Skip to main content
NCBI home page
Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2013 Jul 10;33(9):1362–1372. doi: 10.1038/jcbfm.2013.120

Progesterone treatment for experimental stroke: an individual animal meta-analysis

Raymond Wong 1, Cheryl Renton 1, Claire L Gibson 2, Stephanie J Murphy 3, David A Kendall 4, Philip M W Bath 1,*, for the Progesterone Pre-Clinical Stroke Pooling Project Collaboration
PMCID: PMC3764382  PMID: 23838830

Abstract

Preclinical studies suggest progesterone is neuroprotective after cerebral ischemia. The gold standard for assessing intervention effects across studies within and between subgroups is to use meta-analysis based on individual animal data (IAD). Preclinical studies of progesterone in experimental stroke were identified from searches of electronic databases and reference lists. Corresponding authors of papers of interest were contacted to obtain IAD and, if unavailable, summary data were obtained from the publication. Data are given as standardized mean differences (SMDs, continuous data) or odds ratios (binary data), with 95% confidence intervals (95% CIs). In an unadjusted analysis of IAD and summary data, progesterone reduced standardized lesion volume (SMD −0.766, 95% CI −1.173 to −0.358, P<0.001). Publication bias was apparent on visual inspection of a Begg's funnel plot on lesion volume and statistically using Egger's test (P=0.001). Using individual animal data alone, progesterone was associated with an increase in death in adjusted analysis (odds ratio 2.64, 95% CI 1.17 to 5.97, P=0.020). Although progesterone significantly reduced lesion volume, it also appeared to increase the incidence of death after experimental stroke, particularly in young ovariectomized female animals. Experimental studies must report the effect of interactions on death and on modifiers, such as age and sex.

Keywords: individual animal data, meta-analysis, neuroprotection, progesterone, steroid hormones, systematic review

Introduction

Stroke is the third leading cause of death and the most common cause of chronic disability in the UK and US.1, 2, 3 The majority of cerebral strokes (∼85%) are ischemic in nature, yet few effective treatments currently exist for acute ischemic stroke. Although tissue plasminogen activator is effective, its use is limited to patients with ischemic stroke, presenting within 4 to 5 hours. An additional prospective approach to improving outcome after stroke, whether ischemic or hemorrhagic, is to use neuroprotective strategies that aim to reduce chronic neuronal damage.

Preclinical studies have indicated that progesterone, an endogenous steroid hormone, is neuroprotective, in that it reduces lesion volume and improves functional outcome, after cerebral ischemia.4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 Progesterone has already been shown to be safe and effective for several clinical applications, e.g., hormone replacement therapy, and a variety of preparations are available for different modes of administration.15 Furthermore, no severe adverse effects have been reported even when progesterone was used at high dose.16, 17 In two clinical trials involving patients with traumatic brain injury, progesterone was shown to be well tolerated.18, 19 However, there have been no clinical trials examining the effect of acute administration of progesterone after ischemic stroke.

Studies assessing the safety and efficacy of progesterone in preclinical models of stroke4, 5, 6, 7, 8, 9, 13, 20, 21, 22 have previously been integrated in a systematic review and meta-analysis based on published summary (‘group') data.14 However, this systematic review suggested that key questions remained to be answered, including demonstrating efficacy as a function of sex and age, and evaluating the dose and timing of progesterone administration. The gold standard for assessing effects across studies within and between subgroups is to use a meta-analysis based on individual animal data (IAD), as previously performed for NXY-059, another putative neuroprotectant.23 The present study reports the results of a systematic review and meta-analysis based on IAD of preclinical studies of progesterone in experimental models of stroke.

Materials and methods

Ethics

No ethics approval was needed for the present study, which follows a protocol defined at the beginning of the project based on a previous study using IAD from experimental stroke studies of NXY-059.23

Search Strategy

Completed studies that investigated the effect of progesterone in animal models of focal stroke were sought via searches of electronic databases (last search: 9 October 2012), including ‘Web of Knowledge' and ‘PubMed'. Search strategies used the following key words: progesterone, stroke, ischemia. Reference lists from the existing systematic and non-systematic reviews, and identified study publications, were also searched.24, 25 Where duplicate publications were identified, information from the primary report was used. Publications could be in any language.

Selection of Studies

Completed controlled studies, randomized, pseudo-randomized, or nonrandomized, and whether published or unpublished, were included if experimental focal stroke injury was induced in animal models and progesterone was administered. Stroke models could include transient or permanent focal cerebral ischemia, and progesterone or its metabolite allopregnanolone had to be administered exogenously. Studies had to include data on at least one outcome of interest: lesion volume, progesterone plasma concentration, vital status and/or functional outcome. Studies were excluded if they did not report induction of focal cerebral ischemia or administration of progesterone, or did not contain original data or involved just global ischemia.

Validity Assessment

Studies were identified based on the inclusion/exclusion criteria listed above. The methodological quality of each study was assessed, using a nine-point score modified from the original eight-point STAIR (Stroke Therapy Academic Industry Round Table) system.26 A point was given for published evidence (supplemented with information from the corresponding author) for each of the following criteria: presence of randomization (pseudo-randomized 0.5), monitoring of physiologic parameters, assessment of dose–response relationship, assessment of optimum time window, masked outcome measurement, assessment of outcome at 1–3 days, assessment of outcomes 1–30 days, combined lesion volume and functional outcome, and for blinding of surgery.23

Data Abstraction

Two reviewers (RW and CG) identified studies. One author resolved disagreements by discussion and review (PB).

Data Extraction

Corresponding authors of papers of interest were contacted to ask whether they would be willing to join the pooling project collaboration and, if so, to share IAD with data fields listed in the project's protocol (outline of methods in this manuscript).

Individual data for each animal was sought for species, gender (male, young intact female, old female, ovariectomized young female), age (or weight as a surrogate), lesion volume (total, cortical, and subcortical), and vital status (including information on timing and cause of death—surgery, culling owing to poor health, spontaneous). Information on treatment was also obtained: time of treatment from occlusion time (hours before/after), loading and maintenance dose of progesterone, treatment duration, and plasma progesterone concentration.

The following study design information was extracted either from the publication or via the corresponding author: experimental model—transient, permanent; randomization—randomized, pseudo-randomized, not randomized; blinding of surgeon to treatment; and blinding of outcome assessors to treatment. Studies were considered randomized if animals were numbered before commencement of the study and a randomization code was used to allocate animals to treatment groups; if animals were ‘picked at random' from a cage, then these studies were considered pseudo-randomized, as this type of approach is open to bias. The estimated study quality from the publication was confirmed with the corresponding authors.

Quantitative Data Synthesis

IAD were transferred to the project's coordinating center in Nottingham by e-mail attachment, e.g., Excel file. Study data sets were merged into a single Microsoft Excel sheet using common field names with one row per animal. Where individual data could not be obtained, summary data were obtained from the publication and entered into a second Excel sheet. Summary data were also created from IAD and added to this second spreadsheet to allow summary meta-analysis of all studies.

Lesion volume was recorded as either absolute volume or as a percentage of the intact contralateral hemisphere. To combine the different units, lesion volume was standardized, i.e., mean score/s.d. of score. Data from animals that have died before study completion is commonly excluded from meta-analyses of animal data, but to allow its inclusion, lesion volume was given a ‘worst-case' value. For studies in which lesion volume was collected as a percentage, a value of 100% was assigned for death; for studies in which lesion volume was collected as an absolute value, species-dependent maximal values were assigned −400 mm3 for rats and 225 mm3 for mice. Analyses of lesion volume were performed with death imputed and excluded to confirm the robustness of conclusions. Taking account of the role of progesterone as an endogenous sex steroid, sex was trichotomized into male, young ovariectomized female (age <12 months), and old females. Progesterone dose was standardized to mg/kg.

Meta-analysis of individual animal data was carried out using random-effects mixed modeling with covariate adjustment in SAS (version 9.3). Meta-analysis of summary data was carried out using random-effects analysis in STATA (version 11). Random-effects analysis was used to take account of the expected heterogeneity between studies caused by variations in study design, i.e., differences in species, stroke model, dosing, and study practice.

All analyses were performed unadjusted (no covariates). In addition, analyses using IAD were adjusted for the following covariates, chosen for their biologic relevance or relationship to treatment: model (transient and permanent), age (young ⩽12, aged >12 months), sex (male, young overiectomized female, and aged female), time to treatment, loading progesterone dose, total progesterone dose (sum of loading and maintenance), and anesthetic agent (halothane and isoflurane). Interaction tests between treatment and covariates were assessed if the covariate was significantly associated with the outcome.

Analyses were performed by the backwards elimination procedure. This procedure starts with all potential predictor variables in the regression model and, successively, variables are dropped one at a time, such that the resulting model has the lowest value of the information criteria. Variables are dropped on the basis of their contribution to the reduction of error sum squares or ‘worst' included variable. This is stopped when the best model is reached according to the Akaike Information Criterion (AIC). The AIC is an alternative significance test to estimate quantities of interest and judges a model by how closely its fitted values tend to be to the true values. AIC serves the purpose of model comparison only and does not provide diagnostics about the fit of the model to the data.

Data are given as standardized mean differences (SMD, continuous data) or odds ratios (binary data) with 95% confidence intervals (95% CIs), P-value for effect, P-value for heterogeneity, and P-value for interaction; P-values <0.05 are considered significant. Negative coefficients imply a reduction in lesion volume. An odds ratio less than one implies a reduction in death.

Results

Study Flow

Nineteen completed studies fulfilled the inclusion criteria (Figure 1, Table 1); as one study included two data sets,27 20 separate data sets were used in the analyses. Authors were contacted and were asked about sharing available individual animal data for analysis. In total, IAD were received from 12 published and 2 unpublished studies; one published study was split into two data sets, giving 15 data sets overall. Shared data included animals excluded in publications, in particular those that died before study completion. Altogether, IAD were available for 689 animals. IAD were not shared for six studies in spite of repeated contact with authors;6, 7, 12, 13, 28, 29 nevertheless, data from these studies were included in analyses based on summary data. Five studies were excluded, mostly because they did not have data on total lesion volume or vital status, e.g., one study only had data on blood volume collected.4

Figure 1.

Figure 1

Open in a new tab

Flow chart showing study search, and included and excluded studies. Summary data were used for studies where individual animal data were not made available. Studies were excluded if they did not report induction of focal cerebral ischemia, administration of progesterone, or measurement of lesion volume, or did not contain original data; n=number of studies.

Table 1. Studies included in the analysis of lesion volume, deaths, and progesterone plasma concentration.

Study Lab Species Sex Anesthetic Random Surgery masked Outcome masked Temperature control Model Time prog loading dose to occlusion (min) Prog load (mg/kg) Prog maintenance (mg/kg/hour)
Responders with IAD
 Toung et al27 Baltimore, USA Rat F Halothane (4–5% induction, 1.25–1.5% maintenance delivery in O2 enriched air) y n n y Focal (transient) −30 5 i.p. 5+5 i.p. for 24.5 hours
 Toung et al27 (Prog+Est)                   −30 5 i.p. 5+5 i.p. for 24.5 hours
 Parker et al30 Portland, USA Mice F Halothane (4–5% induction, 1.25–1.5% maintenance delivery in O2 enriched air) y n n y Focal (transient) −7 Days None 15/30 mg s.c. 21 day release implant for 7–8 days
 Murphy et al8 Baltimore, USA Rat F Halothane (4–5% induction, 1.25–1.5% maintenance delivery in O2 enriched air) y n n y Focal (transient) −30 5/10/20 i.p. None
                    −30 5 i.p. 5+5 i.p. for 24.5 hours
 Murphy, 2002 (unpublished) Baltimore, USA Rat F Halothane (4–5% induction, 1.25–1.5% maintenance delivery in O2 enriched air) y n n y Focal (transient) −30 5 i.p. 5+5 i.p. for 24.5 hours
 Murphy et al21 Baltimore, USA Rat F Halothane (4–5% induction, 1.25–1.5% maintenance delivery in O2 enriched air) y n n y Focal (transient) −30 30/60 i.p. None
                    −7 to −10 days 30/60 i.p. 30/60 i.p. daily for 7–10 days
 Gibson et al5 Nottingham, UK Mice M Isoflurane 4% (NO2/O2 70/30% mixture) y y y y Focal (transient) 60 8 i.p. 8+8 i.p. for 23 or 47 hours
 Gibson et al9 Nottingham, UK Mice M Isoflurane 4% (NO2/O2 70/30% mixture) y y y y Focal (permanent) 60 8 i.p. 8+8 i.p. for 47 hours
 Sayeed et al52 Atlanta, USA Rat M Isoflurane 4% (NO2/O2 70/30% mixture) y n n y Focal (Transient) 115 8 i.p. 8s.c. for 72 hours
 Sayeed et al10 Atlanta, USA Rat M Isoflurane 4% (NO2/O2 70/30% mixture) y n y y Focal (permanent) 120 8 i.p. 8+8 +8s.c. for 72 hours
 Sayeed and colleagues12 Atlanta, USA Rat M Isoflurane 4% (NO2/O2 70/30% mixture) y n n y Focal (transient) 115 8 i.p. 8s.c. for 72 hours
 Coomber et al33 Leicester, UK Mice F Isoflurane 4% (NO2/O2 70/30% mixture) y y y y Focal (transient) −7 days None 50 mg s.c. 21 day release implant for 7 days
 Gibson et al34 Leicester, UK Mice F Isoflurane 4% (NO2/O2 70/30% mixture) y y y y Focal (transient) 60 8 i.p. 8+8 i.p. for 23 or 47 hours
 Wong, 2012 (unpublished) Nottingham, Uk Mice M Isoflurane (100% 02) y y y y Focal (transient) 30,60 8 i.p. 50 mg s.c. 3 day release implant for 48 hours
 
Nonresponders/no IAD
 Jiang et al6 Atlanta, USA Rat M Halothane (3.5% induction, 1–2% maintenance delivery 70% N2) and 30% 02) y n n y Focal (transient) −30 4 i.p. 4+4 i.p. for 48 hours
 Chen and Chopp13 Rochester, USA Rat M Halothane (3.5% induction, 1–2% maintenance delivery 70% N2) and 30% 02) n n n y Focal (transient) 120 4/8/32 i.p. 4+4/ 8+8/ 32+32 i.p. for 7 days
 Ishrat et al12 Atlanta, USA Rat M Isoflurane 5% induction, 1.5–2% maintenance, 2:1 NO2 and O2 mix n n n y Focal (permanent) 60 8 i.p. 8 +8+8s.c. for 72 hours
 Kumon et al7 Ehime, Japan Rat M Halothane (3% induction, 1–2% maintenance delivery 70% N20 and 30% O2) y y y y Focal (transient) 120 4/8 i.p. None
 Choi et al28 Pusan, Korea Rat M Sodium thiopental (50 mg/kg) n n n y Focal (transient) −24 hours 4 i.p. 4 i.p. for 48 hours
 Liu et al29 Paris, France Mice M Ketamine (50 mg/kg) and xylazine hydrochloride (6 mg/kg) y n y y Focal (transient) 60 8 i.p. 8+8 i.p. for 23 or 47 hours
Open in a new tab

Abbreviations: EST, estrogen; F, female; IAD, individual animal data; i.p.; intraperitoneal; M, male; n, no; Pro, progesterone; S.C., subcutaneous; unpub, unpublished; y, yes.

Data Set Characteristics

Data set characteristics are reported in Tables 1 and 2. The data sets used compared the effect of exogenously administered progesterone versus no progesterone, or vehicle, on lesion volume after acute focal cerebral ischemia, as well as data sets involved in the analysis of death and progesterone plasma concentration. All studies used a monofilament to induce focal cerebral ischemia in the middle cerebral artery territory. Seven data sets used mice (permanent ischemia in 1, transient ischemia in 6) and 13 data sets used rats (permanent ischemia in 1, transient ischemia in 12). The data sets included 12 using adult male animals, 4 using adult overiectomized females, 1 using aged males (age >12 months) and 3 using aged females. Insufficient data were available on cortical or subcortical lesion volume measurements to permit analysis and only total or hemispheric lesion volume was analyzed. Also, insufficient data was available for the analysis of functional outcomes. Dosing methods included intraperitoneal injection (13 data sets), intraperitoneal with subcutaneous injection (4 data sets) and subcutaneous implants of slow release pellets/minipumps (3 data sets). A variety of regimens were used for progesterone administration with dosing commencing from 7 to 10 days before middle cerebral artery occlusion (MCAO) to 2 days afterwards (10 data sets with pre-MCAO dosing and 10 data sets with post-MCAO dosing start times). Also, the majority of pre-MCAO dosing start times did continue treatment through to occlusion and reperfusion (eight data sets with pre-MCAO dosing start times with continued dosing through to reperfusion). Lesion volume, assessed from histologically stained brain slices, was reported in mm3, % cross-sectional area, or % of intact contralateral hemisphere. Study quality ranged from 2 to 7/9. Publication bias was apparent on visual inspection of a Begg's funnel plot based on lesion volume (Figure 2), and when assessed statistically using Egger's test (P=0.001).

Table 2. Experimental results.

Study Exclusions from analysis (prog: control) Death (prog: control) Lesion volume timing (hour) Total lesion animals (prog: control) Total lesion volume (prog: control) Measure of lesion Quality/9 Comments for progesterone and study
Responders with IAD
 Toung et al27 5 (4:1) 5 (4:1) 24 20 (10:10) 18.61 (13.45): 24.06 (13.31) % 3 Combined hormone reduced infarct volume and progesterone does not attenuate estrogen effect.
 Toung et al27 (Prog+Est) 2 (1:1) 2 (1:1)   20 (10:10) 7.59 (5.69): 8.92 (9.15) %    
 Parker et al30 0 0 23.5 24 (16:8) 49.77 (12.9): 53.46 (18.68) % 4 Chronic, exogenous progesterone before MCAO alters ischemic brain injury in ovariectomized female mice
 Murphy et al8 28 (15:13) 2 24 61 (39:22) 17.85 (10.78): 18.9 (14.37) % 5 Progesterone both before MCAO and during reperfusion decreases ischemic brain injury
 Murphy, 2002 (unpub) 0 0 None None None NA 3 Progesterone plasma concentration measured and data used for death analysis
 Murphy et al21 29 (19:10) 11 24 56 (42:14) 13.32 (8.26): 8.42 (8.09) % 5 Progesterone does not ameliorate histologic injury after MCAO in previously ovariectimised adult female rats. Chronic progesterone administration can exacerbate infarction in subcortical regions
 Gibson et al5 4 4 24, 48 20 (10:10) 13.97 (5.21): 18.64 (5.9) mm3 7 Beneficial effects of progesterone after cerebral ischemia
 Gibson et al9 6 4 (2:2) 48 24 (12:12) 82.5 (12.58): 90.87 (15.88) mm3 5 Progesterone is neuroprotective in both permanent and transient ischemia and effect is related to suppression of the inflammatory response
 Sayeed et al52 9 2 72 13 (6:7) 15.96 (3.6): 24.38 (6.69) % 4 Progesterone is effective at reducing infarct pathology
 Sayeed et al10 3 1 72 15 (7:8) 13.81 (5.68): 27.8 (6.27) % 5 Progesterone is neuroprotective
 Sayeed and colleagues12 0 0 72 16 (8:8) 9.32 (2.29): 20.12 (2.91) % 4 Progesterone is neuroprotective
 Coomber et al33 9 4(3:1) 48 13 (6:7) 20.62 (9.22): 23.41 (10.18) mm3 5 No effect
 Gibson et al34 10 8 (5:3) 48 27 (13:14) 21.59 (7.42): 24.67 (5.68) mm3 7 Progesterone is not beneficial in ovariectomized mice but is in aged female mice for lesion volume
 Wong, 2012 (unpub) 32 29(14:15) none none none n/a 3 Progesterone plasma concentration measured and data used for death analysis
 
Nonresponders/no IAD
 Jiang et al6 — — 48 36 (24:12) 24.43 (10.39): 35.1 (15.59) % 5 Progesterone administered before or after MCAO reduces ischemic cell damage and improves physiologic and neurologic function 2 days after stroke.
 Chen and Chopp13 — — 7 days post MCAO 28 (7:7,7,7) 26.57 (11.67): 34.4 (10.5) % 5 8 mg/kg of progesterone reduce brain lesion and improved neurologic functional deficit
 Ishrat et al12 — — 72 16 (8:8) 9.24 (2.96): 20.11 (3.1) % 3 Progesterone treated have reduced infarct volume and improved ability to stay on rotarod and grip test
 Kumon et al7 — — 2 or 7 days after MCAO 48 (32:16) 31.15 (14.13): 41.2 (10.4) mm3 7 Treatment with high dose of 8 mg/kg of progesterone results in reduction of lesion size, neurologic deficits and body weight
 Choi et al28 — — 24 11 (5:6) 210 (67.6): 231.4 (154.07) mm3 2 No effect
 Liu et al29 — — 48 64 (32:32) 43.25 (22.75): 52.25 (17.96) mm3 5 Progesterone was neuroprotective in wild-type mice
Open in a new tab

Abbreviations: IAD, individual animal data; MCAO, middle cerebral artery occlusion; NA, not appropriate; unpub, unpublished.

Exclusion from Analysis: total number (progesterone: control), death: total number (progesterone: control), total lesion volume: mean (s.d.). Exclusions refer to animals excluded from analysis in manuscripts and include animal deaths. Death refers to animals that died for whatever reason, whether they were treated or not. Lesion volume was reported in mm3, % cross-sectional area, or % of intact contralateral hemisphere.

Figure 2.

Figure 2

Open in a new tab

Begg's funnel plot for studies of progesterone on lesion volume when combining individual animal and summary data. The funnel plot relates precision (reciprocal of s.e.) to the standardized mean difference (SMD). Asymmetry is present indicating publication bias (Egger's test,53 P=0.001).

Quantitative Data Synthesis

Lesion volume—combined individual and summary data

Because of the lack of data on individual animals that died after the start of treatment in summary data (where IAD were not shared), it was not possible to account for death in combined IAD and summary data analysis.

In an unadjusted analysis of 18 data sets (512 animals), progesterone reduced standardized lesion volume (SMD −0.766, 95% CI −1.173 to −0.358, P<0.001) (Table 4, Figure 3). In predefined subgroups of animals, significant reductions in lesion volume were seen with progesterone in mice, rats, male animals, male mice, male rats, and in both transient and permanent models of ischemia. When assessed by predefined study design and quality markers, progesterone reduced lesion volume in studies that were randomized, had or did not have masked surgery, had or did not have masked outcome assessment, used isoflurane as the anesthetic agent, and those that were of high quality (STAIR scale=7) (Table 3). Some subgroup analyses were of low statistical power because of limited data, e.g., sodium thiopenthal as the anesthetic agent (1 study, 11 animals). Heterogeneity was present for some analyses confirming the need for the use of a random effects statistical model.

Figure 3.

Figure 3

Open in a new tab

Forrest plot of effect of progesterone on lesion volume using individual animal and summary data. Studies are ordered by animals' sex: male; young ovariectomized female; old female.

Table 3. Effect of progesterone on standardized total lesion volume using combined individual animal and summary data, both combined and in predefined subgroups.
Model Data sets Animals SMD 95% CI Effect P Inter-action P
Total 18 512 −0.766 −1.173, −0.358 <0.001a  
Species
 Mice 6 172 −0.492 −0.881, −0.102 0.013 0.44
 Rats 12 340 −0.958 −1.539, −0.376 0.001a  
 
Sex
 Male 11 291 −1.316 −1.902, −0.730 <0.001a  
 Female, adult 3 130 0.113 −0.437, 0.662 0.688  
 Female, agedb 4 91 −0.333 −0.754, 0.088 0.121  
 
Mice
 Male 3 108 −0.697 −1.308, −0.086 0.025  
 Female, adult            
 Female, agedb            
 
Rats
 Male 8 183 −1.547 −2.303, −0.792 <0.001a  
 Female, adult            
 Female, agedb            
 
Model
 Transient 15 457 −0.549 −0.928, −0.171 0.004a 0.075
 Permanent 3 55 −2.058 −3.850, −0.266 0.024a  
 
Treatment time
 Before MCAO            
 After MCAO            
 
Loading dose            
Maintenance dose *          
Total dose *          
             
Randomized
 Yes 15 457 −0.674 −1.088, −0.260 0.001a  
 No 3 55 −1.348 −3.046, 0.350 0.120a  
 
Masked surgery
 Yes 5 132 −0.589 −0.946, −0.233 0.001  
 No 13 380 −0.934 −1.537, −0.332 0.002a  
 
Masked outcome
 Yes 7 211 −0.725 −1.134, −0.316 0.001  
 No 11 301 −0.813 −1.413, −0.213 0.008a  
 
Anesthetic
 Isoflurane 8 144 −1.512 −2.324, −0.701 <0.001a  
 Halothane 8 293 −0.323 −0.700, 0.054 0.093a  
 Sodium thiopental 1 11 −0.173 −1.363, 1.016 0.775  
 Ketamine+xylazine 1 64 −0.430 −0.931, 0.064 0.173  
 
Quality
 2 1 11 −0.173 −1.363, 1.016 0.775  
 3 3 56 −1.224 −2.843, 0.395 0.138†  
 4 3 53 −1.828 −3.824, 0.168 0.073†  
 5 8 297 −0.534 −1.120, 0.052 0.074†  
 7 3 95 −0.636 −1.061, 0.211 0.003  
Open in a new tab

Abbreviations: CI, confidence interval; MCAO, middle cerebral artery occlusion; SMD, standardized mean differences.

Data are SMD, 95% CI and significance for effect and interaction. Analyses are adjusted for random effects. Result in bold are statistically significant.

a

Heterogeneity present between studies.

b

Heterogeneity present between ovariectomized.

When assessing the effect of interactions between treatment and subgroups, progesterone had greater effects on reducing lesion volume in males than young females (Figure 3), and when the anesthetic was isoflurane rather than halothane (Table 3).

Adjustments for analyses involving individual animal data

Using individual animal data alone, backwards elimination of covariates revealed that maintenance dose alone led to a minimum in AIC for analyses of lesion volume. For death, AIC was minimized with the combination of sex, time to treatment, loading dose, total dose, and anesthetic agent as covariates. Subsequent adjusted analyses were made using these covariates.

Lesion volume—individual animal data, death included

Clinical trials include patients who die after treatment in their outcome analyses; hence, lesion volume was imputed for animals that died after treatment. In 12 data sets (337 animals), treatment with progesterone did not show any benefit on standardized lesion size, whether in an adjusted analysis (SMD −0.322, 95% CI −0.779 to +0.135, P=0.16) (Table 4) or unadjusted analysis (SMD −0.208, 95% CI −0.542 to 0.125, P=0.20). In view of the neutral result, subgroup analyses were not performed.

Table 4. Effect of progesterone on standardized total lesion volume, including animals that died, using individual animal data overall and in predefined subgroups.
Model SMD 95% CI Effect P Interaction P
Overall, adjusted −0.322 −0.779, 0.135 0.16 NA
Species, mice versus rats −0.033 −0.679, 0.613 0.91 NA
Age, aged versus adult −0.071 −0.637, 0.495 0.80 NA
Sex, adult female versus male versus aged females 0.167 −0.508, 0.842 0.86 NA
Model −0.138 −0.705, 0.678 0.97 NA
Time to treatment (per hour) −0.001 −0.005, 0.004 0.72 NA
Loading dose (per mg/kg) −0.002 −0.020, 0.016 0.83 NA
Total dose (per mg/kg) 0.003 −0.002, 0.001 0.18 NA
Anesthetic, halothane versus isoflurane −0.034 −0.789, 0.721 0.93 NA
Open in a new tab

Abbreviations: CI, confidence interval; NA, not appropriate; SMD, standardized mean differences.

Animals that died were assigned a lesion volume of 100%, 1000*mm3 for rats or 225 mm3 for mice. Data are SMD, 95% CI, and significance for effect and interaction. Analyses are adjusted for random effects and species, age, sex, model, time to treatment, loading dose, total dose, and anesthetic agent. Result in bold are statistically significant.

Lesion volume—individual animal data, death excluded

Using individual animal data alone and ignoring animals that died (12 data sets, 309 animals), progesterone reduced standardized lesion volume in both adjusted (SMD −0.585, 95% CI −0.954 to −0.215, P=0.004) (Table 5) and unadjusted (SMD −0.451, 95% CI −0.735 to −0.166, P=0.004) analyses. Progesterone reduced standardized lesion volume in predefined subgroups: males, male rats, animals anesthetized with isoflurane, and when outcome was measured masked to outcome.

Table 5. Effect of progesterone on standardized total lesion volume, excluding animals that died, using individual animal data overall and in predefined subgroups.
Model SMD 95% CI Effect P Interaction P
Overall, adjusted −0.585 −0.954, −0.215 0.004 NA
Species, mice versus rats −0.031 −0.540, 0.478 0.90 NA
Age, Aged versus young −0.037 −0.616, 0.541 0.90 NA
Sex, adult female versus male versus aged females 0.130 −0.445, 0.706 0.87 NA
Model, permanent versus transient 0.004 −0.535, 0.543 0.99 NA
Time to treatment (per hour) −0.001 −0.005, 0.003 0.58 NA
Loading dose (per mg/kg) 0.011 −0.006, 0.028 0.20 NA
Total dose (per mg/kg) −0.001 −0.005, 0.004 0.80 NA
Anesthetic (halothane versus isoflurane) −0.000 −0.589, 0.588 1.00 0.80
Open in a new tab

Abbreviations: CI, confidence interval; NA, not appropriate; SMD, standardized mean differences.

Data are SMD, 95% CI, and significance for effect and interaction. Analyses are adjusted for random effects and maintenance dose of progesterone. Result in bold are statistically significant.

Death

Using individual animal data alone (14 data sets, 503 animals, 58 deaths), progesterone was associated with an increase in death, significantly so in an adjusted analysis (odds ratio 2.64, 95% CI 1.17 to 5.97, P=0.020) (Table 6), and with a trend in an unadjusted analysis (odds ratio 1.81, 95% CI 0.70 to 4.66, P=0.22). When assessed in predefined subgroups, the increase in death rate was most prominent in mice, older animals, young intact females (versus males or old females/ovariectomized young females), and with later administration of drug. A significant interaction was present between treatment and species (Table 6). Analysis of death included data sets from two unpublished studies (Murphy et al (2002), unpublished, and Wong et al (2012), unpublished).

Table 6. Effect of progesterone on death using individual animal *and summary* data overall and in predefined subgroups.
Model OR 95% CI Effect P Interaction P
Overall, adjusted 2.64 1.17–5.97 0.020 NA
Species, mice versus rats 10.35 2.14–49.93 0.004 0.028
Age, aged versus young 11.70 1.70–80.20 0.012 0.76
Sex, adult female versus male versus aged females 11.68 1.79–76.34 0.028 ND
Model, transient versus permanent — — NA ND
Time to treatment (per hour) 1.01 1.00–1.01 0.025 NA
Loading dose (per mg/kg) 0.89 0.69–1.15 0.36 NA
Total dose (load+maintenance, per mg/g) 1.03 0.99–1.06 0.16 NA
Anesthetic, halothane versus isoflurane 1.11 0.25–4.97 0.89 NA
Open in a new tab

Abbreviations: CI, confidence interval; NA, not appropriate; ND, Not done—no deaths in a comparator group; OR, odds ratio.

Data are OR, 95% CI, and significance for effect and interaction. Analyses are adjusted for random effects and species, age, sex, model, time to treatment, loading dose, total dose, and anaesthetic agent. Result in bold are statistically significant.

Progesterone blood concentration

The relationship between progesterone-loading dose (mg/kg) and blood concentration (ng/mL) was assessed in four studies on rats (Murphy et al,8, 21 Parker et al,30 and Murphy (2002), unpublished) and one in mice (Wong et al31). Some studies did not involve stroke models (Murphy (2002), unpublished and Wong et al31). Blood progesterone concentration was measured 1 hour from the start of treatment. A linear concentration–dose relationship was seen (P<0.0001, Figure 4) with:

Figure 4.

Figure 4

Open in a new tab

Relationship between blood concentration and loading dose of progesterone. Progesterone blood concentration was measured at 1 hour from the start of treatment. Equation of best fit [Progesterone] in blood (ng/mL)=4.156 × progesterone loading dose (mg/kg)+1.304.

[Progesterone]=1.304+4.156 × progesterone loading dose.

Discussion

In our earlier systematic review and meta-analysis, based on published summary data, progesterone was associated in a dose-dependent manner, with reduced lesion volume after experimental brain injury.24 However, meta-analysis based on summary data does not necessarily allow subgroup analyses to be performed, whereas a meta-analysis based on IAD usually does. Therefore, it was not possible to assess the effect of treatment on death or in subgroups of animals or experimental designs. In the present systematic review and meta-analysis, data for individual animals, including those that died, was assessed from both published and unpublished studies focusing on progesterone in surgically induced stroke. Such meta-analyses are considered to be the gold standard for assessment of intervention effects.32

Individual subject-based meta-analysis is recognized as providing additional important insights for patients in clinical trials, and the same may be true for meta-analysis of IAD for animals. However, there are critical differences to consider when considering meta-analysis based on individual patients to IAD. Human studies, in general, are small numbers of large studies looking at a single treatment effect, with heterogeneity introduced by unavoidable differences in patients recruited, whereas animal studies are generally large numbers of small studies, looking at different models, doses, and species, with heterogeneity introduced deliberately in the design of different experiments. Study heterogeneity is large for clinical studies but is likely to be smaller for animal studies, and the benefits of IAD analysis are thus less clear but worth seeking.

The main findings were that progesterone reduced lesion volume but increased the incidence of stroke-related death. In a pharmacokinetic substudy, progesterone concentration in the blood was proportional to loading dose (Figure 4). The presence of progesterone in plasma, an hour after dosing, confirms animals have been successfully dosed.

Progesterone was found to reduce lesion volume in analyses based on both combined IAD and summary data (which excluded animals that died post treatment), or using IAD alone where animals that died were excluded. Although analyses based on summary data or IAD varied somewhat, lesion volume was reduced regardless of rodent species (mice and rats) or model of occlusion (transient and permanent), yet only in males. However, when animals that died were included in the analysis of lesion volume (amounting to analysis of the composite outcome of lesion volume or death), progesterone exerted no beneficial effect. The explanation is that progesterone increased the incidence of death, particularly in mice versus rats, older animals versus adult, adult overiectomized females versus males or older females, and when administered late in the protocol.

Several drug–subgroup interactions bear further comment. The administration of exogenous progesterone appeared to be detrimental in adult overiectomized females as compared with males (as seen previously24) and older females, indicating other factors involved, other than endogenous levels of progesterone. This finding has been observed previously in individual studies, e.g., Gibson and Murphy5, who found progesterone to aid survival after ischemia in young male mice, although others found no effect.4, 33, 34 Other studies have either not investigated survival or the reported numbers of deaths are small, thereby preventing detailed analysis. Importantly, only death post treatment should be included, and earlier culling, e.g., because of inadequate occlusion (as determined using laser Doppler), does not amount to attrition bias. Equally, animals that have to be killed humanely after treatment, because they are in poor condition, e.g., they exhibit barrel-rolling, must be included in the numbers that died. The application of humane endpoints to inform decisions regarding termination of animals will vary between institutions and, hence, it is important to include these animals in this type of analysis.

The finding that progesterone appears to reduce lesion size but increase death is important, as most preclinical studies only report the former. This may not be surprising, as such studies are usually small and, therefore, do not have sufficient statistical power to individually assess death. However, the issue of low numbers of deaths also applies to clinical trials, and they typically report both death alone, and the combination of death and poor nonfatal outcome (lesion size in this study). The problem is that published studies don't report death and that actually this effect on death has been revealed though analysis of IAD by contacting authors. Several potential neuroprotectants have been reported to be protective in preclinical studies but hazardous in clinical trials, these including diaspirin cross-linked haemoglobin, enlimomab, selfotel, and tirilazad.35, 36, 37, 38 It is interesting to speculate on whether these agents increased death in preclinical studies, and whether clinical trials would have proceeded if IAD meta-analyses had been performed beforehand. Preclinical studies and analysis should strive to reflect the design of clinical trials to screen out ineffective and even deleterious interventions before reaching human testing. Nevertheless, IAD meta-analyses of preclinical studies may not successfully predict the results of clinical trials. In the only other IAD meta-analysis performed to date, NXY-059 was found to reduce stroke lesion volume but it failed to be effective in clinical trial.23, 39, 40 Our results, and previous experience in experimental stroke and clinical trials, suggest that it may be beneficial for preclinical studies to be analyzed rigorously, including performing IAD meta-analysis, before clinical trials are initiated.

Several caveats concerning the present study need to be considered; some reflecting issues with the meta-analysis itself and others concerned with the relative paucity of experimental data relating to progesterone. First, there was evidence of significant publication bias on the basis of the effects of progesterone on lesion volume when assessed using summary data assessed both visually (Figure 2) and using Egger's test.41 The Egger's test assumes that larger studies are more likely to have results close to the ‘truth,' whereas small studies will be spread more widely on either side; a variation from this assumption, i.e., missing studies for say neutral effects, can indicate publication bias. Publication bias can lead to effect size to be overestimated if missing studies may well have been neutral, or even negative, raising the possibility that progesterone does not, in fact, have neuroprotective effects in cerebral ischemia. There are also approaches in estimating the impact on the summary estimate, such as the ‘trim and fill' method. However, these were not carried out as the focus of this investigation was the effect of death on individual animal data.

Second, standard mean difference allows comparisons to be made if different methods of measurement or different animal species have been used to measure the same outcome as in this case. In addition, it provides an interpretable value regarding the direction and magnitude of the effect of an intervention, such as progesterone treatment. Although alternate approaches do exist, SMD is commonly used in meta-analyses, but it should be noted that the reliability of the SMD can be affected both by sample size and when comparisons are made between studies that differ significantly on their study design, which might increase or decrease the effect size.

Third, in instances where animals were excluded because of death, maximum lesion volumes were assigned to those animals. Of course, this may introduce further bias as we cannot assume animals did exhibit large infarct volumes; thus, data were analyzed using both adjusted and unadjusted lesion volume measurements. However, by assigning a maximum lesion volume we are attempting, in those animals which died, to assign the worst possible outcome rather than suggesting death may have occurred because of large ischemic damage versus other causes, such as excessive edema formation or hemorrhage.

Fourth, although 18 data sets were identified as being potentially relevant to lesion volume, IAD were only obtained from 12 of these. Data from six trials could not be obtained in spite of repeated requests to the authors. In the absence of any response from the authors, it is difficult to gauge whether these studies were unusual in any respect. However, only one of these studies had extremely positive data for lesion volume (Figure 2); thus, it is unlikely that the overall results for lesion volume would have changed significantly if IAD had been available for all studies.

Sixth, data were only available for rodents (mice and rats); thus, effects in primates or any other ‘second species' could not be assessed (in contrast to NXY-059 for which marmosets were also studied23). Differences in stroke outcomes and the effect of potential neuroprotectants on them, between agyric and gyric brains, have been well discussed previously.42

Seventh, most data involved young male animals (n=11 for lesion volume) and few related to aged males, overiectomized females, and none in hormonally intact females. Further experiments in older animals and hormonally intact females are required before the translational potential of progesterone treatment can be predicted. Nevertheless, the data suggest that progesterone may be hazardous in adult overiectomized females.

Eighth, few animals had any comorbidities, such as hypertension or diabetes, as are commonly present in human stroke victims.43 The presence of comorbidities, such as hypertension, might attenuate neuroprotective effects, as seen with NXY-059.23

Ninth, lesion volume data were only available for total lesion size and not for cortical or subcortical damage. Many putative neuroprotectants appear to exert most of their effect on cortical stroke,44 which is a potential disadvantage, as many human strokes only involve small subcortical (lacunar) lesions.

Tenth, studies should assess functional measures as well as lesion volume and few of the included studies provided such data. This contrasts with our previous IAD meta-analysis of NXY-059 in which data were also available on motor function.23 Lesions do not necessarily correlate with functional outcome,45 as histologic analyses only differentiate between live and dead brain cells but not between viable cells that are fully functional and functionally compromised.46 The inclusion of only one measure, be it lesion volume or functional outcome, cannot by itself fully assess whether a treatment is effective. Therefore, a combination of both measures in a single study is insightful and would be a good indication of study quality. Ideally functional measures should include death, as does the modified Rankin Scale, the preferred outcome in acute stroke trials.47

Eleventh, although both transient and permanent models of stroke were represented, all experimental protocols involved stroke induction with a monofilament. In general, it is better if experiments use a number of different systems for inducing ischemia. The pathophysiological mechanisms and size of ischemic lesion can vary according to the model used,48 which may affect the efficacy of treatment.

Twelfth, although a wide range of time between stroke induction to initiation of treatment (ranging from 7 to 10 days before MCAO to 2 hours afterwards), only post-stroke treatment is relevant to human stroke, so that many of the experiments do not really contribute data relevant to the decision on whether it is appropriate to take this treatment into humans.

Thirteenth, it has been suggested that anesthetics without their own neuroprotective activity should be used to avoid confounding the effects of the potential neuroprotectant under examination with those of the anesthetic agent. However, isoflurane, an aesthetic with well-described neuroprotective potential49 was used in most experiments, and was associated with a lower lesion volume that in experiments using halothane. However, halothane use is in decline because of its potential hepatotoxic effects and the increased availability of other inhalational agents with fewer systemic side effects.50 Systematic reviews to date have not analyzed the neuroprotective potential of isoflurane in animal data.

Finally, the studies were of varying quality about randomization and blinding of surgery and outcome assessment, so that selection, performance, and observer bias may have been present. However, where adjusted, analysis of lesion volume appeared to be related to high rather than low study quality, as found in our earlier summary-based meta-analysis of progesterone.24 This contrasts with the common finding in meta-analyses of preclinical studies of neuroprotection being reported in low-quality studies.51 Other quality markers were missing in many of the included studies, including the presence of a sample size calculation.

In conclusion, although progesterone might reduce ischemic lesion volume, it also appears to increase the incidence of stroke-related death in adult overiectomized females. Its negative effects appear to be particularly evident in adult overiectomized female animals, highlighting the fact that endogenous hormone background needs to be taken into account in experimental stroke studies. These findings suggest that clinical trials, for any potential neuroprotectant, should not be commenced until an IAD-based meta-analysis of preclinical data has been performed. Publications and meta-analysis should include death, and combined death and lesion volume as outcomes, as interventions may have both positive and negative effects. To enable such analysis, authors of preclinical studies should be encouraged to share their data with IAD pooling projects as is common practice in clinical medicine.

Acknowledgments

We thank the collaborators who participated in this study: Stephanie Murphy (Murphy et al 2000, 2002,8, 21; Parker et al 2005,30; Murphy et al 2002 unpublished); Claire Gibson (Gibson et al 2004,5 2005,9 2011,34; Coomber et al 201033); Raymond Wong (Wong et al 2012,31; Wong et al 2012 unpublished); Iqbal Sayeed (Sayeed et al 2006,52 2007,10 200912).

The authors declare no conflict of interest.

Footnotes

This work was supported by the Medical Research Council of the United Kingdom (grant number G800129).

References

  1. Wolfe C.The burden of strokeIn: Wolfe C, Rudd T, Beech R (eds). Stroke services and research London: The Stroke Association; 199613–39. [Google Scholar]
  2. Adamson JBA, Ebrahim S. Is stroke the most common cause of disability. J Stroke Cerebrovasc Dis. 2004;13:171–177. doi: 10.1016/j.jstrokecerebrovasdis.2004.06.003. [DOI] [PubMed] [Google Scholar]
  3. Feigin VL. Stroke epidemiology in the developing world. Lancet. 2005;365:2160–2161. doi: 10.1016/S0140-6736(05)66755-4. [DOI] [PubMed] [Google Scholar]
  4. Alkayed NJ, Murphy SJ, Traystman RJ, Hurn PD. Neuroprotective effects of female gonadal steroids in reproductively senescent female rats. Stroke. 2000;31:161–168. doi: 10.1161/01.str.31.1.161. [DOI] [PubMed] [Google Scholar]
  5. Gibson CL, Murphy SP. Progesterone enhances functional recovery after middle cerebral artery occlusion in male mice. J Cereb Blood Flow Metab. 2004;24:805–813. doi: 10.1097/01.WCB.0000125365.83980.00. [DOI] [PubMed] [Google Scholar]
  6. Jiang N, Chopp M, Stein D, Feit H. Progesterone ss neuroprotective after transient middle cerebral artery occlusion in male rats. Brain Res. 1996;735:101–107. doi: 10.1016/0006-8993(96)00605-1. [DOI] [PubMed] [Google Scholar]
  7. Kumon Y, Kim SC, Tompkins P, Stevens A, Sakaki S, Loftus CM. Neuroprotective effect of postischemic administration of progesterone in spontaneously hypertensive rats with focal cerebral sschemia. J Neurosurg. 2000;92:848–852. doi: 10.3171/jns.2000.92.5.0848. [DOI] [PubMed] [Google Scholar]
  8. Murphy SJ, Littleton-Kearney MM, Hurn PD. Progesterone administration during reperfusion, but not preischemia alone, reduces injury in ovariectomized rats. J Cereb Blood Flow Metab. 2002;22:1181–1188. doi: 10.1097/01.WCB.0000037990.07114.07. [DOI] [PubMed] [Google Scholar]
  9. Gibson CL, Constantin D, Prior MJW, Bath PMW, Murphy SP. Progesterone supresses the inflammatory response and nitric oxide synthase-2 expression following cerebral ischemia. Exp Neurol. 2005;193:522–530. doi: 10.1016/j.expneurol.2005.01.009. [DOI] [PubMed] [Google Scholar]
  10. Sayeed I, Wali B, Stein DG. Progesterone inhibits ischemic brain injury in a rat model of permanent middle cerebral artery occlusion. Restor Neurol Neurosci. 2007;25:151–159. [PubMed] [Google Scholar]
  11. Sayeed I, Parvez S, Wali B, Siemen D, Stein DG. Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism for better neuroprotective effects of allopregnanolone over progesterone. Brain Res. 2009;1263:165–173. doi: 10.1016/j.brainres.2009.01.045. [DOI] [PubMed] [Google Scholar]
  12. Ishrat T, Sayeed I, Atif F, Stein DG. Effects of progesterone administration on infarct volume and functional deficits following permanent focal cerebral ischemia in rats. Brain Res. 2009;1257:94–101. doi: 10.1016/j.brainres.2008.12.048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chen J, Chopp M. Neuroprotective effects of progesterone after transient middle cerebral artery occlusion in rat. J Neurol Sci. 1999;171:24–30. doi: 10.1016/s0022-510x(99)00247-6. [DOI] [PubMed] [Google Scholar]
  14. Gibson CL, Gray LJ, Bath PMW, Murphy S. Progesterone for the treatment of experimental brain injury; a systematic review. Brain. 2007;131:318–328. doi: 10.1093/brain/awm183. [DOI] [PubMed] [Google Scholar]
  15. Goletiani NV, Keith DR, Gorsky SJ. Progesterone: review of safety for clinical studies. Exp Clin Psychopharm. 2007;15:427–444. doi: 10.1037/1064-1297.15.5.427. [DOI] [PubMed] [Google Scholar]
  16. Little BC, Matta RJ, Zahn TP. Physiological and psychological effects of progesterone in man. J Nerv Ment Dis. 1974;159:256–262. doi: 10.1097/00005053-197410000-00004. [DOI] [PubMed] [Google Scholar]
  17. Gaver RC, Pittman KA, Reilly CM, Smyth RD, Goodson PJ, Fenzi E, et al. Bioequivalence evaluation of new megestrol-acetate formulations in humans. Semin Oncol. 1985;12:17–19. [PubMed] [Google Scholar]
  18. Wright DW, Kellermann AL, Hertzberg VS, Clark PL, Frankel M, Goldstein FC, et al. Protect: a randomized clinical trial of progresterone for acute traumatic brain injury. Ann Emerg Med. 2007;49:391–402. doi: 10.1016/j.annemergmed.2006.07.932. [DOI] [PubMed] [Google Scholar]
  19. Xiao GM, Wei J, Yan WQ, Wang WM, Lu ZH. Improved outcomes from the administration of progesterone for patients with acute severe traumatic brain injury: a randomized controlled trial. Crit Care. 2008;12:10. doi: 10.1186/cc6887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Roof RL, Duvdevani R, Heyburn JE, Stein DG. Progesterone rapidly decreases brain edema: treatment delayed up to 24 Hours is still effective. Exp Neurol. 1996;138:246–251. doi: 10.1006/exnr.1996.0063. [DOI] [PubMed] [Google Scholar]
  21. Murphy SJ, Traystman RJ, Hurn PD, Duckles SP. Progesterone exacerbates striatal stroke injury in progesterone deficient female animals. Stroke. 2000;31:1173–1178. doi: 10.1161/01.str.31.5.1173. [DOI] [PubMed] [Google Scholar]
  22. He J, Evans CO, Hoffman SW, Oyesiku NM, Stein DG. Progesterone and allopregnanolone reduce inflammatory cytokines after traumatic brain injury. Exp Neurol. 2004;189:404–412. doi: 10.1016/j.expneurol.2004.06.008. [DOI] [PubMed] [Google Scholar]
  23. Bath PMW, Gray LJ, Bath AJG, Buchan A, Miyata T, Green AR. Effects of NXY-059 in experimental stroke: an individual animal meta-analysis. Br J Pharmacol. 2009;157:1157–1171. doi: 10.1111/j.1476-5381.2009.00196.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gibson CL, Gray LJ, Bath PMW, Murphy SP. Progesterone for the treatment of experimental brain injury; a systematic review. Brain. 2008;131:318–328. doi: 10.1093/brain/awm183. [DOI] [PubMed] [Google Scholar]
  25. Roof RL, Hall ED. Gender differences in acute cns trauma and stroke: neuroprotective effects of estrogen and progesterone. J Neurotrauma. 2000;17:367–388. doi: 10.1089/neu.2000.17.367. [DOI] [PubMed] [Google Scholar]
  26. Stroke Therapy Academic Industry Roundtable (Stair) Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke. 1999;30:2752–2758. doi: 10.1161/01.str.30.12.2752. [DOI] [PubMed] [Google Scholar]
  27. Toung TJ, Chen T-Y, Littleton-Kearney MT, Hurn PD, Murphy SJ. Effects of combined estrogen and progresterone on brain infarction in reproductively senescent female rats. J Cereb Blood Flow Metab. 2004;24:1160–1166. doi: 10.1097/01.WCB.0000135594.13576.D2. [DOI] [PubMed] [Google Scholar]
  28. Choi YC, Lee JH, Hong KW, Lee KS. 17 beta-estradiol prevents focal cerebral ischemic damages via activation of Akt and creb in association with reduced pten phosphorylation in rats. Fundam Clin Pharm. 2004;18:547–557. doi: 10.1111/j.1472-8206.2004.00284.x. [DOI] [PubMed] [Google Scholar]
  29. Liu AL, Margaill I, Zhang SD, Labombarda F, Coqueran B, Delespierre B, et al. Progesterone receptors: a key for neuroprotection in experimental stroke. Endocrinology. 2012;153:3747–3757. doi: 10.1210/en.2012-1138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Parker SM, Hurn PD, Murphy SJ. Progesterone and allopregnanolone as neuroprotectants in ischemic ovariectomized mouse brain. J Cereb Blood Flow Metab. 2005;25:S21–S21. [Google Scholar]
  31. Wong R, Ray D, Kendall DA. Progesterone pharmacokinetics in the mouse: implications for potential stroke therapy. J Pharm Pharmacol. 2012;64:1614–1620. doi: 10.1111/j.2042-7158.2012.01537.x. [DOI] [PubMed] [Google Scholar]
  32. Stewart LA, Parmar MK. Meta-analysis of the literature or of individual patient data: is there a difference. Lancet. 1993;341:418–422. doi: 10.1016/0140-6736(93)93004-k. [DOI] [PubMed] [Google Scholar]
  33. Coomber B, Gibson CL. Sustained levels of progesterone prior to the onset of cerebral ischemia are not beneficial to female mice. Brain Res. 2010;1361:124–132. doi: 10.1016/j.brainres.2010.09.037. [DOI] [PubMed] [Google Scholar]
  34. Gibson CL, Coomber B, Murphy SP. Progesterone Is neuroprotective following cerebral ischaemia in reproductively ageing female mice. Brain. 2011;134:2125–2133. doi: 10.1093/brain/awr132. [DOI] [PubMed] [Google Scholar]
  35. Saxena R, Wijnhoud AD, Carton H, Hacke W, Kaste M, Przybelski, et al. Controlled safety study of a hemoglobin-based oxygen carrier, dclhb, in acute ischemic stroke. Stroke. 1999;30:993–996. doi: 10.1161/01.str.30.5.993. [DOI] [PubMed] [Google Scholar]
  36. Furuya K, Takeda H, Azhar S, McCarron RM, Chen Y, Ruetzler, et al. Examination of several Potential mechanisms for the negative outcome in a clinical stroke trial of enlimomab, a murine anti-human intercellular adhesion molecule-1 antibody: a bedside to bench Study. Stroke. 2001;32:2665–2674. doi: 10.1161/hs3211.098535. [DOI] [PubMed] [Google Scholar]
  37. Tirilazad Int Steering Comm Tirilazad mesylate in acute ischemic stroke: a systematic review. Stroke. 2000;31:2257–2265. doi: 10.1161/01.str.31.9.2257. [DOI] [PubMed] [Google Scholar]
  38. Davis SM, Lees KR, Albers GW, Diener HC, Markabi S, Karisson G, et al. Selfotel in acute ischemic stroke- possible neurotoxic effects of an NMDA antagonist. Stroke. 2000;31:347–354. doi: 10.1161/01.str.31.2.347. [DOI] [PubMed] [Google Scholar]
  39. Shuaib A, Lees KR, Lyden P, Grotta J, Davalos A, Davis SM, et al. NXY-059 for the treatment of acute ischemic stroke. N Eng J Med. 2007;357:562–571. doi: 10.1056/NEJMoa070240. [DOI] [PubMed] [Google Scholar]
  40. Diener HC, Lees KR, Lyden P, Grotta J, Davalos A, Davis S, et al. NXY-059 for the treatment of acute stroke: pooled analysis of the Saint I and II trials. Stroke. 2008;39:1751–1758. doi: 10.1161/STROKEAHA.107.503334. [DOI] [PubMed] [Google Scholar]
  41. Egger M, Smith GD. Bias in location and selection of studies. Br Med J. 1998;316:61–66. doi: 10.1136/bmj.316.7124.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Mehra M, Henninger N, Hirsch JA, Chueh J, Wakhloo AK, Gounis MJ. Preclinical acute ischemic stroke modeling. J Neurointerv Surg. 2012;4:307–313. doi: 10.1136/neurintsurg-2011-010101. [DOI] [PubMed] [Google Scholar]
  43. Ankolekar S, Rewell S, Howells DW, Bath PMW. The influence of stroke risk factors and comorbidities on assessment of stroke therapies in humans and animals. Int J Stroke. 2012;7:386–397. doi: 10.1111/j.1747-4949.2012.00802.x. [DOI] [PubMed] [Google Scholar]
  44. Bailey EL, Mcculloch J, Sudlow C, Wardlaw M. Potential animal models of lacunar stroke a systematic review. Stroke. 2009;40:E451–E458. doi: 10.1161/STROKEAHA.108.528430. [DOI] [PubMed] [Google Scholar]
  45. Pineiro R, Pendlebury ST, Smith S, Flitney D, Blamire AM, Styles P, et al. Relating MRI changes to motor deficit after ischemic stroke by segmentation of functional motor pathways. Stroke. 2000;31:672–679. doi: 10.1161/01.str.31.3.672. [DOI] [PubMed] [Google Scholar]
  46. Aronowski J, Samways E, Strong R, Rhoades HM, Grotta JC. An alternative method for the quantitation of neuronal damage after experimental middle cerebral artery occlusion in rats: analysis of behavioral deficit. J Cereb Blood Flow Metab. 1996;16:705–713. doi: 10.1097/00004647-199607000-00022. [DOI] [PubMed] [Google Scholar]
  47. Lees KR, Bath PM, Schellinger PD, Kerr DM, Fulton R, Hacke W, et al. Contemporary outcome measures in acute stroke research: choice of primary outcome measure. Stroke. 2012;43:1163–1170. doi: 10.1161/STROKEAHA.111.641423. [DOI] [PubMed] [Google Scholar]
  48. Durukan A, Talisumak T. Acute ischemic stroke: overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral sschemia. Pharmacol Biochem Behav. 2007;87:179–197. doi: 10.1016/j.pbb.2007.04.015. [DOI] [PubMed] [Google Scholar]
  49. Sanders RD, Ma D, Maze M. Anaesthesia induced neuroprotection. Best Pract Res Clin Anaesthesiol. 2005;19:461–474. doi: 10.1016/j.bpa.2005.01.005. [DOI] [PubMed] [Google Scholar]
  50. Kitano H, Kirsch JR, Hurn PD, Murphy SJ. Inhalational anesthetics as neuroprotectants or chemical preconditioning agents in ischemic brain. J Cereb Blood Flow Metab. 2007;27:1108–1128. doi: 10.1038/sj.jcbfm.9600410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Crossley NA, Sena E, Goehler J, Horn J, van der Worp B, Bath PM, et al. Empirical evidence of bias in the design of experimental stroke studies: a metaepidemiologic approach. Stroke. 2008;39:929–934. doi: 10.1161/STROKEAHA.107.498725. [DOI] [PubMed] [Google Scholar]
  52. Sayeed I, Guo QM, Hoffman SW, Stein DG. Allopregnanolone, a progesterone metabolite, is more effective than progesterone in reducing cortical infarct volume after transient middle cerebral artery occlusion. Ann Emerg Med. 2006;47:381–389. doi: 10.1016/j.annemergmed.2005.12.011. [DOI] [PubMed] [Google Scholar]
  53. Egger M, Smith GD, Schneider M, Minder C. Bias in meta-analysis detected by a simple, graphical test. BMJ. 1997;315:629–634. doi: 10.1136/bmj.315.7109.629. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Cerebral Blood Flow & Metabolism are provided here courtesy of SAGE Publications

RESOURCES