35
Developmental Brain Research, 68 (1992) 35-40 Elsevier Science Publishers B.V.
BRESD 51474
Behavioral effects of continuous hippocampal stimulation in the developing rat S a m u e l T h u r b e r , A n t o m a C h r o n o p o u l o s , Carl E. S t a f s t r o m a n d G r e g o r y L. H o l m e s Department of Neurology, Harvard Medical School, Children's Hospital, Boston, MA 02115 (USA) (Accepted 10 March 1992)
Key words: Continuous hippocampal stimulation; Epilepsy; Seizure
There is controversy as to whether prolonged seizures are more detrimental to the immature than the mature brain. To evaluate this question continuous hippocampal stimulation was used to induce prolonged limbic seizures in 20-, 30- and 60-day-old rats. The long-term effects on learning and activity level were then studied at age 80 days using the Morris water maze, a test of spatial learning and memory, and the open field test, a test of an animal's reaction to a novel environment. Limbic status epilepticus in 60-day-old but not 20- and 30-day-old rats caused long-term impairment of learning in the Morris water maze. No differences were noted between the control and the experimental animals in the open field test. These results suggest that the age of seizure onset is an important determinant of long-term cognitive sequelae.
INTRODUCFION
A frequent and important question asked by parents of children with epilepsy is whether seizures can lead to brain damage. It is clear that children with epilepsy are at significant risk for cognitive impairment ~'j°'~t'3~. While in some children this cognitive impairment may be explained by the etiological factors associated with the seizures, there is now evidence that some children with poorly controlled epilepsy have progressive declines of IQ on serial intelligence tests ~°'3~. One of the reported risk factors for cognitive impairment in epilepsy is an early age of seizure onset ~'~3. Several authors have observed that seizures beginning in early childhood are associated with a higher risk of intellectual impairment than when seizures begin in late childhood or the teenage years 4-6'38. However, it is not clear whether these adverse effects are secondary to damage caused by the seizures or a reflection of the insult that produced the seizures. While studies in animals t6'2~-26.4°m have paralleled clinical studies 9'2° demonstrating that prolonged seizures can cause long-term neurological sequelae, there is controversy as to whether prolonged seizures
in the immature brain are more detrimental than in the mature brain. Previous work in our laboratory, using the kainic acid (KA) model of epilepsy, has suggested that the immature animal has fewer long-term sequelae to prolonged seizures than the mature animal !~'34-3~' However, one of the inberent challenges in studying the long-term effects of seizures is differentiating the effects of seizures from the effects of the agent causing the seizure. Since there are changes in KA receptors with age, it is difficult to compare the effects of administration of this agent to animals of various ages. To circumvent this problem we compared the effects of prolonged seizures on learning, memory and activity level in immature and mature animals using continuous hippocampal stimulation (CHS), a model in which low amperage electrical stimulations are administered through a hippocampal electrode using a 10 s on, 1 s off paradigm over 90 min ~'t9'27. This pattern of stimulation results in nearly continuous 'limbic' seizures characterized by immobility, repetitive chewing, head nodding and vibrissae twitching, with intermittent intense seizure consisting of forelimb clonus and rearing. The CHS model has several attractive features: seizures are induced by the same intensity of current in each age
Correspondence: G.L. Holmes, Clinical Neurophysiology Laboratory, Hunneweil 2, Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.
36 group, the seizures are relatively uniform from rat to rat, are not associated with cyanosis, and result in a very low mortality rate. Sequelae resulting from these CHS-induced seizures were investigated using behavioral testing when the rats were fully mature.
Histology All animals were sacrificed with a lethal dose of sodium pentobarbital and then perfused transcardially with phosphate buffered saline followed by 10% buffered formalin phosphate. Brains from four animals randomly selected from each CHS-treated group were cut in 25-/zm sections and stained with Cresyl violet. These brains were examined for histological lesions. All animals had electrode placement verified by gross examination.
MATERIALS AND METHODS
Statistics Male Sprague-Dawley rats of ages 15-58 days were used in the study. All animals were housed in plastic cages with free access to food and water with 12 h light-dark cycles until they were 80 days old. Bipolar electrodes, used for both stimulating and recording, were placed in animals in three age groups: 18 (n ffi 22), 28 (n = 24) and 58 (n = 28)days old using techniques previously described ~4'~'~s. Animals were anesthetized with 50 mg/kg of pentobarbital sodium intraperitoneally, and electrodes were stereotaxically implanted in the right ventral hippocampus using the following coordinates; 18day-old (intra-aurai line, tooth bar = 0): AP, 0.16: ML, 0.45; DV, 0.'~8 cm; 28-day-old: AP, 0.18, ML, 0.50; DV, 0.41 cm, 58-day-old (bregma, tooth bar = + 0.5): AP, 0.32; ML, 0.56; DV, 0.65 cm 3~. The electrode was then secured to the skull with aluminum screws (one screw in 18- and 28-day-old and three screws in 58-day-old rats) and dental acrylic. Rats were then allowed a 2-day recovery period. A minimum of 2 rain of baseline electrocorticographic (ECG) activity was recorded immediately prior to stimulation in each rat. The animals were then stimulated with a Grass stimulator connected serially to two constant current stimulus isolation units. The stimulus consisted of 10-s trains of 400 /zA, 1 ms, 50 Hz, biphasic current pulses followed by I s with the stimulus off. This stimulus paradigm was employed for 30 rain followed by a 3-rain rest period during which the EEO activity was recorded for the first 2 rain. This cycle was repeated three times for a total of 90 min of actual stimulation. Each stimulation session was videotaped for future analysis. Behavioral studies began at age 80 days and continued for 2 weeks. Water maze testing was performed during the first week and the open field test was performed the following week. The behavioral studies employed in this study have been previously used in our laboratory ''t~. All animals were tested in a blinded fi~shion.
Water maze testing A circular swimming pool made from a galvanized stock water tank (ll7 cm diameterxS0 em high) was filled to a depth of 25 cm with water at a temperature of 26:1: l°C, The pool was illuminated by overhead fluorescent lights and kept in a permanent location throughout the study, Milk was added to make the water opaque and prevent visualization of the platform, Four points on the rim of the pool were designated as north, south, east and west. On day i the rats were placed in the pool for 60 s of habituation to the apparatus with no platform present ('free swim'), On days 2-5, rats were trained for 24 trials (6 trials per day) to locate and escape onto a wooden platform (8 crux8 cm) placed 1.5 cm under the water. At the start of each trial, the rat was held facing the perimeter and dropped into the pool to ensure immersion. Entry from the N, S, E or W points was varied in a quasi-random order. Latencies to escape onto the platform were recorded 's'~''~.
Time to reach the water maze platform was compared in the controls and CHS-treated animals using the ANOVA with repeated measures. Time to platform was compared both over 24 trials and by the 4 test days (6 trials per day). When the ANOVA demonstrated significance ( P < 0.05) the t-test was then used to compare time to platform for each test day. In the open field test central blocks, peripheral blocks, total blocks, ~ears and time investigating objects were compared in the controls and CHS-treated animals |n each of the three groups using the ANOVA with repeated measures 42.
RESULTS
Behavioral changes with CHS All rats receiving hippocampal stimulations displayed behavioral manifestations of limbic seizures. Behavior during the stimulation varied as a function of age. All age groups initially manifested running, hyperactivity, or both. Isolated myoclonic jerks and scratching were noted throughout the stimulation period. In the 20-day-old group, the first 60 rain of CHS typically produced a period of wet dog shakes that progressively intensified and became more frequent but then gradually decreased in duration and intensity. The final 30 rain were characterized by the appearance of facial and unilateral forelimb clonus, contralateral to the stimulating electrode with occasional full body tonus or wild running. In the 30-day-old group the period of wet dog shakes typically lasted between 45 and 50 rain. During the final 30-45 rain of stimulation of bilateral forelimb elonus occurred and rearing was noted. However, forelimb clonus and rearing rarely occurred simultane-
Baeeilne
30 Minutes
Immoblle
Open field test A square piece of plywood (61 cmx61 cm) with wooden side boards (30 cm high) was divided into 64 squares (7,6 cm-7,6 cm). For testing purposes, the open field was divided into a central area of 16 squares and 48 peripheral squares. Several objects (battery, bottle cap, felt marker) were placed in the middle of the field, The rats were placed in the open field for trials of 60 s each, separated by a rest period of 15 s during which the animal was removed from the open field. The number of central and peripheral squares crossed, number of rears, and the time investigating objects were counted and recorded. The animals were tested on 4 consecutive days.
60 Minutes
Quiet
|,
90 Minutes 20 day-old
,
,
i
i
300 m v L ~ . , . 2 Secs
Fig. ]. F~xample of EFGs from 20-day-old rat at four time points during continuous hippocampal stimulation.
37 Time (Secs) to Platform 80
Baseline
60
30 Minutes
Quiet let
•
40
60 Minutes
Controls
Immobile
90 Minutes 30 day-old
300 mv Lm,,m 2 Secs
0
Fig. 2. Example of EEGs from 30-day-old rat at four time points
,
2
,
4
.
1
6
ously. In the 60-day-old group wet dog shakes were not as pronounced as in the younger age groups and lasted for only the first 15-20 min. The remaining stimulation time was characterized by periods of ataxia, immobility or full body tonus. Intermittently, the animals had bilateral.forelimb clonus with rearing, similar to stage 5 seizures seen in kindling. No animals died during the stimulation nor was any cyanosis noted.
Electroencephalographic changes with CHS No epileptiform activity was seen during baseline recordings of the CHS-stimulated or sham-stimulated controls. EEG ictai discharges were reviewed during three sampling periods; 30, 60 and 90 rain after the onset of the stimulation. While all rats receiving CHS had ictal discharges during at least one of the sampling periods, the presence or absence of ictal discharges was variable. Both the ictal duration for the three sampling periods and total ictal duration during the rest period are listed in Table I. No statistical differences were noted in the three groups. Therefore, seizure duration, as judged by ictal electroencephalographic criteria, was similar in the three groups. There were no changes in the morphology or frequency of the
30 Minutes
60 Minutes
"90'MinutesI,
,
8
,
i
i
,
,
,
,
.
,
,
,
,
,
,
,
,
10 12 14 16 18 20 22 24 Trial Number
during continuous hippocampai stimulation.
Baseline
,
Fig. 4. Mean time to platform across trials in water maze of controls and rats treated with continuous hippocampal stimulation at age 60 days.
ictal discharges from the sampling period at 30-min to the 90-min sample. While the 60-day-old rats usually had more rapid spikes than animals that received CHS at 20 or 30 days old, this was not a consistent finding. Figs. 1-3 are examples of EEGs from one animal from each age group.
Behavioral testing Water maze. Figs. 4 and 5 demonstrate mean escape latencies in the water maze as a function of trial number in the three age groups. There were no differences in the animals receiving CHS and controls in the, 20- and 30-day-old animals (20 d.o.: F - 0.507, P-0.484; df-1,20; 30 d.o.: F=0.968, P--0.336; d f = 1,22). In the 60 d.o. animals the differences between the CHS rats and controls were close to significance Time (89ce) to Platform 300 I
~,.' I
Immobile
.
Chewing
J~' Wet Dog Shakes 3 = ' m v L m 60 day.old 2 Sacs
Fig. 3. Example of EEGs from 60-day-old rat at four time points during continuous hippocampai stimulation.
1
2
3
4
Day Number I I C o n t r o l . I~ICHS I Fig. 5. Mean time to platform across days of testing in water maze of controls and rats treated with continuous hippocampal stimulation at age 60 days ( * P < 0.05).
38 TABLE i
lctal duration (s) during rest periods in animals receh'ing CHS Age group
30 min
60 m&
90 rain
Total
20-day-old 30-day-old 60-day-old P
80 ± 20 5 0 ± 17 52 ± 23 0.580
77 ± 22 104± 16 80 :i: 15 0.455
51 + 24 96+24 74 ± 22 0.455
2 !0 ± 55 256+55 183 + 43 0.498
( F - 3.943; P = 0.0577; df--1,26) (Fig. 4). The CHS rats and controls in the 60 d.o. group were then compared for total time to platform for each of the test days (Fig. 1). With this analysis there was a significant difference between the two groups (F -- 6.56, P - 0.017; df = 1,26). The CHS-treated and sham-treated groups were then compared on each day using the t-test. As can be seen in Fig. 5, significant differences between the two groups were seen on day 2 and day 4. Open field test. No significant differences were noted in the control and CHS animals with any of the measures in the open field test (20 d.o.: peripheral blocks crossed, F = 0.081, P = 0.779; central blocks crossed, F = 0.879, P=0.361; rearing, F = 3.801, P = 0.067; time investigating objects, F = 0.011, P = 0.917; 30 d.o.: peripheral blocks crossed, F = 0.011, P = 0.919; central blocks crossed, F---0.022, P = 0.885; rearing, F - 0.008, P 0.931; time investigating objects, F - 0.664, P = 0.429; 60 d.o.: peripheral blocks crossed, F = 0.058, P = 0.814; central blocks crossed, F = 4.427, P = 0.062; rearing, F = 0.335, P ffi 0.576; time investigating objects, F 1.796, P ffi 0.210). No significant differences were noted between the controls and CHS-stimulated animals in any of the three age groups.
Histology No gross lesions were detected in the. 20- or 30-dayold animals receiving CHS. In one of the four 60-dayold rats examined there was partial loss of cells in the CA3 region of the hippocampus. DISCUSSION We found that CHS produced status epilepticus in both immature and mature animals. In all three age groups the electrical stimulations produced limbic activity with periods of immobility, scratching, hyperactivity, wet dog shakes and facial and forelimb clonus. Ictal electroencephalographic activities were seen in all three age groups. However, frequency, amplitude or duration of ictai discharges did not correlate with behavioral changes. While the seizure severity and EEG changes were similar in the three age groups, there were differences
in the neurological sequelae following the status epilepticus. Limbic status epilepticus in 60-day-old but not 20- and 30-day-old rats caused long-term impairment of learning in the Morris water maze, a test of spatial learning and memory 2.28.3°. The design of the study tests the ability of an animal to learn and remember the spatial location of the escape platform using only visual cues and measures hippocampal integrity 3°'37. The better performance of the younger rats suggests two possibilities; one, that the mature brain may be more susceptible to the long-term cognitive effects of prolonged seizures than the immature brain, or two, that the plastic properties of the immature brain have allowed functional recovery by the day of testing. We did not find any differences in the performance of the three age groups in the open field test. The open field test measures the animal's reaction to a novel environment a'39. We did not observe any apparent difference in behavior between the CHS-treated and sham-treated controls during routine handling. The effects of CHS therefore do not appear to alter emotionality, exploratory behavior, or fear. Although we did not detect any differences in behavior or duration of ictai discharges between the three study groups, it remains possible that the electrical stimulations affected the brains of the three age groups differently. For example, spread of the electrical discharge, through volume conduction and impulse propagation, is dependent on tissue characteristics such as neuronal density and myelination. While the same electrical stimulus was applied to all age groups, the local and diffuse effects of such stimulations may have varied as a function of age. There have been few studies examining the effects of prolonged seizures on the developing brain. For example, while Meldrum et al. 22 demonstrated neuronal cell loss in the neocortex, hippocampus and cerebellum in baboons undergoing 1.5-5 h of status epilepticus induced by bicucuUine, only adolescent baboons were used. In a similar study using neonatal marmoset monkeys, S6derfeldt et al. 33 found that these results could not be extrapolated to younger animals. The authors administered bicuculline to neonatal marmoset monkeys (14-17 days of age) and produced generalized seizures lasting 1.5-4.3 h. Only minimal neuropatho. logical changes were detected using light and electron microscopy. In a study examining the effects of serial seizures on brain growth, Wasterlain and Plum 41 demonstrated reductions in cell number in brains of newborn rats subjected to ten daily electroconvulsive seizures induced by a l-s, 150-V electrical stimulus between the
39 ages of 2 and 11 days. In rats receiving electroconvulsive shocks between days 9 and 18 a reduction of cell weight was reported. Daily seizures in older rats (age 19-28 days) did not affect the brain in a measurable way. However, some of the adverse effects of seizures in the developing brain may be transient. This was demonstrated by Wasterlain et al.4° who examined the brains of rat pups exposed to flurothyl or bicuculline and found that profound adverse effects noted in brain growth at age 7 days had almost dissipated when the brains were reexamined at age 30 days. Using another animal model, Cavalheiro et al. 3 reported resistance to pilocarpine-induced seizures in rats under the age of 12 days. Previous work in our laboratory has demonstrated that the long-term effects of KA-induced status epilepticus are highly dependent on the age at which the animals receive the drug. When administered intraperitoneally, KA results in status epilepticus in animals as young as one day of age m6.We have found that KA administered to 5-, 10- and 20-day-old rats cau,';es no long-term alteration in learning, memory, activiq, level, behavior or fiurothyl seizure susceptibility 35. However, KA administered to rats 30 days and older resulted in significant impairment in learning, memory, behavior and activity level when tested as adults ~6'35. Adult rats receiving KA had a higher rate of spontaneous seizures, more rapid kindling, and a greater susceptibility to flurothyl seizures than young rats 14'35. We concluded from these studies that prepubescent and mature rats had significant long-term behavioral deficits and alterations in brain excitability after KA. induced status epilepticus. However, younger rats, despite having a higher mortality rate with KA, had no detectable behavioral abnormalities and minimal alterations in seizure susceptibility. The results of this study should be interpreted with caution. Only one behavioral test showed a difference between groups. Further studies, using additional measures of learning, memory and behavior will be necessary to confirm these findings. An important variable that must be addressed is the time interval between CHS and testing. Since all of our animals were not tested at a uniform period after the CHS, the 60-day-old rats were tested 20 days after the stimulation while the group receiving CHS at age 20 days had a 60-day period before testing was performed. In this regard, however, in unpublished observations we have found age at time of KA administration to be a more important variable than interval between drug administration and testing. We chose to study animals of 20, 30 and 60 days because our work with KA demonstrated that 30-day-old rats, but not 20-day-old rats, had neu-
rological deficits after KA-induced status epilepticus. While the results of this study cannot be directly extrapolated to the human situation, *.he accumulated evidence suggests that the immature brain may be less vulnerable to the prolonged effects of seizures than the mature brain. Acknowledgements. Supported by grants to G.L.H. from the NINDS (RO1 NS27984) and Steven Linn Research Fund.
REFERENCES 1 Bertram, E.H., Lothman, E.W. and Lenn, N.J., The hippocampus in experimental chronic epilepsy: a morphometric analysis, Ann. Neurol., 27 (1990) 43-48. 2 Bures, J. and Buresova, O., Spatial memory in animals. In R.J. John (Ed.), Machinery of the Mind, Birk~iuser, Boston, 1990, pp. 291-310. 3 Cavalheiro, E.A., Silva, D.F., Turski, W.A., Calderazzo-Filhi, L.S., Bortolotto, Z.A. and Turski, L., The susc¢.~tibility of rats to pilocarpine-induced seizures is age-dependent, Dev. Brain Res., 37 (1987) 43-58. 4 Chevrie, J.J. and Aicardi, J., Childhood epileptic encephalopathy with slow spike-wave. A statistical study of 80 cases, Epilepsia, 13 (1972) 259-271. 5 Dikmen, S., Matthews, C.G. and Harley, J.P., Effect of early versus late onset of major motor epilepsy upon cognitive-intellectual performance, Epilepsia, 16 (1975) 73-81. 6 Dikmen, S., Matthews, C.G. and Harley, J.P., Effect of early versus late onset of major motor epilepsy upon cognitive-intellectual performance: further considerations, Epilepsia, 18 (1977) 31-36. 7 Farwell, J.R., Dodrill, C.B. and Batzel, L.W., Neuropsychological abilities of children with epilepsy, Epilepsia, 26(5) (1985) 395-400. 8 Foshee, D.P., Vierck, C.J., Meier, G.W. and Federspiel, C., Simultaneous measure of general activity and exploratory behavior, Percept. Mot. Skills, 2083 (1965)445-451. 9 Fowler, M., Brain damage after febrile convulsions, Arch. Dis. Child., 32 (1957) 67-76. 10 Funakoshi, A., Morikawa, T., Muramatsu, R., Yagi, K. and Seino, M., A prospective WISC-R study in children with epilepsy, Jpn. J. Psychiatry Neurol., 42 (1988) 562-564. 11 Holmes, G.L., Diagnosis and Management of Seizures in Children, W.B. Saunders, Philadelphia, 1987, pp. 1-293. 12 Holmes, G.L., Do seizures cause brain damage?, Epilepsia, 32 (Suppl. 5) (1991) S14-$28. 13 Holmes, G.L., The long-term effects of seizures on the developing brain: clinical and laboratory issues, Brain Det,., 13 (1991) 393-409. 14 Holmes, G.L. and Thompson, J.L., Effect of kainic acid on seizure susceptibility in the developing brain, Brain Res., 467 (1988) 51-59. 15 Holmes, G.L., Thompson, J.L., Carl, G.F., Gailagher, B.S., Hoy, J. and McLaughlin, M., Effect of 2-amino.7-phosphonoheptanoic acid (APH) on seizure susceptibility in the prepubescent and mature rat, Epilepsy Res., 5 (1990) 125-130. 16 Holmes, G.L., Thompson, J.L., Marchi, T. and Feldman, D.S., Behavioral effects of kainic acid administratioi: on the immature brain, Epilepsia, 29 (1988) 721-730. 17 Holmes, G.L., Thompson, J.L., Marchi, T.A., Gabriel, P.S., Hogan, M.A., Carl, F.G. and Feldman, D.S., Effects of seizures on learning, memory, and behavior in the genetically epilepsyprone rat, Ann. Neurol., 27 (1990) 24-32. 18 Holmes, G.L. and Weber, D.A., Increased susceptibility to pentylenetetrazol-induced seizures in adult rats following electrical kindling during brain development, Brain Res., 313 (1983) 312-314.
40 19 Lothman, E.W., Bertram, E.H., Bekenstein, J.W. and Perlin, J.B., Self-sustaining limbic status epilepticus induced by 'continuous' hippocampal stimulation: electrographic and behavioral characteristics, Epilepsy Res., 3 (1989) 107-119. 20 Maytal, J., Shinnar, S., Mosh~, S.L. and Alvarez, L.A., Low morbidity and mortality of status epilepticus in children, Pediatrics, 83(3) (1989) 323-331. 21 Meldrum, B., Physiological changes during prolonged seizures and epileptic brain damage, Neuropaediatrie, 9 (1978) 203-212. 22 Meldrum. B.S., Metabolic factors during prolonged seizures and their relation to nerve cell death. In A.V. Delgado-Escueta, C.G. Wasterlain, P.M. Treiman and R.J. Porter (Eds.), Advances in Neurology. Vol. 34, Status Epilepticus: Mechanisms of Brain Damage and Treatment, Raven, New York, 1983, pp. 261-275. 23 Meldrum, B.S. and Brierley, J.B., Prolonged epileptic seizures in primates: Ischaemic cell change and its relation to ictal physiological events. Arch. NeuroL, 28 (1973) 10-17. 24 Meldrum, B.S., Horton, R.W. and Brierley, J.B., Epileptic brain damage in adolescent baboons following seizures induced by allylglycine, Brain Res., 97 (1974) 417-428. 25 Meldrum, B.S., Vigouroux, R.A. and Brierley, J.B., Systemic factors and epileptic brain damage. Prolonged seizures in paralysed artificially ventilated baboons. Arch. Neurol., 29 (1973) 8287. 26 Menini, C., Meldrum, B.S., Richie, D.S., Silva-Comte, C. and Stutzmann, J.M., Sustained limbic seizures induced by intraamygdaloid kainic acid in the baboon: symptomatology and neuropathological consequences, Ann. NeuroL, 8 (1980) 501-509. 27 Milgram, N.W., Green, !., Liberman, M., Riexinger, K. and Petit, T.L., I-.:stablishment of status epilepticus by limbic system stimulation in previously unstimulated rats, Exp. Neurol., 88 (1985) 253-264. 28 Morris, R., Development of a water maze procedure for studying spatial learning in the rat, J. NeuroscL Methods, 11 (1984)47-60. 29 Morris, R.G.M., Synaptic plasticity and learning: selective impairment of learning in rats and blockade of long-term potentiation in vivo by the N-methyI-D-aspartate receptor antagonist AP5, J. Neurosci., 9 (1989) 3040-3057.
30 Morris, R.G.M., Garrud, P., Rawlins, J.N.P. and O'Keefe, J., Place navigation is impaired in rats with hippocampal lesions, Nature, 297 (1982) 681-683. 31 Rodin, E.A., Schmaltz, S. and Twitty, G. Intellectual functions of patients with childhood-onset epilepsy, Dev. Med. Child Neurol., 28 (1986) 25-33. 32 Sherwood, N.M. and Timiras, P.S., A Stereotaxic Atlas of the Developing Rat Brain, University of California, Berkely, CA, 1970. 33 S6derfeldt, B., Fujikawa, D.G. and Wasterlain, C.G., Neuropathology of status epilepticus in the neonatal marmoset monkey. In C.G. Wasterlain and P. Vert (Eds.), Neonatal Seizures, Raven, New York. 1991, pp. 91-98. 34 Stafstrom, C., Edwards, M. and Holmes, G., Effect of age on spontaneous seizure frequency following systemic kainic acid administration, Epilepsia, 30 (1989) 722. 35 Stafstrom, C., Thompson, .'L., Chronopoulos, A., Thurber, S. and Holmes, G.L., Effect of age on behavioral abnormalities following kainic acid-induced status epilepticus, Ann. Neurol., 28 (1990) 467-468. 36 Stafstrom, C.E., Thompson, J.L. and Holmes, G.L., Kainic acid seizures in the developing brain: status epilepticus and spontaneous recurrent seizures, Dev. Brain Res., 65 (1992) 237-246. 37 Sutherland, R.J., Whishaw, I.Q. and Kolb, B., A behavioural analysis of spatial localization following electrolytic, kainate or colchicine-induced daraage to the hippocampus, Behav. Brain Res., 7 (1983) 133-153. 38 Wada, J.A., Sato, M. and Corcoran, M.E., Persistent seizure susceptibility and recurrent spontaneous seizures in kindled cats, Epilepsia, 15 (1974) 465-478. 39 Walsh, R.N. and Cummins, R.A., The open field test: a critical review, Psychol. Bull., 83 (1976) 482-504. 40 Wasterlain, C.G., Effects of neonatal status epileptieus on rat brain development, Neurology, 26 (1975) 975-986. 41 Wasterlain, C.G. and Plum, F., Vulnerability of developing rat brain to electroconvulsive seizures, Arch. Neurol., 29 (1973) 38-45. 42 Zar, J.H., Biostatistical Analysis, Prentice-Hall, Englewood Cliffs, NJ, 1974, pp. 1-718.
Developmental Brain Research, 68 (1992) 35-40 Elsevier Science Publishers B.V.
BRESD 51474
Behavioral effects of continuous hippocampal stimulation in the developing rat S a m u e l T h u r b e r , A n t o m a C h r o n o p o u l o s , Carl E. S t a f s t r o m a n d G r e g o r y L. H o l m e s Department of Neurology, Harvard Medical School, Children's Hospital, Boston, MA 02115 (USA) (Accepted 10 March 1992)
Key words: Continuous hippocampal stimulation; Epilepsy; Seizure
There is controversy as to whether prolonged seizures are more detrimental to the immature than the mature brain. To evaluate this question continuous hippocampal stimulation was used to induce prolonged limbic seizures in 20-, 30- and 60-day-old rats. The long-term effects on learning and activity level were then studied at age 80 days using the Morris water maze, a test of spatial learning and memory, and the open field test, a test of an animal's reaction to a novel environment. Limbic status epilepticus in 60-day-old but not 20- and 30-day-old rats caused long-term impairment of learning in the Morris water maze. No differences were noted between the control and the experimental animals in the open field test. These results suggest that the age of seizure onset is an important determinant of long-term cognitive sequelae.
INTRODUCFION
A frequent and important question asked by parents of children with epilepsy is whether seizures can lead to brain damage. It is clear that children with epilepsy are at significant risk for cognitive impairment ~'j°'~t'3~. While in some children this cognitive impairment may be explained by the etiological factors associated with the seizures, there is now evidence that some children with poorly controlled epilepsy have progressive declines of IQ on serial intelligence tests ~°'3~. One of the reported risk factors for cognitive impairment in epilepsy is an early age of seizure onset ~'~3. Several authors have observed that seizures beginning in early childhood are associated with a higher risk of intellectual impairment than when seizures begin in late childhood or the teenage years 4-6'38. However, it is not clear whether these adverse effects are secondary to damage caused by the seizures or a reflection of the insult that produced the seizures. While studies in animals t6'2~-26.4°m have paralleled clinical studies 9'2° demonstrating that prolonged seizures can cause long-term neurological sequelae, there is controversy as to whether prolonged seizures
in the immature brain are more detrimental than in the mature brain. Previous work in our laboratory, using the kainic acid (KA) model of epilepsy, has suggested that the immature animal has fewer long-term sequelae to prolonged seizures than the mature animal !~'34-3~' However, one of the inberent challenges in studying the long-term effects of seizures is differentiating the effects of seizures from the effects of the agent causing the seizure. Since there are changes in KA receptors with age, it is difficult to compare the effects of administration of this agent to animals of various ages. To circumvent this problem we compared the effects of prolonged seizures on learning, memory and activity level in immature and mature animals using continuous hippocampal stimulation (CHS), a model in which low amperage electrical stimulations are administered through a hippocampal electrode using a 10 s on, 1 s off paradigm over 90 min ~'t9'27. This pattern of stimulation results in nearly continuous 'limbic' seizures characterized by immobility, repetitive chewing, head nodding and vibrissae twitching, with intermittent intense seizure consisting of forelimb clonus and rearing. The CHS model has several attractive features: seizures are induced by the same intensity of current in each age
Correspondence: G.L. Holmes, Clinical Neurophysiology Laboratory, Hunneweil 2, Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.
36 group, the seizures are relatively uniform from rat to rat, are not associated with cyanosis, and result in a very low mortality rate. Sequelae resulting from these CHS-induced seizures were investigated using behavioral testing when the rats were fully mature.
Histology All animals were sacrificed with a lethal dose of sodium pentobarbital and then perfused transcardially with phosphate buffered saline followed by 10% buffered formalin phosphate. Brains from four animals randomly selected from each CHS-treated group were cut in 25-/zm sections and stained with Cresyl violet. These brains were examined for histological lesions. All animals had electrode placement verified by gross examination.
MATERIALS AND METHODS
Statistics Male Sprague-Dawley rats of ages 15-58 days were used in the study. All animals were housed in plastic cages with free access to food and water with 12 h light-dark cycles until they were 80 days old. Bipolar electrodes, used for both stimulating and recording, were placed in animals in three age groups: 18 (n ffi 22), 28 (n = 24) and 58 (n = 28)days old using techniques previously described ~4'~'~s. Animals were anesthetized with 50 mg/kg of pentobarbital sodium intraperitoneally, and electrodes were stereotaxically implanted in the right ventral hippocampus using the following coordinates; 18day-old (intra-aurai line, tooth bar = 0): AP, 0.16: ML, 0.45; DV, 0.'~8 cm; 28-day-old: AP, 0.18, ML, 0.50; DV, 0.41 cm, 58-day-old (bregma, tooth bar = + 0.5): AP, 0.32; ML, 0.56; DV, 0.65 cm 3~. The electrode was then secured to the skull with aluminum screws (one screw in 18- and 28-day-old and three screws in 58-day-old rats) and dental acrylic. Rats were then allowed a 2-day recovery period. A minimum of 2 rain of baseline electrocorticographic (ECG) activity was recorded immediately prior to stimulation in each rat. The animals were then stimulated with a Grass stimulator connected serially to two constant current stimulus isolation units. The stimulus consisted of 10-s trains of 400 /zA, 1 ms, 50 Hz, biphasic current pulses followed by I s with the stimulus off. This stimulus paradigm was employed for 30 rain followed by a 3-rain rest period during which the EEO activity was recorded for the first 2 rain. This cycle was repeated three times for a total of 90 min of actual stimulation. Each stimulation session was videotaped for future analysis. Behavioral studies began at age 80 days and continued for 2 weeks. Water maze testing was performed during the first week and the open field test was performed the following week. The behavioral studies employed in this study have been previously used in our laboratory ''t~. All animals were tested in a blinded fi~shion.
Water maze testing A circular swimming pool made from a galvanized stock water tank (ll7 cm diameterxS0 em high) was filled to a depth of 25 cm with water at a temperature of 26:1: l°C, The pool was illuminated by overhead fluorescent lights and kept in a permanent location throughout the study, Milk was added to make the water opaque and prevent visualization of the platform, Four points on the rim of the pool were designated as north, south, east and west. On day i the rats were placed in the pool for 60 s of habituation to the apparatus with no platform present ('free swim'), On days 2-5, rats were trained for 24 trials (6 trials per day) to locate and escape onto a wooden platform (8 crux8 cm) placed 1.5 cm under the water. At the start of each trial, the rat was held facing the perimeter and dropped into the pool to ensure immersion. Entry from the N, S, E or W points was varied in a quasi-random order. Latencies to escape onto the platform were recorded 's'~''~.
Time to reach the water maze platform was compared in the controls and CHS-treated animals using the ANOVA with repeated measures. Time to platform was compared both over 24 trials and by the 4 test days (6 trials per day). When the ANOVA demonstrated significance ( P < 0.05) the t-test was then used to compare time to platform for each test day. In the open field test central blocks, peripheral blocks, total blocks, ~ears and time investigating objects were compared in the controls and CHS-treated animals |n each of the three groups using the ANOVA with repeated measures 42.
RESULTS
Behavioral changes with CHS All rats receiving hippocampal stimulations displayed behavioral manifestations of limbic seizures. Behavior during the stimulation varied as a function of age. All age groups initially manifested running, hyperactivity, or both. Isolated myoclonic jerks and scratching were noted throughout the stimulation period. In the 20-day-old group, the first 60 rain of CHS typically produced a period of wet dog shakes that progressively intensified and became more frequent but then gradually decreased in duration and intensity. The final 30 rain were characterized by the appearance of facial and unilateral forelimb clonus, contralateral to the stimulating electrode with occasional full body tonus or wild running. In the 30-day-old group the period of wet dog shakes typically lasted between 45 and 50 rain. During the final 30-45 rain of stimulation of bilateral forelimb elonus occurred and rearing was noted. However, forelimb clonus and rearing rarely occurred simultane-
Baeeilne
30 Minutes
Immoblle
Open field test A square piece of plywood (61 cmx61 cm) with wooden side boards (30 cm high) was divided into 64 squares (7,6 cm-7,6 cm). For testing purposes, the open field was divided into a central area of 16 squares and 48 peripheral squares. Several objects (battery, bottle cap, felt marker) were placed in the middle of the field, The rats were placed in the open field for trials of 60 s each, separated by a rest period of 15 s during which the animal was removed from the open field. The number of central and peripheral squares crossed, number of rears, and the time investigating objects were counted and recorded. The animals were tested on 4 consecutive days.
60 Minutes
Quiet
|,
90 Minutes 20 day-old
,
,
i
i
300 m v L ~ . , . 2 Secs
Fig. ]. F~xample of EFGs from 20-day-old rat at four time points during continuous hippocampal stimulation.
37 Time (Secs) to Platform 80
Baseline
60
30 Minutes
Quiet let
•
40
60 Minutes
Controls
Immobile
90 Minutes 30 day-old
300 mv Lm,,m 2 Secs
0
Fig. 2. Example of EEGs from 30-day-old rat at four time points
,
2
,
4
.
1
6
ously. In the 60-day-old group wet dog shakes were not as pronounced as in the younger age groups and lasted for only the first 15-20 min. The remaining stimulation time was characterized by periods of ataxia, immobility or full body tonus. Intermittently, the animals had bilateral.forelimb clonus with rearing, similar to stage 5 seizures seen in kindling. No animals died during the stimulation nor was any cyanosis noted.
Electroencephalographic changes with CHS No epileptiform activity was seen during baseline recordings of the CHS-stimulated or sham-stimulated controls. EEG ictai discharges were reviewed during three sampling periods; 30, 60 and 90 rain after the onset of the stimulation. While all rats receiving CHS had ictal discharges during at least one of the sampling periods, the presence or absence of ictal discharges was variable. Both the ictal duration for the three sampling periods and total ictal duration during the rest period are listed in Table I. No statistical differences were noted in the three groups. Therefore, seizure duration, as judged by ictal electroencephalographic criteria, was similar in the three groups. There were no changes in the morphology or frequency of the
30 Minutes
60 Minutes
"90'MinutesI,
,
8
,
i
i
,
,
,
,
.
,
,
,
,
,
,
,
,
10 12 14 16 18 20 22 24 Trial Number
during continuous hippocampai stimulation.
Baseline
,
Fig. 4. Mean time to platform across trials in water maze of controls and rats treated with continuous hippocampal stimulation at age 60 days.
ictal discharges from the sampling period at 30-min to the 90-min sample. While the 60-day-old rats usually had more rapid spikes than animals that received CHS at 20 or 30 days old, this was not a consistent finding. Figs. 1-3 are examples of EEGs from one animal from each age group.
Behavioral testing Water maze. Figs. 4 and 5 demonstrate mean escape latencies in the water maze as a function of trial number in the three age groups. There were no differences in the animals receiving CHS and controls in the, 20- and 30-day-old animals (20 d.o.: F - 0.507, P-0.484; df-1,20; 30 d.o.: F=0.968, P--0.336; d f = 1,22). In the 60 d.o. animals the differences between the CHS rats and controls were close to significance Time (89ce) to Platform 300 I
~,.' I
Immobile
.
Chewing
J~' Wet Dog Shakes 3 = ' m v L m 60 day.old 2 Sacs
Fig. 3. Example of EEGs from 60-day-old rat at four time points during continuous hippocampai stimulation.
1
2
3
4
Day Number I I C o n t r o l . I~ICHS I Fig. 5. Mean time to platform across days of testing in water maze of controls and rats treated with continuous hippocampal stimulation at age 60 days ( * P < 0.05).
38 TABLE i
lctal duration (s) during rest periods in animals receh'ing CHS Age group
30 min
60 m&
90 rain
Total
20-day-old 30-day-old 60-day-old P
80 ± 20 5 0 ± 17 52 ± 23 0.580
77 ± 22 104± 16 80 :i: 15 0.455
51 + 24 96+24 74 ± 22 0.455
2 !0 ± 55 256+55 183 + 43 0.498
( F - 3.943; P = 0.0577; df--1,26) (Fig. 4). The CHS rats and controls in the 60 d.o. group were then compared for total time to platform for each of the test days (Fig. 1). With this analysis there was a significant difference between the two groups (F -- 6.56, P - 0.017; df = 1,26). The CHS-treated and sham-treated groups were then compared on each day using the t-test. As can be seen in Fig. 5, significant differences between the two groups were seen on day 2 and day 4. Open field test. No significant differences were noted in the control and CHS animals with any of the measures in the open field test (20 d.o.: peripheral blocks crossed, F = 0.081, P = 0.779; central blocks crossed, F = 0.879, P=0.361; rearing, F = 3.801, P = 0.067; time investigating objects, F = 0.011, P = 0.917; 30 d.o.: peripheral blocks crossed, F = 0.011, P = 0.919; central blocks crossed, F---0.022, P = 0.885; rearing, F - 0.008, P 0.931; time investigating objects, F - 0.664, P = 0.429; 60 d.o.: peripheral blocks crossed, F = 0.058, P = 0.814; central blocks crossed, F = 4.427, P = 0.062; rearing, F = 0.335, P ffi 0.576; time investigating objects, F 1.796, P ffi 0.210). No significant differences were noted between the controls and CHS-stimulated animals in any of the three age groups.
Histology No gross lesions were detected in the. 20- or 30-dayold animals receiving CHS. In one of the four 60-dayold rats examined there was partial loss of cells in the CA3 region of the hippocampus. DISCUSSION We found that CHS produced status epilepticus in both immature and mature animals. In all three age groups the electrical stimulations produced limbic activity with periods of immobility, scratching, hyperactivity, wet dog shakes and facial and forelimb clonus. Ictal electroencephalographic activities were seen in all three age groups. However, frequency, amplitude or duration of ictai discharges did not correlate with behavioral changes. While the seizure severity and EEG changes were similar in the three age groups, there were differences
in the neurological sequelae following the status epilepticus. Limbic status epilepticus in 60-day-old but not 20- and 30-day-old rats caused long-term impairment of learning in the Morris water maze, a test of spatial learning and memory 2.28.3°. The design of the study tests the ability of an animal to learn and remember the spatial location of the escape platform using only visual cues and measures hippocampal integrity 3°'37. The better performance of the younger rats suggests two possibilities; one, that the mature brain may be more susceptible to the long-term cognitive effects of prolonged seizures than the immature brain, or two, that the plastic properties of the immature brain have allowed functional recovery by the day of testing. We did not find any differences in the performance of the three age groups in the open field test. The open field test measures the animal's reaction to a novel environment a'39. We did not observe any apparent difference in behavior between the CHS-treated and sham-treated controls during routine handling. The effects of CHS therefore do not appear to alter emotionality, exploratory behavior, or fear. Although we did not detect any differences in behavior or duration of ictai discharges between the three study groups, it remains possible that the electrical stimulations affected the brains of the three age groups differently. For example, spread of the electrical discharge, through volume conduction and impulse propagation, is dependent on tissue characteristics such as neuronal density and myelination. While the same electrical stimulus was applied to all age groups, the local and diffuse effects of such stimulations may have varied as a function of age. There have been few studies examining the effects of prolonged seizures on the developing brain. For example, while Meldrum et al. 22 demonstrated neuronal cell loss in the neocortex, hippocampus and cerebellum in baboons undergoing 1.5-5 h of status epilepticus induced by bicucuUine, only adolescent baboons were used. In a similar study using neonatal marmoset monkeys, S6derfeldt et al. 33 found that these results could not be extrapolated to younger animals. The authors administered bicuculline to neonatal marmoset monkeys (14-17 days of age) and produced generalized seizures lasting 1.5-4.3 h. Only minimal neuropatho. logical changes were detected using light and electron microscopy. In a study examining the effects of serial seizures on brain growth, Wasterlain and Plum 41 demonstrated reductions in cell number in brains of newborn rats subjected to ten daily electroconvulsive seizures induced by a l-s, 150-V electrical stimulus between the
39 ages of 2 and 11 days. In rats receiving electroconvulsive shocks between days 9 and 18 a reduction of cell weight was reported. Daily seizures in older rats (age 19-28 days) did not affect the brain in a measurable way. However, some of the adverse effects of seizures in the developing brain may be transient. This was demonstrated by Wasterlain et al.4° who examined the brains of rat pups exposed to flurothyl or bicuculline and found that profound adverse effects noted in brain growth at age 7 days had almost dissipated when the brains were reexamined at age 30 days. Using another animal model, Cavalheiro et al. 3 reported resistance to pilocarpine-induced seizures in rats under the age of 12 days. Previous work in our laboratory has demonstrated that the long-term effects of KA-induced status epilepticus are highly dependent on the age at which the animals receive the drug. When administered intraperitoneally, KA results in status epilepticus in animals as young as one day of age m6.We have found that KA administered to 5-, 10- and 20-day-old rats cau,';es no long-term alteration in learning, memory, activiq, level, behavior or fiurothyl seizure susceptibility 35. However, KA administered to rats 30 days and older resulted in significant impairment in learning, memory, behavior and activity level when tested as adults ~6'35. Adult rats receiving KA had a higher rate of spontaneous seizures, more rapid kindling, and a greater susceptibility to flurothyl seizures than young rats 14'35. We concluded from these studies that prepubescent and mature rats had significant long-term behavioral deficits and alterations in brain excitability after KA. induced status epilepticus. However, younger rats, despite having a higher mortality rate with KA, had no detectable behavioral abnormalities and minimal alterations in seizure susceptibility. The results of this study should be interpreted with caution. Only one behavioral test showed a difference between groups. Further studies, using additional measures of learning, memory and behavior will be necessary to confirm these findings. An important variable that must be addressed is the time interval between CHS and testing. Since all of our animals were not tested at a uniform period after the CHS, the 60-day-old rats were tested 20 days after the stimulation while the group receiving CHS at age 20 days had a 60-day period before testing was performed. In this regard, however, in unpublished observations we have found age at time of KA administration to be a more important variable than interval between drug administration and testing. We chose to study animals of 20, 30 and 60 days because our work with KA demonstrated that 30-day-old rats, but not 20-day-old rats, had neu-
rological deficits after KA-induced status epilepticus. While the results of this study cannot be directly extrapolated to the human situation, *.he accumulated evidence suggests that the immature brain may be less vulnerable to the prolonged effects of seizures than the mature brain. Acknowledgements. Supported by grants to G.L.H. from the NINDS (RO1 NS27984) and Steven Linn Research Fund.
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