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A Comparison of caffeine and pemoline models of self-injury in rats

University of Florida Institutional Repository

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A COMPARISON OF CAFFEINE AND PEMOLI NE MODELS OF SELF-INJURY IN RATS By STACI DENISE KIES A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Staci D. Kies

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This document is dedicated to my parents, Ken and Debbie Kies, for their emotional support during the writing of this thesis.

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ACKNOWLEDGMENTS I would like to thank my thesis committee members, Dr. Tim Vollmer and Dr. Mark Lewis, for their helpful comments on this thesis. I would especially like to thank my advisor and committee chair, Dr. Darragh Devine, for creating this particular project, as well as spending the time to make revisions to my many rough drafts. iv

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................viii INTRODUCTION...............................................................................................................1 METHODS..........................................................................................................................5 Animals.........................................................................................................................5 Drugs.............................................................................................................................5 Experimental Procedure................................................................................................6 Histology.......................................................................................................................6 Statistical Analyses.......................................................................................................7 RESULTS..........................................................................................................................10 Tissue Trauma............................................................................................................10 Hypothalamic-Pituitary-Adrenal Axis Functioning...................................................13 DISCUSSION....................................................................................................................19 LIST OF REFERENCES...................................................................................................23 BIOGRAPHICAL SKETCH.............................................................................................28 v

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LIST OF TABLES Table page 1. Qualitative SIB scale........................................................................................................9 2. Topography of SIB.......................................................................................................15 vi

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LIST OF FIGURES Figure page 1. Incidence of SIB...........................................................................................................15 2. Caffeineand Pemoline-Induced Self-Injury.................................................................16 3. Basal Levels of Stress Hormones..................................................................................17 4. Alterations in Glandular and Body Weight..................................................................18 vii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science A COMPARISON OF CAFFEINE AND PEMOLINE MODELS OF SELF-INJURY IN RATS By Staci D. Kies May 2003 Chair: Darragh P. Devine Major Department: Psychology Chronic and stereotyped self-injurious behavior (SIB) is a maladaptive and debilitating behavior disorder, which can often have life-threatening consequences. It is exhibited predominantly by autistic and intellectually handicapped individuals, including those with a variety of specific genetic disorders. Disregulation of dopamine neurotransmission appears to be an important neurochemical feature of a variety of disorders in which SIB is observed, and several animal models have been developed in which dopamine function is altered. We have investigated the etiology of SIB in two of these models, using caffeine, an adenosine antagonist, and pemoline, an indirect dopamine agonist, in rats. In these investigations, we identified that caffeine produces only mild self-injury, and the effective doses are highly toxic. Pemoline was effective across a range of doses, and the expression of pemoline-induced self-injury occurred in a dose-orderly manner. Furthermore, effective pemoline doses were identified at which self-injury was only seen in a subset of the rats. This suggests that there may be viii

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individual differences in vulnerability to self-injure in the pemoline model of SIB. These individual differences are reminiscent of the fact that individuals with specific clinical disorders (e.g., autism) differ in their vulnerability to self-injure. Accordingly, research with the pemoline model of self-injury may help to uncover the biological factors that underlie individual differences in vulnerability to exhibit self-injury. ix

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INTRODUCTION Self-injurious behavior (SIB) is a devastating, chronic and usually stereotyped behavior disorder in which tissue damage is self-inflicted. This maladaptive behavior disorder is commonly seen in intellectually handicapped populations, wherein the severity of the SIB can range from mild to life-threatening. The expression of self-injury differs between clinical groups of intellectually handicapped individuals. Self-injury to the head and/or hands is often seen in autistic and other intellectually handicapped populations (Symons & Thompson 1997), skin-picking is commonly seen in Prader-Willi syndrome (Hellings & Warnock, 1994; Schepis et al., 1994), and lip-, tongue-, and digit-biting is seen in Lesch-Nyhan syndrome (Anderson & Ernst, 1994). In these groups, the behavior disorder is often highly resistant to treatment (Anderson & Ernst, 1994). In some instances, SIB is co-expressed with other behavior disorders, especially stereotypy. In fact, it has been proposed that SIB occurs on a continuum with stereotypy, wherein SIB is a severe or sensitized expression of stereotypy (Barron & Sandman, 1984; Guess & Carr, 1991) and the basal ganglia have been specifically implicated in this co-morbidity because of the apparently overlapping neural mechanisms involved in the expression of both of these behavioral abnormalities (Turner & Lewis, 2002). It has been estimated that 8-20% of a general population of intellectually handicapped individuals exhibit some form of SIB (Schroeder et al., 1978; Oliver et al., 1987) and the incidence of SIB is higher in institutionalized populations than it is in community-based groups (Oliver et al., 1987). Furthermore, the incidence of SIB varies 1

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2 between groups of individuals that differ in the nature of their intellectual handicaps. In Lesch-Nyhan patients, all (Nyhan, 1968a; Nyhan, 1968b; Partington & Hennen, 1967) or nearly all individuals (Mitchell & McInnes, 1984) exhibit self-biting behavior and the severity of the SIB varies from individual to individual. These individual differences in expression of SIB in Lesch-Nyhan patients appear to be related to the age of onset of the SIB (Anderson & Ernst, 1994). In Prader-Willi syndrome, skin-picking has been reported in 81% of individuals (Symons et al., 1999). Among individuals afflicted with Cornelia de Lange syndrome, approximately 44% exhibit some form of self-injury (Berney et al., 1999), and 34 % of autistic individuals exhibit SIB (Matson et al., 1996). The high incidence of SIB in these various disorders suggests that there is something about intellectual handicaps in general that predisposes individuals to exhibit SIB. Furthermore, even within these disorders, some but not all exhibit SIB, and the severity of SIB may vary between afflicted individuals. Accordingly, there appear to be individual differences in vulnerability to acquire this devastating behavior disorder both between groups with different disorders, and within specific types of disorder. Dysregulation of dopamine neurotransmission appears to be an important neurochemical feature of a variety of disorders in which SIB is common. Dopaminergic innervation is reduced in the caudate, putamen, nucleus accumbens, globus pallidus, frontal cortex, substantia nigra, and ventral tegmental area of Lesch-Nyhan patients (Ernst et al., 1996; Lloyd et al., 1981). Saito et al. (1999) further identified that the reduced dopamine content in the caudate and putamen was accompanied by an increase in D1 and D2 receptors. Taken together, these data suggest that dopamine receptor supersensitivity may be involved in the expression of SIB. Additional neurochemical

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3 disregulation has been found in adenosine (Page & Coleman, 1998; Rosenberger-Debiesse & Coleman, 1986; Sweetman & Nyhan, 1970) opioid (Coid et al., 1983; Gillberg et al., 1985; Saito et al., 1999; Sandman, 1988; Sandman et al., 1990; Willemsen-Swinkels et al., 1996) and serotonin (Castells et al., 1979; Jankovic et al., 1988) systems in Lesch-Nyhan syndrome, autism, and other disorders in which SIB is expressed. Investigation of the neurobiological mechanisms that participate in the development and expression of SIB has been facilitated by the identification of a variety of animal models of this behavior disorder. These models include social isolation in early development (Harlow & Harlow, 1962; Harlow et al., 1965; Seay & Harlow, 1965), neonatal 6-hydroxydopamine (6-OHDA) lesions followed by dopamine agonist administration in adulthood (Breese et al., 1984), and administration of pharmacological agents that block adenosine receptors, (Hoefnagel, 1968; Mardikar et al., 1969; Sakata & Fuchimoto, 1973) or augment dopamine function (Genovese et al., 1969; Sivam, 1995). We have investigated the etiology of SIB in caffeineand pemoline-treated rats. Caffeine is a non-selective adenosine receptor antagonist and chronic caffeine administration has been reported to induce self-injury in rats (Kasim & Jinnah, 2002; Mueller et al., 1982; Mueller & Nyhan, 1983; Minana et al., 1984; Minana & Grisolia 1986; Peters, 1967) if extremely high doses are administered repeatedly. Pemoline is an indirect dopamine agonist that acts by blocking the reuptake of dopamine. Administration of pemoline at a very high dose is known to produce a rapid onset of stereotypy and SIB (Cromwell et al., 1997; Cromwell et al., 1999; Mueller & Hsiao, 1980) whereas moderately high doses of pemoline are known to produce SIB after

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4 repeated administration across several days (Mueller & Hsiao, 1980; Mueller & Nyhan, 1982; Mueller et al., 1986; Turner et al., 1999). In these investigations of the caffeine and pemoline models, we identified that caffeine produces self-injury only when administered repeatedly at doses that are highly toxic. Pemoline was effective across a range of doses, and self-induced tissue trauma was only seen in a subpopulation of the rats. This suggests that there may be individual differences in vulnerability to self-injure in this animal model of SIB.

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METHODS Animals Male Long Evan rats weighing 100-125g were housed in a climatically-controlled vivarium with a 12 hr light: 12 hr dark cycle (lights on at 3:00 p.m. for the caffeine experiment, and 7:00 a.m. for the pemoline experiment). All the rats had free access to food and water. The rats were pair-housed for 1 week in standard polyethylene cages (43 x 21.5 x 25.5 cm) prior to the repeated caffeine or pemoline administration. Starting on the first day of caffeine or pemoline treatment, each rat was individually housed in standard caging (to ascertain that any recorded injuries were self-inflicted). All the procedures in these experiments were pre-approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida, and all procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Drugs Caffeine was obtained from Sigma-Aldrich Co. and pemoline was obtained from Spectrum Chemicals. The caffeine was suspended in warm saline at a concentration of 20mg/ml, and the pemoline was suspended in warm peanut oil at a concentration of 50mg/ml. Both suspensions were kept warm and stirred constantly right up to the injection time. Independent groups of rats were given daily subcutaneous (s.c.) injections of caffeine (140 or 185 mg/kg/day for 15 days; n = 6 or 12 rats per group) or pemoline (100 mg/kg/day for 15 days, or 200mg/kg/day for 5 days, or 300 mg/kg/day for 4 days; n 5

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6 = 12 rats per group). Additional groups of rats were injected daily with saline for 15 days or peanut oil for 5 days (1.0 ml/kg; n = 6 rats per group). All injections (caffeine, pemoline, and both vehicles) were administered daily between 8:30 and 9:30 a.m. Experimental Procedure Each morning, the rats were checked for injuries, weighed, and injected with caffeine, pemoline, or vehicle. The rats were checked for injuries again every evening. A self-injury score was recorded each morning and evening, according to the presence and extent of injuries (see table 1 for the scale that was used to evaluate self-inflicted tissue damage). The placement of each self-inflicted injury was also recorded, and the size of each injury was measured with a ruler. In any case where an open lesion was identified, the rat expressing the open lesion was immediately euthanized. On the final day (day 16 for 140 mg/kg caffeine, 185 mg/kg caffeine, 100 mg/kg pemoline, and saline groups; day 6 for 200 mg/kg pemoline and peanut-oil groups; day 4 for 300 mg/kg pemoline group) of the experiment (unless the rat had to be euthanized early), each rat was rapidly decapitated one hour after lights on. The trunk blood was collected, and plasma was isolated by centrifugation at 2800 rpm / 1,000 rcf for 5 minutes at 4C. The adrenal and thymus glands were removed from each rat. The isolated plasma and glands were frozen on dry ice, and stored at -80C. Histology The adrenal and thymus glands were weighed. Plasma adrenocorticotrophic hormone (ACTH) concentrations were quantified by immunoradiometric assay (IRMA), using a kit from Nichols Institute Diagnostics. Plasma corticosterone (CORT) concentrations were quantified by radioimmunoassay (RIA) using a kit from Diagnostic Products Corporation.

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7 Statistical Analyses The saline and peanut oil vehicle control groups were compared using 2 x 15 repeated measures analyses of variance (ANOVA) for measurements of self-injury (SIB score, size of tissue damage, and number of sites) and body weight. These vehicle-treated groups were also compared using t-tests for the adrenal and thymus weights, and for the plasma ACTH and CORT concentrations. Neither group of vehicle-treated rats exhibited any self-injury, and there were no significant between-groups differences in any of the other measures (results not shown), and so the groups were combined and used as the common control group for the caffeine and pemoline experiments (vehicle group n = 12). The self-injury scores, number of self-injury sites and total size of self-injuries were each analyzed using 2-way repeated measures ANOVAs. These scores were analyzed with a 3 x 15 (group x day) ANOVA to compare the 185, 140, and control groups for the caffeine experiment. In light of the fact that the pemoline experiments had to be terminated on differing days for each dose group, three ANOVA procedures were conducted. The first ANOVA was a 4 x 4 (group x day) procedure comparing the 300 mg/kg, 200 mg/kg, 100 mg/kg and vehicle control groups across the four days when all groups were run. A 3 x 6 ANOVA was used to compare the 200 mg/kg, 100 mg/kg, and vehicle control groups across the six days that these three groups were all run, and a 2 x 15 ANOVA was used to compare the 100 mg/kg and vehicle control groups across the 15 days when these remaining two groups were run. The vehicle-treated group was used as the control group in both experiments. Data for tissue damages were utilized from the morning recordings only the evening scorings (which generally resembled the morning scorings quite closely) were simply used to make certain that no animal was allowed to

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8 seriously injure itself overnight without intervention. All significant effects were further analyzed with Newman-Keuls post tests, comparing values for each drug-treated group, with the corresponding value for the vehicle-treated control group, and comparing relevant between-groups differences among the various doses for each drug treatment. Between-groups differences in adrenal and thymus weights, and in plasma ACTH and CORT concentrations were each analyzed using one-way ANOVAs for each experiment (caffeine and pemoline), followed by Newman-Keuls post tests for all significant between groups differences. Between-groups differences in the rats body weights were analyzed with 2-way repeated measures ANOVAs in the same manner as were the self-injury scores.

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9 Table 1. Qualitative SIB scale: The rats were scored using these rankings (0-4), based on the severity of self-inflicted tissue damage. SCORE SEVERITY DESCRIPTION 0 no SIB None 1 very mild SIB slight edema, pink moist skin, involves small area 2 mild SIB moderate edema, slight erythema, slightly denuded skin involves medium area, and/or involves multiple sites 3 moderate SIB substantial edema and erythema, large area substantially denuded skin, and/or minor tissue loss 4 severe SIB amputation of digits, and/or clear open lesions requires euthanasia

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RESULTS Tissue Trauma Some, but not all, of the caffeine-treated rats exhibited self-induced tissue damage (see Figure 1a). The number of rats in each group that exhibited tissue damage increased across days of treatment, reaching a peak around day 8 in the group that was treated with 185 mg/kg/day, and around day 12 in the rats that were treated with 140 mg/kg/day. The experiment was terminated on day 16 because of the health conditions of the caffeine-treated rats In fact, one rat in the 185 mg/kg group died on day 14, apparently due to the toxic effects of chronic caffeine administration. Furthermore, when we used a higher dose of caffeine, the dose was lethal early in the course of treatment, and the experiment had to be discontinued before any self-injury was observed (data not shown). Administration of pemoline also produced self-inflicted tissue trauma, and in contrast to the caffeine-induced self-injury, these effects were dose-orderly (see Figure 1b). The self-induced tissue trauma occurred in a greater number of the rats, and onset earlier in the rats that were treated with the higher doses of pemoline. In this experiment, the group that was treated with 300 mg/kg/day was terminated on day 4 because a significant number of the rats exhibited one or more open lesions. The experiment was terminated on day 6 for the group that was treated with 200 mg/kg/day because of the tissue trauma, and the experiment was terminated on day 16 for the group that was treated with 100 mg/kg/day, although these rats did not exhibit open lesions. 10

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11 The daily scoring of caffeine-induced self-inflicted tissue damage revealed that there were significant between-groups differences in the severity of tissue damage across the 15 days of the experiment (interaction effect: F(28, 35) = 1.73, p < 0.05; see Table 1 for the ranked scale of tissue damage scores). Furthermore, the group that was treated with 185 mg/kg/day exhibited significantly higher tissue trauma scores than did the vehicle-treated controls near the end of the experiment (see Fig. 2a). There were no significant differences in tissue trauma scores between the group that was treated with 140 mg/kg/day, and the group that was treated with 185 mg/kg/day, and so the severity of the caffeine-induced self-injury did not occur in a dose-orderly manner. The severity of tissue-damage in the pemoline-treated rats was dose-orderly, with the higher doses producing significantly higher tissue trauma scores than did the lower doses across the days that each group of rats was tested (see Fig. 2d). The rats that were tested with 300 mg/kg/day of pemoline exhibited significantly higher trauma scores than did the other groups of rats during the 4 days (interaction effect: F(9,47) = 10.88, p < 0.01). The group of rats that was treated with 200 mg/kg/day of pemoline exhibited significantly higher trauma scores than did the 100 mg/kg and vehicle group during the 6 days that they were tested (interaction effect: F(10,35) = 11.97, p < 0.01). In addition, the group of rats that was treated with 100 mg/kg/day of pemoline exhibited significantly higher trauma scores than did the vehicle-treated group of rats during the 15 days that they were tested (interaction effect: F(14,23) = 2.87, p < 0.01). The measures of the total size of tissue damage and the number of tissue damage sites revealed a pattern of results that resembled the results using the ranked scores of tissue trauma. The caffeine-treated rats exhibited greater sizes of tissue damage

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12 (interaction effect: F(28, 35) = 1.58, p < 0.05), and greater numbers of sites of tissue damage (interaction effect: F(28,35) = 1.93, p < 0.01) than did the vehicle-treated controls (which did not exhibit tissue trauma), but the effects did not differ between the two groups of caffeine-treated rats, and hence were not dose orderly (see Fig 2b and 2c). On the other hand, the pemoline-treated rats exhibited dose-orderly between-groups differences in size and numbers of self-induced tissue damages (see Fig. 2e and 2f). The rats that were tested with 300 mg/kg/day of pemoline exhibited significantly larger (interaction effect: F(9, 47) = 8.39, p < 0.01) and more numerous (interaction effect: F(9,47) = 8.22, p 0<.01) damages than did the other groups of rats across the 4 days that they were tested. The group of rats that was treated with 200 mg/kg/day of pemoline exhibited significantly larger (interaction effect: F(10,35) = 4.93, p<.01) and more numerous (interaction effect: F(10,35) = 5.40, p <0.01) damages than did the 100 mg/kg and vehicle groups of rats across the 6 days that they were tested. The group of rats that was treated with 100 mg/kg/day of pemoline exhibited significantly larger (interaction effect: F(14,23) = 3.17, p < 0.01) and more numerous (interaction effect: F(14,23) = 2.97, p < 0.01) damages than did the vehicle-treated group of rats across the 15 days that they were tested. Interestingly, there was a tendency that the rats that were treated with caffeine exhibited injury sites on their tails, and did not injure other body sites. The rats that were treated with 200 and 300 mg/kg/day of pemoline primarily injured their forepaws and ventrum (both thorax and abdomen), whereas the rats that were treated with 100 mg/kg/day primarily injured their tails (see table 2).

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13 Hypothalamic-Pituitary-Adrenal Axis Functioning Repeated caffeine administration produced substantial alterations in HPA axis activity, in that basal ACTH levels were significantly increased (F(2,35) = 88.92, p < 0.01) in both doses of caffeine (see Figure 3a), however these effects were not dose-orderly since the 185 and 140 mg/kg groups did not differ significantly. In addition, basal CORT levels were significantly increased in the higher dose of caffeine (F(2,35) = 8.40, p < 0.01) compared to the vehicle group (see Figure 3b). Repeated pemoline administration produced substantial alterations in HPA axis activity, so that 200 mg/kg/day produced significantly higher elevations in circulating ACTH (F(2,33) = 15.80, p<.01) and CORT (F(2,33) = 7.14, p<.01) concentrations (see Figure 3c and 3d). Hormonal data are not available for the rats that were treated with 300 mg/kg/day because they were euthanized at a different time of the day (early evening) than were the other groups (early morning) owing to the severity of injury that had developed at that time. Repeated caffeine administration also produced substantial alterations in adrenal and thymus gland masses (see Fig. 4a and 4b), producing significant hypertrophy of the adrenal glands (F(2,35) = 4.15, p < 0.05) in the 185 mg/kg group and significant atrophy of the thymus glands (F(2,35) = 41.85, p < 0.01) in both 140 mg/kg and 185 mg/kg groups, when these glandular weights were adjusted for between-groups differences in body weights (see description of body weight differences, below). The repeated administration of pemoline produced adrenal hypertrophy only at the highest (300 mg/kg/day) dose (F(3, 44) = 11.66, p < 0.01), and did not significantly alter thymus weights in any of the groups of rats (F(3,44) = 1.74, p > 0.05) (see Figure 4c and 4d).

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14 In the experiment with repeated caffeine administration, the caffeine-treated rats did not gain weight as rapidly as did the rats that were treated with vehicle (interaction effect: F(28,35) = 11.44, p < 0.01), and there were differences between weight gain of the rats in the groups that were treated with the 140 and 185 mg/kg/day doses (see Fig. 4c). In the pemoline experiment, there were also significant between group differences in the weight gains in the four treatment groups (see Fig. 4f). In fact, the rats that were treated with the highest dose (300 mg/kg/day, F(9,47) = 27.26, p<.01) exhibited weight loss, and the rats in the 200 mg/kg/day group (F(10,35) = 14.34, p<.01) exhibited suppressed weight gain, but the rats that were treated with 100 mg/kg/day only differed in their body weights from the vehicle group during the early days of the experiment (F(14,23) = 2.86, p<.05)

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15 Caffeine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 25 50 75 100 vehicle 140 mg/kg 185 mg/kg daypercentage of rats thatshowed tissue damage Pemoline 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 25 50 75 100vehicle 100 mg/kg 200 mg/kg 300 mg/kg daypercentage of rats thatshowed tissue damageAB Figure 1. Incidence of SIB: Pemoline, but not caffeine, administration produced self-injury in a dose-orderly manner. a) Some, but not all, of the caffeine-treated rats self-injured. b) In pemoline-treated rats, the onset of SIB occurred earlier and the total number of rats that self-injured was greater in rats treated with the higher doses of pemoline. None of the rats treated with saline or peanut oil exhibited any signs of tissue damage. Table 2. Topography of SIB: Rats that self-injured in the caffeine-treated group predominantly exhibited raw skin on their tails, with little damage on the paws and no damage on the ventrum. The rats that self-injured in the pemoline-treated group, exhibited tissue damage on the tails, paws or ventrum, depending upon the dose administered. The number of rats exhibiting tissue damage on the tails, paws or ventrum are listed for each of the pharmacologically-treated groups. All the groups have 12 rats, except for the 140 mg/kg caffeine group, which has 6 rats. GROUP forepaws hindpaws ventrum tail vehicle 0 0 0 0 185 caffeine 0 2 0 3 140 caffeine 1 0 0 2 300 pemoline 9 1 5 2 200 pemoline 4 2 7 1 100 pemoline 1 0 0 4

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16 Caffeine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 vehicle 140 mg/kg 185 mg/kg daytissue injury score Pemoline 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4vehicle 100 mg/kg 200 mg/kg 300 mg/kg daytissue injury score 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4daytotal size of injury (cm) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4daytotal size of injury (cm) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0.0 0.5 1.0 1.5 2.0 2.5daynumber of injuries 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0.0 0.5 1.0 1.5 2.0 2.5daynumber of injuries ADBECF Fig. 2. Caffeineand Pemoline-Induced Self-Injury: The rats that were treated with caffeine exhibited significant self-inflicted tissue trauma, as indicated by a) tissue trauma scores, b) overall measures of tissue trauma size, and c) total number of tissue damages across 15 days of treatment. The rats that were treated with pemoline also exhibited self-inflicted tissue trauma, as indicated by d) tissue trauma scores, e) overall measures of tissue trauma size, and f) total number of tissue damages across 4, 6, or 15 days of treatment. V6alues expressed are group means the standard error of the mean (SEM). Significant between-groups differences are depicted as follows: p < 0.01 comparing 185 mg/kg caffeine with vehicle; p < 0.01 comparing 140 mg/kg caffeine with vehicle; p < 0.01 comparing 300 mg/kg pemoline with vehicle; p < 0.01 comparing 300 mg/kg pemoline with 100 mg/kg pemoline; p < 0.01 comparing 300 mg/kg pemoline with 200 mg/kg pemoline; p < 0.01 comparing 200 mg/kg pemoline with vehicle; p < 0.01 comparing 200 mg/kg pemoline with 100 mg/kg pemoline; p < 0.05, p < 0.01 comparing 100 mg/kg pemoline with vehicle.

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17 Pemoline vehicle100200300 0 25 50 75datanotavailabledose (mg/kg) plasma ACTH [pg/ml] vehicle100200300 0 50 100 150 200datanotavailabledose (mg/kg) plasma CORT [ng/ml]ADBC vehicle140185 0 50 100 150 200dose (mg/kg)plasma CORT [ng/ml] Caffeine vehicle140185 0 25 50 75 dose (mg/kg)plasma ACTH [pg/ml] Figure 3. Basal Levels of Stress Hormones: The rats that were treated with caffeine exhibited significant increases in a) basal ACTH levels, however, only the higher dose of caffeine significantly altered b) basal CORT levels. The rats that were treated with 200 mg/kg/day pemoline also exhibited significant alterations in basal stress hormones, as indicated by c) ACTH levels and d) CORT levels. Values expressed are group means SEM. Significant between-groups differences are depicted as follows: p < 0.01 comparing 185 mg/kg caffeine with vehicle; p < 0.01 comparing 140 mg/kg caffeine with vehicle; p < 0.01 comparing 200 mg/kg pemoline with vehicle; p < 0.01 comparing 200 mg/kg pemoline with 100 mg/kg pemoline.

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18 Caffeine vehicle140185 0 10 20 30dose (mg/kg)relative adrenal wt(mg/100g) Pemoline vehicle100200300 0 10 20 30Day 16Day 16Day 6Day 4dose (mg/kg)relative adrenal wt(mg/100g) vehicle140185 0 100 200 300dose (mg/kg)relative thymus wt(mg/100g) vehicle100200300 0 100 200 300dose (mg/kg)Day 16Day 16Day 6Day 4relative thymus wt(mg/100g) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 150 200 250 300vehicle 140 mg/kg 185 mg/kg daybody weight (g) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 150 200 250 300vehicle 100 mg/kg 200 mg/kg 300 mg/kg daybody weight (g) ADBECF Figure 4. Alterations in Glandular and Body Weight: The rats that were treated with caffeine exhibited alterations in glandular weight, as indicated by a) adrenal hypertrophy in the 185 mg/kg group and b) thymus atrophy in both groups. c) The caffeine-treated rats did not gain weight as rapidly as did the rats that were treated with vehicle. The rats that were treated with the highest dose of pemoline showed d) adrenal hypertrophy, but repeated pemoline administration did not alter e) thymus weights. Administration of pemoline affected f) body weight, in that the 300 mg/kg group lost weight, while the 200 mg/kg group did not gain weight; however, the 100 mg/kg group did not significantly differ from the vehicle towards the end of the experiment. Values expressed are group means SEM. Significant between-groups differences are depicted as follows: p < 0.01 comparing 185 mg/kg caffeine with vehicle; p < 0.01 comparing 140 mg/kg caffeine with vehicle; p < 0.01 comparing 300 mg/kg pemoline with vehicle; p < 0.01 comparing 300 mg/kg pemoline with 100 mg/kg pemoline; p < 0.01 comparing 300 mg/kg pemoline with 200 mg/kg pemoline; p < 0.01 comparing 200 mg/kg pemoline with vehicle, p < 0.01 comparing 100 mg/kg pemoline vehicle. For body weight, (graphs c and f), significance symbols are shown for treatment groups vs. vehicle group only.

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DISCUSSION Previous reports of caffeineand pemoline-induced self-injury have described the effects of caffeine or pemoline treatments either by reporting the numbers of rats that exhibited tissue damage (Kasim & Jinnah, 2002; Minana et al., 1984; Minana & Grisolia, 1986; Mueller & Nyhan, 1982; Mueller et al., 1982; Mueller & Nyhan, 1983; Mueller et al., 1986; Peters, 1967), or by reporting the severity of the tissue damage using a rating scale (King et al., 1993; King et al., 1995; Mueller & Hsiao, 1980; Turner et al., 1999). In the present experiments, we directly compared the effectiveness of caffeine and pemoline treatments. We measured the numbers of rats that exhibited tissue trauma daily during treatment with each dose of each drug, and we measured the severity of tissue trauma with a 5-point scale of tissue damage (King et al., 1993; King et al., 1995; Turner et al., 1999). In addition, we assessed the number of trauma sites, and the size of tissue trauma each day during treatment with each of the pharmacological manipulations. This phenomenological evaluation of these two pharmacological models across days of treatment revealed important differences between caffeineand pemoline-induced SIB. In fact, the caffeine-induced SIB was mild (never exceeding on the rating scale), and this mild self-injury occurred only in a small number of the rats, even though the doses that were required to produce these self-injurious outcomes were in the range that produced extreme toxicity. The caffeine-treated rats exhibited severe signs of malaise at all doses tested, including behavioral lethargy, reduced weight gain, porphyrhin secretions around the eyes and snout, alterations in HPA axis function, and 19

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20 even death. One of the twelve rats died during treatment with 185 mg/kg of caffeine, a finding that is consistent with a previous report (Peters, 1967) that this dose administered intragastrically produced approximately 10% mortality. In our preliminary studies, a higher dose produced immediate mortality in more than 50% of the rats so that experiment was immediately discontinued, and we were unable to examine whether doses higher than 185 mg/kg would induce greater self-injury due to these toxic actions. Repeated administration of pemoline produced substantially greater incidence and severity of self-induced tissue trauma than did caffeine, and in contrast to the effects of caffeine, these effects of pemoline were dose-orderly. Furthermore, the pemoline-treated rats exhibited significantly fewer and less severe signs of drug-induced toxicity, especially at the 100 and 200 mg/kg doses. Porphyrhin secretions were not observed in the rats that were treated with the 100 and 200 mg doses of pemoline, all the pemoline-treated rats exhibited hyperactivity rather than lethargy or malaise, and the pemoline was never lethal even at a dose (300 mg/kg/day) that produced very rapid onset of severe self-injury in more than 90% of the rats. Accordingly, the 100-300 mg/kg dose range effectively produced self-injury that was accompanied by minimal impact upon the health status of the rats. However, it should be noted that higher doses of pemoline do appear to produce substantial toxicity, and 500 mg/kg/day has been shown to produce approximately 50% mortality (Genovese et al., 1969). The caffeine and pemoline models also differed in terms of the topographical expression of SIB. In the caffeine-treated rats, tissue damage was generally restricted to the tail; there was very little tissue trauma on the forepaws and no tissue damage on the ventrum (Table 2). The mildness of the caffeine-induced self-injury, coupled with the

PAGE 30

21 fact that it was focused on the tail, contrasts with previous reports that described severe self-inflicted injuries on the paws (Mueller et al., 1982; Mueller & Nyhan, 1983) or on the paws and tails (Peters, 1967) of caffeine-treated rats. The reason for this apparent contradiction is unclear. However, we did observe that the caffeine-treated rats had extensive amounts of dark red porphyrhin secretions on their forepaws, where they spread these secretions from their snouts onto their paws during grooming. These secretions closely resembled blood, and the encrusted secretions on the forepaws of our rats looked like severe injury, until we washed the paws, and found no injury underneath. Furthermore, self-biting behavior was never observed in casual observations in the caffeine-treated rats. In the pemoline-treated rats, the extent of tissue damage was much greater, and was more commonly exhibited on the forepaws and ventrum (thorax and abdomen). In contrast to injuries in the caffeine-treated rats, the tail was the least common area of injury. This is consistent with previous reports of pemoline-induced SIB (Mueller & Hsiao, 1980; Mueller et al., 1986). In the pemoline-treated rats, the self-biting behavior was highly stereotyped, with rats often showing biting that started at the forepaws, and moved on to the ventral thorax and abdomen, and this self-biting behavior was consistently observed in casual observations. Evaluation of pemoline doses that were effective in approximately 50-75% of the rats (100-200 mg/kg) revealed that there are individual differences in vulnerability to self-injure in this pharmacological model. In all three doses of chronic pemoline administration, some of the rats self-injured, whereas some of the rats did not. This is reminiscent of the fact that individuals within clinical populations (e.g. autistic individuals) appear to differ in their vulnerability or predisposition to exhibit self-injury

PAGE 31

22 so that only a subset of afflicted individuals demonstrate self-injurious behaviors. Accordingly, we believe that the pemoline model of self-injury may provide a useful tool to examine the neurobiological basis of individual differences in vulnerability to self-injure, in that this biological vulnerability may have a significant impact upon our understanding of the etiology of clinical SIB in human populations. Individual differences in vulnerability to self-injure also occurred in the caffeine model, but the toxicity of this treatment is problematic, and therefore, the pemoline model appears to be a better model for the study of factors that determine individual differences in this vulnerability. In fact, the 200 mg/kg dose of pemoline appears to be close to the ED50 for induction of SIB and seems to be a reasonable dose to use when investigating individual differences in brain functioning, drug sensitivity, and hormonal responses that may shed light on individual differences in the clinical populations. These investigations could be coupled with studies in genetic models of SIB (Kasim & Jinnah, 2002), in effects of cortical (Cromwell et al., 1999) and other brain lesions, and in pharmacological manipulations (e.g. antagonist challenges) that could alter vulnerability (King et al., 1993; King et al., 1995) to exhibit SIB. In addition, the impact of environmental factors (e.g. stress exposure, environmental enrichment, operant conditioning) that could alter the innate predisposition to self-injure could be studied in the pemoline model of SIB. Ultimately, these studies may help increase our understanding of pathologies that are associated with self-injury, and lead towards improved prevention or treatment of self-injurious behavior.

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24 Genovese E., Napoli P.A., Bolega-Zonta N. (1969) Self-aggressiveness: a new type of behavioral change induced by pemoline. Life Sciences 8(1): 513-515 Gilbert S., Spellacy E., Watts R.W.E. (1979) Problems in the behavioural treatment of self-injury in the Lesch-Nyhan syndrome. Developmental Medicine in Child Neurology 21: 795-800. Gillberg C., Terenius L., Lonnerholm G. (1985) Endorphin activity in childhood psychosis. Spinal fluid levels in 24 cases. Archives of General Psychiatry 42: 780-783. Guess D., Carr E. (1991) Emergence and maintenance of stereotypy and self-injury. American Journal of Mental Retardation 96(3): 299-319 Harlow H.F., Dodsworth R.O., Harlow M.K. (1965) Total social isolation in monkeys. Proceedings of the National.Academy of Sciences U.S.A 54: 90-97. Harlow H.F., Harlow M.K. (1962) Social deprivation in monkeys. Scientific American 207: 136-146. Hellings J.A., Warnock J.K. (1994) Self-injurious behavior and serotonin in Prader-Willi Syndrome. Psychopharmacological Bulletin 30(2): 245-50. Hoefnagel D. (1968) Seminars on the Lesch-Nyhan syndrome: Summary. Federal Proceedings 27: 1042-1046. Jankovic J., Caskey T.C., Stout J.T., Butler I.J. (1988) Lesch-Nyhan syndrome: A study of motor behavior and cerebrospinal fluid neurotransmitters. Annals of Neurology 23: 466-469. Kasim S., Jinnah H. (2002) Pharmacologic thresholds for self-injurious behavior in a genetic mouse model of Lesch-Nyhan disease. Pharmacology Biochemistry and Behavior 73: 583-592 King B.H., Au D., Poland R.E. (1993) Low-dose naltrexone inhibits pemoline-induced self-biting behavior in prepubertal rats. Journal of Child and Adolescent Psychopharmacology 3(2): 71-79. King B.H., Au D., Poland R.E. (1995) Pretreatment with MK-801 inhibits pemoline-induced self-biting behavior in prepubertal rats. Developmental Neuroscience 17: 47-52 Lloyd K.G., Hornykiewicz O., Davidson L., Farley I., Goldstein M., Shibuya M., Kelley W.N., Fox I.H. (1981) Biochemical evidence of dysfunction of brain neurotransmitters in the Lesch-Nyhan syndrome. New England Journal of Medicine 305: 1106-1111.

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25 Mardikar B.R., Srinivasan S., Balwani J.H. (1969) On the phenomenom of caffeine induced automutilation. Indian Journal of Physiological Pharmacology 13(4): 261-263 Matson J.L., Baglio C.S., Smiroldo B.B., Hamilton M., Packlowskyj T., Williams D., Kirk-patrick-Sanchez S. (1996) Characteristics of autism as assessed by Diagnostic Assessment for Severely Handicapped-II DASH-II. Research of Developmental Disabilities 17: 135-43. Minana M.D., Portoles M., Jorda A., Grisolia S. (1984) Lesch-Nyhan Syndrome, Caffeine model: increae of purine and pyrimidine enzymes in rat brain. Journal of Neurochemistry 43: 1556-1560. Minana M.D., Grisolia S. (1986) Caffeine Ingestion by rats increases noradrenaline turnover and results in self-biting. Journal of Neurochemistry 47: 728-732. Mitchell G., McInnes R.R. (1984) Differential diagnosis of cerebral palsy: Lesch-Nyhan syndrome without the self-mutilation. Canadian Medical Association Journal 130(10): 1323-4. Mueller K., Hollingsworth E., Pettit H. (1986) Repeated pemoline produces self-injuroius behavior in adult and weanling rats. Pharmacology Biochemistry and Behavior 25: 933-938. Mueller K., Hsiao S. (1980) Pemoline-induced self-biting in rats and self-mutilation in the deLange syndrome. Pharmacology Biochemistry and Behavior 13(5): 627-631. Mueller K., Nyhan W.L .(1982) Pharmacologic control of pemoline induced self-injurious behavior in rats. Pharmacology Biochemistry Behavior 16: 957-963. Mueller K., Nyhan W.L. (1983) Clonidine potentiates drug-induced self-injurious behavior in rats. Pharmacology Biochemistry Behavior 18: 891-894. Mueller K., Saboda S., Palmour R., Nyhan W.L. (1982) Self-injurious behavior produced in rats by daily caffeine and continuous amphetamine. Pharmacology Biochemistry and Behavior 17: 613-617. Nyhan W.L. (1968a) Clinical features of the Lesch-Nyhan syndrome. Introduction--clinical and genetic features. Federal Proceedings 27(4): 1027-33. Nyhan W.L. Lesch-Nyhan syndrome. (1968b) Summary of clinical features. Federal Proceedings 27(4): 1034-41. Oliver C., Murphy G.H., Corbett J.A. (1987) Self-injurious behavior in people with mental handicap: a total population study. Journal of Mental Deficit Research 31: 147-62.

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26 Page T. Coleman, M. (1998) De novo purine synthesis is increased in the fibroblasts of purine autism patients. Advanced Experimental and Medical Biology 431: 793-796. Partington M., Hennen B.K. (1967) Lesch-Nyhan Syndrome: Self-destructive behavior, mental retardation, neurodisease, and hyperuricaemia. Developmental and Medical Child Neurology 9(5): 563-72. Peters J.M. (1967) Caffeine-induced hemorrhagic automutilation. Archives of International Pharmacodynamic Therapy 169(1): 139-46. Rosenberger-Debiesse J., Coleman M. (1986) Preliminary evidence for multiple etiologies in autism. Journal of Autism Developmental Disorders 16: 385-392. Saito Y., Ito M., Hanaoka S., Ohama E., Akaboshi S., Takashima S. (1999) Dopamine receptor upregulation in Lesch-Nyhan syndrome: a postmortem study. Neuropediatrics 30: 66-71. Sakata T., Fuchimoto H. (1973) Stereotyped and aggressive behavior induced by sustained high doses of theophylline in rats. Japan Journal of Pharmacology 23(6): 781-5. Sandman, C.A. (1988) b-endorphin disregulation in autistic and self-injurious behavior: a neurodevelopmental hypothesis. Synapse 2: 193-199. Sandman C.A., Barron J.L., Chicz-DeMet A., DeMet E.M. (1990) Plasma b-endorphin levels in patients with self-injurious behavior and stereotypy. American Journal of Mental Retardation 95: 84-92. Schepis C., Failla P., Siragusa M., Romano C. (1994) Skin-picking: best cutaneous feature in recognization of Prader-Willi Syndrome. International Journal of Dermatology 33(12): 866-7. Schoeder S.R., Schroeder C.S., Smith B., Dalldorf J. (1978) Prevalence of self-injurious behaviors in a large state facility for the retarded: a three year follow-up study. Journal of Autism and Childhood Schizophrenia 8(3): 261-269. Seay B., Harlow H.F. (1965) Maternal separation in the rhesus monkey. Journal of Nervous Mental Disorders 140: 434-441. Sivam S.P. (1989) D1 dopamine receptor-mediated substance P depletion in the striatonigral neurons of rats subjected to neonatal dopaminergic denervation: implications for self-injurious behavior. Brain Research 500(1-2): 119-30. Sweetman L., Nyhan W.L. (1970) Detailed comparison of the urinary excretion of purines in a patient with the Lesch-Nyhan syndrome and a control subject. Biochemical Medicine 4: 121-134.

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27 Symons F.J., Butler M.G., Sanders M.D., Feurer I.D., Thompson T. (1999) Self-injurious behavior and Prader-Willi syndrome: behavioral forms and body locations. American Journal of Mental Retardation 104(3): 260-9. Symons F.J., Thompson T. (1997) Self-injurious behavior and body site preference. Journal of Intellectual Disabilities Research 41(6): 456-68. Turner C.A., Lewis M.H. (2002) Dopaminergic mechanisms in self-injurious behavior and related disorders. American Psychological Association, Washington, D.C. In Self-injurious behavior: gene-brain-behavior relationships (Editors: Schroeder S.R., Oster-Granite M.L., Thompson T.) 165-179. Turner C.A., Panksepp J., Bekkedal M., Borkowski, Burgdorf (1999) Paradoxial effects of serotonin and opiods in pemoline-induced self-injurious behavior. Pharmacology Biochemistry and Behavior 63(3): 361-366. Willemsen-Swinkels S.H.N., Buitelaar J.K., Weijnen F.G., Thijssen J.H.H., Van Engeland H. (1996) Plasma beta-endorphin concentrations in people with learning disability and self-injurious and/or autistic behaviour. British Journal of Psychiatry 168: 105-109.

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BIOGRAPHICAL SKETCH Staci Kies was born on February 5 th 1978, in Joliet, IL. In 1986, she moved to Melbourne, FL. After graduating high school in 1996, she attended Brevard Community College, where she received her Associate of Arts in May 1997. Staci then went on to attend the University of Central Florida, where she obtained her Bachelor of Science in psychology in May 1999. From August 1999 to the present, she has been pursuing a graduate degree in behavioral neuroscience in the Psychology Department of the University of Florida. 28


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A COMPARISON OF CAFFEINE AND PEMOLINE MODELS OF SELF-INJURY IN
RATS
















By

STACI DENISE KIES


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2003















Copyright 2003


by

Staci D. Kies















This document is dedicated to my parents, Ken and Debbie Kies, for their emotional
support during the writing of this thesis.















ACKNOWLEDGMENTS

I would like to thank my thesis committee members, Dr. Tim Vollmer and Dr.

Mark Lewis, for their helpful comments on this thesis. I would especially like to thank

my advisor and committee chair, Dr. Darragh Devine, for creating this particular project,

as well as spending the time to make revisions to my many rough drafts.
















TABLE OF CONTENTS

Page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TA BLES ............... ............................................ ............ .......... vi

L IST O F F IG U R E S .... ....... .................................................... ..... ....... .............. vii

A B ST R A C T .................................................... viii

IN TRODU CTION ............................................... ........ .. ...... .............. .. 1

M E T H O D S .......................................................................... . 5

A nim als .................................................. 5
D rug s.................................................................. . 5
Experim ental Procedure.................................................. 6
H isto lo g y ................................................................................. 6
S statistical A n aly ses ................................................................................ 7

R E S U L T S ................................................................................10

Tissue Trauma .........................................0.................10
Hypothalamic-Pituitary-Adrenal Axis Functioning ...................................... 13

D IS C U S S IO N ....................................................................................................... 19

L IST O F R E FE R E N C E S .............................................................................. 23

B IO G R A PH IC A L SK E T C H ........................................................................................ 28















v















LIST OF TABLES

Table p

1. Q ualitative SIB scale............. .... .............................................................. ......... .... .9

2. Topography of SIB ................................... .. .......... .......... .... 15















LIST OF FIGURES

Figure page

1. Incidence of SIB ................................................................15

2. Caffeine- and Pemoline-Induced Self-Injury.....................................................16

3. B asal Levels of Stress H orm ones. ................................. ...................... ...................17

4. Alterations in Glandular and Body Weight. ..................................... ............... 18















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

A COMPARISON OF CAFFEINE AND PEMOLINE MODELS OF SELF-INJURY IN
RATS

By

Staci D. Kies

May 2003

Chair: Darragh P. Devine
Major Department: Psychology

Chronic and stereotyped self-injurious behavior (SIB) is a maladaptive and

debilitating behavior disorder, which can often have life-threatening consequences. It is

exhibited predominantly by autistic and intellectually handicapped individuals, including

those with a variety of specific genetic disorders. Disregulation of dopamine

neurotransmission appears to be an important neurochemical feature of a variety of

disorders in which SIB is observed, and several animal models have been developed in

which dopamine function is altered. We have investigated the etiology of SIB in two of

these models, using caffeine, an adenosine antagonist, and pemoline, an indirect

dopamine agonist, in rats. In these investigations, we identified that caffeine produces

only mild self-injury, and the effective doses are highly toxic. Pemoline was effective

across a range of doses, and the expression of pemoline-induced self-injury occurred in a

dose-orderly manner. Furthermore, effective pemoline doses were identified at which

self-injury was only seen in a subset of the rats. This suggests that there may be









individual differences in vulnerability to self-injure in the pemoline model of SIB. These

individual differences are reminiscent of the fact that individuals with specific clinical

disorders (e.g., autism) differ in their vulnerability to self-injure. Accordingly, research

with the pemoline model of self-injury may help to uncover the biological factors that

underlie individual differences in vulnerability to exhibit self-injury.















INTRODUCTION

Self-injurious behavior (SB) is a devastating, chronic and usually stereotyped

behavior disorder in which tissue damage is self-inflicted. This maladaptive behavior

disorder is commonly seen in intellectually handicapped populations, wherein the

severity of the SIB can range from mild to life-threatening. The expression of self-injury

differs between clinical groups of intellectually handicapped individuals. Self-injury to

the head and/or hands is often seen in autistic and other intellectually handicapped

populations (Symons & Thompson 1997), skin-picking is commonly seen in Prader-Willi

syndrome (Hellings & Warnock, 1994; Schepis et al., 1994), and lip-, tongue-, and digit-

biting is seen in Lesch-Nyhan syndrome (Anderson & Ernst, 1994). In these groups, the

behavior disorder is often highly resistant to treatment (Anderson & Ernst, 1994).

In some instances, SIB is co-expressed with other behavior disorders, especially

stereotypy. In fact, it has been proposed that SIB occurs on a continuum with stereotypy,

wherein SIB is a severe or sensitized expression of stereotypy (Barron & Sandman, 1984;

Guess & Carr, 1991) and the basal ganglia have been specifically implicated in this co-

morbidity because of the apparently overlapping neural mechanisms involved in the

expression of both of these behavioral abnormalities (Turner & Lewis, 2002).

It has been estimated that 8-20% of a general population of intellectually

handicapped individuals exhibit some form of SIB (Schroeder et al., 1978; Oliver et al.,

1987) and the incidence of SIB is higher in institutionalized populations than it is in

community-based groups (Oliver et al., 1987). Furthermore, the incidence of SIB varies









between groups of individuals that differ in the nature of their intellectual handicaps. In

Lesch-Nyhan patients, all (Nyhan, 1968a; Nyhan, 1968b; Partington & Hennen, 1967) or

nearly all individuals (Mitchell & Mclnnes, 1984) exhibit self-biting behavior and the

severity of the SIB varies from individual to individual. These individual differences in

expression of SIB in Lesch-Nyhan patients appear to be related to the age of onset of the

SIB (Anderson & Ernst, 1994). In Prader-Willi syndrome, skin-picking has been

reported in 81% of individuals (Symons et al., 1999). Among individuals afflicted with

Cornelia de Lange syndrome, approximately 44% exhibit some form of self-injury

(Berney et al., 1999), and 34 % of autistic individuals exhibit SIB (Matson et al., 1996).

The high incidence of SIB in these various disorders suggests that there is something

about intellectual handicaps in general that predisposes individuals to exhibit SIB.

Furthermore, even within these disorders, some but not all exhibit SIB, and the severity

of SIB may vary between afflicted individuals. Accordingly, there appear to be

individual differences in vulnerability to acquire this devastating behavior disorder both

between groups with different disorders, and within specific types of disorder.

Dysregulation of dopamine neurotransmission appears to be an important

neurochemical feature of a variety of disorders in which SIB is common. Dopaminergic

innervation is reduced in the caudate, putamen, nucleus accumbens, globus pallidus,

frontal cortex, substantial nigra, and ventral tegmental area of Lesch-Nyhan patients

(Ernst et al., 1996; Lloyd et al., 1981). Saito et al. (1999) further identified that the

reduced dopamine content in the caudate and putamen was accompanied by an increase

in Dl and D2 receptors. Taken together, these data suggest that dopamine receptor

supersensitivity may be involved in the expression of SIB. Additional neurochemical









disregulation has been found in adenosine (Page & Coleman, 1998; Rosenberger-

Debiesse & Coleman, 1986; Sweetman & Nyhan, 1970) opioid (Coid et al., 1983;

Gillberg et al., 1985; Saito et al., 1999; Sandman, 1988; Sandman et al., 1990;

Willemsen-Swinkels et al., 1996) and serotonin (Castells et al., 1979; Jankovic et al.,

1988) systems in Lesch-Nyhan syndrome, autism, and other disorders in which SIB is

expressed.

Investigation of the neurobiological mechanisms that participate in the

development and expression of SIB has been facilitated by the identification of a variety

of animal models of this behavior disorder. These models include social isolation in

early development (Harlow & Harlow, 1962; Harlow et al., 1965; Seay & Harlow, 1965),

neonatal 6-hydroxydopamine (6-OHDA) lesions followed by dopamine agonist

administration in adulthood (Breese et al., 1984), and administration of pharmacological

agents that block adenosine receptors, (Hoefnagel, 1968; Mardikar et al., 1969; Sakata &

Fuchimoto, 1973) or augment dopamine function (Genovese et al., 1969; Sivam, 1995).

We have investigated the etiology of SIB in caffeine- and pemoline-treated rats.

Caffeine is a non-selective adenosine receptor antagonist and chronic caffeine

administration has been reported to induce self-injury in rats (Kasim & Jinnah, 2002;

Mueller et al., 1982; Mueller & Nyhan, 1983; Minana et al., 1984; Minana & Grisolia

1986; Peters, 1967) if extremely high doses are administered repeatedly. Pemoline is an

indirect dopamine agonist that acts by blocking the reuptake of dopamine.

Administration of pemoline at a very high dose is known to produce a rapid onset of

stereotypy and SIB (Cromwell et al., 1997; Cromwell et al., 1999; Mueller & Hsiao,

1980) whereas moderately high doses of pemoline are known to produce SIB after






4


repeated administration across several days (Mueller & Hsiao, 1980; Mueller & Nyhan,

1982; Mueller et al., 1986; Turner et al., 1999). In these investigations of the caffeine

and pemoline models, we identified that caffeine produces self-injury only when

administered repeatedly at doses that are highly toxic. Pemoline was effective across a

range of doses, and self-induced tissue trauma was only seen in a subpopulation of the

rats. This suggests that there may be individual differences in vulnerability to self-injure

in this animal model of SIB.















METHODS

Animals

Male Long Evan rats weighing 100-125g were housed in a climatically-controlled

vivarium with a 12 hr light: 12 hr dark cycle (lights on at 3:00 p.m. for the caffeine

experiment, and 7:00 a.m. for the pemoline experiment). All the rats had free access to

food and water. The rats were pair-housed for 1 week in standard polyethylene cages (43

x 21.5 x 25.5 cm) prior to the repeated caffeine or pemoline administration. Starting on

the first day of caffeine or pemoline treatment, each rat was individually housed in

standard caging (to ascertain that any recorded injuries were self-inflicted). All the

procedures in these experiments were pre-approved by the Institutional Animal Care and

Use Committee (IACUC) at the University of Florida, and all procedures were carried out

in accordance with the National Institutes of Health Guide for the Care and Use of

Laboratory Animals.

Drugs

Caffeine was obtained from Sigma-Aldrich Co. and pemoline was obtained from

Spectrum Chemicals. The caffeine was suspended in warm saline at a concentration of

20mg/ml, and the pemoline was suspended in warm peanut oil at a concentration of

50mg/ml. Both suspensions were kept warm and stirred constantly right up to the

injection time. Independent groups of rats were given daily subcutaneous (s.c.) injections

of caffeine (140 or 185 mg/kg/day for 15 days; n = 6 or 12 rats per group) or pemoline

(100 mg/kg/day for 15 days, or 200mg/kg/day for 5 days, or 300 mg/kg/day for 4 days; n









= 12 rats per group). Additional groups of rats were injected daily with saline for 15 days

or peanut oil for 5 days (1.0 ml/kg; n = 6 rats per group). All injections (caffeine,

pemoline, and both vehicles) were administered daily between 8:30 and 9:30 a.m.

Experimental Procedure

Each morning, the rats were checked for injuries, weighed, and injected with

caffeine, pemoline, or vehicle. The rats were checked for injuries again every evening.

A self-injury score was recorded each morning and evening, according to the presence

and extent of injuries (see table 1 for the scale that was used to evaluate self-inflicted

tissue damage). The placement of each self-inflicted injury was also recorded, and the

size of each injury was measured with a ruler. In any case where an open lesion was

identified, the rat expressing the open lesion was immediately euthanized.

On the final day (day 16 for 140 mg/kg caffeine, 185 mg/kg caffeine, 100 mg/kg

pemoline, and saline groups; day 6 for 200 mg/kg pemoline and peanut-oil groups; day 4

for 300 mg/kg pemoline group) of the experiment (unless the rat had to be euthanized

early), each rat was rapidly decapitated one hour after "lights on". The trunk blood was

collected, and plasma was isolated by centrifugation at 2800 rpm / 1,000 rcf for 5 minutes

at 40C. The adrenal and thymus glands were removed from each rat. The isolated

plasma and glands were frozen on dry ice, and stored at -800C.

Histology

The adrenal and thymus glands were weighed. Plasma adrenocorticotrophic

hormone (ACTH) concentrations were quantified by immunoradiometric assay (IRMA),

using a kit from Nichols Institute Diagnostics. Plasma corticosterone (CORT)

concentrations were quantified by radioimmunoassay (RIA) using a kit from Diagnostic

Products Corporation.









Statistical Analyses

The saline and peanut oil vehicle control groups were compared using 2 x 15

repeated measures analyses of variance (ANOVA) for measurements of self-injury (SIB

score, size of tissue damage, and number of sites) and body weight. These vehicle-

treated groups were also compared using t-tests for the adrenal and thymus weights, and

for the plasma ACTH and CORT concentrations. Neither group of vehicle-treated rats

exhibited any self-injury, and there were no significant between-groups differences in any

of the other measures (results not shown), and so the groups were combined and used as

the common control group for the caffeine and pemoline experiments (vehicle group n =

12).

The self-injury scores, number of self-injury sites and total size of self-injuries

were each analyzed using 2-way repeated measures ANOVAs. These scores were

analyzed with a 3 x 15 (group x day) ANOVA to compare the 185, 140, and control

groups for the caffeine experiment. In light of the fact that the pemoline experiments had

to be terminated on differing days for each dose group, three ANOVA procedures were

conducted. The first ANOVA was a 4 x 4 (group x day) procedure comparing the 300

mg/kg, 200 mg/kg, 100 mg/kg and vehicle control groups across the four days when all

groups were run. A 3 x 6 ANOVA was used to compare the 200 mg/kg, 100 mg/kg, and

vehicle control groups across the six days that these three groups were all run, and a 2 x

15 ANOVA was used to compare the 100 mg/kg and vehicle control groups across the 15

days when these remaining two groups were run. The vehicle-treated group was used as

the control group in both experiments. Data for tissue damages were utilized from the

morning recordings only the evening scoring (which generally resembled the morning

scoring quite closely) were simply used to make certain that no animal was allowed to









seriously injure itself overnight without intervention. All significant effects were further

analyzed with Newman-Keul's post tests, comparing values for each drug-treated group,

with the corresponding value for the vehicle-treated control group, and comparing

relevant between-groups differences among the various doses for each drug treatment.

Between-groups differences in adrenal and thymus weights, and in plasma ACTH

and CORT concentrations were each analyzed using one-way ANOVAs for each

experiment (caffeine and pemoline), followed by Newman-Keul's post tests for all

significant between groups differences. Between-groups differences in the rats' body

weights were analyzed with 2-way repeated measures ANOVAs in the same manner as

were the self-injury scores.







9


Table 1. Qualitative SIB scale: The rats were scored using these rankings (0-4), based on
the severity of self-inflicted tissue damage.

SCORE SEVERITY DESCRIPTION
0 no SIB None

1 very mild SIB slight edema, pink moist skin, involves small area

2 mild SIB moderate edema, slight erythema, slightly denuded skin
involves medium area, and/or involves multiple sites
3 moderate SIB substantial edema and erythema, large area
substantially denuded skin, and/or minor tissue loss
4 severe SIB amputation of digits, and/or clear open lesions
requires euthanasia















RESULTS

Tissue Trauma

Some, but not all, of the caffeine-treated rats exhibited self-induced tissue damage

(see Figure la). The number of rats in each group that exhibited tissue damage increased

across days of treatment, reaching a peak around day 8 in the group that was treated with

185 mg/kg/day, and around day 12 in the rats that were treated with 140 mg/kg/day.

The experiment was terminated on day 16 because of the health conditions of the

caffeine-treated rats In fact, one rat in the 185 mg/kg group died on day 14, apparently

due to the toxic effects of chronic caffeine administration. Furthermore, when we used a

higher dose of caffeine, the dose was lethal early in the course of treatment, and the

experiment had to be discontinued before any self-injury was observed (data not shown).

Administration of pemoline also produced self-inflicted tissue trauma, and in

contrast to the caffeine-induced self-injury, these effects were dose-orderly (see Figure

lb). The self-induced tissue trauma occurred in a greater number of the rats, and onset

earlier in the rats that were treated with the higher doses of pemoline. In this experiment,

the group that was treated with 300 mg/kg/day was terminated on day 4 because a

significant number of the rats exhibited one or more open lesions. The experiment was

terminated on day 6 for the group that was treated with 200 mg/kg/day because of the

tissue trauma, and the experiment was terminated on day 16 for the group that was treated

with 100 mg/kg/day, although these rats did not exhibit open lesions.









The daily scoring of caffeine-induced self-inflicted tissue damage revealed that

there were significant between-groups differences in the severity of tissue damage across

the 15 days of the experiment (interaction effect: F(28, 35) = 1.73, p < 0.05; see Table 1

for the ranked scale of tissue damage scores). Furthermore, the group that was treated

with 185 mg/kg/day exhibited significantly higher tissue trauma scores than did the

vehicle-treated controls near the end of the experiment (see Fig. 2a). There were no

significant differences in tissue trauma scores between the group that was treated with

140 mg/kg/day, and the group that was treated with 185 mg/kg/day, and so the severity of

the caffeine-induced self-injury did not occur in a dose-orderly manner.

The severity of tissue-damage in the pemoline-treated rats was dose-orderly, with

the higher doses producing significantly higher tissue trauma scores than did the lower

doses across the days that each group of rats was tested (see Fig. 2d). The rats that were

tested with 300 mg/kg/day of pemoline exhibited significantly higher trauma scores than

did the other groups of rats during the 4 days (interaction effect: F(9,47) = 10.88, p <

0.01). The group of rats that was treated with 200 mg/kg/day of pemoline exhibited

significantly higher trauma scores than did the 100 mg/kg and vehicle group during the 6

days that they were tested (interaction effect: F(10,35) = 11.97, p < 0.01). In addition,

the group of rats that was treated with 100 mg/kg/day of pemoline exhibited significantly

higher trauma scores than did the vehicle-treated group of rats during the 15 days that

they were tested (interaction effect: F(14,23) = 2.87, p < 0.01).

The measures of the total size of tissue damage and the number of tissue damage

sites revealed a pattern of results that resembled the results using the ranked scores of

tissue trauma. The caffeine-treated rats exhibited greater sizes of tissue damage









(interaction effect: F(28, 35) = 1.58, p < 0.05), and greater numbers of sites of tissue

damage (interaction effect: F(28,35) = 1.93, p < 0.01) than did the vehicle-treated

controls (which did not exhibit tissue trauma), but the effects did not differ between the

two groups of caffeine-treated rats, and hence were not dose orderly (see Fig 2b and 2c).

On the other hand, the pemoline-treated rats exhibited dose-orderly between-groups

differences in size and numbers of self-induced tissue damages (see Fig. 2e and 2f). The

rats that were tested with 300 mg/kg/day of pemoline exhibited significantly larger

(interaction effect: F(9, 47) = 8.39, p < 0.01) and more numerous (interaction effect:

F(9,47) = 8.22, p 0<.01) damages than did the other groups of rats across the 4 days that

they were tested. The group of rats that was treated with 200 mg/kg/day of pemoline

exhibited significantly larger (interaction effect: F(10,35) = 4.93, p<.01) and more

numerous (interaction effect: F(10,35) = 5.40, p <0.01) damages than did the 100 mg/kg

and vehicle groups of rats across the 6 days that they were tested. The group of rats that

was treated with 100 mg/kg/day of pemoline exhibited significantly larger (interaction

effect: F(14,23) = 3.17, p < 0.01) and more numerous (interaction effect: F(14,23) = 2.97,

p < 0.01) damages than did the vehicle-treated group of rats across the 15 days that they

were tested.

Interestingly, there was a tendency that the rats that were treated with caffeine

exhibited injury sites on their tails, and did not injure other body sites. The rats that were

treated with 200 and 300 mg/kg/day of pemoline primarily injured their forepaws and

ventrum (both thorax and abdomen), whereas the rats that were treated with 100

mg/kg/day primarily injured their tails (see table 2).









Hypothalamic-Pituitary-Adrenal Axis Functioning

Repeated caffeine administration produced substantial alterations in HPA axis

activity, in that basal ACTH levels were significantly increased (F(2,35) = 88.92, p <

0.01) in both doses of caffeine (see Figure 3a), however these effects were not dose-

orderly since the 185 and 140 mg/kg groups did not differ significantly. In addition,

basal CORT levels were significantly increased in the higher dose of caffeine (F(2,35) =

8.40, p < 0.01) compared to the vehicle group (see Figure 3b).

Repeated pemoline administration produced substantial alterations in HPA axis activity,

so that 200 mg/kg/day produced significantly higher elevations in circulating ACTH

(F(2,33) = 15.80, p<.01) and CORT (F(2,33) = 7.14, p<.01) concentrations (see Figure 3c

and 3d). Hormonal data are not available for the rats that were treated with 300

mg/kg/day because they were euthanized at a different time of the day (early evening)

than were the other groups (early morning) owing to the severity of injury that had

developed at that time.

Repeated caffeine administration also produced substantial alterations in adrenal

and thymus gland masses (see Fig. 4a and 4b), producing significant hypertrophy of the

adrenal glands (F(2,35) = 4.15, p < 0.05) in the 185 mg/kg group and significant atrophy

of the thymus glands (F(2,35) = 41.85, p < 0.01) in both 140 mg/kg and 185 mg/kg

groups, when these glandular weights were adjusted for between-groups differences in

body weights (see description of body weight differences, below). The repeated

administration of pemoline produced adrenal hypertrophy only at the highest (300

mg/kg/day) dose (F(3, 44) = 11.66, p < 0.01), and did not significantly alter thymus

weights in any of the groups of rats (F(3,44) = 1.74, p > 0.05) (see Figure 4c and 4d).









In the experiment with repeated caffeine administration, the caffeine-treated rats

did not gain weight as rapidly as did the rats that were treated with vehicle (interaction

effect: F(28,35) = 11.44, p < 0.01), and there were differences between weight gain of the

rats in the groups that were treated with the 140 and 185 mg/kg/day doses (see Fig. 4c).

In the pemoline experiment, there were also significant between group differences in the

weight gains in the four treatment groups (see Fig. 4f). In fact, the rats that were treated

with the highest dose (300 mg/kg/day, F(9,47) = 27.26, p<.01) exhibited weight loss, and

the rats in the 200 mg/kg/day group (F(10,35)= 14.34, p<.01) exhibited suppressed

weight gain, but the rats that were treated with 100 mg/kg/day only differed in their body

weights from the vehicle group during the early days of the experiment (F(14,23) = 2.86,

p<.05)












Caffeine


Pemoline


-o- vehicle
- 140 mg/kg
-- 185 mg/kg


((




0).
0. 0)


A
100-

g 75-

S50-

25-
0. o)
0-


1 2 3 4 5 6 7 8 9 101112131415
day


Figure 1. Incidence of SIB: Pemoline, but not caffeine, administration
produced self-injury in a dose-orderly manner. a) Some, but not all, of the
caffeine-treated rats self-injured. b) In pemoline-treated rats, the onset of SIB
occurred earlier and the total number of rats that self-injured was greater in
rats treated with the higher doses of pemoline. None of the rats treated with
saline or peanut oil exhibited any signs of tissue damage.













Table 2. Topography of SIB: Rats that self-injured in the caffeine-treated group
predominantly exhibited raw skin on their tails, with little damage on the paws
and no damage on the ventrum. The rats that self-injured in the pemoline-
treated group, exhibited tissue damage on the tails, paws or ventrum,
depending upon the dose administered. The number of rats exhibiting tissue
damage on the tails, paws or ventrum are listed for each of the
pharmacologically-treated groups. All the groups have 12 rats, except for the
140 mg/kg caffeine group, which has 6 rats.


GROUP forepaws hindpaws ventrum tail
vehicle 0 0 0 0
185 caffeine 0 2 0 3
140 caffeine 1 0 0 2
300 pemoline 9 1 5 2
200 pemoline 4 2 7 1
100 pemoline 1 0 0 4


1 2 3 4 5 6 7 8 9101112131415
day








16





A Caffeine D Pemoline
4- 4-
vehicle ** -o-vehicle
o 140 mg/kg 3 --- 100 mg/kg
---185 mg/kg + ----200 mg/kg
*t* ** --300 mg/kg
S2- 2- **



1 2 3 4 5 6 7 8 9101112131415 1 2 3 4 5 6 7 8 9 101112131415
day day
B E
4- 4-
C F
15- ** 4, **


E E tt
5 2 o 2- 1 **
B i


1 2 3 4 5 6 7 8 9 101112131415 1 2 3 4 5 6 7 8 9 101112131415
day day
C F *


Fig. 2. Caffeine- and Pemoline-Induced Self-Injury: The rats that were treated wi 20th
*E ._ *
nu1 5- 15 *da ** T
1 tt tissue trauma tt e) overall measures of tissue trauma size, and
0 5 ^ m & ^ 0 5 -

1 2 3 4 5 6 7 8 9101112131415 1 2 3 4 5 6 7 8 9 101112131415
day day



Fig. 2. Caffeine- and Pemoline-Induced Self-Injury: The rats that were treated with
caffeine exhibited significant self-inflicted tissue trauma, as indicated by a)
tissue trauma scores, b) overall measures of tissue trauma size, and c) total
number of tissue damages across 15 days of treatment. The rats that were
treated with pemoline also exhibited self-inflicted tissue trauma, as indicated
by d) tissue trauma scores, e) overall measures of tissue trauma size, and f)
total number of tissue damages across 4, 6, or 15 days of treatment. Values
expressed are group means + the standard error of the mean (SEM).
Significant between-groups differences are depicted as follows: p < 0.01
comparing 185 mg/kg caffeine with vehicle; tt p < 0.01 comparing 140
mg/kg caffeine with vehicle; p < 0.01 comparing 300 mg/kg pemoline
with vehicle; tt p < 0.01 comparing 300 mg/kg pemoline with 100 mg/kg
pemoline; p < 0.01 comparing 300 mg/kg pemoline with 200 mg/kg
pemoline; ** p < 0.01 comparing 200 mg/kg pemoline with vehicle; 4 p <
0.01 comparing 200 mg/kg pemoline with 100 mg/kg pemoline; p < 0.05,
*-- p < 0.01 comparing 100 mg/kg pemoline with vehicle.







17



A Caffeine C Pemoline
75 75-
E tt
i. ++C '.
50- 50-


E 25- 25- data
S= not
C 0- T available
0- ----- 0- -------
vehicle 140 185 vehicle 100 200 300
dose (mg/kg) dose (mg/kg)


B D
200- ** 200-



0 100- 0 100-
o/0

E E data
50- 50- ** n
_M not
S__ available

vehicle 140 185 vehicle 100 200 300
dose (mg/kg) dose (mg/kg)



Figure 3. Basal Levels of Stress Hormones: The rats that were treated with caffeine
exhibited significant increases in a) basal ACTH levels, however, only the
higher dose of caffeine significantly altered b) basal CORT levels. The rats
that were treated with 200 mg/kg/day pemoline also exhibited significant
alterations in basal stress hormones, as indicated by c) ACTH levels and d)
CORT levels. Values expressed are group means + SEM. Significant
between-groups differences are depicted as follows: ** p < 0.01 comparing
185 mg/kg caffeine with vehicle; tt p < 0.01 comparing 140 mg/kg caffeine
with vehicle; ** p < 0.01 comparing 200 mg/kg pemoline with vehicle; 99
p < 0.01 comparing 200 mg/kg pemoline with 100 mg/kg pemoline.








18



A Caffeine D Pemoline


g 20- E g 20-



0 .0-i
vehicle 140 185 vehicle 100 200 300
dose (mg/kg) dose (mg/kg)

B E


E 2.200- E 200-

-- v oo. 100-

ve 140 185 vehicle 100 200 300
dose (mg/kg) dose (mg/kg)

C F
300- -0-vehicle tt 300
--140 mg/kg tt tttt*
125085 mg/kg t tt 2 5 0-
250 ttt *2 ***5
tt 200 vehicle
S200- 200- -- 100 m g/kg
-200 mg/kg
S150 ---300 mg/kg
150 1' -. --- i- -- -i -- -i --- i 150 -!.- -i --- i- ----- -i --- i
1 2 3 4 5 6 7 8 9 101112131415 1 2 3 4 5 6 7 8 9 101112131415
day day




Figure 4. Alterations in Glandular and Body Weight: The rats that were treated with
caffeine exhibited alterations in glandular weight, as indicated by a) adrenal hypertrophy
in the 185 mg/kg group and b) thymus atrophy in both groups. c) The caffeine-treated
rats did not gain weight as rapidly as did the rats that were treated with vehicle. The rats
that were treated with the highest dose of pemoline showed d) adrenal hypertrophy, but
repeated pemoline administration did not alter e) thymus weights. Administration of
pemoline affected f) body weight, in that the 300 mg/kg group lost weight, while the 200
mg/kg group did not gain weight; however, the 100 mg/kg group did not significantly
differ from the vehicle towards the end of the experiment. Values expressed are group
means + SEM. Significant between-groups differences are depicted as follows: ** p <
0.01 comparing 185 mg/kg caffeine with vehicle; tt p < 0.01 comparing 140 mg/kg
caffeine with vehicle;* p < 0.01 comparing 300 mg/kg pemoline with vehicle; tt p <
0.01 comparing 300 mg/kg pemoline with 100 mg/kg pemoline; p < 0.01 comparing
300 mg/kg pemoline with 200 mg/kg pemoline; ** p < 0.01 comparing 200 mg/kg
pemoline with vehicle, *-* p < 0.01 comparing 100 mg/kg pemoline vehicle. For body
weight, (graphs c and f), significance symbols are shown for treatment groups vs. vehicle
group only.















DISCUSSION

Previous reports of caffeine- and pemoline-induced self-injury have described the

effects of caffeine or pemoline treatments either by reporting the numbers of rats that

exhibited tissue damage (Kasim & Jinnah, 2002; Minana et al., 1984; Minana & Grisolia,

1986; Mueller & Nyhan, 1982; Mueller et al., 1982; Mueller & Nyhan, 1983; Mueller et

al., 1986; Peters, 1967), or by reporting the severity of the tissue damage using a rating

scale (King et al., 1993; King et al., 1995; Mueller & Hsiao, 1980; Turner et al., 1999).

In the present experiments, we directly compared the effectiveness of caffeine and

pemoline treatments. We measured the numbers of rats that exhibited tissue trauma daily

during treatment with each dose of each drug, and we measured the severity of tissue

trauma with a 5-point scale of tissue damage (King et al., 1993; King et al., 1995; Turner

et al., 1999). In addition, we assessed the number of trauma sites, and the size of tissue

trauma each day during treatment with each of the pharmacological manipulations.

This phenomenological evaluation of these two pharmacological models across

days of treatment revealed important differences between caffeine- and pemoline-induced

SIB. In fact, the caffeine-induced SIB was mild (never exceeding "2" on the rating

scale), and this mild self-injury occurred only in a small number of the rats, even though

the doses that were required to produce these self-injurious outcomes were in the range

that produced extreme toxicity. The caffeine-treated rats exhibited severe signs of

malaise at all doses tested, including behavioral lethargy, reduced weight gain,

porphyrhin secretions around the eyes and snout, alterations in HPA axis function, and









even death. One of the twelve rats died during treatment with 185 mg/kg of caffeine, a

finding that is consistent with a previous report (Peters, 1967) that this dose administered

intragastrically produced approximately 10% mortality. In our preliminary studies, a

higher dose produced immediate mortality in more than 50% of the rats so that

experiment was immediately discontinued, and we were unable to examine whether doses

higher than 185 mg/kg would induce greater self-injury due to these toxic actions.

Repeated administration of pemoline produced substantially greater incidence and

severity of self-induced tissue trauma than did caffeine, and in contrast to the effects of

caffeine, these effects of pemoline were dose-orderly. Furthermore, the pemoline-treated

rats exhibited significantly fewer and less severe signs of drug-induced toxicity,

especially at the 100 and 200 mg/kg doses. Porphyrhin secretions were not observed in

the rats that were treated with the 100 and 200 mg doses of pemoline, all the pemoline-

treated rats exhibited hyperactivity rather than lethargy or malaise, and the pemoline was

never lethal even at a dose (300 mg/kg/day) that produced very rapid onset of severe self-

injury in more than 90% of the rats. Accordingly, the 100-300 mg/kg dose range

effectively produced self-injury that was accompanied by minimal impact upon the health

status of the rats. However, it should be noted that higher doses of pemoline do appear to

produce substantial toxicity, and 500 mg/kg/day has been shown to produce

approximately 50% mortality (Genovese et al., 1969).

The caffeine and pemoline models also differed in terms of the topographical

expression of SIB. In the caffeine-treated rats, tissue damage was generally restricted to

the tail; there was very little tissue trauma on the forepaws and no tissue damage on the

ventrum (Table 2). The mildness of the caffeine-induced self-injury, coupled with the









fact that it was focused on the tail, contrasts with previous reports that described severe

self-inflicted injuries on the paws (Mueller et al., 1982; Mueller & Nyhan, 1983) or on

the paws and tails (Peters, 1967) of caffeine-treated rats. The reason for this apparent

contradiction is unclear. However, we did observe that the caffeine-treated rats had

extensive amounts of dark red porphyrhin secretions on their forepaws, where they

spread these secretions from their snouts onto their paws during grooming. These

secretions closely resembled blood, and the encrusted secretions on the forepaws of our

rats looked like severe injury, until we washed the paws, and found no injury underneath.

Furthermore, self-biting behavior was never observed in casual observations in the

caffeine-treated rats. In the pemoline-treated rats, the extent of tissue damage was much

greater, and was more commonly exhibited on the forepaws and ventrum (thorax and

abdomen). In contrast to injuries in the caffeine-treated rats, the tail was the least

common area of injury. This is consistent with previous reports of pemoline-induced SIB

(Mueller & Hsiao, 1980; Mueller et al., 1986). In the pemoline-treated rats, the self-

biting behavior was highly stereotyped, with rats often showing biting that started at the

forepaws, and moved on to the ventral thorax and abdomen, and this self-biting behavior

was consistently observed in casual observations.

Evaluation of pemoline doses that were effective in approximately 50-75% of the

rats (100-200 mg/kg) revealed that there are individual differences in vulnerability to

self-injure in this pharmacological model. In all three doses of chronic pemoline

administration, some of the rats self-injured, whereas some of the rats did not. This is

reminiscent of the fact that individuals within clinical populations (e.g. autistic

individuals) appear to differ in their vulnerability or predisposition to exhibit self-injury









so that only a subset of afflicted individuals demonstrate self-injurious behaviors.

Accordingly, we believe that the pemoline model of self-injury may provide a useful tool

to examine the neurobiological basis of individual differences in vulnerability to self-

injure, in that this biological vulnerability may have a significant impact upon our

understanding of the etiology of clinical SIB in human populations. Individual

differences in vulnerability to self-injure also occurred in the caffeine model, but the

toxicity of this treatment is problematic, and therefore, the pemoline model appears to be

a better model for the study of factors that determine individual differences in this

vulnerability. In fact, the 200 mg/kg dose of pemoline appears to be close to the ED50

for induction of SIB and seems to be a reasonable dose to use when investigating

individual differences in brain functioning, drug sensitivity, and hormonal responses that

may shed light on individual differences in the clinical populations. These investigations

could be coupled with studies in genetic models of SIB (Kasim & Jinnah, 2002), in

effects of cortical (Cromwell et al., 1999) and other brain lesions, and in pharmacological

manipulations (e.g. antagonist challenges) that could alter vulnerability (King et al.,

1993; King et al., 1995) to exhibit SIB. In addition, the impact of environmental factors

(e.g. stress exposure, environmental enrichment, operant conditioning) that could alter the

innate predisposition to self-injure could be studied in the pemoline model of SIB.

Ultimately, these studies may help increase our understanding of pathologies that are

associated with self-injury, and lead towards improved prevention or treatment of self-

injurious behavior.















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BIOGRAPHICAL SKETCH

Staci Kies was born on February 5th, 1978, in Joliet, IL. In 1986, she moved to

Melbourne, FL. After graduating high school in 1996, she attended Brevard Community

College, where she received her Associate of Arts in May 1997. Staci then went on to

attend the University of Central Florida, where she obtained her Bachelor of Science in

psychology in May 1999. From August 1999 to the present, she has been pursuing a

graduate degree in behavioral neuroscience in the Psychology Department of the

University of Florida.