in vitro study of the long-term cortisol treatment effects on the growth rate and...

In vitro study of the long-term cortisoltreatment effects on the growth rate andproliferation of the neural stem/precursorcells

Alireza Abdanipour1, Mohsen Sagha2, Ali Noori-Zadeh1, Iraj Pakzad3,Taki Tiraihi1

1Shefa Neuroscience Research Center, Khatam Al-Anbia Hospital, Tehran, Iran, 2Department of AnatomicalSciences and Pathology, Research Laboratory for Embryology and Stem Cells, School of Medicine, ArdabilUniversity of Medical Sciences, Ardabil, Iran, 3Department of Microbiology, Faculty of Medicine, Ilam Universityof Medical Sciences, Ilam, Iran

Adult neural stem/precursor cells (NSPCs) residing in the subventricular zone of the lateral ventricles andthe subgranular zone of the dentate gyrus of the hippocampus are involved in the memory formations andpsychological problems. It is believed that basal levels of glucocorticoids are essential for neuronaldevelopment, plasticity, and survival, while stress-mediated levels of glucocorticoids produce neuronalloss. Degeneration of NSPCs by the apoptotic and necrotic stimuli have great devastating outcomes on thebrain and contributes to the pathophysiology of neurological as well as psychological disorders. Using MTTassay, acridine orange, and TUNEL assay, we have demonstrated that cortisol at high and excessive (morethan 5 mM) levels had anti-proliferative effects on the NSPCs derived from subventricular and subgranularzones in a dose- and time-dependent manner through apoptosis as well as necrosis. These outcomes canhighlight the role of stress-mediated decline of adult neurogenesis in the aging brain and interconnectstress-mediated cortisol secretion with brain aging diseases.

Keywords: Cortisol, NSPCs, Adult neurogenesis, Subventricular zone, Dentate gyrus, Neurosphere

IntroductionAdult neurogenesis or the production of new neural

stem/precursor cells (NSPCs) has been shown to

occur in two major regions of the brain including

the subventricular zone of the lateral ventricles and

the subgranular zone of the dentate gyrus of the

hippocampus. In the dentate gyrus, thousands of

granule cells were born daily; however, only a small

portion of the NSPCs survives and develops into

mature granule cells.1 It has been shown that adult

neurogenesis is the key component of the pattern

separation function of the dentate gyrus, and recent

studies also shed light on the significant relationships

between adult neurogenesis and cognition.2–4 The

functional theories of new NSPCs production have

consolidated several aspects of adult neurogenesis

and maturation.5 The immature granule cells demon-

strate an increased intrinsic excitability and plasti-

city,6,7 and interestingly, it is believed that this

immature state of granule cells represents a critical

developmental period in which they encode signifi-

cant features of their environments.8,9 Dentate gyrus

and cornu ammonis 3 interact via the mossy fibers

to process high-resolution spatial information.10–12

Further, the dentate gyrus has been proposed to

mediate spatial pattern separation of similar spaces

for the detection of small changes within an environ-

ment. It is presumed that cornu ammonis 3 mediates

a similar pattern separation process on much larger

scale spaces,11 and they may contribute to neurolo-

gical problems such as Alzheimer’s disease (AD).

Moreover, NSPCs located within the subventricular

zone exert an innate homeostatic regulatory function

protecting striatal neurons from glutamate-mediated

excitotoxicity13, and thus adult neurogenesis may

potentially play a crucial role in Huntington’s disease

(HD) as well, in which loss of the striatal projection

neurons occurs.14 Moreover, striatum is the main

input nucleus of the basal ganglia and a key neural

substrate for procedural learning and memory.15 The

vast majority of the striatal neurons are GABAergic

Correspondence to: Alireza Abdanipour, Shefa Neuroscience ResearchCenter, Khatam Al-Anbia Hospital, Tehran, Iran. Email: [email protected]

� W. S. Maney & Son Ltd 2014DOI 10.1179/1743132814Y.0000000431 Neurological Research 2014 VOL. 000 NO. 000 1

projection neurons, which exhibit slow but, tempo-

rally precise spiking. In Parkinson’s disease (PD), a

broad spectrum of non-motor symptoms and deficits

exist and they may partly rely on hippocampal

function and indeed it is assumed that some of them

are related to defective adult neurogenesis.16 In a

general sense, according to the roles of NSPCs in the

adult brain functions, any disturbances in the adult

brain ability to regenerate NSPCs may contribute to

the pathophysiological conditions, and it is believed,

however, that adult neurogenesis steadily declines

during brain aging causing diseases such as AD, PD,

HD, innate anxiety, major depressive disorder (MDD),

etc. The progressive decline of NSPCs proliferation

during aging raises the questions of whether the

precursor cells become unresponsive to cellular niche

cues or whether the cellular niche does not provide the

stimuli for evoking proliferation or a niche factor itself

is already dampening this process. However, adult

neurogenesis is regulated by a variety of factors such as

learning,17 exercise,18 seizures,19 age,20,21 and stress.22

It has been shown that in response to stress, cortisol

secretion from the zona faciculata of the adrenal gland

at high concentrations suppresses the proliferation of

the neurons.23,24 We hypothesized that cortisol secre-

tion at high concentrations may suppress the prolifera-

tion of NSPCs, and this phenomenon can contribute to

the brain aging during adulthood and senescence. In

this regard, we have evaluated the chronological effects

of cortisol application in the cell culture medium at

different concentrations on the apoptosis and necrosis

rate of NSPCs using MTT and TUNEL assays as well

as acridine orange staining. We found that long-term

treatments of NSPCs by high and excessive levels of

cortisol decreased the viability and proliferation rate of

NSPCs. Also, the number of apoptotic and necrotic

cells increased in a dose- and time-dependent manner.

These outcomes can highlight the role of stress-

mediated decline of adult neurogenesis in the aging

brain and interconnect stress-mediated cortisol secre-

tion with the brain pathophysiology.

MethodsIsolation of NSPCs and neurosphere formationNeural stem/precursor cells were isolated from sub-

granular and subventricular zones of the rats (8–

12 weeks old) under deep ketamine/xylazine anesthesia.

The brains were removed quickly and washed in the ice-

cold phosphate buffered saline (PBS) supplemented

with 4.5 g/L glucose. The dissected hippocampi and

subventricular zones were washed with a 10 mL PBS/

glucose solution and then centrifuged down for

5 minutes at 1600 g and 4uC. After removal of the

supernatant, the tissues were homogenized with scissors

and scalpels. The collected tissues were dissociated by

digestion with a mixture of papain (Sigma), dispase II

(Sigma) and accutase (Invitrogen). The tissues were

incubated for 30 minutes at room temperature (RT)

and the solution was mixed gently every 10 minutes.

The cell mixture was passed through a 70 mm cell

strainer (Falcon) and then, centrifuged for 5 minutes, at

800 g and 4uC. Following the centrifugation, the pellets

were resuspended in a 1 mL Dulbecco modified eagle

medium/F12 medium (DMEM/F12; Invitrogen) sup-

plemented with B27 (Gibco), 100 units/mL penicillin,

and 100 units/mL streptomycin; 20 ng/mL epidermal

growth factor (EGF, Sigma); and 20 ng/mL fibroblast

growth Factor-2 (bFGF, Millipore).25 The isolated cells

were seeded in a six-well non-adherent plate and then

incubated at 37uC in 5% CO2 to form neurospheres for

1–2 weeks. For the preparation of a single-cell layer

from the neurospheres, they were centrifuged for

5 minutes, at 300 g and 4uC. Then, the cells were

washed in the basic media and dissociated with 0.02%

PBS-EDTA and then expanded on the poly-L-lysine

(Sigma) coated six-well chambers and passaged up to

three times.

Immunofluorescence stainingThe NSPCs were seeded on the poly-L-lysine (Sigma)-

coated cover slips and cultured for 4 hours at 37uC.

The cells were fixed (4% paraformaldehyde, 20 min-

utes, RT), washed with PBS, and then permeabilized

(0.2% Triton X-100, 10 minutes, RT). Subsequently,

they were treated with the blocking buffer (PBS with

0.1% goat serum; 30 minutes, RT) before the incuba-

tion with the primary antibodies. Then, the cells were

incubated overnight at 4uC with the primary polyclonal

antibodies against rat nestin (1 : 100; Santa Cruz) and

Sox2 (1 : 200; Santa Cruz) proteins, separately. After

washing with PBS, 60 minutes at RT, incubations with

the species-relevant fluorescent-labeled secondary anti-

bodies were followed. The nuclei were counterstained

with ethidium bromide, and the images were obtained

using a fluorescence microscope. For negative controls,

the immunocytochemistry was performed as for the

samples with the except that the primary antibody was

omitted. For positive controls, neonatal rat dentate

gyrus was used for the Sox2 and nestin expression.

MTT assay and cell viability evaluationSubconfluent cultures of the NSPCs at the third

passage were seeded on the 96-well microtiter plates

and maintained in the DMEM/F12 supplemented with

B27, 20 ng/mL EGF, 20 ng/mL bFGF, and 3% FBS

for 24 hours. To determine the cell viability at various

concentrations of the cortisol, the NSPCs were treated

with cortisol prepared in water (hydrocortisone-water

soluble, synonym: cyclodextrin-encapsulated hydro-

cortisone, H0396, Sigma) at 0, 0.25, 0.5, 1, 2.5, 5, 10,

15, and 20 mM concentrations at different exposure

time courses (24, 48, 72, 96, and 120 hours) and

then, cortisol toxicity were evaluated using MTT

Abdanipour et al. Long-term cortisol treatment effects

2 Neurological Research 2014 VOL. 000 NO. 000

(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium

bromide) assay.26 Briefly after treatment, the medium

was removed and replaced with 200 mL of 0.5 mg/mL

MTT in 10% FBS-containing medium, and the cells

were incubated in the CO2 incubator at 37uC for

4 hours. The supernatants were removed from the

wells, and the reduced MTT dye was solubilized in the

200 mL/well of the DMSO. Absorbance at 570 nm was

determined by a plate reader, and the percentage of the

relative cell viability was calculated as follows: A570 of

the treated samples/A570 of the untreated samples 6100.

Acridine orange/ethidium bromide stainingCell morphological changes in the necrosis were

assessed by acridine orange/ethidium bromide staining.

The NSPCs were treated with cortisol at 5, 10, 15, and

20 mM concentrations at each aforementioned exposure

time courses. The cells were washed with the PBS buffer,

and fixed with 4% paraformaldehyde for 15 minutes

and then, stained with acridine orange/ethidium bro-

mide (Sigma), 100 mg/mL of each, for 5 minutes at

RT.27 Two hundred of the cells were counted and the

number of the necrotic cells was assessed as their

cytoplasm had been stained orange/yellow, and also

had a nuclear morphology resembling that of the

viable cells without any condensed chromatin and

fragmented nuclei. The cells were observed using a

fluorescence microscope.

TUNEL assayPotential DNA fragmentation and thus apoptosis was

visualized with a fluorescent staining method using In

Situ Cell Death Detection Kit (Roche, Germany)

according to the manufacturer’s instructions. After

each exposure time course treatment at 5, 10, 15, and

20 mM concentrations of cortisol, the cells were fixed

with a 4% paraformaldehyde solution prepared in the

PBS buffer for 15 minutes at RT and then permeabi-

lized with a 0.4% Triton X-100 solution for 10 minutes

at RT. Then, the cells were subjected to a terminal

deoxynucleotidyl transferase-mediated deoxyuridine

triphosphate nick end labeling assay (TUNEL assay).

In order to stain the nuclei, DAPI (4’, 6-diamidino-2-

phenylindole) was subsequently added. Hematoxylin

was also used for counterstaining. The percentage of

TUNEL-positive cells was assessed in the five ran-

domly selected fields for each well.28

Statistical analysisThe quantification was performed based on three

independent experiments for the MTT and TUNEL

and acridine orange assays, separately. The results

were normalized with the control groups, which were

presented as a mean percentage ¡ SEM and analyzed

by one-way ANOVA followed by Tukey’s post hoc

multiple group comparison test. A difference between

groups was considered statistically to be significant if

P , 0.05.

ResultsNeurosphere formationThe isolated NSPCs were grown in the appropriate

medium and clusters of the dividing cells formed into

the neurospheres. After 5–6 days of the incubation,

average diameter of the floating neurospheres grew up

to around 120 mm (Fig. 1A). Then, the individual

neurospheres were transferred into a fresh growth

medium containing bFGF and EGF. They formed

new neurospheres within the next 24 hours, eventually

reaching sizes close to that of the primary neu-

rospheres. After 6–10 days in the culture medium,

primary neurospheres were used for further experi-

ments (Fig. 1B). The results of the immunocytochem-

ical staining of the neurospheres showed that the

NSPCs were positive for nestin (98.34 ¡ 2.45) and

Sox2 (90.66 ¡ 3.12) markers (Fig. 1C and D) as we

performed control stainings confirming the specificity

of the labeling (Fig. 1E and F).

MTT assayMTT assay was used to show the toxicity effects of

cortisol on the NSPCs. As shown in Fig. 2, cortisol

caused a significant decline in the cell viability rate of

the NSPCs and the highest inhibition of cell prolifera-

tion was observed at the highest concentration and

longer duration time course of cortisol, i.e. 20 mM for

120 hours (30.16 ¡ 3.42). However, we did not detect

any decline in the cell viability between 0 and 5 mM

concentrations of cortisol at any aforementioned time

courses.

Assessment of necrosisThe results of the necrosis evaluation of the NSPCs in a

dose–response and exposure time courses of corti-

sol were shown in Figs. 3A and 4A. As it has been

demonstrated in these two figures, higher concentration

of cortisol and longer exposure time courses resulted in

a significant increase in the acridine orange/ethidium

bromide-positive cells compared to the control (0 mM).

Assessment of apoptosisAs cortisol concentrations and time exposures were

increased, TUNEL staining results showed that there

were significant increases in the percentage of the

apoptotic cells (shown in Figs. 3B and 4B). TUNEL

staining results showed that there were significant

increases in the percentage of the apoptotic cells as

the cortisol concentration were increased (shown in

Table 1).

DiscussionIn the present study, the stress hormone, cortisol,

significantly decreased the in vitro cell viability and

proliferation of the NSPCs expressing nestin and

Sox2 in a dose- and time-dependent manner through


Neurological Research 2014 VOL. 000 NO. 000 3

two different mechanisms: necrosis and apoptosis. The

first direct evidence for the existence of the NSPCs

with regenerative power came from the culture studies

when brain cells grown in the presence of growth

factors. In such a condition, a small population of

the adult central nervous system (CNS) cells divides

actively to produce large numbers of progeny that

accumulate into so-called neurospheres as we per-

formed for the isolated NSPCs from rat in this study.29

However, in order to generalize the effects of cortisol

Figure 1 Representative photomicrographs of the morphology, differentiation, and proliferation of neurospheres and neural

stem/precursor cells (NSPCs). (A) represents neurospheres after 6 days of culture; (B) represents NSPCs at the third passage;

(C) represents NSPCs stained for nestin with anti-nestin first antibody followed by FITC-conjugated secondary antibody; the

nuclei were counterstained by ethidium bromide. (D) represents NSPCs stained for Sox2 with anti-Sox2 first antibody followed

by FITC-conjugated secondary antibody. (E) and (F) represent positive control stainings for nestin and Sox2, respectively. A–F

magnification: 2006.



(the major glucocorticoid stress hormone in the human)

to the NSPCs, we used cortisol instead of corticosterone

(the major glucocorticoid stress hormone in the rats).

Stressful stimuli invoke cortisol responses by the

hypothalamic–pituitary–adrenal (HPA) axis triggering

cortisol release from the zona faciculata of the adrenal

gland, and hippocampus is the primary target of the

cortisol in the brain.30 Cortisol is a steroid hormone,

which readily crosses the blood–brain barrier, and its

action on the hippocampus is mediated by its interac-

tion with glucocorticoid (GR, low affinity) or miner-

alocorticoid receptors (MR, high affinity). It is believed

that these receptors are necessary for normal cellular

metabolism and crucial for CNS functions, including

memory and learning processes.31 In this research, we

used MTT assay, which measures mitochondrial reduc-

tion potency to produce a colored formazan. Hence, it

is an integrated measure of viability, proliferation, and

Figure 2 MTT assay evaluation of the dose–response and exposure time courses of cortisol at 0, 0.25, 0.5, 1, 2.5, 5, 10, 15, and

20 mM concentrations. Results show the mean% viability (relative to the untreated cells). As the time and the concentration of

cortisol increase, the neural stem/precursor cells’ (NSPCs) viability decreases. However, we did not detect any cortisol cell

toxicity below 5 mM. The bars indicate the mean ¡ SEM from three replicates. ‘*’ represents the significant difference in

comparison with zero concentration (untreated control group). ‘Q’ represents the significant difference in comparison with

120 hours at the same concentration of cortisol (P , 0.05).

Figure 3 TUNEL and acridine orange staining of neural stem/precursor cells (NSPCs) at different concentrations of cortisol

and exposure time courses. The NSPCs were treated with cortisol at 0, 5, 10, 15, and 20 mM concentrations for 24, 48, 72, 96, and

120 hours exposure time courses. (A) represents TUNEL assay of cortisol-treated NSPCs. Cortisol at high concentrations had

an anti-proliferative effect on NSPCs derived from subventricular and subgranular zones in a dose- and time-dependent manner

through apoptosis as the assay shows DNA fragmentations (black arrow). (B) represents acridine orange staining of cortisol-

treated NSPCs. Late necrotic NSPCs display orange red and non-condensed and non-fragmented nucleus (yellow arrow).

Cortisol at high concentrations had a necrosis effect on NSPCs derived from subventricular and subgranular zones in a dose

and time dependent.



Figure 4 Apoptosis and necrosis quantification of different concentrations of cortisol and exposure time courses. (A) Higher

concentrations of cortisol and longer exposure time courses resulted in a significant increase in the necrosis process. (B) As

cortisol concentrations and time exposures were increased, TUNEL staining results also showed that there were significant

increases in the percentage of the apoptotic cells as well. The results represent the percentage of the necrosis ¡ SEM and also

the apoptosis ¡ SEM. ‘*’ represents the significant difference in comparison with 24 hours. ‘a,’ ‘b,’ and ‘c’ represent the

significant difference compared to 0, 5, and 10 mM at the same time, respectively (P , 0.05).

Table 1 The mean percentages of the apoptotic neural stem/precursor cells (NSPCs) (stained with TUNEL assay)

Cortisol concentrations

Exposure time courses

24 hours 48 hours 72 hours 96 hours 120 hours

0 mM 1.14 ¡ 0.44 1.62 ¡ 0.48 2.45 ¡ 0.29 3.14 ¡ 0.3 2.85 ¡ 0.355 mM 2.57 ¡ 1 4.51 ¡ 0.64 4.88 ¡ 0.62 8.82 ¡ 0.77 11.44 ¡ 1.2410 mM 6.81 ¡ 1.41 13.27 ¡ 1.15 17.02 ¡ 1.53 22.32 ¡ 2.78 29.77 ¡ 0.8415 mM 16.11 ¡ 1.58 20.66 ¡ 1.34 18.94 ¡ 1.76 25.89 ¡ 1.92 35.33 ¡ 2.3220 mM 18.26 ¡ 0.94 27.48 ¡ 1.55 40.96 ¡ 1.32 48.2 ¡ 0.92 49.48 ¡ 1.97

The NSPCs were treated with cortisol at 0, 5, 10, 15, and 20 mM concentrations for 24, 48, 72, 96, and 120 hours time courses.



metabolic activity, and thus, it is a reasonable assay for

cell health assessment. However, cortisol concentra-

tions lower than 5 mM did not show any cell toxicity

effects on the NSPCs in comparison with untreated

group (zero concentration) and thus, for cortisol

toxicity evaluation, concentrations higher than 5 mM

were further considered. Although basal levels of

glucocorticoids are essential for neuronal development,

plasticity, and survival, stress-mediated and excessive

levels of glucocorticoids produce NSPCs loss (in the

case of cortisol, higher than 5 mM). We showed that

high and excessive doses of cortisol and longer periods

of treatment had profound inhibitory effects on the

NSPCs viability and proliferation through both of the

necrosis and apoptosis processes. Glucocorticoids are

known to be able to inhibit cell differentiation and

synaptic development in the various brain regions at

high concentrations.32–35 The outstanding result of

cortisol neurotoxicity is brain functionality decline.

Because any decline in the adult neurogenesis may

underlie cognitive impairments associated with the

brain aging and the neurodegenerative disorders. If

stress alters the set point for growth of NSPCs in the

adult brain, it could give credence to many studies that

learning disabilities and even mental illnesses might

have association with cortisol effects on neurogenesis in

the human. However, of the hippocampal structures,

dentate gyrus sensitivity to glucocorticoids is of par-

ticular concern that is associated with learning and

memory formation.36 Indeed, in support of this hypo-

thesis, studies showed that the plasma cortisol levels are

increased in AD.37–40 Corticotrophin-releasing hor-

mone (CRH) hyperdrive occurs when the mRNA levels

of CRH increase in the hypothalamic paraventricular

nucleus (PVN), and this phenomenon causes the

hyperactivity of the HPA axis in AD.41 Also, the

evidence for the primary hyperdrive of CRH involv-

ing MDD comes from multiple sources: biochemical

studies, functional HPA-axis tests, neuroimaging, and

postmortem studies, as well as clinical trials with HPA-

axis-related compounds.42 In this research, glucocorti-

coid toxicity was investigated in the neural stem cells;

however, data from other studies showed that gluco-

corticoids have toxic effects on the adult neurons as

well.23,24 As the hippocampus has a pivotal role in the

negative feedback inhibition of cortisol secretion,43 its

neuronal damage may lead to the inefficient inhibitory

control of the CRH producing cells resulting in the

increased circulating cortisol and further hippocampus

damages44 and NSPCs toxicity as well. This feed-

forward cascade are known as the ‘glucocorticoid

cascade hypothesis’ and was proposed to be the major

pathogenetic mechanism in the human neurodegenera-

tive diseases associated with HPA-axis alterations.45

The subject is more attractive as it has been shown that

the NSPCs of the subventricular zone exert an ‘innate’

homeostatic regulatory role by protecting striatal neu-

rons from glutamate-mediated excitotoxicity.13 More-

over, memory and the HPA-axis dysfunction are

synchronously present in HD, AD, and MDD46 and

also adult neurogenesis in the animal models and

postmortem studies is severely affected.16 We found

that the majority of the NSPCs promoted to necrosis as

well as apoptosis after exposure to high concentrations

of cortisol. Thus, cortisol toxicity may underlie such

brain diseases. Necrosis and apoptosis concomitantly

occurs as these phenomena were detected by TUNEL

and acridine orange assays. Acridine orange can detect

necrotic cells and TUNEL assay also is a sensitive

method for in situ detection of DNA fragmentation in

the apoptotic cells based on labeling of the blunt ends of

DNA breaks. So, cortisol may have the lion’s share in

the degeneration of the NSPCs residing in the brain

albeit at higher than 5 mM concentration. At molecular

level, it has been shown that dexamethasone inhibited

the cell cycle through ubiquitin-mediated degradation

of Cyclin D147 and also it has been proposed that

glucocorticoids endanger hippocampal neurons through

exacerbating the excitotoxic glutamate-calcium-reac-

tive oxygen species cascade.48 Further investigations

are needed to determine the exact mechanisms of

cortisol-mediated NSPCs apoptosis and also necrosis

at molecular levels. Understanding the underlined

mechanisms can pave the way to design new ther-

apeutic drugs targeting brain aging processes.

Disclaimer StatementsContributors Alireza Abdanipour: designer, monitoring

projects, projects, corresponding author; Mohsen

Sagha: cooperative research project; Ali Noori-Zadeh:

cooperative research project, molecular biology advi-

sor; Iraj Pakzad: main advisor; Taki Tiraihi: supervisor.

Funding Shefa Neuroscience Research Center,

Khatam Al-Anbia Hospital, Tehran, Iran.

Conflicts of interest The authors have no conflict of

interest to declare with respect to this manuscript.

Ethics approval

AcknowledgementsWe are grateful to all experts and students of the

Khatam Al-Anbia Hospital for their help to carry out

this project.

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