Modulation of traumatic brain injury using progesterone and the role of glial cells on its neuroprotective actions

Journal of Neuroimmunology 237 (2011) 4–12

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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Review article

Modulation of traumatic brain injury using progesterone and the role of glial cells on its neuroprotective actions V. Ramana Feeser a,⁎, Roger M. Loria a, b, c a b c

Department of Emergency Medicine, Virginia Commonwealth University Reanimation Engineering Shock Center (VCURES), Richmond, Virginia, United States Department of Microbiology and Immunology, Virginia Commonwealth University Reanimation Engineering Shock Center (VCURES), Richmond, Virginia, United States Pathology Virginia Commonwealth University Medical Center, Virginia Commonwealth University Reanimation Engineering Shock Center (VCURES), Richmond, Virginia, United States

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 12 January 2011 Received in revised form 13 June 2011 Accepted 15 June 2011

TBI is a complex disease process caused by a cascade of systemic events. Attention is now turning to drugs that act on multiple pathways to enhance survival and functional outcomes. Progesterone has been found to be beneficial in several animal species, different models of brain injury, and in two preliminary human clinical trials. It holds promise as a treatment for TBI. Progesterone's multiple mechanisms of action may work synergistically to prevent the death of neurons and glia, leading to reduced morbidity and mortality. This review highlights the importance of glial cells as mediators of progesterone's actions on the CNS and describes progesterone's pleiotrophic effects on immune enhancement and neuroprotection in TBI. © 2011 Elsevier B.V. All rights reserved.

Keywords: Progesterone Traumatic brain injury Neuroprotection Glial cells Microglia

Contents 1. 2. 3. 4. 5.

Background . . . . . . . . . . . . Biology of progesterone . . . . . . . Animal research . . . . . . . . . . Human research . . . . . . . . . . Neuroprotective mechanism of action 5.1. Decreased cerebral edema . . 5.2. Decreased inflammation . . . 5.3. Decreased apoptosis . . . . . 5.4. Anti-oxidant effects . . . . . 5.5. Up-regulation of GABA . . . . 5.6. Pro-coagulant effects . . . . . 5.7. Systemic effects . . . . . . . 5.8. Multifunctional effects . . . . 6. Biomarkers in traumatic brain injury. 7. Role of glial cells . . . . . . . . . . 8. Conclusion . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . References . . . . . . . . . . . . . . .

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1. Background ⁎ Corresponding author at: Main Hospital, 2nd Flr, Suite 500, 1250 E. Marshall Street, PO Box 980401, Richmond, VA 23298-0401, United States. Tel.: + 1 804 828 5250; fax: + 1 804 828 8597. E-mail address: [email protected] (V.R. Feeser). 0165-5728/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2011.06.007

Traumatic Brain Injury (TBI) is an important public health problem and it remains a leading cause of injury-related death and disability. TBI is the number one cause of death among people ages 1 to 44 years

V.R. Feeser, R.M. Loria / Journal of Neuroimmunology 237 (2011) 4–12

(Thurman et al., 1999) and is the primary cause of death among all trauma-related deaths (Sousin et al., 1995; Adekoya et al., 2002). Each year in the United States, an estimated 1.7 million people sustain TBI and of these, 275,000 are hospitalized with fatal outcomes in one in five patients (Faul et al., 2010). Annually, an estimated 80,000 Americans are left with permanent neurological disabilities from TBI. In the year 2000, total costs including those from lost productivity totaled 60 billion dollars (Finkelstein et al., 2006). In 1993, the Brain Trauma Foundation developed evidence based guidelines for the treatment of TBI and with the exception of mannitol and barbiturates, no other agents were found to enhance recovery from TBI. Despite substantial efforts, no new treatment for TBI has entered into clinical practice for more than 30 years (Roberts et al., 1998). During this time, however, a large body of research has been done examining progesterone's role in the central nervous system (CNS). Progesterone's effects on the reproductive system are well known but it is only in recent decades that its neuroprotective effects have been identified. Baulieu first coined the term “neurosteroids” when he discovered that the nervous system is an endocrine organ where steroid hormones like progesterone are synthesized de novo or from circulating precursors in the brain (Baulieu et al., 1996). Previous focus had been on the neurons of the brain but recent research highlights the importance of glial cells as mediators of progesterone's actions on the CNS (Freeman, 2010; Graeber, 2010). Glial cells, in particular astrocytes and microglia, have been identified to have specific actions including biosynthesis and metabolism of progesterone in the normal and injured brain and are direct targets for the neuroprotective actions of this hormone on the nervous system (Jordan, 1999). In this article, we review the laboratory and clinical evidence for the use of progesterone in TBI, describe the biology of progesterone, discuss the multiple mechanisms of action by which progesterone provides neuroprotection, discuss the role of biomarkers, and focus on this hormone's target specific actions on glial cells of the CNS. Most work on progesterone had focused on its reproductive functions but recent work has broadened to include research on its neuroprotective functions in the brain. Progesterone is locally synthesized in the brain from cholesterol and this hormone can be metabolized within the brain to reduced derivatives 5α-DHP and 3α,5α THP (tetrahydroprogesterone which is often referred to as allopregnanolone) which may actually be the active neuroprotective agent (De Nicola et al., 2009; Herson et al., 2009). Progesterone is synthesized in the brains of males and females at similar levels and it plays a key role in the brain development during gestation. Progesterone levels are ten times higher in pregnant females compared to nonpregnant ones. Just as higher levels are progestational and protect the fetus during development, higher levels of this neurosteroid may also protect the brain during times of injury (Stein and Wright, 2010). 2. Biology of progesterone Progesterone, also known as P4 (pregn-4-ene-3,20-dione) is a C21 steroid hormone and the biosynthesis of this hormone is a multistep process (Fig. 1), (Garcia-Segura and Melcangi, 2006). The starting point for all steroid hormones including progesterone is cholesterol. The first enzymatic step uses cholesterol side-chain cleavage enzyme and converts cholesterol to pregnenolone. Pregnenolone is converted to progesterone by 3β-hydroxy-steroid dehydrogenase. Pregnenolone can be converted via 17-hydroxy-pregnenolone to dehydroepiandrosterone (DHEA) and progesterone can be converted via 17hydroxy-progesterone to androstenedione both by the enzyme 17βhydroxylase/C17-20-lyase. DHEA can be converted to androstenediol (AED) and androstenetriol (AET). Androstenedione converted to testosterone by the enzyme 17β-hydroxylase/C17-20-lyase. Andro-

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stenedione can be converted to estrone and testosterone to estradiol both by the enzyme aromatase. In addition, progesterone can be converted to the glucocorticoid corticosterone and progesterone's derivative 17-hydroxy-progesterone can be converted to the glucocorticoid cortisol. Finally, cortisol can be converted by 11β-hydroxysteroid dehydrogenase to cortisone and corticosterone can be converted via 18hydroxy-corticosterone to the mineralocorticoid aldosterone. These multiple conversions in the biosynthesis of steroids, many of which are bidirectional, reveal that progesterone's effects not only comes from progesterone itself but may also be a function of these other related hormones. Interestingly, the biochemical structures of these steroid metabolites are closely similar yet have different independent functions. Many of these derivative hormones including DHEA, AED and AET have documented immunological effects against systemic insults including lethal viral and bacterial infections (Loria, 2009), tumors (Loria, 2002), radiation injury (Loria et al., 2000), trauma-hemorrhage (Marcu et al., 2007), and wound healing (Feeser et al., 2009). These steroid derivatives may indirectly contribute to progesterone's pleiotrophic effects on immune enhancement and neuroprotection in TBI. 3. Animal research First reported in 1980, Stein showed that female rats recovered better than males following TBI suggesting the possibility of a hormonal influence (Stein, 2001). These initial findings were confirmed using a calibrated contusion to the medial frontal cortex of a rat TBI model. Animals were sacrificed at 24 h, and samples of injured brain were examined for their extent of cerebral edema (Roof et al., 1993). The female rats in proestrus (high estrogen levels but low progesterone levels) developed significantly less edema than male rats. The key finding, however, was in the third group of rats, pseudopregnant females (high progesterone levels), which developed only minor cerebral edema. To test the hypothesis that treatment with progesterone after TBI would decrease cerebral edema, progesterone 4 mg/kg was given to male and female rats subcutaneously at 1 h after injury and then by intraperitoneal injections at 6, 24 and 48 h after injury in the same model of TBI (Roof et al., 1992). At 72 h, animals were sacrificed and rats treated with progesterone developed significantly less cerebral edema than vehicle-treated controls. This study also showed significantly smaller brain lesions and decreased secondary loss of neurons. An important finding in this study was that males responded just as well as females suggesting that progesterone receptors are present in the brains of both sexes. The above study illustrates significant differences regarding the severity of cerebral edema at different time points with and without treatment with progesterone. In control animals in this model, cerebral edema after TBI developed within 2 h of injury, peaked at 24 h and decreased by 7 days. Progesterone decreased levels of cerebral edema at 3 days down to that normally seen at 7 days. In the same study, progesterone treatment was examined (2, 6, 24, or 48 h post-injury) and severity of cerebral edema was measured. Treatment at 2 h was best but significant decreases in cerebral edema were seen up to 24 h. Cerebral edema typically increases intracranial pressure which causes secondary cell loss and this finding supports the conclusion that controlling cerebral edema earlier is better (Roof et al., 1992). The effects of progesterone treatment in the TBI model on the cognitive functions were also examined by Stein's group. Progesterone 4 mg/kg or vehicle was given at 1, 6, 24, 48, 72, 96, and 120 h postinjury. One week after injury, each group of rats was tested in a Morris water maze. Rats with TBI treated with progesterone did better than placebo and approached the levels achieved in the sham surgery groups (Roof et al., 1994). Another study showed that while 3 days of post-injury treatment with progesterone decreased cerebral edema

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Fig. 1. Metabolism of steroids in the nervous system.

but 5 days of treatment was required to see functional recovery using spatial learning tasks (Shear et al., 2002). Whereas previous studies focused on the extent of cerebral edema, these studies were important in that they showed that treatment with progesterone improved cognitive and functional recovery which is a primary goal of any neuroprotective agent. A series of experiments examining ideal timing and dosing of progesterone in this model showed that progesterone still produced significant benefit when administered as late as 24 h after injury (Roof et al., 1996). The degree of positive benefit was maximized however, the earlier progesterone was given. Regarding dosing, progesterone levels correlated with severity of cerebral edema with higher progesterone levels resulting in less cerebral edema (Wright et al., 2001). The most effective doses were in the 8 to 16 mg/kg range with lower doses and higher doses (as high as 32 mg/kg) being less effective (Goss et al., 2003). Although higher doses of progesterone appear to be better at decreasing cerebral edema, there appears to be an upper limit for optimum dose. Rebound effects can be observed if progesterone is abruptly withdrawn so tapering of progesterone dose may be important (Cutler et al., 2006). Progesterone treatment in a variety of brain injury models was reported to be effective and these included four different models. The first consisted of diffuse brain injury, where both estrogen and progesterone attenuated brain edema (O'Connor et al., 2005). The second model consisted of bilateral medial frontal cortex aspiration, where progesterone treatment minimized neuronal loss (Asbury et al., 1998). In the third model, progesterone treatment in penetrating brain injury resulted in decreased astrocytic infiltration to the injury site (Garcia-Estrada et al., 1993; Garcia-Estrada et al., 1999). In a

fourth model, diffuse axonal injury after blunt impact injury to the skull, progesterone decreased the extent of brain injury (Vink and Van Den Heuval, 2004). The results demonstrate the effectiveness of progesterone treatment in a variety of different brain injury models and further emphasize its pleiotrophic effects for the treatment of TBI and brain injuries. A multitude of other studies have also found beneficial effects using progesterone in other models of neurologic injuries including stroke, multiple sclerosis, neuropathy, and spinal cord injury but a review of these is outside the scope of this article. Animal studies conducted by at least 20 different research groups working with 4 species (mice, rats, rabbits and cats) and different models of injury have reported that progesterone exerts neuroprotective effects (Stein et al., 2008). Only a few studies have shown negative results using progesterone for neuroprotection. One study in a stroke model failed to show positive effects for progesterone (Toung et al., 2004). Another study using a model of chemical injury to the brain showed no protection with progesterone (Azcoitia et al., 1998). One study using a moderate unilateral contusion of the cerebral cortex in rats found no beneficial effects after 3 days of progesterone treatment (Gilmer et al., 2008). Finally, only one study showed that progesterone worsened outcomes but this was after use in a stroke model. This model used pre-treatment with high doses of progesterone (30 mg/kg) for 3 weeks and it was abruptly stopped before inducing stroke by middle cerebral artery occlusion and progesterone treatment was found to worsen stroke injury (Murphy et al., 2000). This report confirms previous findings that too high doses of progesterone are ineffective and that tapering of progesterone is important to prevent

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“withdrawal” effects. This might have been prevented by gradual tapering-off of progesterone dose (Cutler et al., 2005). Attention should be made to a systematic review of 18 animal studies that administered progesterone in acute cerebral injury (stroke and TBI) and measured brain lesion volume (Gibson et al., 2008). Progesterone was effective at reducing lesion volume only in those TBI studies that achieved the highest quality scores. Five of the seven included studies for TBI had high quality scores of 4 or 5 and these did show beneficial effects for progesterone. This review only included studies that used lesion volume as their primary outcome and there were too few studies for other outcomes to be systematically reviewed. A few recent reports question the therapeutic window for progesterone since the majority of animal studies evaluated treatment less than 2 h after injury which is an unrealistic timeframe in human clinical trials (Loane and Faden, 2010). The design of clinical trials is based on animal studies especially when establishing safety and optimizing dosing and timing of progesterone administration and this review suggests gaps exist in the basic science literature. Well-designed bi-directional experimental and clinical research protocols will need to be instituted to answer the final question on whether progesterone should be used clinically in the treatment of TBI. 4. Human research Two independent human trials have been done at two different sites using progesterone in TBI. The ProTECT (Progesterone for Traumatic Brain Injury, Experimental Clinical Treatment) study done in a single location in the United States, a double blind Phase II clinical trial, studied 100 adult patients with moderate and severe TBI randomized to receive continuous infusion of intravenous (IV) progesterone or placebo within 11 h of injury (Wright et al., 2007). Progesterone (in 95% ethanol) or ethanol alone (placebo) was mixed in Intralipid 20% and an initial bolus dose of 0.71 mg/kg of progesterone was given during the first hour and reduced to 0.5 mg/kg per hour for the next 71 h (total dose for a 70 kg patient is 2535 mg IV). Severity of brain injury was classified using Glasgow Coma Scale (GCS) with scores of 4–8 categorized as severe and 9–12 as moderate. Limited studies in the United States in humans using intravenous progesterone had been done previously (56–58), none of which involved TBI, so the primary goal of the study was to establish safety of the drug. For this reason, randomization was done at a 4:1 ratio resulting in 77 patients in progesterone arm and 23 patients in placebo arm. The primary goal was achieved and progesterone was shown to be safe even in the multi-system injured patient. With the exception of 30-day mortality, there were no differences in serious adverse events between groups. The limitation of this study is that the number of controls was too small to achieve statistical power to assess the efficacy of progesterone treatment however promising trends were observed. Overall, there was a 50% reduction in 30-day mortality with 13.0% mortality in the progesterone arm and 30.4% in placebo arm (relative risk RR 0.43, 95% CI 0.18 to 0.99, p = 0.06) and this decreased 30-day mortality was concentrated in patients with severe TBI. In addition, improved 30-day functional outcomes as measured by Glasgow-Outcome Scale-Extended (GOS-E) and Disability Rating Scale (DRS) were observed in treated patients with moderate TBI with 55.6% in progesterone group has moderate to good recovery (GOS-E) compared to 0% in the placebo group (no RR estimate possible; p = .0202). A second trial done in China in 159 patients with severe TBI (GCS ≤ 8) was a double-blind placebo-controlled study that randomized using a 1:1 ratio resulting in 82 patients in progesterone arm and 77 patients in placebo arm (Xiao et al., 2008). The treatment arm had to be initiated within 8 h of injury and used progesterone 1.0 mg/kg intramuscular (IM) injections every 12 h for 5 days (total dose for a 70 kg patient is 700 mg IM). Unlike the ProTECT study, this study

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followed patients out to 6 months. At 3 month follow-up, 47% of progesterone-treated patients showed better functional outcome as measured by Glasgow Outcome Scale as compared to 31% of placebotreated patients (p = 0.034). Similarly, at 6 month follow-up, 58% of progesterone treated patients showed better function compared with 42% of the placebo group (p = 0.048). The modified Functional Independence Measure (FIM) scores in the progesterone group were higher than those in the placebo groups at both 3-month and 6-month follow-ups (p b 0.05 and p b 0.01, respectively). The key finding, however, was a reduction in 6- month mortality where the progesterone-treated arm had 18% mortality compared to placebo arm which had 32% mortality (p = 0.039). Again, no complications or adverse events were associated with administration of progesterone. Interestingly, neither of these two human studies found statistically significant differences in intracranial pressure (ICP) measurements. One of progesterone's main mechanisms of action is decreasing cerebral edema and the higher the progesterone level, the lower the level of cerebral edema (Wright et al., 2001). Surprisingly, progesterone appears to have little effect on directly reducing the ICP of patients with severe TBI. This is in contrast to a recent animal study which showed increased ICP after TBI but found ICP decreased significantly at 4 h and 24 h in progesterone treated groups and this correlated with decreased cerebral edema (Shahrokhi et al., 2010). These animals were given progesterone 8 mg/kg by intraperitoneal injection 30 min after TBI and this early treatment may account for this positive finding. Both of these Phase II clinical studies were small, single center studies that focused on safety of progesterone. Interestingly, consistent positive effects were seen at total doses as low as 700 mg and as high as 2535 mg in a 70 kg patient suggesting there is a wide dosing range for efficacy. The results, however, are promising since both not only showed reduced mortality but also showed reduced morbidity as shown by improved functional outcomes. Currently, two major Phase III clinical trials that began in 2010 (ProTECT III and SyNAPSe ) have set out to provide a more robust answer on the effectiveness of progesterone treatment in TBI (Stein and Wright, 2010). The ProTECT III trial is a NIH/NINDS-supported multicenter trial that plans to enroll 1140 patients with moderate to severe TBI (GCS 4–12) and administer continuous intravenous progesterone or placebo within 4 h of injury to be given for 3 days with an additional one day taper. The SyNAPSe study (Study of the Neuroprotective Activity of Progesterone in Severe Traumatic Brain Injuries) sponsored by BHR Pharma (Herndon, Virginia) is an international multicenter trial that plans to enroll 1200 patients with severe TBI (GCS 4–8) and administer continuous intravenous progesterone or placebo within 8 h of injury to be given for 5 days. 5. Neuroprotective mechanism of action In addition to the direct damage done to the brain by traumatic injury, a number of pathophysiologic processes occur which result in a secondary injury cascade. Pathophysiologic changes of TBI include cerebral edema, release of excitatory neurotransmitters, oxidative stress, inflammation and apoptosis. Progesterone neuroprotection in TBI appears to be mediated primarily by reducing cerebral edema, decreasing inflammation and apoptosis; it also has an anti- oxidant effect and up regulates the inhibitory neurotransmitter GABA (Fig. 2). 5.1. Decreased cerebral edema In the normal brain, water is distributed between several compartments including cerebrospinal fluid (CSF), blood, intracellular and interstitial brain parenchyma and water flows between these compartments based on osmotic and hydrostatic pressure gradients. Brain injury and tissue breakdown leads to cytotoxic cerebral edema with resultant increased fluid inside neurons and astrocytes resulting

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Potential mechanisms in TBI Remyelination

Reduces Inflammatory Cytokines

Progesterone

Reduces Cerebral Edema Cytotoxic Edema

Mitochondrial Recoupling Decreases free radicals & lipid peroxidation

Antagonizes Sigma Receptor Vasogenic Edema

Increases Bcl2 AkT-P

Reduce Apoptosis

5.3. Decreased apoptosis

Blocks Excitotoxicity

Enhances GABA

Fig. 2. Neuroprotective mechanisms of progesterone in TBI. Modifed from: Sayeed and Stein, 2009, Progesterone as a neuroprotective factor in traumatic and ischemic brain injury, Prog Brain Res, 175(15), 219–237.

in cell loss. Disruption of the blood brain barrier (BBB) produces vasogenic edema and the resultant swelling in the brain increases intracranial pressure which compromises cerebral blood flow and results in brain ischemia, herniation, and death. Cytotoxic edema appears to be the more likely following TBI (Marmarou et al., 2006). Progesterone did not repair the BBB following frontal contusion but prevented the influx of water and other inflammatory molecules (Duvdevani et al., 1995). Progesterone reduces both vasogenic and cytotoxic edema after TBI (Roof et al., 1996). Membrane progesterone binding protein 25-Dx is thought to play a role in progesteronemediated reduction in cerebral edema. After TBI in rats, 25-Dx expression was up-regulated in reactive astrocytes adjacent to the site of brain injury (Meffre et al., 2005). Progesterone's reduction in cerebral edema is also modulated by its effects on aquaporins which are membrane water channels acting as osmosensors and control water fluxes into and out of the brain (Amiry-Moghaddam et al., 2003). Aquaporin-4 (AQP 4) is the primary water channel found in the brain located on astrocyte foot processes at the borders between the major fluid compartments (CSF and blood) and brain parenchyma. Early in cytotoxic edema, AQP4 facilitates edema formation and later when vasogenic edema occurs, AQP4 increases the rate of edema elimination (Papadopoulos and Verkman, 2007). After only two injections of progesterone in a rat TBI model, cerebral edema was significantly decreased and this correlated to lowered expression of AQP-4 in the zone of injury (Guo et al., 2006). Decrease in cerebral edema has been shown to be beneficial in animal models up to 24 h and this characteristic opens a long therapeutic window to make progesterone treatment practical for use in humans.

Nuclear factor κB (NFκB) has been implicated in inflammation and apoptosis after TBI and progesterone reduces NFκB and prevents neuronal apoptosis. In addition to inhibition of apoptosis, progesterone also inhibits gliosis. NFκB-regulated factors such as IL-1β, C3, cyclooxygenase-2 and nitric oxide synthase are reduced by treatment with progesterone (Djebaili et al., 2004; He et al., 2004; Djebaili et al., 2005; Pettus et al., 2005). Pro-apoptotic enzymes caspase-3, Bax, Bad, and AKT are reduced (Djebaili et al., 2005) and anti-apoptotic proteins Bcl-2 and ERK are up-regulated by treatment with progesterone (Djebaili et al., 2005; Stein, 2008). 5.4. Anti-oxidant effects Lipid membrane peroxidation is a major contributor to damage to the BBB. Levels of 8-epiPGF2α, a marker of lipid peroxidation, are reduced in progesterone-treated TBI rats (Roof and Hall, 2000). Progesterone reduces lipid peroxidation by membrane stabilization (Roof et al., 1997) and reduces the damage by free radicals resulting in reduced oxidative stress (Pajovic et al., 1999). Reducing the presence of free radicals can improve cell survival adjacent to the injury and stabilize the BBB. 5.5. Up-regulation of GABA Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the brain and progesterone acts by increasing GABA transmission which results in GABA-mediated inhibition of neuronal activity and decreased excitatory input. Brain injury causes the release of glutamate and other excitatory molecules. Therefore, progesterone which up-regulates GABA inhibition causes a decrease in injurymediated excitotoxicity (Lambert et al., 2003; Pierson et al., 2005; Mani, 2006) and also weakens excitatory amino acid responsiveness (Smith, 1991). Metabolites of progesterone such as allopregnanolone demonstrate many of the same neuroprotective benefits but they may be actually be more potent at modulating this GABA transmission. 5.6. Pro-coagulant effects TBI-induced coagulopathy is another important factor causing secondary neuronal damage. Progesterone (16 mg/kg), allopregnanolone (8 mg/kg) and vehicle were compared for their effects on hemostatic proteins in a rat TBI model (VanLandingham et al., 2008). Progesterone acted as a procoagulant by increasing thrombin, fibrinogen, and coagulation factor XIII. Allopregnanolone acted as an anticoagulant by increasing tissue-type plasminogen activator. This differential effect on the coagulation cascade may make progesterone the ideal choice in TBI where blood loss can be detrimental and may make allopreganolone more suitable for ischemic strokes (Sayeed and Stein, 2009).

5.2. Decreased inflammation

5.7. Systemic effects

TBI produces a marked inflammatory reaction with influx of neutrophils and macrophages and heavy gliosis surrounding the zone of injury. Progesterone and its derivatives, DHEA and AED, are potent anti-inflammatory agents which work by inhibiting cytokine release and immune cell activation and migration (Chao et al., 1994; Ehring et al., 1998). The key mechanism is inhibiting proinflammatory cytokines most notably interleukin-1 (IL-1), IL-6, and tumor necrosis factor (Arvin et al., 1996, Grossman et al., 2004). Inflammatory proteins C3 complement and Nfκβ p65 were significantly decreased after progesterone treatment (Pettus et al., 2005). Inflammation is a significant contributor to secondary injury and microglia activation and control of the inflammatory response benefits the immediate zone of injury as well as more distal sites and even other organs (Chen et al., 2008).

TBI has systemic effects and can cause oxidative damage in heart, kidney, lung, liver and other body tissues (Shohami et al., 1999). TBI produces widespread systemic inflammatory effects and progesterone reduces the expression of inflammatory cytokines (Chen et al., 2007, Chen et al., 2008). Brain injuries induce marked expression of inflammatory factors in the intestinal mucosa of the gut including TNF-α, Nf-κβ, and IL-6. TBI-associated systemic inflammation can lead to multiple organ failure and infection which can cause death. Multiple organ injury is most pronounced with moderate and severe TBI but also occurs in mild TBI indicating that any degree of TBI can result in multiple organ failure. (Utagawa et al., 2008). Therefore, using a drug like progesterone which has pleiotrophic sites of action may be advantageous in the treatment of a complex systemic process like TBI, a fact further discussed below.

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5.8. Multifunctional effects TBI is caused by both primary and secondary injury mechanisms. Primary injury occurs from the forces at the time of injury that cause mechanical damage to neurons, axons, glia and blood vessels. Primary injury is known to be irreversible. Secondary injury is the cellular, biochemical, and metabolic alterations that occur over time in response to the primary insult. Typically, this secondary injury results in neurological deficits and there is a narrow therapeutic window whereby treatment can stop or at least attenuate these events and result in improved outcomes. It is this multifactorial secondary injury process that has been described above whereby treatments like progesterone hold promise for success. Targeting a single factor in TBI is unlikely to improve outcome but instead targeting multiple injury factors is the approach most likely to lead to a successful management strategy. Pleiotrophic progesterone which influences several brain functions demonstrates extensive neuroprotection. Multipotential drugs like progesterone which modulate multiple injury mechanisms should be the focus of future clinical trials in TBI. (Faden and Stoica, 2007; Vink and Nimmo, 2009; Loane and Faden, 2010). The NIH consensus panel report calls for more studies to be done using combination drugs or using drugs with multiple sites of action for the treatment of TBI (Margulies and Hicks, 2009). 6. Biomarkers in traumatic brain injury TBI includes a heterogenous spectrum of pathologies (hemorrhage, axonal injury, ischemic injury) that affects a heterogenous patient population (age, gender, pre-existing comorbidities, presence of isolated TBI or multisystem trauma, etc.) and biomarkers may be useful in capturing the complexity of this insult. Biomarkers that identify the diagnosis, prognosis, or treatment response for TBI have become a topic of increased interest. Three types of biomarkers in TBI which include imaging modalities, hormone levels, and measure of brain-specific proteins have shown promise but have yet to be validated. Conventional neuroimaging in TBI is the computed tomography scan (CT scan) and this imaging modality has been limited in its diagnosis, prognosis and evaluation of treatment efficacy for brain injury. With advances in magnetic resonance imaging (MRI), techniques including susceptibility-weighted imaging (SWI) for diagnosing microhemorrhages, diffusion-weighted imaging and diffusion tensor imaging (DWI/DTI) for edema quantification and identification of white matter and axonal injuries, magnetic resonance spectroscopy (MRS) for measuring metabolites, perfusion-weighted imaging to measure cerebral blood flow and perfusion, and functional MRI to detect cortical activation patterns used in cognitive tasks have been studied in TBI (Kou et al., 2010). They have demonstrated capacity as serving as biomarkers of TBI and will need to be studied for their usefulness in evaluating treatment efficacy with progesterone. Hormones play an important role in TBI and research has shown that sex hormones can determine outcome after brain injury, with estradiol and progesterone having neuroprotective effects and testosterone having potentially damaging effects. After injury, as many as 80% develop pituitary gland dysfunction and hypoendocrine disorders. This can happen due to direct damage to the pituitary and hypothalamus but also may arise from stress hormone abnormalities seen in acute critical illnesses which can affect sex steroid levels. A recent study from the University of Pittsburgh examined hormone levels of 117 patients with severe TBI in order to determine if certain hormones may act as biomarkers (Wagner et al., 2011). They found that elevated estradiol levels were associated with poor outcome which contradicted past research but confirmed high testosterone levels were associated with worse outcome. Progesterone levels were initially higher in males with TBI compared to controls but over time, progesterone levels in both men and women declined likely due to the uniform development of hypogonadism with severe TBI. The rapidly

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declining progesterone levels provide the rationale for why both genders may benefit from acute progesterone treatment. Interestingly, older age was associated with a higher stress response and worsened outcome. Hormone levels measured during the acute stages of TBI may be used as biomarkers to determine if a specific hormonal strategy like progesterone can improve overall outcome. Tissue breakdown in TBI results in the release of structural proteins in the bloodstream that can be used to assess trauma severity and response to treatment. The brain expresses more proteins than any organ in the body and the detection of brain-specific proteins in the blood holds promise. The list of potential biomarkers in TBI is long and includes α-II-Spectrin BDPs (breakdown products), α-Synuclean, C-tau (cleaved tau), 3'5’cAM/2’,3'cAMP, Ceruloplamin/CU (copper), CK-BB (creatine kinase-BB), CRP (C-reactive protein), SAA (serum amyloid A protein), CRMP-2 (collapsin response mediator protein-2), FABP (brain type-fatty acid binding proteins), F2-Isoprostane, 4-HNE (4Hydroxynonenal), 5-HIAA (5-hydroxy indol acetic acid), GFAP (glial fibrillary acidic protein), HVA (homovanillic acid), ICAM (intercellular adhesion molecule), IL-1β, IL-6, IL-8, IL-10, IL-12p70, lactate, magnesium, MBP (myelin basic protein), MCP-1 (monocyte chemo-attractant protein-1), MIP-1α (macrophage inflammatory protein), NAA (Nacetylaspartate), phospo-neurofilament, NSE (neuron-specific enolase), NE (norepinephrine), S100B, TGF-β (transforming growth factor-beta), TNF-α (tumor necrosis factor-alpha) and UCH-L1 (ubiquitin C-terminal hydrolase), (Dash et al., 2010). Although some of these changes have been reported to correlate with mortality and outcome, further research is required. Biomarkers of Injury and Outcome in ProTECT III (BIO-ProTECT) is designed to validate preliminary data assessing biomarker levels in patients enrolled in the ProTECT III trial (http://www/ researchgrantdatabase.com/g/1R01NS071867-01A1/BIOMARKERSOF-INJU…). Preliminary data from Michael Ross's group at Emory University suggest that serum levels of S100B, GFAP, UCH-L1 and SBDP150 are more accurate predictors of severity of injury than GCS and CT scan. The primary aim of BIO-ProTECT will be to measure these four biomarker levels at 24 and 48 h after randomization and correlate with outcome prediction at 6 months. Additionally, it will examine whether decreased release of these biomarkers can evaluate treatment response to progesterone. The secondary aim will measure progesterone levels at 24 and 48 h after randomization and correlate these with 6 month outcomes. 7. Role of glial cells There is increasing evidence that brain injury not only affects neurons but interplays with endothelial cells, astroglia, microglia, oligodendroglia and other precursor cells. Neuroprotection strategies recognize the importance of optimizing non-neuronal functioning while inhibiting neuron cell death pathways. Progesterone is produced by the brain and for the brain by neurons and glial cells in both males and females. In addition to its direct action on neurons, progesterone displays neuroprotective effects by it actions on glial cells. Traditional thinking was that glial cells assisted the functions of neurons but it is now evident that neurons and glial cells function as a single unit. Brain function depends on neuron to glia and glia to neuron signaling and each component influence the differentiation and metabolism of each other. Glial cells are the macrophages of the brain and act as the first and primary form of active immune defense in the central nervous system. Glial cells are subdivided into two main categories: macroglia and microglia (Garcia-Segura et al., 1996). Macroglia include ependymal cells, astroglia and oligodendroglia with the main subtype being astroglia. Astroglia are responsible for the regulating neuron metabolism and activity, guiding migrating neurons and modulating neuron differentiation. Microglia constitute 20% of the total glial cell population within the brain and are in charge of protecting brain

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tissue against damage. Glial cells are distributed throughout the brain and are constantly scavenging the CNS for damaged neurons and consequently have a major role in TBI. Glial cells play a central role in the synthesis and metabolism of progesterone. The steps in steroidogenesis (Fig. 1) take place in neurons and glia. Astrocytes and oligodendrocytes can transform cholesterol into pregnenolone and metabolize this to progesterone. Progesterone may then be transformed into its metabolites DHP and THP (allopregnanolone) by the enzymes 5α-reductase and 3α-hydroxysteroid dehydrogenase respectively (Garcia-Segura and Melcangi, 2006). Progesterone synthesis and metabolism by glial cells is affected by cellular interactions which stimulate the expression and activities of these enzymes. Proteins involved in the intramitochondrial trafficking of cholesterol (the first step in steroidogenesis) are upregulated in the nervous system after injury. A local increase in the quantity of steroids is observed after TBI. This finding suggests that increased steroidogenesis is part of the mechanism used by the brain to respond to TBI. Steroidogenesis in the brain is coordinated by glia-neuron crosstalk (Garcia-Ovejero et al., 2005). Some of the steroids produced by glia are neuroprotective but glial cells also serve as targets for steroids and regulate neuron survival. Steroid hormones act in the brain as molecular signals for neuroendocrine effects and affect brain development and plasticity. Neurosteroids refers to steroids made in the brain from cholesterol and neuroactive steroids refer to steroids that change neuron excitability and regulate neural function. Steroids acting on glial cells or produced by the brain affect neuron development and function and responses to brain injury. Steroids are involved in the communication of glia and neurons in the nervous system. In addition to being a source of progesterone, glial cells are also targets of these molecules. Several studies show the importance of glial cells as targets for the neuroprotective and anti-inflammatory actions of progesterone (Cekic et al., 2009; De Nicola et al., 2009; Kipp and Beyer, 2009). Progesterone downregulates the expression of proinflammatory cytokines by microglia and astroglia and thereby reduces brain inflammation. As stated earlier, progesterone also attenuates brain edema in part by regulating the expression of aquaporin 4 which is a water channel present in astrocytes that plays a key role in regulating water balance in damaged brain tissue. By acting on microglia and astroglia, progesterone reduces reactive gliosis, and the release of pro-inflammatory cytokines, brain edema and modulates the inflammatory process. Steroids signal on glial cells by multiple mechanisms. Classical steroid hormone receptors are ligand activated transcription factors that regulate target genes and gene transcription. Steroid hormone receptors are expressed by glia but not all glia cell types have the same receptors and change expression based on whether the glia are resting or reactive. In addition to regulating transcription, steroids exert rapid signaling events in glia. Sex steroids regulate gene expression, neuron survival, neuron and glial cell differentiation, and control synaptic transmission (Garcia-Ovejero et al., 2005). The effects of sex steroids like progesterone may be different based on whether they are acting under physiologic or pathologic conditions. Progesterone acts on glial cells by regulating the synthesis of other steroid hormones which may affect neuronal and glial cell function. Progesterone regulates responses in each of the major glial cell types. Astrocyte size is dependent on the expression of glial fibrillary acidic protein (GFAP). Progesterone and its metabolite DHP elevate GFAP mRNA levels in astrocytes within hours of exposure and regulate the production of multiple proteins including ApoE which is important in synaptic remodeling (Brinton et al., 2008). Astrocytes also convert the neurosteroid pregnenolone into progesterone. In addition, astrocytes can metabolize progesterone to allopregnanolone and can metabolize pregnenolone to 7α-hydroxy-pregnenolone and DHEA to androstenedione or to 7α-hydroxy-DHEA (Akwa et al., 1993). DHEA

and derivatives AED and AET have been shown to have immune regulatory activities. Astrocytes therefore can deliver a multitude of neurosteroids to other cell types in the CNS. These neuron-glia interactions are an important factor in the regulation of CNS function. Astrocytes control ion concentration, the energy supply and other metabolites and thereby modulate function at the synapse (Jordan, 1999). By taking up glutamate, astrocytes shorten the synapse and decrease the potentially excitotoxic effects from glutamate. Microglia are normally at rest but are rapidly activated by changes in potassium homeostasis in the brain. When injury occurs, activated microglia proliferate and migrate to the site of injury. After extreme activation, microglia act as phagocytes and can secrete the neurotransmitter glutamate, reactive oxygen species and nitric oxide all of which may be detrimental to the already injured brain tissue. Progesterone, however, has been found to regulate this microglia activation and inhibit cytokine-induced microglia proliferation in vitro (Ganter et al., 1992). This effect was also observed in a rat model of penetrating brain injury, where administration of progesterone decreased accumulation and proliferation of astrocytes in the injured brain (Garcia-Estrada et al., 1993). 8. Conclusion Traumatic brain injury (TBI) remains a leading cause of morbidity and mortality worldwide. Despite extensive research, no new treatment for TBI has entered clinical practice. A large and growing body of literature has examined the role of progesterone as treatment for TBI. TBI is a complex disease process caused by a cascade of systemic events and attention is now turning to drugs that act on multiple pathways to enhance survival and functional outcomes. Progesterone has been found to be beneficial in several animal species, different models of brain injury, and two preliminary human clinical trials, and holds promise as a treatment for TBI. Progesterone's multiple mechanisms of action may work synergistically to prevent the death of neurons and glia, leading to reduced morbidity and mortality. TBI is heterogeneous, one that varies in clinical presentation, severity, and pathophysiology and the pleiotrophic nature of progesterone, with its multiple functions and mechanisms of action, suggests promise for progesterone as a neuroprotective treatment in TBI. Acknowledgements This work was supported in part by the U.S. Army Institute of Surgical Research and the U.S. Army Medical Research Acquisition Activity (USAMRAA), in part by the National Institute of Neurological Disorders and Stroke–ProTECT III (Progesterone for the Treatment of Traumatic Brain Injury), in part by SCDR Cancer Research Fund, and in part by Virginia Commonwealth University Reanimation Engineering Shock Center (VCURES). We have no interests or affiliation with BHR Pharma. References Adekoya, N., Thurman, D., White, D., Webb, K., 2002. Surveillance for traumatic brain injury deaths—United States, 1989—1998. MMWR 51 (SS10), 1–16. Akwa, Y., Sananès, N., Gouèzou, M., Robel, P., Baulieu, E., Le Goascogne, C., 1993. Astrocytes and neurosteroids: metabolism of pregnenolone and dehydropepiandrosterone Regulation by cell density. J. Cell Biol. 121 (1), 135–143. Amiry-Moghaddam, M., Otsuka, T., Hurn, P., Traystman, R., Haug, F., Froehner, S., Adams, M., Neely, J., Agre, P., Ottersen, O., Bhardwaj, A., 2003. An alpha-syntrophindependent pool of AQP4 in astroglial en-feet confers bidirectional water flow between blood and brain. Proc. Natl Acad. Sci. U S A 100 (4), 2106–2111. Arvin, B., Neville, L., Barone, F., Feuerstein, G., 1996. The role of inflammation and cytokines in brain injury. Neurosci. Biobehav. Rev. 20 (3), 445–452. Asbury, E., Fritts, M., Horton, J., Isaac, W., 1998. Progesterone facilitates the acquisition of avoidance learning and protects against subcortical neuronal death following prefrontal cortex ablation in the rat. Behav. Brain Res. 97 (1–2), 99–106. Azcoitia, I., Sierra, A., Garcia-Segura, L., 1998. Estradiol prevents kainic acid-induced neuronal loss in the rat dentate gyrus. NeuroReport 9 (13), 3075–3079.

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