Abstract
Background: Pathologic changes in the motor cortex and corticospinal tracts in ALS may be reflected by abnormal signal intensities on conventional MRI. The sensitivity of these changes in detecting underlying pathology remains unclear.
Method: The authors used automated image analysis to quantify volumes of cerebral gray and white matter in 16 patients with ALS (eight limb onset, eight bulbar onset) and eight normal controls. Previously they had demonstrated a reduction in N-acetyl aspartate/creatine + phosphocreatine (NAA/[Cr + PCr]) measured by 1H-MRS in the subcortical white matter in the motor cortex region in the patients with bulbar-onset ALS. To determine whether this resulted from axonal degeneration, they also compared gray and white matter volumes in the patients with limb- and bulbar-onset ALS.
Results: There were no differences in the total brain volumes of gray or white matter for the three subject groups (p > 0.23). Comparison of the total ALS group and controls revealed localized deficits in gray matter volume centered on Brodmann areas 8, 9, and 10 bilaterally. Comparison of the patients with limb- and bulbar-onset ALS revealed deficits in the white matter volume in the bulbar-onset group, extending bilaterally from the precentral gyrus into the internal capsule and brainstem, consistent with the course of the corticospinal tract. There was no loss in gray matter volume in the precentral gyri.
Conclusions: The loss of gray matter in the frontal regions (total ALS group) provides further support that ALS is a multisystem disorder. In addition, there is in vivo evidence of axonal degeneration in the subcortical white matter in the motor region in patients with bulbar-onset ALS. This is consistent with a “dying back” process affecting cortical motoneurons in bulbar-onset ALS.
ALS is characterized by the progressive degeneration of upper and lower motoneurons leading to weakness of the bulbar, limb, thoracic, and abdominal muscles. In the cerebral cortex, regional neuronal loss can be prominent in the precentral gyrus as the giant pyramidal Betz cells and their axons degenerate.
Neuroimaging in ALS is currently performed clinically to exclude other pathologies rather than for definitive diagnosis. On conventional MRI, hyperintensities along the corticospinal tracts are described using T2-weighted,1,2⇓ proton density–weighted,3 and fluid-attenuated inversion recovery pulse sequences,4 although these changes are neither sensitive nor specific to the pathologic effects of ALS.5-7⇓⇓ Low-intensity lesions in the motor cortex on T2-weighted images are also reported5 but become more common with advancing age in neurologically normal subjects and can occur in both central and peripheral neurologic disorders.8
Although there is little doubt that the motor system is the cardinal target of the pathologic insult, it is now well recognized that extramotor systems can be involved in ALS. Evidence from histologic,9,10⇓ neuroimaging,11,12⇓ and neuropsychological studies13,14⇓ suggests that other brain regions can be involved in the pathologic process. However, the extent of the extramotor involvement remains unclear.
The primary aim of this study was to determine significant differences in maps of cortical and cerebellar gray and white matter in ALS patients and neurologically normal controls using automated image analysis techniques.15-17⇓⇓
1H-MRS is a potentially useful method for assessing upper motoneuron (UMN) pathology in ALS. In the first report using this technique in ALS, the motor cortex of 12 ALS patients was compared with that of six normal controls.18 Using a single-slice chemical shift imaging technique, a significant reduction in N-acetyl aspartate/creatine + phosphocreatine (NAA/[Cr + PCr]) was found not only in the primary motor cortex but also in the primary sensory cortex and the superior parietal gyrus, most marked in those patients with definite UMN involvement. Further studies applied 1H-MRS to the motor cortex in ALS examining metabolite ratios within a single voxel19,20⇓ or using a multislice chemical shift imaging technique.21 These again demonstrated a reduction in NAA measured relative to Cr + PCr, choline, or both, suggesting loss of or damage to motoneurons. Although the use of metabolite ratios allows better control for partial voluming by CSF, a drawback of using such ratios is that both metabolites may be affected by the disease process. Estimation of the absolute concentration of the three major resonances using an internal water reference method was used to address this issue.22 The authors found a decreased concentration of NAA in an 8-cm3 volume of interest in the motor cortex in patients with El Escorial definite or probable ALS. Cr + PCr and choline were unaffected. Another report also demonstrated reduced NAA in the precentral gyrus in ALS patients but with an elevation in choline.23 Of note, one study showed a reduction in NAA in ALS patients without alteration in other metabolites compared with controls but failed to show this reduction when measuring metabolite ratios from the same volumes of interest.24 Therefore, the method used in analysis may affect the results.
If 1H-MRS is to provide an objective and quantitative measurement of UMN function in ALS, the abnormal values should correlate with UMN loss. Pathologic correlates provide the gold standard but only reflect end-stage disease. Although several groups divided patients into those with UMN signs and those without, few studies have specifically tried to correlate metabolite ratios with clinical measures of UMN dysfunction. Maximum finger tap rate has been used as an estimate of UMN dysfunction and correlates with NAA/Cho + (Cr + PCr) in the motor cortex.21 The region of interest extended beyond the regions of finger control, suggesting diffuse motoneuron loss in the cortex.21
All these studies explored motor system metabolites measured by 1H-MRS at a single point in the disease process. Sequential changes in metabolite ratios over 2 years were investigated from a volume of interest comprising both cortical gray matter and subcortical white matter in the motor cortex region. Eight of nine patients (five ALS, four lower motoneuron involvement only) demonstrated the most marked reduction over time in the NAA/Cho ratio, with lesser reductions in the NAA/Cr + PCr, Cho/Cr + PCr, and inositol/Cr + PCr ratios.20
The range of metabolite results from normal to pathologic is likely to reflect the heterogeneity of the pathologic characteristics of the motor cortex in ALS.
We previously demonstrated a reduction in the NAA/(Cr + PCr) ratio in subcortical white matter in the motor region of bulbar-onset ALS patients.25 This may represent axonal degeneration and astrocytic gliosis within this region, but 1H-MRS is unable to distinguish between neuronal loss and neuronal dysfunction.
Our second aim was to explore the hypothesis that the reduction in NAA/(Cr + PCr) metabolite peak area ratio demonstrated in the subcortical white matter in the motor cortex region of bulbar-onset patients previously25 was caused by neuronal loss. To examine this, we quantified and compared the volumes of gray and white matter in the bulbar- and limb-onset ALS patients studied previously with 1H-MRS.25
Methods.
Clinical base.
Patients were recruited from the King’s Motor Neuron Disease Care and Research Center, London. Sixteen patients with El Escorial definite, probable or possible ALS were studied,26 eight with limb onset (mean age, 55.5 ± 15.2 years; six men, two women; mean disease duration, 17.9 ± 9.0 months) and eight with bulbar onset (mean age, 54.3 ± 14.2 years; three men, five women; mean disease duration 18.5 ± 14.7 months), and compared with eight healthy, age-matched controls (mean age, 55.8 ± 14.9 years; three men, five women). All patients were seen regularly in the clinic, allowing us to ensure the diagnosis of ALS was unchanged over the period of follow-up. Control subjects were unrelated friends or spouses of the patients.
Clinical assessments.
Before inclusion in the study, all patients were assessed in the King’s Motor Neuron Disease Care and Research Center and were recruited if they had clinical and electrophysiologic evidence of combined UMN and lower motoneuron involvement in at least one region. The clinical findings on the patients were described in detail previously25 and are shown in table 1.
Details of patients with ALS
No patient had a history of cerebrovascular disease and none was taking psychoactive drugs. Written informed consent was obtained from all participants, and the study was approved by the ethics committees of King’s Healthcare and the Bethlem & Maudsley Trust.
Image acquisition.
Images were acquired on a 1.5-T General Electric Signa MR system (Milwaukee, WI). A quadrature head coil was used for radiofrequency transmission and reception. Dual-echo fast spin-echo near axial images were acquired for structural analysis, covering the whole brain, providing both proton density–weighted and T2-weighted images simultaneously (repetition time, TR = 4000 milliseconds; echo times, TE1 = 17 milliseconds and TE2 = 85 milliseconds; echo train length = 8, field of view = 22 cm, slice thickness = 5 mm, slice gap = 0.5 mm, 256 × 192 acquisition matrix). The acquisition parameters were optimized using a software simulation tool.27
Structural analysis of images.
The dual-echo structural MR data were analyzed using an automated computational method, described in detail previously.15,16⇓ Region-of-interest approaches employed in most structural MRI studies require a priori assumptions about the structures involved and may be insensitive to identifying regions of tissue change, in particular in areas where boundaries are hard to delineate. Computer-based methods of estimating gray and white matter and CSF distributions offer a number of advantages in addition to improved detectability. The entire brain is considered, permitting a more global view of pathologic changes in disease. Because the technique is automated, reliability is increased as well as being more sensitive to local tissue differences compared with traditional region-of-interest approaches.28
Initially, extracerebral tissues, such as skin or bone, were removed automatically by a computational algorithm.15 Some manual editing of the segmented images was then carried out using a custom graphic user interface to ensure common coverage of the cortex, cerebellum, and midbrain. Further processing of the data categorized each voxel in terms of the partial volume occupied by gray matter, white matter, CSF, or dura/blood vessels using the relative intensity information of the two MR signals (proton density and T2 weighting) acquired at each voxel. This resulted in four maps for each individual in the study, representing the distribution of gray matter, white matter, CSF, and dura/blood vessels.
To infer differences in tissue distribution between groups, the resulting gray and white matter probability maps were mapped onto a template image in standard stereotactic space29 so that a voxel approximately represented equivalent anatomic locations in every individual. The template image was constructed by proportional rescaling of a random subset of 10 proton density–weighted images taken from the control (n = 5) and patient (n = 5) groups. Using AFNI software,30 the distances between certain anatomic landmarks (the anterior and posterior commissures, the lateral, superior and inferior convexities of each cerebral hemisphere) were linearly rescaled to approximate each individual image to the reference brain depicted in a standard stereotactic atlas.29 The 10 transformed images were then averaged to produce a single template image in standard space. Each individual gray and white matter probability map was then registered onto the template image using affine transforms, registering them in standard space.
Statistical inference of differences between groups was achieved by initially comparing gray (or white) matter distributions at each voxel level by regression of a general linear model (GLM), which included the potential confounding factors of global tissue volume and age. This generated maps of the coefficient of group membership divided by its standard error, and the statistical significance of this value at each voxel was determined by a two-tailed test using a reference null distribution formed by a randomization procedure.17 Because of the large number of voxel locations tested, the estimated number of type 1 errors at reasonable statistical thresholds (p values) is unacceptably large. Thus, spatial information was included by considering three-dimensional voxel clusters, reducing the number of tests.17 Here, maps of the group membership coefficient divided by its standard error for the observed and randomized distributions were thresholded such that only voxels passing p < 0.05 were retained. The sum of the suprathreshold voxel measures for each of the resulting three-dimensional clusters was then the measure tested, its sign indicating a relative excess or deficit in tissue density in the patient group. Significance testing of the clusters was again performed against a null distribution obtained by randomization.
Results.
Two subjects showed generalized atrophy on their MR images, one limb-onset patient and one control. Although it was possible to segment the patient data adequately, this was not the case for the control data, which was not included in the subsequent analysis. This is because the computational method uses particular anatomic landmarks to register the data into standard space. In the patient data, although there was some atrophy to inspection, this was not sufficient to prevent data analysis. In the case of the control subject, however, the degree of atrophy was sufficient to prevent key anatomic landmarks aligning with others in the cohort, thereby preventing registration into standard space. The excluded control was not examined clinically.
Total brain and tissue class volumes.
The mean total brain volumes and tissue class volumes (gray matter, white matter, and CSF) for the three subject groups are shown in table 2. Using analysis of variance, there were no significant differences between the subject groups (p > 0.23).
Total and tissue class brain volumes in the three subject groups
For assessing localized differences between groups, our choice of statistical threshold was made such that the expected number of false-positive clusters across the image was less than or equal to one. This was achieved with a p value of 0.0025 or less. We analyzed differences between groups at a statistical significance of p = 0.001 and p = 0.0025. If differences were detected at the more conservative significance level (p = 0.001), we used this as the basis for analysis. If differences were not detectable at this level, we used a significance level of p = 0.0025 because both levels met our criteria and we regarded both as conservative levels of significance.
Localized differences between total ALS group (n = 16) and controls (n = 7).
Comparisons performed at p = 0.001 did not reveal differences between the groups. The results reported were performed using a cluster-wise voxel probability of type 1 error p = 0.0025. This probability fulfilled the criteria that the expected number of false-positive clusters across the image was less than one.
Gray matter volumes.
Differences between the ALS patients and controls in the volume of gray matter were detected at three three-dimensional voxel clusters (figure 1). These all represented relative deficits in the gray matter volume in the ALS group and are detailed in table 3.
Figure 1. Differences in gray matter volume between patients with ALS and control subjects displayed on the average gray matter template. Regions of deficit in gray matter volume in the patient group are shown in yellow. Comparisons were performed using a cluster-wise voxel probability of type 1 error p = 0.0025 (estimated number of false positives less than one).
Regional differences in gray matter volumes between patients with ALS (n = 16) and control subjects (n = 7)
White matter volumes.
Differences between the ALS patients and controls were identified in one voxel cluster, representing an excess in white matter in the ALS patients in the right inferior frontal gyrus (volume, 0.9 mL).
Localized differences between patients with limb- (n = 8) and bulbar-onset (n = 8) ALS.
Comparisons were performed using a cluster-wise voxel probability of type 1 error p = 0.001.
Gray matter volumes.
Localized deficits in gray matter volume in the bulbar-onset ALS patients were detected in voxel clusters centered in the brainstem (volume, 0.1 mL) and cerebellum (volume, 2.8 mL). A further region of deficit was identified in the fusiform gyrus (Brodmann area [BA], 34; volume, 0.1 mL).
White matter volumes.
Significant differences between the limb- and bulbar-onset patients were found in two voxel clusters (figure 2). The two cluster deficits in white matter volume in the bulbar-onset group extended bilaterally from the precentral gyrus via the internal capsule to the midbrain (volumes: right, 3.4 mL; left, 2.8 mL) and are likely to represent the corticospinal tract.
Figure 2. Differences in white matter volume between patients with bulbar- and limb-onset ALS displayed on the average white matter template. Regions of deficit in white matter volume in the bulbar-onset patients are shown in yellow. Comparisons were performed using a cluster-wise voxel probability of type 1 error p = 0.001 (estimated number of false positives less than one).
Localized differences between patients with limb-onset ALS (n = 8) and control subjects (n = 7).
Gray matter volumes.
No deficits in gray matter volume were detected at significance p = 0.001. By testing at a significance of p = 0.0025, clusters were identified indicating a deficit in gray matter volume in the limb-onset patients centered on the right cingulate gyrus (BA 32) (volume, 0.24 mL), and the medial frontal gyrus (BA 10) (volume, 0.8 mL).
White matter volumes.
No localized differences in white matter volume were identified between limb-onset patients and controls.
Localized differences between patients with bulbar-onset ALS (n = 8) and control subjects (n = 7).
Gray matter volumes.
No differences in gray matter volume were detected at p = 0.001. Using p = 0.0025, a deficit in gray matter volume in the bulbar-onset patients centered in the right superior frontal gyrus (BA 10) was identified (volume, 1.5 mL). A region of excess gray matter in the patient group was also identified in the right midtemporal gyrus (volume, 0.5 mL).
White matter volume.
At significance p = 0.0025, one region of deficit in white matter volume (1.4 mL) was identified on the left of the brain, extending from the precentral gyrus to the internal capsule and brainstem. By relaxing the p value further, bilateral changes were seen along the corticospinal tract, but this introduced an unacceptable number of possible false-positive clusters.
Discussion.
In this study, we analyzed brain volumes of gray and white matter in ALS patients and compared them with normal controls using an automated analysis program.15-17⇓⇓
We failed to show differences in the gray matter volume in the precentral gyrus in the ALS group compared with controls. The pathologic hallmark of the motor cortex in ALS is loss of the giant pyramidal Betz cells.31 These cells contribute 5% of the total pyramidal cells in the precentral gyrus, and macroscopically the brain often appears normal. Microscopic changes in the motor cortex vary from severe Betz cell loss with astrogliosis and atrophy of the motor cortex to no detectable pathology. With conventional MRI, the precentral gyrus may show qualitative low signal intensity on T2-weighted images in ALS.32-35⇓⇓⇓ However, cortical low intensity areas are often reported in patients with other neurologic diseases, such as AD, particularly with increasing age,8 and are also found in normal controls.5 Our failure to detect gray matter atrophy in the ALS group may reflect the heterogeneity of the changes in the motor cortex in this disorder. This does not exclude primary motor cortex involvement but does raise the question of what constitutes the pathologic process in the motor cortex. Although in some cases extensive cell loss may occur, in others, a dying back process along the corticospinal tract motoneurons without direct cell loss in the motor cortex may underlie the UMN signs found clinically. This is also evident in histologic techniques. A stereologic approach failed to show a reduction in the total number of neurons in the motor cortex in patients with ALS compared with controls,36 whereas a previous study demonstrated histometric evidence of abnormal primary motor cortex neurons.37 However, this change was in the size and shape of the cell bodies rather than the number.37
We demonstrated deficits in gray matter in the frontal regions in the ALS group compared with controls centered on the right superior and medial frontal gyri and the left midfrontal gyrus. Although the centroids for these regions lie in BA 9 and 10, as the regions are traced along the two-dimensional images (see figure 1), they can be seen to involve BA 8, 9, and 10 bilaterally. Examination of limb-onset patients compared with controls and bulbar-onset patients with controls revealed frontal deficits in gray matter volume in both subsets of patients, suggesting that this is not just a chance finding. Pathologically, astrocytic gliosis has been described in the midfrontal, inferior parietal, temporal, cingulate, and occipital subcortical white matter in ALS,9 but in one quantitative study, there was no reduction in the total number of neurons in the neocortex in eight ALS patients compared with nine healthy controls.36
Neuroimaging studies have provided evidence of extramotor involvement. Structural assessment of the cortical and subcortical volumes in patients with ALS revealed a significant reduction in the amount of underlying white matter in the anterior frontal lobes without a reduction in the cortical surface area when compared with controls.12 This would be consistent with degeneration of axons projecting to the frontal cortex from elsewhere. However, these measurements were made by tracing the regions of interest taken from five 4-mm sections superior to the corpus callosum and therefore did not include whole brain measures. The manual delineation of boundaries may reduce the sensitivity to detect subtle changes and introduces investigator bias.
Functional neuroimaging using PET has demonstrated cortical involvement extending beyond the motor cortex,14,38-40⇓⇓⇓ and by combining neuropsychological and PET activation studies, reduced activation of the prefrontal and premotor cortex was seen in nondemented ALS patients with impaired verbal fluency.41
Cognitive changes in ALS predominantly affect executive-type functions, mediated by the prefrontal cortex.13,14⇓ Although this may be more common in patients with bulbar involvement clinically, the frontal deficits in gray matter volume in this study were identified in both bulbar- and limb-onset patients. Our findings directly confirm the focal nature of early extramotor involvement in ALS, with loss of gray matter volume in BA 8, 9, and 10 being entirely consistent with the cognitive deficits detected in ALS patients. The fact that volumetric changes were seen in relatively few extramotor areas probably reflects the relative insensitivity of this approach compared with quantitative cellular morphometry.
No differences were found in the volumes of subcortical white matter in the motor cortex region in the ALS patients as a whole compared with controls. However, the bulbar-onset group showed a deficit in the volume of white matter in the motor cortex region compared with the limb-onset group and with controls. Our current findings support the hypothesis that the reduced NAA/(Cr + PCr) metabolite peak area ratio demonstrated in this region25 was caused by axonal loss with astrogliosis rather than neuronal dysfunction. The deficit in white matter volume extended from the precentral gyrus to the internal capsule and brainstem bilaterally and is likely to represent the course of the corticospinal tracts. Our findings are suggestive of axonal loss in the subcortical white matter in bulbar-onset ALS patients. The changes occurred in the absence of a deficit in gray matter volume in the motor cortex, consistent with a dying back pathology of the cortical motoneurons.
The deficit in white matter volume was only detected in the bulbar-onset patients, suggesting more severe UMN involvement in this group. The bulbar-onset patients all fulfilled criteria for El Escorial definite or probable disease at the time of the scan, whereas the limb-onset patients had El Escorial probable or possible ALS.26 This difference is significant (p = 0.004) and suggests a greater burden of UMN damage in the bulbar-onset group. Bulbar-onset ALS is associated with a female predominance and cognitive disturbance, whereas limb-onset ALS is typically shown to occur more commonly in men. These findings also suggest the bulbar-onset disease differs from other ALS types. The fact that we did not detect differences in the limb-onset patients compared with controls may reflect less severe UMN involvement in this subset.
Bilateral T2-hyperintense lesions were identified in the posterior limb of the internal capsule in two bulbar-onset patients, three limb-onset patients, and one control. These regions were classified by the segmentation software as CSF with an apparent absence of white matter. The obvious potential for type 1 errors was negated by the conservative nature of the statistical inference. Only voxels to which all individuals contributed nonzero tissue density were considered; thus, voxels at the location of these lesions were ignored.
On comparison of the gray matter between the limb- and bulbar-onset ALS patients, we identified a deficit in the bulbar-onset patients in the brainstem and cerebellum. The brainstem deficit may represent the cranial nerve nuclei involved in the pathologic process in ALS, which are clinically more severely involved in this subgroup of patients. However, the volume of deficit was very small (0.1 mL). Cerebellar deficits are not generally described in ALS. It is noteworthy that one patient with bulbar-onset disease had a positive family history of ALS. In familial ALS, degeneration of the posterior columns, Clarke’s column, and the spinocerebellar tracts are described neuropathologically,42-45⇓⇓⇓ and in one patient with familial ALS who had been on respiratory support for 5 years before death, degeneration within the cerebellar cortex was reported.45 Patients with sporadic ALS of long duration, usually owing to respiratory support, can also show multisystem degeneration at autopsy.46,47⇓ However, the cerebellar deficits were not demonstrated in the comparison of the limb-onset or bulbar-onset patients and controls, and further studies are needed to determine their significance.
Our findings raise important questions about selective vulnerability and the nature of the pathologic process in ALS. They support the notion that prefrontal areas (e.g., BA 9 and 10 in particular) are affected early in ALS, as suggested by PET activation and cognitive studies.13,14,41⇓⇓ The atrophy of these areas may be selective compared with other cortical areas including (to our surprise) the primary motor cortex. Other imaging studies48 indicate that wider prefrontal areas may be involved in the disease process, and atrophy is likely to be an insensitive marker of cellular pathology. Further longitudinal studies of the evolution and distribution of atrophy using this approach are warranted. Our failure to show atrophy of the primary motor cortex is unlikely to be artifactual because we could show atrophy in the distribution of the corticospinal tract and in prefrontal regions. Although a dying back process affecting corticospinal tract motoneurons may account for this observation, this is unlikely to be the full explanation. Better understanding of clinical and pathologic heterogeneity and of cortical selective vulnerability in ALS is needed.
Footnotes
-
Dr. Ellis was an Action Research Training Fellow.
- Received November 27, 2000.
- Accepted July 2, 2001.
References
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