Progesterone in experimental permanent stroke: a dose-response and therapeutic time-window study

Abstract

Currently, the only approved treatment for ischaemic stroke is tissue plasminogen activator, a clot-buster. This treatment can have dangerous consequences if not given within the first 4 h after stroke. Our group and others have shown progesterone to be beneficial in preclinical studies of stroke, but a progesterone dose-response and time-window study is lacking. We tested male Sprague-Dawley rats (12 months old) with permanent middle cerebral artery occlusion or sham operations on multiple measures of sensory, motor and cognitive performance. For the dose-response study, animals received intraperitoneal injections of progesterone (8, 16 or 32 mg/kg) at 1 h post-occlusion, and subcutaneous injections at 6 h and then once every 24 h for 7 days. For the time-window study, the optimal dose of progesterone was given starting at 3, 6 or 24 h post-stroke. Behavioural recovery was evaluated at repeated intervals. Rats were killed at 22 days post-stroke and brains extracted for evaluation of infarct volume. Both 8 and 16 mg/kg doses of progesterone produced attenuation of infarct volume compared with the placebo, and improved functional outcomes up to 3 weeks after stroke on locomotor activity, grip strength, sensory neglect, gait impairment, motor coordination and spatial navigation tests. In the time-window study, the progesterone group exhibited substantial neuroprotection as late as 6 h after stroke onset. Compared with placebo, progesterone showed a significant reduction in infarct size with 3- and 6-h delays. Moderate doses (8 and 16 mg/kg) of progesterone reduced infarct size and improved functional deficits in our clinically relevant model of stroke. The 8 mg/kg dose was optimal in improving motor, sensory and memory function, and this effect was observed over a large therapeutic time window. Progesterone shows promise as a potential therapeutic agent and should be examined for safety and efficacy in a clinical trial for ischaemic stroke.

Keywords: dose response; functional recovery; progesterone; stroke; therapeutic time window.

Figure 1

Dose-response effect of progesterone on permanent MCAO-induced grip strength and motor deficits. (A) Grip strength: the rats subjected to permanent MCAO + vehicle showed significantly (P < 0.05) lower grip-strength scores at all time points compared with sham + vehicle rats. Progesterone-treated rats showed significant improvement in average scores compared to permanent MCAO + vehicle-treated animals. (B) Motor impairment as assessed using the Rotarod after permanent MCAO: rotometric performance significantly (P < 0.05) decreased in rats subjected to permanent MCAO + vehicle compared with sham-operated + vehicle rats. Progesterone-treated rats were significantly less impaired than vehicle-treated rats at all time points. Values are expressed as mean ± SE.

Figure 2

Dose-response effect of progesterone on permanent MCAO-induced sensory neglect and locomotor activity. (A) Sensory neglect: mean latencies to remove stickers from contralateral forepaws (sensory neglect task) after permanent MCAO: progesterone reduces sensory impairment after permanent MCAO. (B) Locomotor activity: total distance travelled after permanent MCAO. Mean post-stroke locomotor activity at Days 2, 6, 9 and 22 showed increased total distance in progesterone-treated animals. Values are expressed as mean ± SE.

Figure 3

Dose-response effect of progesterone on a memory task as assessed by per cent time spent in the platform quadrant during Morris water maze probe trial. Values are expressed as mean ± SE. *Permanent MCAO + vehicle versus sham + vehicle; #Permanent MCAO + vehicle versus permanent MCAO + progesterone.

Figure 4

Dose-response effect of progesterone on gait impairment after permanent MCAO. (A) Stride length; (B) print area; (C) swing speed as assessed at 2, 6 and 21 days after permanent MCAO. Values are expressed as mean ± SE.

Figure 5

Dose-response effect of progesterone on infarct volume. (A) Cresyl violet-stained coronal sections from representative animals given either vehicle or progesterone, with brains harvested at 23 days post-occlusion. Infarcts are shown as pale (unstained) regions involving the cortex. The infarct area in progesterone-treated animals is substantially reduced. (B) Infarct volumes after 23 days of occlusion. Compared to vehicle alone, P8 and P16 significantly reduced infarct volumes (% of contralateral hemisphere). The data are represented as mean ± SE; *P < 0.05 = significant difference compared with permanent MCAO + vehicle.

Figure 6

Effect of delayed progesterone on permanent MCAO-induced grip strength and motor deficit. (A) 3-h; (B) 6-h; and (C) 24-h delay of progesterone treatment. Values are expressed as mean ± SE. Motor impairment as assessed using the Rotarod. (D) 3-h; (E) 6-h; and (F) 24-h delayed progesterone treatment. Time spent on the Rotarod is expressed as per cent of pre-surgery value (mean ± SE). *Permanent MCAO + vehicle versus sham + vehicle; ^#Permanent MCAO + vehicle versus permanent MCAO + progesterone.

Figure 7

Effect of delayed progesterone treatment on permanent MCAO-induced sensory neglect and locomotor deficit. Mean latencies to remove stickers from contralateral forepaws (sensory neglect task): (A) 3-h; (B) 6-h; (C) 24-h delay of progesterone treatment. Total distance travelled after permanent MCAO: (D) 3-h; (E) (6-h); (F) 24-h delayed progesterone treatment. Values are expressed as mean ± SE. *Permanent MCAO + vehicle versus sham + vehicle; ^#Permanent MCAO + vehicle versus permanent MCAO + progesterone.

Figure 8

Delayed progesterone treatment effects on a memory task as assessed by per cent time spent in the platform quadrant during Morris water maze probe trial. Values are expressed as mean ± SE. *Permanent MCAO + vehicle versus sham + vehicle; ^#Permanent MCAO + vehicle versus permanent MCAO + progesterone.

Figure 9

Delayed progesterone treatment effect on permanent MCAO-induced gait impairments. (A–C) Stride length; (D–F) print area; (G–I) swing speed following 3-, 6-, and 24- h delayed progesterone treatment. Values are expressed as mean ± SE. *Permanent MCAO + vehicle versus sham + vehicle; ^#Permanent MCAO + vehicle versus permanent MCAO + progesterone.

Figure 10

Delayed progesterone treatment effects on infarct volume at 23 days post-occlusion. (A) Cresyl violet-stained coronal sections from representative animals given either vehicle or progesterone, with brains harvested at 22 days post-occlusion. Infarcts are shown as pale (unstained) regions involving the cortex. (B) Delayed progesterone treatment reduces infarct volume at 3 h and 6 h at 23 d post-permanent MCAO. Compared to vehicle alone, progesterone significantly reduced infarct volumes (% of contralateral hemisphere). Progesterone treatment begun at 24 h did not reduce the infarct volume. The data are represented as mean ± SE; *P < 0.01 = significant difference compared to permanent MCAO + vehicle.