Progesterone treatment in two rat models of ocular ischemia

Abstract

Purpose: To determine whether the neurosteroid progesterone, shown to have protective effects in animal models of traumatic brain injury, stroke, and spinal cord injury, is also protective in ocular ischemia animal models.

Methods: Progesterone treatment was tested in two ocular ischemia models in rats: a rodent anterior ischemic optic neuropathy (rAION) model, which induces permanent monocular optic nerve stroke, and the middle cerebral artery occlusion (MCAO) model, which causes transient ischemia in both the retina and brain due to an intraluminal filament that blocks the ophthalmic and middle cerebral arteries. Visual function and retinal histology were assessed to determine whether progesterone attenuated retinal injury in these models. Additionally, behavioral testing and 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining in brains were used to compare progesterone's neuroprotective effects in both retina and brain using the MCAO model.

Results: Progesterone treatment showed no effect on visual evoked potential (VEP) reduction and retinal ganglion cell loss in the permanent rAION model. In the transient MCAO model, progesterone treatment reduced (1) electroretinogram (ERG) deficits, (2) MCAO-induced upregulation of glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP), and (3) retinal ganglion cell loss. As expected, progesterone treatment also had significant protective effects in behavioral tests and a reduction in infarct size in the brain.

Conclusions: Progesterone treatment showed protective effects in the retina following MCAO but not rAION injury, which may result from mechanistic differences with injury type and the therapeutic action of progesterone.

Figure 1

Fundus photographs from control, progesterone-treated AION, and vehicle-treated AION rats. In control retinas, distinct boundaries (arrow) and a reddish flush indicative of intact microvasculature (*) were observed. Lack of distinct boundaries in AION retinas is a sign of optic nerve edema, and lack of a reddish flush suggests obliteration of microvasculature. Additionally, vascular dilation (arrowhead) was observed in AION retinas. No differences were observed between vehicle- and progesterone-treated AION retinas. ON, optic nerve; A, artery; V, vein.

Figure 2

Visual evoked potentials for control and progesterone- and vehicle-treated rAION rats at 3 days post AION. (A) Averaged VEP waveforms. Arrows designate P1 and N1 for controls. (B) Mean P1 and N1 amplitudes at 3 days post AION. A significant main effect of group was observed for N1 amplitudes [Kruskal-Wallis one-way ANOVA on ranks, P < 0.05], with both progesterone- and vehicle-treated rAION groups showing reduced N1 amplitudes. (C) No differences were observed in mean P1 and N1 implicit times at 3 days post AION.

Figure 3

Immunohistochemistry with the retinal ganglion cell–specific marker, Brn3a. (A) Representative flat mount petals stained with Brn3a for retinas from control (top left), vehicle-treated AION (top right), and progesterone-treated AION rats (bottom left). Retinas from both progesterone- and vehicle-treated rats had petals showing extensive retinal ganglion cell loss (#) and petals that appeared “healthy” (*). (B) Both progesterone- and vehicle-treated AION retinas showed a significant reduction in retinal ganglion cells as compared with control retinas (P < 0.001).

Figure 4

Representative ERG waveforms from sham, progesterone-, and vehicle-treated MCAO rats. Vehicle-treated rats show reduced amplitudes in both MCAO and contralateral eyes at 2 days post MCAO in response to 137 cd s/m² flash stimuli. Progesterone treatment reduces these deficits, particularly in contralateral eyes.

Figure 5

Quantification of ERG a-wave, b-wave, and oscillatory potential amplitudes and implicit times. Significant reductions in dark-adapted a-wave (A), b-wave (C), and oscillatory potential amplitudes (E) were observed in MCAO eyes from vehicle-treated rats compared with shams. Significant reductions in a-wave and b-wave amplitude were also observed in contralateral eyes from vehicle-treated MCAO rats compared with shams (P < 0.05). Progesterone-treated MCAO rats showed significant increases over vehicle-treated MCAO rats in b-wave amplitude at 0.249 and 4.1 cd s/m² flash stimuli in contralateral retinas (P < 0.05) and a trend for an increase in MCAO retinas. *Significantly different from Veh MCAO and Prog MCAO. **Significantly different from Veh MCAO, Prog MCAO, and Veh Contra. #Significant difference between Prog Contra and Veh Contra at this flash intensity. A significant main effect of group was observed for dark-adapted a-wave implicit times (B). Significant delays in ERG implicit times were observed for dark-adapted b-waves (D) and oscillatory potentials (F) for MCAO eyes from vehicle-treated rats compared with shams. *P < 0.05. Prog, progesterone; Veh, vehicle; Contra, contralateral.

Figure 6

Representative photographs from immunohistochemistry with GFAP and GS after MCAO. Three days after MCAO, GFAP was upregulated in Müller cells in MCAO (and some contralateral) retinas from vehicle-treated rats, but not progesterone-treated rats, compared with shams. Glutamine synthetase was upregulated in Müller cells in MCAO and contralateral retinas from vehicle-treated rats, with slight upregulation in MCAO and contralateral retinas from progesterone-treated rats, compared with shams. GCL, ganglion cell layer; lPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments.

Figure 7

Mean GS intensity as percent of sham for vehicle- and progesterone-treated MCAO rats. Significant increases in GS intensity were observed in MCAO retinas from vehicle-treated rats compared with shams (P < 0.01), with a trend for an increase in contralateral retinas. Both MCAO and contralateral retinas from progesterone-treated rats showed significantly reduced levels of GS intensity compared with vehicle-treated rats (P < 0.05), but did not differ significantly from shams.

Figure 8

Representative photographs of cresyl violet staining and quantification of retinal ganglion cells. (A) Cresyl violet staining in retinal sections from sham, vehicle-treated MCAO, and progesterone-treated MCAO rats. (B) At 3 days post MCAO, ipsilateral retinas from vehicle-treated MCAO rats showed significantly reduced retinal ganglion cell numbers compared to all other groups (P < 0.001). Ipsilateral retinas from progesterone-treated MCAO rats showed significant increases in retinal ganglion cell counts over ipsilateral retinas from vehicle-treated MCAO rats (P < 0.001), and significant decreases compared with all other groups (P < 0.001). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments.

Figure 9

Behavioral assessment at 1 day post MCAO. (A) Vehicle-treated MCAO rats showed reduced grip strength compared with shams (P < 0.001). Progesterone-treated MCAO rats showed significant increases in grip strength over vehicle-treated MCAO rats (P < 0.05). (B) Vehicle-treated MCAO rats showed increased time to notice on sticky-tape task compared with shams (P < 0.001). Progesterone-treated MCAO rats showed reduced time to notice compared with vehicle-treated MCAO rats (P < 0.01).

Figure 10

Representative TTC staining and quantification at 3 days post MCAO. Representative TTC-stained brain slices for (A) vehicle-treated and (B) progesterone-treated MCAO rats with ∼70% occlusion. (C) Reduced infarct size was observed in progesterone- versus vehicle-treated MCAO rats (45%, P = 0.054).