde axoplasmic transport: neuroprotection with brimonidine. Exp Neurol 2002; 178: 243-258.
12. Aviles-Trigueros M, Mayor-Torroglosa S, Garcia-Aviles A, et al. Transient ischemia of the retina results in massive degeneration of the retinotectal projection: long-term neuroprotection with brimonidine. Exp Neurol 2003; 184: 767-777.
13. Mayor-Torroglosa S, De la Villa P, Rodriguez ME, et al. Ischemia results 3 months later in an altered ERG, degeneration of inner retinal layers, and a deafferented tectum: neuroprotection with brimonidine. IOVS 2005; 46: 3825-3835.