Keywords:
Brain ischemia - blood-brain barrier
Palavras-chave:
Isquemia encefálica - barreira hematoencefálica
Ischemic stroke is one of the leading causes of disability and mortality[1]. On the one hand, stroke leads to complex processes, including intracellular calcium
overload, free radicals-mediated toxicity and disruption of the blood brain barrier
(BBB), which lead to acute neurological deficit[2]. On the other hand, stroke can also lead to neurodegeneration[3]. The tissue plasminogen activator (t-PA) is the only FDA-approved drug for clinical
treatment of acute ischemic stroke. However, the t-PA treatment often induces secondary
impairments, and can cause severe neurodegeneration, such as cerebral ischemia-reperfusion
injury[4]. Therefore, the main priority is to find new drugs for ischemic stroke treatment.
Many plant extracts and/or constituents are being proven to have protective effects
in various neurodegenerative disorders[3]. Camellia japonica L. is a member of the tea family, widely cultivated in China, Japan, Korea and other
parts of the world. Previous studies have demonstrated that C. japonica possesses various biological activities, such as an anti-allergic effect[5], antibacterial activity[6], antioxidant activity[7],[8],[9], and endothelium-dependent relaxation of the coronary artery. Particularly, the
extracts obtained from the fruit of C. japonica have strong cardiovascular protection effects[3],[10]. Our previous study demonstrated that the extract of C. japonica obtained from fresh flowers has a protective effect on hippocampal neurons subjected
to an anoxia-reoxygenation injury[11]. But it is not clear whether the extract of C. japonica has a protective effect on cerebral ischemia-reperfusion injuries in rats.
Thus the present study was designed to demonstrate the possible effect of extract
of C. japonica obtained from flowers on a rat cerebral ischemia-reperfusion injury and its mechanism,
with normal saline and nimodipine used as negative and positive controls, respectively,
according to our previous study[12]. Nimodipine has also been used as a positive control in another experimental cerebral
ischemia-reperfusion study[13]. The infarct volume and lactate dehydrogenase (LDH) activity in serum were considered
as indicators for the neuron damage index after ischemia-reperfusion injury; the Morris
maze test and the Step-down test were used to evaluate learning and memory deficits;
the presence of serum immunoglobulin G (IgG) in brain parenchyma was measured as a
marker of BBB damage; and the serum malondialdehyde (MDA) level was measured as an
oxidative stress marker.
METHODS
Preparation of extract of C. japonica
The ethanolic extracts obtained from fresh flowers of C. japonica was purchased from Xian Wanfang Biological Technology Co., Ltd (Xi'an, China). According
to the supplier, the flowers of C. japonica were washed, air-dried at 70°C and extracted at 30°C for three days in 100% methanol.
The extract solution was then filtered and vacuum-evaporated until dry.
Drugs and reagents
Nimodipine was purchased from the Shandong Fangming Pharmaceutical Group Co. Ltd.
(Shandong, China). The 2,3,5-triphenyltetrazolium chloride (TTC), MDA and LDH test
kits were purchased from Nanjing Jiancheng Biological Company (Nanjing, China). The
rat IgG ELISA kit was purchased from Abcam Trading (Shanghai) Company, Ltd. (Shanghai,
China).
Animals
Adult Sprague Dawley rats (male to female: 1:1) weighing 220-250g were purchased from
Anhui Medical University Animal Center. The animals were housed in the Animal Center
of Hefei Technical College with free access to food and water, with a relative humidity
of 54 ± 2% and a controlled temperature of 22 ± 2°C. All experimental procedures conformed
to the Ethics Review Committee of Hefei Technical College, which follows the protocol
outlined in the Guide for the Care and Use of Laboratory Animals published by the
US National Institutes of Health (NIH publication number 86-23, revised 2011).
Experimental designation
Rats were randomly divided into the following seven groups: sham, model, control,
extract of C. japonica pretreatment (20, 40, 80 mg/kg) and nimodipine (2mg/kg) groups. In the extract of
C. japonica pretreatment and nimodipine groups, the rats were given extract of C. japonica at a dose of 20, 40 or 80 mg/kg (prepared with 0.9% sodium chloride) or nimodipine
at a dose of 2 mg/kg by intragastric administration, respectively, once a day for
three consecutive days before ischemia-reperfusion; the rats in the control group
were administered 0.9% sodium chloride; rats of the model group did not receive any
drug or reagent.
Rat cerebral ischemia-reperfusion model[12]
The rat cerebral ischemia-reperfusion injury was created by 90 minutes of middle cerebral
artery occlusion and 48 hours of reperfusion. Rats were deeply anesthetized with chloral
hydrate (350mg/kg). A nylon suture was introduced through the internal carotid artery
to block the origin of the middle cerebral artery branch for 90 minutes. The suture
was then withdrawn from the middle cerebral artery for reperfusion for 48 hours. Body
temperature was maintained at 37°C throughout the surgery on a heated platform. The
rats of the sham group were not exposed to ischemic stroke. The animals of the extract
of C. japonica, control and nimodipine groups received a once-a-day extract of C. japonica, nimodipine or saline for three consecutive days by intragastric administration before
ischemic stroke.
Step-down test
At 48 hours after the reperfusion, the rat was placed on the floor of the jumping
apparatus; the floor was made of parallel stainless steel bars[14],[15]. After adapting for five minutes, the rat was exposed to a 36V electric foot shock.
The latency of the rat jumping onto the elevated platform, and the frequency of it
jumping down from platform to the floor, receiving electric shocks within five minutes,
were assessed as the learning latency and the number of learning errors, respectively.
The following day, the rat was placed directly on the platform, and the latency and
frequency of it jumping down onto floor to receive electric foot shocks within five
minutes, were recorded for assessing memory latency and number of memory errors, respectively.
Morris water maze test
The pool (150 cm × 150 cm × 60 cm) of the Morris water maze was divided into SW, NW,
NE and SE quadrants; the platform was located in the center of the SW quadrant. The
pool was filled with water (22 ± 2°C) to a depth of 21cm and 1 cm higher than the
platform. Nontoxic titanium dioxide powder was added to the water to make the water
opaque. The movement of the rat in the maze was recorded by a computerized video tracking
system[14],[16],[17]. The procedure was divided into two phases: place navigation and spatial probe.
In the place navigation trial, all rats received four trials each day with an inter-trial
interval of five minutes for four consecutive days; each rat was put into the water
at each of the four quadrants, respectively. The rat was allowed to swim freely until
it found and stayed on the platform. The time required to find and stay on the platform
is known as the escape latency. If a rat failed to find the escape platform within
60 seconds, it was gently guided to the platform and allowed to stay there for 15
seconds before the next trial, and the escape latency was recorded as 60 seconds.
The starting point of the subsequent trial was carried out in a clockwise direction.
The result of the four trials of each day was expressed as the average escape latency.
In the probe trial, the platform was removed. The rats were put into the water at
the NE quadrant and allowed to swim freely for 60 seconds. The number of entries into
the SW quadrant (platform location), time and distance of the rat crossing the SW
quadrant were recorded.
Determination of the percentage of infarct volume[12],[18]
To determine the percentage of infarct volume, the brain was sliced coronally at 2
mm intervals using a brain matrix. Subsequently, the tissue slices were incubated
in the dark in 2% TTC in phosphate-buffered solution at 37°C for 30 minutes, and then
placed in 4% paraformaldehyde for 10 minutes. The nonischemic area was stained red;
the infarct area was not stained and presented as pale. Stained brain slices were
photographed to delineate the area of infarct size using Image J, version 1.6 software
(National Institutes of Health, Bethesda, MD, USA). The total infarct volume was calculated
by summation of the infarcted area (mm2) of each slice and multiplied by the thickness of the slice (2 mm). The percentage
of infarct volume was then determined.
Measurement of LDH activity and MDA level
After the Step-down test and Morris water maze test, the blood of each rat was collected
for centrifugation for 10 minutes at 4,000 g, and the serum transferred into another
tube. The MDA level and the LDH activity were measured using a biochemistry assay
kit (Jiancheng Bioengineering Ltd, Nanjing, China) following the manufacturer's manual.
Measurement of serum IgG present in brain parenchyma
A commercial rat IgG ELISA kit (Abcam Trading (Shanghai) Company Ltd. Shanghai, China)
was used to measure the blood IgG in brain parenchyma, providing an indirect measurement
of the BBB permeability, as a compromised BBB influences the IgG present in brain
parenchyma. We therefore measured the IgG in 100 μg protein of brain parenchyma, prepared
from the cortex and subcortex of the rat brain, respectively.
Statistical analysis
All experimental data are presented as mean ± SD. The SPSS 17.0 software was used
for statistical analysis, the significance of differences between groups analyzed
by Dunnett's test. Statistical significance was considered when p < 0.05.
RESULTS
Effect of extract of C. japonica on impairment of rat learning and memory function after cerebral ischemia-reperfusion
injury
The Step-down test in rats is a classical method to study the dysfunction of learning
and memory after cerebral injury[19]. Likewise, in this study, we followed the same approach to investigate the effect
of extract of C. japonica on the dysfunction of learning and memory in rats injured by cerebral ischemia-reperfusion.
The results are shown in [Figure 1]. Cerebral ischemia-reperfusion significantly increased learning latency and the
number of learning and memory errors (compared with the sham group, p < 0.01), which
were notably alleviated when rats were pretreated with 40 or 80 mg/kg extract of C. japonica (compared with the model group, p < 0.01). Pretreatment with 2 mg/kg nimodipine had
a similar protective effect on learning and memory deficits.
Figure 1 Effect of extract of C. japonica on the impairment of rat learning and memory function (means ± SD, n = 8). A. Effect
of extract of C.japonica on impairment of rat learning and memory latency; B. Effect of extract of C. japonica on the increase of the number of learning errors and memory errors.#p < 0.01 vs Sham;
*p > 0.05 vs Control; **p < 0.01 vs Model; ECJ: extract of C.japonica; Nim: nimodipine
Effects of extract of C. japonica on impairment of rat place navigation and spatial probe ability
The Morris maze test is a well-proven method for evaluating place navigation and spatial
ability to assess spatial reference memory in rodents. The procedure is divided into
two phases: place navigation and spatial probe[20]. In the place navigation trial, rats were trained to find the escape platform. Each
group of rats exhibited a similar ability of finding the escape platform on the first
day of training; the average escape latencies among all groups did not show obvious
differences ([Figure 2A]). As shown in [Figure 2B-2D], the average escape latencies of all groups decreased markedly from the second to
the fourth day of training, but the average latency of the model group was obviously
longer than that of the sham group on the third and fourth days. While, pretreatment
with extract of C. japonica (40 and 80 mg/kg) markedly inhibited the prolongation of the escape latency of rats,
the pretreatment with 2 mg/kg nimodipine had a similar effect.
Figure 2 Effects of extract of C. japonica on the impairment of rat place navigation (Morris water maze test, means ± SD, n
= 8). A. Day 1 training; B. Day 2 training; C. Day 3 training; D. Day 4 training.#p
< 0.01 vs Sham; *p > 0.05 vs Control; **p < 0.01 vs Model; ECJ: extract of C. japonica; Nim: nimodipine.
In the spatial probe trial, the number of entries, the time and distance of crossing
to the platform location were measured. As shown in the [Table], the number of entries and both proportions of time and distance of crossing to
the platform in the model rat group decreased significantly compared with those in
the sham rat group. However, pretreatment with 40 and 80mg/kg extract of C. japonica, or 2 mg/kg nimodipine had a significantly protective effect on the decrease in the
spatial probe ability in rats.
Table
Protective effect of extract of C. japonica on impairment of rat spatial probe ability induced by cerebral ischemia-reperfusion.
|
Group
|
Dose (mg/kg)
|
Number of entries
|
Proportion of time (%)
|
Proportion of swim distance (%)
|
|
Model
|
/
|
1.38 ± 0.52*.**
|
22.94 ± 0.11*.**
|
24.23 ± 0.09*.**
|
|
Control
|
/
|
1.50 ± 0.53
|
21.94 ± 0.08
|
23.12 ± 0.07
|
|
Sham
|
/
|
2.57 ± 0.52
|
38.07 ± 0.09
|
33.80 ± 0.06
|
|
80
|
2.50 ± 0.53***
|
33.47 ± 0.07***
|
33.05 ± 0.05***
|
|
Extract of C. japonica
|
40
|
2.25 ± 0.71***
|
30.4 ± 0.08***
|
30.5 ± 0.07***
|
|
20
|
1.63 ± 0.52
|
23.33 ± 0.10
|
22.73 ± 0.11
|
|
Nimodipine
|
2
|
2.63 ± 0.52***
|
33.21 ± 0.08***
|
32.4 ± 0.06***
|
*p < 0.01 vs Sham;
**p > 0.05 vs Control;
***p < 0.01 vs Model.
Effect of extract of C. japonica on the percentage of infarct volume
The cerebral infarct volume measured using the TTC staining method is usually used
as a direct indicator of middle cerebral artery occlusion injury. The results are
shown in [Figure 3]; the average percentage of the infarct volume of the model group rats was 29%, and
the control group was 30%. Pretreatment with extract of C. japonica (40, 80 mg/kg) or nimodipine (2 mg/kg) markedly decreased the percentage of infarct
volume in these rats (compared with the model group, p < 0.01).
Figure 3 Effect of extract of C. japonica on the percentage of cerebral infarct in rats. A. Representative TTC-stained coronal
brain sections; B. Effect of extract of C. japonica on the percentage of infarct (means ± SD, n = 8).#p < 0.01 vs Sham; *p > 0.05 vs
Control; **p < 0.01 vs Model; ECJ: extract of C.japonica; Nim: nimodipine.
Effect of extract of C. japonica on serum MDA level and LDH activity
Like LDH leakage from brain cells to serum, MDA, a product of lipid peroxidation,
has also been used to assess cerebral ischemia-reperfusion injury, indicated by increases
of its activity in serum. As shown in [Figure 4], in the model group and the control group, marked increases in serum MDA level and
LDH activity were detected. Pretreatment with 40, 80 mg/kg extract of C. japonica significantly inhibited the elevation of serum MDA levels and LDH activity. The pretreatment
of 2 mg/kg nimodipine had a similar protective effect.
Figure 4 Effect of extract of C. japonica on increases of LDH activity and MDA content in rat serum (means ± SD, n = 8). A.
Changes in the LDH activity; B. Changes in the MDA level.#p < 0.01 vs Sham; *p > 0.05
vs Control; **p < 0.01 vs Model; ECJ: extract of C.japonica; Nim: nimodipine.
Determination of serum IgG present in brain parenchyma
To demonstrate the mechanism of the protective effect of extract of C. japonica on cerebral ischemia-reperfusion injury, the BBB integrity was evaluated by detecting
the serum IgG extravasation into the brain parenchyma. There is a minimal amount of
serum IgG detected in brain parenchyma unless the BBB integrity is damaged. The results
are shown in [Figure 5], where pretreatment with 40, 80 mg/kg extract of C. japonica or 2 mg/kg nimodipine significantly reduced the increase of IgG in the ipsilateral
cortex, ipsilateral subcortex, contralateral cortex and contralateral subcortex induced
by cerebral ischemia-reperfusion (p < 0.01 compared with the model group).
Figure 5 Determination of serum IgG present in brain parenchyma (means ± SD, n = 8). A. Effect
of extract of C. japonica on extravasation of serum IgG in the ipsilateral cortex; B. Effect of extract of
C.japonica on extravasation of serum IgG in the contralateral cortex; C. Effect of extract of
C. japonica on extravasation of serum IgG in the ipsilateral subcortex; D. Effect of extract
of C. japonica on extravasation of serum IgG in the contralateral subcortex.*p < 0.01 vs Sham; *p
> 0.05 vs Control; **p < 0.01 vs Model; ECJ: extract of C. japonica; Nim: nimodipine.
DISCUSSION
Camellia japonica is a popular garden plant, the leaves, seeds and flowers of which are widely used
as traditional medicine. A previous study revealed that extracts of C. japonica obtained from leaves, seed and flowers have various biological activities[8]. Our previous study also showed that the extract of C. japonica from flowers has a protective effect on neuronal damage injured by anoxia-reoxygenation
using a preconditioning method[11]. Moreover, pretreatment with a drug is a frequently-used method in the experimental
study of cerebral ischemia-reperfusion injury[21]. Therefore, the present study was designed to evaluate the effect of extract of
C. japonica on cerebral ischemia-reperfusion injury using the preconditioning method.
Middle cerebral artery occlusion is a widely-used model of focal cerebral ischemia-reperfusion
injury according to previous reports[18]. In this study, we found that middle cerebral artery occlusion significantly impaired
learning and memory functions of rats. Compared with the sham group, cerebral ischemia-reperfusion
markedly increased learning and memory latency, number of learning errors and memory
errors. The results of the place navigation trial also showed that a cerebral ischemia-reperfusion
injury could markedly induce the impairment of place navigation ability. Moreover,
cerebral ischemia-reperfusion injury also impaired the spatial probe ability of rats.
Interestingly, these changes could significantly be inhibited by pretreatment with
40, 80mg/kg extract of C. japonica or 2 mg/kg nimodipine.
In order to further confirm the protection of extract of C. japonica against the rat ischemia-reperfusion injury, we measured the percentage of cerebral
infarct volume, serum MDA (an oxidative stress biomarker) level and LDH activity.
As we know, MDA is an indicator of lipid peroxidation[22], and LDH is a metabolic enzyme in neurons and is released into the blood from an
injured neuron[23]. Therefore, an increase of serum MDA level and LDH activity may reflect the damage
of neurons. Our results also showed that cerebral ischemia-reperfusion markedly increased
the percentage of infarct volume, serum MDA level and LDH activity. Compared with
the model group or control group, pretreatment with extract of C. japonica (40, 80 mg/kg) or nimodipine (2 mg/kg) could significantly decrease the percentage
of infarct volume, serum MDA level and LDH activity (p < 0.01). These results provided
direct evidence that extract of C. japonica has a significant neuroprotective effect on cerebral ischemia-reperfusion injury.
Moreover, disruption of the BBB is also one of the pathophysiological mechanisms of
ischemic stroke, with elevated permeability and compromised barrier function[24]. The presence of serum IgG in brain parenchyma was assessed as an index of BBB damage[25]. As shown in our study, due to BBB disruption, extravasation of serum IgG into the
brain parenchyma induced by ischemia-reperfusion injury was detected with ELISA. Not
surprisingly, the pretreatment with 40, 80 mg/kg extract of C. japonica or 2 mg/kg nimodipine prior to the ischemic insult significantly inhibited the extravasation
of serum IgG into the brain parenchyma, suggesting a protective effect on ischemic
BBB damage.
In this study, we found that extract of C. japonica had protective effects on cerebral ischemia-reperfusion injury in rats. The mechanism
of the protective effect may be related to the inhibition of lipid peroxidation and
BBB compromise. A previous study reported that the constituents in the flowers of
C. japonica contain several hydrolyzable tannins, triterpenes, acylated anthocyanins, and purine
alkaloids[10]. Further studies are of crucial importance to elucidate the role of the constituents
of extract of C. japonica in the neuroprotective effect.
