doi:10.1093/brain/awm183
Brain (2008), 131, 318 ^328
REVIE W ARTICLE
Progesterone for the treatment of experimental
brain injury; a systematic review
Claire L. Gibson,1 Laura J. Gray,2 Philip M. W. Bath2 and Sean P. Murphy3
1
School of Psychology, University of Leicester, Lancaster Road, Leicester, LE1 9HN, UK, 2Division of Stroke Medicine,
University of Nottingham, City Hospital Campus, Nottingham, UK and 3Department of Neurological Surgery,
University of Washington School of Medicine, Seattle, WA, USA
Correspondence to: Dr Claire L. Gibson, School of Psychology, Henry Wellcome Building, Lancaster Road, Leicester,
LE1 9HN, UK
E-mail: [email protected]
Steroid sex hormones are potential neuroprotective candidates following CNS injury. All clinical trials to date
have examined the effects of oestrogen alone or oestrogen-progestin combination therapy. Experimental studies have suggested that progesterone, in its own right, is a potential neuroprotective agent following acute cerebral injury. We performed a systematic review of controlled animal studies that administered progesterone
before, or after, acute cerebral injury and measured lesion volume. Relevant studies were found from searching
PubMed, Embase and Web of Science. From 119 identified publications, data from 18 studies using 480 experimental subjects met specific criteria and were analysed using the Cochrane Review Manager software.
Following cerebral ischaemia, a significant benefit of progesterone was observed regardless of the assigned
study quality score (P = 0.0002) whereas, following traumatic brain injury (TBI) a significant benefit of progesterone was only observed in studies that obtained the highest quality score of 5 (P = 0.02). Progesterone reduced
lesion volume in a dose-dependent manner following either cerebral ischaemia (P < 0.001) or TBI (P = 0.03) with
the most effective progesterone dose varying according to experimental injury model used. Progesterone treatment was only effective at reducing lesion volume when administered immediately following (i.e. 0^2 h) cerebral
ischaemia (P = 0.0008). No studies using models of cerebral ischaemia or TBI assessed efficacy when progesterone was administered at later than 6 h following the onset of cerebral injury. Limited data were available for
different groups of animals according to age/hormonal status and the full dose^response relationship was not
available in all experimental groups. Although this systematic review provides some supporting evidence for a
neuroprotective role of progesterone following either cerebral ischaemia or TBI importantly it highlights areas
which need further pre-clinical investigation.
Keywords: progesterone; stroke; traumatic brain injury; systematic review; neuroprotection
Abbreviations: SMD = standardized mean difference; TBI = traumatic brain injury
Received March 15, 2007. Revised June 25, 2007. Accepted July 10, 2007. Advance Access publication August 21, 2007
Introduction
Stroke is the leading cause of neurological disability and a
major cause of death in the western world (Lo et al., 2003).
In addition, traumatic brain injury (TBI) is the leading
cause of disability in adults under the age of 40 (Djebaili
et al., 2004). Despite advances in our understanding of the
pathophysiological and pathochemical damage that occurs
following either of these types of brain injury current
treatments are limited both in their efficacy and utility.
Both stroke and TBI result in massive neuronal loss
and research has primarily focused on developing neuroprotective strategies in order to prevent/reduce this.
Evidence exists for a gender difference in vulnerability to
either stroke or TBI in humans both in terms of risk and
outcome. For example, pre-menopausal women have a
lower risk of stroke (Kannel and Thom, 1994; Sacco et al.,
1997) and a better outcome following stroke (Thorvaldsen
et al., 1995) or TBI (Groswasser et al., 1998; Bounds et al.,
1995) relative to men of the same age. Following the
menopause, the incidence of stroke in women rapidly
ß The Author (2007). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
Progesterone; experimental brain injury
increases (Wenger et al., 1993) coincident with diminished
circulating levels of the sex steroid hormones oestrogen and
progesterone.
Whilst the majority of research has focused on oestrogen
as the main source of neuroprotection seen in female
animals (Gibson et al., 2006), there is increasing evidence
that progesterone also plays a beneficial role in the injured
brain. The neuroprotective potential of progesterone was
initially revealed by the observation that female rats recover
better from TBI than male rats in terms of functional
impairment (Atella et al., 1987; Stein, 2001). Importantly,
these studies were extended to demonstrate that females
high in endogenous progesterone (i.e. pseudo-pregnant) at
the time of injury recovered better in comparison to
untreated males. The evidence for the neuroprotective effect
of progesterone following TBI is substantial (Roof et al.,
1993; Stein, 2001, 2005) and has led to the first randomized
clinical trial for acute TBI (ProTECT). This trial has just
been successfully completed and the investigators report
that the use of progesterone after TBI was well tolerated in
terms of safety (Wright et al., 2007). Although experimental
evidence is beginning to reveal a neuroprotective effect of
progesterone following cerebral ischaemia no clinical trials
have examined the effects of acute administration of
progesterone following cerebral stroke to date.
To evaluate the neuroprotective potential of progesterone
we have conducted a systematic review of animal studies to
investigate the neuroprotective properties of exogenously
applied progesterone on lesion volume after experimental
cerebral injury (i.e. cerebral ischaemia or TBI). Lesion
volume was chosen as the most relevant outcome for the
purpose of this review although some may argue it has
limited value when considering whether a treatment is
beneficial. The Stroke Academic Industry Roundtable
(STAIR, 1999) suggested that drug efficacy in animals
should be assessed against behavioural rather than volumetric
end points. However, direct evidence is lacking to prove this
approach results in better prediction of efficacy in clinical
trial, and in fact in humans lesion volume determined by
MRI diffusion-weighted imaging shows correlation both with
impairment at 24 h (Tong et al., 1998) and with clinical
outcome (Wardlaw et al., 2002; Engelter et al., 2003). The
purpose of this review is to identify key factors, such as
timing of treatment, therapeutic dose and effectiveness
according to sex and age, which require further experimental
investigation. Such experimental studies will help inform the
design of future clinical trials aimed at assessing the
neuroprotective potential of acute progesterone treatment
following cerebral stroke or TBI.
Methods
Study identification
Experimental controlled studies of the effects of exogenously
applied progesterone on lesion size in animal models of cerebral
injury (i.e. stroke or TBI) were identified from PubMed, Embase,
Brain (2008), 131, 318 ^328
319
Fig. 1 Search process showing reasons for exclusions of studies.
A total of 18 studies were included. Studies were excluded if they
did not report the following: induction of experimental brain
injury, administration of progesterone, measurement of lesion
volume or contain primary data. N, number of studies.
and Web of Science by searching for all articles published from
1980 to December 31, 2006. The earliest study included for
analysis was from 1994 (Roof et al., 1994). Additional publications
were identified from reference lists of all identified publications
and non-systematic (i.e. literature-based reviews) review articles
(Roof and Hall, 2000; Stein, 2001, 2005; Simpkins et al., 2005;
Singh, 2005, 2006). The search strategy employed the following
keywords: progesterone, traumatic or ischaemia, cerebral.
However, studies were only included if they met all of the
following criteria: experimental cerebral injury induced
(i.e. cerebral ischaemia or TBI), progesterone administered and
infarct volume measured (Fig. 1).
Data extraction
Two authors (C.L.G. and L.J.G.) independently extracted data
from relevant publications on animal species, number, gender,
model of brain injury, intervention (dose of progesterone, timing
relevant to induction of injury) and infarct volume (mm3,% of
normal brain, mean,SD). A comparison (C) was defined as
assessment of outcome (i.e. lesion volume) in treatment and
control groups after treatment with an administered dose of
progesterone starting at a stated time before or after the onset of
cerebral injury. For each comparison, the data for mean outcome,
SD and number of animals per group were extracted. If published
studies (S) used multiple groups, for example to assess
dose–response relationships, then the data from each group were
320
Brain (2008), 131, 318 ^328
C. L. Gibson et al.
Fig. 2 Funnel plot showing the existence of publication bias. Publication bias was assessed by visually examining a funnel plot of precision
(reciprocal of SE) against the SMD and asymmetry was formally assessed using the Egger test. (A) Publication bias was absent for studies
reporting the effect of progesterone administration on lesion volume in models of cerebral ischaemia (Egger’s test statistic = 0.64,
P = 0.53). (B) However, publication bias was present for studies using models of TBI (Egger’s test statistic = 2.31, P = 0.038).
individually extracted for analysis. Infarct volumes were classified
as total and, if available, cortical and subcortical. Occasionally,
numerical data were not reported in the text and these were
extracted from enlarged, photocopied figures.
The methodological quality of each study was assessed using an
eight-point rating as described previously (Macleod et al., 2005;
Wilmot et al., 2005a, b; Gibson et al., 2006). One point was given
for written evidence of each of the following criteria: presence of
randomization, monitoring of physiological parameters, assessment of dose–response relationship, assessment of optimal time
window, masked outcome measurement, assessment of outcome at
Days 1–3, assessment of outcome at Days 1–30, combined
measurement of lesion volume and functional outcome.
Data analysis
The data were analysed using Cochrane Review Manager (version
4.2) and Stata (version 7). The effect of progesterone compared
with control on total lesion volume was assessed using the
standardized mean difference (SMD); here the difference in effect
between the progesterone and control group is divided by the total
SD. This allows comparisons to be made if different methods of
measurement or different animal species have been used. These
estimates were pooled using the DerSimonian and Laird (1986)
random effects model, which is a more conservative method than
using a fixed-effects model and takes into account any statistical
heterogeneity found between studies.All analyses were carried out
by type of cerebral injury, ischaemia and TBI. To examine the
effects of study characteristics and potential sources of heterogeneity on outcome, stratified meta-analyses were performed
with experiments grouped according to: (i) study quality score;
(ii) population grouping—all animals, adult males, ovariectomized females, intact females, aged females, aged males;
(iii) progesterone dose and (iv) timing of progesterone administration in relation to onset of brain injury.
Publication bias was assessed by visually examining a funnel
plot (Fig. 2) of precision (reciprocal of SE) against the SMD, with
asymmetry being formally assessed using the Egger test (STATA
function ‘metabias’; Egger et al., 1997). Significance was set at
P < 0.05 and 95% confidence intervals (CI) are quoted
throughout.
Results
Methodological design
The literature search identified 119 potential studies,
although most had to be excluded for reasons given in
Fig. 1. The characteristics of the remaining 18 studies are
reported in Table 1. All of the included studies reported the
effect of exogenously administered progesterone on lesion
volume after acute cerebral injury. All included studies
reported the effect of administering progesterone versus no
progesterone (or vehicle) on infarct volume. The 18 studies
represented the outcome from seven independent research
groups. Five of the studies came from four different
research groups, while the remaining 13 studies represent
work from three research groups. Within the 18 studies,
data from a total of 480 experimental subjects were
included for analysis.
The majority of studies employed a model of transient
focal ischaemia (10 studies), with one study using a model
of permanent focal ischaemia (Gibson et al., 2005) and
seven studies using a model of TBI. Various rat strains
(Wistar, Sprague-Dawley, Spontaneously Hypertensive and
Reproductively Senescent) were used in 15 out of the 18
studies; three studies used mice. Methodological design was
variable as far as drug administration was concerned.
Several routes of administration (subcutaneous, intraperitoneal, intravenous) were used with first-dose timings
in relation to onset of cerebral injury ranging from
7 days before, to 6 h after. In terms of outcome measure,
lesion volume was visualized by histological staining and
reported as; lesion volume (mm3), percentage of total crosssectional area, or percentage of ipsilateral non-ischaemic
total/region.
Publication bias was absent for studies reporting the
effect of lesion volume in models of stroke (Egger’s test
statistic = 0.64, P = 0.53, Fig. 2A) but present for studies
using models of TBI (Egger’s test statistic = 2.31,
P = 0.038, Fig. 2B).
Progesterone; experimental brain injury
Brain (2008), 131, 318 ^328
321
Table 1 Characteristics of included studies
Study
Year Species Sex/hormonal Age Model of Dose range Preparation
status
injury
(mg/kg)
First dose Route
timing
Measure Other
of infarct outcome(s)
Alkayed et al.
Chen et al.
Choi et al.
Cutler et al.
Djebali et al.
Gibson and Murphy
Gibson et al.
Goss et al.
He et al.
Jiang et al.
Jones et al.
Kumon et al.
Murphy et al.
Murphy et al.
Robertson et al.
Roof et al.
Sayeed et al.
Toung et al.
2000
1999
2004
2006
2004
2004
2005
2003
2004
1996
2005
2000
2000
2002
2006
1994
2006
2004
7d
+2 h
24 h
+1h
+1h
+1h
+1h
+1h
+1h
0.5 h
+0.03 h
+2 h
0.5 h
7d
7d
+1h
+2 h
0.5 h
%
%
mm3
%
%
mm3
mm3
%
%
%
mm3
%
%
%
%
%
%
%
WR
WR
SDR
SDR
SDR
CM
CM
SDR
SDR
WR
CM
SHRSP
WR
WR
SDR
SDR
SDR
WR
RSF
M
M
M
M
M
M
M
M
M
M
M
OF
OF
OF
M
M
RSF
A
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
A
FCI (T)
FCI (T)
FCI (T)
TBI
TBI
FCI (T)
FCI (P)
TBI
TBI
FCI (T)
TBI
FCI (T)
FCI (T)
FCI (T)
TBI
TBI
FCI (T)
FCI (T)
10
4^32
4
16
16
8
8
8^32
4
4
8
4^ 8
5^20
30^ 60
NR
4
8
5
Progesterone
Progesterone
Progesterone
Progesterone
Progesterone
Progesterone
Progesterone
Progesterone
Metabolites
Progesterone
Progesterone
Progesterone
Progesterone
Progesterone
Progesterone
Progesterone
Progesterone
Progesterone
s.c.
i.v.
i.p.
i.p.
i.p., s.c.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
s.c.
i.p.
i.p., s.c.
i.p.
Function
Function
Function
Function
Function
NS
Function
NS
Function
WR = Wistar rats; SDR = Sprague-Dawley rats; CM = C57 mice; SHRSP = stroke-prone spontaneously hypertensive rats;
RSF = reproductively senescent females; M = males; OF = ovariectomised females; A = aged; N = normal adult; FCI (T) = focal cerebral
ischemia (transient); TBI = traumatic brain injury; FCI (P) = focal cerebral ischemia (permanent); NR = not reported; s.c. = subcutaneous;
i.v. = intravenous; i.p. = intra-peritoneal; NS = neurological score.
Reported study quality
The median quality rating for included articles that used a
model of cerebral ischaemia was 4 out of 8 (range 2–7)
whilst the median quality rating for included articles using
a model of TBI was 5 (range 2–5). Animals were allocated
treatment by randomization in six out of the 11 included
ischaemia articles (Jiang et al., 1996; Kumon et al., 2000;
Gibson and Murphy 2004; Toung et al., 2004; Gibson et al.,
2005; Sayeed et al., 2006) and all but one (Robertson et al.,
2006) of the seven included TBI articles did so. Whereas
only one TBI article that was included (Goss et al., 2003)
reported the monitoring of physiological parameters, all of
the ischaemia articles did so (although the majority of these
only monitored body temperature). Only four ischaemia
articles (Chen et al., 1999; Kumon et al., 2000; Murphy
et al., 2000, 2002) and three TBI articles (Goss et al., 2003;
Djebali et al., 2004; Robertson et al., 2006) assessed
dose–response relationships. Three ischaemia articles
(Jiang et al., 1996; Murphy et al., 2000, 2002) investigated
the optimal time window of administering progesterone
whereas no included articles using a TBI model reported
doing so. However, one published study has demonstrated
that progesterone is still effective at reducing oedema
volume when administered at 24 h following TBI (Roof
et al., 1996). All 11 included articles that used a model of
ischaemia assessed outcome at Days 0–3; three of those also
assessed outcome at Days 7–30 (Chen et al., 1999; Kumon
et al., 2000; Gibson and Murphy 2004) and four included
articles reported the combined measurement of lesion
volume and functional outcome (Jiang et al., 1996;
Chen et al., 1999; Kumon et al., 2000; Gibson and
Murphy 2004). All of the seven included articles that
used a model of TBI assessed outcome at Days 7–30
whereas only three (Djebali et al., 2004; Jones et al., 2005;
Cutler et al., 2006) assessed outcome at Days 1–3 and all
but one (Robertson et al., 2006) of those included
combined lesion volume measurements with measures of
functional outcome. Only four of the ischaemia articles
(Gibson et al., 2005; Kumon et al., 2000) and three of the
TBI articles (He et al., 2004; Jones et al., 2005; Cutler et al.,
2006) reported outcome measures being blinded to
treatment.
Following cerebral ischaemia, a beneficial effect of
progesterone treatment was observed when the effects of
quality score are discounted (effect size = 0.51, 95%
CI = 0.82 to 0.19, P = 0.0002). The beneficial effect of
administering progesterone increased with increasing
reported quality score (Fig. 3A); indeed, there was no
apparent beneficial effect of administering progesterone in
studies that obtained a quality score of 4 or less. Following
TBI progesterone did not have a beneficial effect on
lesion volume when the effects of quality score are
discounted ( 0.37, 0.79 to 0.06, P = 0.09); however, this
result was dependent on a low quality study (score 3)
reporting a detrimental effect of progesterone (P = 0.02).
Higher quality studies (median quality score 5) reported a
beneficial effect of progesterone administration (P = 0.02,
Fig. 3B).
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C. L. Gibson et al.
Fig. 3 Standardized mean difference and 95% CI by reported quality score after cerebral ischaemia and TBI. (A) Effect size was independent of quality score after cerebral ischaemia. (B) However, a beneficial effect of progesterone was only present after TBI in studies with a
reported quality score of 4 or 5. N, number of animals; C, number of comparisons; S, number of published studies.
have a detrimental effect when administered to ovariectomized females although this was not significant (P = 0.07).
Overall, significantly different estimates were found within
hormonal status groups (males, ovariectomized females,
intact females) for lesion volume after cerebral ischaemia
(2 = 57.44, df = 24, P = 0.0001). Following TBI progesterone treatment had a trend to benefit when administered to
males (P = 0.07).
Dose of progesterone
Fig. 4 Standardized mean difference and 95% CI for lesion volume
following either cerebral ischaemia or TBI. Data shown are
grouped according to hormonal status/age of animals; males,
ovariectomized females, intact females and aged females.
When grouped according to hormonal status/age progesterone
only had a significant beneficial effect in male animals that had
undergone cerebral ischaemia. However, studies were limited
to similar groups of animals being used in terms of hormonal
status/age. Ov, ovariectomized; N, number of animals; C, number
of comparisons; S, number of published studies.
Infarct volume according to
hormonal status/age
The effect of progesterone on lesion volume following
cerebral ischaemia or TBI was analysed according to
population groupings based on hormonal status i.e.
males, ovariectomized females, intact females, aged females
and aged males (Fig. 4).
Following cerebral ischaemia progesterone treatment had
the largest beneficial effect when administered to males
(P 4 0.00001), a trend to benefit was also seen in aged
females (P = 0.21). In contrast, progesterone appeared to
The effects of progesterone dose on lesion volume were also
analysed irrespective of hormonal status (Fig. 5). For
articles that utilized a model of cerebral ischaemia a
beneficial effect of progesterone on lesion volume was only
observed in the lowest dose treatment group, i.e. 1–15 mg/
kg ( 0.77,
1.09 to
0.45, P < 0.00001, Fig. 5A).
Following TBI, a significant effect of progesterone was
seen in a higher dose range i.e. 16–30 mg/kg than that seen
for cerebral ischaemia ( 0.85, 1.59 to 0.10, P = 0.03,
Fig.5A). Significantly different estimates were found within
progesterone dose groups for cerebral ischaemia (2 = 57.39,
df = 24, P = 0.0001) and TBI (2 = 27.55, df = 12, P = 0.006).
In order to identify any gender differences in response to
different progesterone doses, the data were analysed for
males (Fig. 5B) and ovariectomized females (Fig. 5C). For
males, the only dose range to have any beneficial effect on
lesion volume was 1–15 mg/kg for cerebral ischaemia
(P < 0.00001) and 16–30 mg/kg for TBI (P = 0.03).
Following cerebral ischaemia progesterone appeared to
have an increasing detrimental effect with increasing dose
when administered to ovariectomized females (Fig. 5C)
however this effect was not significant and not all dose
ranges have been explored in all groups.
Timing of progesterone administration
in relation to onset of ischaemia
Only post-injury administration of progesterone was
effective at reducing lesion volume when comparing all
Progesterone; experimental brain injury
Brain (2008), 131, 318 ^328
323
Fig. 5 Standardized mean difference and 95% CI for lesion volume following cerebral ischaemia or TBI in all animals (A), males (B) and
ovariectomized females (C). Data are grouped according to the dose of progesterone administered (mg/kg, where reported). Following
cerebral ischaemia progesterone had a significant effect on reducing lesion volume after administration of the lowest dose range of
progesterone reported i.e. 1^15 mg/kg whereas a beneficial effect of progesterone following TBI was reported following progesterone
administration within the dose range of 16 ^30 mg/kg. N, number of animals; C, number of comparisons; S, number of published studies.
animals (Fig. 6). Progesterone was effective at reducing
lesion volume when administered 0–2 h ( 1.18, 1.97 to
0.38, P = 0.004, Fig. 6A) or 2–6 h ( 0.86, 1.23 to 0.49,
P = 0.0008, Fig. 6A) following cerebral ischaemia. In males,
progesterone treatment also had a significant effect when
administered immediately prior to the onset (i.e. 2 to 0 h)
of cerebral ischaemia ( 0.70, 1.29 to 0.11, P = 0.004,
Fig. 6B). In contrast, progesterone appeared to have a
detrimental effect (non-significant) on lesion volume when
administered prior to ischaemia in ovariectomized females
(Fig. 6C). No studies reported administering progesterone
to ovariectomized females following the onset of cerebral
ischaemia. No studies reported the effects of administering
progesterone on lesion volume beyond 6 h following the
onset of ischaemia.
Although progesterone appeared to be effective when
administered between 0 and 2 h post-TBI this effect was not
significant (P = 0.07, Fig. 6A). Additionally, no studies
reported the effects of administering progesterone on lesion
volume at later than 2 h following the onset of TBI or
immediately prior (i.e. 2 to 0 h) to the onset of TBI. The
majority of TBI studies used male animals (Fig. 6B) with
ideal timing of administration not being fully investigated
for other groups. Significantly different estimates were
found within the timing of progesterone groups for cerebral
ischaemia (2 = 57.44, df = 24, P = 0.0001) and TBI
(2 = 29.19, df = 14, P = 0.01).
Discussion
This systematic review indicates that progesterone is
neuroprotective, in terms of reducing lesion volume,
following either cerebral ischaemia or TBI. However,
progesterone was only effective at reducing lesion volume
following TBI in those studies that achieved the highest
quality score whereas progesterone was effective following
cerebral ischaemia regardless of quality score. To identify any
limitations of the effectiveness of progesterone, data were
grouped according to hormonal status and age, dose and
timing of progesterone treatment in relation to the onset of
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C. L. Gibson et al.
Fig. 6 Standardized mean difference and 95% CI for lesion volume following cerebral ischaemia or TBI in all animals (A), males (B) and
ovariectomized females (C). Data are grouped according to the time of the first administration of progesterone in relation to the onset of
either cerebral ischaemia or TBI. Progesterone was only effective when administered following the onset of cerebral ischaemia. N, number
of animals; C, number of comparisons; S, number of published studies.
cerebral injury. When data were grouped according to
hormonal status and age, progesterone treatment following
cerebral ischaemia was only effective when administered to
males; an effect not seen following TBI. However, there were
insufficient studies of progesterone in other various groups
such as ovariectomized females, intact females, and aged
animals. Although this review did identify potentially
efficacious dose ranges following cerebral ischaemia and
TBI, adequate dose–response relationships were not fully
examined in all experimental groups according to hormonal
status. In addition, no included studies administered
progesterone treatment at a time point later than 6 h
following the onset of cerebral ischaemia or TBI.
Systematic reviews and meta-analyses are fundamental
tools in the interpretation of the effectiveness of a particular
treatment across a large number of studies. Historically they
have been used exclusively for reviewing clinical trials and
only a few have applied systematic review techniques to
data obtained from animal experiments. However, the need
to conduct systematic reviews on animal experiments aimed
at modelling clinically relevant problems, such as stroke and
TBI, has been highlighted recently (Sandercock and
Roberts, 2002; Dirnagl, 2006). Systematic reviews allow
for a more objective appraisal of the research evidence than
is allowed by the traditional narrative reviews more
commonly associated with animal research. It would
appear, from the papers analysed in this review, that the
methodological quality of a large number of animal studies
is poor; in particular few studies randomize animals to
treatment groups. Systematic reviews allow an insight into
the cause of any bias present in animal experiments. The
long-term aim of animal studies examining candidate
neuroprotective factors is to inform the design of clinical
trials. Some would argue the need for a much more
systematic review of animal data before proceeding to
clinical trials (Pound et al., 2004; Dirnagl, 2006) to reveal
not only that a drug can have neuroprotective activity
under certain conditions but also to give an insight into
potential limitations of the drug (e.g. time to treatment),
which are likely to affect its clinical usefulness.
However, systematic reviews do have various limitations.
Firstly, analyses can only include available data, usually only
Progesterone; experimental brain injury
available in published studies. Negative or neutral studies
are less likely to be published so results may overstate effect
size. Interestingly, positive studies of recently patented
compounds may also not be published. In this respect,
Egger’s asymmetry test revealed that publication bias was
absent in studies of cerebral ischaemia but present in
studies of TBI. Consequently, the benefits of progesterone
on lesion volume following TBI may have been overestimated which may be attributable to the low number of
TBI studies included for analysis. Additionally, nonpublication will limit available information on the effect
of treatment within certain protocol aspects such as dose or
time of administration. The technique of extracting multiple pieces of information from single publications has the
potential to introduce bias into the review because the
results have been generated from the same laboratories and
investigators.
This review focuses only on the effect of progesterone on
lesion volume following either cerebral stroke or TBI,
largely due to insufficient data regarding other outcomes
such as behaviour. When comparing TBI with focal
ischaemia, there may appear to be a large amount of
literature supporting the neuroprotective effect of progesterone following injury. However, only those studies that
measured lesion volume were included in this review
whereas a large number of papers support the neuroprotective properties of progesterone following TBI when
looking at various outcomes including oedema volume or
various molecular and functional tests. To date, only one
paper (Gibson et al., 2006) has measured oedema volume
following cerebral ischaemia and very few have measured
functional ability. Functional outcome, in combination with
effects on histopathology, may be as important in terms of
assessing benefit (STAIR, 1999). In terms of cerebral
ischaemia, infarct size has been reported to correlate
(Rogers et al., 1997) or not correlate (Hattori et al., 2000;
Reglodi et al., 2003) with neurological impairment.
The aim of this review is to demonstrate whether
progesterone is effective at reducing lesion volume following cerebral injury, i.e. ischaemia or TBI. Importantly, this
review also aims to give an insight into the limitations of
the effectiveness of progesterone. We assessed quality score
in accordance with previously published protocols
(Macleod et al., 2005; Wilmot et al., 2005a, b; Gibson
et al., 2006) which are all based upon recommendations by
the STAIR (1999). The publication of these standards for
pre-clinical neuroprotective drug development were organized by an expert panel of stroke researchers in order to
address why so many clinical trials of neuroprotective drugs
for acute ischaemic stroke have failed. Although this panel
of experts recognized the importance of pre-clinical testing
of neuroprotective drugs guidelines concerning how to
perform pre-clinical development of potential neuroprotective treatments were lacking. Without rigorous, robust and
detailed pre-clinical evaluation, it is unlikely that novel
neuroprotective drugs will prove to be effective when tested
Brain (2008), 131, 318 ^328
325
in large, time-consuming and expensive clinical trials. The
current study is the first to apply such an assessment to
experimental studies using models of TBI. Reassuringly, the
majority of TBI papers did rate relatively high in terms of
quality score and all eight measures of quality were deemed
relevant to TBI studies. However, we could only assess the
studies as reported, so a low quality score may indicate
either that the authors did not undertake a procedure, for
example randomization, or that they did not report it. Lack
of randomization can introduce bias and may result in
overestimation of treatment efficacy. Randomization is
necessary in order to exclude systematic errors of sampling.
As with randomization, blinding was only reported in a
fraction of included studies. In order to remove experimenter bias all outcome measures (e.g. lesion volume)
should be assessed blinded to treatment of the experimental
groups. Factors such as randomization and blinded
assessment seem to be important in terms of affecting
outcome in experimental stroke studies. For example, a
recent systematic review of hypothermia in experimental
stroke studies found that non-randomized studies overstated the decrease in lesion volume and studies which were
not blinded to outcome overstated efficacy when compared
to randomized and blinded outcome studies respectively
(van der Worp et al., 2007). Although sources of variation
between experimental animals might be small the literature
does support the notion that randomization can influence
outcome in experimental animal stroke studies (Macleod
et al., 2004, 2005; Sena et al., 2007). In addition, it is
important that the experimenter has no knowledge of the
assigned experimental group when inducing ischaemia as
knowledge of experimental group may affect management,
albeit subtly, of any complications associated with the
ischaemia (e.g. ability to induce sufficient ischaemia,
perioperative hypotension) and therefore influence outcome. Additionally, while most studies reported monitoring
physiological parameters, the majority only measured body
temperature. Although body temperature is a useful
parameter, additional physiological parameters also give
invaluable information about the physiological effects of a
particular treatment, which could be crucial when considering the design of clinical trials.
In order to assess the limitations of the effectiveness of
progesterone we evaluated the effects of hormonal status
and age. The majority of studies used young adult males.
This is in contrast to the majority of experimental studies
evaluating the neuroprotective effect of oestrogens which
tend to use ovariectomized females (Gibson et al., 2006).
It is important that the effectiveness of steroid hormones,
such as progesterone, are assessed in males as they represent
the population group most at risk of TBI and, in
conjunction with post-menopausal women, represent the
group at highest risk of a cerebral stroke. However, clinical
trials aimed at testing the effectiveness of hormone therapy
on the occurrence and outcome following vascular events
have reported conflicting results. In fact, a systematic review
326
Brain (2008), 131, 318 ^328
of completed clinical trials found that hormone therapy was
associated with an elevated risk of stroke, which was
ischaemic in type and of increased severity (Bath and Gray,
2005). However, all clinical trials have assessed the effects of
either combined (oestrogen–progestin) hormone therapy or
oestrogen-only hormone therapy following stroke. Thus,
the effects of progestin-only hormone therapy on incidence
and outcome following stroke are unknown.
This review highlights that very few studies have assessed
the neuroprotective effectiveness of progesterone in either
female animals (ovaroectomized or intact) or aged animals.
Although cerebral stroke is considered a disease of aging
experimental studies using aged animals are rarely undertaken because of financial and logistical costs. However, it is
important that the effectiveness of progesterone treatment
is assessed in aged animals, especially following stroke, prior
to the initiation of any clinical trials. This review also
highlights the lack of studies, which fully explore the
effectiveness of progesterone with regards to timing of first
administration. As the majority of patients at greatest risk
from TBI or cerebral stroke (i.e. men and post-menopausal
women) will not be receiving hormone therapy it is
clinically relevant to extend the first time of application.
Although one study has shown that progesterone treatment
delayed up to 24 h following TBI was still effective at
reducing brain oedema (Roof et al., 1996) no studies
looking at lesion volume have initiated progesterone
treatment at later than up to 6 h following the onset of
cerebral ischaemia or TBI. All included studies applied
progesterone within the dose range of 4–60 mg/kg but the
majority of studies did not measure circulating plasma
progesterone levels. The few that have indicate that
intraperitoneal absorption of 4 mg/kg resulted in plasma
progesterone levels between 41.9 and 70.7 ng/ml 4 following injection (Jiang et al., 1996) whereas intraperitoneal
administration of 30 mg/kg resulted in an average plasma
progesterone level of 133 ng/ml (Murphy et al., 2000). In
the rat, plasma progesterone levels range from basal levels
of 2–18 ng/ml to 120–130 ng/ml in pregnancy, with
intermediate values of 40–90 ng/ml during late proestrus
(Wiest, 1970; Nequin et al., 1979). In the current study,
those studies which used a lower dose of progesterone
would have achieved physiological levels in intact female
animals but supraphysiological levels in male animals. Thus,
there is a need for adequate dose-response relationships to
be examined more fully in all experimental groups.The
effects of progesterone following various types of CNS
injury are described as being pleiotropic in that it has been
shown to reduce necrotic damage and cell loss, reduce
oedema formation, and restore cognitive performance
(Gibson et al., 2005; Stein, 2005). In terms of the
mechanisms of progesterone-induced neuroprotection
in vitro studies or studies using models of TBI or spinal
cord injury have revealed that progesterone; has antioxidant
properties (Roof et al., 1997); regulates the expression of
trophic factors such as brain-derived neurotrophic factor
C. L. Gibson et al.
(Gonzalez et al., 2004); activates intracellular signalling
pathways involved in the promotion of cell survival (Singh,
2001); increases the expression of anti-apoptotic molecules
such as Bcl-2 and Bcl-XL (Yao et al., 2005); and, decreases
the expression of pro-apoptotic molecules such as Bax, Bad
and caspase-3 (Djebali et al., 2004). Some of these effects of
progesterone may be mediated by the activation of classical
progestin receptors, which are widely expressed in the brain
(Guerra-Araiza et al., 2003). In addition, rapid membrane
effects of progesterone may be mediated by membrane
progestin receptors, or by the membrane-associated progesterone-binding protein 25-Dx (Meffre et al., 2005).
Despite all these beneficial effects of progesterone in the
CNS its specific mechanisms following acute cerebral injury
are not completely understood.
Overall this review has emphasized the need for
bi-directional translational research between experimental
and clinical studies. Progesterone is a potential neuroprotective treatment following either cerebral stroke or TBI.
In fact, the first clinical trial aimed at assessing the
neuroprotective effect of progesterone following TBI has
just been completed (Wright et al., 2007). In that clinical
trial, the investigators report that progesterone resulted in
improved outcome and was well tolerated in terms of safety
after TBI. Larger scale trials to assess outcome following
progesterone treatment for TBI are planned although
clinical trials following cerebral stroke have yet to be
initiated. The design of such clinical trials relies fundamentally upon animal studies to inform them with regards to
both potential hazards and the optimal progesterone
treatment in terms of dosing and timing among different
populations. This review has identified fundamental areas
where experimental evidence is still required.
Acknowledgements
PMWB is Stroke Association Professor of Stroke Medicine.
Supported by NIH grant NS29226 (to S.P.M.).
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