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A COMPARISON OF CAFFEINE AND PEMOLINE MODELS OF SELF-INJURY IN
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
Staci D. Kies
This document is dedicated to my parents, Ken and Debbie Kies, for their emotional
support during the writing of this thesis.
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
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
LIST OF TABLES
1. Q ualitative SIB scale............. .... .............................................................. ......... .... .9
2. Topography of SIB ................................... .. .......... .......... .... 15
LIST OF FIGURES
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
Staci D. Kies
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.
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
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
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.
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
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.
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.
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
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 =
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.
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
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
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,
- 140 mg/kg
-- 185 mg/kg
1 2 3 4 5 6 7 8 9 101112131415
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
A Caffeine D Pemoline
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
15- ** 4, **
E E tt
5 2 o 2- 1 **
1 2 3 4 5 6 7 8 9 101112131415 1 2 3 4 5 6 7 8 9 101112131415
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
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.
A Caffeine C Pemoline
i. ++C '.
E 25- 25- data
C 0- T available
0- ----- 0- -------
vehicle 140 185 vehicle 100 200 300
dose (mg/kg) dose (mg/kg)
200- ** 200-
0 100- 0 100-
E E data
50- 50- ** n
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.
A Caffeine D Pemoline
g 20- E g 20-
vehicle 140 185 vehicle 100 200 300
dose (mg/kg) dose (mg/kg)
E 2.200- E 200-
-- v oo. 100-
ve 140 185 vehicle 100 200 300
dose (mg/kg) dose (mg/kg)
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
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
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
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-
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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.