Int J Mol Med (髓鞘 年龄)_图文

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 32: 1021-1028, 2013

Age-related decline of myelin proteins is highly correlated

with activation of astrocytes and microglia in the rat CNS

FANG XIE

1,2*

, JIU-CONG ZHANG

3*

, HAN FU

1*

and JUN CHEN

1

1

Institute for Biomedical Sciences of Pain, Tangdu Hospital, The Fourth Military Medical University,

Xi'an, Shaanxi 710038;

2

Department of Stress Medicine, Institute of Basic Medical Sciences,

Academy of Military Medicine Sciences, Beijing 100850;

3

Department of Gastroenterology,

Lanzhou General Hospital of Lanzhou Military Command, Lanzhou, Gansu 730050, P.R. China

Received May 18, 2013; Accepted August 19, 2013

DOI: 10.3892/ijmm.2013.1486

Abstract. It has been shown that aging can greatly influence

the integrity and ultrastructure of white matter and the myelin

sheath; however, studies regarding the effects of aging on the

expression of myelin proteins are still limited. In the present

study, immunohistochemical mapping was used to investigate

the overall expression of myelin basic protein (Mbp) and

myelin oligodendrocyte glycoprotein (Mog) in the central

nervous system (CNS) of rats in postnatal months 2, 5, 18

and 26. Astrocyte and microglia activation was also detected

by glial fibrillary acidic protein (GFAP) or ionized calcium-

binding adaptor molecule 1 (Iba1) staining and western

blotting. A significant decline of Mbp and Mog was identi-

fied as a universal alteration in the CNS of aged rats. Aging

also induced significant astrocyte and microglial activation.

Correlation analysis indicated a negative correlation between

the reduction of age-related myelin proteins and glial activa-

tion in aging. This correlation of myelin breakdown and glial

activation in aging may reveal new evidence in connecting

the inflammation and myelin breakdown mechanism of

age-related neurodegenerative diseases.

Introduction

White matter of the central nervous system (CNS) is named

after its glistening pale appearance visible on the cut surface

of the brain and spinal cord. This feature is attributed to the

myelin sheath, which is a multiple laminar membrane struc-

ture wrapped around the axons and plays an important role,

Correspondence to:

Dr Jun Chen, Tangdu Hospital, The Fourth

Military Medical University, 1st Xinsi Road, Baqiao District, Xi'an,

Shaanxi 710038, P.R. China

E-mail: vancoxie@

Contributed equally

*

Key words:

aging, myelin proteins, astrocyte, microglia, central

nervous system, mapping

as an insulate cable-coat, in maintaining the fast saltatory

conduction of action potentials along the nerve fibers (1). The

lipid-rich feature of the myelin sheath makes it very fragile

to the environmental changes in the brain, such as metabolic

disorders and aging. Due to the sensitivity of myelin sheath to

senescence, it is thought to be one of the major targets of brain

aging (2). Age-related alterations in the myelin sheath were

identified in the light microscopy study of Lintl and Braak

(3), who observed a reduction in the intensity of hematoxylin

staining in the aged myelin. Since then, increasing evidence

has suggested the existence of a progressive myelin breakdown

in the aged nervous system. Many neuroimaging studies,

especially the diffusion tensor imaging and the voxel-based

morphometric analysis, showed an obvious age-related decline

in human brain white matter integrity, in other words a reduc-

tion in the structural integrity of the myelin sheath (4,5).

Ultrastructure electron microscopy studies demonstrated that

age-related myelin breakdown could be identified in both the

central and peripheral nervous system of humans, non-human

primates, rodents and other species (6,7). Furthermore, the

alterations of the myelin sheath in an aging brain are consid-

ered to be involved in the development of age-related disorders,

such as Alzheimer's and Parkinson's disease (8,9). However,

the mechanism of these age-related alterations in the myelin

sheath has not been fully understood.

Myelin-associated proteins, such as myelin basic

protein (Mbp) and proteolopid protein (Plp), are key compo-

nents of the myelin sheath and play a very important role in

maintaining the integrity of myelin multi-lamellar structure.

However, investigations concerning the expression level of

myelin proteins in aged CNS are limited, for both humans

and animals. On the other hand, there is general agreement

that astrocyte and microglia undergo activation with age

in rodents, monkeys and even humans. These two types of

glial cells become hypertrophic in an aged brain, express

more senescence markers and occasionally increase their

numbers (10,11). Nevertheless, it is not fully understood

whether this activation of astrocytes and microglia contributes

to the age-related myelin breakdown. In the present study,

we investigated the age-related expression of myelin proteins

using a whole CNS mapping. The downregulation of Mbp and

myelin oligodendrocyte glycoprotein (Mog) was observed as

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XIE et al: AGE-RELATED DECLINE OF MYELIN PROTEINS AND GLIAL CELL ACTIVATIONS

a general alteration of

activation of astrocyte

the

myelin sheath in aging. Age-related

negative correlation between the myelin protein breakdown

s and microglia was also analyzed. The

and glial cell activation was identified.

Materials and methods

Animals.

from postnatal month (PNM) 2-26 were used. The rats were

Male Sprague-Dawley albino rats ranging in age

divided into

26. All the animals were provided by

4 groups of 7 animals each, aged PNM2, 5, 18 and

Center of the Fourth Military Medical University (FMMU).

the Laboratory Animal

Animals were housed in plastic boxes at 22-26˚C on a 12

light/dark cycle. Food and water were provided

Drinking water was chlorinated and acidified to pH 6.2-6.8.

ad libitum

-h

.

Cages were cleaned with autoclaving and the

was

ir environment

were approved by the

maintained strictly steady. The experimental protocols

Committee of FMMU (permit no.: SCXK2007-007). The

Institutional Animal Care and Use

present study was performed in accordance with the National

Institute of Health Guide for the Care and Use of Laboratory

Animals (NIH Publications no. 80-23) revised in 1996.

Immunohistochemistry.

anesthetized

Three rats from each group

Sigma,

with pentobarbital sodium (100 mg/kg,

were

i.p.;

with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M

St. Louis, MO, USA) and transcardially perfused

phosphate-

were dissected, post-fixed at 4˚C for

buffered saline (PBS). Whole brain and spinal cord

the same fixative solution and transferred into 30% sucrose

an additional 90 min in

0.01 M PBS overnight. Serial 40 µm cryo-sections (coronal

brain sections and transversal spinal sections

CM1900 freezing microtome (Leica, Mannheim, Germany),

) were cut on

incubated for 1 h

10%

followed by incubation with

goat serum

with

in 0.01

0.05% Triton X-100 and

M PBS at room temperature,

then with

overnight. The primary antibodies were mouse anti-myelin

the primary antibody at 4˚C

oligodendrocyte glycoprotein (anti-Mog) (1:200, Millipore,

Billerica, MA, USA), rabbit anti-Mbp (1:200, Sigma), rabbit

anti-glial fibrillary acidic protein (anti-GFAP) (GFAP, 1:400,

Millipore) and rabbit anti-ionized calcium-binding adaptor

molecule 1 (anti-Iba1) (Iba1, 1:300, Wako, Osaka, Japan).

Diamidino-phenyl-indole

administ

(DAPI, 1 µg/ml, Sigma)

fluorescen

ered for at least 30 sec to stain the cell nucleus for

was

under

ce labeling. Micrographic images were obtained

Iba1-positive cells were counted by Image-pro Plus software

the microscope (Olympus, Tokyo, Japan). GFAP or

in the

campal formation, corpus callosum, internal capsule, spinal

rat cerebral cortex S1, dentate gyrus (DG) of hippo-

cord dorsal horn and spinal cord posterior funiculus

tively. The number of DAPI-labeled cell nucle

, respec-

callosum and posterior funiculus were also obtained through

i in the corpus

Image-pro Plus software. Cell densities were calculated in the

prementioned regions.

Western blotting.

with pentobarbital sodium (100 mg/kg, i.p.). The whole brain

Four rats from each group were anesthetized

without

,

lysed in an ice-cold RIPA lysis buffer (Applygen Technologies

the cerebellum, was dissected, homogenized and

Inc., Beijing, China) of 1% NP-40, 0.1% sodium dodecyl

sulphate (SDS) in 50 mM Tris-HCl, pH 7.4 and containing

protease inhibitor. Protein concentrations were determined by

the BCA protein assay kit (Thermo Scientific, Pittsburgh, PA,

USA). After 10 min of incubation at 98˚C with SDS-PAGE

buffer,

SDS-PAGE gel and transferred to

protein samples (40 µg) were

membrane with a semi-dry transfer system (Bio-Rad, Hercules,

a polyvinylidene difluoride

separated by 10%

CA, USA). Membranes were blocked at room temperature

for

followed by incubation with antibody. Mouse anti-Mog (1:500,

1 h with 5% milk in PBS containing 0.2% Tween-20,

Millipore), rabbit anti-Mbp (1:500, Sigma), rabbit anti-GFAP

(1:800, Millipore) and rabbit anti-Iba1 (1:600, Wako) antibod

served

antibody (1:8,000, Sigma) was used as

as the primary antibodies. Mouse anti-rat

β

-tubulin

ies

membranes were developed with Pierce ECL Western blotting

an internal control. The

substrate kit (Thermo Scientific) and the signals were capture

with FluorChem

®

USA). Scanned images were analyzed by Quantity One 1-D

FC2 (Alpha Innotech, San Leandro, CA,

d

analysis software (Bio-Rad).

Statistical analysis.

Differences among groups were compared using one-way

Data are expressed as means ± SEM.

ANOVA, followed by Bonferroni's or Fisher's PLSD post

analysis when appropriate. Correlation between the expression

-hoc

levels

analysis

of GFAP, Iba1, Mbp and Mog based on western blot

multiple comparisons

results was analyzed by Pearson's coefficients and

analysis

dure

. Multiple regression analys

were corrected by

es, with stepwise proce-

using Bonferroni's

myelin protein decline in aged rat brain

, were performed to investigate the determinants of the

performed

significance was indicated by P<0.05.

using the SPSS statistical

s

package.

. All analyses were

Statistical

Results

Age‑related down‑regulation of myelin proteins.

CNS immunohistochemical mapping, we observed a marked

Using whole

downregulation

proteins, such as Mog and Mbp (Fig. 1 and

in the expression of myelin associated

This alteration of myelin protein expression is universal in

data not shown).

the CNS,

to the medulla and c

it appears from the prefrontal section of the brain

spinal cord (Fig. 1). This myelin

an be identified in any segment of the

not occur only in very old rat CNS. In fact, this alteration

-protein downregulation does

was first identified in PNM5 in our study (data not shown).

As Mog and Mbp

matter,

more obvious in

the reduction

are usaully

of Mog

highly expressed in the white

and Mbp levels

in the brain and the

the white matters of the corpus callosum

was much

(Fig. 2A-H). Immunohistochemical data showed that Mog

posterior funiculus in the spinal cord

and Mbp were highly

the

expression of Mog markedly

nerve fibers in PNM2 corpus callosum. However, the

expressed and distributed parallel to

and

(Figs. 2A and B, and 3A and B), while Mbp

corrugated appearance in

decreased and had a twisted

PNM18 corpus

able

was hardly detect-

callosum

cord in PNM18 lost most response to Mog and Mbp antibodies

(Fig. 2C and D). The posterior funiculus of the spinal

and only some Mog- and Mbp-positive cell

(Figs. 2E-H, and 3D and E). The changes of myelin protein

s were present

expression were a little complicated in the grey matters.

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 32: 1021-1028, 2013

1023

Figure 1. Myelin oligodendrocyte glycoprotein (Mog) expression altered with age in rat brain and spinal cord. Mog expression in (A) PNM2 and (B) PNM18

rat brain and spinal cord. Scale bar, 1 mm.

Figure 2. Age-related alteration in myelin protein expression. (A-D) Decreased expression of myelin oligodendrocyte glycoprotein (Mog) and myelin basic

protein (Mbp) in aged corpus callosum (cc). (E-H) Downregulation of Mog and Mbp in spinal cord posterior funiculus (pf). (I-L) Expression of Mog and Mbp

in postnatal month 2 (PNM2) and PNM18 primary somatosensory cortex (S1). (M-P) Alteration of Mog and Mbp expression in aged spinal cord dorsal horn

(DH). Scale bar, 100 µm.

Compared with the PNM2 rat CNS, the amount of Mbp seems

to be at the same level in the primary somatosensory cortex

(Fig. 2K and L) and spinal cord dorsal horn (Fig. 2O and P)

of PNM18 rat. Specifically, Mog was expressed even more

in the aged somatosensory cortex (Fig. 2I and J) and dorsal

horn (Fig. 2M and N). DAPI labeling showed that the number

of glial cells, including oligodendrocytes, astrocytes and

microglia, increased significantly in the aged corpus callosum.

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XIE et al: AGE-RELATED DECLINE OF MYELIN PROTEINS AND GLIAL CELL ACTIVATIONS

Figure 3. Myelin oligodendrocyte glycoprotein (Mog) altered with age in the rat corpus callosum and dorsal column. Expression of Mog in (A) postnatal

month 2 (PNM2) and (B) PNM18 rat corpus callosum. The density of DAPI-labeled nucleus in the corpus callosum was evident in (C-E), showing the level

of Mog in the PNM2 and PNM18 rat dorsal column, respectively. The density of DAPI-labeled nucleus in the spinal cord dorsal column is shown in F. Scale

bar, 50 µm;

*

P<0.05;

**

P<0.01; Error bars: ± SEM.

glial nuclei decreased significantly in the posterior funiculus

of aged spinal cords (Fig. 3E and F). However, linked nuclei

could still be observed (Fig. 3E).

Similar to the results of immunohistochemical mapping

and immunofluorescence, results of western blotting also

showed an age-related alteration in the expression of Mog and

Mbp in the rat brain (Fig. 4). The downregulation of Mog and

Mbp is age-dependent. Compared with PNM2, the expres-

sion level of Mog and Mbp in PNM26 decreased almost 50%

(Fig. 4B).

Activation of astrocytes and microglia in aged rat CNS.

The immunohistochemical mapping of the rat CNS was

conducted to investigate the activation of astrocytes and

microglia. GFAP and Iba1 were used as activation markers

of astrocytes and microglia, respectively. The mapping

results showed a significant age-related increase in the

expression of GFAP and Iba1 (Figs. 5 and 6). In PNM2 rat,

the pale appearance of tissue slides indicated the weak-

ness in CNS response to anti-GFAP or Iba1 antibody. The

upregulation of GFAP and Iba1 was first detected in PNM5

and was also observed in PNM18 and PNM26 rat CNS. The

number of GFAP-positive cells markedly increased in the

grey and white matters of PNM18 rat (Fig. 5A-L and N).

In the primary somatosensory cortex and dentate gyrus of

hippocampal formation of PNM18, a 10-fold increase in

the density of astrocyte was identified (Fig. 5A-D and M).

Similarly, the GFAP labeling indicated that the density of

astrocyte increased three to seven times in aged corpus

callosum, internal capsule and spinal cord posterior funic-

ulus (Fig. 5E-H and K-M). Of note, although the number of

GFAP-positive cells increased in the white matters of PNM18

Figure 4. The downregulation of myelin protein in aging brain. (A) Expression

of myelin basic protein (Mbp) and myelin oligodendrocyte glycoprotein

(Mog) based on western blot analysis in postnatal month 2 (PNM2), 5, 18 and

26 rat brain tissue homogenate (n=4 per time point). (B) Statistical analysis

of western blot results.

*

P<0.05;

**

P<0.01; Error bars: ± SEM.

Contrary to the arrangement of glial nuclei in rows between

nerve fibers in PNM2, the glial nuclei in PNM18 corpus

callosum were distributed more irregularly and some of them

were

grouped together as in a nest (Fig. 3B). By contrast, the

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 32: 1021-1028, 2013

1025

Figure 5. Astrocyte activated in aged rat brain and spinal cord. (A), (C), (E), (G), (I) and (K) show the GFAP immunochemistry staining in PNM2 rat cerebral

cortex S1, dentate gyrus of hippocampal formation, corpus callosum, internal capsule, spinal cord dorsal horn and spinal cord posterior funiculus, respectively.

(B), (D), (F), (H), (J) and (L) show the astrocyte in postnatal month 18 (PNM18) rat brain and spinal cord positions mentioned above. The number of activated

astrocyte per square millimeter in young and aged rat brains and spinal cords are shown in (M). (N) shows the relative intensity of astrocyte activation in

aged rats (normalized by comparing with PNM2 number of activated astrocyte per square millimeter) within the grey and white matters, respectively. Scale

bar, 100 µm;

*

P<0.05;

**

P<0.01 compared with PNM2; Error bars: ± SEM.

Figure 6. Microglia activated in aged rat brain and spinal cord. Ionized calcium-binding adaptor molecule 1 (Iba1) was used to visualize microglias. For region

annotation see Fig. 5. Scale bar, 100 µm;

*

P<0.05;

**

P<0.01 compared with postnatal month 2 (PNM2); Error bars: ± SEM.

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XIE et al: AGE-RELATED DECLINE OF MYELIN PROTEINS AND GLIAL CELL ACTIVATIONS

Figure 7. The upregulation of glial protein in the aging brain. (A) Expression

of glial fibrillary acidic protein (GFAP) and Iba1 based on western blot

ysis

anal-

(n=4 per time point). (B) Statistic

in postnatal month 2 (PNM2), 5, 18 and 26 rat brain tissue homogenate

al analysis of western blot results.

*

**

P<0.01; Error bars: ± SEM.

P<0.05;

rat, the non-cell-shaped staining was greater in PNM2 white

matter (Fig. 5E-H, K and L). This dark background of white

matter in PNM2 is probably cause

neuropil that was formed by inactivated astrocytes. On the

d by the staining of a

other hand, the activation of microglia was also significant

in the

of Iba1 in aged grey matter was greater than

grey and white matters (Fig. 6) and the upregulation

matter (Fig. 6N). Iba1

that in white

able

-positive microglia were hardly detect-

and achromatous appearance of slides. However, the number

in the whole CNS of PNM2, which yielded the pale

of Iba1-lab

brain and spinal cord. A 2- to 6-fold increase in the density

eled microglia was markedly elevated in PNM18

of activated microglia was identified in the cerebral cortex,

dentate gyrus of hippocampal formation, corpus callosum,

internal capsule, spinal cord dorsal horn and spinal cord

posterior funiculus, respectively (Fig. 6A-M).

tion of GFAP and Iba1 in

Western blotting data showed an age-related accumula

of GFAP and Iba1 increased progressively with age, which

the brain (Fig. 7). The expression

-

doubled the level

rat brains compared with

s of GFAP and Iba1 in PNM18 and PNM26

those in PNM2 (Fig. 7B).

Correlation between age‑related glial activation and myelin

protein decline in aging.

was present both in the reduction of myelin proteins and the

Considering that age-dependence

increase of glial markers, we evaluated whether these two

alterations were correlated with each other. The main findings

of the correlational analyses were that: i) the downregulation

of Mbp showed a significant negative correlation with the

expression of GFAP (Fig. 8A) and Iba1 (Fig. 8B) in

ii) the level of expression of Mog in

the brain;

correlated with the concentration of GFAP (Fig. 8C) and Iba1

the brain was negatively

(Fig. 8D), respectively. Stepwise multiple regression analysis,

with the expression levels

variables and the expression levels

of GFAP and Iba1 as independent

dependent variables, identified the concentration of GFAP

of Mog and Mbp as

and Iba1 in the

the expression level of Mog (Adj. R-square = 0.861, P<0.001)

brain as independent factors for predicting

and Mbp (Adj. R-square = 0.848, P<0.001). These data indi

cated that the upregulation of GFAP and Iba1,

-

the activation of astrocyte

in other words,

the decline of myelin proteins in

s and microglia, may contribute to

the aging process.

Discussion

Although

aging on the

there is evidence to show the effects of normal

tions have focused on the limited

myelin sheath in human and animals, investiga-

expression of myelin proteins in CNS. Different experimental

age-related changes in the

methods, different animals and different regions of interest

in CNS could induce confusing and even conflicting results

(12-14). Therefore, a comprehensive detailed investigation of

the expression of myelin proteins in aged CNS is still needed.

In the current study, a universal reduction in the expression of

Mog and Mbp was identified in the CNS of aged rat

well-

proteins was consistent with Mbp, P0 decline in peripheral

controlled mapping. This downregulation of

s through

myelin

nerves

the reduced hematoxylin staining of aged myelin, which was

(15,16) and was likely to be a reason accounting for

identified in early studies on humans (3).

sheath,

Mbp is one of the major abundant proteins in

species (17). Mbp is

comprising >30% of the total proteins

the

in

myelin

most

at the cytoplasmic surface in the major dense line of myelin

an extrinsic protein localized exclusively

sheath and is believed to be the principal protein stabilizing

the major dense line of CNS myelin (17). Splits of the lamellae

at the major dense line w

morphological alteration

ere found to be the most common

of ultrastructural investigations (11,18) and could probably

s in aged myelin sheath after a series

due to the age-related Mbp

On the other hand, Mog is a CNS myelin protein of great

decrease observed in our study.

be

neuroimmunological interest, but its function remains to be

determined (19).

this age-related decrease

As yet, no other investigations have shown

were

of Mog. However, what changes

remain a mystery and should be further examined.

induced by this downregulation of Mog in aged CNS

cated a significant increase of

DAPI labeling of the nuclei in the corpus callosum indi-

brains

cell type in corpus callosum, we assumed that elevation in

. Considering that oligodendrocyte

the glial cell number in aging

s were the major

the number of DAPI-labeled nucle

the increase of oligodendrocyte

i was mainly attribut

ultrastructural studies that were administ

s. This is consistent with the

ed to

and visual cortex

and Sethares and by Peters

es of aging primates in the studies by Peters

ered in the cerebral

of

our study (Fig. 3)

glial nuclei in the aged corpus

et al (20,21). The aggregation

callosum, observed in

oligodendrocytes

using electron microscop

, which was found in aged monkey cortex

, and the similar pair- or row-together of

es

drocytes proliferat

y (20,22), suggested that oligoden-

an increase in their number. However, DAPI labeling also

e with age and this proliferation leads to

indicated a decrease in the number of glial cells in aged

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 32: 1021-1028, 2013

1027

Figure 8. The negative correlation between the myelin protein breakdown and glia cell activation. (A) and (B) Expression of myelin basic protein (Mbp) and

ionized calcium-binding adaptor molecule 1 (Iba1), respectively, in the brain were negatively correlated with the expression of GFAP. (C and D) Expression

of myelin oligodendrocyte glycoprotein (Mog) in brain exhibits an inverse correlation to the expression of glial fibrillary acidic protein (GFAP) and Iba1.

spinal cord posterior funiculus. Further studies are needed to

determine whether the increase in the number of oligoden-

drocytes in aging is universal in CNS.

Results of the immunohistochemical mapping and western

blotting indicated a significant upregulation of GFAP and Iba1

in aged rat CNS. This increase of age-related GAFP (mRNA

and protein expression levels) and hypertrophy of astrocytes in

aged rodent brains were also identified in other studies (23,24).

However, previous reports have suggested that the number of

astrocytes did not appear to increase or increased slightly in

number during normal aging (11,25), which was not consistent

with the significant age-related increase in the number of

astrocytes in the present study. The dark neuropil staining in

the white matter, which was observed in PNM2 in our study

(Fig. 5E-H, K and L) suggested that the inactive astrocytes

expressed low levels of GFAP that were not easily detected

by GFAP immunohistochemistry. Therefore, we assumed that

the great changes of GFAP in our study were a good marker

of astrocyte activation, but could not be used to evaluate the

proliferation of astrocytes in aging.

On the other hand, Iba1 was widely used as a microglial

activation marker, since it is greatly upregulated in acti-

vated microglia (26). Based on Iba1 immunohistochemical

staining, age-related activation of microglia was identified

in our study, which was also detected in the brain of aged

rats (27) and primates (28) using other microglial activation

markers.

Our correlation analysis revealed that the age-related

decrease of Mbp and Mog was highly correlated with the

activation of astrocytes and microglia in aged rat CNS. As

previous attention was mostly focused on the interaction

between astrocytes/microglia and neurons, this negative

correlation is relatively novel. A decreased neuroprotective

capacity of aged astrocyte was also found by a previous

in vitro study (29). This compromised neuroprotection of

astrocytes, during aging, was believed to be associated with

the reduced nerve growth factors in aging, such as FGF-2 and

BDNF, which are released by astrocytes (30). Considering

that sufficient nerve growth factors are also needed in main-

taining the integrity of the myelin sheath (31), we assumed

that the reduced nerve growth factors in aging astrocytes may

contribute to the decline of myelin proteins. On the other

hand, chronic activated microglia and astrocytes can induce

damage by releasing highly toxic products, such as reactive

oxygen intermediates, inflammatory cytokines and comple-

mentary factors (32). There is evidence showing that myelin

proteolysis, in aging, is linked to calpain-1 and the comple-

ment system expressed in microglia (33). However, the exact

mechanism of the decrease of age-related myelin proteins

and the contribution of astrocytes and microglial activation

in this decrease remain unknown. Further studies must be

performed to explore the links between the downregulation

of myelin proteins and activation of astrocytes and microglia.

In summary, a significant decline of myelin proteins in

the whole CNS of aged rats was identified by immunohis-

tochemical staining and western blotting. The decrease in

myelin proteins was highly correlated with the age-related

activation of astrocytes and microglia. Mog and Mbp

mapping could be used as a good model to investigate the

aging effects on myelin sheath in CNS. The correlation of

myelin breakdown and glial activation in aging is able to

provide new evidence concerning the connection of inflam-

mation and myelin breakdown mechanism in age-related

neurodegenerative diseases.

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XIE et al: AGE-RELATED DECLINE OF MYELIN PROTEINS AND GLIAL CELL ACTIVATIONS

Acknowledgements

This

Research

study was supported by grants from the Major State Basic

(no. 2011CB504100, 2013BAI04B04) and the National Natural

Development Program of China (973 Program)

Science Foundation of China (no. 81171049).

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