u

Interim report to the European Society of Contraception and Reproductive Health

Title of the project: Neuroprotective properties of desogestrel

Introduction

In April 2015 the ESCRH agreed to support our project by means of 10 000 EURO.

Neuroprotective effects of the natural progestin progesterone are well-known. However, this progestin is hardly ever used in women with need for contraception or HRT.

Goal of the present project was to investigate if the widely used progestin desogestrel would exert similar protective effects on ischemic stroke and motoric recovery in a rodent model.

Infarct size and functional deficits were analyzed in mice subjected to transient middle cerebral artery occlusion (MCAO). Potential mechanisms of this effect will be further characterized.

Outcomes

Results:

1) Desogestrel treatments result in lesion volume reduction in male mice and improved functional outcome after MCAO in male and ovariectomized mice.

Lesion volume

In adult male mice, ischemia induced a brain infarction of 80.6±8 mm3 on average in mice treated with sesame oil (Figure 1). Administration of progesterone was associated with a reduction in infarct volume to 10.1±5mm3; P<0.001. Desogestrel dose 1 (1.5 mg/kg b.w.) reduced infarct volume to 24.2±14mm3; P<0.01 while desogestrel 2 (3 mg/kg bw) had no significant effect (63.0±15mm3). Ischemia induced a brain infarction of 69.4±11 mm3 in female ovariectomized mice treated with sesame oil. Administration of progesterone, desogestrel after ischemia tended to reduce the infarct volume but this effect was not significant.

Ischemia induced a brain infarction of 46.2±3 mm3 in female premenopausal mice treated daily with sesame oil. Administration of progesterone and desogestrel after ischemia had no significant effect. However, lesion volume was significantly smaller in female treated sesame oil compared to male treated with sesame oil mice (p<0.05), indicating a potential protective role of estrogens in the on-ovariectomised animals.

Figure 1: infarct size in male, ovariectomised female and premenopausal female mice

Red bars: control. Black bars: progesterone treatment. Violet Desogestrel dose 1; desogestrel dose 2 .

Figure 1: Lesion volume after middle cerebral artery occlusion (MCAO) in male, ovariectomized and female mice C57BL/6J mice, given as cubic millimeter. Different colors indicate different treatment groups. Three days after ischemia, infarction was determined by TTC staining and the stroke size was evaluated using the NIH ImageJ software. Data are given as mean ± SEM. Desogestrel1 = desogestrel 1.5 mg/kg/d, Desogestrel2 = desogestrel 3 mg/kg/d,

Sensorymotor function tests: Sticky Tape

Deficits in sensorimotor function after MCAO were assessed in the sticky tape test. The time to removal of the tape on the affected right side was measured. On d1 after left-sided MCAO, deficits to remove the tape on the right forepaw were noted in all male mice, in sesame oil treated mice to a similar degree on d1 and d3 (153.5±16s and 166.8±14s). Progesterone had no significant effect on removal time. On average, deso1 animals performed significantly faster on d1 and d3, (62.5±37s and 66.2±15s for the deso1 respectively; P<0.05 ). In ovariectomized mice, progesterone and desogestrel treated mice tended to perform faster, however these effects were not significant.

In female mice, deficits to remove the tape on the right side at d1 were noted in animals treated with sesame oil, causing an increase in the time to remove the tape on the right forepaw (89.0±30s). The time to removal of the tape on d3 was (114.0±31s). Progesterone treated mice performed significantly faster at d3, which shows less deficits in these animals (20.2±2s; P<0.05). However, desogestrel treated mice showed no significant effect.

Sensorymotor function tests: Composite neurological score

Motor deficits were also evaluated by a composite neurological score (Wahl et al., 1992). The neurological score was obtained by the examination of 4 items and the scores for each item were summed: (1) Forelimb and hind limb grasping reflexes, (2) placing reactions and visual placing, (3) abnormal postures after tail suspension, (4) spontaneous ipsilateral circling. The lower the neurological score the more severe the deficit.

Before surgery, mice had a maximum score of 13 and MCAO induced motor deficits in male sesame oil treated mice at d1 (6.3±1) and d3 (7.1±0.5, Figure 3). Progesterone treatment significantly increased the neurological score at d1 and d3 (10.2±2 and 11.0±2 respectively; P < 0.01). The administration of desogestrel1 significantly increased the neurological score at d1 and d3. (10.0±1 and 11.8±0.5 respectively; P< 0.01 and P<0.001). In ovariectomized mice, MCAO induced motor deficits in sesame oil treated mice at d1 (7.1±1) and d3 (6.8±1). Progesterone treatment significantly increased the neurological score at d3 (10.4±1; P < 0.01). The administration of desogestre1 and desogestrel2 significantly increased the neurological score at d3 (11.0±1 and 9.6±1 respectively; P< 0.05 and P<0.01). In female mice, MCAO induced motor deficits in sesame oil treated mice at d1 (7.7±0.7) and d3 (8.6±0.5). Progesterone treatment significantly increased the neurological score at d1 and d3 (10.5±1 and 11.8±0.3; P < 0.05). The administration of desogestrel had no significant effect on the neurological score at d1 and d3.

Summary of current status

In the first part of the project, we were able to demonstrate a neuroprotective effect of progestereone and desogestrel and after cerebral ischemia in male and less pronounced in female ovariectomized mice. Female, premenopausal mice had smaller infarcts than males and ovariectomized female mice even in the absence of any additional treatment. The smaller infarct size might be attributable to neuroprotective effects of estrogen. In this group, only progesterone had a (small) additional protective effect.

For desogestrel, dose 1 (1.5mg/kg b.w.) seemed to be most effective.

Step 1 (part of the project)

Now that a neuroprotective property of the new progestin desogestrel has been shown, the next step will be to study the mechanisms of this effect. We wish to analyze the influence of GABAA receptor subtypes in the brain, since both have been implicated to contribute to effects of progesterones on brain injury and stress (Maguire et al., 2005). To do so, we will analyze the expression of these receptor subtypes in male mice (most pronounced effect) using the dose of desogestrel leading to the observed neuroprotective effects by Western Blotting. We will also analyze the reason for differential effects of the substances on infarct size versus neurological scores; and are planning to include a few more animals for histological analysis of the brain.

Step 2 (next project)

Non ovariectomised mice showed smaller lesions after MCAO (before hormonal treatment) and no significant reductions with treatment with progestins. These findings might results from the small number of animals involved for each group. As baseline infarction size is smaller, more animals would be necessary to be able to demonstrate a protective effect. We will therefore in a future study focus on this subgroup and include as a result of our power analysis 19 animals.

References

Gibson CL, Murphy SP (2004) Progesterone enhances functional recovery after middle cerebral artery occlusion in male mice. J Cereb Blood Flow Metab 24:805-813.

Maguire JL, Stell BM, Rafizadeh M, Mody I (2005) Ovarian cycle-linked changes in GABA(A) receptors mediating tonic inhibition alter seizure susceptibility and anxiety. Nat Neurosci 8:797-804.

Wahl F, Allix M, Plotkine M, Boulu RG (1992) Neurological and behavioral outcomes of focal cerebral ischemia in rats. Stroke 23:267-272.

Date: May 11th 2016

Prof. Dr. Gabriele Merki

XXX