The Study Of Neuroprotective Effects Of Progesterone And Allopregnanolone In Traumatic Brain Injury And Its Potential Mechanisms Background

Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality in young people for which there is still no effective clinical treatment to counteract the secondary neurodegenerative processes. Recent studies demonstrate that progesterone and some of its metabolites can significantly reduce cerebral edema and enhance functional recovery from TBI and stroke in several animal models.Using the controlled cortical impact (CCI) experimental model of TBI, the extent of edema formation can be reduced by progesterone treatment, and the same group has demonstrated a variable dose range and wide-ranging time scale to achieve this protective effect. In addition to reducing edema, progesterone administration can reduce lesion volume, attenuates free radical damage such as lipid peroxidation, reduce neuronal loss, increase blood-brain barrier integrity and confer cognitive improvements after traumatic brain injury, and improve neurological recovery after experimental spinal cord injury. Progesterone also reduces neuronal cell death and attenuates neurological abnormalities after cerebral ischemia. As a result of these and related findings, progesterone is currently being tested in a phase II clinical trial in humans with moderate to severe, blunt TBI. While these studies further indicate the neuroprotective properties of the steroid, the mechanism(s) by which progesterone achieves these benefits is unclear.Allopregnanolone (ALLO), a progesterone metabolite, is also known to be a potent endogenous positive modulator of central nervous system (CNS) receptor functions. Injections of ALLO reduce memory deficits and stem the loss of cholinergic neurons in rats given bilateral damage to the pre-frontal cortex. As a metabolite of progesterone in the brain, ALLO could be the molecule through which progesterone acts to induce its neuroprotective effects.The mechanism of action of neurosteroids involves two kinds of responses: a rapid one through membrane or surface effects, and a slower one mediated by cytosolic receptors activating traditional genomic expression. The fast steroid responses involve the modulation of neurotransmitter receptor activity, inparticular the type A of GABA, the N-methyl-D-aspartate (NMDA) and the 81 receptors, leading to a decrease of excitotoxicity. It is still unclear which pathway is more crucial for progesterone to bring into being its beneficial effects after brain injury.In this study, we focus on some of the cellular mechanisms mediating the neuroprotective effects of progesterone, aiming to learn more about how functional recovery occurs after this neurosteroid was injected. First, we established a modified Feeney's TBI rat model and observed the effects of progesterone on the morphological and functional recovery. Some indicators related to the pathophysiological process of TBI, including posttraumatic inflammation, free radical damage and cell apoptosis, were also examined. Meanwhile, in order to clarify whether progesterone acts through the classical cytosolic progesterone receptor to exert its neuroprotective effects, we studied the role of specific progesterone receptor antagonist mifepristone in the progesterone-treated TBI models. Subsequently, an in vitro mechanical injury model of cultured rat cortical neurons was set up to investigate the effects of progesterone and its metabolite ALLO on mechanical neuron damage.PART I The study of neuroprotective effects of progesterone in experimental traumatic brain injuryThe aim of this study was to investigate whether progesterone could exert neuroprotective effects on the experimental rat TBI models. Some indicators related to the pathophysiological process of TBI, including posttraumatic inflammation, free radical damage and cell apoptosis, were also examined. Meanwhile, in order to clarify whether progesterone acts through the classical cytosolic progesterone receptor to exert its neuroprotective effects, we studied the role of specific progesterone receptor antagonist mifepristone in the progesterone-treated TBI models.Materials and MethodsA total of 165 adult male Sprague-Dawley rats served as subjects. The rat TBI model was made based on Feeney's method, with some modification. Based on previous studies determining optimal dose response, all progesterone-treated animals received 10mg/kg progesterone in 25% 2-hydroxypropyl-β-cyclodextrin (HBC). The animals received the first dose of either progesterone or an equalvolume of HBC 1h after the injury. Subsequent injections were given at 6h, 12h and 24h after injury.The experimental animals were divided randomly into five groups: sham operate+vehicle(SV), sham operate+progesterone(SP), injury+vehicle(IV), injury+ progesterone(IP) and injury+progesterone+mifepristone(IPM). For IPM group, an injection of mifepristone (25mg/kg) was administrated intraperitoneally 12h prior to the injury. After the trauma impact, mifepristone of the same dose was injected 30min before every injection of progesterone. All of the indicators were examined 48h after the injury.Bcl-2 and Bax protein expression in the injury region of the brain were detected by immunohistochemical analysis (streptavidin-peroxidase method). Apoptotic cells were detected by terminal dUTP nick-end labeling (TUNEL) techniques.48h after the impact, 5 rats of each group were decapitated. Brains were extracted, divided at bregma, and then sagittally bisected. The dorsal half of the right posterior quarter, which included the injured area, was used for reverse transcriptase PCR (RT-PCR) and western blot analysis of the target gene Bcl-2, Bax and GFAP. A semi-quantitative analysis of the PCR products and western blot results were conducted against the house-keeping gene/protein β-actin or GAPDH. In addition, the concentrations of major inflammatory cytokines TNF-α and IL-1β in the injured brain tissue were examined by enzyme-linked immunosorbent assay (ELISA).To investigate the lipid peroxidization level in the injured area, a malondialdehyde (MDA) assay kit was used to examine the thiobarbituric acid reacting substances (TBARS) concentrations. Homogenate SOD activity was determined by a xanthine oxidase system. The total glutathione and glutathione disulfide were measured by a Glutathione Quantification Kit and the level of reduced glutathione was calculated consequently.48h after the injury, 2% Evans blue dye (4 ml/kg) was injected into the caudal vein, 1h later, after decapitation, the Evans blue content in brain tissue was measured to evaluate the blood-brain barrier permeability compromise. Brain water content was determined by the dry-wet weight method. A battery of behavioral tests was performed before and at 24h, 7, 14 and 21d after the injurybased on the modified Neurological Severity Score (mNSS). The mNSS is a composite of motor, sensory, reflex, and balance tests, graded on a scale of 0 to 18.ResultsIn HE staining, normal morphology was observed in the brain sections of sham-operates (SV and SP groups). The sections of injured rats receiving vehicle (IV group) showed that neurons and glial were shrunken, and extensive neuronal loss and neutrophil infiltration were evident 48 h after traumatic brain injury. In the sections of injured rats treated with progesterone (IP and IPM group), the level of neural damage was to a certain extent attenuated, compared to those of IV group.Immunohistochemical analysis revealed that Bcl-2 protein was slightly expressed in the sham operate groups (7.19±1.07% for SV and 6.02±1.21% for SP). The positive percentages were 6.34±1.21% for IV group, 4.12±1.66% for IP group and 6.28±2.02% for IPM group. Bax protein was not detected in the non-injured control group, while it reached a relatively high level in the injury region 48 after the trauma (9.65±2.15%). Progesterone/mifepristone administration didn't alter the expression level of Bax.Only a small amount of TUNEL labeled cells were detected in the sham-operate groups (1.36±1.03% for SV, 0.44±0.20% for SP). After TBI, the labeling index increased significantly (12.21 ±2.00% for IV). Progesterone and mifepristone administration didn't alter the TUNEL labeling intensity (9.98±1.67% for IP, 11.23±2.89% for IPM) to a statistical significance.Semi-quantitative RT-PCR and western blot analysis revealed that the mRNA and protein of GFAP were moderately expressed in the sham-operate groups. After TBI, the GFAP level increased significantly and was not influenced by progesterone (and mifepristone) injection. No obvious difference was found among the transcription and expression level of Bcl-2 in the five groups. In the sham-operate groups, mRNA and protein expression of Bax were not detected. 48h after the injury, the transcription and expression level of Bax increased significantly. Progesterone and mifepristone injection could not inhibit the increase of Bax expression.The inflammatory cytokines IL-1β and TNF-α remained at a low level in sham-operate animals. When the brain trauma occurred, their concentrations increased to a significantly high level. Progesterone could effectively down-regulate the concentrations of these two cytokines, though they still remained higher than normal values. The accompanied administration of mifepristone didn't restrain the inhibitory action of progesterone.48h after TBI, the concentration of TBARS became significantly higher than the sham-operate controls. Injection of progesterone reduced the increase of TBARS considerably, though it was still about 40% higher than the non-injured controls. The accompanied use of mifepristone showed no inhibitory effect on progesterone's action. Progesterone significantly increased the reduced levels of SOD and GSH activity after TBI, and this effect was not hindered by its receptor's antagonist mifepristone.After TBI, compared to the sham-operate controls, the amount of the dye Evan blue in the injured hemisphere increased 4-7 times, suggesting a compromised blood-brain barrier. Unpredictably, injection of 10mg/kg progesterone did not down-regulate the amount of Evan blue in the injured brain. Nevertheless, progesterone administration could reduce the increased water content after TBI from 84.27% to 81.12%. In addition, progesterone significantly decreased the mNSS score at 2 weeks and 3 weeks after TBI. Moreover, mifepristone could not encounter these neuroprotective effects of progesterone.Conclusions(1)Progesterone have neuroprotective actions after brain injury, improve the morphological conditions in the injured area; (2)Progesterone can not reduce cell apoptosis after TBI, neither can it change the transcription and expression level of apoptosis related gene Bcl-2 and Bax; (3)Progesterone can to some extent inhibit the increase of cytokines IL-1β and TNF-α, while it can not change the GFAP level after TBI; (4)Progesterone can increase the reduced levels of SOD and GSH activity after TBI, and therefore inhibit lipid peroxidation. However, it can not lessen the blood-brain barrier compromise; (5)Progesterone can reduce edema formation and promote neurofunctional recovery; (6)The specific progesterone receptor antagonist mifepristone can not inhibit progesterone's neuroprotective effects.PART II The neuroprotective effects of progesterone and allopregnanolone in a mechanically injured model of rat corticalneurons in vitroThe mechanism of secondary brain damage after TBI is very complicated. A variety of issues influence the outcome of TBI, including neutrophil infiltration, activation of different glial cells and the pathological response of cerebral vessels. Most previous studies concerning the neuroprotective effects of progesterone were based on in vivo researches, such as rodent TBI models. In this study, we established an in vitro model of mechanically injured rat cortical neurons and observed the effects of progesterone and its metabolite alloprognanolone (ALLO) on the injured neurons. Moreover, Some indicators related to the pathophysiological process of TBI, including free radical damage and cell apoptosis, were also examined in attempting to clarify the mechanism by which progesterone exert its beneficial effects.Materials and MethodsCortical cells were dissociated and collected from newborn Sprague-Dawley rats and cultured in poly-L-lysine coated 6-well plates. The culture medium was Neurobasal-A containing 2% B27supplement. At the 3rd day, in order to suppress the proliferation of glial cells, cytarabine was added. At the 10th day, immunohistochemical staining with neurofilament-200 (NF-200) and glial fibrillary acidic protein (GFAP) antibody was conducted to identify neurons.After continuous 10 days of primary culture in vitro, mechanical injuries were delivered to the neurons by 20G syringe needle transecting. The experiment subjects consist of 5 groups: (1) non-injured controls (CTRL); (2) vehicle added controls (VEH); (3) mechanically injured neurons (INJ); (4) progesterone added neurons (PROG); and (5)ALLO added neurons (ALLO). In VEH group, no mechanical transection was conducted and 20μl 1% alcohol was added to each well. In INJ group, moderate mechanical transecting was done to the neurons by needles and 20μl 1% alcohol was added to each well. In PROG group, mechanical transecting was conducted and 20μl 1mg/ml progesterone in alcoholsolution was added to each well. In ALLO group, mechanical transecting was conducted and 20μl 0.375mg/ml ALLO in alcohol solution was added to each well. Annexin V-PI flow cytometry analysis of apoptosis was conducted at 24h after the injury, the other examinations were all carried out at 12h after the injury.Trypan blue exclusion test was used to examine the cell viability. Cells were also stained by Hoechst 33342/Propidium Iodide (PI), and then observed under a fluorescence microscope. The degree of injury to neurons was evaluated by the ratio of PI-positive cells to the total cells counted. The nuclear morphology stained by Hoechst was observed to assess the intensity of apoptotic cell death. 24h after the mechanical injury, apoptotic cell death in CTRL, INJ and PROG group was examined with Annexin V-PI labeling and flow cytometry analysis.Using a lactate dehydrogenase (LDH) assay kit, the concentration of LDH in the culture medium and the cells were examined respectively, subsequently the LDH leakage rate was calculated to evaluate the extent of neuron damage.To investigate the lipid peroxidization level in the injured neurons, a malondialdehyde (MDA) assay kit was used to examine the thiobarbituric acid reacting substances (TBARS) concentrations. Homogenate SOD activity was determined by a xanthine oxidase system. The total glutathione and glutathione disulfide were measured by a Glutathione Quantification Kit and the level of reduced glutathione was calculated consequently.12h after the mechanical injury, RT-PCR and western blot analysis were used to examine the transcription and expression level of the apoptosis related gene Bcl-2, Bax. Semi-quantitative analysis of the PCR products and western blot results were conducted against the house-keeping gene/protein β-actin or GAPDH.ResultsAfter 10 days of primary culture, immunohistochemical staining showed that above 85% cells were NF-200 positive, and only a small number of cells were GFAP positive. This suggested that the purity of cultured neurons was qualified enough for further experiment.Mechanical injuries induced degeneration and death of the neurons. Within 2-3 min after the mechanical injury was delivered, many neuronal cell bodiesnear a transection developed pronounced swelling and granularity under phase-contrast microscopy. With time passing by, the swelling occurred not only in neurons immediately adjacent to the transection, but also in those up to 1-2 mm away from the transection. Over the next 24h, many swollen neurons further degenerated, and the axonal processes appeared loose instead of having their normal tight, cabled appearance. More and more neurons displayed membrane blebbing and lysis, cell body shrinkage, chromatin condensation, even necrosis.12h after the mechanical injury, Trypan blue exclusion test showed that the cell survival rate declined to 55.21±8.17%. Progesterone and ALLO increased the cell survival rate to 74.17±8.69% and 70.08±l 1.96% respectively. After the injury, LDH leakage ratio increased significantly to 41.13±8.65%, about 4-6 times higher than the non-injured control groups. Progesterone and ALLO decreased the LDH leakage ratio to 31.22±5.87% and 32.87±4.61% respectively.Double labeling with Hoechst 33342 and PI showed that most cells in CTRL and VEH groups were Hoechst positive, with the nuclei uniformly and hazily emitting blue light, while the red-light emitting dead cells were rare. 12h after the mechanical injury, the percentage of PI positive cells increased significantly in the INJ group (25.1± 11.2%). Among the Hoechst positive cells, some condensed or fragmented nuclei characteristic of apoptosis were observed. Progesterone and ALLO decreased the PI positive ratio to 17.2±8.9% and 19.3±7.8% respectively, while neither of them could change the proportion of apoptotic cells.12h after the mechanical injury, the concentration of TBARS became significantly higher than the non-injured controls. Progesterone reduced the increase of TBARS considerably, though it was still about two times higher than the non-injured controls. ALLO also reduced the concentration of TBARS, with a similar level compared to progesterone. In addition, progesterone and ALLO could significantly increase the reduced levels of SOD and GSH activity after the mechanical injury.Semi-quantitative RT-PCR and western blot analysis revealed no obvious difference among the transcription and expression level of Bcl-2 in the five groups. In the non-injured control groups, mRNA and protein expression of Baxwere not detected. 12h after the injury, the transcription and expression level of Bax increased significantly. Progesterone or ALLO could not inhibit the increase of Bax expression.The flow cytometry analysis with Annexin V-PI labeling revealed that most cells in the non-injured controls were FITC and PI negative. 24h after the injury, FITC+/PI+ cells in the INJ group increased to 31.21 ±4.45%, and FITC+/PI- also increased to 12.7±3.75%, which were significantly higher than that of CTRL group. Progesterone reduced the FITC+/PI+ cells significantly to 24.57±6.43%, while FITC+/PI- still remained at a relatively high level (14.33±6.44%).Conclusions(1)Progesterone and its metabolite ALLO can reduce LDH leakage and increase the cell survival rate after the in vitro cultured neurons are mechanically injured; (2) Progesterone can not reduce cell apoptosis in this in vitro model of mechanically injured neurons, neither can it change the transcription and expression level of apoptosis related gene Bcl-2 and Bax; (3)Progesterone and ALLO can increase the activity of SOD and GSH, and reduce the extent of lipid peroxidation.