the dynamic changes of capillary permeability and upregulation of vegf in rats following...

The Dynamic Changes of Capillary Permeability andUpregulation of VEGF in Rats Following Radiation-Induced Brain Injury

XUELONG JIN,* BIN LIANG,* ZEQUN CHEN,* XINGJU LIU,* AND ZHIWEN ZHANG†

*Department of Physiology, Tianjin Medical University, Tianjin, China; †Department of Neurosurgery, First Affiliated Hospital of Chinese PLA General

Hospital, Beijing, China

Address for correspondence: Xuelong Jin, Department of Physiology, Tianjin Medical University, Tianjin 300070, China. E-mail: [email protected]

Received 2 September 2013; accepted 25 October 2013.

ABSTRACT

Objective: To explore the dynamic changes of capillary perme-

ability and the expression of VEGF in cerebral cortex after RIBI.

Methods: Male SD rats were randomly divided into the RIBI

group and control group, and the RIBI group was randomly

subdivided into five groups for analysis on day 1, 3, 7, 14, and 28,

respectively. We established an RIBI model, and then evaluated BBB

permeability by EB. We also measured the expression of VEGF with

IHC stain and western blot.

Results: EB extravasation in injured cortex of RIBI group was

increased at five time points compared with the control group. The

western blot results and IHC revealed that the levels of VEGF

expression in the RIBI groups was significantly increased at day 1

compared with the control group, then rose to a maximum at day 7,

and subsequently the levels of expression recovered from day 14 to 28.

Conclusions: The increases in both BBB permeability and VEGF

expression in the brain cortex of RIBI groups at same time period

confirmed the possibility of brain injury following irradiation of

6 Gy.

KEY WORDS: radiation-induced brain injury, vascular endothelial

growth factor, capillary permeability, blood–brain barrier

Abbreviations used: BBB, blood–brain barrier; BCA, bicinchoninic

acid; BSA, bovine serum albumin; CNS, central nervous system; CT,

electronic computer X-ray tomography technique; DNA, deoxyri-

bonucleic acid; EB, Evans blue; ECL-plus reagent, electro-

chemiluminescence-plus reagent; IHC, immunohistochemistry;

IOD, integrated optical density; LSD, least significant difference;

PBS, phosphate buffered saline; PVDF, polyvinylidene fluoride

membrane; RIBI, radiation-induced brain injury; RIPA, radio

immunoprecipitation assay; SD rat, Sprague–Dawley rat SDS-

PAGE, sodium dodecyl sulfate-polyacrylamide gelelectrophoresis;

TBST, tris-buffered saline with tween; VEGF, vascular endothelial

growth factor.

Please cite this paper as: Jin X, Liang B, Chen Z, Liu X, Zhang Z. The dynamic changes of capillary permeability and upregulation of VEGF in rats following

radiation-induced brain injury. Microcirculation 21: 171–177, 2014.

INTRODUCTION

The brain is one of the most commonly irradiated sits for

radiation therapy, which is used extensively to treat primary

brain tumors and metastases and to prevent intracranial

relapse in many malignancies [12,29]. RIBI is a serious

complication which is commonly associated with significant

functional morbidity and decreased quality of life in patients

treated with brain radiotherapy. Historically, radiation-

induced injury to normal tissue has been attributed to

DNA damage and the subsequent death of replicating cells

[19]. These suggest that radiation-induced brain damage is

perpetuated by surviving cells [29].

BBB consists in part of a highly specialized set of cells

which separates the brain from the vascular system [22], and

it is a highly specialized region of the vascular tree, which

preserves the integrity of the nervous system by limiting the

passage of harmful substances and inflammatory cells into

the brain [16]. Dysfunction of the BBB allows intravascular

proteins and fluid to penetrate into the cerebral parenchymal

extracellular space, leading to vasogenic cerebral edema and

reduced blood flow to neurons and, finally, causing irrevers-

ible apoptosis. In contrast to the highly permeable capillaries

in the systemic circulation [14], blood–CNS vascular barriersare formed by continuous endothelial cells with tight

junction protein complexes and low rates of vesicular

transport [28].

At present, VEGF has been identified as the most

important proangiogenic factor [27]. VEGF family comprises

a group of potent endothelial cell mitogens [26]. In several

physiologically and pathologically experiment setting, VEGF

has been demonstrated to act as a survival factor for the

DOI:10.1111/micc.12103

Original Article

ª 2013 John Wiley & Sons Ltd 171

vasculature in various tissues and organs [18]. VEGF

increases the expression of adhesion molecules and coagu-

lation factors and increases vascular permeability [9,23].

Among neuronal cell-derived growth factors, VEGF is

abundantly expressed in CNS [25]. In fact, VEGF was first

discovered from the brain tissue 20 years ago [6], and

displays broad biological functions including modulation of

angiogenesis, vasculogenesis, vascular permeability, vascular

remodeling, vascular survival, arterial differentiation, neuro-

trophic activity, hematopoiesis, and inflammatory responses

[4,5]. Deletion of only one allele of the VEGF gene in mice

leads to early embryonic lethality due to lack of hematopoi-

etic and vascular systems, suggesting that the levels of VEGF

are crucial for embryonic development and maintenance of

the physiological functions [20]. Some researches suggest

that VEGF and its receptors regulate brain angiogenesis

[2,21,31].

Vascular injury has been hypothesized to play a critical

role in the development of late radiation brain injury,

including radiation necrosis [3,30]. Shortly after radiation,

vascular structure and function can be altered; these alter-

ations include blood vessel dilatation, endothelial cell

enlargement, capillary loss, and peri-vascular astrocyte

hypertrophy which can lead to BBB disruption, increased

permeability, and edema [7].

MATERIALS AND METHODS

All animal procedures were approved by Tianjin Medical

University Institutional Animal Care and Use Committee.

Male SD rats were given a standard laboratory diet and

distilled water ad libitum and kept in cages at 22 � 2°C. Therats (n = 72, body weight 210–230 g) were used in this study,

and randomly divided into the RIBI group (n = 60) and

control group (n = 12). Rats in the RIBI group were

randomly subdivided into five groups (each n = 12) for

analysis on days 1, 3, 7, 14, and 28, respectively.

Preparing for RIBI Rat ModelIn this experiment, CT injury device was used to induce RIBI.

In brief, rats in control and RIBI groups were intraperito-

neally anesthetized with 30% urethane (5 mL/kg) and then

placed in a dedicated self-shielded lead to limit radiation

exposure. A specified area (between the coronal suture and

the lambdoid suture) was exposed. Irradiation was per-

formed using CT (PET CT, somatom emotion 16, Siemens,

Munich, Germany). To compensate for the affects of tissue

attenuation, the prescribed radiation dose was administered

in three consecutive 2 Gy fractions. All rats of RIBI group

underwent CT scan with a dose of 6 Gy (2 Gy 9 3 times).

Radiation doses were confirmed in initial experiments. All

controls were prepared in the same way and subjected to

sham radiation.

Evaluating BBB PermeabilityBBB permeability was assessed by measurement of EB

extravasation in the brain of rats as described [8]. Three

rats of each group were anesthetized as described above, and

3% EB (3 mL/kg) was injected via direct cannulation of the

right internal jugular vein. After 60 minutes, the animals

were perfused with saline. The animals were then killed by

decapitating, the brain was removed, and the cerebellum was

dissected away from the cortical tissue. Subsequently, each

brain cortex was incubated for 48 hours in 5 mL of

dimethylformamide at 60°C to allow dye extraction. After

centrifugation, the optical density of each sample was

measured at 632 nm using a spectrophotometer. EB content

was calculated as described previously [22] and was

expressed in lg/g. We also observed the capillary permeabil-

ity in vivo using Microscope vascular camera device and

microcirculation video recording system (Technology &

Market Co, Ltd of Chengdu, Sichuan, China).

Investigating the Expression of VEGF

Semi-quantitative western blot. Rats were killed at different

time points postoperatively, the brain tissue (2–3 mm in the

lesion site, weighting 80–100 mg) was dissected out and

immediately frozen at�80°Cuntil use. The tissue sampleswere

lysed with RIPA buffer, and then centrifuged at 10,000 g and

4°C for 30 minutes. The concentration of total protein in each

sample was quantitated by BCA method, as per the manufac-

turer’s instructions. Equal amounts of protein were separated

by 10% SDS-PAGE and then transferred to PVDF for

80 minutes. Non-specific binding of the membrane was

blocked by TBST (tris-buffered saline containing 0.1% Tween

20) containing 3% non-fat milk for 60 minutes, and then

incubated for 60 minutes with anti-VEGF antibody (1:100;

Abcam, Cambridge, UK) at room temperature. The mem-

branes were washed three times for 10 minutes each with

TBST, and then incubated for 60 minutes with secondary

antibody (1:40000; Boster, Wuhan, Hubei, China) at room

temperature. After washing, the signal was detected using an

ECL-plus reagent (Boster).

Immunohistochemical staining for VEGF. Rats in each

group (n = 6) were killed by decapitating and transcardially

perfused with 0.01 M PBS, (pH 7.4) to remove blood from

the vasculature, followed by 4% paraformaldehyde in 0.1 M

PBS (pH7.4). After removed, the brain was fixed in 4%

paraformaldehyde for two days at room temperature and

processed for 4 mm coronal paraffin sections through the

RIBI zone, then 6 lm sections were cut. After deparaffini-

zation and redehydration, nonspecific endogenous peroxi-

dase activity was blocked by treating sections with 3%

hydrogen peroxide in methanol for 30 minutes, and then

washed three times in PBS. Antigen was exposed by boiling

X. Jin et al.

172 ª 2013 John Wiley & Sons Ltd

the sections for 20 minutes in 0.01 M citrate buffer (pH 6.0).

After further washing, nonspecific binding was blocked with

5% BSA for 30 minutes in 37°C. Mouse monoclonal

anti-VEGF antibody (1:100; Abcam) was applied on tissue

slides overnight at 4°C. The next day, the slides were washedin PBS and then incubated with secondary biotin-labeled

antibody (goat anti-mouse IgG, Boster) for 30 minutes at

37°C. After further washing, sections were incubated with

peroxidase-labeled avidin complex prior to color develop-

ment using diaminobenzidine. Negative controls were

processed identically except that PBS replaced the primary

antibody.

We examined tissue slice under an Nikon Ti-U inverted

microscope (Nikon Corporation, Tokyo, Japan). For quanti-

fication of immunohistochemical data, five non-overlapping

high-power (9400) images of each section were captured.

The overall expression level of VEGF was ascertained by

measuring the IODof each image (9400) using Image-Proplus

5.0 medical image analysis software (Media Cybernetics,

Bethesda, MD, USA).

Statistical AnalysisAll experiments were repeated at least three times. Data were

calculated with Graphpad prism 5.0 and expressed as

A

B

a b

c d

Figure 1. EB extravasation is obvious by using Microscope vascular camera device and microcirculation video recording system. (A). (a) the image of

control group, EB was limited in the blood vessels; (b) the image of control group, analyzed by Image Pro Plus; (c) the image of RIBI group at day 3, EB

leaked out of the blood vessels; (d) the image of RIBI group at day 3, analyzed by Image Pro Plus; (B). EB extravasation. Quantification of EB (lg/g brain

tissue). Compared to control group, the EB extravasation in RIBI groups was significantly increased.

The Cerebral Microcirculation Changes After RIBI


means � standard deviation. BBB permeability and VEGF

expression level in RIBI group were analyzed using one-way

ANOVA. The LSD t-test was used to compare EB content in

RIBI and control groups. A p-values of <0.05 were consid-

ered statistically significant.

RESULTS

The Change of BBB PermeabilityEB extravasation in injured cortex of the RIBI group was

increased at five time points compared to the control group

(Figure 2). At day 3, the EB extravasation was obvious when

viewed through microscope equipped with vascular camera

device and Microcirculation video recording system (the

image was analyzed using Image Pro Plus, Figure 1). We

used Spectrophotometer to measure the EB extravasation,

and the peak was at day 7 (4.09 � 0.09, p < 0.05) before

declining (3.56 � 0.08, p < 0.05; 3.05 � 0.08, p < 0.05,

Figure 2, Table 1).

Expression of VEGF

Western blot. Western blot analysis for VEGF showed that

the expression of VEGF significantly increased at day 1

compared with controls and remained higher at day 7

Table 1. The change of EB extravasation and VEGF expression following RIBI (�X� s). EB extravasation increased gradually in RIBI groups, and

compared to control group were considered significant; compared to control group, the expression of VEGF was significantly increased in RIBI groups

Group N

EB extravasation

(lg/g)Relative expression

of VEGF by IHC

Relative expression

of VEGF using western blot

Control 12 1.65 � 0.07 5162 � 236.2 0.8538 � 0.031

1 d 12 2.26 � 0.07* 22859 � 2043* 1.140 � 0.044*

3 d 12 3.06 � 0.10* 65962 � 4959* 1.430 � 0.039*

7 d 12 4.09 � 0.09* 110576 � 4373* 1.592 � 0.069*

14 d 12 3.56 � 0.08* 96683 � 4119* 1.569 � 0.044*

28 d 12 3.05 � 0.08* 81389 � 6038* 1.351 � 0.035*

*p < 0.05, compared with control group.

Figure 2. EB extravasation. Quantification of EB (lg/g). EB extravasation

increased gradually in RIBI groups, and the peaking was at day 7.

A

B

Figure 3. The expression of VEGF following RIBI. (A) Representative

bands of VEGF expression in RIBI groups detected by western blot; (B)

Quantification of the optical density of the VEGF bands, normalized to

GAPDH. There was a significant upregulation of VEGF expression in RIBI

groups.

X. Jin et al.


(1.592 � 0.069, p < 0.05), and then declined at day 14

(1.569 � 0.044, p < 0.05) after irradiation but still higher

than the control (Figure 3, Table 1).

IHC. IHC revealed that RIBI is also accompanied by a

significant change in VEGF expression levels (p < 0.05). In

the RIBI animals, the level of VEGF was significantly

increased at day 1 compared with the control group

(5162 � 236.2 versus 22859 � 2043, p < 0.05), and further

rose to a maximum at day 7 (110576 � 4373, p < 0.05). The

expression levels subsequently decreased from day 14 to day

28 (96683 � 4119, 81389 � 6038), but still higher than the

control (Figure 4, Table 1).

DISCUSSION

BBB is mainly composed of vascular cells, astroglial cells, and

a basement membrane [13]. Some studies suggest that

B

A

Figure 4. Immunohistochemical analysis of VEGF. (A) Immunohistochemical analysis in the brains of control rats showed normal appearance of cells in

the cerebral cortex. In RIBI groups, VEGF positive cells (brown) in cerebral cortex increased significantly compared with control group; (B)

Immunoexpression for VEGF was analyzed semi-quantitatively and higher scores were determined in areas of RIBI brain structures with controls.

Importantly, the peak of VEGF expression was noted at day 7.



dysfunction of BBB is associated with disruption of basal

lamina around cerebral capillaries after radiation [15,24].

Other authors have indicated that dose-related changes in

endothelial cell enlargement, vessel dilation, and basement

membrane thickening, and the changes in vascular perme-

ability and associated hypoxia could be observed after

irradiation [13]. The increased vascular permeability may

be responsible for tissue injury. For example, irradiation

mediates disruption of the BBB by altering the structural and

functional integrity of the microvasculature in brain [1].

Several studies showed that irradiation causes loss of

proteoglycans of the vascular basement membrane, resulting

in edema and transudation of proteins into the parenchyma

[1,10,11,17]. Here, we examined the BBB integrity using EB

extravasation. The results showed that the EB was increased

significantly in RIBI groups, which demonstrated the

disruption of BBB.

VEGF plays a significant role in BBB breakdown and

angiogenesis after brain injury [31]. The association of

increased VEGF expression and EB extravasation suggests

that VEGF upregulation is associated with an increase in

vascular permeability resulting from irradiation injury. VEGF

has an essential role in angiogenesis and the repair process

after brain injury. VEGF was in a standstill or wild activated

state in normal tissues of human and animals, which could

have a low expression. In this experiment, we adopt western

blot and IHC to evaluate the expression of VEGF, and we got

the same results through the two different methods.

In this study, we used CT to irradiate the rats with the dose

of 6 Gy. The study showed the BBB permeability gradually

increased till the seventh day after irradiation, and then it

began to decrease, but still higher than the control group.

VEGF expression in the irradiated brain tissue was also

increased gradually till the seventh day, and then decreased

subsequently. Through this experiment, we observed that

both BBB permeability and VEGF expression were up-

regulated progressively, and had their peak at day 7. Based on

the results, the increased BBB permeability and VEGF

expression in the specific brain area of rats in control and

RIBI groups demonstrated that irradiation of 6 Gy could

lead to changes of cerebral microcirculation.

PERSPECTIVE

The increased BBB permeability following radiation is both

the result of RIBI and the resource of secondary brain injury

after irradiation. Thus, to alleviate the BBB permeability and

to control the pathophysiological process effectively would

be significantly important for both treatment and prognosis

of RIBI. Our study showed that the increased VEGF

expression was time-depended, but whether the brain injury

or the cell apoptosis would be alleviated with the upregu-

lation of VEGF needs to be further studied.

ACKNOWLEDGMENTS

We acknowledge the support of Ministry of Science and

Technology of the People’s Republic of China (no.

2011DFA30550).

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