Due to the increasing cases of neurodegenerative diseases in recent years, the eventual goal of nerve repair is very important. One approach for achieving a neuronal cell induction is by regenerative pharmacology. Nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are neurotrophins that play roles in neuronal development, differentiation, and protection. On the other hand, dehydroepiandrosterone (DHEA) is a neurosteroid which has multiple actions in the nervous system. DHEA could be an important agent in regenerative pharmacology for neuronal differentiation during tissue regeneration. In this study, we investigated the possible role of DHEA to modulate NGF and BDNF production. The
The central nervous system (CNS) is composed of an orchestrated control of cell proliferation, motility and maturation of neuronal and glial cells, axonal growth, neurite outgrowth, and the design of synapses. Neurotrophins are originally identified as important peptides involved in the development of nervous system and could determine neuronal differentiation phenotype. The neurotrophins that influence neural development include nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, and neurotrophin-6 (NT-6) [
DHEA is an adrenal, glial, and neuronal derived steroid. Although DHEA is produced by the human adrenal, it is not produced by the rodent adrenal. It has multiple actions in the nervous system but no specific receptor has been reported for this neurosteroid. DHEA could be an important agent in neuronal differentiation during development [
The successful regeneration of the neurons is dependent on the cells survival and their progenitors proliferation [
This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the Tehran University of Medical Sciences. The protocol was approved by the Institutional Animal Care and Use Committee at the Research Center for Science and Technology in Medicine, Tehran University of Medical Sciences. Fifty-four male Wistar rats (aged between 15 and 45 days) were purchased from Pasteur institute, Tehran, Iran. The animals were housed in the polypropylene cages, three per cage, in a controlled temperature (22°C), under a 12 h light: dark cycle. Food and water were available
DHEA was used in the concentrations of 8 mg/kg daily subcutaneously under anesthesia for 2 weeks. The treatment dose was chosen according to several studies conducted on adult rats. This dose is in the middle of the range found effective in many studies and that might show oversensitivity to the higher doses [
Bromodeoxyuridine (BrdU) incorporation was assessed as described by Pechnick et al., in 2008 [
DHEA basal level in brain was measured using Radioimmunoassay (RIA) kit (Diagnostic Systems Laboratories, USA). One mL of cortex region homogenates was extracted with 1 mL di-ethyl-ether, centrifuged at 300 g for 10 min, and kept for about 15 min at −70°C to freeze. The ether phase was decanted into a new glass tube, evaporated till dryness, and dissolved in 120
The levels of NGF, BDNF, NT-3, and NT-4 were determined using Emax ImmunoAssay System (Promega, USA) as previously described [
Cortical neurons were isolated from cerebral cortex of rats. Isolated cerebral cortex was suspended in neurobasal medium (GIBCO, USA) containing B-27 supplement (Invitrogen, USA). The suspension was placed on poly-D-lysine coated plates. The cultures were kept in the 90% humidity and 10% CO2 atmosphere at 37°C for 14 days and media were half-replaced with fresh media every 3 days.
DHEA was used as a series of concentration (10−4–10−8 mol/mL) in neurobasal medium. Cortical neurons were isolated from cerebral cortex of rats suspended in this treated medium. The cultures were kept in the 90% humidity and 10% CO2 atmosphere at 37°C for 2 weeks as described above. Anti-NGF and anti-BDNF antibodies (each 2
The amount of released neurotrophins was quantified from medium of treated neuron (Promega kit, USA). ELISA was performed according to the manufacturer’s manual. Briefly, the wells of plates were coated with anti-NGF/anti-BDNF mAb diluted with carbonate coating buffer at 4°C for 24 h. After 1 h blockade with blocking buffer, the plates were incubated with standard and culture medium sample for 2 h followed by incubation with anti-rat BDNF/anti-rat NGF polyclonal Ab. After 1 h incubation of Anti-Ig HRP conjugate, the reaction was developed with tetra-methyl-benzidine and the absorbance was read at 450 nm with a plate reader after stopping the reaction with 1 N HCl.
Isolated cortical neurons from treated rats in all groups were plated on 24-well plates (Greiner, Germany). These cells were fixed in 4% paraformaldehyde (PFA in PBS) and washed in PBS. The cells were then incubated in 2 M HCl for 30 min at 37°C. Then they was neutralized with 0.1 M NaOH and washed several times in PBS. Fixed cells were blocked in 5% bovine serum albumin (BSA) with 0.3% Triton X-100 and incubated with anti-MAP-2 (microtubule associated protein 2) antibody (monoclonal 1 : 1000, Chemicon). After incubation with the primary antibodies, FITC conjugated secondary antibody (1 : 500, AbCam, UK) was used to visualize the signal with Ceti immunofluorescence microscopy [
Data are presented as means ± SD. DHEA and BDNF basal levels were analyzed by independent
The basal levels of DHEA (Figure
Mean basal levels of DHEA of 15–30–45-day old Wistar rats (a). Mean basal levels of NGF, BDNF, NT-3, and NT-4 neurotrophins in the brain cortex of 30-day old Wistar rats and Mean levels of neurotrophins after DHEA treatment in the brain cortex of Wistar rats. A comparison between basal levels of NGF, BDNF, NT-3, and NT-4 neurotrophines before and after DHEA treatment in the brain cortex of Wistar rats. The levels of NGF, BDNF, NT-3, and NT-4 neurotrophines were measured using ELISA (b). In each part, the data are obtained from nine animals.
Rats treated with DHEA daily showed increase in neural cells proliferation. BrdU-labeled neurons were found in all treatment groups (Figure
Number of BrdU+ cells. Representative image showing BrdU+ cells in the brain cortex under DHEA treatment and using antineurotrophins after DHEA treatment (a). The majority of BrdU+ cells which survive in brain cortex of Wistar rats (b). In each experiment, the data were obtained from nine animals.
Considering the roles of NGF and BDNF in neurons, we investigated the role of DHEA on the neurite outgrowth in rat cortical neurons. The cultured cortical neurons were treated with DHEA and visualized by a neural cell marker, MAP-2 (Figure
DHEA induced upregulation of NGF and BDNF facilitated the differentiation of rat cortical neurons. Neurons from the DHEA treated rats were immunostained against neuronal marker MAP-2 (a). The number of neurite extending from each neuron is demonstrated. Anti-NGF and anti-BDNF antibodies (1
The rat cortical neuron cultures treated with DHEA maintained for one month in incubator refreshing the media every 3 days. Extending DHEA treatment throughout the 30 d survival period increased the numbers of viable cells (Figure
In this study, we provided evidences that DHEA increased NGF and BDNF production, neuronal cell proliferation, neuronal cell survival, and neurite outgrowth. The microenvironment of the CNS plays a major role in controlling neurogenesis. Levels of neurosteroids in the blood and/or CNS are a significant determinant in the formation of new neuronal cells. Our results add a new point to this map: the neurosteroid DHEA promotes overexpression of NGF and BDNF.
DHEA is an important regulator for the proliferation of neural stem cells [
We showed that DHEA in the absence of NGF and BDNF did have a little effect on neurite outgrowth, but activation of the cells by NGF and BDNF is necessary to the increase of neuronal specific biomarkers (Figure
Despite the Ziegler results that showed that DHEA decreases NGF-induced cell survival and shifts the cells toward a neuroendocrine phenotype [
Activation of cells by NGF and/or BDNF is necessary for DHEA to mediate the neurite outgrowth (Figures
The steroids amount in the blood or brain is sensitive to a variety of external events or intrinsic processes. So, the steroid dependent control is a significant determinant of variations in the differentiation or the formation of new cells. In 2000, Åberg et al. showed that DHEA may activate the expression of growth factors like systemic IGF that are contributing in neurogenesis [
The neurotrophins exert both long-term cell survival effects and short-term effects on neuronal cells. It is confirmed that BDNF promotes long-term effects on neurons [
In a study by Gubba et al., they showed that DHEA (100 nM) upregulated NGF after 3 h, but not at other time points (12, 24, or 48 h). Also they showed that DHEA had no effect on the other neurotrophins in mixed primary cultures [
Another hypothesis that has an important part in our conclusion is that DHEA could bind to the NGF and BDNF receptors and in this way exert these effects. Lazaridis et al. have shown that DHEA binds to the NGF receptor [
DHEA stimulates NGF and BDNF neurotrophins overexpression and release. It enhances neuronal cell survival, neuronal cell proliferation, and neurite outgrowth via these neurotrophins. If our results could be generalized for human, so formation of new cells in the brain via regenerative pharmacology could be important in treating neurodegenerative diseases such as Alzheimer and Parkinson.
The authors have no conflict of interests.
The authors thank the editors and referees for their time.