AMYOTROPHIC lateral sclerosis (ALS) is a progressive neurodegenerative disease with selected both upper and lower motor neuron lesions.1-2 In ALS, many voxel-based morphometry (VBM) studies found that gray matter(GM) volume decreased widely, especially in motor cortex,3-4 frontal and temporal regions,4-6 corpus callosum,7 amygdala,8 caudate nucleus head.9 However, Mezzapesa et al’s study showed that no volume reduction of primary motor cortices in ALS patients.10 Voxel-wise meta-analysis11-12 has revealed that ALS is a complex degenerative disease involving multi-systems besides motor system, and right precentral GM atrophy is a common finding and prominent feature of brain structural changes in ALS.13 However, GM changes over the whole brain were not comprehensively studied in bulbar-onset and spinal-onset ALS patients.

The aim of this study is to investigate brain structural imaging signatures in clinical subtypes of ALS.We hypothesized that some brain regions may intrinsically suffered from volume loss in the patients with ALS. To address this hypothesis, we obtained brain structure images of 65 patients with ALS (ALS group)and 65 normal controls (NC) from 3.0T MRI system. All the ALS patients were classified into bulbar-onset group(ALS-bulbar, n=15) and spinal-onset group (ALS-spinal,n=50) according to the initial onset location. We firstly identified the alteration of GM changes over the whole brain between the ALS group and the NCs, and then the GM volume analysis was performed among the ALS-bulbar group, ALS-spinal group and the control group to identify the pattern of different GM volume loss.

PATIENTS AND METHODS

Participants

This study was approved by the institutional ethic review board and written informed consents were obtained from all participants. Sixty-five patients, including 34 cases with definite diagnosis of ALS and 31 cases with suspicious diagnosis of ALS according to the revised El Escorial,14 were consecutively recruited from the Chinese PLA General Hospital from 2007 to 2010.The subjects were excluded for the following conditions:1. history of cerebrovascular disease, long-standing hypertension, diabetes mellitus, inflammatory disease of the central nervous system and cranium trauma; 2. taking psychoactive drugs or hormone; 3. structural damage observed on the conventional MR images. All the subjects were right handed. ALS functional rating score(ALSFRS)15 and Norris Scale16 were administered to all the patients for clinical rating their ALS symptoms, and mini-mental state examination (MMSE)17 was applied to evaluate the cognitive function of all subjects.

MRI examination and acquisition

MR structural images were obtained from a 3.0T MR imaging system (SIGNA EXCITE, GE Healthcare, Milwaukee, WI, USA) and a conventional eight-channel quadrature head coil. Three-dimensional T1-weighted fast spoiled gradient recalled echo (3D T1-FSPGR)sequence was used, and the parameters were as follows: TR (repetition time) = 6.3 ms, TE (echo time) =2.8 ms, flip angle = 15°, FOV (field of view) = 24 cm×24 cm, Matrix = 256×256, in-plane resolution of 0.9375×0.9375 mm2, NEX (number of acquisition) =1. Conventional T2-weighted images, T1-weighted images and diffusion weighted images were also obtained to exclude the patients with diseases other than ALS that inflecting brain morphometry. The imaging protocol was identical to each subject.

Data processing

All MR structural image data were processed using Statistical Parametric Mapping 12 (SPM 12) (http://www.fil.ion.ucl.ac.uk/spm/) running under MATLAB 7.6 (The Mathworks, Natick, MA, USA) to perform VBM.18 The preprocess of VBM was as follows: (1) setting the image origin at the anterior commissure (AC); (2) normalizing the individual structural images to the DARTEL (Diffeomorphic Anatomical Registration using Exponentiated Lie algebra) templates space; (3) segmenting the normalized images into GM, white matter and cerebrospinal fluid (CSF); (4) calculating the volume of 142 brain regions based on SPM Neuromorphometrics atlas; total GM volume (TGMV), total white matter volume (TWMV),and total CSF volume (TCSFV) were recorded. Total intracranial volume (TIV) was the sum of the volume of segmented brain tissue (TIV = TGMV + TWMV +TCSFV), and fraction of GMV (fGMV) was the ratio of TGMV to TIV (fGMV = TGMV/TIV); (5) smoothing the segmented GM images with a kernel of 8 mm FWHM (full width at half maxima) before statistical analysis.

Statistical analysis

Further analysis in the clinical subtypes demonstrated that bulbar-onset and spinal-onset ALS presented different patterns of GM atrophy. For the bulbar-onset ALS, only extra-motor brain regions were involved, and the motor cortex was spared. The involved brain regions were located in frontal lobe (left medial orbital gyrus) and temporal lobe (left inferior temporal gyrus and right middle temporal gyrus).These findings may be associated with the poorer cognitive function in bulbar-onset ALS patients than those of spinal-onset subtype.21 Therefore, bulbar subtype may be considered as fronto-temporal pattern ALS,and the longitudinal observe will help to understand the association between ALS and fronto-temporal dementia.

The quantitative data with normal distribution were presented as mean ± standard deviation, and the quantitative data with nonnormal distribution were presented as median ± interquartile range. The partial correlation analysis was applied between the clinical variables (ALSFRS, disease duration, Norris score,MMSE) and volume of abnormal brain regions in ALS patients. The group difference for the abnormal brain regions was compared using ANCOVA covarying with age and sex, and the Welch test was used because of variance non-homogeneity. The statistical analysis was performed using SPSS 19.0.

为了验证所提出的频率协调控制的有效性,通过搭建MATLAB/Simulink仿真模型进行验证。柴储混合电力系统模型参数取值如表1所示。

2.4 关注突出成就和社会热点，培养学生的社会责任意识 生物学知识与生产、生活和社会息息相关，如健康知识、饮食常识、环保知识、遗传病的预防、吸烟的危害、转基因食品的安全问题等；生命科学的突出成就与人类的健康生活和国家的社会经济利益密切相关，如诺贝尔生理学或医学奖对维护人类健康的意义、袁隆平杂交水稻对人类社会的贡献等。教学中，教师要鼓励学生运用所学的生物学知识去参与个人和社会事务的讨论，尝试解决生活中的生物学问题，激发学生热爱自然、热爱生命、热爱生物科学、关心和维护地球生态环境的责任意识，树立建设祖国和家乡的义务感和责任感。

RESULTS

Demography and clinical scale test

The participants came from 10 provinces and included 63 sporadic cases and 2 familial cases. There were 28 female and 37 males in the ALS group, 29 females and 36 males in the NC group (χ2=0.03, P=0.86). The age of the two group were 49.63±10.25 years old and 43.48±12.22 years old (t=1.98, P=0.002). The MMSE of the ALS patients and NCs were comparable (both 28.00±3.00, t=1.98, P=43). For ALS patients, the average of disease duration, ALSFRS and Norris scores were 17.74±21.46 months, 30.00±7.00 (normal value 48)and 69.00±28.50 (normal value 99), respectively.

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（1）滤筒增重称量。将现场采回的滤筒在105～110℃烘箱中烘干1h，取出后放干燥器内冷却至室温，用天平进行称重。（带去现场前滤筒在同样条件下已经称好初重。）用终重减去初重就是滤筒的总增重量Δm 。

化工装置连续生产的特点，因此，上下班生产交接是个重要环节，交接不清会给安全生产埋下隐患，导致“放炮”。为此，无论严冬酷暑、风霜雪雨，只要当班，董松江都要提前15分钟到岗，接班前先仔细了解设备运行以及生产状况，常年不辍。

GM volume changes over the whole brain of ALS patients

Compared with the NC group, the decreased GM volume of brain regions in the ALS-spinal group were located in the rPrcGy, left medial precentral gyrus, left postcentral gyrus, bilateral superior frontal gyrus, right middle frontal gyrus, bilateral middle occipital gyrus,bilateral angular gyrus, right middle temporal gyrus,left thalamus proper (lThaPro), left central operculum,left supramarginal gyrus, left precuneus, left inferior frontal angular gyrus, left fusiform gyrus, and left superior parietal lobule (Table 3, Fig. 3) .

ANCOVA demonstrated the GM volume of the rPrcGy were significant different between the ALS and the NC groups (P=0.03). No significant difference of TGMV, TIV and fGMV was observed for the rMidFroGy between the ALS and NC group (all P＞0.05) (Table 1).

Figure 1. Brain region with decreased gray matter (GM)volume in ALS group compared with NC group. Colored areas represent the involved brain areas: right precentral gyrus and middle frontal gyrus respectively. The value of the color bar represents T value.

Table 1. Volume comparisons of rPrcGy, rMidFroGy, TGMV, TIV and fGMV between the ALS and NC groups (10-3 ml, n=65)

*analysis of covariance was performed for comparisons adjusted by age. NC: normal control; ALS: amyotrophic lateral sclerosis; rPreGy: right precentral gyrus; rMidFroGy: right middle frontal gyrus; TGMV: total gray matter volume; TIV:total intracranial volume; fGMV: fraction of gray matter volume (=TGMV/TIV).

Groups　 Volume of rPrcGy 　 Volume of rMidFroGy means±SD　95% CI　means±SD　95% CI NCs　12.14±1.33　11.68-12.30　18.71±2.41　17.84-18.97 ALS　11.36±1.34　11.19-11.81　17.64±2.61　17.38-18.51 F value　4.76　1.26 P value*　0.03　0.27 Groups　 TGMV　 TIV　 fGMV means±SD　95% CI　means±SD　95% CI　means±SD　95% CI NCs　610.75±56.74　589.83-617.29　1423.72±127.38　1385.11-1453.02　0.43±0.02　0.42-0.43 ALS　574.72±64.43　578.18-605.64　1393.96±144.74　1364.66-1432.57　0.42±0.02　0.41-0.43 F value　1.36　0.69　0.41 P value*　0.25　0.41　0.52

GM volume changes between ALS-bulbar and NC

In patients with ALS-bulbar, compared with NCs, the decreased GM volume was located in the left medial orbital gyrus ［MNI coordinate (x, y, z): -11, 41, -30;K value: 125; T value: 4.09; P value: 0.000］, the left inferior temporal gyrus ［MNI coordinate (x, y, z): -53,-32, -21; K value: 418; T value: 4.87; P value: 0.000］and the right middle temporal gyrus ［MNI coordinate(x, y, z): 65, -32, -14; K value: 134; T value: 4.16;P value: 0.000］ (Fig. 2). No increased GM volume in ALS-bulbar was recognized.

Among the above three regions with loss of GM,only the volume of the left inferior temporal gyrus in the ALS-bulbar was found to have significant difference from the NCs (F=6.19, P=0.02). The GMV, fGMV and TIV were not significantly different between the ALS-bulbar and NC groups (Table 2).

GM volume changes between ALS spinal and NC

In patients with ALS, the decreased GM volume of brain regions, compared with NC, located in the right precentral gyrus (rPrcGy) ［MNI coordinate (x, y, z): 39, -12,53; K value: 624; T value: 4.38; P value: 0.000］ and right middle frontal gyrus (rMidFroGy) ［MNI coordinate (x,y, z): 42, 56, 11; K value: 150; T value: 3.88; P value:0.000］. No increased GM volume of brain region in ALS was recognized (Fig. 1).

Differences of GM volume alterations between ALS-spinal and ALS-bulbar

Figure 2. Brain regions with decreased GM volume in bulbar-onset ALS patients compared with NC group. Colored areas represent the involved brain areas, including left medial orbital gyrus, left inferior temporal gyrus and right middle temporal gyrus group respectively. The value of the color bar represents T value.

Table 2. Volume comparisons of the brain regions with decreased GMV and TGMV, TIV, fGMV between the ALS-bulbar and NC groups (10-3 ml)

*Analysis of covariance was performed for the group comparisons covarying with age. lMedOrGY: left medial orbital gyrus;lInfTemGy: left inferior temporal gyrus; rMidTemGy: right middle temporal gyrus.

Groups　n　 Volume of lMedOrbGy　 Volume of lInfTemGy　 Volume of rMidTemGy means±SD　95% CI　means±SD　95% CI　means±SD　95% CI NCs　65　4.40±0.57　4.08-4.64　10.00±1.00　9.39-10.59　14.14±1.97　13.09-14.76 bulbar-onset ALS　15　4.15±0.51　3.91-4.47　8.94±1.17　8.35-9.55　13.49±1.46　12.87-14.54 F value　0.70　6.19　0.14 P value*　0.41　0.02　0.71 Groups　n　 TGMV　 TIV　 fGMV means±SD　95% CI　means±SD　95% CI　means±SD　95% CI NCs　65　610.75±56.74　589.83-617.29　1423.72±127.38　1385.11-1453.02　0.43±0.02　0.42-0.43 bulbar-onset ALS　15　577.18±65.12　554.32-614.58　1366.53±133.84　1309.04-1441.14　0.42±0.02　0.41-0.44 F value　0.07　0　0.32 P value*　0.80　0.98　0.58

Table 3. Brain regions with decreased GM volume in patients with ALS-spinal compared with NCs

MNI: Montreal Neurological Institute; X, Y, Z: coordinates of the primary maximum of the cluster. (T＞3.18, Puncorr value＜0.001, expected 122 voxels per cluster)

Anatomic region MNI-space　K value　P value　T value X Y Z Left Thalamus Proper　-21　-21　12　928　0.000　5.58 Right Precentral Gyrus　48　-9　57　2753　0.000　5.46 Left Superior Frontal Gyrus　-18　54　6　4582　0.000　5.35 Left Postcentral Gyrus　-54　-8　33　4032　0.000　5.15 Right Middle Frontal Gyrus　42　53　9　1015　0.000　4.88 Left Central Operculum　-54　2　3　633　0.000　4.27 Right Superior Frontal Gyrus　23　54　21　351　0.000　4.24 Right Middle Occipital Gyrus　32　-75　41　215　0.000　4.15 Left Supramarginal Gyrus　-45　-48　24　141　0.000　4.14 Left Precuneus　-5　-65　27　1157　0.000　4.06 Left Middle Occipital Gyrus　-29　-80　15　293　0.000　4.00 Left Medial Precentral Gyrus　0　-18　75　220　0.000　3.97 Right Superior Frontal Gyrus　26　33　47　123　0.000　3.96 Right Middle Temporal Gyrus　60　-20　-8　274　0.000　3.95 Right Angular Gyrus　57　-48　18　166　0.000　3.87 Left Angular Gyrus　-48　-60　35　153　0.000　3.78 Left Inferior Frontal Angular Gyrus　-48　26　6　130　0.000　3.74 Left Fusiform Gyrus　-32　-33　-29　177　0.000　3.70 Left Superior Frontal Gyrus　-17　39　48　300　0.000　3.69 Left Superior Parietal Lobule　-26　-54　56　176　0.000　3.67

Figure 3. Brain regions with decreased GM volume in ALS-spinal patients compared with NC group. Multiple colored areas represent the involved brain regions, including bilateral precentral gyrus (right precentral gyrus dominance) and the other multiple brain regions. The value of the color bar represents T value.

Compared with ALS-bulbar, the ALS-spinal presented decreased GM volume in the left postcentral gyrus［MNI coordinate (x, y, z): -65, -5, 29; K value: 335; T value: 4.32; P value: 0.000］; meanwhile, the ALS-bulbar presented decreased GM volume in the left middle temporal gyrus (MNI coordinate (x, y, z): -59, -29, -18;K value: 281; T value: 4.07; P value: 0.000) compared with ALS-spinal (Fig. 4).

Correlation analysis between regional volume changes and the clinical features

In ALS patients, the volume of rPrcGy was positively correlated with ALSFRS (r=0.299, P=0.017). In ALS-bulbar patients, volume of the left medial orbital gyrus, left inferior temporal gyrus, right middle temporal gyrus was not found to correlate with any of clinical variables (disease duration, ALSFRS or Norris score).In ALS-spinal patients, there was a significant negative correlation between the volume of the lThaPro and the disease duration (r=-0.383, P=0.007), and no correlation was found between the volume of lThaPro and ALSFRS, or Norris score.

DISCUSSION

Figure 4. Brain regions with altered volume of GM in the ALS-spinal and ALS-bulbar. Hot cluster in yellow, located in the left postcentral gyrus, represented the decreased volume in ALS-spinal compared with ALS-bulbar; cold cluster in green, located in the left middle temporal gyrus,represented decreased volume in ALS-bulbar compared with ALS-spinal.

In patients with spinal-onset ALS, our study demonstrated that motor and extra-motor brain regions were both involved compared with NCs. The involved motor brain regions included the rPreGy (2753 voxels) and left medial precentral gyrus (220 voxels),which was consistent with previous studies,4,22-25 and could underlie the pathological characteristics26-27 and MRS findings28 in ALS. However, some studies reported no atrophy of motor cortex,5,10,29 and some study found the metabolic changes took place in motor cortex of ALS patients, with no concomitant anatomical change.30 The reasons for the inconsistence might be associated with heterogeneity course of pathologic process and the different clinical features, such as initial onset of the ALS patients in this study. The result of this study indicated that motor cortex atrophy was the MRI signature of spinal-onset ALS.

VBM was performed using two-sample t test with TIV,age and sex as covariates by the factorial design specification tools of SPM. Significance was set if a Puncorr value＜0.001. The cluster size was listed as K value,and the difference of the cluster between two groups was represented by T value. The minimal number of contiguous voxels was set based on the expected voxels per cluster.

However, the analysis of the whole brain region involvement for the bulbar-onset ALS patients showed that only left inferior temporal gyrus was significantly different from the controls. This finding suggested that the left inferior temporal gyrus was the key damaged brain region in bulbar-onset ALS patients.

在实验确定的最佳色谱条件下，选取1#果酒样品，分别加入10，50，100 mg/L标准混合溶液，平行进行6次实验，实验结果见表3。回收率为81.6%～102.8%，相对平均偏差不大于4.4%，说明方法精密度高，准确度好。

This study demonstrated that the rPreGy presented volume loss in the ALS patients. This result was consistent with the previous studies,11-13 which suggested that the rPreGy atrophy may be a MRI structural signature for ALS, disregarding its clinical type. VBM analysis demonstrated that atrophy of rPreGy presented only in ALS-spinal patients, but not in ALS-bulbar patients. Therefore, VBM technique could elucidate the different nuromechanism for the different ALS subtypes, and provide a valuable method to understand the structure changes of motor cortex in ALS.The finding of rPreGy atrophy on voxel-level might be explained by the decreased number and shape of cortical neurons19-20 from the pathological viewpoints.

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Extra-motor GM involvement was also demonstrated in this study, which could be considered as a substantial feature of ALS. The extra-motor GM damage involved bilateral frontal cortex, right temporal cortex, as well as the other brain region where GM volume loss. Bilateral superior frontal gyrus presented significant GM loss, indicating that these brain regions may play a critical role in motor response inhibition31 and recovery of language function32 in ALS patients.Since left inferior frontal gyrus also plays a critical role in integrating individual word meanings to coherent sentence-level messages33 and language production,34 the volume loss of frontal gray matter we found in this study may help to understand the verbal fluency deficits of ALS.35-37

Regional atrophy of temporal lobe mainly located in the right middle temporal gyrus, which was partially consistent with previous studies.10,24 The asymmetric atrophy in temporal lobe may be associated with heterogeneous pathological process and the cognitive state.

In this study we found that GM deficits distributed widely in the whole brain of spinal-onset ALS compared with bulbar-onset ALS. In clinical practice, the bulbar-onset ALS is less common than spinal-onset ALS;38 however, 48% of ALS patients were identified with cognitive impairment,39 and the abnormalities indicated fronto-temporal involvement. Our results suggest that widespread GM loss of extra-motor cortex such as frontal lobe and temporal lobe exists in spinal-onset ALS patients compared with bulbar-onset ALS patients.The extensive atrophy of extra-motor cortex also suggests that more upper motor neurons may be involved in spinal-onset ALS than bulbar-onset ALS patients.These findings may also reflect the heterogeneity of clinical onsets in ALS patients, and provide useful information to better understand the cognitive impairment in ALS.

VBM analysis demonstrated the differences existed between bulbar- and spinal-onset ALS. The volume of left postcentral gyrus in spinal-onset ALS and left middle temporal gyrus in bulbar-onset ALS were significantly different compared to each other. This finding indicates the intrinsic difference may exist between the clinical sub-types of ALS, and its clinical significance should be investigated in future.

The limits of this study included: (1) The samples presented heterogeneity, and the bulbar-onset ALS sample was relatively small; (2) Although MMSE was performed to exclude dementia, the mild cognitive impairment was not evaluated in detail.

To conclude, these findings supported that the atrophy of rPreGy might be used as a feasible imaging biomarker for diagnosis of ALS. Extra-motor GM atrophy (fronto-temporal pattern) is a MRI structural signature in bulbar-onset ALS patients. Motor and extra-motor (fronto-temporal and extra-fronto-temporal pattern) GM atrophy were structural signatures on MRI in spinal-onset ALS patients. VBM is a sensitive and effective tool for brain structure evaluation in patients with ALS.

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