Progesterone treatment normalizes the levels of cell proliferation and cell death in the dentate gyrus of the hippocampus after traumatic brain injury
Research highlights
► Progesterone improves outcome following traumatic brain injury in humans and rodents. ► Traumatic brain injury increased cell proliferation and cell death in hippocampus. ► Progesterone normalized the injury-induced increase in cell proliferation and death. ► Improved cognitive outcome could be related to progesterone effects on neurogenesis.
Introduction
Progesterone, a naturally occurring steroid hormone, is present in both male and female brains. Its actions in the central nervous system are not limited to the control of neuroendocrine regulation and reproduction (Baulieu and Schumacher, 2000, Birzniece et al., 2006); it influences several neural survival functions including neuronal and glial differentiation, hippocampal neurogenesis, and synaptic stability (Ghoumari et al., 2005, Smith et al., 1987, Tsutsui et al., 2004, Zhang et al., 2010a, Zhang et al., 2010b). There are now more than 160 publications showing that progesterone is an effective neuroprotective agent against a number of different insults including blunt and penetrating traumatic brain injury (TBI) (De Nicola et al., 2009, Garcia-Estrada et al., 1999, Labombarda et al., 2010, Schumacher et al., 2007, Stein, 2007, Stein and Sayeed, 2010, Stein and Wright, 2010), diffuse TBI (O'Connor et al., 2007), stroke (Ishrat et al., 2010, Sayeed and Stein, 2009), anoxic brain injury, glutamate toxicity (Ogata et al., 1993), and spinal cord injury (Labombarda et al., 2010) in both males and females. Progesterone exerts its beneficial effects in the central nervous system via multiple pathways that are not always dependent on the activation of the classical intranuclear progesterone receptor (Bottino et al., 2010, Falkenstein et al., 2000, Losel et al., 2003). These effects include, among others, protecting the blood–brain barrier, reducing cerebral edema, decreasing the inflammatory cascade, and decreasing cellular necrosis and apoptosis (Djebaili et al., 2005, Gibson et al., 2005, He et al., 2004, Ishrat et al., 2010, Majewska, 1992, Mani, 2006, Roof et al., 1997, Smith and Woolley, 2004).
Treatment with progesterone following TBI also improves functional outcome. For example, cognitive deficits, a hallmark of TBI, are attenuated following progesterone treatment (Roof et al., 1994). Systemic treatment with progesterone facilitates recovery of spatial learning and memory, which involves the hippocampus, in brain-injured male rats compared to placebo-treated male rats (Djebaili et al., 2004, Roof et al., 1994, Shear et al., 2002). The neuroprotective properties of progesterone in response to TBI are also seen in humans. Two recently completed phase IIa single-center clinical trials found a beneficial effect of progesterone as a treatment in moderate to severe TBI in male and female adults, with fewer mortalities and better functional outcome seen in progesterone-treated patients (Wright et al., 2007, Xiao et al., 2008). These encouraging findings have led to two phase III double-blind randomized clinical trials now enrolling patients (ProTECT III: NCT00822900; SyNAPSe: NCT01143064).
Hippocampal neurons may be the most vulnerable to TBI-induced degeneration and are among the first population of neurons in the brain to die following experimental TBI (Ariza et al., 2006, Kotapka et al., 1991). The dramatic upregulation in apoptotic and necrotic cell death following TBI can contribute to cognitive deficits, and drugs that prevent cell death also reduce cognitive impairment (Sinson et al., 1997).
Progenitor cells in the dentate gyrus of the hippocampus retain the ability to proliferate into neurons during adulthood in most mammalian species studied including humans (Eriksson et al., 1998, Gould et al., 1997, Gould et al., 1999, Gould et al., 2001, Huang and Sato, 1998, Lavenex et al., 2000). Hippocampal neurogenesis consists of at least four processes: cell proliferation, cell migration, cell differentiation, and cell survival. Cell proliferation in the dentate gyrus is the production of new cells from the division of progenitor cells within the subgranular zone. Daughter cells migrate from the subgranular zone to the inner layers of the granule cell layer while undergoing a process of fate determination, differentiating into either neurons or glial cells (Brown et al., 2003, Cameron et al., 1993). These new cells continue to mature over a period of weeks to months resulting in new functionally mature neurons (Esposito et al., 2005, van Praag et al., 2002). Cell survival is the number of new cells that survive to maturity with the majority of these new cells becoming neurons (Cameron et al., 1993).
In addition to its effects on cell death and hippocampus-dependent learning and memory, TBI also affects different aspects of adult hippocampal neurogenesis (Chirumamilla et al., 2002, Gao et al., 2009, Lu et al., 2005, Richardson et al., 2007). Different experimental models of TBI, including lateral fluid percussion and controlled cortical impact (CCI) injury, induce a 3- to 4-fold increase in the number of BrdU-ir cells in the dentate gyrus as early as 2 days postinjury that peaks within the first 7 days following injury (Dash et al., 2001, Emery et al., 2005, Kernie et al., 2001, Lu et al., 2003, Lu et al., 2005, Rice et al., 2003, Sun et al., 2005, Yoshimura et al., 2003) and returns to baseline levels by 35 days post-injury (Kleindienst et al., 2005). However it is important to note that in the majority of these studies cell proliferation was not independently assessed from cell survival as multiple BrdU injections were given and thus the number of BrdU-ir cells were an index of both proliferating cells and the short-term survival of newly-produced cells. The capacity of TBI to increase cell proliferation and subsequent neurogenesis in the dentate gyrus may not necessarily be beneficial and could actually contribute to injury-induced cognitive deficits. Some authors have suggested that too much or too little neurogenesis may interfere with proper functioning of the hippocampus, as predicted by computer modeling (Butz et al., 2008). Indeed, prolonged and continuous seizures lead to an increase in neurogenesis which can then directly contribute to cognitive decline seen after the seizures (Jessberger et al., 2007). Here we offer the hypothesis that the long-term beneficial effects of progesterone on cognitive outcome after TBI are associated with progesterone's influence on modulating potentially pathological levels of neurogenesis.
The aim of the present study was to determine the effects of progesterone treatment on cell death, cell proliferation and immature neuron survival in the hippocampus after TBI. We tested the idea that TBI would increase cell proliferation, the survival of immature neurons and cell death and that progesterone treatment would reduce this TBI-induced augmentation.
Section snippets
Animals
A total of twenty-four adult male Sprague–Dawley rats (8 to 12 weeks; Charles River Laboratories, Wilmington, MA) weighing 275–300 g were used according to procedures approved by the Institutional Animal Care and Use Committee, Emory University, Atlanta, GA, USA (protocol 164–2008). The rats were handled for at least 5 days before surgery, and were individually housed. Food and water were provided ad libitum throughout the experiment and the animals lived in a reversed 12-h light/12-h dark cycle
Brain injury increases cell proliferation compared to sham operations. Progesterone decreases cell proliferation in TBI rats but increases cell proliferation in intact rats
The total number of Ki67-ir cells in the granule cell layer and the hilus for all groups are shown in Fig. 3A. A repeated-measures ANOVA conducted on the total number of Ki67-ir cells in the granule cell layer and the hilus showed a significant interaction between group and region [F(3, 16) = 8.05, p < 0.002]. Post-hoc analysis revealed that the sham group had significantly fewer total Ki67-ir cells in the granule cell layer compared to the sham + progesterone group (p < 0.004) and the TBI group (p <
Discussion
The results from the present study demonstrate that TBI increased cell proliferation and cell death in the dentate gyrus of the adult male rat, while progesterone treatment normalized the injury-induced levels of both cell proliferation and cell death to sham levels. Progesterone's ability to influence cell proliferation was dependent on whether it was administered to a healthy or injured brain. In the present study neither injury nor progesterone treatment influenced short-term cell survival
Conclusions
Severe TBI is often very debilitating and leads to alterations in hippocampal function and structure. Progesterone is an effective neuroprotective agent that improves functional outcome following TBI. The results of our study extend the evidence for the beneficial effects of progesterone following TBI, and show that progesterone administration following contusion reduces the contusion-induced increases in cell proliferation, number of immature neurons, and cell death in the dentate gyrus of
Acknowledgments
We would like to thank Stephanie Lieblich for assistance with this work. Financial support for this research was provided by the Canadian Institutes of Health Research (LAMG) and U01-NSO-62676-02A2 (DGS). CKB and JRE are funded by Michael Smith Foundation for Medical Research senior trainee awards. LAMG is a Michael Smith Senior Scholar. DGS receives research support from BHR Pharma, LLC and Allen & Company.
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