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The effect of population density on the development of experimental atherosclerosis in female mice
ATHEROSCLEROSIS ELSEVIER
Atherosclerosis 115 (1995) 85-88
The effect of population density on the development of experimental atherosclerosis in female mice A.H. Lin*, C.K. Castle, G.W. Melchior, K.R. Marotti Molecular Biology Research and Endocrine Pharmacology and Metabolism Research, Upjohn Laboratories, Kalamazoo, MI, USA
Received 11 August 1994; revision received 14 December 1994; accepted 15 December 1994
Abstract
The effect of cage population density on plasma lipids and the development of atherosclerosis was examined in female C57BL/6 mice. Mice were housed at a density of one, two or five animals per cage and fed an atherogenic diet for 28 weeks. Subsequently, the animals were bled, sacrificed, the hearts removed and the extent of fatty lesion development in the aorta examined and quantified. As the population density increased, there was a statistically significant increase in total cholesterol levels, VLDL + LDL cholesterol levels, the VLDL + LDL/HDL ratio and lesion severity. These differences are due to the psychosocial stress associated with living within a confined space with high population density over an extended period of time. Keywords: Psychosocial; Atherosclerosis; C57BL/6 mice; Female; Population density; Plasma cholesterol
1. Introduction
With the ability to express h u m a n genes in mice has come a renewed interest in those animals as models of h u m a n disease. One such disease, atherosclerosis, can be produced in certain strains of inbred mice [1-5], and a variety of transgenic mice have been created to test which metabolic processes contribute to, or protect from, development of that disease [6-11]. However, in addition to genetic determinants, a number of environmental factors that could contribute to experimental
* Corresponding author.
atherosclerosis in mice may complicate interpretation of the results especially when the experiments are done in different laboratories under various environmental conditions. Stress induced by housing conditions, for example, appears to affect the severity of the experimental atherosclerosis produced in non-human primates [12-17], and the same m a y be true of mice. The purpose of the study reported here was to test the effect of housing density on the severity of experimental atherosclerosis in female C57BL/6 mice. We have chosen female mice because of the difficulty encountered with fighting in multiply housed male mice. The results indicate that housing density can have a marked effect on the rate at which the
0021-9150/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0021-9150(94)05502-N
A.H. Lin et al. / Atherosclerosis 115 (1995) 85-88
86
experimental disease develops in the C57BL/6 mouse.
2. Materials and methods 2.1. Animals and diets Prior to the start of the experiment all the mice were housed under identical conditions. Female C57BL/6 mice were housed 1 mouse per cage, 2 mice per cage or 5 mice per cage in a room containing only C57BL/6 mice and kept at 22°C. All cages were changed twice per week regardless of population density. All mice were housed on a single rack and therefore subjected to similar exposures of light, noise and ventilation. The animals were at least 3 months old when the experiment was initiated and were fed a high fat high cholesterol diet for 28 weeks [18,19]. The animals were bled via the retro-orbital sinus every seven weeks to assess plasma cholesterol levels. 2.2. Plasma cholesterol measurements Total plasma cholesterol, H D L cholesterol and V L D L + L D L cholesterol levels were determined as previously described [20]. 2.3. Quantification o f atherosclerosis At the end of the study, the mice were anesthetized via i.p. ~avertin and exsanguinated through the brachial artery. The hearts were removed, fixed in phosphate buffered formaldehyde
and sectioned as described by Paigen et al. [21]. The sections were stained with hematoxylin and Oil Red O and counterstained with Fast Green. The lesion area of every eighth section was quantified as previously described [10].
2.4. Statistics One-way analysis of variance (ANOVA) of the plasma lipids and lesion severity was performed using the SigmaStat statistical program (Jandel Scientific, San Rafael, CA). 3. Results and discussion Table 1 summarizes the effect of housing density on plasma lipids and fatty lesion severity. The total plasma cholesterol and the V L D L + L D L cholesterol increased incrementally as the number of mice per cage increased from one to five. The difference in total and V L D L + L D L plasma cholesterol levels between the animals housed at one mouse per cage and those housed five per cage was statistically significant (P = 0.004). Conversely, the H D L plasma cholesterol levels in the C57BL/6 mice decreased incrementally, although the changes were not statistically significant. The combined effect of an increasing V L D L + L D L cholesterol and a decreasing H D L cholesterol resulted in a statistically significant increase in the V L D L + L D L / H D L ratio and the severity of lesion formation. Since these mice were
Table 1 Effect of housing density on the development of atherosclerosis in female C57BL/6 mice Number of cages
Number of mice/cage
Total cholesterol (mg/dl)
VLDL + LDL cholesterol (mg/dl)
HDL cholesterol (mg/dl)
(VLDL + LDL)/ HDL
Lesion area (/tm2 x 10-4)
10 5 2
1 2 5
150 -+_9 174_+9 200 -+ 11
83 _+14 116_+ 10 145 _+12
67 _+6 58 _+3 55 -+ 3
1.50 + 0.48 2.20+0.29 2.76 _+0.34
1.86 _+0.51 4.11 -+0.81 5.16 _+0.83
0.092 0.004 0.102
0.240 0.082 0.504
0.266 0.044 0.257
0.028 0.004 0.397
P values 1 vs. 2 1 vs. 5 2 vs. 5
0.085 0.003 0.108
Cholesterol and lesion values were determined after 28 weeks on diet and are expressed as mean _+S.E.M. One mouse housed 1/cage and 2 mice (in the same cage) housed 2/cage died just prior to the termination of the experiment.
A.H. Lin et al. / Atherosclerosis 115 (1995) 85-88
o f the same genetic b a c k g r o u n d and fed the same diet, these effects are attributed to the population density in the cage. There was always an ample supply o f f o o d and water for the mice, therefore the competition for nutrients was not a factor. The differences in lesion severity m a y be due to the psychosocial stress associated with living within a confined space with high population density over an extended period o f time. K a p l a n et al. [12,13,17] and Clarkson et al. [14-16] have reported the effect o f psychosocial influences on the development o f atherosclerosis in n o n - h u m a n primates. Their studies indicated that d o m i n a n t females within a primate colony tend to develop less atherosclerosis while the socially subordinate females tend to develop a m o r e severe disease. Additional studies have indicated that differences in p o p u l a t i o n density and other psychosocial factors can also have a variety o f physiological effects on mice. F o r example, a relationship has been established between social rank, hypertension and aortic arteriosclerosis in C B A mice [22,23]. Renne has reported that population density and cage type can affect weight gain in mice [24], and Peng et al. have d e m o n s t r a t e d that increased population density results in an increase in corticosterone levels and reduced peripheral lymphocyte populations [25]. C h a m p l i n has observed suppression o f oestrus in multiply housed female C57BL/6 mice [26]. O u r studies indicate that housing density has a m a r k e d effect on b o t h the plasma lipid composition and the severity o f fatty lesion development in C57BL/6 mice. The reason for these changes is u n k n o w n but they can only be attributed to a physiological response to the increase in population density. Nonetheless, housing density appears to have an effect in the standard C57BL/6 mouse, and should be taken into account when interpreting studies o f experimental atherosclerosis in this model.
References [1] Morrisett JD, Kim H-S, Patsch JR, Datta SK, Trentin JJ. Genetic susceptibility and resistance to diet-induced atherosclerosis and hyperlipoproteinemia. Arteriosclerosis 1982;2:312.
87
[2] Paigen B, Morrow A, Brandon C, Mitchell D, Holmes P. Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis 1985;57:65. [3] Paigen B, Holmes PA, Mitchell D, Albee D. Comparison of atherosclerotic lesions and HDL-lipid levels in male, female and testosterone-treated female mice from strains C57BL/6, BALB/c and C3H. Atherosclerosis 1987;64:215. [4] Paigen B, Mitchell D, Reue K, Morrow A, Lusis AJ, LeBoeuf RC. Ath-1, a gene determining atherosclerosis susceptibility and high density lipoprotein levels in mice. Proc Natl Acad Sci 1987;84:3763. [5] Nishina PM, Wang J, Touofuku W, Kuypers FA, Ishida BY. Atherosclerosis and plasma and liver lipids in nine inbred strains of mice. Lipids 1993;28:599. [6] Rubin EM, Krauss RM, Spangler EA, Verstuyft JG, Clift SM. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein A1. Nature 1991;353:265. [7] Plump AS, Smith JD, Hayek T, Alto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein Edeficient mice created by homologous recombination in es cells. Cell 1992;71:343. [8] Marotti KR, Castle CK, Murray RW, Rehberg EF, Polites HG, Melchior GW. The role of cholesteryl ester transfer protein in primate apolipoprotein A-I metabolism. Arteriosclerosis Thromb 1992;12:736. [9] Melchior GW, Castle CK, Murray RW, Blake WL, Dinh DM, Marotti KR. Apolipoprotein A-I metabolism in cholesteryl ester transfer protein transgenic mice. J Biol Chem 1994;269:8044. [10] Marotti KR, Castle CK, Boyle TP, Lin AH, Murray RW, Melchior GW. Severe atherosclerosis in transgenic mice expressing simian cholesteryl transfer protein. Nature 1993;364:73. [11] Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression, Arteriosclerosis Thromb 1994;14:141. [12] Kaplan JR, Clarkson TB, Manuck SB. Pathogenesis of carotid bifurcation atherosclerosis in cynomolgus monkeys. Stroke 1984;15:994. [13] Kaplan JR, Adams MR, Clarkson TB, Koritnik DR. Psychosoical influences on female 'protection' among cynomolgus macaques. Atherosclerosis 1984;53:283. [14] Clarkson TB, Adams MR, Kaplan JR, Koritnik DR. Psychosocial and reproductive influences on plasma lipids, lipoproteins, and atherosclerosis in nonhuman primates. J Lipid Res 1984;25:1629. [15] Clarkson TB, Weingand KW, Kaplan JR, Adams MR. Mechanisms of atherogenesis. Circulation 1987;76:I20. [16] Clarkson TB, Kaplan JR, Adams MR, Manuck SB. Psychosocial influences on the pathogenesis of atherosclerosis among nonhuman primates. Circulation 1987;76:I29. [17] Kaplan JR, Adams MR, Clarkson TB, Manuck SB, Shively CA. Social behavior and gender in biomedical investigations using monkeys: studies in atherogenesis. Lab Anim Sci 1991;41:334.
88
A.H. Lin et al. / Atherosclerosis I15 (1995) 85-88
[18] Nishina PM, Verstuyft J, Paigen B. Synthetic low and high fat diets for the study of atherosclerosis in the mouse. J Lipid Res 1990;31:859. [19] Nishina PM, Lowe S, Verstuyft J, Naggert JK, Kuypers FA, Paigen B. Effects of dietary fats from animal and plant sources on diet-induced fatty streak lesions in C57BL/6 mice. J Lipid Res 1993;34:1413. [20] Castle CK, Colca JR, Melchior GW. Lipoprotein profile characterization of the KKA y mouse, a rodent model of type II diabetes, before and after treatment with the insulin-sensitizing agent pioglitazone. Arteriosclerosis Thromb 1993;13:302. [21] Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis 1987;68:231.
[22] Henry JP, Ely DL, Stephens PM, Ratcliffe HL, Santisteban GA, Shapiro AP. The role of psychosocial factors in the development of arteriosclerosis in CBA mice. Atherosclerosis 1971;14:203. [23] Ely DL. Hypertension, social rank, and aortic arteriosclerosis in CBA/J mice. Physiol Behav 1981;26:655. [24] Renne U. Influence of different cages and cage population density on body weight gain of laboratory mice. Z Versuchstierkd 1989;32:153. [25] Peng X, Lang CM, Drozdowicz CK, Ohlsson-Wilhelm BM. Effect of cage population density on plasma corticosterone and peripheral lymphocyte populations of laboratory mice. Lab Anim 1989;23:302. [26] Champlin AK. Suppression of oestrus in grouped mice: the effects of various densities and the possible nature of the stimulus. J Reprod Fert 1971;27:233.
Atherosclerosis 115 (1995) 85-88
The effect of population density on the development of experimental atherosclerosis in female mice A.H. Lin*, C.K. Castle, G.W. Melchior, K.R. Marotti Molecular Biology Research and Endocrine Pharmacology and Metabolism Research, Upjohn Laboratories, Kalamazoo, MI, USA
Received 11 August 1994; revision received 14 December 1994; accepted 15 December 1994
Abstract
The effect of cage population density on plasma lipids and the development of atherosclerosis was examined in female C57BL/6 mice. Mice were housed at a density of one, two or five animals per cage and fed an atherogenic diet for 28 weeks. Subsequently, the animals were bled, sacrificed, the hearts removed and the extent of fatty lesion development in the aorta examined and quantified. As the population density increased, there was a statistically significant increase in total cholesterol levels, VLDL + LDL cholesterol levels, the VLDL + LDL/HDL ratio and lesion severity. These differences are due to the psychosocial stress associated with living within a confined space with high population density over an extended period of time. Keywords: Psychosocial; Atherosclerosis; C57BL/6 mice; Female; Population density; Plasma cholesterol
1. Introduction
With the ability to express h u m a n genes in mice has come a renewed interest in those animals as models of h u m a n disease. One such disease, atherosclerosis, can be produced in certain strains of inbred mice [1-5], and a variety of transgenic mice have been created to test which metabolic processes contribute to, or protect from, development of that disease [6-11]. However, in addition to genetic determinants, a number of environmental factors that could contribute to experimental
* Corresponding author.
atherosclerosis in mice may complicate interpretation of the results especially when the experiments are done in different laboratories under various environmental conditions. Stress induced by housing conditions, for example, appears to affect the severity of the experimental atherosclerosis produced in non-human primates [12-17], and the same m a y be true of mice. The purpose of the study reported here was to test the effect of housing density on the severity of experimental atherosclerosis in female C57BL/6 mice. We have chosen female mice because of the difficulty encountered with fighting in multiply housed male mice. The results indicate that housing density can have a marked effect on the rate at which the
0021-9150/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0021-9150(94)05502-N
A.H. Lin et al. / Atherosclerosis 115 (1995) 85-88
86
experimental disease develops in the C57BL/6 mouse.
2. Materials and methods 2.1. Animals and diets Prior to the start of the experiment all the mice were housed under identical conditions. Female C57BL/6 mice were housed 1 mouse per cage, 2 mice per cage or 5 mice per cage in a room containing only C57BL/6 mice and kept at 22°C. All cages were changed twice per week regardless of population density. All mice were housed on a single rack and therefore subjected to similar exposures of light, noise and ventilation. The animals were at least 3 months old when the experiment was initiated and were fed a high fat high cholesterol diet for 28 weeks [18,19]. The animals were bled via the retro-orbital sinus every seven weeks to assess plasma cholesterol levels. 2.2. Plasma cholesterol measurements Total plasma cholesterol, H D L cholesterol and V L D L + L D L cholesterol levels were determined as previously described [20]. 2.3. Quantification o f atherosclerosis At the end of the study, the mice were anesthetized via i.p. ~avertin and exsanguinated through the brachial artery. The hearts were removed, fixed in phosphate buffered formaldehyde
and sectioned as described by Paigen et al. [21]. The sections were stained with hematoxylin and Oil Red O and counterstained with Fast Green. The lesion area of every eighth section was quantified as previously described [10].
2.4. Statistics One-way analysis of variance (ANOVA) of the plasma lipids and lesion severity was performed using the SigmaStat statistical program (Jandel Scientific, San Rafael, CA). 3. Results and discussion Table 1 summarizes the effect of housing density on plasma lipids and fatty lesion severity. The total plasma cholesterol and the V L D L + L D L cholesterol increased incrementally as the number of mice per cage increased from one to five. The difference in total and V L D L + L D L plasma cholesterol levels between the animals housed at one mouse per cage and those housed five per cage was statistically significant (P = 0.004). Conversely, the H D L plasma cholesterol levels in the C57BL/6 mice decreased incrementally, although the changes were not statistically significant. The combined effect of an increasing V L D L + L D L cholesterol and a decreasing H D L cholesterol resulted in a statistically significant increase in the V L D L + L D L / H D L ratio and the severity of lesion formation. Since these mice were
Table 1 Effect of housing density on the development of atherosclerosis in female C57BL/6 mice Number of cages
Number of mice/cage
Total cholesterol (mg/dl)
VLDL + LDL cholesterol (mg/dl)
HDL cholesterol (mg/dl)
(VLDL + LDL)/ HDL
Lesion area (/tm2 x 10-4)
10 5 2
1 2 5
150 -+_9 174_+9 200 -+ 11
83 _+14 116_+ 10 145 _+12
67 _+6 58 _+3 55 -+ 3
1.50 + 0.48 2.20+0.29 2.76 _+0.34
1.86 _+0.51 4.11 -+0.81 5.16 _+0.83
0.092 0.004 0.102
0.240 0.082 0.504
0.266 0.044 0.257
0.028 0.004 0.397
P values 1 vs. 2 1 vs. 5 2 vs. 5
0.085 0.003 0.108
Cholesterol and lesion values were determined after 28 weeks on diet and are expressed as mean _+S.E.M. One mouse housed 1/cage and 2 mice (in the same cage) housed 2/cage died just prior to the termination of the experiment.
A.H. Lin et al. / Atherosclerosis 115 (1995) 85-88
o f the same genetic b a c k g r o u n d and fed the same diet, these effects are attributed to the population density in the cage. There was always an ample supply o f f o o d and water for the mice, therefore the competition for nutrients was not a factor. The differences in lesion severity m a y be due to the psychosocial stress associated with living within a confined space with high population density over an extended period o f time. K a p l a n et al. [12,13,17] and Clarkson et al. [14-16] have reported the effect o f psychosocial influences on the development o f atherosclerosis in n o n - h u m a n primates. Their studies indicated that d o m i n a n t females within a primate colony tend to develop less atherosclerosis while the socially subordinate females tend to develop a m o r e severe disease. Additional studies have indicated that differences in p o p u l a t i o n density and other psychosocial factors can also have a variety o f physiological effects on mice. F o r example, a relationship has been established between social rank, hypertension and aortic arteriosclerosis in C B A mice [22,23]. Renne has reported that population density and cage type can affect weight gain in mice [24], and Peng et al. have d e m o n s t r a t e d that increased population density results in an increase in corticosterone levels and reduced peripheral lymphocyte populations [25]. C h a m p l i n has observed suppression o f oestrus in multiply housed female C57BL/6 mice [26]. O u r studies indicate that housing density has a m a r k e d effect on b o t h the plasma lipid composition and the severity o f fatty lesion development in C57BL/6 mice. The reason for these changes is u n k n o w n but they can only be attributed to a physiological response to the increase in population density. Nonetheless, housing density appears to have an effect in the standard C57BL/6 mouse, and should be taken into account when interpreting studies o f experimental atherosclerosis in this model.
References [1] Morrisett JD, Kim H-S, Patsch JR, Datta SK, Trentin JJ. Genetic susceptibility and resistance to diet-induced atherosclerosis and hyperlipoproteinemia. Arteriosclerosis 1982;2:312.
87
[2] Paigen B, Morrow A, Brandon C, Mitchell D, Holmes P. Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis 1985;57:65. [3] Paigen B, Holmes PA, Mitchell D, Albee D. Comparison of atherosclerotic lesions and HDL-lipid levels in male, female and testosterone-treated female mice from strains C57BL/6, BALB/c and C3H. Atherosclerosis 1987;64:215. [4] Paigen B, Mitchell D, Reue K, Morrow A, Lusis AJ, LeBoeuf RC. Ath-1, a gene determining atherosclerosis susceptibility and high density lipoprotein levels in mice. Proc Natl Acad Sci 1987;84:3763. [5] Nishina PM, Wang J, Touofuku W, Kuypers FA, Ishida BY. Atherosclerosis and plasma and liver lipids in nine inbred strains of mice. Lipids 1993;28:599. [6] Rubin EM, Krauss RM, Spangler EA, Verstuyft JG, Clift SM. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein A1. Nature 1991;353:265. [7] Plump AS, Smith JD, Hayek T, Alto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein Edeficient mice created by homologous recombination in es cells. Cell 1992;71:343. [8] Marotti KR, Castle CK, Murray RW, Rehberg EF, Polites HG, Melchior GW. The role of cholesteryl ester transfer protein in primate apolipoprotein A-I metabolism. Arteriosclerosis Thromb 1992;12:736. [9] Melchior GW, Castle CK, Murray RW, Blake WL, Dinh DM, Marotti KR. Apolipoprotein A-I metabolism in cholesteryl ester transfer protein transgenic mice. J Biol Chem 1994;269:8044. [10] Marotti KR, Castle CK, Boyle TP, Lin AH, Murray RW, Melchior GW. Severe atherosclerosis in transgenic mice expressing simian cholesteryl transfer protein. Nature 1993;364:73. [11] Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression, Arteriosclerosis Thromb 1994;14:141. [12] Kaplan JR, Clarkson TB, Manuck SB. Pathogenesis of carotid bifurcation atherosclerosis in cynomolgus monkeys. Stroke 1984;15:994. [13] Kaplan JR, Adams MR, Clarkson TB, Koritnik DR. Psychosoical influences on female 'protection' among cynomolgus macaques. Atherosclerosis 1984;53:283. [14] Clarkson TB, Adams MR, Kaplan JR, Koritnik DR. Psychosocial and reproductive influences on plasma lipids, lipoproteins, and atherosclerosis in nonhuman primates. J Lipid Res 1984;25:1629. [15] Clarkson TB, Weingand KW, Kaplan JR, Adams MR. Mechanisms of atherogenesis. Circulation 1987;76:I20. [16] Clarkson TB, Kaplan JR, Adams MR, Manuck SB. Psychosocial influences on the pathogenesis of atherosclerosis among nonhuman primates. Circulation 1987;76:I29. [17] Kaplan JR, Adams MR, Clarkson TB, Manuck SB, Shively CA. Social behavior and gender in biomedical investigations using monkeys: studies in atherogenesis. Lab Anim Sci 1991;41:334.
88
A.H. Lin et al. / Atherosclerosis I15 (1995) 85-88
[18] Nishina PM, Verstuyft J, Paigen B. Synthetic low and high fat diets for the study of atherosclerosis in the mouse. J Lipid Res 1990;31:859. [19] Nishina PM, Lowe S, Verstuyft J, Naggert JK, Kuypers FA, Paigen B. Effects of dietary fats from animal and plant sources on diet-induced fatty streak lesions in C57BL/6 mice. J Lipid Res 1993;34:1413. [20] Castle CK, Colca JR, Melchior GW. Lipoprotein profile characterization of the KKA y mouse, a rodent model of type II diabetes, before and after treatment with the insulin-sensitizing agent pioglitazone. Arteriosclerosis Thromb 1993;13:302. [21] Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis 1987;68:231.
[22] Henry JP, Ely DL, Stephens PM, Ratcliffe HL, Santisteban GA, Shapiro AP. The role of psychosocial factors in the development of arteriosclerosis in CBA mice. Atherosclerosis 1971;14:203. [23] Ely DL. Hypertension, social rank, and aortic arteriosclerosis in CBA/J mice. Physiol Behav 1981;26:655. [24] Renne U. Influence of different cages and cage population density on body weight gain of laboratory mice. Z Versuchstierkd 1989;32:153. [25] Peng X, Lang CM, Drozdowicz CK, Ohlsson-Wilhelm BM. Effect of cage population density on plasma corticosterone and peripheral lymphocyte populations of laboratory mice. Lab Anim 1989;23:302. [26] Champlin AK. Suppression of oestrus in grouped mice: the effects of various densities and the possible nature of the stimulus. J Reprod Fert 1971;27:233.