Keywords:
Cognitive Dysfunction - Stroke - Hippocampus
Palavras-chave:
Disfunção Cognitiva - Acidente Vascular Cerebral - Hipocampo
INTRODUCTION
Post-stroke cognitive impairment (PSCI) is a common complication of ischemic stroke.
About 25% of patients has severe PSCI three months after stroke, including memory,
orientation, language and attention[1]. Cognitive impairment is a major contributor to longer duration of hospital stay,
lower quality of life and difficulty returning to social life[2]. Prevention for PSCI can be implemented by lowering blood pressure, administration
of statin, neuroprotective and anti-inflammatory drugs. However, there is no evidence
of convincing efficacy. Recent studies also suggested that lifestyle interventions,
physical activity and cognitive training may improve cognition of PSCI, but without
sufficient controlled clinical trials[3].
Enriched environment (EE) is an effective rodent rehabilitation treatment method,
in which several animals are accommodated in a large space equipped with various toys
and receive more sensorial movements, perceptions and social stimulation than in standard
conditions. EE has a neuroprotective effect on animal models of cerebral ischemia[4]. There is significant evidence that EE also benefits the clinical rehabilitation
of post-stroke patients, including promoting greater exercise, social interaction
and personal control[5]. Exploring the underlying mechanisms that EE improves cognitive function in PSCI
is of great significance for providing more individualized cognitive rehabilitation
programs in stroke patients.
α7-nicotinic acetylcholine receptor (α7-nAChR) is a cholinergic receptor that is abundantly-expressed
in the hippocampus and the frontal cortex, and has been confirmed to play a critical
role in improving cognitive function of learning and memory[6],[7]. As the biological foundation of cognitive processes, synaptic transmission can
be modulated by cholinergic pathway and evaluated by long-term potentiation (LTP).
Activation of α7-nAChR contributes to a better induction of LTP[8], which is also essential for inhibiting cytokine synthesis, such as IL-1β, IL-6,
by the cholinergic anti-inflammatory pathway (CAP)[9]. Activation of α7-nAChR inhibits inflammation in patients with stroke[10]. In the aging process of mice, EE not only increases the expression of choline acetyltransferase
(ChAT) and α7-nAChR in the hippocampus but also improves spatial memory[11]. EE also significantly improves cognitive impairment in PSCI mice, induces hippocampal
LTP, and enhances ChAT promoter acetylation[12].
These studies suggest that activation of cholinergic signals after stroke enhances
neuroplasticity and cognitive function. However, it is still unclear whether EE can
regulate α7-nAChR and the role of α7-nAChR in CAP and LTP of PSCI.
To explore the effects of EE on cognitive function, neuroplasticity and underlying
mechanism in PSCI rats, a rat model of ischemic stroke was induced by middle cerebral
artery occlusion and reperfusion (MCAO/R). We have characterized the effects of EE
on cognitive function, expression of α7-nAChR protein, induction of LTP in hippocampus,
and serum cytokine levels.
MATERIALS AND METHODS
Animals
Adult male Sprague-Dawley rats (200±20 g, 10 weeks) were purchased from Shanghai SLRC
Laboratory Animal Co., Ltd. (Shanghai, China). Rats were housed under pathogen-free
conditions and maintained at controlled temperature (20-24°C) and humidity (40-70%)
on a 12h light/dark schedule, with food and water freely available. The experimental
procedures were approved by the Animal Ethics Committee of Zhoupu Hospital.
Post-stroke cognitive impairment model
Ischemic stroke model was established through MCAO/R in rats with minor modifications[13]. Briefly, animals were anesthetized with an intraperitoneal injection of sodium
pentobarbital (45 mg/kg) and their body were maintained at 37°C of temperature via
a thermal pad throughout the process. The midline neck incision was made for each
rat to expose the right common carotid artery, internal carotid artery, and external
carotid artery. The common carotid artery was ligated near the bifurcation of the
carotid artery. A lysine-coated nylon monofilament (0.32±0.02 mm) (Beijing Sunbio
Biotech Co., Ltd., Beijing, China) was inserted into right internal carotid artery
via the common carotid artery, and was gently advanced to the origin of the middle
cerebral artery. The filaments were pull out 1h later to restore blood flow (reperfusion).
Twenty-four hours after MCAO/R, all surviving rats underwent neurobehavioral examination
using a longa scoring system: score 0: no neurological deficit; score 1: completely
unable to extend right forepaw; score 2: rotated right; score 3: toward right; score
4: can not walk spontaneously and consciousness level declines. Rats with scores of
1-3 were considered successful MCAO/R model and then divided into standard environment
(SE) or EE housing condition according to the scores. Sham surgery received the same
procedures except for the filament insertion.
Experimental design
Rats were divided into 3 groups according to surgery and housing condition: (1) Control
group: sham surgery rats were housed in SE condition, n=16; (2) SE group: MCAO/R rats
were housed in SE condition, n=16; (3) EE group: MCAO/R rats were housed in EE condition,
n=16. In EE housing condition, 6-8 rats were housing together in a large cage (90
cm long × 75 cm wide × 50 cm high) with various objects, including climbing ladders,
wood platform, toys or tunnels of different shape and color. These subjects changed
3 times a week to keep novelty. Four rats were housed together in each SE housing
condition, which consisted of a standard cage (44 cm long × 32 cm wide × 20 cm high)
with no objects. To further investigate the role of α7-nAChR in LTP induction by EE,
nicotine (α7-nAChR activator, 0.5 mg/kg, i.p.) or α-BGT (α7-nAChR inhibitor, 1.0 μg/kg,
i.v.) were administered daily for 28 days according to a previously method[7], 4 rats for each administration. Rats were housed in EE or SE condition for 4 weeks.
Behavioral tests
Assessment of neurological function
Modified neurological severity scores (mNSS)[14] were evaluated on 7, 14, 28 days after MCAO/R to assess the degree of neurological
deficits. mNSS is a comprehensive test including motor, sensory, reflex and balance
tests and scaled from 0 to 18 score (score 0: normal; score 18: maximal deficit).
The higher scores reflect more severe deficits. The scores were evaluated by two researchers
who were blinded to the experiment grouping.
Morris water-maze test
Morris water-maze test was performed to measure the spatial learning and memory including
two phases. The apparatus consisted of a circular tank (160 cm in diameter and 60
cm in height) that was filled with water at 25±1°C to a depth of 30 cm, and a circular
hidden platform (12 cm diameter) was located in the middle of northeast quadrant of
the pool and 2 cm below the water level. The first phase involved space learning trainings
prior to experimental trail from 3 days after MCAO/R. Rats were randomly placed into
pool facing the wall of tank from southeast and southwest directions, and allowed
90 s to reach the hidden platform. Each rat received 4 trainings prior to trail per
day for 3 consecutive days. The second phase involved place navigation trial and probe
trial at 7, 14 and 28 days after MCAO/R. The place navigation trial was carried out
like space learning training and the duration reaching the platform was recorded as
the escape latency (maximum 90 s). In the probe trail, platform was removed from the
pool and the number of crossing over original platform position were recorded as platform
crossing times.
Quantification of infarct volume
At 28 days after MCAO/R, rats were decapitated, and the brains were quickly frozen
and sliced into coronal sections (2 mm thickness), and sections were stained with
1.2% 2,3,5-triphenyl tetrazolium chloride (TTC) (Sigma, St. Louis, Mucun, USA) at
37°C for 30 min, and then fixed in 4% paraformaldehyde overnight. Then, sections were
observed and photographed. The infarcted tissue showed unstained (white) and normal
tissue showed stained (red), and were analyzed using digital image analysis software
(SigmaScan Pro, Jandel, San Rafael, CA, USA). The percentage of infarct volume was
calculated using the following equation: percentage of infarct volume = (total infarct
volume/whole brain section volume) × 100%. The volume was quantified by summing areas
of sections multiplied by the section thickness (2 mm).
Expression of α7-nAChR in the hippocampus
Rats were decapitated at 28 days after MCAO/R, and the hippocampus was fixed overnight
with 4% paraformaldehyde overnight and embedded with paraffin wax to acquire serial
sections (5 μm thickness). After dewaxing, dehydration, incubation with 3% H2O2, and microwave antigen retrieval, the hippocampus serial sections were then incubated
with rabbit anti-α7-nAChR primary antibody (1:100, ab10096, Abcam, UK) at 4°C overnight.
After washing with phosphate buffered saline (PBS), sections were incubated with goat-anti-rabbit
IgG labelled with HRP as secondary antibody (1:100, ab6721, Abcam, UK) at room temperature
for 2h, and then were stained with diaminobenzidine (DAB) (Leica, Germany) and hematoxylin.
The DAB stained brown cells are α7-nAChR positive neurons, which were counted from
five random high magnification vision in CA1 region under a microscope (BX51, Olympus,
Japan). The cell density was average number of α7-nAChR positive neurons per high
magnification vision (cells/HP).
ELISA for cytokines and cholinergic proteins
Blood samples were obtained from caudal vein of rats at 7, 14 and 28 days after MCAO/R,
and the ipsilateral hippocampal tissue at 28 days after MCAO/R. Both serum and hippocampal
homogenates were separated by centrifugation at 5000 g for 10 min at 4°C to acquire
serum and hippocampal homogenates. ELISA assay kits were used to measure serum levels
of interleukin-1β (IL-1β), interleukin-6 (IL-6), neuron-specific enolase (NSE) and
brain-derived neurotrophic factor (BDNF) (R&D Systems, Minneapolis, MN, USA). Moreover,
the ACh content (A105-1), ChAT (A079) and acetylcholinesterase (AChE) (A024) activities
in the hippocampus were measured by commercial assay kits (Nanjing Jiancheng Biological
Engineering Institute, Nanjing, China). The values of above proteins were determined
by measuring the wavelength at 450 nm using a microplate reader (Ricso RK201, Shenzhen
Ricso Technology Co., Ltd, China).
Electrophysiology
Rats were decapitated at 28 days after MCAO/R, brains were rapidly removed and the
ipsilateral hippocampus region was cut into transverse slices (400 μm thickness).
Slices were continuously perfused with ice-cold ACSF composed of the following: NaCl
124 mM, CaCl2 2.0 mM, KCl 4.5 mM, MgCl2 1.0 mM, NaHCO3 26 mM, NaH2PO4 1.2 mM, D-glucose 10 mM, and pH 7.4. A bipolar electrode was inserted into the ipsilateral
Schaffer collaterals using microscope (Olympus BX50-wI,Olympus,Japan) to deliver the
orthorhombic stimulus by a stimulator (SEN-3301, Nihon Kohden, Japan), and a recording
electrode was inserted into the ipsilateral CA1 region to record the field excitatory
postsynaptic potential (fEPSP). Baseline responses were recorded for 20 minutes prior
to beginning the experiment with a constant current pulse (frequency 0.1 Hz, pulse
duration 0.25 ms). Then, LTP was induced by high frequency stimulation (HFS, 100 Hz).
LTP was recorded every 5 min for 120 min with the same stimulation intensity as pre-HFS.
To further investigated the role of α7-nAChR in LTP induction by EE, α-BGT (1.0 μg/kg,
i.v.) and nicotine (0.5 mg/kg, i.p.) were administered daily for 28 days.
Statistical analysis
All quantitative data were presented as mean±standard deviation (SD) and analyzed
by SPSS 19.0 statistical software (SPSS, USA). The differences among groups in Morris
water-maze test and serum cytokines were analyzed using two-way analysis of variance
(ANOVA) with repeated measures, with following Bonferroni test for multiple comparisons.
One-way ANOVA was applied to compare the differences in other data, followed by Bonferroni’s
post hoc. For statistically significance, p<0.05 was considered as criteria.
RESULTS
Enriched environment ameliorated cognitive deficits in ischemic stroke rats
At 28 days after MCAO/R, the infarct volume of EE group (38.13±1.44%; n=4) was slightly
smaller than SE group (40.48±2.69%; n=4). However, with no significant difference
(p=0.189) ([Figure 1A]). In terms of the functional recovery, mNSS scores were significantly decreased
by 16.5, 19.6 and 21.1% at 7, 14 and 28 days after MCAO/R, respectively, compared
with SE group (p<0.001; n=8 rats per group) ([Figure 1B]). In terms of learning and memory, data of Morris water-maze test suggested that,
compared with control group, escape latency time was significantly increased and crossing
platform times were significantly decreased in rats of SE group at different time
points. Moreover, results were reversed significantly by EE treatment (p<0.05; n=8
per group) ([Figures 1C and 1D]). These results indicate that EE is capable of alleviating cognitive impairment
induced by ischemic lesion.
Figure 1 Effects of enriched environment on infarct volume, neurological deficit scores and
cognitive impairment.(A) Representative photographs of brain slices that are stained
with TTC at 28 days after MCAO/R. The white areas are infracted tissue. EE slightly
reduces infarct size, with no significant difference (n=4 rats per group). (B) EE
significantly decreases mNSS score at 7, 14 and 28 days after MCAO/R. Date are expressed
as mean±SD. **p<0.01, ***p<0.001 vs. SE group of the same time points (n=8 rats per group). (C-D) EE significantly decreases
escape latency time and increases crossing platform times. Date are expressed as mean±SD.
***p<0.001 vs. control group of the same time points; #p<0.05, ##p<0.01, ###p<0.001 vs. SE group of the same time points (n=8 rats per group). SE: standard environment;
EE: enriched environment, mNSS: Modified neurological severity scores.
Enriched environment enhanced α7-nAChR expression and cholinergic pathway of hippocampus
The density of α7-nAChR in the hippocampal CA1 region was detected by immunohistochemistry.
Represent pictures were shown for the α7-nAChR positive neurons in control group ([Figure 2A, photo 1]), SE group ([Figure 2A, photo 2]) and EE group ([Figure 2A, photo 3]). Compared with the control group (46.75±4.88%), the cell density of α7-nAChR positive
neurons was decreased in SE group (18.08±3.16%; p<0.001), and then restored by EE
(36.43±2.73%; p<0.001 vs. SE group). It was then measured the ACh content, ChAT and AChE activities of hippocampal
tissues with ELISA. MCAO/R significantly decreased the ACh content (43.93%; p<0.001)
([Figure 2B]) and ChAT activity (49.10%; p<0.001) ([Figure 2C]), but increased AChE activity (153.83%; p<0.001) ([Figure 2D]) in the hippocampal tissues. Moreover, these changes were significantly reversed
by EE (ACh content, p<0.001; ChAT activity, p=0.003; AChE activity, p<0.001), n=4
per group. All these findings suggest that EE enhances α7-nAChR expression and cholinergic
pathway of hippocampus.
Figure 2 The effects of enriched environment on cholinergic pathway in the hippocampal tissue
of middle cerebral artery occlusion and reperfusion rats.(A) Representative images
of hippocampal α7-nAChR positive neurons in the hippocampal CA1 region are shown in
rats of control group, SE group and EE group at 28 days after MCAO/R (×200). Scale
bar=10 μm. EE increases the cell density of α7-nAChR positive neurons in the hippocampus
(p<0.05). EE increases ACh content (B) and ChAT activity (C), and decreases AChE activity
(D) in hippocampal tissue of MCAO/R rats. Date are expressed as mean±SD, ***p<0.001 vs. control group; ##p<0.01, ###p<0.001 vs. SE group. n=4 samples from 4 rats per group. ACh, acetylcholine; ChAT, choline acetyltransferase;
AChE, acetylcholinesterase.
Enriched environment suppressed proinflammatory cytokines, brain damage and improved
nerve regeneration
ELISA immunoassay were performed to detect the expression levels of serum proinflammatory
cytokines (IL-1β and IL-6) ([Figures 3A and 3B]), NSE ([Figure 3C]) and BDNF ([Figure 3D]) at 7, 14 and 28 days after MCAO/R. Compared with control group, the level of proinflammatory
cytokines and NSE were much higher (p<0.001) and BDNF was decreased significantly
(p<0.001) at all time points after MCAO/R. EE significantly attenuated MCAO/R-induced
change of proinflammatory cytokines, NSE and BDNF in serum, n=8 per group. These results
suggest that EE is capable to suppress proinflammatory cytokines, brain damage and
improve nerve regeneration.
Figure 3 Effects of enriched environment on serum cytokine levels in middle cerebral artery
occlusion and reperfusion rats.EE significantly decreases serum IL-1β (A), IL-6 (B)
and NSE (C) levels, and increases BDNF (D) level in rats at 7, 14 and 28 days after
MCAO/R. Date are expressed as mean±SD, ***p<0.001 vs. control group of the same time points; ##p<0.01, ###p<0.001 vs. SE group of the same time points. n=8 samples from 8 rats per group. NSE, c.
Enriched environment enhanced LTP in α7-nAChR dependent manner
To investigate the effect of EE on neural plasticity, hippocampal slices were stimulated
by HFS to measure LTP. In the 5 min after HFS, the fEPSP slope of control group sharply
increased compared baseline and then slowly decreased. EE could increase fEPSP slope
in MCAO/R rats, especially before 90 min compared with SE group ([Figure 4A]). This indicates that ischemic stroke induces obvious damage on LTP, while EE enhances
LTP after HFS. Average fEPSP amplitude at 60 min after HFS was significantly increased
by 20.50% with EE, compared with SE group (p=0.002) ([Figure 4B]), and this increase was further enhanced by cotreatment with nicotine (19.10%; p=0.002
vs. EE group), and attenuated by cotreatment with α-BGT (12.15%; p=0.026 vs. EE group) ([Figure 4C]), n=4 per group. These results suggest that EE enhances synaptic plasticity and
the effect is dependent on α7-nAChR.
Figure 4 The effects of enriched environment on long-term potentiation of middle cerebral
artery occlusion and reperfusion rats.(A) Time course and extent of LTP induction
in rats 28 days after MCAO/R. fEPSP amplitude is normalized to average baseline. (B)
EE increases the fEPSP slope. Date are expresses as mean±SD, ***p<0.001 vs. control group, ##p<0.01 vs. SE group. (C) The effects of EE on LTP are dependent on α7-nAChR. Average fEPSP
amplitude at 60 min after HFS by EE are enhanced by cotreatment with nicotine, and
attenuated by cotreatment with α-BGT. Date are expressed as mean±SD, *p<0.05, **p<0.01
vs. EE group. n=4 slices from 4 rats per group. α-BGT, α-bungarotoxin; LTP, long-term
potentiation; HFS: high frequency stimulation; fEPSP: field excitatory postsynaptic
potential.
DISCUSSION
The present study demonstrated effects and related mechanisms of EE in PSCI rats induced
by MCAO/R. EE significantly improves neurological function and attenuates cognitive
impairment in MCAO/R rats. In addition, α7-nAChR expression, hippocampal ACh content
and ChAT activity was increased due to EE, and AChE activity was decreased due to
EE, compared with SE group. EE also suppresses neuroinflammation, as evidenced by
reduced serum levels of IL-1β and IL-6. NSE levels and BDNF level were reversed by
EE after MCAO/R. Moreover, LTP was enhanced by EE in hippocampal slices, which was
further enhanced by nicotine and attenuated by α-BGT. Together, these results provide
evidence that EE has neuroprotective effects on PSCI rats. What’s more, the mechanisms
underlying this protection may be associated with enhanced expression of α7-nAChR,
suppression of inflammation, and enhanced induction of LTP.
No significant difference in infraction volume was observed between EE and SE groups
in our study, which is similar to recent studies in PSCI. For example, infarct volume
on day 31 after MCAO in EE group is also smaller slightly, but no statistically significant
difference compared with SE group[15]. This result is also observed at 14 days after MCAO[16], possibly due to that the neuronal death is irreversible. Moreover, studies also
found that pretreatment with EE for 2 or 5 weeks significantly decreases infract volume
induced by ischemic stroke in rats, suggesting that pretreatment with EE protect neuronal
death from stroke[17],[18]. However, a previous systematic review found that EE increases infract volume of
some degree without significantly statistic difference compared with SE group in two
third of stroke-related studies, possibly due to the stress of new environment and
cortical hyperexcitability[19]. Given that the small sample size in our study and no consensus on the effect of
infarct volume by EE in PSCI, the effect of EE on infarct volume still need further
research.
The anti-inflammatory effects of EE at different time point after stroke in this study
is in accordance with some previous studies. For example, at four weeks after ischemic
stroke, EE attenuates histopathological and oxidative damage to the brain, thereby
improving cognitive function in chronic cerebral hypoperfusion (CCH) rats[20]. In an earlier phase of stroke, EE significantly decreases the expression of IL-1βat
5d after MCAO[21]. These show similar results with our study and indicate that suppressed neuroinflammation
is an important mechanism of EE. The hippocampus is likely a good target of anti-inflammatory
responses given that hippocampus is the main brain area responsible for cognitive
function.
The present study shows that EE activates cholinergic pathway in MCAO/R rats. Agonists
of α7-nAChR has an improvement effect on cognition, while reduced α7-nAChR expression
has been found in various neurological disorders[22]. α7-nAChR is a major component of CAP and controls inflammation in central nervous
system[23]. In ischemic stroke, α7-nAChR activates CAP and suppresses neuroinflammation, thus
improving cognitive impairment[24]. These indicate that enhanced hippocampal α7-nAChR expression in our study may contribute
to inhibition of neuroinflammation and improvement of cognitive function. α7-nAChR
also might mediate the cognitive effect of EE. To our knowledge, our study is the
first report on the up-regulation of α7-nAChR by EE in ischemic stroke. However, the
intermediate links between EE and α7-nAChR remain largely unknown.
This study shows that EE increased ACh content and ChAT activity, and decreased AChE
activity in the hippocampus of MCAO/R rats. ACh is a neurotransmitter and are associated
with cognitive ability[25], who is regulated by two enzymes: ACh is synthesize by ChAT and degraded by AChE[26]. One report showed that acetylation of histones bound to the ChAT gene promoter
and cholinergic circuits are enhanced by EE in PSCI mice, which supports our study.
Increased AChE activity in the prefrontal cortex is associated with age-related cognitive
decline[27], while decreased AChE activity and enhanced memory are found in physically enriched
rats compared with rats reared in social environment[28]. These suggest that EE increases hippocampal ACh contents by increasing ChAT and
decreasing AChE activities, thereby improving cognitive function.
EE enhances LTP in MCAO/R rats in this study. LTP manifests persistent strengthening
of synapses and subsequent enhanced signaling between two neurons. Suppressed hippocampal
LTP can result in cognitive impairment[29]. Hippocampal LTP is reduced in rats of CCH and reversed by EE[30]. Enhanced LTP indicates higher synaptic plasticity, and might be associated with
increased BDNF by EE[31]. Furthermore, there is inter-regulation between BDNF and α7-nAChR. Choline could
induce higher α7-nAChR activity and then lead to increased BDNF level[32]. Conversely, BDNF up-regulated α7-nAChR levels on subpopulations of hippocampal
interneurons, which are important target cells that participate LTP[33],[34]. The regulatory role of α7-nAChR in LTP was supported by our study, which is that
LTP enhancement in hippocampal slices by EE was dependent on α7-nAChR.
In summary, EE demonstrates a marked preventive effect against cognitive impairment
of MCAO/R rats. The mechanism underlying EE may be associated with enhanced expression
of α7-nAChR, activation of CAP, and enhanced synaptic plasticity. The study reveals
that EE may be a promising therapeutic method for PSCI patients, and provides potential
possibility for the combination of EE with cholinergic activating agents.
