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© 2008 American Society for Nutrition J. Nutr. 138:1165-1171, June 2008

Ingestive Behavior and Neurosciences

Fish Oil Supplementation of Control and (n-3) Fatty Acid-Deficient Male Rats Enhances Reference and Working Memory Performance and Increases Brain Regional Docosahexaenoic Acid Levels1–3,

Wan-Ling Chung, Jen-Jui Chen and Hui-Min Su*

Department of Physiology, National Taiwan University College of Medicine, Taipei 100, Taiwan

* To whom correspondence should be addressed. E-mail: hmsu1203{at}ntu.edu.tw.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Most previous studies have focused on improved reference memory and recovery of whole brain docosahexaenoic acid [DHA, 22:6(n-3)] levels in DHA-deficient animals supplemented with fish oil (FO) or switched to an adequate DHA-enriched diet. The aims of this study were to determine whether reference and working memory performance can be enhanced in control male rats and improved in (n-3) fatty acid-deficient male rats given an FO supplement and whether brain DHA accumulation, deficiency, and recovery are region specific. From the embryo to postnatal d 140, 4 groups of rats were fed a nonpurified or sunflower oil-based (n-3) fatty acid-deficient diet alone or supplemented with (n-3) fatty acids from FO representing ~0.3% of the energy source. The male rats were tested at postnatal d 102–130 for spatial learning memory performance in the Morris water maze. The fatty acid composition of different brain regions was analyzed by GC. Rats fed the (n-3) fatty acid-deficient diet showed significantly poorer reference and working memory, and FO supplementation partially rescued both memory performances. Furthermore, FO supplementation during brain development and adulthood in normal rats resulted in significant enhancement of both memories. Following dietary DHA repletion, the hippocampus and olfactory bulbs accumulated more DHA, were more resistant to dietary DHA deprivation, and showed better DHA recovery than the visual cortex, frontal cortex, and cerebellum. These results suggest that DHA is critical for the development and maintenance of learning memory performance.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Docosahexaenoic acid [DHA, 22:6(n-3)]4 is essential for normal neurological function (1). In the mammalian brain, lipids make up 10% of the fresh weight and 50% of the dry weight, and DHA and arachidonic acid [20:4(n-6)] are the major PUFA in neuronal membranes (2). Most DHA accumulation in the brain takes place during brain development from the beginning of the 3rd trimester of gestation to 2 y after birth in humans and from prenatal d 7 to postnatal d 21 in rats (3–5). However, brain DHA levels reduce with age (6,7) and are reduced in the brain disorder Alzheimer's disease (AD) (8,9). Deficiency of brain DHA is associated with reduced learning ability and cognition in rats (1) and with memory loss in AD patients (10).

Most previous studies have focused on whole brain DHA recovery and improved reference memory in DHA-deficient animals switched to an adequate (n-3) fatty acid diet or supplemented with fish oil (FO). Progression in AD patients and cognitive decline in elderly men are delayed by FO consumption (11–13). In studies of DHA supplementation in an AD mouse model or aged animals, learning memory performance was improved by DHA (14–17). Reduced brain DHA levels are associated with poor spatial reference memory performance (18), which is not restored by a compensatory increase in docosapentaenoic acid [22:5(n-6)] levels in the brain (19), whereas recovery of brain DHA levels in DHA-deficient rats leads to recovery of reference memory (20). However, it is not known whether learning memory can be enhanced in normal animals by FO supplementation. Furthermore, although brain DHA accumulation is region specific, information is lacking on DHA loss and recovery.

Because of the motivation of escaping from the water as quickly as possible, the Morris water maze is a good tool for assessing spatial learning memory performance (21,22). The water maze also allows many aspects of behavior performance to be studied. Two types of memory, reference memory and working memory, are tested. Spatial reference memory is a standard task involving finding a submerged platform located at the same position in different sessions. In contrast, working memory is a task involving finding a submerged platform located at different positions in different sessions (23). Because most previous studies have examined spatial reference memory in DHA-deficient animals (20), it is important to determine whether spatial reference memory or working memory can be enhanced in normal animals or improved in DHA-deficient animals by FO supplementation.

This study was designed to evaluate whether FO supplementation improved reference and working memory not only in DHA-deficient rats but also in normal rats fed a sunflower oil-based (n-3) fatty acid-deficient diet or nonpurified diet alone or supplemented with (n-3) fatty acid-rich FO from the embryo through postnatal d 140. We also determined whether brain DHA deficiency, recovery, and accumulation were region specific.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Animals and study design. Pregnant Sprague-Dawley rats at 2 d of gestation were obtained from BioLasco Taiwan, a technology licensee of Charles River Laboratories in Taiwan, and were immediately randomly assigned to 3 experimental diets (study design is shown in Fig. 1). The pregnant dams were fed a normal nonpurified diet (5001, LabDiet) [control (C group)], nonpurified diet supplemented by oral gavage with 0.15 mL of FO (180 mg of eicosanoid acid [20:5(n-3)] and 120 mg of DHA in 1 mL of FO, Leiner Health Products) (C+FO group), or a sunflower oil-based (n-3) fatty acid-deficient diet prepared in our laboratory (D group). Only male pups were used, so as to avoid the hormonal effect of the estrus cycle on spatial learning memory performance (24,25). After weaning at postnatal d 21, the C group pups were maintained on the same diet as their dams throughout the study; the C+FO pups were maintained on the nonpurified diet until postnatal d 60, then on the same diet supplemented with 0.1 mL of FO by oral gavage once a day until they were killed at postnatal d 140. The D group was maintained on the same diet as the dams to postnatal d 60, when one-half were assigned to the D+FO group and received the sunflower oil-based (n-3) fatty acid-deficient diet supplemented with 0.1 mL of FO as above, while the other one-half continued on the same diet. To avoid bias due to additional interaction between the rats and the experimenter, each rat in the C group and the remainder of the D group were given 0.1 mL of water by oral gavage.


Figure 1
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  		FIGURE 1  Study design for rats fed a nonpurified diet or sunflower oil-based (n-3) fatty acid-deficient diet alone or supplemented with FO or water. C, Nonpurified diet for dam and pup; C+FO, nonpurified diet plus FO, where indicated, for dam and pup; D, (n-3) fatty acid-deficient diet for dam and pup; D+FO, as for the D group, but supplemented with FO from age 60 d. The spatial reference and working memory tests were performed at 102 d of age and memory consolidation was measured at 130 d old in the Morris water maze. The rats were killed at 140 d of age and the hippocampus, olfactory bulb, visual cortex, frontal cortex, and cerebellum were removed.

 
Spatial learning memory performance (spatial reference and working memory) was measured at postnatal d 102 after 6 wk of FO or water supplementation. All rats were housed in a humidity-controlled room at 24 ± 1°C on a 12-h-light/-dark cycle with free access to tap water and diet. The protocols and animal treatments used in this study were approved by the Animal Care and Use Committee of the National Taiwan University College of Medicine.

    Diet composition. The (n-3) fatty acid-deficient diet (Table 1) containing 20% of oil by weight was modified from the AIN 76 purified diet to maintain the same nutrition density (26); the oil used was sunflower oil containing 60% linoleic acid [18:2(n-6)] and no (n-3) fatty acids (Table 2). All diet ingredients were obtained from MP Biomedicals, except the methionine and choline, which were from Sigma-Aldrich, and the sunflower oil, corn starch, and sucrose, which were purchased from a local supermarket.


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  		TABLE 1 Ingredients of the (n-3) fatty acid-deficient and nonpurified diets

 

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  		TABLE 2 Fatty acid composition of the diets and FO1

 
    Morris water maze test. The Morris water maze was used to examine spatial learning memory performance. The apparatus and procedure were modified from previous studies (22,27). The maze consisted of a circular swimming pool (55 cm high and 183 cm diameter) with 3 posters of different colors and shapes on the wall and was filled to a height of 35 cm with tap water (22 ± 2°C) stained with powdered milk. A circular (11 cm diameter) transparent platform placed in the middle of 1 quadrant of the pool was submerged 1.5 cm below the water surface. The starting position for each trial was a different randomized spot. The rat was released with its head pointing toward the side of the pool and the time required for it to reach the submerged platform was recorded as the escape latency using a timer. If the rat did not find the submerged platform within 120 s, it was led to it and allowed to remain there for 15 s.

For the spatial reference memory test, the submerged platform was always located at the same position and the rats underwent 4 trials per session and 5 sessions in a 3-d period with at least a 3-h rest between sessions. The mean escape latency for all rats in each group in each session was calculated as the spatial reference memory for that session.

One day after the spatial reference memory test, working memory was measured as the ability to find a submerged platform located at different positions in different sessions, the rats undergoing 12 trials per session for 3 sessions on 3 d. We calculated the mean escape latency for each rat for each trial number (e.g. trial 1) for the 3 sessions and used this as the working memory performance for that trial number. Immediately after the working memory performance, a visual test was performed to determine whether the rat could find a visible platform with a flag located 5 cm above the water surface in 30 s; rats that failed this test were excluded from the study.

Memory consolidation was evaluated as the mean escape latency in 4 trials performed 3 wk after the last water maze test with the platform located at the same position as in the reference memory test. The rats were killed on postnatal d 140, 10 d after the memory consolidation test to avoid effects of intensive swimming on biochemical assays.

    Lipid analysis. The rats were anesthetized with CO2 and decapitated. Blood was collected in heparinized tubes and plasma prepared immediately by centrifugation. The brain was rapidly removed and the hippocampus, olfactory bulb, frontal cortex, visual cortex, and cerebellum dissected on ice, frozen in liquid nitrogen, and stored in a –80°C freezer until analysis. Total lipids were extracted from aliquots of tissue homogenate according to the method of Blight and Dyer (28), dried with nitrogen gas, and then, as described previously (29), were converted to their methyl esters and analyzed by Hewlett-Packard 5890 GC using flame ionization detection on a DB-1 fused silica capillary column (60 m x 0.25 mm x 0.1 µm, Agilent) with nitrogen as carrier gas. The oven temperature program was set at 60°C for 2 min, then increased by 10°C per minute to 170°C, then by 3°C per minute to 270°C, and finally held at 270°C for 15 min. The fatty acid peaks were identified by comparison of the retention times with those of a standard mixture of 68A (Nu-Chek Prep), 37 FAME, PUFA2, and PUFA3 (all from SUPELCO). The fatty acid composition was expressed as the weight of a percentage of the total weight of carbon 14 to carbon 22 fatty acids (wt%).

    Statistical analysis. Data are presented as the means ± SEM. Statistical differences between the 4 groups and the main effects were tested by 2-way ANOVA. When the interaction was significant, comparisons among groups were performed using Duncan's multiple range tests. The reference and working memory data were tested by 2-way ANOVA for repeated measures and further analyzed by multiple marginal regression models for repeated measures of response variable using the generalized estimating equations (GEE) method to examine differences between the 4 groups over learning sessions or trials and the effects of the treatments on the 4 groups. Basic model-fitting techniques for regression analysis, including variable selection, goodness-of-fit assessment, and regression diagnostics, were used to ensure the quality of the analysis results. If the first-order autocorrelation [i.e. AR(1)] structure fit the repeated-measures data well, the model-based SEE were used in the GEE analysis; if not, the empirical standard error estimates were reported. These regression analyses were performed using the Reg and GenMod procedures in the SAS statistical software (version 9.1, SAS Institute). Two-sided P < 0.05 was considered significant.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Spatial learning memory performance. Spatial learning memory performance was evaluated using the Morris water maze in which the rats needed to remember the position of a submerged platform. The escape latency was the time taken for the rat to climb onto the submerged platform. The effect on reference and working memory of FO supplementation of C and (n-3) fatty acid-deficient rats is shown in Figure 2.


Figure 2
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  		FIGURE 2  Effect of FO supplementation of the nonpurified or (n-3) fatty acid-deficient diet on spatial learning memory in the Morris water maze. The time required for rats to reach the submerged platform was measured as the escape latency, presented as the means ± SEM, n = 9–11. (A) Spatial reference memory in a standard task to find a submerged platform located at the same position, with 4 trials per session for 5 sessions. The mean escape latency in each session was calculated as the spatial reference memory. (B) Working memory was evaluated as a memory-relevant information ability to find the submerged platform at different locations in different sessions, with 12 trials per session for 3 sessions. The mean escape latency in each trial (e.g. trial 1) over the 3 sessions was calculated as the working memory performance. Groups without a common letter differ, P < 0.05. The detailed statistical analysis is presented in Supplemental Tables 1 and 2.

 
Reference memory was evaluated by the time required for the rats to find a submerged platform located at the same position, with 4 trials per session for 5 sessions. Two-way ANOVA for repeated measures revealed main effects of both session (P < 0.0001) and group (P < 0.0001) with a session x group interaction (P = 0.01). The repeated measures data were further analyzed by marginal regression models using the GEE method to assess the effects of treatment, session, and trial on the escape latency (the detailed result of the final regression model is presented in Supplemental Table 1). In short, the rats in all 4 groups gradually learned the task, but their learning speeds varied among the groups. The sessions 1 and 2 together were the reference sessions for comparison. Conditioning on the effects of the other covariates in this multiple regression model, the escape latency decreased (P < 0.0001) in sessions 3 (Formula = –25.8) and 4 (Formula = –66.6), with no further decrease in session 5 (Formula = –64.5). We chose the C group as the reference group for comparison. Conditioning on the other covariates, the escape latency was shorter in the C+FO group (Formula = –10.6; P = 0.043) than in the C group and was longer in the D+FO (Formula = 13.3; P = 0.0047) and D groups (Formula = 40.5; P < 0.0001) than in the C group. In addition, the escape latency was longer in the D group than in the D+FO group (P < 0.0001). Moreover, the escape latency was shorter in the D group in the 5th session after the learning in the previous 4 sessions. In contrast, the escape latency was particularly shorter in the C+FO group in the 2nd session after the learning in the first session. These results show that reference memory was enhanced in C rats and partially improved in (n-3) fatty acid-deficient rats supplemented with FO.

Working memory was assessed by the time required for the rats to find a submerged platform at different locations in each session, with 12 trials per session and 3 sessions. As with reference memory, 2-way (trials and groups) ANOVA with repeated measures revealed significant main effects of both trial (P < 0.0001) and group (P < 0.0001) with a trial x group interaction (P = 0.0114). When followed by marginal regression analysis using the GEE method, conditioning on the other covariates, escape latency decreased from trail 2 to the last trial (Formula = –7.1 to –23.2; P < 0.01) (Supplemental Table 2), showing that the rats in all 4 groups learned the skill. We chose the C group as the reference group for comparison. Conditioning on the other covariates, the escape latency for the C+FO group was shorter than that for the C group (Formula = –4.7; P = 0.0007), whereas the escape latencies for the D+FO (Formula = 20.1; P < 0.0001) and D groups (Formula = 35.4; P < 0.0001) were longer than those for the C group over the trials. In addition, the escape latency in the D+FO group was less than that in the D group (P = 0.0003). Working memory was therefore also enhanced in C rats and partially improved in (n-3) fatty acid-deficient rats supplemented with FO.

Memory consolidation was measured at 3 wk after the last reference memory test, with the submerged platform in the same position. The mean escape latencies in the 5th session in the reference memory and memory consolidation tests did not differ (P = 0.11) and were, respectively, 9 s and 11 s in the C+FO group, 18 s and 25 s in the C group, 28 s and 35 s in the D+FO group, and 33 s and 46 s in the D group. Two-way (diet and FO) ANOVA revealed main effects of both diet (P = 0.0001) and FO supplementation (P = 0.0024) with no diet x FO interaction (P = 0.5579).

The groups did not differ in escape latency in the visual test, in which all rats climbed onto the visible platform within 30 s, indicating no difficulties with visual and motor performance in the water maze.

    DHA levels in different brain regions. Although the diet ingredients (Table 1) and dietary fatty acid composition (Table 2) differed, body weight did not differ between the 4 groups either at postnatal d 102 when rats performed the water maze or at postnatal d 140 when they were killed (data not shown).

As shown by the major PUFA composition analysis (Table 3, levels of other fatty acids are presented in Supplemental Table 3), feeding the (n-3) fatty acid-deficient diet from the embryo throughout the entire life caused depletion of brain DHA. The greatest DHA depletion in the D group was seen in the frontal cortex and visual cortex (75 and 68% loss, respectively), followed by the cerebellum (61% loss), hippocampus (60% loss), and olfactory bulb (51% loss). DHA levels in the 5 brain regions in the D group were 3.8–6.9% of total fatty acids compared with 11.1–17.7% in the C group. 22:5(n-6) levels increased to 6.7–8.2% of total fatty acids in the D group compared with <0.5% in the C group. In the hippocampus and olfactory bulb, the reduced DHA levels in the D group were completely compensated by 22:5(n-6), the sum of the 22:5(n-6) and DHA levels being similar in the D and C groups. However, in the frontal cortex, visual cortex, and cerebellum, the sum of the 22:5(n-6) and DHA levels was significantly lower in the (n-3) fatty acid-deficient diet groups than in the nonpurified diet groups.


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  		TABLE 3 Major PUFA composition of different brain regions in rats fed the nonpurified diet or the (n-3) fatty acid-deficient diet with or without FO supplementation12

 
The DHA-deficient rats that received FO from postnatal d 60 for 80 d (D+FO group) showed considerable recovery of brain DHA levels. The greatest recovery of DHA levels was in the hippocampus (92% of C group levels) and olfactory bulb (91%), followed by the cerebellum (74%), visual cortex (65%), and frontal cortex (61%). Recovery of brain DHA levels was associated with reduced 22:5(n-6) levels from 7–8% to 1–3% of total fatty acids in the 5 brain regions. Again, in the hippocampus and olfactory bulb, the sum of the 22:5(n-6) and DHA levels was the same as in the C group, whereas in the frontal cortex, visual cortex, and cerebellum, it was significantly lower in the D+FO group than the C group.

FO supplementation during brain development and adulthood (C+FO group) resulted in significantly greater DHA accumulation in the hippocampus and olfactory bulbs but not in the frontal cortex, visual cortex, and cerebellum. DHA levels were ~27–29% higher in the hippocampus and olfactory bulb in the C+FO group than the C group but did not change in the frontal cortex, visual cortex, and cerebellum.


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Most previous studies have focused on whole brain DHA recovery and improved reference memory in DHA-deficient animals given an FO supplement. In this study, we compared the D+FO and D groups and found that recovery of brain DHA levels significantly improved both spatial reference and working memory performance. Moreover, comparison of the escape latencies in the C and C+FO groups showed that FO supplementation of normal rats also enhanced spatial reference and working memory. The hippocampus and olfactory bulbs showed faster DHA recovery, a greater capability for DHA accumulation, and more resistance to DHA deprivation than the frontal cortex, visual cortex, and cerebellum.

The energy intakes of the nonpurified diet and the (n-3) fatty acid-deficient diet were estimated to be similar. During adulthood, the intake of the nonpurified diet, with an energy density of 12.6 kJ/g, was ~24 g/d, whereas the intake of the (n-3) fatty acid-deficient diet, with an energy density of 18.8 kJ/g, was ~17 g/d. According to our analysis of the FO, the supplemented (n-3) fatty acids provided by FO accounted for ~0.26–0.28% of the energy source in this study. This amount of (n-3) fatty acid supplementation for 80 d in adulthood resulted in recovery of brain regional DHA levels to 61–92% of those in the C group and rescued reference and working memory performance compared with the DHA-deficient rats. However, the rescued memory performance did not reach the same level as in C rats and the escape latency in the reference memory (Formula = 13.3; P < 0.001) and working memory (Formula = 20.1; P < 0.0001) tests was still significantly higher in the D+FO group than in the C group. It would be interesting to know whether full brain DHA recovery would be associated with full rescue of memory performance. (n-3) Fatty acid supplementation during brain development and during 80 d of adulthood induced more DHA accumulation in the hippocampus and olfactory bulb and enhanced memory performance. Whether these effects of FO supplementation occur only in adulthood requires further study.

Neurons originating in the subventricular zone travel to the olfactory bulb where they differentiate into mature neurons (30). The olfactory bulb is a unique area that can be easily dissected. However, the subventricular zone is situated along the lateral wall of the lateral ventricle and is not easy accessible and defined for dissection. We therefore analyzed the olfactory bulb to represent adult neurogenesis in the subventricular zone-olfactory bulb system. The frontal and visual cortexes are the sites for working memory and sense of vision, respectively, and are important for memory performance (31,32), whereas the cerebellum is the region for motor control related to water maze performance. This is why we analyzed these brain regions in this study. The hippocampus and olfactory bulbs were more effective in DHA accumulation, more resistant to DHA deprivation, and showed better DHA recovery after dietary DHA repletion than the visual cortex, frontal cortex, and cerebellum. This suggests that brain areas capable of undergoing neurogenesis, such as the hippocampus and olfactory bulb, may have a greater ability to accumulate or retain DHA. This finding is strengthened by 2 recently published studies demonstrating that neurogenesis is decreased in the DHA-deficient embryonic rat brain (33) and upregulated in the brains of juvenile lobsters fed a (n-3) fatty acid-enriched diet (34), suggesting that (n-3) fatty acids enhance both prenatal and adult neurogenesis. In adult rodents, the hippocampus and olfactory bulb are the only 2 brain regions in which continuous neurogenesis is present (35), whereas in monkeys (36) and humans (37), neurogenesis occurs only in the hippocampus.

With a normal diet, such as nonpurified diet, brain DHA levels are region specific, ranging from 11.1–17.7% of total fatty acids in 5 selected brain regions in our study in rats, 10.7–16.3% in 7 brain regions in another study in rats (38), 6.6–22.1% in 11 brain regions in mice (39), and 4.5–15.8% in 26 brain regions in baboon neonates (40). The present study showed that recovery of DHA levels in the brain was also region specific. In DHA-deficient rats supplemented with FO during adulthood for 80 d (D+FO group), DHA levels recovered to 90 and 92% of those in the C group in the hippocampus and olfactory bulb, respectively, whereas only 61–64% recovery occurred in the frontal and visual cortexes and 75% in the cerebellum. DHA recovery is not only brain region specific but also tissue specific; e.g. when an (n-3) fatty acid-deficient diet is switched to an (n-3) fatty acid-adequate diet, full recovery occurs in the liver, kidney, lung, retina, and testes by 2, 3, 3, 6, and 10 wk, respectively (41), and in the serum, liver, and retina by 2, 1, and 8 wk, respectively. Approximately 80% recovery of brain DHA occurs at 8 wk (42) and by 12 wk, 90–100% recovery occurs in the cerebellum, cortex, hippocampus, hypothalamus, striatum, and midbrain, but only ~62% in the medulla (38). It is inferred that DHA recovery is more efficient in the peripheral tissues and circulation than in the central nervous system.

In summary, recovery of brain DHA levels improved spatial reference and working memory. Moreover, FO supplementation during brain development and adulthood in normal rats resulted in enhancement of both memories. The hippocampus and olfactory bulbs, 2 brain areas showing neurogenesis, showed faster DHA recovery, a greater capability for DHA accumulation, and more resistance to DHA deprivation than the frontal cortex, visual cortex, and cerebellum. These results suggest that DHA is important for the development and maintenance of spatial learning memory performance.


   ACKNOWLEDGMENTS
 
We thank Dr. Fu-Chang Hu and Soa-Yu Chan from the National Center of Excellence for General Clinical Trials and Research, National Taiwan University Hospital and the College of Public Health, National Taiwan University for assistance with the statistical analysis.


   FOOTNOTES
 
1 Supported by the National Science Council of Taiwan (grant NSC93-2320-B-002-093) and by the Department of Health (grant DOH-94-TD-F-113-031). Back

2 Author disclosures: W.-L. Chung, J.-J. Chen, and H.-M. Su, no conflicts of interest. Back

3 Supplemental Tables 1–3 are available with the online posting of this paper at jn.nutrition.org. Back

4 Abbreviations used: AD, Alzheimer's disease; C, control, nonpurified diet; C+FO, nonpurified diet supplemented with fish oil; D, (n-3) fatty acid-deficient diet; D+FO, (n-3) fatty acid-deficient diet supplemented with fish oil; DHA, docosahexaenoic acid, [22:6(n-3)]; FO, fish oil; GEE, generalized estimating equation. Back

Manuscript received 17 January 2008. Initial review completed 30 January 2008. Revision accepted 22 March 2008.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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