|Year : 2014 | Volume
| Issue : 1 | Page : 69-73
Association between serum cholesterol, brain serotonin, and anxiety: A study in simvastatin administered experimental animals
Jaya Mary Thomas1, Joyamma Varkey1, Bibin Baby Augustine2
1 Department of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Medical College, Trivandrum, Kerala, India
2 Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Guwahati, Assam, India
|Date of Submission||18-Sep-2013|
|Date of Acceptance||10-Dec-2013|
|Date of Web Publication||8-Jan-2014|
Jaya Mary Thomas
Department of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Medical College, Trivandrum, Kerala
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: The present research was designed to investigate the association between serum cholesterol, brain serotonin, and anxiety in simvastatin administered experimental animals. Materials and Methods: Both mice and rats were used for the study. Simvastatin (50 and 100 mg/kg in rats and 70 and 140 mg/kg in mice) was administered orally for one month. Serum cholesterol and brain serotonin levels were monitored. Two behavioral models, elevated plus maze test and mirrored chamber test were used to evaluate anxiety in the animals. Results: Simvastatin caused a significant reduction (P < 0.01) in serum cholesterol and brain serotonin level compared with the control group of animals. Animals treated with simvastatin showed significant level of anxiety in the behavioral models when compared with the control group. Conclusion: Hence these experimental results have established the pharmacological evidence to the hypothesis for the relation between serum cholesterol, brain serotonin, and symptoms of anxiety and also confirm that long-term administration of lipophilic statins can lead to anxiety.
Keywords: Anxiety, cholesterol, elevated plus maze test, mirrored chamber test, serotonin, simvastatin
|How to cite this article:|
Thomas JM, Varkey J, Augustine BB. Association between serum cholesterol, brain serotonin, and anxiety: A study in simvastatin administered experimental animals. Int J Nutr Pharmacol Neurol Dis 2014;4:69-73
|How to cite this URL:|
Thomas JM, Varkey J, Augustine BB. Association between serum cholesterol, brain serotonin, and anxiety: A study in simvastatin administered experimental animals. Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2017 Dec 14];4:69-73. Available from: http://www.ijnpnd.com/text.asp?2014/4/1/69/124617
| Introduction|| |
Lowering of serum cholesterol level and decreased incidence of coronary heart disease is well established.  In spite of the effect on cardiovascular mortality there are reports relating to lowered serum cholesterol level and incidence of anxiety-related behaviors. , Cholesterol plays an important role in the development, function, and stability of synapses. Evidence shows that low cholesterol influences 5-HT function.  Studies also show that subjects having low cholesterol exhibit suicidal, agitated, and criminal behavior.  The altered behaviors on lowered serum cholesterol have been attributed to the reduced number of serotonin receptors in brain as a result of low cholesterol in membrane.  As with all preventive treatments, it is important to ensure that long-term cholesterol-lowering treatments do not lead to any harmful outcomes. Hence we analyzed whether, there is any relation between lowered serum cholesterol, brain serotonin level, and symptoms of anxiety in experimental animals on long-term statin administration. Simvastatin a highly lipophilic statin, easily cross the blood-brain barrier and reduces the brain cholesterol level has been chosen for the current study. 
| Materials and Methods|| |
Drugs and chemicals
Simvastatin and serotonin creatinine sulfate were purchased from Sigma Aldrich, Germany. Cholesterol estimation kit was obtained from Alibaba suppliers. All other chemicals used were of analytical grade.
Swiss albino mice of either sex weighing between 20 and 25 g and wistar rats of either sex weighing between 150 and 300 g were used for the present study. They were obtained from the animal house (Reg No: 752/02/a/CPCSEA), Govt. Medical college, Thiruvananthapuram. All animals were housed in poly propylene cages under a 12 h light/dark cycle and in controlled room temperature (22 ± 2°C) with free access to pellets of standard rodent diet and drinking water.
All experimental pharmacological studies were done after getting permission from the Institutional Animal Ethical Committee, Medical College, Thiruvananthapuram (IAEC Approval No 05/06/2011 MCT) and care of animals was taken as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
Evaluation of anxiogenic effect of simvastatin
All experiments were conducted on the very next day after administration of simvastatin for 30 days.
Elevated plus maze paradigm
The apparatus consist of two open arms (5 × 10 cm) and two closed arms (5 × 10 × 15 cm) radiating from a platform (5 × 5 cm) to form a plus-sign figure. The apparatus was situated 40 cm above the floor. The open arms edges were 0.5 cm in height to keep the mice from falling and the closed-arms edges were 15 cm in height. Animals were individually placed on the center of the elevated plus maze with its head facing the open arm. Every precaution was taken to ensure that no external stimuli, other than the height of the plus maze could invoke maze anxiety. An entry was rated once an animal was on an arm with all four feet. 
The stop watch was started and the following parameters were noted for 5-min period.
- Number of entries into the open arm
- Number of entries into the closed arm
- Time spent in the open arm
- Time spent in the closed arm
- Latency to enter open arm.
Group IA (6 mice) - Received 0.3% CMC (0.5 ml/100g) orally daily for 30 days
Group IB (6 mice) - Received simvastatin 70 mg/kg orally daily for 30 days
Group IC (6 mice) - Received simvastatin 140 mg/kg orally daily for 30 days.
Mirrored chamber paradigm
A mirrored arena in the mirrored chamber creates an aversive environment to the animal. The apparatus consisted of a mirrored cube open on one side placed in a square wooden box. The mirrored cube measuring 30 cm on one side was constructed of five pieces of mirrored glass with one mirrored side and an opposite side painted dark brown. The mirrored cube was placed in the centre of the wooden container to form a 5 cm corridor that completely surrounded the mirrored chamber. The other walls of the container were painted brown. Animals were individually placed at a single fixed starting point at the same corner of the corridor of the mirrored chamber and the stop watch was immediately started. During a 5-min section, the time taken by the animal to enter in to the chamber and time the animal spends inside the chamber were noted. The criterion for entering the chamber was all four feet placed on the floor panel of the mirrored chamber. 
Group IIA (6 rats) - Received 0.3% CMC (0.5 ml/100g) orally for daily 30 days
Group IIB (6 rats) - Received simvastatin 50 mg/kg orally daily for 30 days
Group IIC (6 rats) - Received simvastatin 100 mg/kg orally for daily for 30 days.
Biochemical and neurochemical estimation
Estimation of biochemical and neurochemical parameters were done in wistar rats following the mirrored chamber test. Blood were collected from the animals by retro orbital bleeding and then the animals were humanely sacrificed and brain was removed.
Serum cholesterol level estimation
Collected blood was kept aside for 45 min and centrifuged at 3000 rpm for 5 min to separate the serum. To 1 ml of the serum 1 ml of cholesterol working reagent was added, mixed well, and kept the tubes immediately in water bath at 55°C for 90 s. Cooled the tube contents and measured the absorbance at 560 nm using UV Visible spectrophotometer (Thermoscientific). Serum cholesterol level was calculated using the following formula: Cholesterol concentration in mg/dl = (absorbance of test/absorbance of standard) ×200. 
Brain serotonin level estimation
One part of the brain tissue was homogenized in two parts of 0.1N HCl using motor driven homogenizer. Homogenate obtained was transferred to a 60 ml of glass stoppered bottle and adjusted to pH 10 by the addition of anhydrous sodium carbonate. Five milliliter of borate buffer (pH 10) was then added. Diluted the bottle contents with water to a volume of 15 ml and then added 5 g of NaCl and 20 ml of n ?- butanol. It was shaken for 15 min, centrifuged the bottle, and decanted the fluid from solid material in to another bottle. Removed the aqueous layer by aspiration and washed the butanol phase twice by shaking with equal volume of borate buffer. Transferred 15 ml of butanol phase to another bottle containing 30 ml of n ?- heptane and 3 ml of 0.1N HCl. Centrifuged the bottle and removed the supernatant solvent. One milliliter of the acid layer was added to 0.3 ml of concentrated HCl in quartz cuvette. Activated the solution at 295 nm in the spectro fluorimeter and measured the fluorescence at 550 nm. 
Values were expressed as mean ± SEM. Statistical analysis was performed using one - way analysis of variance (ANOVA) (Graphpad software, Inc, California) followed by Dunnets post hoc test and values of P < 0.05 were considered to be statistically significant.
| Results|| |
Elevated plus maze test
The anxiogenic effect on long-term administration of simvastatin was evaluated using elevated plus maze test in mice. Simvastatin 70 and 140 mg/kg caused a decrease in the time spend in open arm (26.33 ± 1.5 (P < 0.01) and 24.33 ± 1.73 P < 0.001), respectively) and the number of entries in the open arms (3.17 ± 0.16 (P < 0.0001) and 2.167 ± 0.30 (P < 0.0001), respectively) compared with control (33.73 ± 1.2 and 4.50 ± 0.2, respectively). On the contrary, Simvastatin 70 and 140 mg/kg significantly increased the time spend in closed arms (237.83 ± 2.6 (P < 0.01) and 251.83 ± 1.8 (P < 0.001), respectively), number of entries in the closed arms, (26.83 ± 1.10 (P < 0.01) and 30.50 ± 0.34 (P < 0.001), respectively) and latency to enter the open arm (210.00 ± 2.7 (P < 0.05) and 260.50 ± 2.4 (P < 0.01), respectively) compared with the control group (222.66 ± 1.2, 21.83 ± 0.9 and 48.38 ± 0.5, respectively) [Figure 1] and [Figure 2].
|Figure 1: Evaluation of anxiogenic potential of simvastatin using Elevated Plus Maze Test. All values are expressed as mean (n=6) **P < 0.01, ***P < 0.001 compared with control|
Click here to view
|Figure 2: Evaluation of anxiogenic potential of simvastatin using Elevated Plus Maze Test. **P < 0.01, ***P < 0.001 compared with control|
Click here to view
Mirrored chamber test
The anxiogenic effect on long-term administration of simvastatin was evaluated using mirrored chamber test in rats. Simvastatin 50 and 100 mg/kg caused a significant decrease in the time spent in mirrored chamber (14.50 ± 0.42 (P < 0.05) and 11.33 ± 0.49 (P < 0.01), respectively) compared with control (16.83 ± 0.79). On the contrary, Simvastatin 50 and 100 mg/kg caused an increase in the latency to enter inside the mirrored chamber (181.83 ± 1.078 (P < 0.01) and 242.5 ± 0.885 (P < 0.01), respectively) compared with control (22.66 ± 0.80) [Figure 3].
|Figure 3: Evaluation of anxiogenic potential of simvastatin using Mirrored Chamber Test. *P < 0.05, **P < 0.01 compared with control|
Click here to view
Serum cholesterol level estimation
Simvastatin 50 mg/kg caused significant reduction in serum cholesterol level (53.14 ± 1.21) (P < 0.01) when compared with control (73.45 ± 2.34). Simvastatin 100 mg/kg also caused significant reduction in serum cholesterol level (41.23 ± 2.54) (P < 0.01) when compared with control (73.45 ± 2.34) [Figure 4].
|Figure 4: Evaluation of effect of long term administration of simvastatin on serum cholesterol level. *P < 0.05 and **P < 0.01 compared with control|
Click here to view
Brain serotonin level estimation
Simvastatin 100 mg/kg significantly reduced the brain serotonin level (920.41 ± 42.02) (P < 0.01) when compared with the control (1121.3 ± 15.23) [Figure 5].
|Figure 5: Evaluation of effect of long-term administration of simvastatin on Brain serotonin level. **P < 0.01 compared with control|
Click here to view
| Discussion|| |
To our knowledge, this is the first study on, anxiogenic effect of simvastatin during its long-term administration in experimental animals. In this study, we could find evidence in support of the hypothesis that cholesterol lowering significantly affects brain serotonin level and in turn it leads to symptoms of anxiety. Elevated plus maze test and mirrored chamber test are based on the assumption that unfamiliar, nonprotective, and brightly lit environmental stress provokes inhibition of normal behavior. This normal behavioral inhibition is further augmented in the presence of fear or anxiety-like state. The ultimate manifestation of anxiety and fear in the animals is exhibited by decrease in the motor activity and preference to remain at safer places.  Both low and high dose of simvastatin caused an augmented anxiogenic effect in both models of anxiety.
Long-term administration of simvastatin caused a significant reduction in serum cholesterol level. Lowered serum cholesterol and incidence of anxiety-related behavior has been previously reported. Suarez et al. reported that in healthy young adult women, low lipid and lipoprotein concentrations are inversely associated with trait measures of anxiety. 
It has been proposed that cholesterol can modulate the function of GPCRs (G-Protein Coupled Receptor) either through a direct (specific) interaction with GPCRs, which could induce a conformational change in the receptor, or through an indirect way by altering the membrane physical properties in which the receptor is embedded, or through a combination of both.  Shrivastava et al. reported the chronic cholesterol depletion induced by mevastatin on the function of human serotonin 1A receptor expressed in Chinese hamster ovary cells. Their study showed a significant reduction in the level of specific ligand binding and G-protein coupling to serotonin 1A receptor up on chronic cholesterol depletion.  Ramboz et al. reported an increased level of anxiety in serotonin 1A receptor knockout mice in elevated plus maze model.  Thus in our study, the reduction in cholesterol might have caused a decrease in serotonin 1A receptor activity and increased anxiety in elevated plus maze model.
A significant reduction in brain serotonin was obtained in the study. The brain's biogenic amines, especially the neurotransmitter serotonin, are considered to play a fundamental role in anxiety-related behaviors.  Results of behavioral models as well as brain serotonin level were consistent with the previous studies conducted by Schwarting et al., where they reported that inter individual differences in plus maze behavior of male wistar rats are related to different tissue levels of 5-HT in the ventral striatum. 
Going through the results of the above study an evidence for the association between lowered serum cholesterol, brain serotonin, and symptoms of anxiety can be seen. Further advanced studies are required to confirm it.
| Conclusion|| |
In the light of above findings we may conclude that, increased anxiety shown by the experimental animals in the behavioral models might be due to decreased serum cholesterol and associated reduction in brain neurotransmitter serotonin since, cholesterol affect the membrane fluidity, leading to altered 5-HT metabolism. The decrease in brain 5-HT neurotransmission following simvastatin administration may be the cause of anxiety reported in subjects taking simvastatin for chronic use.
| References|| |
|1.||Frick MH, Elo O, Haapa K, Heinonen OP, Heinsalmi P, Helo P, et al. Helsinki heart study: Primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia. N Engl J Med 1987;317:1237-45. |
|2.||Suarez EC. Relations of trait depression and anxiety to low lipid and lipoprotein concentrations in healthy young adult women. Psychosom Med 1999;61:273-9. |
|3.||Huang TL, Wu SC, Chiang YS, Chen JF. Correlation between serum lipid, lipoprotein concentrations and anxious state, depressive state or major depressive disorder. Psychiatry Res 2003;118:147-53. |
|4.||Buydens-Branchey L, Branchey M, Hudson J, Fergeson P. Low HDL cholesterol, aggression and altered central serotonergic activity. Psychiatry Res 2000;93:93-102. |
|5.||Golomb BA, Stattin H, Mednick S. Low cholesterol and violent crime. J Psychiatr Res 2000;34:301-9. |
|6.||Kaplan JR, Klein KP, Manuck SB. Cholesterol meets Darwin: Public health and evolutionary implications of the cholesterol-serotonin hypothesis. Evol Anthropol 1997;6:28-37. |
|7.||Joshi HN, Fakes MG, Serajuddin AT. Differentiation of 3-hydroxy-3-methylglutaryl-coenzyme:A reductase inhibitors by their relative lipophilicity. Pharm Pharmacol Commun 1999;5:269-71. |
|8.||Chen SW, Kong WX, Zhang YJ, Li YL, Mi XJ, Mu XS. Possible anxiolytic effects of taurine in the mouse elevated plus-maze. Life Sci 2004;75:1503-11. |
|9.||Kulkarni SK. Hand Book of Experimental Pharmacology, 3 rd ed.Pitampura, Delhi: Vallabh Prakashan; 1999. p. 24-25. |
|10.||Mallika V, Goswami B, Rajappa M. Atherosclerosis pathophysiology and the role of novel risk factors: A clinicobiochemical perspective. Angiology 2007;58:513-22. |
|11.||Bogdanski DF, Pletscher A, Brodie BB, Undenfriend S. Identification and assay of serotonin in brain. J Pharmacol Exp Ther 1956;117:82-8. |
|12.||Paila YD, Tiwari S, Chattopadhyay A. Are specific nonannular cholesterol binding sites present in G-protein coupled receptors? Biochim Biophys Acta 2009;1788:295-302. |
|13.||Shrivastava S, Pucadyil TJ, Paila YD, Ganguly S, Chattopadhyay A. Chronic cholesterol depletion using statin impairs the function and dynamics of human serotonin (1A) receptors. Biochemistry 2010;49:5426-35. |
|14.||Ramboz S, Oosting R, Amara DA, Kung HF, Blier P, Mendelsohn M, et al. Serotonin receptor 1A knockout: An animal model of anxiety-related disorder. Proc Natl Acad Sci U S A 1998;95:14476-81. |
|15.||Pich EM, Samanin R. Disinhibitory effects of buspirone and low doses of sulpiride and haloperidol in two experimental anxiety models in rats: Possible role of dopamine. Psychopharmacol (Berl) 1986;89:125-30. |
|16.||Schwarting RK, Thiel CM, Müller CP, Huston JP. Relationship between anxiety and serotonin in the ventral striatum. Neuroreport 1998;9:1025-9. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]