Chunling Zhao, Yan Chen, Chunlai Zhang, Linya Lü, Qian Xu

1Department of Physiology, Luzhou Medical College, Luzhou 646000, Sichuan Province, China

2Department of Pediatric Surgery, Affiliated Hospital of Luzhou Medical College, Luzhou 646000, Sichuan Province, China

lNTRODUCTlON

A large number of clinical studies demonstrate that obstructive sleep apnea syndrome (OSAS)can induce brain injury and permanently decrease cognitive function[1-3]. Although the precise mechanism by which OSAS induces cognitive impairment in unknown, chronic intermittent hypoxia (CIH) has been shown to be involved in this process. CIH can induce damage in multiple systems. The hippocampus, essential for the regulation of spatial learning and memory processes, is the most sensitive brain region to hypoxia[4].

Recent evidence from experimental animals shows that CIH-induced impairments of learning and memory correlate with down-regulation of neuronal acetylcholine(ACh) in related brain areas[5-7].

Pharmacological experiments have demonstrated learning and memory deficits after anticholinergic treatments[8-9].

Hippocampal ACh release increases in cue-rich condition training compared with that in cue-poor condition training, and high-altitude hypoxia or brain ischemia can induce cognitive impairment and reductions of neuronal ACh release[10-12]. ACh is intimately involved with learning and memory processes.

Choline acetyl transferase (ChAT), a rate-limiting enzyme during ACh synthesis,can be used to mark cholinergic neurons and to indirectly reflect levels of ACh. ACh functions by binding to its receptors;muscarinic acetylcholine receptors and nicotinic acetylcholine receptors (nAChRs) are the two main kinds of ACh receptors. Recent studies have demonstrated an important role of nAChR in cognitive function[13-14]. Alpha4 is a major nAChR subunit (nAChRα4), which is mainly distributed in the hippocampus, cortex and other learning and memory-associated brain areas. Decreased levels of nAChRα4in the brain correlate with cognitive function disorders, such as Alzheimer’s disease and Parkinson’s disease[15-17], indicating a role of nAChRα4in cognitive function.

Very limited data are available regarding changes in nAChR expression with changes in ACh expression during CIH-induced cognitive impairment. We hypothesized that after CIH-induced cognitive impairment,morphological changes in hippocampal neurons would be related to cholinergic system changes and that changes in nAChR expression would correlate with decreases in ACh after CIH-induced cognitive impairment.

Therefore, in the present study we established a CIH model in rats to simulate the characteristics of OSAS and we observed changes in cognitive function, hippocampal neurons and the cholinergic system.

RESULTS

Quantitative analysis of experimental animals

Twenty Sprague-Dawley rats were randomly assigned to control and model groups. Model group rats were placed in a hypoxia cabin to induce CIH. Control group rats were placed in the same cabin with air. All 20 rats were included in the final analysis.

Effects of ClH on spatial cognitive function

The Morris water maze was used to assess place navigation and spatial probing abilities. Compared with the control group, the latency to escape was significantly prolonged on training days 1, 2 and 3, after 5 weeks of CIH (P ＜ 0.05; Figure 1). In the spatial probing test, the percentage of time spent in the target quadrant(northwest, the original quadrant) was significantly less in the CIH group compared with the control group (P ＜0.05), indicating decreased ability of CIH rats to search the target quadrant compared with control rats. The percentage of time spent in the northeast quadrant was significantly increased in the CIH group compared with the control group (P ＜ 0.05; Table 1).

Figure 1 Latency of escape in place navigation test between control and chronic intermittent hypoxia (CIH)groups. Learning and memory was decreased in the CIH group. aP ＜ 0.05, vs. control group. Data are expressed as mean ± SD and there are 10 rats in each group.

Table 1 Percentage of the time spent in each quadrant for control and chronic intermittent hypoxia (CIH) groups in the spatial probing test

Effect of ClH on hippocampal morphology

Conventional hematoxylin-eosin staining revealed no pathological changes in hippocampal neurons in control rats at 5 weeks. However, degeneration and necrosis of hippocampal neurons, manifested by cell swelling,vacuolization, nuclear shrinkage and darkly stained cell bodies, were observed in the CIH group. The degree of neuronal injury varied among the different regions of the hippocampus (CA1, CA2, CA3, CA4 and dentate gyrus),with injury being most prominent in the dentate gyrus(Figure 2).

Figure 2 Histopathological changes of the hippocampal dentate gyrus at 5 weeks, observed by light microscopy(hematoxylin-eosin staining, × 400).

Effects of ClH on the ChAT and nAChRα4 expression in the hippocampus

Immunohistochemistry showed ChAT staining,brown-yellow granules, in the cytoplasm but not in the nucleus, which was stained blue by hematoxylin.ChAT-positive cells were observed in both groups. No staining was observed in the negative control. Compared with the control group (absorbance value: 0.27 ± 0.04),levels of hippocampal ChAT were significantly reduced in the CIH group (absorbance value: 0.18 ± 0.03, P ＜ 0.05;Figure 3).

Figure 3 Choline acetyl transferase (ChAT) expression in hippocampal CA3 at 5 weeks (immunohistochemical staining, × 400).ChAT expression was manifested as brown-yellow stains in the cytoplasm. Compared with the control group (A), the chronic intermittent hypoxia group (B) presented lightly stained cytoplasm and a weaker level of expression. Arrow represents ChAT-positive cells. ChAT-positive expression in CA1, CA2 and dentate gyrus is shown in supplementary Figures 1 and 2 online.

nAChRα4staining, brown-yellow particles, was mainly located in the cell membrane, with a small amount in the cytoplasm. The nucleus was stained blue by hematoxylin.

nAChRα4-positive cells were observed in both groups.

No staining was observed in the negative control.

Hippocampal nAChRα4expression (absorbance value:0.30 ± 0.06) was significantly less in the CIH group compared with the control group (absorbance value:0.44 ± 0.12, P ＜ 0.05; Figure 4).

Figure 4 Nicotinic acetylcholine receptor α4 (nAChRα4)expression in hippocampal CA3 at 5 weeks(immunohistochemical staining, × 400).nAChRα4 expression, brown-yellow staining, was mainly located in cell membranes, with a small amount of staining in the cytoplasm.Compared with the control group (A), the chronic intermittent hypoxia group (B) had a more lightly stained cytoplasm and weaker expression in hippocampal CA3.Arrow represents nAChRα4 positive cells. nAChRα4 expression in the CA2 region is shown in supplementary Figure 3 online.

DlSCUSSlON

Hypoxia is a major cause of cognitive impairment in OSAS patients; however, the mechanism by which hypoxia causes this effect this remains poorly understood. The central nervous system is the most sensitive organ of the body to hypoxia and hypoxia can induce dysfunction in learning, memory, behavior and emotion. The cholinergic system is involved in neuroprotection and in learning and memory functions and increasing numbers of animal studies have focused on the relationship between the cholinergic system and cognitive function[6,13-15].

The present study examined the spatial learning and memory abilities of rats using the Morris water maze test.

The latency to escape of rats in both control and CIH groups reduced over the 7 days of training, which indicates that rats in both groups have a certain degree of learning and memory ability. In the place navigation and probe searching tests, the latency at 1, 2 and 3 days was prolonged for CIH animals, while the percent of time spent in the target position was shortened for CIH rats compared with controls, suggesting that CIH can lead to cognitive impairment in rats. These results are consistent with previous studies in mice[16].

In addition, decreased hippocampal ChAT and nAChRα4expression was observed in the CIH group, indicating CIH-induced cognitive impairment my correlate with changes in the hippocampal cholinergic system. The decrease in hippocampal ChAT and nAChRα4expression may be because CIH induced hippocampal neuronal injury, thereby affecting ChAT and nAChRα4expression. In addition, CIH has been shown to induce a reduction in ACh release, resulting in a decrease in nAChRα4expression[18]. However, nAChR regulation is a very complex process involving multiple factors, such as desensitization, receptor functional deactivation, and cholinergic afferent fiber loss[16,19]. Therefore, further studies are required to thoroughly demonstrate the mechanism.

In conclusion, CIH exposure-induced cognitive impairment may associate with hippocampal neuronal degeneration and necrosis and with reduced expression of ChAT and nAChRα4.

MATERlALS AND METHODS

Design

A randomized, controlled, animal experiment.

Time and setting

This experiment was performed at the Laboratory of Physiology and Pathology, Luzhou Medical College,China from May 2007 to April 2008．

Materials

Twenty male, healthy, Sprague-Dawley rats, weighing 230-270 g, were provided by the Animal Department of Luzhou Medical College (License No. SYXK 2004-065).

The rats were all housed in the same conditions and allowed free access to food and water. Experimental protocols were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[20].

Methods

CIH model establishment and intervention

The 20 rats were equally and randomly assigned to control and CIH groups. The CIH model was established according to a previously described method with modifications[21-22]. Briefly, CIH group rats were placed in an in-house built polycarbonate cabin containing silica-gel to absorb moisture and soda lime to absorb CO2and vapor. There were several holes in the cabin wall for maintaining atmospheric pressure inside the cabin. The cabin was filled with nitrogen gas and the oxygen concentration was continuously measured using an O2analyzer. O2concentration was maintained at 8-10%. CIH group rats were exposed to hypoxia in the cabin 8 hours every day for 5 weeks. Rats in the control group were placed under the same conditions as the CIH group except they were not exposed to hypoxia.

Behavioral testing using the Morris water maze

The Morris water maze test was performed for 7 consecutive days for both groups, and consisted of three experiments as follows. (1) Place navigation test(platform in water): the maze was divided into four quadrants, northwest, northeast, southwest and southeast. A platform was submerged 2 cm below the water surface in the middle ring of the northwest quadrant. The rats were placed into the water in each quadrant. The swim path and time taken to find the platform (latency of escape, maximum 120 seconds)were recorded. Rats were allowed to stay on the platform for 30 seconds, and were then removed for another trial 4 minutes later. The test was performed for 5 consecutive days. (2) Place navigation test (platform above water): the experiments were conducted in the same way as for the test with the platform in water except that the platform was 2 cm above the water; (3)Spatial navigation test (no platform): the test was performed 4 hours after the place navigation test. The platform was removed and the memory of the platform position was evaluated by placing the rats in the southeast and southwest quadrants, which were far away from the original platform location quadrant. The rat was permitted to explore the tank for 60 seconds. There was a 4 minute inter-trial interval between two trials. The duration of time spent in various quadrants and the swim path were recorded. In the place navigation test, the mean value of latency of four quadrants was used as the latency of one rat at that day.

Hippocampal histopathology

Rats were anesthetized with 4% ether (inhalational anesthesia) and rapidly transcardially perfused (through the left ventricle) with normal saline, followed by 4%paraformaldehyde (500 mL) for 40 minutes. The brains were removed immediately, cut coronally into three blocks at the juncture of the posterior optic chiasm,cerebral peduncle and pons, and fixed in 4%paraformaldehyde for 4 additional hours. The brain tissues were routinely dehydrated with a graded series of alcohol, cleared with xylene, embedded in paraffin and sectioned (4 μm thickness). Morphological changes in the hippocampus were observed using a light microscope (Olympus, Tokyo, Japan) after hematoxylin-eosin staining.

Immunohistochemical staining of hippocampal ChAT and nAChRα4

Serial sections were prepared (4 μm thickness, two serial sections every seven sections), deparaffinized and rehydrated, and then immunohistochemically stained using ChAT or nAChRα4 polyclonal antibodies.

Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) was used to analyze results. Three high-power fields of view (× 400) were randomly selected from dentate gyrus, CA1, CA2, CA3 and CA4 regions of the hippocampus, and absorbance of positive staining was determined and averaged.

Immunohistochemistry was performed as follows. After deparaffinization and rehydration, sections were washed with PBS (5 minutes × 3), immersed in 3% H2O2for 10 minutes at room temperature, washed with PBS(5 minutes × 3), immersed in warm sodium citrate buffer for 15 minutes, washed with PBS (5 minutes × 3) and then blocked with normal goat serum for 20 minutes at room temperature. 50 μL rabbit anti-ChAT (1: 200;Shanghai Lenton Bioscience, Shanghai, China) or rabbit anti-nAChRα4(1: 200; Boster, Wuhan, China) polyclonal antibodies were added and incubated overnight at 4°C(PBS was added to the negative control). The sections were rewarmed to 37°C for 45 minutes, washed with PBS (2 minutes × 3), incubated with reagent 1 of ready-to-use-two-step non-biotin detection kit (50 μL,1: 1; Zhongshan Goldenbridge, Beijing, China) at 37°C for 20 minutes, washed with PBS (2 minutes × 3),followed by incubation with horseradish peroxidase-labeled anti-rabbit IgG (50 μL, 1: 1;Zhongshan Goldenbridge) at 37°C for 20 minutes. The sections were then developed with diaminobenzidine for 5-10 minutes, washed with tap water for 10 minutes,hematoxylin counterstained for 2 minutes, differentiated in hydrochloric acid-alcohol, washed with tap water, dried in a graded series of alcohol, cleared with xylene and mounted with neutral gum.

Statistical analysis

Data are expressed as the mean ± SD and were analyzed using SPSS 13.0 software (SPSS, Chicago, IL,USA). Repeated measures analysis of variance[23]was used for comparing differences in the latency of escape among different days. Independent-sample t tests were used for intergroup comparisons. The t-test (two-tailed)was used for other data analysis. A P value ＜ 0.05 was considered statistically significant.

Author contributions:Chunling Zhao conceived and designed this study, wrote the manuscript and provided and integrated data. Yan Chen also designed this study and provided,integrated and analyzed data. Chunlai Zhang provided experimental data and technical support. Linya Lü conceived and guided this study and provided experimental data and technical support. Qian Xu provided experimental data.

Conflicts of interest:None declared.

Funding:This study was supported by a grant from Luzhou Medical College, No. 2009.

Ethical approval:This study received permission from the Animal Care and Research Committee of Luzhou Medical College, China.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 17, 2011 after selecting the “NRR Current Issue” button on the page.

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