Psychoneuroendocrinology (2015) 56, 1—11
Available online at www.sciencedirect.com
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Correlation of brain levels of progesterone and dehydroepiandrosterone with neurological recovery after traumatic brain injury in female mice Ana Belen Lopez-Rodriguez a,b,∗, Estefania Acaz-Fonseca a, Silvia Giatti c, Donatella Caruso c, Maria-Paz Viveros b, Roberto C. Melcangi c, Luis M. Garcia-Segura a a
Instituto Cajal, CSIC, Avenida Doctor Arce 37, 28002 Madrid, Spain Department of Animal Physiology (Animal Physiology II), Faculty of Biology, Complutense University of Madrid, Madrid, Spain c Department of Pharmacological and Biomolecular Sciences, Center of Excellence on Neurodegenerative Diseases, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy b
Received 25 November 2014; received in revised form 20 February 2015; accepted 24 February 2015
KEYWORDS Tetrahydroprogesterone; 17-Estradiol; Dehydroepiandrosterone; Isopregnanolone; Progesterone; Testosterone
∗
Summary Traumatic brain injury (TBI) is an important cause of disability in humans. Neuroactive steroids, such as progesterone and dehydroepiandrosterone (DHEA), are neuroprotective in TBI models. However in order to design potential neuroprotective strategies based on neuroactive steroids it is important to determine whether its brain levels are altered by TBI. In this study we have used a weight-drop model of TBI in young adult female mice to determine the levels of neuroactive steroids in the brain and plasma at 24 h, 72 h and 2 weeks after injury. We have also analyzed whether the levels of neuroactive steroids after TBI correlated with the neurological score of the animals. TBI caused neurological deficit detectable at 24 and 72 h, which recovered by 2 weeks after injury. Brain levels of progesterone, tetrahydroprogesterone (THP), isopregnanolone and 17-estradiol were decreased 24 h, 72 h and 2 weeks after TBI. DHEA and brain testosterone levels presented a transient decrease at 24 h after lesion. Brain levels of progesterone and DHEA showed a positive correlation with neurological recovery. Plasma
Corresponding author at: Instituto Cajal, CSIC, Avenida Doctor Arce 37, 28002 Madrid, Spain. Tel.: +34 915854730. E-mail address: [email protected] (A.B. Lopez-Rodriguez).
http://dx.doi.org/10.1016/j.psyneuen.2015.02.018 0306-4530/© 2015 Elsevier Ltd. All rights reserved.
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A.B. Lopez-Rodriguez et al. analyses showed that progesterone was decreased 72 h after lesion but, in contrast with brain progesterone, its levels did not correlate with neurological deficit. These findings indicate that TBI alters the levels of neuroactive steroids in the brain with independence of its plasma levels and suggest that the pharmacological increase in the brain of the levels of progesterone and DHEA may result in the improvement of neurological recovery after TBI. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Steroidogenesis is initiated with the transport of cholesterol from the outer to the inner mitochondrial membrane, where P450scc, the enzyme that converts cholesterol in pregnenolone, is located (Papadopoulos and Miller, 2012). The brain is a steroidogenic organ and both neurons and glial cells have steroidogenic activity and express enzymes involved in steroid synthesis and steroid metabolism (Baulieu et al., 2001; Melcangi et al., 2008). The steroids synthesized in the brain are known as neurosteroids (Baulieu et al., 2001). In addition, the brain is a target for hormonal steroids produced in the gonads, the adrenal glands and other peripheral organs (Melcangi et al., 2008). The term neuroactive steroid is used to denominate those steroids that, independently of its central or peripheral origin or whether they are natural or synthetic, are able to modify the activity of neural cells (Melcangi et al., 2008). Recent studies indicate that brain or cerebrospinal fluid levels of neuroactive steroids are altered under different pathological conditions, such as traumatic brain injury (Meffre et al., 2007), diabetes (Pesaresi et al., 2010), experimental Parkinson’s disease (Melcangi et al., 2012), Alzheimer’s pathology (Caruso et al., 2013a), experimental autoimmune encephalomyelitis (Caruso et al., 2010; Giatti et al., 2010) and multiple sclerosis (Caruso et al., 2014). These modifications in the brain levels of neuroactive steroids are not fully reflected by changes in their level in plasma (Meffre et al., 2007; Giatti et al., 2010), indicating that pathological conditions may alter steroid synthesis and metabolism in the brain. The changes in the brain levels of neuroactive steroids under pathological conditions may contribute to the neurological outcome of such conditions, since several neuroactive steroids have neuroprotective activity. One of the neurodegenerative conditions in which the neuroprotective potency of neuroactive steroids has been tested in animal models and clinical trials is traumatic brain injury (TBI), which is the result of a mechanical insult to the brain. The incidence of TBI in humans, which is similar around the world, is approximately of 200 cases per 100,000 inhabitants per year (Bruns and Hauser, 2003; Bondanelli et al., 2005). Most TBI cases (85—89%) are the result of close-head injuries (Masson et al., 2001; Wu et al., 2008) and therefore, animal models of close-head injuries have been developed to imitate the situation in humans. One of such models is the weight-drop model (Cernak, 2005), which reproduces some of the outcomes of TBI in humans and results in body weight loss, neurological alterations, brain edema, neuroinflammation and neurodegeneration (Xiong et al., 2007; Homsi et al., 2009; Lopez-Rodriguez et al., 2015; Siopi et al., 2012b). Some neuroactive steroids, such as progesterone
(Jones et al., 2005; O’Connor et al., 2005; Pascual et al., 2013), have shown to have neuroprotective properties in this TBI model. Since TBI is an acute event, the possibility exists for an intervention in humans during the first hours after the lesion and neuroactive steroids are potential candidates for such an intervention. However, the promising results of phase II clinical trials using progesterone in patients with TBI (Wright et al., 2007; Xiao et al., 2008; Aminmansour et al., 2012) have not been confirmed in phase III clinical trials (Skolnick et al., 2014; Wright et al., 2014), indicating that we need to improve our knowledge on the role of neuroactive steroids on brain injury. In particular, we need more information on the consequences of TBI for the endogenous brain levels of neuroactive steroids. In the present study we have determined the levels of neuroactive steroids in the brain and plasma of young female mice at different times after TBI. We have also analyzed whether the levels of neuroactive steroids after TBI correlated with the neurological score of the animals.
2. Materials and methods 2.1. Animals One of the limitations of the weight-drop model used in the present study is that it has been standardized on the basis of the body weight of the animals. For females, the range of body weight (28—32 g) corresponds to young adult animals, but for males this range of body weight corresponds to peripuberal animals. Given the complexity of the hormonal changes during the peripuberal period, which may affect neuroactive steroid levels and complicate the analysis and the interpretation of the results, we have restricted our study to female animals. Therefore, experiments were performed in 28—32 g (63 days old) Swiss female mice (Harlan, Spain). Animals were housed with controlled temperature (22 ± 2 ◦ C), 12 h light/dark cycle and with free access to food and water. Animal care and procedures were approved by our institutional animal use and care committee and followed the European Parliament and Council Directive (2010/63/EU) and the Spanish regulation (Ley 6/2013, 11th June) on the protection of animals for experimental use. Swiss strain was used because previous studies have characterized the molecular and cellular changes occurring in this mouse strain after TBI and have demonstrated that it reproduces the damages observed in the human brain, such as contusion, axonal damage, brain edema or hemorrhage (Homsi et al., 2009, 2010; Siopi et al., 2012b; Lopez-Rodriguez et al., 2015) as well as the secondary injury mechanisms, such as modifications in metabolic cascades, cell death, excitotoxicity and neuroinflammation (Xiong
Correlation of progesterone and dehydroepiandrosterone with neurological recovery after TBI et al., 2013). A total number of 27 females were included in the study. Naïve animals (n = 6) were left undisturbed. Injured animals were sacrificed at 24 h (n = 7), 72 h (n = 8) and 2 weeks (n = 6) after traumatic brain injury (TBI) by cervical dislocation. Blood was obtained from the trunk at the moment of sacrifice for posterior plasma analyses.
2.2. Body weight control Animals were weighted 24 h before being subjected to TBI model and once again immediately before the sacrifice in order to characterize their general status and well-being of the animals. This parameter is used to describe the severity of the model, taking into account that 5—10% b.w. loss is associated with a moderate lesion, 10—20% b.w. loss is associated with a severe lesion and more than 20% b.w. loss represents and endpoint criteria (Directive 2010/63/EU).
2.3. Estrous cycle monitoring Estrous cycle stage was monitored by analysis of cell types in vaginal smears at the moment of sacrifice. Smears were spread out on gelatinized slides and were observed under optic microscope for cell type identification. Since the majority of the animals were in estrus, only data for animals in this phase of the estrous cycle were included in the study. The N for other phases of the estrous cycle was too small for statistical analysis.
2.4. Traumatic brain injury Prior to the protocol, each animal was randomly assigned to one of the different groups of the study. Mice were anesthetized with 2% isofluorane (IsoFlo, Esteve) before being subjected to TBI. Closed-head trauma was induced by a 50 g weight dropped from a 36 cm height, along a stainless steel rod, on the right frontal side of the head (Lopez-Rodriguez et al., 2015). This experimental paradigm creates a limited contra-coup lesion in the right hemisphere (orbitofrontal cortex and perirhinal cortex), accompanied with functional deficit and a 5—15% mortality rate within the first 5 min following the impact (Homsi et al., 2009, 2010; Siopi et al., 2012a).
2.5. Neurological deficit assessment An 8 points neurological score was used for the assessment of the functional outcome; it was performed 24 h, 72 h and 2 weeks after TBI under blind code. This test is a variation of a previous one which considered 10 essential parameters easy to evaluate, objective in interpretation and independent to the subjective evaluation of the researcher (Flierl et al., 2009; Stahel et al., 2000). The behavioral device consisted on an open circular plastic arena (16 cm height and 30 cm diameter) illuminated 50—50% that contained an exit aperture (2 × 2.5 cm) located in the brighter area. The animal was placed in the darker zone and was allowed to explore freely for 2 min. Table 1 resumes the scoring system; theoretically, naïve animals should score 8 points.
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2.6. Cerebral edema evaluation Previous studies have shown that significant increases in blood brain barrier (BBB) breakdown and edema are also detected in the contralateral hemisphere after TBI (Lin et al., 2012; Lopez-Rodriguez et al., 2015). Therefore, edema was evaluated in the contralateral hemisphere, since it was not possible to assess this parameter in the ipsilateral hemisphere, which was used for steroid analysis. Animals were sacrificed by cervical dislocation and the brain was gently removed. A region of tissue (75—100 mg) from the left hemisphere (3—0 mm from bregma) was punched-out with a cannula of 5 mm inner diameter and immediately weighed in order to obtain the wet weight (WW) and then heated at 100 ◦ C for 24 h. Then, samples were weighed again to obtain the dry weight (DW). BWC was calculated as follows: % H2 O = [(WW − DW)/WW] × 100.
2.7. Quantitative analysis of neuroactive steroids by LC—MS/MS Neuroactive steroid levels were assessed in brain and plasma. A region of tissue (75—100 mg) from the surrounding injured area in the right hemisphere (3—0 mm from bregma) was punched-out with a cannula of 5 mm inner diameter and immediately frozen. Blood was obtained at the moment of sacrifice from the trunk by exsanguination and collected in heparinized tubes. Blood was then centrifuged 15 min, at 3000 rpm and 4 ◦ C to obtain plasma. Extraction and purification of the samples were performed as previously described (Caruso et al., 2013b). Briefly, samples were spiked with 13 C3 -17-E (1 ng/sample), D9 -PROG (0.2 ng/sample) and D4 -PREG (5 ng/sample), as internal standards (IS) and homogenized in MeOH/acetic acid (99:1 v/v) using a tissue lyser (Qiagen, Italy). After an overnight extraction at 4 ◦ C, samples were centrifuged at 12,000 rpm for 5 min and the pellet was extracted twice with 1 ml of MeOH/acetic acid (99:1 v/v). The organic residues were resuspended with 3 ml of MeOH/H2 0 (10:90 v/v) and passed through SPE cartridges; the steroids were eluted in MeOH, concentrated and transferred in autosampler vials before the LC—MS/MS analysis. Quantitative analysis was performed on the basis of calibration curves daily prepared and analyzed as previously described (Caruso et al., 2013b). Linear least-square regression analysis was performed and in addition, a blank (non-spiked sample) and a zero sample (only spiked with IS) were run to demonstrate the absence of interferences at the retention times and m/z corresponding to all the analytes. Moreover, the precision of the assay, inter-assay accuracy, precision and reproducibility are calculated as described in (Caruso et al., 2013b) and are within tolerance range for all the neuroactive steroids. Positive atmospheric pressure chemical ionization (APCI+) experiments were performed with a linear ion trap—mass spectrometer (LTQ, ThermoElectron Co, San Jose, CA, USA) using nitrogen as sheath, auxiliary and sweep gas. The instrument was equipped with a Surveyor liquid chromatography (LC) Pump Plus and a Surveyor Autosampler Plus (ThermoElectron Co, San Jose, CA, USA).
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Neurological Score test for mice. Circle exit task and physiologic parameters.
Task
Description
Points
Circle exit
Exit the device 2 min. The animal performed risk evaluation behaviors like head-dipping or stretched attend posture Exit the device 6).
17-estradiol [F(3,23) = 3.139]. TBI did not significantly affect the levels of pregnenolone and DHT. However the results of power analysis indicated that a higher number of animals would be necessary to detect significant differences. Post-hoc comparisons revealed a significant decrease in the brain levels of these neuroactive steroids at 24 h and/or 72 h after TBI (Figs. 2 and 3). Brain levels of progesterone (p = 0.008), THP (p = 0.001), isopregnanolone (p = 0.020) and 17-estradiol (p = 0.037) remained decreased by 2 weeks after TBI, compared to naïve animals (Figs. 2 and 3). Data from all the groups were pooled and Spearman’s rho was used to identify bivariant associations between brain neuroactive steroid levels and neurological score followed by linear regression test. Spearman’s test showed a significant correlation between neurological scores and the brain levels of progesterone (rho = 0.389, p = 0.50) and DHEA (rho = 0.479, p = 0.015). Linear regression test revealed a positive linear regression: r2 = 0.269, n = 26, p = 0.007, for progesterone (Fig. 2D) and r2 = 0.175, n = 25, p = 0.038, for DHEA (Fig. 3B).
3.5. Plasma neuroactive steroid levels One way ANOVA revealed a significant effect of time after TBI on the plasma levels of progesterone [F(3,17) = 6.820] and 17-estradiol [F(3,20) = 7.895]. Post-hoc comparisons showed a decrease of plasma progesterone levels that was significant at 72 h (p = 0.035) after TBI. Progesterone levels were also decreased at 24 h and 2 weeks compared to controls, but the difference was not statistically significant. Plasma 17-estradiol levels were significantly increased at 24 h (p = 0.009) after TBI. Plasma 17-estradiol levels in injured animals from 24 h group also differed from those sacrificed at 72 h (p = 0.002) and 2 weeks (p = 0.040) after TBI. Spearman’s test did not reveal a significant correlation of plasma neuroactive steroid levels and neurological score (Fig. 4).
4. Discussion In this study we have assessed the changes in neuroactive steroid levels in the brain and plasma after TBI in female
mice. We have used a murine weight-drop model that mimics some human symptoms after TBI. In agreement with previous studies, we have found that this model results in temporary neurological impairment as determined by a significant transient decrease in neurological score (Siopi et al., 2012b; Lopez-Rodriguez et al., 2015). As shown in previous studies (Xiong et al., 2007), TBI resulted also in a transitory decrease in the percentage of body weight change. Both neurological score and the percentage of body weight change were decreased by 24 and 72 h after TBI and recovered to control values by 2 weeks. In contrast with previous results obtained in male mice (Lopez-Rodriguez et al., 2015), we did not detect a significant effect of TBI on brain edema. The reason for this difference in brain edema after TBI in male and female mice is unknown. Previous studies have shown that acute mortality rate after TBI models is lower in female mice (Kupina et al., 2003) and rats (Roof and Hall, 2000) compared to males. In addition, females show in general reduced lesion volumes after TBI than males (Shahrokhi et al., 2010). It is known that the expression of molecules involved in the initiation of steroidogenesis, such as steroidogenic acute regulatory protein (StAR) and translocator protein of 18KD (TSPO), is increased in the nervous tissue after brain injury (Chen and Guilarte, 2008; Lavaque et al., 2006; Mirzatoni et al., 2010; Sierra et al., 2003). Brain injury also increases the expression of steroidogenic enzymes in the brain (Mirzatoni et al., 2010), such as aromatase, which results in increased intracerebral production of 17estradiol, which is neuroprotective (Azcoitia et al., 2001; Arevalo et al., 2015; Zhang et al., 2014). These findings suggest that steroidogenesis is enhanced after brain injury to increase the brain levels of neuroprotective steroids. However, in contrast to our expectations, the brain levels of several neuroprotective steroids, such as progesterone, THP, DHEA and 17-estradiol were decreased, transiently or permanently, after TBI in female mice. The decrease in brain progesterone levels after TBI was particularly a surprising finding since previous studies have shown a significant transient increase in brain progesterone levels after TBI in male rats (Meffre et al., 2007). This may represent sex or species differences that may impact in the outcome of progesterone therapy after TBI. In fact, in contrast to male rats, pseudopregnant female rats did not show significant changes in
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Fig. 2 Effects of TBI on brain levels of pregnenolone (A and B), progesterone (C and D), dihydroprogesterone (E and F), tetrahydroprogesterone (G and H) and isopregnanolone (I and J). (A, C, E, G, I) Brain levels of neuroactive steroids at 24 h, 72 h and 2 weeks after TBI. (B, D, F, H, J) Analysis of correlation between brain levels of neuroactive steroids and neurological score. Data are mean ± SEM. * p < 0.05 versus Naïve group (n > 6).
Correlation of progesterone and dehydroepiandrosterone with neurological recovery after TBI
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Fig. 3 Effects of TBI on brain levels of dehydroepiandrosterone (A and B), testosterone (C and D), dihydrotestosterone (E and F), 17-alpha-estradiol (G and H) and 17-beta-estradiol (I and J). (A, C, E, G, I) Brain levels of neuroactive steroids at 24 h, 72 h and 2 weeks after TBI. (B, D, F, H, J) Analysis of correlation between brain levels of neuroactive steroids and neurological score. Data are mean ± SEM. * p < 0.05 versus Naïve group; # p < 0.05 versus TBI 2 weeks animals (n > 6).
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Fig. 4 Effects of TBI on plasma levels of progesterone (A and B), dehydroepiandrosterone (C and D) and 17-estradiol (E and F). (A, C, E) Plasma levels at 24 h, 72 h and 2 weeks after TBI. (B, D, F) Analysis of correlation between plasma levels and neurological score. Data are mean ± SEM. * p < 0.05 versus Naïve group; # p < 0.05 versus TBI 2 weeks animals; & p < 0.05 versus TBI 72 h animals (n > 4).
the brain levels of progesterone after TBI (Meffre et al., 2007). In our study, the brain levels of pregnenolone, the precursor of progesterone and DHEA and the first steroid produced from cholesterol, were not significantly affected by TBI, suggesting that the decrease in the brain levels of progesterone, DHEA and its neuroprotective metabolites was not due to a decreased steroidogenic activity caused by brain injury. The decrease in brain progesterone levels after TBI may reflect its reduced plasma levels, which in turn may be the consequence of an alteration in the hypothalamo—pituitary—gonadal and the hypothalamo—pituitary—adrenal axes, since pituitary dysfunction is a common consequence of TBI (Lorenzo et al., 2005; Popovic, 2005). This further supports the potential therapeutic value of the treatment with neuroactive steroids, such as progesterone, to prevent the decline on its levels in the brain after TBI. However, the brain levels of the progesterone metabolite THP, which mediates at least part of the neuroprotective actions of progesterone (Ciriza et al., 2004, 2006), were decreased after TBI in the brain, but not in plasma, indicating that changes in
brain levels of this neuroactive steroid after TBI are not due to changes in plasma levels. A similar conclusion can be reached for the progesterone metabolite isopregnanolone, for DHEA and for testosterone, whose levels were decreased in the brain but not in plasma after TBI. A discrepancy between brain and plasma levels after TBI was also observed for 17-estradiol, which was decreased in the brain but was increased in plasma after TBI. Therefore, the changes of the brain levels of some steroids after TBI cannot be explained by changes in their plasma levels. Since the BBB is disrupted after TBI and its activity and functionality is altered (Greve and Zink, 2009; Chodobski et al., 2011; Li et al., 2014), the observed modifications in steroid levels suggest that the brain regulates steroid synthesis and metabolism after TBI with independence of peripheral steroids, as shown in other models of brain pathology (Meffre et al., 2007; Giatti et al., 2010). This opens the question on whether the systemic treatment with neuroactive steroids necessarily results in a parallel increase in these molecules and in its neuroprotective metabolites in the brain. Thus, a complementary approach to the administration of neuroactive steroids for the treatment of TBI could be the use of molecules that
Correlation of progesterone and dehydroepiandrosterone with neurological recovery after TBI enhance brain steroidogenesis, such as TSPO ligands or liver X receptors (LXR) (Mitro et al., 2012). Our present findings, showing that brain levels of progesterone and DHEA positively correlate with neurological score, suggest that the endogenous levels of these neuroactive steroids may contribute to reduce brain damage after TBI. Indeed, numerous studies have shown that progesterone and DHEA elicit neuroprotective mechanisms in the brain (Borowicz et al., 2011; Melcangi et al., 2014) and that progesterone (Jones et al., 2005; O’Connor et al., 2005; Pascual et al., 2013), DHEA sulfate and a DHEA analog (Hoffman et al., 2003; Malik et al., 2003; Juhász-Vedres et al., 2006; Milman et al., 2008) improve functional recovery after TBI and other forms of brain injury. Thus, the positive correlation of brain progesterone and DHEA levels with neurological score may reflect the endogenous protective actions of these steroids.
5. Conclusion Our findings indicate that the levels of several neuroprotective steroids, such as progesterone, DHEA, THP and 17-estradiol, are decreased in the brain of female mice after TBI. Furthermore, the levels of progesterone in plasma were also decreased after TBI. Interestingly, the brain levels of progesterone and DHEA positively correlated with the neurological score, suggesting that the animals that after TBI preserve higher levels of these two neuroprotective steroids in their brain could be better protected against neural damage. These findings further support the potential therapeutic value of the administration of neuroactive steroids, such as progesterone and DHEA, and of molecules that enhance brain steroidogenesis, such as TSPO or LXR ligands, for the treatment of TBI.
Role of the funding source Founding sources did not participate in study design, collection, analyses, interpretation of data, in the writing of the report neither in the decision to submit the article for publication.
Conflict of interest statement Authors declare no conflict of interest.
Acknowledgements We acknowledge financial support from the Ministerio de Economía y Competitividad, Spain to L.M.G.S. (BFU201130217-C03-01) and M.P.V. (BFU2012-38144), Instituto de Salud Carlos III, Redes temáticas de Investigación Cooperativa en Salud, Red de Trastornos Adictivos to M.P.V. (RD2012/0028/0021), GRUPO UCM to M.P.V. (951579) and Fondazione Cariplo to R.C.M. (grant number 2012-0547).
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Correlation of brain levels of progesterone and dehydroepiandrosterone with neurological recovery after traumatic brain injury in female mice Ana Belen Lopez-Rodriguez a,b,∗, Estefania Acaz-Fonseca a, Silvia Giatti c, Donatella Caruso c, Maria-Paz Viveros b, Roberto C. Melcangi c, Luis M. Garcia-Segura a a
Instituto Cajal, CSIC, Avenida Doctor Arce 37, 28002 Madrid, Spain Department of Animal Physiology (Animal Physiology II), Faculty of Biology, Complutense University of Madrid, Madrid, Spain c Department of Pharmacological and Biomolecular Sciences, Center of Excellence on Neurodegenerative Diseases, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy b
Received 25 November 2014; received in revised form 20 February 2015; accepted 24 February 2015
KEYWORDS Tetrahydroprogesterone; 17-Estradiol; Dehydroepiandrosterone; Isopregnanolone; Progesterone; Testosterone
∗
Summary Traumatic brain injury (TBI) is an important cause of disability in humans. Neuroactive steroids, such as progesterone and dehydroepiandrosterone (DHEA), are neuroprotective in TBI models. However in order to design potential neuroprotective strategies based on neuroactive steroids it is important to determine whether its brain levels are altered by TBI. In this study we have used a weight-drop model of TBI in young adult female mice to determine the levels of neuroactive steroids in the brain and plasma at 24 h, 72 h and 2 weeks after injury. We have also analyzed whether the levels of neuroactive steroids after TBI correlated with the neurological score of the animals. TBI caused neurological deficit detectable at 24 and 72 h, which recovered by 2 weeks after injury. Brain levels of progesterone, tetrahydroprogesterone (THP), isopregnanolone and 17-estradiol were decreased 24 h, 72 h and 2 weeks after TBI. DHEA and brain testosterone levels presented a transient decrease at 24 h after lesion. Brain levels of progesterone and DHEA showed a positive correlation with neurological recovery. Plasma
Corresponding author at: Instituto Cajal, CSIC, Avenida Doctor Arce 37, 28002 Madrid, Spain. Tel.: +34 915854730. E-mail address: [email protected] (A.B. Lopez-Rodriguez).
http://dx.doi.org/10.1016/j.psyneuen.2015.02.018 0306-4530/© 2015 Elsevier Ltd. All rights reserved.
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A.B. Lopez-Rodriguez et al. analyses showed that progesterone was decreased 72 h after lesion but, in contrast with brain progesterone, its levels did not correlate with neurological deficit. These findings indicate that TBI alters the levels of neuroactive steroids in the brain with independence of its plasma levels and suggest that the pharmacological increase in the brain of the levels of progesterone and DHEA may result in the improvement of neurological recovery after TBI. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Steroidogenesis is initiated with the transport of cholesterol from the outer to the inner mitochondrial membrane, where P450scc, the enzyme that converts cholesterol in pregnenolone, is located (Papadopoulos and Miller, 2012). The brain is a steroidogenic organ and both neurons and glial cells have steroidogenic activity and express enzymes involved in steroid synthesis and steroid metabolism (Baulieu et al., 2001; Melcangi et al., 2008). The steroids synthesized in the brain are known as neurosteroids (Baulieu et al., 2001). In addition, the brain is a target for hormonal steroids produced in the gonads, the adrenal glands and other peripheral organs (Melcangi et al., 2008). The term neuroactive steroid is used to denominate those steroids that, independently of its central or peripheral origin or whether they are natural or synthetic, are able to modify the activity of neural cells (Melcangi et al., 2008). Recent studies indicate that brain or cerebrospinal fluid levels of neuroactive steroids are altered under different pathological conditions, such as traumatic brain injury (Meffre et al., 2007), diabetes (Pesaresi et al., 2010), experimental Parkinson’s disease (Melcangi et al., 2012), Alzheimer’s pathology (Caruso et al., 2013a), experimental autoimmune encephalomyelitis (Caruso et al., 2010; Giatti et al., 2010) and multiple sclerosis (Caruso et al., 2014). These modifications in the brain levels of neuroactive steroids are not fully reflected by changes in their level in plasma (Meffre et al., 2007; Giatti et al., 2010), indicating that pathological conditions may alter steroid synthesis and metabolism in the brain. The changes in the brain levels of neuroactive steroids under pathological conditions may contribute to the neurological outcome of such conditions, since several neuroactive steroids have neuroprotective activity. One of the neurodegenerative conditions in which the neuroprotective potency of neuroactive steroids has been tested in animal models and clinical trials is traumatic brain injury (TBI), which is the result of a mechanical insult to the brain. The incidence of TBI in humans, which is similar around the world, is approximately of 200 cases per 100,000 inhabitants per year (Bruns and Hauser, 2003; Bondanelli et al., 2005). Most TBI cases (85—89%) are the result of close-head injuries (Masson et al., 2001; Wu et al., 2008) and therefore, animal models of close-head injuries have been developed to imitate the situation in humans. One of such models is the weight-drop model (Cernak, 2005), which reproduces some of the outcomes of TBI in humans and results in body weight loss, neurological alterations, brain edema, neuroinflammation and neurodegeneration (Xiong et al., 2007; Homsi et al., 2009; Lopez-Rodriguez et al., 2015; Siopi et al., 2012b). Some neuroactive steroids, such as progesterone
(Jones et al., 2005; O’Connor et al., 2005; Pascual et al., 2013), have shown to have neuroprotective properties in this TBI model. Since TBI is an acute event, the possibility exists for an intervention in humans during the first hours after the lesion and neuroactive steroids are potential candidates for such an intervention. However, the promising results of phase II clinical trials using progesterone in patients with TBI (Wright et al., 2007; Xiao et al., 2008; Aminmansour et al., 2012) have not been confirmed in phase III clinical trials (Skolnick et al., 2014; Wright et al., 2014), indicating that we need to improve our knowledge on the role of neuroactive steroids on brain injury. In particular, we need more information on the consequences of TBI for the endogenous brain levels of neuroactive steroids. In the present study we have determined the levels of neuroactive steroids in the brain and plasma of young female mice at different times after TBI. We have also analyzed whether the levels of neuroactive steroids after TBI correlated with the neurological score of the animals.
2. Materials and methods 2.1. Animals One of the limitations of the weight-drop model used in the present study is that it has been standardized on the basis of the body weight of the animals. For females, the range of body weight (28—32 g) corresponds to young adult animals, but for males this range of body weight corresponds to peripuberal animals. Given the complexity of the hormonal changes during the peripuberal period, which may affect neuroactive steroid levels and complicate the analysis and the interpretation of the results, we have restricted our study to female animals. Therefore, experiments were performed in 28—32 g (63 days old) Swiss female mice (Harlan, Spain). Animals were housed with controlled temperature (22 ± 2 ◦ C), 12 h light/dark cycle and with free access to food and water. Animal care and procedures were approved by our institutional animal use and care committee and followed the European Parliament and Council Directive (2010/63/EU) and the Spanish regulation (Ley 6/2013, 11th June) on the protection of animals for experimental use. Swiss strain was used because previous studies have characterized the molecular and cellular changes occurring in this mouse strain after TBI and have demonstrated that it reproduces the damages observed in the human brain, such as contusion, axonal damage, brain edema or hemorrhage (Homsi et al., 2009, 2010; Siopi et al., 2012b; Lopez-Rodriguez et al., 2015) as well as the secondary injury mechanisms, such as modifications in metabolic cascades, cell death, excitotoxicity and neuroinflammation (Xiong
Correlation of progesterone and dehydroepiandrosterone with neurological recovery after TBI et al., 2013). A total number of 27 females were included in the study. Naïve animals (n = 6) were left undisturbed. Injured animals were sacrificed at 24 h (n = 7), 72 h (n = 8) and 2 weeks (n = 6) after traumatic brain injury (TBI) by cervical dislocation. Blood was obtained from the trunk at the moment of sacrifice for posterior plasma analyses.
2.2. Body weight control Animals were weighted 24 h before being subjected to TBI model and once again immediately before the sacrifice in order to characterize their general status and well-being of the animals. This parameter is used to describe the severity of the model, taking into account that 5—10% b.w. loss is associated with a moderate lesion, 10—20% b.w. loss is associated with a severe lesion and more than 20% b.w. loss represents and endpoint criteria (Directive 2010/63/EU).
2.3. Estrous cycle monitoring Estrous cycle stage was monitored by analysis of cell types in vaginal smears at the moment of sacrifice. Smears were spread out on gelatinized slides and were observed under optic microscope for cell type identification. Since the majority of the animals were in estrus, only data for animals in this phase of the estrous cycle were included in the study. The N for other phases of the estrous cycle was too small for statistical analysis.
2.4. Traumatic brain injury Prior to the protocol, each animal was randomly assigned to one of the different groups of the study. Mice were anesthetized with 2% isofluorane (IsoFlo, Esteve) before being subjected to TBI. Closed-head trauma was induced by a 50 g weight dropped from a 36 cm height, along a stainless steel rod, on the right frontal side of the head (Lopez-Rodriguez et al., 2015). This experimental paradigm creates a limited contra-coup lesion in the right hemisphere (orbitofrontal cortex and perirhinal cortex), accompanied with functional deficit and a 5—15% mortality rate within the first 5 min following the impact (Homsi et al., 2009, 2010; Siopi et al., 2012a).
2.5. Neurological deficit assessment An 8 points neurological score was used for the assessment of the functional outcome; it was performed 24 h, 72 h and 2 weeks after TBI under blind code. This test is a variation of a previous one which considered 10 essential parameters easy to evaluate, objective in interpretation and independent to the subjective evaluation of the researcher (Flierl et al., 2009; Stahel et al., 2000). The behavioral device consisted on an open circular plastic arena (16 cm height and 30 cm diameter) illuminated 50—50% that contained an exit aperture (2 × 2.5 cm) located in the brighter area. The animal was placed in the darker zone and was allowed to explore freely for 2 min. Table 1 resumes the scoring system; theoretically, naïve animals should score 8 points.
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2.6. Cerebral edema evaluation Previous studies have shown that significant increases in blood brain barrier (BBB) breakdown and edema are also detected in the contralateral hemisphere after TBI (Lin et al., 2012; Lopez-Rodriguez et al., 2015). Therefore, edema was evaluated in the contralateral hemisphere, since it was not possible to assess this parameter in the ipsilateral hemisphere, which was used for steroid analysis. Animals were sacrificed by cervical dislocation and the brain was gently removed. A region of tissue (75—100 mg) from the left hemisphere (3—0 mm from bregma) was punched-out with a cannula of 5 mm inner diameter and immediately weighed in order to obtain the wet weight (WW) and then heated at 100 ◦ C for 24 h. Then, samples were weighed again to obtain the dry weight (DW). BWC was calculated as follows: % H2 O = [(WW − DW)/WW] × 100.
2.7. Quantitative analysis of neuroactive steroids by LC—MS/MS Neuroactive steroid levels were assessed in brain and plasma. A region of tissue (75—100 mg) from the surrounding injured area in the right hemisphere (3—0 mm from bregma) was punched-out with a cannula of 5 mm inner diameter and immediately frozen. Blood was obtained at the moment of sacrifice from the trunk by exsanguination and collected in heparinized tubes. Blood was then centrifuged 15 min, at 3000 rpm and 4 ◦ C to obtain plasma. Extraction and purification of the samples were performed as previously described (Caruso et al., 2013b). Briefly, samples were spiked with 13 C3 -17-E (1 ng/sample), D9 -PROG (0.2 ng/sample) and D4 -PREG (5 ng/sample), as internal standards (IS) and homogenized in MeOH/acetic acid (99:1 v/v) using a tissue lyser (Qiagen, Italy). After an overnight extraction at 4 ◦ C, samples were centrifuged at 12,000 rpm for 5 min and the pellet was extracted twice with 1 ml of MeOH/acetic acid (99:1 v/v). The organic residues were resuspended with 3 ml of MeOH/H2 0 (10:90 v/v) and passed through SPE cartridges; the steroids were eluted in MeOH, concentrated and transferred in autosampler vials before the LC—MS/MS analysis. Quantitative analysis was performed on the basis of calibration curves daily prepared and analyzed as previously described (Caruso et al., 2013b). Linear least-square regression analysis was performed and in addition, a blank (non-spiked sample) and a zero sample (only spiked with IS) were run to demonstrate the absence of interferences at the retention times and m/z corresponding to all the analytes. Moreover, the precision of the assay, inter-assay accuracy, precision and reproducibility are calculated as described in (Caruso et al., 2013b) and are within tolerance range for all the neuroactive steroids. Positive atmospheric pressure chemical ionization (APCI+) experiments were performed with a linear ion trap—mass spectrometer (LTQ, ThermoElectron Co, San Jose, CA, USA) using nitrogen as sheath, auxiliary and sweep gas. The instrument was equipped with a Surveyor liquid chromatography (LC) Pump Plus and a Surveyor Autosampler Plus (ThermoElectron Co, San Jose, CA, USA).
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A.B. Lopez-Rodriguez et al. Table 1
Neurological Score test for mice. Circle exit task and physiologic parameters.
Task
Description
Points
Circle exit
Exit the device 2 min. The animal performed risk evaluation behaviors like head-dipping or stretched attend posture Exit the device 6).
17-estradiol [F(3,23) = 3.139]. TBI did not significantly affect the levels of pregnenolone and DHT. However the results of power analysis indicated that a higher number of animals would be necessary to detect significant differences. Post-hoc comparisons revealed a significant decrease in the brain levels of these neuroactive steroids at 24 h and/or 72 h after TBI (Figs. 2 and 3). Brain levels of progesterone (p = 0.008), THP (p = 0.001), isopregnanolone (p = 0.020) and 17-estradiol (p = 0.037) remained decreased by 2 weeks after TBI, compared to naïve animals (Figs. 2 and 3). Data from all the groups were pooled and Spearman’s rho was used to identify bivariant associations between brain neuroactive steroid levels and neurological score followed by linear regression test. Spearman’s test showed a significant correlation between neurological scores and the brain levels of progesterone (rho = 0.389, p = 0.50) and DHEA (rho = 0.479, p = 0.015). Linear regression test revealed a positive linear regression: r2 = 0.269, n = 26, p = 0.007, for progesterone (Fig. 2D) and r2 = 0.175, n = 25, p = 0.038, for DHEA (Fig. 3B).
3.5. Plasma neuroactive steroid levels One way ANOVA revealed a significant effect of time after TBI on the plasma levels of progesterone [F(3,17) = 6.820] and 17-estradiol [F(3,20) = 7.895]. Post-hoc comparisons showed a decrease of plasma progesterone levels that was significant at 72 h (p = 0.035) after TBI. Progesterone levels were also decreased at 24 h and 2 weeks compared to controls, but the difference was not statistically significant. Plasma 17-estradiol levels were significantly increased at 24 h (p = 0.009) after TBI. Plasma 17-estradiol levels in injured animals from 24 h group also differed from those sacrificed at 72 h (p = 0.002) and 2 weeks (p = 0.040) after TBI. Spearman’s test did not reveal a significant correlation of plasma neuroactive steroid levels and neurological score (Fig. 4).
4. Discussion In this study we have assessed the changes in neuroactive steroid levels in the brain and plasma after TBI in female
mice. We have used a murine weight-drop model that mimics some human symptoms after TBI. In agreement with previous studies, we have found that this model results in temporary neurological impairment as determined by a significant transient decrease in neurological score (Siopi et al., 2012b; Lopez-Rodriguez et al., 2015). As shown in previous studies (Xiong et al., 2007), TBI resulted also in a transitory decrease in the percentage of body weight change. Both neurological score and the percentage of body weight change were decreased by 24 and 72 h after TBI and recovered to control values by 2 weeks. In contrast with previous results obtained in male mice (Lopez-Rodriguez et al., 2015), we did not detect a significant effect of TBI on brain edema. The reason for this difference in brain edema after TBI in male and female mice is unknown. Previous studies have shown that acute mortality rate after TBI models is lower in female mice (Kupina et al., 2003) and rats (Roof and Hall, 2000) compared to males. In addition, females show in general reduced lesion volumes after TBI than males (Shahrokhi et al., 2010). It is known that the expression of molecules involved in the initiation of steroidogenesis, such as steroidogenic acute regulatory protein (StAR) and translocator protein of 18KD (TSPO), is increased in the nervous tissue after brain injury (Chen and Guilarte, 2008; Lavaque et al., 2006; Mirzatoni et al., 2010; Sierra et al., 2003). Brain injury also increases the expression of steroidogenic enzymes in the brain (Mirzatoni et al., 2010), such as aromatase, which results in increased intracerebral production of 17estradiol, which is neuroprotective (Azcoitia et al., 2001; Arevalo et al., 2015; Zhang et al., 2014). These findings suggest that steroidogenesis is enhanced after brain injury to increase the brain levels of neuroprotective steroids. However, in contrast to our expectations, the brain levels of several neuroprotective steroids, such as progesterone, THP, DHEA and 17-estradiol were decreased, transiently or permanently, after TBI in female mice. The decrease in brain progesterone levels after TBI was particularly a surprising finding since previous studies have shown a significant transient increase in brain progesterone levels after TBI in male rats (Meffre et al., 2007). This may represent sex or species differences that may impact in the outcome of progesterone therapy after TBI. In fact, in contrast to male rats, pseudopregnant female rats did not show significant changes in
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A.B. Lopez-Rodriguez et al.
Fig. 2 Effects of TBI on brain levels of pregnenolone (A and B), progesterone (C and D), dihydroprogesterone (E and F), tetrahydroprogesterone (G and H) and isopregnanolone (I and J). (A, C, E, G, I) Brain levels of neuroactive steroids at 24 h, 72 h and 2 weeks after TBI. (B, D, F, H, J) Analysis of correlation between brain levels of neuroactive steroids and neurological score. Data are mean ± SEM. * p < 0.05 versus Naïve group (n > 6).
Correlation of progesterone and dehydroepiandrosterone with neurological recovery after TBI
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Fig. 3 Effects of TBI on brain levels of dehydroepiandrosterone (A and B), testosterone (C and D), dihydrotestosterone (E and F), 17-alpha-estradiol (G and H) and 17-beta-estradiol (I and J). (A, C, E, G, I) Brain levels of neuroactive steroids at 24 h, 72 h and 2 weeks after TBI. (B, D, F, H, J) Analysis of correlation between brain levels of neuroactive steroids and neurological score. Data are mean ± SEM. * p < 0.05 versus Naïve group; # p < 0.05 versus TBI 2 weeks animals (n > 6).
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A.B. Lopez-Rodriguez et al.
Fig. 4 Effects of TBI on plasma levels of progesterone (A and B), dehydroepiandrosterone (C and D) and 17-estradiol (E and F). (A, C, E) Plasma levels at 24 h, 72 h and 2 weeks after TBI. (B, D, F) Analysis of correlation between plasma levels and neurological score. Data are mean ± SEM. * p < 0.05 versus Naïve group; # p < 0.05 versus TBI 2 weeks animals; & p < 0.05 versus TBI 72 h animals (n > 4).
the brain levels of progesterone after TBI (Meffre et al., 2007). In our study, the brain levels of pregnenolone, the precursor of progesterone and DHEA and the first steroid produced from cholesterol, were not significantly affected by TBI, suggesting that the decrease in the brain levels of progesterone, DHEA and its neuroprotective metabolites was not due to a decreased steroidogenic activity caused by brain injury. The decrease in brain progesterone levels after TBI may reflect its reduced plasma levels, which in turn may be the consequence of an alteration in the hypothalamo—pituitary—gonadal and the hypothalamo—pituitary—adrenal axes, since pituitary dysfunction is a common consequence of TBI (Lorenzo et al., 2005; Popovic, 2005). This further supports the potential therapeutic value of the treatment with neuroactive steroids, such as progesterone, to prevent the decline on its levels in the brain after TBI. However, the brain levels of the progesterone metabolite THP, which mediates at least part of the neuroprotective actions of progesterone (Ciriza et al., 2004, 2006), were decreased after TBI in the brain, but not in plasma, indicating that changes in
brain levels of this neuroactive steroid after TBI are not due to changes in plasma levels. A similar conclusion can be reached for the progesterone metabolite isopregnanolone, for DHEA and for testosterone, whose levels were decreased in the brain but not in plasma after TBI. A discrepancy between brain and plasma levels after TBI was also observed for 17-estradiol, which was decreased in the brain but was increased in plasma after TBI. Therefore, the changes of the brain levels of some steroids after TBI cannot be explained by changes in their plasma levels. Since the BBB is disrupted after TBI and its activity and functionality is altered (Greve and Zink, 2009; Chodobski et al., 2011; Li et al., 2014), the observed modifications in steroid levels suggest that the brain regulates steroid synthesis and metabolism after TBI with independence of peripheral steroids, as shown in other models of brain pathology (Meffre et al., 2007; Giatti et al., 2010). This opens the question on whether the systemic treatment with neuroactive steroids necessarily results in a parallel increase in these molecules and in its neuroprotective metabolites in the brain. Thus, a complementary approach to the administration of neuroactive steroids for the treatment of TBI could be the use of molecules that
Correlation of progesterone and dehydroepiandrosterone with neurological recovery after TBI enhance brain steroidogenesis, such as TSPO ligands or liver X receptors (LXR) (Mitro et al., 2012). Our present findings, showing that brain levels of progesterone and DHEA positively correlate with neurological score, suggest that the endogenous levels of these neuroactive steroids may contribute to reduce brain damage after TBI. Indeed, numerous studies have shown that progesterone and DHEA elicit neuroprotective mechanisms in the brain (Borowicz et al., 2011; Melcangi et al., 2014) and that progesterone (Jones et al., 2005; O’Connor et al., 2005; Pascual et al., 2013), DHEA sulfate and a DHEA analog (Hoffman et al., 2003; Malik et al., 2003; Juhász-Vedres et al., 2006; Milman et al., 2008) improve functional recovery after TBI and other forms of brain injury. Thus, the positive correlation of brain progesterone and DHEA levels with neurological score may reflect the endogenous protective actions of these steroids.
5. Conclusion Our findings indicate that the levels of several neuroprotective steroids, such as progesterone, DHEA, THP and 17-estradiol, are decreased in the brain of female mice after TBI. Furthermore, the levels of progesterone in plasma were also decreased after TBI. Interestingly, the brain levels of progesterone and DHEA positively correlated with the neurological score, suggesting that the animals that after TBI preserve higher levels of these two neuroprotective steroids in their brain could be better protected against neural damage. These findings further support the potential therapeutic value of the administration of neuroactive steroids, such as progesterone and DHEA, and of molecules that enhance brain steroidogenesis, such as TSPO or LXR ligands, for the treatment of TBI.
Role of the funding source Founding sources did not participate in study design, collection, analyses, interpretation of data, in the writing of the report neither in the decision to submit the article for publication.
Conflict of interest statement Authors declare no conflict of interest.
Acknowledgements We acknowledge financial support from the Ministerio de Economía y Competitividad, Spain to L.M.G.S. (BFU201130217-C03-01) and M.P.V. (BFU2012-38144), Instituto de Salud Carlos III, Redes temáticas de Investigación Cooperativa en Salud, Red de Trastornos Adictivos to M.P.V. (RD2012/0028/0021), GRUPO UCM to M.P.V. (951579) and Fondazione Cariplo to R.C.M. (grant number 2012-0547).
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