Metabolite profile in the basal ganglia of children with cerebral palsy: a proton magnetic resonance spectroscopy study

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1 Metabolite profile in the basal ganglia of children with cerebral palsy: a proton magnetic resonance spectroscopy study Wojciech Kulak* MD, Associate Professor; Wojciech Sobaniec MD, Professor; Joanna Smigielska-Kuzia MD, Department of Paediatric Neurology and Rehabilitation; Bozena Kubas MD; Jerzy Walecki MD, Professor, Department of Radiology, Medical University of Bialystok, Bialystok, Poland. *Correspondence to first author at Department of Paediatric Neurology and Rehabilitation, Medical University of Bialystok, Waszyngtona 17, Bialystok, Poland. kulak@hot.pl This prospective study determined metabolite profile in the left and right basal ganglia of children with spastic cerebral palsy (CP) compared with children without disabilities, by using proton magnetic resonance spectroscopy ( 1 HMRS). Twenty-three patients with spastic CP (12 males, 11 females; mean age 11y 9mo [SD 4y 2mo], range 4 17y) were examined. Twenty children had spastic diplegia and three had quadriplegia. Twenty-four normally developing children (13 females, 11 males; mean age 10y 3mo [SD 4y 8mo], range 4 17y) served as a comparison group. The relative concentrations of N-acetylaspartate (NAA), choline (Cho), myo-inositol (mi), and gamma-aminobutyric acid (GABA) were measured relative to creatine (Cr) and different combinations of metabolites within 8cm 3 brain voxels. Children with CP showed reduced ratios of NAA:Cr, NAA:Cho, NAA:mI, and GABA:Cr in the basal ganglia relative to a matched comparison group. Patients demonstrated a significant age-dependent increase in NAA:Cr and NAA:Cho in the basal ganglia. No sex-dependent difference was shown in children with CP nor in the comparison group for all tested metabolite ratios. Significant correlation between Apgar score and ratio of mi:cr in the group with CP was found. None of the tested metabolite ratios were correlated with the severity scale of CP in children with CP. NAA:Cr ratios were negatively correlated with learning disability* in patients with CP. Results indicate the association of the metabolite ratios in basal ganglia with learning disability. *North American usage: mental retardation. See end of paper for list of abbreviations. Patients with cerebral palsy (CP) may have problems other than motor impairment, including learning disability, epilepsy, and sensory disturbance. 1 4 The basal ganglia, and more specifically the corpus striatum, play a central role in the feedback loop that modulates cerebral cortical function. 5 Disruption of cortico-striatal pathways seems to be of particular importance in neurobehavioural abnormalities. 6 This is highly relevant in children with CP because, for several reasons, the basal ganglia are vulnerable to injury during a restricted period of brain development. 7,8 Magnetic resonance imaging (MRI) has been used to trace the development of surface landmarks and the progress of myelination, and measure volumetric changes affecting major brain regions as a function of postnatal age. 7,9 However, the imaging-based study of functional changes during development, including physiological events and brain activation, has been more limited. Proton magnetic resonance spectroscopy ( 1 HMRS) is a safe, non-invasive technique for studying the chemistry of the living brain This has proved to be a useful tool for the early evaluation of brain injury in neonates who experienced asphyxia. 10,12 In vivo 1 HMRS studies of the brain are capable of quantifying steady-state metabolic levels of neurotransmitters such as glutamate (Glu), N-acetyl aspartate (NAA), and gamma-aminobutyric acid (GABA), and small molecules such as choline (Cho), glutamine (Gln), glucose (Glu), myo-inositol (mi), creatine (Cr), and lactate (Lac). 13,14 N-acetyl aspartate (NAA) is a marker of neuronal loss; the other metabolites, Cr and Lac, can be viewed as cerebral indicators of energy metabolism. mi is a precursor for the phosphatidyl-inositol second messenger system, regulates osmotic processes within the brain, and regulates mood states. Pronounced elevation of mi may reflect demyelination and glial proliferation. Only one study, that of Krägeloh-Mann et al., 7 performed 1 HMRS on two children with dyskinetic CP. Voxels were located in the macroscopic lesions in the basal ganglia. They found a decrease in NAA:Cr and Cho:Cr ratios in the basal ganglia. In our previous report on 19 children with CP, 15 we found reduced ratios of NAA:Cr, NAA:Cho, NAA:mI, Cho:Cr, and Cho:mI in the left basal ganglia, relative to comparison groups. Furthermore, to our knowledge, apart from our previous report, no 1 HMRS studies have been conducted in children with CP. The objective of this study was to examine the metabolite profile in the left and right basal ganglia of children with spastic diplegia and quadriplegia compared with normally developing (ND) children using 1 HMRS. However, previous MRS studies did not report any changes, or found relatively small sex differences 20 that did not affect the general age-related trends in the observed variable, NAA:Cho. We studied sex-dependent changes in the metabolite ratios in both groups. Method PATIENTS The present study included 23 children with spastic CP (20 with spastic diplegia, 3 with quadriplegia) and 24 ND, matched comparison children. Details of the groups are summarized in Table I. The children with spastic CP were under the care of the Department of Paediatric Neurology and Rehabilitation, Medical University of Bialystok. A group of 24 healthy right-handed children matched for age and sex were recruited as a comparison group. All participants in the Developmental Medicine & Child Neurology 2006, 48:

2 comparison group were free from neurological or psychiatric disorders, had normal intellectual development, and their brain MRIs were normal. Spastic diplegia was defined as motor disability caused by non-progressive damage of the developing brain, with more pronounced spasticity in the lower limbs. 1 Spastic quadriplegia was defined as motor disability with spasticity in upper and lower limbs. The degree of severity of CP was assessed in our patients according to Arguelles et al.: 22 4, very severe=patients do not have any postural control; 3, severe=can walk with maximum support; 2, moderate=can walk with some support; 1, mild=can walk unaided. Seven patients were classified with score 1, eight with score 2, five with score 3, and three with score 4. All the children in this group had formal psychological assessments (the Wechsler Intelligence Scale for Children, Polish version, was used in children from 8 17y). 4 The development of children between 4 and 7 years (n=8) was assessed by the Terman Merrill method. 4 Learning disability was categorized into mild (IQ 70 84), moderate (IQ 50 69), or severe (IQ<50). ND children had IQ>90. Eight children with CP had normal intelligence, four had mild learning disability, and 11 had moderate difficulty. Patients with severe learning disability or who unable to cooperate in obtaining a standardized score were not included in this study. MRI AND 1 HMRS All MR images were obtained with a 1.5T MR scanner (Picker Edge Ecse) by using a standard circularly polarized head coil. Imaging sequences were as follows: T 1 -weighed FAST scan (TR 300ms, TE 4.5ms, 5mm sections) pre- and post-intravenous contrast media administration (0.1mmol/kg Megnevist, Schering) was performed; FSE T 2 -weighed and FLAIR series (TR 5000ms, TE 127.6ms, 5mm sections) were used; 1 HMRS, twovoxel PRESS sequence (TR 1500ms, TE 35ms, NEX 192), 2kHz bandwidth, two voxels (2cm 2cm 2cm) located in the left and right basal ganglia (thalamus, capsula interna; Fig. 1a) from an axial section with no visible changes in MRI. The MOIST method for suppressing the water signal was applied. Spectroscopic data were analyzed by using the Via 2.0 C (Picker) software package. In the spectrum estimation, signals were obtained at the following concentrations (in parts per million): NAA, 2.01; Cr, 3.0; Cho, 3.22; mi, 3.55; GABA 2.30 (Fig. 1b). Peak area metabolite ratios (NAA:Cr, NAA:Cho, NAA:mI, Cho:Cr, mi:cr, and GABA:Cr) were calculated. The total study time averaged 30 to 40 minutes. Barbiturate anaesthesia does not interfere with the main visible brain metabolites measured during 1 HMRS. 21 Five children required sedation. We used thiopental (3mg/kg body weight intravenous) for anaesthesia. STATISTICAL ANALYSIS Non-parametric statistics were used to assess the significance between CP and ND children, owing to abnormal distributions of the mi concentrations in the group with CP in a Shapiro-Wilks test. Wilcoxon matched-pairs signed ranks tests were used to compare the means in the 1 HMRS groups. Spearman s rank correlation coefficient was used to measure the age dependence, sex, Apgar score, learning disability, and severity of CP and metabolite ratios in the CP and comparison groups. Results are presented as means (SD). For all analyses we used two-tailed significance (p<0.05). The Ethics Committee at the Medical University of Bialystok, a b NAA Cr Cho Cr ml GABA ml G si NAA lac PPM Figure 1: (a) Axial a T 2 -weighted magnetic resonance imaging from an 11-year-old female with spastic diplegia. White rectangles in left and right basal ganglia are voxels of interest. (b) 1 HMRS spectra of same patient (chemical shifts in parts per million [ppm]): N-acetlaspartate (NAA) at 2.01, creatine (Cr) at 3.0, choline (Cho) at 3.22, gamma-aminobutyric acid (GABA) at 2.30, and myo-inositol (mi) at 3.55 were obtained., glutamate and glutamine;, ids; lac, lactate. 286 Developmental Medicine & Child Neurology 2006, 48:

3 Poland, approved the study. Informed consent was obtained from participants parents. Results Nineteen children with CP had hypoxic ischaemic lesions with patterns of periventricular leucomalacia in MRI. Four patients had normal MR scans. All ND children had normal MRI. The reduced ratios of NAA:Cr, NAA:Cho, and NAA:mI in the left and right basal ganglia of children with CP and comparison group differed significantly (Table II). Similarly, we found significant differences in the ratios of Cho:Cr and GABA:Cr between children with CP and the comparison group. No significant difference in the mi:cr ratio in the patients with CP compared with comparison group was found. No significant left/right basal ganglia asymmetries were noted for any metabolite ratios in children with CP and the comparison group (Table II). All the tested metabolite ratios were not related to the severity of CP in children with CP (data are not shown). No sex-dependent difference was shown in children with CP for all tested metabolite ratios (e.g. NAA:Cho in males, r= , p=0.858; in females, r=0.0396, p=0.857). No sex-dependent difference was shown in children in the comparison group for all tested metabolite ratios (e.g. NAA:Cho in males, r= 0.271, p=0.198; in females, r=0.271, p=0.198). Significant correlation between Apgar score and mi:cr ratio in the CP group was found (r=0.540, p<0.01). No correlation between Apgar score and mi:cr was noted in the comparison group (r= 0.195, p=0.360). Age dependencies of the ratios NAA:Cr and NAA:Cho in the basal ganglia of patients with CP were found (r=0.648, p<0.001; and r=0.632, p<0.01 respectively). Similar results were obtained for NAA:Cr and NAA:Cho in the comparison group (r=0.603, p<0.001; and r=0.585, p<0.01 respectively; Table III). NAA:Cr ratios (r= 0.574, p<0.01) were negatively correlated with learning disability in patients with CP (Table III). Discussion 1 HMRS, which is based on the phenomenon of chemical shift, can detect metabolite changes in the brain regions with no visible alterations. In accordance with previous studies of various brain disorders, the two most prominent pathological processes in brain tissue that give rise to 1 HMRS-detectable alterations are a loss of or damage to neuroaxonal tissue and the occurrence of glial proliferation The present findings suggest that a relation clearly exists between normal brain function and NAA, and that monitoring the level of NAA may provide important information about brain function. It has been suggested that NAA may also be lost from surviving neurons because of alterations in the brain. 8 No sex-dependent difference was shown in children with CP for all tested metabolite ratios. These findings are in agreement with earlier reports on ND children. 16,17,20 Previous studies of perinatal asphyxia using 1 HMRS have shown that NAA:Cho ratios are significantly lower in patients with an adverse outcome than in survivors without disabilities 23 and that this ratio relates to neuromotor and cognitive performances at 12 months of age. Our results also support earlier reports. 10,24 As cerebral maturation proceeds, NAA levels rise. The NAA:Cr ratio increases rapidly from age 1 month, to 1 or 2 years. 8 In infants, the Cho:Cr ratio is higher than that of adults. The Cho:Cr ratio declines rapidly from 1 month to 1 year of age, then declines more slowly until age 2 years. 17 These findings are in agreement with our results. Furthermore, NAA and Cho are independently associated with the full-scale IQ in normal adults. 25 Together, these Table I: Clinical data of children with spastic cerebral palsy (CP)and comparison group Data CP (n=23) Comparison (n=24) Age, mean (SD) y (4.16) (4.80) Sex Males Females Gestation, range wks Mean (SD) (5.43) 38.1 (1.82) Delivery Term <37 weeks 10 0 Apgar score (asphyxia) 10 0 Birthweight, range g Mean (SD) 2620 (1130) 3350 (470) Low birthweight (<2500g) 11 0 Normal birthweight (>2500g) Table II: Metabolite ratios (mean [SD]) in left and right basal ganglia of children with spastic cerebral palsy (CP) and comparison group; Wilcoxon signed ranks two-tailed test Metabolites CP group (n=23) Comparison group (n=24) p value Left Right Left Right Vs left CP Vs right CP NAA:Cr 1.65 (0.19) 1.69 (0.19) 1.98 (0.22) 1.93 (0.24) <0.001 <0.001 NAA:Cho 2.02 (0.43) 1.99 (0.40) 2.24 (0.25) 2.20 (0.23) <0.05 <0.05 NAA:mI 3.17 (0.82) 3.14 (0.79) 3.66 (0.65) 3.60 (0.61) <0.05 <0.05 Cho:Cr 0.81 (0.10) 0.84 (0.11) 0.91 (0.08) 0.94 (0.09) <0.01 <0.01 mi:cr 0.57 (0.11) 0.59 (0.10) 0.55 (0.09) 0.54 (0.12) ns ns GABA:Cr 0.45 (0.04) 0.48 (0.08) 0.41 (0.05) 0.42 (0.04) <0.05 <0.01 NAA, N-acetylaspartate; Cr, creatine; Cho, choline; mi, myo-inositol; GABA, gamma-aminobutyric acid; ns, not significant. Metabolite Profile in the Basal Ganglia of Children with CP Wojciech Kulak et al. 287

4 metabolites account for a large proportion of the variance in intelligence. Our research on NAA and learning disability is in accordance with the earlier report of Hashimoto et al. 25 They found a decrease in the NAA:Cho ratio in children with learning disability relative to comparison groups with normal intelligence. The NAA:Cr ratio tended to be lower in the learning disability group. The authors suggested that in patients with learning disability, NAA decreases and a disorder or dysfunction of neurons in the brain exists. NAA, which is present in neuronal cells, is considered a marker for neuronal development. 11 It is the most sensitive central nervous system metabolite, and neuronal damage or loss will result in decreased NAA levels. 11,16 Previous studies 17,20 have reported that children older than 2 years with mild developmental delay had markedly abnormal proton MR spectra compared with those of comparison groups. All these children had significant decreases in the NAA:Cr ratio in frontal white matter and in parieto-occipital white matter. In the present study, we detected similar decreases in the NAA:Cr ratio in the basal ganglia. It was hypothesized that children with developmental delay and normal brain MR images may have hypomyelination or decreased synaptic density as an underlying cause, which was detected with proton MR spectroscopy. 8,20 The observed decrease in the NAA:Cr ratio may be the result of hypomyelination, decreased numbers of normal neurons, or decreased synaptic density as an underlying cause, which may be missed on routine MR images. Our findings suggest that NAA is age-dependent within the studied age groups, and an increased NAA concentration is observed in patients with CP and healthy children. It is known that NAA is localized within neurons and involved in the synaptic processes. 13 Subsequent breakdown of NAA leads to aspartate, which is an excitatory amino acid neurotransmitter. In this study, we noted a decreased ratio of GABA:Cr. GABA is the most important inhibitory neurotransmitter in the brain and brainstem/spinal cord. It acts as a transmitter; the inhibition results Table III: Relation between metabolite ratio and sex, age, Apgar score, and learning disability in cerebral palsy and comparison group Groups Variable r-value p value Cerebral palsy (n=23) Males vs NAA:Cho ns Females vs NAA:Cho ns Apgar score vs mi:cr <0.01 Age vs NAA:Cr <0.001 Age vs NAA:Cho <0.01 Learning disability vs NAA:Cr <0.01 Comparison (n=24) Males vs NAA:Cho ns Females vs NAA:Cho ns Apgar score vs mi :Cr ns Age vs NAA:Cr <0.001 Age vs NAA:Cho <0.01 Learning disability vs NAA:Cr r, Spearman s rank correlation coefficient; ns, not significant; NAA, N-acetylaspartate; Cr, creatine; Cho, choline; mi, myo-inositol. from a hyperpolarization of the synaptic transmembrane potential of the inhibited neuron, which is elicited by the binding of GABA molecules to their receptors in the post-synaptic membrane. No sex-dependent difference was shown in children with CP for all tested metabolite ratios. These results are in accordance with previous studies. 16,18,20 On the other hand, we noted sex-related differences due to an increased ratio of NAA:Cho in females in the comparison group. It has been suggested that the elevation in white matter NAA:Cho must be related to the increase in neuronal content relative to the density of axons, which may correspond to neuronal maturation. 20 Cho (the choline-containing compound glycerylphosphorylcholine) is considered a product of sphingomyelin. The Cho level has been regarded as a marker of cellular density, because Cho is the precursor for phosphatidylcholine, which is a major constituent of the cell membrane. 8,19,25 Thus, the NAA:Cho ratio is regarded as a significant indicator in the assessment of neuronal activities, because it represents a relative ratio of neuronal density to cellular density. In the present report, we noted the age-dependent increase of the NAA:Cho ratio in the CP patients and comparison groups. This may suggest the existence of plasticity processes in the developing brain. On the other hand, alterations in the NAA:Cho ratio have been reported in neuronal disorders (Alzheimer s disease, chronic hypoxia, epilepsy, brain tumours, and hepatic encephalopathy). 11,13,17,24 In conclusion, the present findings showed reduced values of NAA and Cho in the basal ganglia of children with CP. This may be indicative of neuronal loss subsequent to an anoxic episode during the prenatal or perinatal periods. The age-dependent increase of NAA:Cr and NAA:Cho ratios were noted in both groups. No sex-dependent difference was shown in children with CP and comparison groups for all tested metabolite ratios. The severity of CP was not related to the tested metabolite ratios. NAA:Cr ratios were negatively correlated with learning disability in patients with CP. We suggest that early detection of the described metabolite deterioration can be helpful in starting more intensive rehabilitation and education of children with perinatal asphyxia with suspicion of CP. Our results indicate the association of the metabolite ratios in basal ganglia with learning disability. DOI: /S Accepted for publication 13th July References 1. Mutch L, Alberman E, Hagberg B, Kodama K, Perat MV. (1992) Cerebral palsy epidemiology: where are we now and where are we going? Dev Med Child Neurol 34: Kulak W, Sobaniec W. (2004) Cerebral palsy in children in northeastern Poland. J Pediatr Neurol 2: Kulak W, Sobaniec W. (2003) Risk factors and prognosis of epilepsy in children with cerebral palsy in north-eastern Poland. Brain Dev 25: Fennell EB, Dikel TN. (2001) Cognitive and neuropsychological functioning in children with cerebral palsy. J Child Neurol 16: Sherman SM, Guillery RW. (2002) The role of the thalamus in the flow of information to the cortex. 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