months of treatment with haloperidol, but was not affected by sulpiride. Chronic administration of sulpiride does not induce identical changes in striatal dopamine ...
Psycho0harmacology
Psychopharmacology (1984) 84:503-511
9 Springer-Verlag 1984
Differential effects of continuous administration for 1 year of haloperidol or sulpiride on striatal dopamine function in the rat N. M. J. Rupniak 1, S. Mann 2, M. D. Hall 1, S. Fleminger 1, G. Kilpatrick 1, P. Jenner 1, and C. D. Marsden 1 1 MCR Movement Disorders Research Group, University Department of Neurology, and Parkinson's Disease Society Research Centre, Institute of Psychiatry & King's College Hospital Medical School, Denmark Hill, London SE5 8AF, UK and 2 Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, UK
Abstract. Administration of haloperidol (1.4-1.6 mg/kg/day) for up to 12 months or sulpiride (102-109 mg/kg/day) for between 6 and 12 months increased the frequency of purposeless chewing jaw movements in rats. N,n-propylnorapomorphine (NPA) (0.25-2.0 mg/kg SC) did not induce hypoactivity in haloperidol-treated rats at any time; sulpiride treatment for 9 and 12 months caused a reduction in the ability of NPA to induce hypoactivity. Haloperidol, but not sulpiride, treatment enduringly inhibited low dose apomorphine effects (0.125 mg/kg SC). After 12 months, stereotypy induced by high doses of apomorphine (0.5-1.0 mg/kg) was exaggerated in haloperidol-, but not sulpiride-treated rats. Bmax for specific striatal 3H-spiperone binding was increased by haloperidol, but not sulpiride, treatment throughout the study. Bmax for 3H-NPA binding was elevated only after 12 months of both haloperidol and sulpiride treatment. Bmax for 3H-piflutixol binding was not altered by chronic haloperidol or sulpiride treatment. Striatal dopamine-stimulated adenylate cyclase activity was inhibited for the 1st month of haloperidol treatment, thereafter returning to control levels; dopamine stimulation was increased after 12 months of sulpiride treatment. Striatal acetylcholine content was increased after 3 and 12 months of treatment with haloperidol, but was not affected by sulpiride. Chronic administration of sulpiride does not induce identical changes in striatal dopamine function to those caused by haloperidol. Key words: Haloperidol - Sulpiride - Striatum Supersensitivity - Dopamine receptors - Rat
The induction of striatal dopamine receptor supersensitivity by repeated administration of neuroleptic drugs in rodents may be related to the emergence of drug-induced extrapyramidal side-effects such as tardive dyskinesia in man (Klawans 1973). During chronic administration of typical neuroleptic drugs such as trifluoperazine, striatal dopamine receptor supersensitivity may develop despite continued neuroleptic intake (Clow et al. 1979a, 1979b). These animals display an exaggerated stereotyped response to apomorphine, an increase in striatal dopamine receptor sites (Bmax) identified by 3H-spiperone, and an increase in striatal acetylcholine concentrations (Murugaiah et al. Offprint requests to: C. D. Marsden
1982). These changes may reflect an increase in dopaminergic tone, since dopamine exerts inhibitory control over striatal acetylcholine release (Sethy and van Woert 1974). The substituted benzamide drug sulpiride differs from classical neuroleptic agents in causing few extrapyramidal side-effects (Alberts 1983). Sulpiride also differs from classical neuroleptic agents in its generally weak activity in models of certain dopamine-mediated behaviours in rodents. For example, sulpiride does not induce dose-dependent catalepsy (Costall and Naylor 1975; Elliot et al. 1977) or inhibit apomorphine-induced stereotypy (Costall and Naylor 1975; Puech et al. 1976; Jenner et al. 1978). Sulpiride does, however, induce profound inhibition of apomorphine-induced climbing in mice (Puech et al. 1976) and rats (Sokoloff et al. 1981, unpublished data presented at 8th International Congress of Pharmacology, Tokyo), suggesting a selective effect on certain motor events. Biochemically, sulpiride may interact with dopamine receptors in a manner distinct from other neuroleptics. Thus, sulpiride is unable to antagonise dopamine stimulation of adenylate cyclase activity via D-1 receptors (Trabucchi et al. 1975; Elliot et al. 1977) and only weakly displaces specific 3H-spiperone binding to D-2 receptors by comparison to a classical neuroleptic drug such as haloperidol (Creese et al. 1979). In addition, specific binding of 3H-sulpiride differs from that of 3H-spiperone in its absolute dependence on the presence of sodium ions (Theodorou et al. 1980). The evidence that repeated administration of sulpiride, when followed by drug withdrawal, induces striatal dopamine receptor supersensitivity as do classical neuroleptic drugs is equivocal. Repeated sulpiride administration, followed by drug withdrawal, is reported to cause an exaggerated stereotyped response to apomorphine (Costall et al. 1978; Jenner et al. 1982). In contrast, such treatment, unlike h aloperidol, may not increase the number of specific striatal 3H-spiperone binding sites (Trabucchi et al. 1980; Bannet et al. 1980; Fuxe et al. 1980). However, in two previous studies from our laboratories, repeated administration of sulpiride, followed by drug withdrawal, like haloperidol, caused an increase in specific striatal 3H-spiperone and 3H-N,n-propylnorapomorphine binding (Jenner et al. 1982; Fleminger et al. 1983a). If changes in striatal dopamine function are of relevance to the genesis of tardive dyskinesia, it might be expected that the chronic, continuous administration of sulpiride to rodents should not induce dopamine receptor supersensitivity. We now compare the effects of continuous
504 treatment with haloperidol or sulpiride for up to 12 months on striatal dopamine function. We find that, unlike haloperidol, continuous sulpiride treatment does not induce an increase in specific striatal 3H-spiperone binding or an exaggerated stereotyped response to apomorphine.
Data are expressed as the mean + 1SEM of the cumulative activity counts in 20 min for six rats at each dose of NPA. The effects of NPA were assessed in strict parallel in control rats and in animals treated with neuroleptics, and on the same occasion.
Apomorphine-induced stereotyped behaviour. Stereotyped Materials and methods
Drug administration. Male Wistar rats (205 + 14 g at the start of the experiment; Bantin and Kingman Ltd.) were housed initially in groups of eight under standard conditions of lighting (12 h light/dark cycle) and temperature (21 + 3~ C). Animals were randomly allocated to one of three groups which received as their daily drinking water distilled water alone (controls), haloperidol solution (target intake 2 mg/kg/day) or sulpiride solution (target intake 100 mg/kg/day) for a continuous period of up to 12 months. Drug doses were based on the average daily clinical doses used in the control of schizophrenia (see Titeler and Seeman 1980), increased five times to offset the generally greater drug metabolising ability of the rat. Haloperidol (Janssen Pharmaceutica, Belgium) was dissolved in a minimum quantity of glacial acetic acid; sulpiride (SESIF, France) was dissolved in a minimum volume of 2% (v/v) sulphuric acid. The resulting solutions were diluted with distilled water to give stock solutions of 10 mg/ml and 50 mg/ml respectively. The pH of the stock solutions was adjusted to between 5.5 and 7.0 using 2 N sodium hydroxide. Solutions were further diluted with distilled water and presented to rats as drinking water. Behavioural and biochemical testing took place at 1, 3, 6, 9, and 12 months after initiation of drug treatment and occurred whilst animals continued to received continuous neuroleptic intake. No animal was used for behavioural testing on more than one occasion within a 3-month period. Animals used for behavioural assessment were not used for biochemical assays at the same time of testing.
Spontaneous chewingjaw movements. Individual rats were observed on a clean table area measuring 45 x 15 cm. Following a 2-min acclimatisation period, the number of individual chewing movements during a 5-min test period was recorded. Chewing movements were recorded only if they appeared to be purposeless, that is, if they were not directed towards any specific object. Observations were made on eight animals per drug treatment group at each time point.
Hypoactivity induced by administration of N,n-propylnorapomorphine (NPA). Locomotor activity in rats was assessed immediately following administration of low doses of the dopamine agonist NPA (0.25-2.0~tg/kg SC; Research Biochemicals Inc.) or saline (0.9% SC). Animals were placed in individual perspex cages (20 x 18 x 18 cm) containing two photocell units spaced equidistant from the centre (designed in the Department by Mr. H. C. Bertoya). The assessment of activity occurred 8 min following placement of rats into the activity cages in order to allow exploratory activity to decline. The number of interruptions of both light beams occurring in each 2-min time period were recorded over the subsequent 20 min for each individual rat.
behaviour was assessed in animals placed in individual perspex cages (20 x 18 x 18 cm). Stereotypy was scored 15 rain following administration of apomorphine hydrochloride (0.125-2.0 mg/kg SC; Sigma) as follows: 0 = indistinguishable from saline-treated animals; 1 = continuous locomotor activity, discontinuous sniffing; 2 = discontinuous locomotor activity, continuous sniffing; 3 = occasional locomotor activity, discontinuous licking, gnawing or biting; 4 = continuous licking, gnawing or biting; 5 = compulsive oral manipulation of faeces. Data are expressed as the mean scores + 1 SEM obtained for 6 - 1 1 animals examined at each dose of apomorphine.
Ligand binding assays. Animals were killed by cervical dislocation and decapitation, and the brain rapidly removed onto ice. The paired striata from five rats were dissected out into ice-cold 50 mM Tris HC1 buffer (pH 7.6) and pooled for in vitro determination of 3H-spiperone or 3H-N,n-propylnorapomorphine (NPA) binding. For 3H-piflutixol binding, tissue pools contained the paired striata of three rats per group. Specific 3H-spiperone binding was determined according to the technique of Leysen et al. (1978), using as the final incubation buffer 50 mM Tris HC1 (pH 7.6), containing 120 mM sodium chloride. 3H-Spiperone (15.5-21.0 Ci/mmole; Amersham International) was added to the incubates in six concentrations between 0.1 and 4.0 nM. Specific binding to D-2 receptors was defined using (-)-sulpiride (10 -5 M; SESIF). Specific binding of 3H-NPA (60 Ci/mmole; New England Nuclear) was carried out using a modification (Hall et al. 1981) of the procedure employed for 3H-apomorphine binding (Leysen and Gommeren 1981) incorporating E D T A (1 mM) in tissue buffers throughout the procedure to reduce non-specific binding. A range of six ligand concentrations between 0.05 and 2.0 nM was employed, and specific binding defined by the incorporation of (+)-ADTN (2-amino-6,7-dihydroxy-l,2,3,4-tetrahydronaphthalene; 10 .6 M, Wellcome Research Laboratories). Specific binding of 3H-piflutixol (11.7 Ci/mmole; Lundbeck) was determined using five ligand concentrations in the range 0.08-1.3 nM. Binding was carried out in the presence of (+)-sulpiride (3 x 10 -5 M) to prevent binding to D-2 receptors. Specific binding to D-1 receptors was defined by incorporation of cis-flupenthixol (10-6M; Lundbeck). All determinations were carried out in triplicate at each ligand concentration. The data for each experiment was subjected to linear regression and Scatchard analysis to determine the number of binding sites (Bmax; pmoles/g wet weight of tissue) and the apparent equilibrium dissociation constant (KD; nM). Limitations on the number of animals available for biochemical tests necessitated that ligand binding assays during the first 6 - 9 months were carried out on only a
505 single tissue pool at each time point. Data from single tissue pools was examined by 2-factor analysis of variance for determination of changes in binding parameters with drug treatment over time. After 9 or 12 months of drug treatment, binding assays for each ligand were carried out on three separate tissue pools from each drug treatment group so that direct comparisons between drug treatment groups could be made at those times.
Dopamine-stimulated adenylate cyclase activity. The ability of dopamine to stimulate striatal adenylate cyclase activity was assessed in striatal homogenates from control and neuroleptic-treated animals according to the method of Miller et al. (1974). Three animals from each treatment group were killed by cervical dislocation and decapitation. The brains were rapidly removed and placed on ice during dissection of the paired corpora striata. The striatal tissue from each animal was homogenised separately in ice-cold buffer (2 mM Tris maleate containing 2 mM E G T A , p H 7.4). Basal and dopamine hydrochloride (1-150 ~tM; Sigma)-stimulated adenylate cyclase activity were determined in duplicate at five dopamine concentrations for each individual homogenate. The results were expressed as pmoles of cyclic AMP formed/2 mg tissue/2.5 rain. The linear portions of the sigmoid concentration response curves were subjected to linear regression analysis in order to determine the increase in cyclic AMP formation over basal induced by the presence of 50 ~tM dopamine.
Statistical analysis. Non-parametric data from chewing jaw movements and stereotypy scores were first analysed for overall group differences using the Kruskal-Wallis analysis of variance of ranks. In cases where the resulting H-scores were associated with a probability of less than 5%, data were subjected to paired Mann-Whitney U-tests. Parametric data from locomotor activity counts and biochemical determinations were compared to control values by analysis of variance followed where appropriate by two-tailed Student's t-test. In cases where ligand binding assays were conducted on single tissue pools data were examined by two-factor analysis of variance to determine changes with drug treatment over time. Results
Drug intake and body weight. Drinking water containing haloperidol or sulpiride was readily accepted by animals over the period of the experiment. The mean daily drug intakes (_+ 1 SEM) achieved over the 12-month period were for haloperidol 1.4-1.6 mg/kg and for sulpiride 102-109 mg/kg. The body weight of sulpiride-treated rats was normal compared to the control group at the end of the 12-month period (control, 512 +_ 9 g; sulpiride-treated, 501 _+ 10 g, P > 0.05, Student's t-test), whilst that of the haloperidol-treated group had fallen slightly below that of the age-matched controls (haloperidol-treated, 447 + 11 g; P < 0.05 compared to control animals, Student's t-test). In other respects drug-treated animals did not differ from control rats in general health or appearance.
Bioassay for acetylcholine in striatal tissue. The paired striata from eight neuroleptic-treated and control animals were rapidly removed and immediately frozen in liquid nitrogen. Tissue from individual animals was weighed and homogenised in 1% acetic ethanol (1 ml glacial acetic acid in 99 ml absolute ethanol) to inhibit acetylcholinesterase activity. Samples were stored at - 7 0 ~ C until assay. On the day of assay, each sample was thawed and then rotary evaporated to dryness at 40 ~ C. The residue was dissolved in 5.0 ml of frog Ringer, and acidified to pH 3.0 where necessary using 0.3 N hydrochloric acid. Portions of each sample (approximately 1.0 ml) were pooled and treated with a few drops of 1 N sodium hydroxide solution prior to boiling. This alkali treatment destroys endogenous acetylcholine. Alkali boiled controls were then neutralised with 1 N hydrochloric acid. Standard solutions containing known amounts of acetylcholine chloride (0.01-0.1 ~tg; Sigma) were added to 1.0 ml of alkali-treated control. Standards were assayed on frog rectus abdominus muscle suspended in eserinised frog Ringer according to the method of Chang and Gaddum (1933) and Feldberg (1945). Muscle contractions were then compared with the effects produced by equal volumes of unknown sample extracts. The preparation of standards in alkali-treated controls ensured that substances other than acetylcholine in the sample capable of producing muscle contraction (such as potassium) were not assayed as acetylcholine. To sensitise and stabilise the muscle preparation, 0.5 mg of Na-ATP (Sigma) was added with each sample and standard to the muscle bath. The assay used in this way has an accuracy of _+ 10%. Results are expressed as the mean acetylcholine concentrations (+_ 1 SEM) in nmol per g of tissue for eight individual animals at each time point.
Spontaneous chewing movements. Animals treated with haloperidol (1.4-1.6 mg/kg/day) displayed an increase in the frequency of purposeless chewing jaw movements by comparison to age-matched control rats after 1, 3, 6, 9, and 12 months. Treatment with sulpiride (102-109 mg/kg/day) for up to 3 months did not induce this behaviour, but chewing was increased following 6 - 1 2 months of sulpiride intake (Table 1). Table 1. Spontaneous chewing behaviour in rats treated continuously for up to 12 months with haloperidol (1.4-1.6 mg/kg/day) or sulpiride (102-109 mg/kg/day) compared to age-matched control animals Duration of treatment (months)
Incidence of chewing/5 rain Control
Haloperidol
Sulpiride
1 3 6 9 12
8.3 + 18.0 + 10.3 + 17.9 + 17.3 +
27.0 + 4.0* 47.4 + 7.3* 26.0 + 2.0* 42.9 _+5.5* 59.3 _+ 5.7*
7.0 _+2.3 22.0 + 4.4 19.4 + 0,9" 27.8 + 3,3* 40.6 + 7,7"
1.7 1.7 2.1 3.2 3.6
Values are mean • 1 SEM scores for observations during a 5-min test period (n = 8). Overall group differences were determined using the Kruskal-Wallis analysis of variance of ranks. The following H scores and associated probalities were obtained: 1 month, H = 11.71, P < 0.01; 3 months, H = 10.42, P < 0.01; 6 months, H = 15.37, P < 0.001; 9 months, H = 10.60, P < 0.01; 12 months, H = 12.56, P < 0.01. For data where P < 0.05, groups were compared by Mann Whitney U-tests. * P < 0.05 vs control, Mann Whitney U-test
506 Table 2. Hypoactivity induced by administration of N,n-propylnorapomorphine (NPA)~I(0.25-2.0 ~g/kg SC) during the course of 12 months continuousI treatment with haloperidol (1.4-1.6 mg/kg/day) or sulpiriOe (102-109 mg/kg/day) compared to age-matched control animals Duration of Dose of Cumulative activity counts in 20 min treatment NPA (months) (/~g/kgS~) Control Haloperidol Sulpiride 1
Saline 0.25 0.50 1.0 2.0
531 + 55 400 _+ 79 163 +_ 34** 217 _+ 38** 244 _+52**
278 + 344 + 211 + 267 + 199 +
3
Saline 0.25 0.50 1.0 2.0
435 + 37 315 _+ 73 130 + 24** 190 + 50** 186 _+ 43**
255 + 47* 286 +_47 338 +_ 39* 246 + 62 215 _+ 41
442 + 55 419 + 56 208 +_ 50** 126 _+ 41"* 278 + 53**
6
Saline 0.25 0.50 1.0 2.0
188 + 94 + ,141 + 125 + 93 +
194 _+ 38 158 + 27 174 + 23 142 + 29 113 + 19
130 _+ 18 204 + 47 180 + 37 96 + 26 221 + 77
9
Saline 0.25 0.50 1.0 2.0
~87 -+ 22 t73 + 24** 122 + 24** 45 + 13"* 76 + 19"*
132 + 208 + 173 + 154 + 120 +
245 + 216 + 166 + 61 + 138 +
Saline 0.25 0.50 1.0 2.0
273 _+ 41 ~88 +- 24 94 _+ 26** 135+27"* 1208_+ 32**
194 + 23 188 + 24 157 + 29 211_+54 201 + 44
12
49 22 22 18 16
43* 66 51 45 66
30* 26 30 30* 25
323 + 46* 343 _+ 68 190 + 67 149 _+ 48 312 + 57
41 28 44 24** 17"*
265 + 22 264 + 29 240 + 32* 211+37 128 + 17"*
Results are expressed as the mean + 1 SEM of cumulative activity scores from six rats at each dose of NPA over a 20-rain test period. Overall differences in activity counts were first subjected to analysis of variance. In cases where the resulting F ratios were associated with a probability of less than 5%, groups were then compared by two-tailed Student's t-test. * P < 0.05 compared to~age-matched control animals, Student's t-test ** P < 0.05 compared to saline treatment, Student's t-test
rats receiving N P A (Table 2). At 6 months, spontaneous locomotor activity was reduced in sulpiride-treated rats by comparison to previous time points, and, as in control animals, administration of N P A did not induce hypoactivity (Table 2). After 9 months of sulpiride intake, administration of only the two highest doses of N P A ( 1 . 0 - 2 . 0 ~tg/kg) reduced locomotor activity and at 12 months, only 2.0 ~tg/kg N P A induced hypoactivity as compared to saline treatment (Table 2).
Apomorphine-induced stereotypy. After 1, 3, 6, and 9 months continuous administration of haloperidol, the stereotyped response to low doses of apomorphine (0.125-0.25 mg/kg SC, 15 rain previously) was inhibited by comparison to control rats (Fig. 1). After 12 months, stereotypy induced only by the lowest dose (0.125 mg/kg) was inhibited. The effects of higher doses of apomorphine ( 0 . 5 - 2 . 0 mg/kg) were in general not inhibited in rats treated for up to 9 months with haloperidol. After 12 months, high doses of apomorphine ( 0 . 5 - 1 . 0 mg/kg) induced an exaggerated stereotyped response in haloperidol-treated rates (Fig. 1). In contrast, stereotypy induced by apomorphine (0.125-2.0 mg/kg) in animals treated for up to 12 months with sulpiride did not differ at any time from that observed in age-matched control rats (Fig. 1).
Specific striatal 3H-spiperone binding. Analysis of variance of the results obtained with single striatal tissue pools taken from animals treated continuously with haloperidol for 1, 3, 6, and 9 months revealed an increase in the number of specific 3H-spiperone binding sites (Bma• o v e r this period compared to values in age-matched control animals (F = 58.96, P < 0.05; two-factor analysis of variance) (Table 3). Bmax remained elevated after 12 months of haloperidol treatment as assessed in three separate tissue pools (Table 3). Administration of sulpiride, however, for up to 9 (F = 2.26, P > 0.05) or 12 months had no effect o n Bmax for striatal 3H-spiperone binding (Table 3). There was no effect of treatment with haloperidol or sulpiride on the apparent dissociation constant (KD) for striatal 3H-spiperone binding up to 9 months (F = 4.82, P > 0.05) or after 12 months of treatment (Table 3).
Specific striatal 3H-NPA binding. The number of specific NPA-induced hypoactivity. Spontaneous locomotor activity following injection of saline was reduced in haloperidol-treated rats after 1, 3 and 9 months by comparison to control animals. Sulpiride-treated rats showed lower spontaneous activity after 1 month of treatment, but not thereafter (Table 2). A t 1, 3, 9, and 12 months, administration of N P A ( 0 . 5 - 2 . 0 ~g/kg SC) to control animals decreased locomotor activity by comparison to saline treatment. However, at 6 months, N P A did not induce hypoactivity owing to an unexplained fall in spontaneous activity in control rats (Table 2). In animals treated for up to 12 months with haloperidol, N P A ( 0 . 2 5 - 2 . 0 ~xg/kg) did not induce hypoactivity by comparison to saline treatment (Table 2). In rats treated for 1 month with sulpiride, hypoactivity was not induced following administration of N P A ( 0 . 2 5 - 2 . 0 ~tg/kg). After 3 months, N P A ( 0 . 5 - 2 . 0 ~tg/kg) induced hypoactivity to the same degree as that in control
binding sites for 3H-NPA in striatal tissue from haloperidol and sulpiride-treated animals did not differ from those in control animals over 9 months of treatment (F = 1.78, P > 0 . 0 5 ) (Table 4). However, after 12 months, animals n'eated with both hatoperidol and sulpiride displayed an increase in Bm~, for 3H-NPA binding compared to age-matched control animals (Table 4). Similarly, KD for striatal 3H-NPA binding was not altered by up to 9 months treatment with either haloperidol or sulpiride (F = 0.28, P > 0.05); after 12 months of treatment, however, KD in both the haloperidol and sulpiride-treated groups was elevated compared to control animals (Table 4).
Specific striatal 3H-piflutixot binding. Analysis of variance of Bmax values for striatal 3H-piflutixol binding in single tissue pools after 1, 3, and 6 months of haloperidol or sulpiride treatment revealed no difference from control values (F = 0.62, P > 0.05). In the three tissue pools examined after 9 and 12 months of haloperidol or sulpiride
507 C
oJ L A
D
4 B
4
* 1.0mg/kg
apdmorph;ne 3
3
21
o
L
~
0.12 0.25 0.5 1 2 Dose of opomorphine (mg/kg S.C.}
0.12mg/kg opomorphine
9
o
o
0.12 0.25 0.5 1 2 Dose of apomorphine (mg/kg S.C.)
0.12 0.25 0.5 1 Dose of apomorphine (mg/kg S.C,)
1
3 6 9 Duration of treotment (months)
12
Fig. 1. Stereotyped response to apomorphine (0.125-2.0 mg/kg SC, 15 min previously) after A 1 month, B 6 months or C 12 months continuous treatment with haloperidol (1.4-1.6 mg/kg/day) or sulpiride (102-109 mg/kg/day). Stereotypy induced by a low dose (0.125 mg/kg) and a high dose (1.0 mg/kg) of apomorphine over the 12-month time course are shown in D. Results are the mean + SEM stereotypy score, n = 6-11 at each dose of apomorphine. Overall group differences were assessed using the Kruskal-Wallis analysis of variance of ranks. The following H scores and associated probability were obtained: Dose of apomorphine (mg/kg)
Duration of treatment (months)
0.125 0.25 0.375 0.50 1.0 2.0
11.61, 11.47, 5.71, 2.82, 0
1
3 P < 0.01 P < 0.01 P > 0.05 P > 0.05 P > 0.05
6
8.55, 10.61, 4.71, 1.41, 3.09,
P < 0.05 P < 0.01 P > 0.05 P > 0.05 P > 0.05
9
10.28, 7.16, 0.61, 0.95, 0
P < 0.01 P < 0.05 P > 0.05 P > 0.05 P > 0.05
10.15, 11.94, 1.41, 1.18, 1.06,
12 P P P P P
< < > > >
0.01 0.01 0.05 0.05 0.05
10.90, 0.32, 3.42, 3.82, 7.15,
P P P P P
< > > >
