Effects of progesterone on the inflammatory response to brain injury in the rat

Brain Research 1008 (2004) 29 – 39 www.elsevier.com/locate/brainres

Research report

Effects of progesterone on the inflammatory response to brain injury in the rat Kimberly J. Grossman a,*, Cynthia W. Goss a,1, Donald G. Stein b,c b

a Department of Psychology, Emory University, Atlanta, GA 30322, USA Department of Emergency Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA c Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA

Accepted 11 February 2004

Abstract The effects of progesterone on the cellular inflammatory response to frontal cortex injury were examined on postsurgical days 1, 3, 5, 7 and 9 in male rats treated with progesterone (4 mg/kg) and/or vehicle. Rats with bilateral contusions showed increased levels of edema on days 1, 3 and 5, more reactive astrocytes on days 3, 5, 7 and 9, and more macrophages/activated microglia on days 1, 3, 5 and 9 compared to shams. The number of neurons in the medial dorsal nucleus (MDN) of the thalamus reduced on days 5 and 9 after injury compared to shams. Progesterone reduced edema levels and increased the accumulation of macrophages/activated microglia compared to vehicle controls ( p < 0.025); however, these changes in the inflammatory response were not related to MDN neuronal survival. Our results confirm the possibility that one way progesterone mediates its neuroprotective effects following injury is through its actions on the inflammatory response. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Trauma Keywords: Progesterone; Traumatic brain injury; Microglia; Astrocyte; Edema; Neuronal survival

1. Introduction Traumatic brain injury (TBI) triggers cytotoxic responses and neuronal loss in areas both adjacent and distal to the site of impact (for reviews, see Refs. [11,14,64]), and this can cause enduring behavioral and cognitive deficits. Because the events triggered by TBI persist for days or longer after the injury, it may be possible to develop pharmacological treatments that can enhance functional recovery and reduce these pathophysiological responses. In this context, progesterone given after brain injury has been demonstrated to decrease cytotoxic responses to brain injury and promotes functional recovery in a variety of * Corresponding author. Present address: Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, 947 E. 58th Street, Chicago, IL MC0926, USA. Tel.: +1-773-702-4447; fax: +1-773702-3774. E-mail address: [email protected] (K.J. Grossman). 1 Present address: Department of Preventive Medicine and Biometrics, University of Colorado Health Sciences Center School of Medicine, 4200 E. 9th Avenue, Campus Box C245, Denver, CO, USA. 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.02.022

injury models. A series of experiments has revealed that progesterone decreases edema [53], attenuates free radical damage [55], reduces neuronal loss [52,55], and promotes behavioral recovery [2,54,57] after cortical contusion injury. Progesterone also reduces neuronal cell death [10] and attenuates neurological abnormalities after ischemia [13, 34,40] and spinal cord injury [62]. Having demonstrated progesterone’s effectiveness in reducing behavioral deficits and anterograde/retrograde degeneration [2,52,57], the next step is to determine how progesterone mediates its neuroprotective effects in the central nervous system. Because progesterone reduces edema after TBI and is considered an immune suppressant due to its role in maintaining pregnancy (e.g., preventing immune-mediated rejection of the fetus) [43], we hypothesized that progesterone might also help to promote recovery from TBI by reducing the inflammatory response to injury. The inflammatory response to brain injury involves edema formation, the recruitment of polymorphonuclear leukocytes (neutrophils) and mononuclear phagocytes (microglia, macrophages), and astrogliosis [1,19]. Traumat-

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ic brain injury initiates this inflammatory response by disrupting the blood brain barrier, creating edema and infiltration of inflammatory cells [9]. Dying neurons signal the release of cytokines and chemokines [26]. Proinflammatory cytokines can initiate the infiltration of inflammatory cells into the brain by activating adhesion molecules. Chemokines are chemoattractant cytokines that facilitate migration of inflammatory cells along concentration gradients [52]. Adhesion molecules facilitate the adhesion of polymorphonuclear leukocytes (neutrophils) to endothelial cells, which allow the leukocytes to infiltrate the brain [9]. These cells then release proinflammatory cytokines, such as IL-1, which stimulate the accumulation of other inflammatory cells such as astrocytes [27]. Activated microglia and reactive astrocytes participate in both neuroprotective processes and neurotoxic processes [3,4,8,48]. Reducing the accumulation of activated microglia is neuroprotective; suppression of activated microglia and macrophages reduces neuronal loss following spinal cord injury [28]. There is still some debate as to whether the suppression of astrocytes after TBI can result in better recovery [3,7,37,47,48]. Given progesterone’s known antiinflammatory properties, we predicted that postoperative administration of the hormone would reduce the glial response to injury and decrease necrosis. We also predicted that decreased accumulation of inflammatory cells near the contusion site would correlate with reduced neuronal degeneration in the medial dorsal nucleus (MDN) of the thalamus.

2. Materials and methods 2.1. Rats Thirty-six male Sprague – Dawley rats, weighing 290 – 340 g at the time of the surgery, were used in this experiment. Six of the animals received sham surgeries and the remaining animals received bilateral frontal contusions. Rats from both groups were randomly assigned to progesterone or control (vehicle) treatment groups. Rats were also randomly assigned to the following survival groups: 24 h, 3, 5, 7 and 9 days after surgery. All rats that received sham surgeries were assigned to an additional 24 h survival group. Each of these survival groups (progesterone and vehicle control) was comprised of six animals. The animals were housed individually in hanging wire cages and maintained on a 12 h dark/light reversed light cycle. Food and water were provided ad libitum. Procedures using animals were approved by the Emory University Institutional Animal Care and Use Committee (Protocol #098-2001). 2.2. Surgical procedures Aseptic surgical and stereotaxic procedures were used. Rats were anesthetized with sodium pentobarbital (Nembu-

tal, 50 mg/kg, intraperitoneal). A midline incision was made, the scalp retracted, and a bilateral 6-mm craniotomy was drilled and centered at 3.0 mm anterior to bregma. The impactor tip was placed over the medial frontal cortex at 3.0 mm A/P and 0.0 mm M/L. These coordinates correspond to the medial frontal cortex described by Paxinos and Watson [49]. The cortical contusion injury was produced using a pneumatically driven contusion device [32]. The brain was impacted for approximately 0.5 s with a 5-mm stainless steel tip with a force of 20 psi, a velocity of 2.25 m/s, and to a depth of 2 mm. After impact, bleeding was controlled and the fascia was sutured closed. The scalp was then closed using surgical staples. Body temperature during surgery was maintained at approximately 37 jC using a homeothermic blanket system (Harvard Apparatus, Holliston, MA). Following surgery, the animals were allowed to recover from the anesthetic in a warm, dry cage until they were fully awake. Sham surgeries were a control for anesthesia and stress. The sham surgeries followed the same procedure as contusion surgeries, except that sham rats did not receive a craniotomy and contusion. 2.3. Injections Depending on their survival time, rats received injections (intraperitoneal for the first and subcutaneously for the remaining six) of either progesterone (4 mg/kg; Sigma) and peanut oil (Sigma) or oil at 1, 6, 24 h, 2, 3, 4 and 5 days after the surgery. This treatment schedule, dose and vehicle were chosen because they have been shown to reduce behavioral deficits and anterograde/retrograde degeneration caused by medial frontal contusion injuries in male rats [54]. A shorter treatment regimen [57], different vehicle [31] and higher doses of progesterone [30] reduce or eliminate progesterone’s neuroprotective effects in this cortical contusion injury model. Progesterone and control (vehicle) treatments were prepared by a member of the laboratory and labeled ‘‘A’’ or ‘‘B’’ so that the experimenter was blind to the treatment. Following surgery, rats were randomly assigned to ‘‘A’’ or ‘‘B’’ treatment groups. 2.4. Histology The animals were killed by decapitation following a lethal dose of Nembutal at the assigned survival times (24 h, 3, 5, 7 or 9 days after injury). One hemisphere of the brain was rapidly collected for edema analysis and the remaining hemisphere was fixed for 24 h in 10% formalin, washed in water and then placed in 70% alcohol until paraffin embedding. The forebrain was sliced on a rotary microtome into 10Am sagittal sections, which were then mounted onto gelatin coated slides (1% gelatin) in preparation for staining and immunohistochemistry. Evenly spaced sections were deparaffinized using xylene (2
10 min) and rehydrated with a graded alcohol series, 100% and 95% alcohol (2
5 min each) and 70% alcohol (1
5 min) and stained for thionin.

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Thionin-stained sections at + 0.40, + 0.90 and + 1.40 mm M/ L relative to bregma were identified. One series of unstained sections adjacent to these three levels was stained with glial fibrillary acidic protein (GFAP) and a second series was stained with ED-1 to visualize reactive astrocytes and macrophages/activated microglia, respectively. 2.5. Edema analysis Four, 3-mm-thick coronal sections of the frontal cortex were cut and the dorsal half of each of the sections were placed in a preweighed (i.e., wet weight) capped container [59]. The containers were uncapped, put into a (vacuum) oven, dried at 60 jC (at 0.3 atm pressure) for 48 h and then recapped and reweighed (i.e., dry weight). Edema (% water content) was measured as the difference between the wet and dry weights divided by the wet weight [53]. Edema was reported as the difference in % water content between the most anterior (lesion area) and most posterior (occipital cortex) dorsal sections of the brain [53]. 2.6. Immunohistochemistry Immunohistochemistry was used to detect the primary monoclonal antibody glial fibrillary acidic protein (GFAP) and to detect the primary monoclonal antibody ED-1. GFAP is a specific protein produced by astrocytes (for a review, see Ref. [18]). ED-1 recognizes a cytoplasmic antigen in lysosomal membranes present in macrophages and activated microglia [17,44]. Following deparaffinization and rehydration, sections were washed for 5 min in 0.1 M phosphate-buffered saline (PBS), pH 7.4 or in dH2O before GFAP or ED-1 immunostaining, respectively. Sections were air-dried for 5 min. A PAP pen (Electron Microscopy Sciences) was used for hydrophobic slide marking and allowed to dry for 5 min. Sections were then incubated in 0.3% H2O2 for 30 min to block endogenous peroxidase activity and washed in PBS for 5 min. Sections for ED-1 labeling were processed for proteolytic antigen retrieval using trypsin (Sigma) for 20 min at 37 jC and washed in PBS. Sections were then incubated in dilutant (PBS with 2% horse serum) for 20 min and then in the primary monoclonal antibodies GFAP (1:500, Serotec) or ED-1 (1:100, Serotec) for 30 min at room temperature. Sections were washed in PBS and incubated for 30 min in biotinylated secondary antibody:horse antimouse IgG (1:250, Vector). Sections were then washed in PBS and incubated in Avidin –Biotin enzyme Complex (ABC, Vectastain Elite ABC Kit, Vector) for 30 min. Following a PBS wash, sections were incubated with 3,3V-diaminobenzidinetetra-hydrochloride (DAB, Peroxidase Substrate Kit, Vector) and washed in TRIS (3
15 min). Sections were dehydrated through a graded alcohol series, 95% and 100% alcohol (2
5 min each), cleared using xylene (2
5 min), and coverslipped with DePeX Mounting Medium (Electron

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Microscopy Sciences). Control sections followed the same procedures, except they were incubated in dilutant without the primary antibody. 2.7. Evaluation of immunohistochemistry Cell profiles, single optical planes of positively stained cells, were counted in six areas (three anterior and three posterior) proximal to the contusion cavity/necrotic area in three evenly spaced sections throughout the contusion area (based on Coogeshall and Lekan [16]). The total area sampled was 0.30 mm2. A grid, where each square was 2
2 cm, was generated using Microsoft Visio. Using the Stereologer program (beta version 1.1.1), the desired area (e.g., area adjacent to the cavity) at 4
was viewed on a computer screen. The grid was placed on the computer screen over the desired area, such that the edge of a row of 2
2 cm grid squares was flush with the cavity/necrotic edge or cortical edge for rats with contusions or sham rats, respectively. Starting 2 cm adjacent to the anterior and posterior lesion/necrotic areas, a 4
2 cm reference space was outlined (based on Fujita et al. [20]). The outlined reference space was divided into six evenly spaced areas and the magnification was increased to 40
. Systematic random sampling was used to determine which areas within the reference space were selected for counting. The six areas within the reference space were assigned a number (one through six) and a die was rolled three times to determine which three of the six areas were selected for counting. A motorized stage was used to move through the reference space to the three selected areas. The Stereologer program generates a 4
4 cm square over each selected area on the computer screen. Profiles of GFAP/ED-1 positive cells that were within the square or on the specified lines of the square were manually counted. The microscope was used to confirm that images on the computer screen were GFAP/ED-1 positive cells. Reactive astrocytes were identified by staining with GFAP and by the presence of their cell bodies and stout processes. ED-1 positive cells (macrophages/activated microglia) were identified by positive staining with ED-1 immunohistochemistry and their size (>10 Am). The number of GFAP/ED-1 positive cells counted for each rat was totaled and then averaged across the three sections. The effect of contusion injury and progesterone treatment on the population of reactive astrocytes and activated macrophages/microglia near the site of injury was measured as the mean number of GFAP/ED-1 positive cells. As noted earlier, the experimenter was blind to the treatment conditions and the survival times of the animals. 2.8. Evaluation of the effects of the inflammatory response on neuronal survival The relationship between any change in edema, astrogliosis and macrophage/activated microglia accumulation

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near the contusion site and neuronal cell survival was evaluated. Because 5 days of progesterone treatment may be necessary to affect behavioral recovery [57], the average number of viable neurons in the medial dorsal nucleus (MDN) of the thalamus was measured and correlated with measures of the cellular inflammatory response on days 5 and 9 after injury. The number of viable neurons in the MDN of the thalamus was counted in three evenly spaced thionin-stained sections (  0.10, + 0.40, + 0.90 M/L relative to bregma) and averaged across the three sections in each rat to assess neuronal survival. The total area sampled was 0.23 mm2. Using the Stereologer program (beta version 1.1.1), the thalamus at 1
was viewed on a computer screen and the MDN was outlined. The outlined reference space was divided into 11 evenly spaced areas and the magnification was increased to 40
. Systematic random sampling was used to determine which areas within the reference space were selected for counting. The 11 areas within the reference space were assigned a number (two through 12) and a pair of dice was rolled six times to determine which six of the 11 areas were selected. A motorized stage was used to move through the reference space to the six selected areas. The Stereologer program generates a 4
4 cm square over each selected area on the computer screen. Profiles of viable neurons that were within the square or on the specified lines of the square were manually counted. The microscope was used to confirm that images on the computer screen were viable neurons. Viable neurons met the criteria of an evenly stained nucleus, having definitive cell bodies with a visible nucleus, and an intact membrane.

3. Results 3.1. Edema Analysis of the effects of lesion and treatment on edema showed that postsurgical mean % difference in water content at specific survival time points was independent of the treatment administered (progesterone or vehicle) as there was no significant Treatment
Group interaction, F(5,24) = 1.47, p>0.05 (Fig. 1A). The statistical power (1-b) was < 0.35, indicating that there was less than a 35% chance of detecting a significant effect if one actually existed. The large effect size ( f = 0.55) indicates that there was a large difference in edema between rats given contusion or sham surgeries and treated with progesterone or vehicle. The significant main effect of Group [ F(5,24) = 10.61, p < 0.01] indicated that the mean % difference in water content was significantly different in animals depending on their survival group, regardless of oil or progesterone treatment (Fig. 1A). Planned comparisons of sham and contusion groups on mean % difference in water content were performed to follow up this main effect. The analyses showed that rats with lesions had higher edema levels on days 1 [t(10) =  2.21, p = 0.05], 3 [t(10) =  6.56, p < 0.01], and 5 [t(10) =  2.98, p < 0.025], compared to

2.9. Statistics All statistical tests were conducted using SPSS (version 10.0 for PC) and used a p value of 0.05, unless otherwise noted. The data was analyzed with a 2
6 (Treatment
Group) ANOVA. Significant main effects were followed up with planned comparisons using independent t-tests to compare sham and contusion groups at each survival time point. For planned comparisons, the modified Bonferroni test was used to correct alpha to reduce familywise error [36]. Power and effect sizes were calculated to determine the probability of having a type II error and to determine the magnitude of the treatment effect, respectively. Pearson’s correlation was used if there was a linear relationship between accumulation of reactive astrocytes and macrophages/activated microglia near the contusion site and edema levels on each postsurgical testing day. Pearson’s correlation was also used to determine if the average number of MDN neurons was related to measures of the cellular inflammatory response on days 5 and 9 after injury. Spearman’s correlation was used if visual inspection of scatterplots indicated heteroscedasticity. Type I error associated with multiple comparisons was reduced with the Bonferroni correction.

Fig. 1. Mean % difference in water content. (A) Effects of lesion and treatment on edema. The mean % difference in water content was increased in rats with contusions compared to shams on days 1, 3 and 5 after injury. (B) Effect of treatment on edema. Progesterone-treated rats showed reduced edema relative to vehicle-treated rats. yp = 0.05; *p < 0.05; **p < 0.01; S.E.M. indicated by vertical error bars.

K.J. Grossman et al. / Brain Research 1008 (2004) 29–39 Table 1 Correlations of edema and accumulation of inflammatory cells Inflammatory cells

Mean % difference in water weight Day 1

Mean macrophages/ activated microglia Mean reactive astrocytes

0.46a 0.54

Day 3 0.47a  0.03

Day 5

Day 7

Day 9

 0.77

 0.16a

 0.6

 0.66

0.03

 0.03

Data represent Spearman’s correlation coefficients, unless indicated otherwise. a Pearson’s correlation coefficient.

shams (Fig. 1A). Analysis of the main effect of Treatment [ F(1,24) =4.61, p < 0.05] indicated that the mean % difference in water content was significantly reduced in progesterone-treated rats relative to rats treated with only the vehicle (Fig. 1B). As displayed in Table 1, Pearson’s and Spearman’s correlations showed that the edema levels were not related to the accumulation of inflammatory cells. 3.2. Reactive astrocytes

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0.90, p>0.05 (Fig. 2A). The statistical power (1-b) was < 0.35, indicating that there was less than a 35% chance of detecting a significant effect if one actually existed. The large effect size ( f = 0.43) indicates that there was a large difference in mean reactive astrocyte accumulation between rats given contusion or sham surgeries and treated with progesterone or vehicle. Analysis of the main effect of Treatment [ F(1,24) = 2.41, p>0.05] indicated that mean reactive astrocytes was not significantly affected in progesterone-treated rats relative to rats treated with only the vehicle (Fig. 2B). The statistical power (1-b) was < 0.38, indicating that there was less than a 38% chance of detecting a significant effect if one actually existed. The medium to large effect size ( f = 0.32) indicates that there was a moderate difference in mean reactive astrocyte accumulation between rats treated with progesterone or vehicle. The significant main effect of Group [ F(5,24) = 8.81, p < 0.01] indicated that mean reactive astrocytes was significantly different in animals depending on their survival group, regardless of oil or progesterone treat-

Analysis of the effects of lesion and treatment on reactive astrocyte accumulation showed that mean reactive astrocytes at specific survival time points was independent of the treatment administered (progesterone or vehicle) as there was no significant Treatment
Group interaction, F(5,24) =

Fig. 2. Mean reactive astrocytes. (A) Effects of lesion and treatment on mean reactive astrocytes. Mean reactive astrocytes increased in rats with contusions compared to shams on days 3, 5, 7 and 9 after injury. (B) Effect of treatment on mean reactive astrocytes. Accumulation of mean reactive astrocytes was not significantly different in progesterone-treated rats relative to sham-treated rats. *p < 0.01; S.E.M. indicated by vertical error bars.

Fig. 3. GFAP immunostaining for astrocytes in the cortex adjacent to the contusion/necrotic area at 40
in a rat with a contusion (A) and in a sham rat (B) on day 5 after injury. Arrow shows reactive astrocyte. Scale bars = 50 Am.

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ment (Fig. 2A). Planned comparisons of sham and contusion groups on mean reactive astrocytes were performed to follow up this significant main effect. The analyses showed that rats with lesions (Figs. 2 and 3A) had higher mean reactive astrocyte levels on days 3 [t(5) =  4.75, p < 0.01], 5 [t(5) =  3.93, p < 0.025], 7 [t(5) =  4.21, p < 0.01], and 9 [t(5) =  14.91, p < 0.01] compared to shams (Figs. 2A and 3B). 3.3. Macrophages/activated microglia Analysis of the effects of treatment and lesion on accumulation of macrophages/activated microglia showed that mean ED-1 positive cells at specific survival time points was independent of the treatment administered (progesterone or vehicle) as there was no significant Treatment
Group interaction, F(5,24) = 1.82, p>0.05 (Fig. 4A). The statistical power (1-b) was 0.40, indicating that there a 40% chance of detecting a significant effect if one actually existed. The large effect size ( f = 0.57) indicates that there was a large difference in mean ED-1 positive cell accumulation between rats given contusion or sham surgeries and treated with progesterone or vehicle. The significant main effect of Group [ F(5,24) = 5.70, p < 0.01] indicated that the mean ED-1 positive cells was

Fig. 5. ED-1 immunostaining for macrophages/activated microglia in the cortex adjacent to the contusion/necrotic area at 40
in a rat with a contusion and treated with vehicle (A) or with progesterone (B) and a sham rat (C) on day 5 after injury. Arrows show ED-1 positive cells. Scale bars = 50 Am. Fig. 4. Mean ED-1 positive cells. (A) Effects of lesion and treatment on accumulation of ED-1 positive cells adjacent to the contusion area. Mean ED-1 positive cells were increased in rats with contusions compared to shams on days 1, 3, 5 and 9 after injury. (B) Effect of treatment on mean ED-1 positive cells. Progesterone-treated rats showed increased accumulation of ED-1 positive cells relative to vehicle-treated rats. *p < 0.01; S.E.M. indicated by vertical error bars.

significantly different in animals depending on their survival group, regardless of oil or progesterone treatment (Fig. 4A). Planned comparisons of sham and contusion groups on mean ED-1 positive cells across days were performed to follow up this significant main effect. The analysis showed

K.J. Grossman et al. / Brain Research 1008 (2004) 29–39

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that rats with lesions (Figs. 4A and 5A,B) had higher mean ED-1 positive cell levels on days 1 [t(5) =  8.26, p < 0.01], 3 [t(5) =  9.52, p < 0.01], 5 [t(5) =  3.39, p < 0.025], and 9 [t(5) =  4.64, p < 0.01] compared to shams (Figs. 4A and 5C). Analysis of the main effect of Treatment [ F(1,24) = 6.47, p < 0.025] indicated that mean ED-1 positive cells was significantly increased in progesterone-treated rats (Figs. 4B and 5B) relative to rats treated with only the vehicle (Figs. 4B and 5A). 3.4. MDN neurons Analysis of the effects of treatment and lesion on mean neurons in the medial dorsal nucleus (MDN) of the thalamus showed that mean MDN neurons at specific survival time points was independent of the treatment administered (progesterone or vehicle) as there was no significant Treatment
Group interaction, F(2,12) = 0.63, p>0.05 (Fig. 6). The statistical power (1-b) was < 0.25, indicating that there was less than a 25% chance of detecting a significant effect if one actually existed. The medium to large effect size ( f = 0.32) indicates that there was a moderate difference in mean neuronal cells in the MDN between rats given contusion or sham surgeries and treated with progesterone or vehicle. Analysis of the main effect of Treatment [ F(1,12) = 0.00, p>0.05] indicated that mean neuronal cells in the MDN was not significantly affected in progesterone-treated rats relative to rats treated with only the vehicle (Fig. 6). The statistical power (1-b) was < 0.34, indicating that there was less than a 34% chance of detecting a significant effect if one actually existed. The small effect size ( f = 0.04) indicates that there was a small difference in mean neuronal cells in the MDN between rats treated with progesterone or vehicle. The significant main effect of Group [ F(2,12) = 4.48, p < 0.05] indicated that the mean neuronal cells in the MDN was significantly different in animals depending on their survival group, regardless of oil or progesterone treat-

Fig. 7. Thionin staining of MDN thalamic neurons at 40
in a rat with a contusion (A) or in a sham rat (B) on day 5 after injury. Arrows show viable neurons. Scale bars = 50 Am.

ment (Fig. 6). Planned comparisons of sham and contusion groups on mean MDN neurons were performed to follow up this significant main effect. The analysis showed that rats with lesions (Figs. 6 and 7A) had lower mean MDN neuron levels on days 5 [t(10) = 2.79, p < 0.025], and 9 [t(10) = 2.34, p < 0.05], after injury compared to shams (Figs. 6 and 7B). As displayed in Table 2, Spearman’s correlations showed that mean MDN neurons were not related to the edema or the Table 2 Correlations of MDN neurons and cellular measures of inflammation Inflammatory measures

Fig. 6. Mean MDN neurons. Mean MDN neurons were reduced in rats with contusions compared to shams on days 5 and 9 after injury. *p < 0.05; S.E.M. indicated by vertical error bars.

Mean % difference in water weight Mean macrophages/activated microglia Mean reactive astrocytes

Mean MDN neurons Day 5

Day 9

 0.37 0.54 0.54

 0.26  0.50 0.09

Data represent Spearman’s correlation coefficients.

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accumulation of macrophages/activated microglia and reactive astrocytes on days 5 and 9 after injury.

4. Discussion The objective of this experiment was to determine if progesterone reduced components of the cellular inflammatory (edema formation, accumulation of reactive astrocytes and macrophages/activated microglia) response to traumatic brain injury (TBI). An additional objective was to determine if changes in the accumulation of inflammatory cells near the contusion site were related to neuronal survival in the medial dorsal nucleus of the thalamus (MDN). Our findings can be taken to suggest that (1) progesterone reduces edema in rats compared to those only given the vehicle; (2) reactive astrocyte accumulation following TBI might not respond to relatively low doses of progesterone (4 mg/kg); (3) progesterone (4 mg/kg) can increase the accumulation of ED-1 positive cells (macrophages/activated microglia) in the cortex compared to vehicle controls; (4) cellular measures of inflammation are not related to neuronal survival in the MDN at 5 and 9 days after injury. Overall, progesterone-treated rats had less edema relative to vehicle-treated rats. This finding that progesterone reduces edema is consistent with previous studies [21,53,57,63]. While it could appear that edema levels were higher in vehicle-treated shams relative to progesterone-treated shams, or the inverse that edema was lower in progesterone-treated shams relative to vehicle-treated shams, our ANOVA analysis showed that these groups are not significantly different. An a posteriori independent sample t-test showed that these groups are not significantly different, [t(4) = 1.80, p>0.05]. An additional question is how edema levels in vehicle and progesteronetreated shams compared to intact rats. Prior to the onset of this experiment, we measured the mean % difference in water weight in three intact rats, (M = 1.15, S.D. = 0.2325). An independent sample t-test comparing edema in intact and vehicle or progesterone-treated shams revealed that edema levels in vehicle-treated shams were not significantly different from intact rats, [t(4) = 1.070, p>0.05] and that edema levels in progesterone-treated shams were not significantly different from intact rats, [t(4) =  1.94, p>0.05]. Taken together, these results confirm that vehicle or progesterone treatment in shams does not affect edema levels relative to each other and relative to intact rats. After a qualitative comparison of the time course of the accumulation of inflammatory cells (e.g., monocytes/macrophages) and the development of edema, Holmin et al. [33] suggested that it is the accumulation of inflammatory cells that causes the swelling rather than trauma to blood vessels. In this experiment, we did not find a significant correlation between edema and the accumulation of macrophages/activated microglia or reactive astrocytes. This was unexpected considering that astrocytes are thought to

contribute to edema formation after injury (for a review, see Ref. [35]). At least two types of trauma-induced edema have been proposed: vasogenic and cellular (cytotoxic) edemas (for a review, see Ref. [38]). Vasogenic edema is swelling caused by an influx of fluid (e.g., water and solute) from the blood into the brain through the compromised blood brain barrier. Cellular edema, previously termed cytotoxic edema, is the influx of fluid into the cell from the extracellular space. Cellular edema is independent of any changes in blood brain barrier permeability. If astrocytes are a major source of cellular swelling, then an increased number of astrocytes would be associated with increased levels of edema. We did not observe this effect in the present experiment. However, our edema measurement method does not distinguish between vasogenic and cellular types of edema. It is possible that the presence of vasogenic edema in our edema measurement overshadowed any changes in cellular edema, making it difficult to detect a relationship between (cellular) edema and mean reactive astrocytes. Perhaps a relationship could be detected if only cellular edema was measured. At 4 mg/kg, progesterone did not affect astrocyte accumulation in rats with medial frontal cortex contusions. This is in contrast to previous studies showing that progesterone decreased astrocyte accumulation after penetrating brain injury [24,25]. However, our results support a recent study by Morali et al. [45] who also found that progesterone treatment (10 mg/kg) did not affect astrocyte accumulation after global ischemia. Three possible explanations for why progesterone did not affect astrocyte accumulation are: (1) differences between the contusion groups simply did not exist, (2) histological measures were not sensitive enough to detect small differences between the groups, (3) the dose or duration of treatment used did not affect reactive astrocyte accumulation at the time we collected the tissue for histology. Reactive astrocytes participate in both neuroprotective and neurotoxic processes [3,48]. The neuroprotective actions of astrocytes include the release of nerve growth factors and the absorption of excitotoxic transmitters (e.g., glutamate) [6]; however, these cells can also contribute to edema formation by potentiating glutamate-induced excitotoxicity physically or chemically blocking regenerative processes (e.g., axonal sprouting), and releasing free radicals and proinflammatory cytokines [5,22,29,58]. There is evidence to suggest that progesterone should decrease the accumulation of astrocytes after TBI. Progesterone (500 Ag/rat) decreased astrocyte accumulation in the cortex at 72 h following penetrating brain injury in ovariectomized, female rats [24]. Additionally, progesterone (10-12 M) administered using osmotic minipumps also decreased astrocyte accumulation and bromodeoxyuridine incorporation into astrocytes in the cortex at 7 days following penetrating brain injury in castrated male rats relative to controls administered the vehicle, containing ethanol (10-12 M) [25].

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However, progesterone treatment (10 mg/kg) did not affect astrocyte accumulation in a global ischemia model [45]. Additionally, a study on the effects of progesterone (4 mg/kg) on astrocyte accumulation following spinal cord injury in male rats did not find an effect of progesterone on injury-induced accumulation GFAP immunoreactive astrocytes near the injury site [41]. The authors suggested this discrepancy could be attributed to differences in the response of GFAP immunoreactive astrocytes to brain and spinal cord injuries. The differences in progesterone’s effects on astrocyte accumulation in these studies and the present study could be due to the type of injury inflicted, the dose of progesterone administered, and/or the length of the treatment regimen. For example, the 3-day progesterone treatment regimen used in the Shear et al. [57] study is not effective in attenuating TBI. Research suggests that progesterone’s effects on brain injury could also depend on the dose administered. A somewhat higher, but moderate, dose of progesterone could affect astrocyte accumulation differently. However, it is important to note that a high dose of progesterone (32 mg/kg) following medial frontal cortex contusions could also disrupt behavioral performance on some tasks [30]. Chronic administration of progesterone could also exacerbate the effects of TBI as Murphy et al. [46] found when they gave pretreatment (7 –10 days) of a high progesterone dose (30 mg/kg) prior to an ischemic stroke. A more definitive answer will require more research because it is not understood how specifically altering accumulation of astrocytes after TBI would affect functional recovery. Research suggests that increased astrocytes (and microglia) could be associated with poor cognitive outcome in rats [51]. Astrocyte (and microglia) accumulation increased in the prefrontal cortex of 8-week old male rats following 90 days administration of a synthetic glucocorticoid, prednisone. This same treatment regimen also resulted in poor performance on the Morris water maze task and created neuronal loss in the prefrontal cortex and CA1 region of the hippocampus. Some TBI research, however, indicates that increasing the accumulation of reactive astrocytes adjacent to the primary injury site might actually prove to be beneficial. For example, GK-11 increased the number of astrocytes near the primary injury site [59] and facilitated recovery on a spatial navigation task, the Morris Water Maze task [60]. We found that progesterone treatment increased ED-1 positive cells (macrophages/activated microglia) compared to the vehicle. Activated microglia participate in both neuroprotective and neurotoxic processes [4,8]. Activated microglia remove tissue debris and release nerve growth factors (for a review, see Ref. [8]), but they also release damaging free radicals [12,15,42], large amounts of glutamate [50] and proinflammatory cytokines (e.g., IL-1, IL-6, TNFa) [29] in vitro. Reducing the accumulation of activated microglia is neuroprotective. Suppression of activated microglia and

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macrophages reduces cell death and improves functional recovery following spinal cord injury [28]. It has been demonstrated that progesterone decreases lipopolysaccharide-stimulated microglia accumulation in vitro [23], but in our study and contrary to the original hypothesis, progesterone increased ED-1 positive cells (macrophages/activated microglia) in the cortex compared to vehicle-treated rats. GK11, a noncompetitive NMDA antagonist, has been observed to facilitate recovery on the Morris Water Maze and increase activated microglia on days 3 and 5 after injury [59,60]. We do not yet know why some neuroprotective agents suppress the accumulation of macrophages and activated microglia, while others seem to enhance it. Additionally, we do not know why progesterone decreases microglia accumulation in vitro, but has the opposite effect in vivo. The effects of microglia accumulation on recovery could vary depending on the dose of neurosteroid and the extent of the accumulation. Much of the research demonstrating a negative effect of activated microglia has been conducted in vitro and it could be that such accumulation in in vitro studies might be much larger compared to what is seen in the in vivo studies [61]. We did not find an effect of progesterone on neuronal survival in the medial dorsal nucleus (MDN) of the thalamus at 5 and 9 days after injury. This is in contrast to previous studies showing that progesterone reduces neuronal death in MDN at later time points [2,54,57]. It is possible that differences between groups do not yet exist at days 5 and 9 when neuronal survival was assessed in the current study. Experiments showing that progesterone reduces neuronal death, assessed cell survival in the MDN at 21 and 16 days after injury [2,54,57]. We did not find a correlation between neuronal survival in the MDN and the accumulation of macrophages/activated microglia, in the area of the lesion on days 5 and 9 after injury. This is unexpected because progesterone affected the accumulation of macrophages/activated microglia and these cells are reputed to participate in both neuroprotective and neurotoxic processes [4,8]. Bruce-Keller [8] has proposed that activated microglia reduce secondary neurodegeneration by removing dying neurons. Removal of the dying neurons could limit secondary degeneration by diminishing the release of cytotoxic substances (e.g., glutamate) into the extracellular environment. An aim of this experiment was to understand how progesterone ameliorates the effects of brain injury to promote functional recovery. Because progesterone can attenuate edema, reduce accumulation of resident inflammatory cells in vitro and in vivo, and is considered an immune suppressant, it was presumed that progesterone would facilitate recovery by reducing the inflammatory response to brain injury. Although we confirmed that progesterone reduces edema and that it increases the accumulation of macrophages/activated microglia after injury, we also found that progesterone did not affect accumulation of reactive astrocytes and did not reduce neuronal degeneration in the

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MDN of the thalamus at 5 and 9 days after injury. Alternatively, progesterone could exert its beneficial effects by targeting other aspects of the inflammatory response, such as reducing levels of proinflammatory cytokines [31,39]. There is some evidence to show that increasing the accumulation of inflammatory cells that modulate increases in the inflammatory response could facilitate behavioral recovery [59,60]. Schwartz et al. [56] have proposed that the duality of the macrophage/microglia response could depend on the time after injury when such accumulation occurs. These authors suggest that activated macrophages help heal tissue in the early stages of injury, but exacerbate pathophysiological processes later on. Our results confirm the possibility that one way progesterone mediates its neuroprotective effects following injury is through its actions on the inflammatory response. More work will be needed to clarify the processes that directly mediate the functional recovery.

Acknowledgements We would like to thank Raphael James for processing the brains in paraffin; Vanessa Bundy for assisting with some of the histology; Drs. David Rye and Teddy Pettus for their advice on cell counting and the Department of Neurology of Emory University School of Medicine for sharing their microscopy equipment. Research supported by NINDS R01-NS3866401A2, General Cologne Reinsurance, GSAS and Department of Psychology of Emory University.

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