Cognitive sequelae in children treated for acute lymphoblastic leukemia with dexamethasone or prednisone.

PURPOSE: The cognitive sequelae of treatment for childhood acute lymphoblastic leukemia (ALL) were compared in a group of patients who received dexamethasone during the intensification and maintenance phases of therapy with those in a historical control group for whom antileukemia therapy was similar, except that the corticosteroid component of therapy was prednisone. METHODS: Patients treated for ALL on Dana-Farber Cancer Institute protocols 87-01 (n = 44) and 91-01 (n = 23) were evaluated by standard cognitive and achievement tests. Corticosteroid therapy was delivered in 5-day pulses given every 3 weeks during intensification and continuation phases of therapy for a total of 2 years. RESULTS: Children treated on protocol 87-01 received prednisone at a dose of 40 mg/m2/d (standard risk, SR) or 120 mg/ m2/d (high risk, HR); those treated on protocol 91-01 received dexamethasone at a dose of 6 mg/m2 per day (SR) or 18 mg/m2 per day (HR). Children treated on protocol 91-01 performed less well on cognitive testing. Subsample analysis indicated that cranial radiation therapy and methotrexate dose did not account for differences in cognitive outcomes. CONCLUSIONS: The findings of this preliminary study are consistent with the hypothesis that dexamethasone therapy can increase risk for neurocognitive late effects in children treated for ALL and indicate that further investigation of this question is warranted.

Neurotoxicity of sodium fluoride in rats.

Fluoride (F) is known to affect mineralizing tissues, but effects upon the developing brain have not been previously considered. This study in Sprague-Dawley rats compares behavior, body weight, plasma and brain F levels after sodium fluoride (NaF) exposures during late gestation, at weaning or in adults. For prenatal exposures, dams received injections (SC) of 0.13 mg/kg NaF or saline on gestational days 14-18 or 17-19. Weanlings received drinking water containing 0, 75, 100, or 125 ppm F for 6 or 20 weeks, and 3 month-old adults received water containing 100 ppm F for 6 weeks. Behavior was tested in a computer pattern recognition system that classified acts in a novel environment and quantified act initiations, total times and time structures. Fluoride exposures caused sex- and dose-specific behavioral deficits with a common pattern. Males were most sensitive to prenatal day 17-19 exposure, whereas females were more sensitive to weanling and adult exposures. After fluoride ingestion, the severity of the effect on behavior increased directly with plasma F levels and F concentrations in specific brain regions. Such association is important considering that plasma levels in this rat model (0.059 to 0.640 ppm F) are similar to those reported in humans exposed to high levels of fluoride.

Radiation effects on growth are altered in rats by prednisone and methotrexate.

CNS therapy for childhood leukemia has adverse effects upon growth and cognition. The cause of these deficits is unknown. In a rat model, we determined which agent, or combination of agents, in CNS therapy affected growth. Young Sprague-Dawley rats were exposed to cranial irradiation (1000 cGy), methotrexate (2 or 4 mg/kg, intraperitoneally), or prednisolone (18 or 36 mg/kg, intraperitoneally) alone or in two- or three-agent combinations. Matched control groups received appropriate sham radiation, intraperitoneal saline, or both. Body weight was recorded from 14 through 150 d of age. After the rats were killed at 150 d, body length was recorded and the head and left femur were removed to determine body and craniofacial proportions. Cranial irradiation alone, but not methotrexate or prednisolone alone, stunted growth permanently and altered craniofacial proportions. When these agents were combined, methotrexate and prednisolone modified the growth response to cranial irradiation. Methotrexate given before cranial irradiation prevented radiation stunting in males. This protection was lost when the dose of methotrexate was increased, when prednisolone was added to the combination, or when females were studied. The protection in males was effective against both growth and behavioral deficits. These results indicate that the physical and behavioral side effects of CNS therapy are better understood in the context of dose, sex, and interactions of the agents.

Interactions of steroid, methotrexate, and radiation determine neurotoxicity in an animal model to study therapy for childhood leukemia.

Children with leukemia receive CNS therapy to improve long-term survival. Neurotoxic effects, such as cognitive impairment, have been associated with this therapy. A rat model was developed to determine which agent, or combination of agents, in CNS therapy causes neurotoxicity. The agents examined were cranial irradiation (1000 cGy), methotrexate (2 or 4 mg/kg, intraperitoneally), and prednisolone (18 or 36 mg/kg, intraperitoneally). Young Sprague-Dawley rats were exposed to each agent alone or to two- or three-agent combinations. Each therapy had matched controls that received sham radiation and/or intraperitoneal saline. Subsequent to exposure, spontaneous behavior was tested using a computer pattern recognition system, which recorded and classified behavior in a novel environment. Behavioral initiations, total times, and time structures were compared in therapy and control groups. Combined rather than single-agent therapies had more behavioral effects, and these were dose- and sex-dependent. Synergistic interactions between agents caused behavioral deficits, and components of the combination determined the abnormality. Some combinations interacted antagonistically, and thus mitigated behavioral deficits. Prednisolone was clearly pivotal to behavioral outcome. A low prednisolone dose antagonized methotrexate preventing deficits, whereas a higher prednisolone dose altered behavior by enhancing effects of methotrexate and radiation. These findings emphasize that steroids are important in agent interactions. Their role in morbidity associated with leukemia treatment protocols may be equally important as that of methotrexate and cranial irradiation.

Stability and reproducibility of time structure in spontaneous behavior of male rats.

The computer pattern recognition system for the study of spontaneous rat behavior has allowed new analytical techniques which expand the definition of experimentally induced changes in behavior. As with any technique, the stability of the measures must be considered when evaluating overall sensitivity. This study evaluates the stability and reproducibility of three behavioral measures: a measure of the number of initiations of specific behavioral acts, a measure of the total time of each act, and a measure of behavioral time structure. Normal statistical parameters are used to evaluate the significance of changes detected using the first two measures, but the third measure utilizes K-functions, the bootstrap and ad hoc criteria to evaluate significance of observed changes. This study compares the stability of results from these three measures as applied to fourteen different groups of control Sprague-Dawley male rats. All three measures provided stable and reproducible results, but the measure of time structure, the K-function analysis, provided the greatest consistency. Behavior, particularly spontaneous behavior, has traditionally been perceived as being intrinsically variable. However, this study shows that the computer pattern recognition system and its analytical techniques provide stable and reproducible values that vary only a few percent.

No change in spontaneous behavior of rats after acute oral doses of aspartame, phenylalanine, and tyrosine.

Spontaneous behavior subsequent to acute oral administration of high doses of aspartame, phenylalanine, or tyrosine was analyzed using a computer pattern recognition system. Sprague-Dawley male rats (250-300 g) were dosed orally with aspartame (500 or 1000 mg/kg), phenylalanine (281 or 562 mg/kg), or tyrosine (309 or 618 mg/kg), and their behavior was analyzed 1 hr after dosing. The computer pattern recognition system recorded and classified 13 different behavioral acts performed by the animals during the first 15-min exploration of a novel environment. Three measures that provide independent information concerning motor output from the central nervous system were taken: the number of behavioral initiations, total time, and time structure. These results were compared with the effects induced by d-amphetamine. Plasma concentrations of phenylalanine and tyrosine were determined from blood samples taken immediately after behavioral examination. Data analysis revealed that these doses of aspartame, phenylalanine, and tyrosine did not induce any significant changes in spontaneous behavior. Unlike low doses of amphetamine and despite high plasma concentrations of phenylalanine and tyrosine, no behavioral alteration was detected by the computer pattern recognition system. Absence of behavioral changes in this study using an objective analysis of behavior raises questions concerning the relationship between amino acid precursor loading and purported anecdotal changes in behavior following aspartame administration.

Progressive behavioral changes in rats after exposure to low levels of ionizing radiation in utero.

The deleterious effects of ionizing radiation on the developing brain may be not only prolonged but progressive. Fetuses were exposed to 0.75 Gy of ionizing radiation on gestational day 15 through whole body exposure of the pregnant rat. Three behavioral tests (gait analysis, continuous corridor activity and photographic analysis of sequences of behavioral acts) were performed at 1 and 3 months, postnatally. Body weight and thickness of the cerebral cortex of irradiated rats were 10-15 percent below controls throughout the period of study. Behavior in all tests was more affected at 3 months than at 1 month of age. Gait of control rats, as measured by the angle of advanced of hind feet, widened about 20 percent for males and 40 percent for females from 1 to 3 months, as expected, while, in irradiated rats, the angle widened only about 10 percent. Continuous corridor activity increased less than 10 percent in controls and about 35 percent in irradiated rats over the same period. In photographic analysis of behavior, controls increased their time spent standing by about 50 percent in males and 20 percent in females from 1 to 3 months of age. Irradiated males increased time standing only about 10 percent and irradiated females decreased about 30 percent over the same period. The data obtained in these experiments support other evidence that some behavioral alterations from perinatal exposure to radiation become more marked with maturation.

An animal model to study toxicity of central nervous system therapy for childhood acute lymphoblastic leukemia: effects on growth and craniofacial proportion.

Many long term survivors of childhood acute lymphoblastic leukemia have short stature, as well as craniofacial and dental abnormalities, as side effects of central nervous system prophylactic therapy. An animal model is presented to assess these adverse effects on growth. Cranial irradiation (1000 cGy) with and without prednisolone (18 mg/kg i.p.) and methotrexate (2 mg/kg i.p.) was administered to 17- and 18-day-old Sprague-Dawley male and female rats. Animals were weighed 3 times/week. Final body weight and body length were measured at 150 days of age. Femur length and craniofacial dimensions were measured directly from the bones, using calipers. For all exposed groups there was a permanent suppression of weight gain with no catch-up growth or normal adolescent growth spurt. Body length was reduced for all treated groups, as were the ratios of body weight to body length and cranial length to body length. Animals subjected to cranial irradiation exhibited microcephaly, whereas those who received a combination of radiation and chemotherapy demonstrated altered craniofacial proportions in addition to microcephaly. Changes in growth patterns and skeletal proportions exhibited sexually dimorphic characteristics. The results indicate that cranial irradiation is a major factor in the growth failure in exposed rats, but chemotherapeutic agents contribute significantly to the outcome of growth and craniofacial dimensions.

An animal model to study toxicity of central nervous system therapy for childhood acute lymphoblastic leukemia: effects on behavior.

Central nervous system prophylactic therapy used in the treatment of acute lymphoblastic leukemia can reduce intelligence quotient scores and impair memory and attention in children. Cranial irradiation, intrathecal methotrexate, and steroids are commonly utilized in acute lymphoblastic leukemia therapy. How they induce neurotoxicity is unknown. This study employs an animal model to explore the induction of neurotoxicity. Male and female Sprague-Dawley rats at 17 and 18 days of age were administered 18 mg/kg prednisolone, 2 mg/kg methotrexate, and 1000 cGy cranial irradiation. Another 18-day-old group was administered 1000 cGy cranial irradiation but no drugs. Matching controls received saline and/or a sham exposure to radiation. All animals at 6 weeks and 4 months of age were tested for alterations in spontaneous behavior. A computer pattern recognition system automatically recorded and classified individual behavioral acts displayed during exploration of a novel environment. Measures of behavioral initiations, total time, and time structure were used to compare treated and control animals. A permanent sex-specific change in the time structure of behavior was induced by the prednisolone, methotrexate, and radiation treatment but not by radiation alone. Unlike hyperactivity, the effect consisted of abnormal clustering and dispersion of acts in a pattern indicative of disrupted development of sexually dimorphic behavior. This study demonstrates the feasibility of an animal model delineating the agent/agents responsible for the neurotoxicity of central nervous system prophylactic therapy.

Time structure analysis of behavioral acts using a computer pattern recognition system.

The spontaneous behavior of rats was monitored, classified and analyzed using a recently developed computer pattern recognition system. The K-function, a parameter developed for statistical research on spatial point processes and patterns, is used to analyze the temporal structure of behavioral acts or the joint relationship of separate acts. Forty male rats were observed in two experiments. In Experiment 1 untreated rats were observed and the temporal structure of their behavior was analyzed to establish an estimate of the false-positive error rate appropriate for use in such analyses. In Experiment 2, 20 control rats and 20 rats exposed to 2.0 mg/kg d-amphetamine were monitored using the computer pattern recognition system. The data from Experiment 1 showed that the false-positive rate was approximately ten percent or less. Experiment 2 demonstrated that the temporal structure of spontaneous motor behavior of amphetamine-treated rats was significantly disrupted.

Evolution of motor activity tests into a screening reality.

Measures of activity have changed in ways that their utility in screening for neurotoxicity has vastly improved. New computer pattern recognition systems now provide rapid, objective tools for identifying animal behavior. Advances in data analysis procedures have replaced the percent change in overall activity level with measures of initiations, total time and temporal structure for multiple behaviors and sequences. These advances can distinguish hyperactivities induced by different mechanisms and rank their neurotoxic potential, a feat not possible with photocell devices generally used in laboratories today. With the recent technological improvements, it is possible that a measure of activity will become one of the best predictors of malfunction in the central nervous system.

Extension of the analysis of the time structure of behavioral acts.

A parameter known as the K-function has previously been used to analyze the temporal structure of behavioral acts. In earlier work the study of these acts or of the joint relationship of separate acts has used a restricted list of activities whose definitions are difficult to explicate. In the present study this technique has been extended to a more extensive list of acts, each meaning of which is intuitively clear. As with the earlier work, the bootstrap method has been used to estimate the uncertainty in these measures, and the usefulness has been examined using data from studies of rats exposed to phenytoin or nitrous oxide.

Analysis of the time distribution and time sequence of behavioral acts.

A technique for analyzing the temporal structure between various initiations of a particular behavioral act has been developed using a parameter known as the K-function, the cornerstone of recent statistical research on spatial point processes and patterns. The technique has been extended to the study of the joint relationship of separate acts. Bootstrap methods are used to estimate the uncertainty in these measures. The usefulness of these techniques is demonstrated using data from studies of rats exposed to phenytoin and nitrous oxide.

Pattern recognition of rat behavior.

Analysis of animal behavior has been an arduous task requiring a human observer to record and classify individual motor acts. A computer pattern recognition system is introduced which simplifies this task by minimizing the need for human intervention. This system uses two video cameras with horizontal and vertical views of the behavior of a control and an experimental rat as they explore a simple environment for 15 minutes. Their behavior is sampled at a rate of one frame/second. Data from the video cameras are then converted into a form acceptable to Micro Vax I and VAX 11/750 computers. Each video picture is reduced to a 256 by 256 array, and ultimately each 15 minute observation session generates 28,800 blocks of information at 512 bytes each. Using a mathematically complete set of moments to the fourth order and the associated scalar invariants, the computer is programmed to identify the five major body positions of the rat including standing, sitting, rearing, walking and lying down. The computer also is programmed to identify the behaviors of grooming, head turning, whole body turning, looking, smelling, sniffing and washing face. This computer pattern recognition system not only speeds up behavioral classification, it alleviates the much criticized subjectivity introduced by human observers.

The effects of nitrous oxide after exposure during middle and late gestation.

Recent evidence has indicated that the anesthetic gas nitrous oxide (N2O) is teratogenic to rats if exposed during the organogenesis phase of gestation. Little is known, however, of the anatomical or functional consequences of exposures occurring later in gestation when the brain is developing. Timed pregnant Sprague-Dawley rats were exposed to 75% N2O/25% O2 using one of the following protocols: 24 hr exposures on gestational days 11-15 or 16-20; or 8 hr exposures on gestational days 9-13, 11-15, 14-15 or day 15 only. Both 24 hr exposure protocols significantly reduced fetal and maternal body weight, an effect not observed after the 8 hr exposures. No N2O exposure resulted in gross morphological or skeletal changes. Likewise, no significant effects on total protein and DNA levels in fetal liver and brain tissues could be found subsequent to 24 hr exposures on days 16-20; or on one, three or six days following an 8 hr exposure on day 15. Evaluations of postnatal growth and neurological development in pups prenatally exposed for 8 hr on days 14 and 15 revealed two noteworthy effects. Their rate of growth in body weight was greater with respect to controls between the ages of 14 and 21 days, especially in the males. Also, reflex suspension was reduced, significantly so in the females. In conclusion, unlike 24 hr exposures, multiple 8 hr exposures to nitrous oxide during the middle to late stages of gestation did not produce effects detectable with standard teratological measures. Subtle differences in growth rate and reflex suspension, however, indicated that normal development had been interrupted, but its clear distinction as a lasting effect requires additional measures of function.

Behavioral toxicity of nitrous oxide in rats following prenatal exposure.

Nitrous oxide (N2O) exposure has been associated with neurotoxicity, especially peripheral neuropathy, in both humans and animals. The effects of this anesthetic gas on the central nervous system (CNS) and spontaneous behavior, however, have yet to be delineated. Timed-pregnant Sprague-Dawley rats were exposed to 75% N2O/25% O2 on gestational days 14 and 15 or day 15 only for eight hours per day. The offspring were tested at one and five months of age; their spontaneous behavior in a novel environment was recorded in the residential maze and using time-lapse photography. The results indicated that in utero exposure to N2O permanently altered the spontaneous motor output of the CNS. This effect was most prominent in 5 month old animals, and females were affected more than males. Exposures on gestational days 14 and 15 produced an effect that was not only greater but also qualitatively different than that produced by exposure on day 15 only. The two-day exposure induced hyperactivity in both sexes, whereas the one-day exposure induced hyperactivity in the males and slight hypoactivity in the females. These behavioral changes were not accompanied by physical abnormalities but nonetheless were lasting effects in need of further consideration.