Effects Of Progesterone On The Neurogenesis And Neuroregeneration After Cerebral Ischemia In Adult Male Mice And Underlying Mechanisms

INTRODUCTIONRecent studies have demonstrated that progenitor cells in the hippocampal dentate gyrus (DG) retain the ability to proliferate and that a significant number of the daughter cells develop into neurons in adult mammalian species—neurogenesis. Newly formed neurons, similar to established ones, are electrically active and make connections to the hippocampal CA3 field, thus suggesting that they are functional. This is reinforced by evidence that increased survival of newborn neurons is strongly correlated with hippocampus-dependent memory, while the inhibition of neurogenesis has adverse effects on hippocampus-dependent behaviors, thus implying that hippocampal neurogenesis contributes, at least in part, to learning and memory. However, little is known about the factors that regulate the processes of neurogenesis including the proliferation of neural progenitor cells and the survival of newborn cells.Past studies have revealed sex difference in the cell proliferation in the DG only when females are in a high estradiol (E2) state in comparison to that of males. The neurogenesis in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the DG significantly increases during pregnancy, and then declines immediately after parturition. Pregnancy, parturition and postpartum coincide with dramatic fluctuations in the levels of steroid hormones. Evidence indicates that in female adult rodents acute exposure to E2 initially enhances cell proliferation, and in male meadow voles E2 enhances the survival of young neurons when administered during their'axon extension'phase. Intriguingly, P4 is naturally present at a similar level in the brains of men and women. Recent studies confirmed that the P4's metabolites influence the process of neurogenesis. To date, however, the specific effect of P4 on adult hippocampal neurogenesis has not yet been fully elucidated.Many studies have shown that global or focal cerebral ischemia significantly increases the progenitor cell proliferation in the adult brain of gerbils, rats, mice, monkeys as well as humans. The newly proliferated cells might migrate to the damaged areas of the brain, particularly after cerebral ischemia—neuroregeneration. However, most of the newly proliferated DG cells die early, making it unlikely that these cells establish correct synaptic connections.Earlier studies have demonstrated that pre-menopausal women have a lower risk of stroke and a better outcome following stroke compared to men of the same age. Our recent study has reported a potential neuroprotective effect of P4 on ischemic brain injury, where the drug not only prevents ischemia-induced brain injury, but also reverses dysfunctions after cerebral ischemia with a longer therapeutic opportunity. Recent studies confirmed that the P4's metabolites allopregnanolone (ALL) is a potent and highly efficacious proliferative agent in vitro and in vivo in both rodent and human neural stem cells. Our more recent study has provided evidence that P4 when administered during'axon extension'phase enhances the survival of young neurons in male adult mice. The aim of this study was to valuate the effects of P4 on post-ischemic neurogenesis in the DG and the subventricular zone (SVZ) in the adult male mice and to explore the underlying molecular mechanisms. Earlier studies have demonstrated that pre-menopausal women have a lower risk of stroke and a better outcome following stroke compared to men of the same age. Gonadal steroid hormone P4 has also been demonstrated to exert potential neuroprotective effects in ischemic brain injury, where the drug not only prevents ischemia-induced brain injury, but also reverses dysfunctions after cerebral ischemia with a longer therapeutic opportunity. However, whether P4 modulate neuroregeneration after cerebral ischemia have not been to date investigated.OBJECTIVE1. This study investigated whether P4 influences cell proliferation and the survival of newborn neurons in the adult mice dentate gyrus, and if so, whether the P4-induced change in neurogenesis is associated with hippocampus-dependent cognitive behavior.2. The aim of this study was to explore the effects of P4 on neuroregeneration after transient middle cerebral artery occlusion (MCAO) in the subventricular zone (SVZ) and the subgranular zone (SGZ) in the DG of the adult male mice and underlying molecular mechanisms. We further investigated the relation between P4-regulated the survival of newborn neurons and P4-improved cognitive and motor deficits after stroke.METHODSPart-I: (1) Bromodeoxyuridine was used for mitotic labeling. The animals were treated with P4 at 0–2nd day (BrdU-D_0-2) after the first BrdU-injection and were perfused with BrdU-staining agents at 48 hr, 7th day, 28th day and 56th day after the first injection of BrdU to investigate the effects of P4 on cell proliferation and the survival of newborn neurons. (2) To determine the administration time– dependency of P4-effect on the survival of newborn neurons, the treatment with P4 was given at BrdU-D_0-2, BrdU-D_5-7, BrdU-D_10-12 or BrdU-D15-17, and BrdU-staining agents was examined at 28th day after the first BrdU-injection. (3) To examine the effects of P4 on the differation and maturation of newborn neurons, P4 was injected at BrdU-D_0-2 and BrdU/NeuN-staining agents was examined at 28th day after the first injection of BrdU. (4) Finasteride and RU486 were given by intraperitoneal injection (i.p.). U0126, PP2, PP3 and LY294002 were infused into the cereboventricle (i.c.v.) at 30 min before P4-injection. (5) All animals were trained in the standard Morris water maze task at BrdU-D28-32 .Part-II: (1) BrdU was injected at 3rd after 60 min transient middle cerebral artery occlusion (MCAO) in the adult male mice. (2) P4 (4 mg/kg) was administered during 2–4 or 7-9 days post-MCAO. Proliferation of progenitor cell, survival and phenotypic maturation in the DG and the subventricular zone (SVZ) was assessed at 24 hr, 7th day, 14th day, 28th day and 56th day after the end of BrdU-injection. (3) Finasteride and RU486 were given by intraperitoneal injection (i.p.) and U0126 was infused into the cereboventricle (i.c.v.) at 30 min before P4-injection. (4) Morris water maze task were performed at 56-60 days post-MCAO.RESULTSPart-I: (1) The administration of P4 (4 mg/kg) at BrdU-D_0-2 produced an approximately 2-fold increase in the number of 28- and 56-day-old BrdU⁺ cells in comparison to the controls, whereas it did not alter the number of 24/48-hr-old BrdU⁺ cells. (2) P4 increased the number of 28-day-old BrdU⁺ cells in comparison to the controls, when administered at BrdU-D_5-7 but not at BrdU-D_10-12 and BrdU15-17. (3) Androstenedione (Ad), testosterone (TE) or estradiol (E2) at the same-dose of P4, when administered at BrdU-D_0-2, could not replicate the effect of P4, while the inhibition of 5α-reductase by finasteride did not affect the P4-action. (4) The P4R antagonist RU486 partially suppressed the P4-effect, while inhibitors for Src, MEK or PI3K totally suppressed the same effect. (5) the mice treated with P4 at BrdU-D_0-2 showed a decrease in the escape-latency to the hidden-platform in comparison to the control mice; and the P4-treated mice spent more swimming time in the quadrant that previously housed the platform than the control mice did; pre-treatment with RU486 completely blocked the effects of P4 .Part-II (1) The progenitor cell proliferation were increased approximately 2-fold followed by MCAO-mice compared to sham-op, whereas the survival of newly proliferated cells were significantly decreased. (2) The administration of P4 at 2–4 post-MCAO decreased the number of 24-hour old BrdU⁺ cells , which was blocked by the 5α-reductase inhibitor finasteride. (3) By contrast, the administration of P4 at 7-9 days post-MCAO increased 2-fold of the number of 28-day old BrdU⁺ and BrdU⁺/NeuN⁺ cells in a P4 receptor (P4R) and extracellular signal-regulated kinase (ERK)-dependent manner. (4) Furthermore, compared to the MCAO-mice, the treatment with P4 improved the swim speed and decreased the escape-latency to reach the hidden-platform.CONCLUSION1. These findings in part-I suggest that P4 enhances the survival of newborn neurons through P4R and/or the Src-ERK and PI3K pathways independent of its influence on cell proliferation, which is well correlated with the potentiated spatial cognitive function of P4-treated animals.2. These results (see in part-II) indicate that the treatment with P4 after ischemia enhances functional recovery, partly through promoting survival and mature of ischemia-induced newly proliferated cells.The present study contributes to the understanding of P4 as a modulator promoting neurogenesis and neuroregeneration after cerebral ischemia in adult male mice, which may help to clarify the influence and importance of P4 in hippocampus-dependent learning and memory in the aging population, neurodegenerative disease and cerebral injury.