Progesterone and allopregnanolone exacerbate hypoxic-ischemic brain injury in immature rats.

Abstract

Progesterone and its metabolite, allopregnanolone, are neurosteroids that are present at high concentrations in fetal brains that decrease right after birth. Allopregnanolone is a potent positive modulator of γ-aminobutyric acid A (GABA(A)) receptor function. We examined the effect of exogenous administration of these steroids on hypoxic-ischemic encephalopathy in immature rats. Progesterone (10mg/kg), allopregnanolone (10mg/kg), or vehicle alone was intraperitoneally administered immediately before and then subcutaneously 6h and 24h after hypoxia-ischemia to postnatal day 7 (P7), day 14 (P14), and day 21 (P21) rats. The effects of the treatments were evaluated using histological analyses (hemispheric volumes and semi-quantitative scoring for neuropathologic injury). Both progesterone and allopregnanolone significantly exacerbated brain injury in P7 and P14 rats, but not in P21 rats. This detrimental effect was similar across the examined brain regions (the cortex, striatum, hippocampus, and thalamus) and showed no sex differences. Co-administration of the GABA(A) receptor antagonist, bicuculline, partially mitigated the exacerbating effect of allopregnanolone. Based on the similarity of the effects of these neurosteroids, we speculate that progesterone accentuates neuronal injury mainly via the activity of allopregnanolone. The present study indicates that the detrimental effects of allopregnanolone were, at least in part, mediated via GABAergic neuroexcitability. This is in line with the notion that GABA is excitatory for immature neurons, while it is inhibitory for mature neurons.

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PROGESTERONE AND ALLOPREGNANOLONE EXACERBATE HYPOXIC-ISCHEMIC
BRAIN INJURY IN IMMATURE RATS
M. Tsuji, A. Taguchi, T. Ikeda
Department of Regenerative Medicine and Tissue Engineering, National Cerebral and
Cardiovascular Center Research Institute, Suita, Japan
Objectives: Progesterone (PROG) and its metabolite, allopregnanolone (ALLO, 3α-hydroxy-5α-
pregnan-20-one, 3α,5α-tetrahydroprogesterone) are neuroactive steroids that are present at
high concentrations in the fetal brains of humans and rats. The PROG and ALLO concentrations
decrease right after birth. ALLO is a potent positive modulator of γ-aminobutyric acid A (GABAA)
receptor function (1). We examined the effect of exogenous administration of these steroids in
an immature rat model of hypoxic-ischemic encephalopathy.

Methods: Seven-day-old (experimental paradigm, P7), 14 day-old (P14), and 21 day-old (P21)
male and female Wistar rats underwent left carotid artery ligation, followed by 120 min, 80 min,
and 50 min of hypoxic (8% oxygen) exposure, respectively. The duration of the hypoxic
exposure was optimized to obtain similar degrees of brain injury in each age groups. PROG (10
mg/kg), ALLO (10 mg/kg), or vehicle were administered intraperitoneally, immediately before,
and then subcutaneously at 6 h and 24 h after the start of the hypoxic exposure. In a different
experimental paradigm, the GABAA receptor antagonist, Bicuculline (2 mg/kg) was administered
intraperitoneally immediately before and subcutaneously, 6 h after each ALLO injection. Seven
days after the hypoxic-ischemic insult, the brains were removed and dissected into coronal 2-
mm sections. We then measured the area of the contralateral and ipsilateral hemispheres in
each brain section. The hemispheric volume for each brain was estimated by summing the
hemispheric area of the brain slices and then multiplying by the section interval thickness. We
evaluated the neuropathological injury in hematoxylin-eosin-stained sections from four brain
regions (cortex, hippocampus, striatum, thalamus) using a semi-quantitative scoring system, as
described previously (2).

Results: Treatment with either PROG or ALLO significantly reduced the ipsilateral hemispheric
volume after the hypoxic-ischemic insult in P7 (p < 0.01) and P14 (p < 0.05), but not P21 rats
compared with vehicle-treated rats. Both steroid treatments increased (exacerbated) the total
neuropathologic injury score in P7 rats, however, only the treatment with PROG showed any
statistically significant changes (p < 0.01). This detrimental effect was similar in the four brain
regions examined. The mortality was significantly higher in the ALLO treated animals (p < 0.05).
Treatment with lower doses of ALLO (3 mg or 1 mg) was less detrimental. The GABAA receptor
antagonist, Bicuculline partially abolished the exacerbating effect of ALLO.

Conclusions: These findings indicate that both PROG and ALLO exhibit adverse effects in
hypoxic-ischemic brain injury in immature rats. Our results in immature rats are in contrast with
the results of an adult stroke model that showed neuroprotective effects of the two compounds
(3). These contrasting results may be due to developmental changes of GABAA receptor
function. GABA depolarizes immature neurons while hyperpolarizing mature neurons (4). Our
studies indicate that GABAA receptors may be involved in the detrimental effect of ALLO in
immature animals.

References:

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1. Paul SM, Purdy RH. FASEB J 1992; 6: 2311-22.

2. Tsuji M, et al. Exp Neurol 2004; 189: 58-65.

3. Sayeed I, et al. Ann Emerg Med. 2006; 47: 381-9.

4. Ben-Ari Y, et al. Physiol Rev. 2007; 87: 1215-84.