ORIGINAL ARTICLE

Ginsenoside Rd maintains adult neural stem cell proliferationduring lead-impaired neurogenesis

Bing Wang • Guodong Feng • Chi Tang •

Li Wang • Haoran Cheng • Yunxia Zhang •

Jing Ma • Ming Shi • Gang Zhao

Received: 25 April 2012 / Accepted: 3 October 2012

� Springer-Verlag Italia 2012

Abstract Lead exposure attracts a great deal of public

attention due to its harmful effects on human health. Even

low-level lead (Pb) exposure reduces the capacity for

neurogenesis. It is well known that microglia-mediated

neurotoxicity can impair neurogenesis. Despite this, few

in vivo studies have been conducted to understand the

relationship between acute Pb exposure and microglial

activation. We investigated whether the acute Pb exposure

altered the expression of a marker of activated microglial

cells (Iba-1), and markers of neurogenesis (BrdU and

doublecortin) in aging rats. As compared to controls, Pb

exposure significantly enhanced the expression of Iba-1

immunoreactivity; increased the expression levels of IL-

1b, IL-6, and TNF-a and decreased the numbers of BrdU?

and doublecortin? cells. Our prior work demonstrated that

ginsenoside Rd (Rd), one of the major active ingredients in

Panax ginseng, was neuroprotective in a variety of para-

digms involving anti-inflammatory mechanisms. Thus, we

further examined whether Rd could attenuate Pb-induced

phenotypes. Compared with the Pb exposure group, Rd

pretreatment indeed attenuated the effects of Pb exposure.

These results suggest that Rd may be neuroprotective in old

rats following acute Pb exposure, which involves limitation

of microglial activation and maintenance of NSC

proliferation.

Keywords Ginsenoside Rd � Pb � BrdU � Iba-1 � Rat

Background

Environmental exposure to lead (Pb) in the general popu-

lation is a major public health issue [1]. Lead is still widely

distributed in the environment, and the consequences of

chronic lead exposure in childhood [2, 3] and in juveniles

[4] have been the subject of extensive research during the

past few decades. Previous studies indicate that chronic

exposure to Pb can modify neurogenesis in the adult hip-

pocampus, and that the elderly are more vulnerable to

neurotoxicity from Pb [5]. With increasing environmental

pollution, the chance of lead exposure to the elderly is

rising [6]. Recent advances have improved our under-

standing of how the toxicology of Pb affects the central

nervous system (CNS) [7]. Research characterizing the

Bing Wang, Guodong Feng, Chi Tang, and Li Wang contributed

equally to this work.

B. Wang � G. Feng � Y. Zhang � M. Shi (&) � G. Zhao (&)

Department of Neurology, Xijing Hospital,

The Fourth Military Medical University, No.169,

West Changle Road, Xi’an 710032, China

e-mail: biomidas@163.com

G. Zhao

e-mail: zhaogang@fmmu.edu.cn

B. Wang

The 538 hospital of PLA, Han Zhong, China

C. Tang

Department of Biomedical Engineering,

Fourth Military Medical University, Xi’an, China

L. Wang

The 421 hospital of PLA, Guangzhou, China

H. Cheng

Lin tong Air Force Aeromedical Training Institute,

Xi’an, China

J. Ma

Department of Traditional Chinese Medicine,

Xijing Hospital, The Fourth Military Medical University,

Xi’an, China

123

Neurol Sci

DOI 10.1007/s10072-012-1215-6

neurotoxicity of this metal has shown that the actions of

lead on glutamate release [8], NMDA receptor function [9,

10] or structural plasticity [11] may underlie the charac-

teristic lead-induced perturbations in synaptic plasticity

and learning impairments.

Additional evidence suggests that neural stem cell (NSC)

proliferation in the hippocampus is modulated by lead

exposure [12, 13]. Numerous studies have indicated a role

for microglia in maintaining the homeostasis of the baseline

neurogenic cascade [14, 15]. However, little is known about

any inflammatory processes involving reactive microglia

after acute Pb exposure in the brain. Thus, we used an old

rat model of acute Pb exposure to determine the effects of

lead on microglial activation and NSC proliferation in the

subventricular zone (SVZ) and the subgranular zone (SGZ)

of the hippocampal dentate gyrus (DG).

There are limited pharmacologic options for treating

lead poisoning using Western medicine [1]. Chelation

therapy remains one of the mainstays of treatment. How-

ever, chelating agents can have adverse effects [16], and

chelation therapy to reduce circulating levels of Pb has

proven ineffective for treating low level environmental

exposure to Pb and has failed to reverse the associated

learning deficits [17]. Therefore, it is proposed that treat-

ment strategies directed at the neuronal actions of Pb may

prove more effective in reversing or alleviating the impact

of Pb on brain function.

In oriental medicine, Sanqi (panax notoginseng) is

commonly used for the treatment of lead poisoning symp-

toms (such as headache, vertigo and dizziness). Ginsenoside

Rd (Rd), one of the major ingredients of the total saponins

from Sanqi, has a molecular formula of C48H82O18�3H2O

with a molecular weight of 1001.2. We previously reported

a neuroprotective role of Rd both in vivo and in vitro [18–

24]. Ginsenoside Rd protects cultured hippocampal neurons

against glutamate-induced excitotoxicity [20]. It is neuro-

protective against transient focal ischemia in the aged brain

[22], and attenuates early oxidative damage and sequential

inflammatory responses after transient focal ischemia [23]

in rats. Although several studies have focused on the ability

of Rd to offer neuroprotection against cerebral ischemic

damage, little is known about any effects of Rd on neuro-

genesis and lead poisoning. Therefore, this study was

designed to test the hypothesis that Rd enhances neuro-

genesis in a rat model of lead exposure.

Materials and methods

Materials

Ginsenoside Rd with a purity of 98 % was obtained from

Tai-He Biopharmaceutical Co. Ltd. (Guangzhou, China)

and prepared in saline containing 10 % 1,3-propanediol (v/

v). BrdU was purchased from Boehringer Mannheim,

Indianapolis, IN. Anti-BrdU antibody was from Sigma

Chemical Co., St. Louis, MO. Anti-doublecortin (DCX)

antibody was from Santa Cruz Biotechnology, Santa Cruz,

CA, and anti-Iba-1 antibody was purchased from Wako

Pure Chemicals, Japan.

Animals and treatment

Retired breeder Sprague–Dawley rats (30–32 weeks,

250–300 g body weight) were used in this study. The rats

were housed under controlled conditions (temperature

23 ± 1 �C, humidity 60 ± 10 %, 12-h/12-h light/dark

rhythm) with free access to water and chow. The animal

experiment protocols were approved by the Animal Care

and Use Committee of the Fourth Military Medical Uni-

versity and were in compliance with the Guidelines for the

Care and Use of Laboratory Animals.

The rats were randomly divided into four groups: the

control group, Pb exposure group, Pb ? Rd group, and Rd

group. In the Pb group, rats received one intraperitoneal

injection of 50 mg/kg Pb acetate (Sigma-Aldrich) dis-

solved in saline at a concentration of 10 mg/ml. This dose

regimen has been shown to produce a significant accu-

mulation of Pb in the cerebrospinal fluid and brain during a

short period of time [25]. In the Pb ? Rd group, the rats

first received 1 week of Rd (once a day, i.p.), and then

received one injection of Pb acetate 30 min after the last

injection of Rd (see Fig. 1). The control rats received an

equal volume of saline, and rats in the Rd group only

received the 1-week injection of Rd. In our previous study

[21], Rd at a concentration of 50 mg/kg was effective in

protecting against cerebral ischemic injury. Therefore, this

concentration was used in the present study.

BrdU labeling

The rats received two pulses of BrdU (Sigma; 50 mg/kg of

body weight) intraperitoneally (see Fig. 1).

Fig. 1 Experimental procedure. The rats received 1 week of 50 mg/kg

Rd (i.p., once a day; green bold line). 0.5 h after Rd treatment, rats were

exposed to Pb (blue arrow). For BrdU labeling, rats received two pulses

of BrdU (as indicated by red arrows in (1) and (2), respectively) and

2 hours later, animals were killed (asterisk) (color figure online)

Neurol Sci

123

Tissue fixation

Under anesthesia with sodium pentobarbital (50 mg/kg,

i.p.), all animals were perfused transcardially with 0.9 %

saline followed by 4 % paraformaldehyde. The brains were

post-fixed for 2 h, and placed in 20 % sucrose until they

sank. Brains were sectioned using a sliding microtome.

20 lm-thick free-floating coronal sections through the

entire lateral ventricle and hippocampus were collected and

stored in PBS.

Immunohistochemical detection of Iba-1 labeling

Immunostaining was performed on floating sections as

described previously [23]. Sections were hydrated for

15 min in PBS, and endogenous peroxidases were blocked

in PBS containing 0.3 % H2O2 for 30 min. Sections were

stained overnight using primary anti-Iba-1 (rabbit, 1:1000),

then washed with PBS and incubated with biotinylated goat

anti-rabbit secondary antibody (1:500; Sigma-Aldrich) for

2 h at room temperature, followed by rinsing in PBS and

incubation with an avidin–biotin-peroxidase complex for

2 h. After a final wash, immunoreactivity was visualized

using 3,3-diaminobenzidine as the chromogen. The speci-

ficity of immunolabeling was verified by controls in which

the primary antibody was omitted.

Immunofluorescence staining

In preparation for BrdU immunocytochemistry, sections

were initially incubated in 50 % formamide/23 SSC buffer

(0.3 M NaCl, 0.03 M sodium citrate) at 65 �C for 2 h, and

then incubated in 2 M HCl at 37 �C for 30 min. As

described earlier [15], after a 10-min wash in 0.1 M borate

buffer (pH 8.5) to neutralize the HCl, sections were incu-

bated with primary mouse anti-BrdU (1:1000) antibody at

4 �C for 36 h. After multiple washes, sections were incu-

bated with FITC-conjugated anti-mouse antibody (1:400;

Chemicon) for 2 h at room temperature. For immunohis-

tochemical detection of single labeling of DCX, we used

mouse anti-DCX antibody (1:1,000). We employed the

same protocols described above, except that pretreatment

with formamide and HCl was omitted. The specificity of

immunolabeling was verified by controls in which the

primary antibody was omitted.

Determination of IL-1b, IL-6, and TNF-a levels

A Rat Cytokine/Chemokine Magnetic Bead Panel kit

(Millipore, Billerica, MA, USA) was used to quantify the

expression levels of IL-1b, IL-6, and TNF-a, according to the

manufacturer’s instructions. In brief, rat hippocampi were

collected at 24 h following Pb exposure and homogenized

in 0.5 mL of cold saline. The homogenate was centrifuged at

12,0009g for 10 min, and then 25-ll aliquots of supernatant

or standards were incubated in a 1.2-lm filter membrane

96-well microtiter plate with multi-cytokine beads for 2 h.

After washing with a vacuum manifold, the plate was incu-

bated with biotinylated reporter for 1.5 h followed by

streptavidin–phycoerythrin for 30 min. After the final wash,

the beads were evaluated in a Luminex 200 instrument, and

the data were collected and analyzed using Milliplex Analyst

software (Millipore, Billerica, MA, USA). A minimum of 50

beads was analyzed. The results were expressed as pico-

grams per milligram of wet tissue (pg/mg).

Cell counting and statistical analysis

At least three to five independent brains were used for

statistical analysis. For each brain, BrdU- or DCX-labeled

cells in lateral ventricle SVZ and hippocampal DG were

counted under a light microscope (5–8 sections/animal) at

2009. The results were expressed as the number of BrdU

or DCX-positive cells per section. Since BrdU-labeled

nuclei were irregular in shape and extensively clustered in

the SVZ and SGZ, the nuclei were counterstained with

DAPI for the quantitation of BrdU-labeled cells (data not

shown). The average number of Iba1-positive cells near the

SVZ and DG was quantitated in a 0.25 mm2 area (6–8

areas/section). The relative optical density (R.O.D.) of Iba-

1 immunoreactivity was measured using NIH software

image J and normalized by dividing by the R.O.D. value of

controls. We used SPSS 17.0 for Windows (SPSS Inc.

Chicago, IL) for statistical analyses and data were pre-

sented as mean ± standard error. One-way analysis of

variance was used to compare among different groups.

A value of p \ 0.05 was considered statistically significant.

Results

Rd inhibits microglia activation after acute lead

exposure

In order to validate whether the acute Pb exposure stimu-

lates microgliosis, we investigated changes in microglial

activation by immunostaining for Iba-1 in all groups.

Following acute Pb exposure, there were clear morpho-

logical changes indicating reactive microgliosis in the

Pb-treated group. Comparing with the controls (Fig. 2a, d),

Iba-1? cells demonstrated enlarged, amoeboid shapes

(Fig. 2b, e). Rd pretreatment significant attenuated

Pb-induced morphological changes (Fig. 2c, f). Although

the total number of Iba-1-immunoreactive microglia was

not different between conditions (Fig. 2g), quantitation of

the optical density of immunoreactivity revealed significant

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123

differences between the Rd and Pb-treated groups in both

the SVZ and the dentate gyrus consistent with an increase

in overall immunoreactivity (Fig. 2h). Furthermore, we

quantified proinflammatory cytokine expression in the rat

hippocampus using Luminex technology, and found that

Pb significantly increased expression of IL-1b, IL-6, and

Fig. 2 Pb exposure induces microglial activation. Compared with the

control group (a, d), Pb significantly activated microglial cells near

the SVZ (b) and DG (e) regions, as indicated in the upper schematics.

Rd markedly attenuated Pb-induced microglial activation (c, f). Insetsshow higher magnification of the cells identified by arrows in

respective panels. Scale bars 100 lm (a–f), 25 lm (insets). Quanti-

tation of Iba-1 positive cells in the SVZ and DG (g) and relative

optical density (R.O.D.) of Iba-1 immunoreactivity in the SVZ and

DG (h) after treatments. Rd itself did not affect Iba-1 immunoreac-

tivity. (i) Pb significantly increased the expression levels of IL-1b,

IL-6 and TNF-a while Rd markedly attenuated the levels of

Pb-induced cytokines in rat hippocampi. *p \ 0.05 (vs. the control);

#p \ 0.05 (vs. the Pb-treated group)

Neurol Sci

123

TNF-a as compared to controls (p \ 0.05). Rd pretreat-

ment attenuated the Pb-induced increase in expression of

these cytokines (p \ 0.05, vs. the Pb group) (Fig. 2i).

Rd increases the number of BrdU? neural progenitors

after Pb exposure

To correlate with the observed changes in microgliosis, we

next examined the effects of acute Pb exposure with and

without Rd treatment on the proliferation of neural pro-

genitors. In control rats, a number of BrdU-positive pro-

liferating cells were identified in the SVZ (Fig. 3a, g) and

hippocampal DG (Fig. 3d, h). After Pb exposure, the

number of BrdU-labeled cells was significantly decreased in

both the SVZ (Fig. 3b, g) and DG (Fig. 3e, h) compared to

controls. However, Rd pretreatment significantly attenuated

the inhibitory effects of Pb on proliferation of neural pro-

genitors (Fig. 3c, f, g, h). Rd itself did not affect the number

of BrdU-labeled cells compared to controls (Fig. 3g, h).

We next investigated the morphology of cells expressing

doublecortin (DCX), a microtubule-binding protein expressed

in neuroblasts in early neuronal differentiation stages.

DCX-positive cells were present in the SVZ (Fig. 4a, g) and

SGZ (Fig. 4d, h) in the controls, Pb exposure significantly

decreased the numbers of DCX-positive cells both in SVZ

(Fig. 4b, g) and DG (Fig. 4e, h). However, Rd pretreatment

attenuated the Pb-induced decrease in DCX-immunoreac-

tive cell numbers (Fig. 4c, f, g, h). The number of DCX?

cells in the Rd group was similar to that of control group

(Fig. 4g, h).

Fig. 3 Pb exposure reduces the numbers of BrdU-positive cells.

Compared with the control group (a, d), Pb treatment significantly

decreased the numbers of BrdU-positive cells in the SVZ (b, arrows)

and DG (e). Rd significantly attenuated Pb-induced decrease in the

numbers of BrdU-positive cells in the SVZ (c) and DG (f). Insets in

(d–f) show higher magnification of the cells in the boxes of respective

panels. Scale bars 100 lm (a–f), 20 lm (insets). cc, corpus callosum;

DG, dentate gyrus; Hil, hilus; LV, lateral ventricle. Scale bar:

100 lm. (g, h) Quantitation of BrdU-positive cells in the SVZ (g) and

DG (h) after Pb exposure. *p \ 0.05 (vs. the saline control);

#p \ 0.05 (vs. the Pb-treated group)

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Discussion

Unlike previous studies which investigated the effects of

chronic Pb exposure in juvenile animals, we instead

examined the effects of acute Pb exposure on the brains of

aging rats. The rates of new cell birth are approximately

30,000 per day in the SVZ and between 3,000 and 9,000

per day in the DG of young adult rats [26]. However, it is

well known that aging correlates with decreased levels of

neurogenesis and associated brain dysfunction [27].

Therefore, we were interested in the possibility that Pb

exposure could further attenuate neurogenesis beyond the

levels observed in normal aging. Our data support not only

an age-associated decrease in neurogenesis, since the

numbers of BrdU-positive cells from control brains were

lower than previous findings observed from young control

animals [15], but also exacerbation of age-dependent

decrease in neurogenesis due to Pb exposure. This is

consistent with an overall ability of Pb to alter neurogen-

esis. Indeed, one study showed that 30 g/mL of Pb acetate

was acutely toxic to rat cerebral cortical precursor cells

[28]. Another demonstrated that continuous exposure to a

low level of Pb (0.2 %) reduced the number of BrdU-

positive cells in the hippocampus [13].

In order to quantify proliferation of the precursor cells,

we used the synthetic thymidine analogue BrdU which

incorporates into the DNA of dividing cells. Based upon

BrdU uptake and labeling, we assessed proliferation of

precursor cells in the SVZ and DG. The BrdU-positive

nuclei were small, irregular, and clustered consistent with

the morphology of precursor cells [28]. DCX is often used

as a marker for migrating neuroblasts [29]. Remarkably,

Pb-treated animals showed decreased numbers of BrdU-

positive proliferating precursor cells as well as DCX-

positive cells. This demonstrated an attenuation of neuronal

differentiation in aged rats. However, this effect was not

Fig. 4 Pb exposure decreases the numbers of DCX-positive cells.

Compared with the control group (a, d), Pb treatment significantly

decreased the numbers of DCX-positive cells in the SVZ (b, arrows)

and DG (e). Rd significantly attenuated Pb-induced reduction of the

numbers of DCX-positive cells in the SVZ (c) and DG (f). cc, corpus

callosum; DG, dentate gyrus; Hil, hilus; LV, lateral ventricle. Scalebar 100 lm. (g, h) Quantitation of DCX-positive cells in the SVZ

(g) and DG (h) after Pb exposure. *p \ 0.05 (vs. the saline control);

#p \ 0.05 (vs. the Pb-treated group)

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123

unique to aged rats as similar findings have been reported

in earlier work demonstrating that Pb treatment retards

proliferation of neural stem cells derived from embryonic

brains [30].

One unique aspect of this study was that the effects of

this classic environmental neurotoxicant, Pb, on prolifer-

ating precursor cells in the SVZ were examined. Prolifer-

ation in the SVZ is affected by age and neurodegenerative

disease suggesting that it is a potential target for therapeutic

intervention [31]. Interestingly, addition to a direct effect of

Pb on neurogenesis, we propose that Pb may also indirectly

decrease neurogenesis by activating microglia. Pb effects

have been studied more extensively on astroglia and neu-

rons, because this neurotoxicant acts directly on neural cells

without prior systemic metabolism or biological activation

[5]. However, less is known about effects of Pb on

microglia. It is well known that microglia undergo age-

related activation [27], and that these cells are the primary

immune cell in the brain. Pb has been shown to have effects

on immune function in human [32, 33]. Therefore, this

study examined Pb-induced alterations in microglial acti-

vation. Our data demonstrated that Pb exposure reduced the

numbers of neuronal precursors in correlation with a

remarkable increase in microglial activation. Acute Pb

exposure may be directly toxic to proliferating precursors

and early migrating neurons, but may also cause indirect

neurotoxicity through increased microgliosis. This likely

has implications for the consequences of Pb exposure on the

repair mechanisms of the aging nervous system.

We acknowledge that there was a lack of direct evidence

for a Pb-induced increase in microgliosis being responsible

for changes in precursor proliferation. However, the cor-

relative findings of increased Iba-1 immunoreactivity in the

SVZ and DG and decreased BrdU labeling are supported

by prior work indicating a clear role of brain inflammatory

change in limiting neurogenesis. It has been shown that

attenuating the inflammatory response secondary to

microglial activation restores neurogenesis in the adult

hippocampus [34, 35]. In addition, recombinant IL-6 and

TNF-a reportedly decrease neurogenesis [35], and co-cul-

ture of hippocampal NSC with activated microglia

decreases immature DCX-expressing neurons in vitro [34].

Activated microglia impair basal hippocampal neurogene-

sis partially through the production of TNF-a [35] with the

degree of impairment correlating with numbers of activated

microglia. Consistently, our results revealed that Pb

exposure increased expression of the proinflammatory

cytokines IL-1b, IL-6 and TNF-a. Taken together, we

suggest that microglial-mediated neurotoxicity is important

for a portion of the reduction in precursor proliferation

induced by the acute Pb exposure.

We report for the first time that Rd promotes neuro-

genesis in the brains of old rats following acute Pb

exposure. Rd pretreatment decreased the level of Iba-1

immunoreactivity, attenuated the Pb-induced increase in

expression levels of IL-1b, IL-6 and TNF-a, and prevented

the Pb-dependent decrease in BrdU- and DCX-positive

cells in the SVZ and SGZ. Our group has previously shown

that Rd is neuroprotective in a variety of paradigms

involving anti-inflammatory mechanisms. For example, we

reported that Rd significantly eliminated inflammatory

injury as indicated by the suppression of microglial acti-

vation after transient focal ischemia in rats [23]. Our

current data and prior work suggest that Rd prevents

Pb-induced decrease in NSC proliferation, in part, by

inhibiting microgliosis. However, we do not exclude the

possibility that Rd may exert still unknown effects which

contributes to NSC protection as well.

In summary, we demonstrated that exposure to Pb

reduced proliferation of NSC both in the SVZ and the DG

of the adult rat hippocampus. Rd attenuated the Pb-induced

microgliosis as well as the decrease in NSC proliferation.

These data suggest that a component of the neuroprotective

mechanism of Rd treatment may involve an anti-inflam-

matory effect to maintain neurogenesis and self-repair.

Acknowledgments The authors thank Prof. Wen Jiang for his

insightful comments and Ms. Dongyun Feng for technical support.

This study was supported by grants from the National Natural Science

Foundation of China (Grant Nos. 31170801, 81070950 and

81171236).

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