Regional homogeneity abnormalities of resting state brain activities in children with growth hormone deficiency

Growth hormone deficiency (GHD) is a common developmental disorder in children characterized by low levels of growth hormone secretion, short stature, and multiple cognitive and behavioral problems, including hyperactivity, anxiety, and depression. However, the pathophysiology of this disorder remains unclear. In order to investigate abnormalities of brain functioning in children with GHD, we preformed functional magnetic resonance imaging and regional homogeneity (ReHo) analysis in 26 children with GHD and 15 age- and sex-matched healthy controls (HCs) in a resting state. Compared with HCs, children with GHD exhibited increased ReHo in the left putamen and decreased ReHo in the right precentral gyrus, reflecting a dysfunction of inhibitory control. Decreased ReHo was also identified in the orbital parts of the bilateral superior frontal gyrus and the medial part of the left superior frontal gyrus, a finding that correlated with the inappropriate anxiety and depression that are observed in this patient population. Our results provide imaging evidence of potential pathophysiologic mechanisms for the cognitive and behavioral abnormalities of children with GHD.

Group comparison results. The two-sample t-tests results were graphically displayed in Fig. 3. The statistical significance threshold was set at p < 0.05, corrected by AlphaSim program (combined a voxel threshold of p < 0.05 and a minimum cluster size of 56 voxels) 24 . There were several regions showing significant differences in ReHo between the children with GHD and HCs ( Table 1). The left PUT showed increased ReHo, while the right PreCG, bilateral orbital parts of the superior frontal gyrus (ORBsup) and left medial part of the superior frontal gyrus (SFGmed) showed decreased ReHo in children with GHD.

Discussion
To the best of our knowledge, this is the first study to analyze the regional homogeneity of brain activity in children with GHD in a resting state. In the current study, we performed rs-fMRI and ReHo analysis in children with GHD and HCs. Compared to HCs, children with GHD had increased ReHo in the left PUT, and decreased ReHo in the right PreCG, bilateral ORBsup and left SFGmed. These findings indicate that children with GHD have altered brain activity compared to HCs.
Compared with the HCs, the GHD group showed significantly increased ReHo in the left PUT. It has been suggested that this brain region plays a vital role in inhibitory control 25 . Several studies have found that the PUT is more active in more impulsive subjects than in less impulsive subjects [26][27][28][29] . Furthermore, in recent fMRI studies, motivated behavior has been shown to be accompanied by a weakened cognitive control of impulses 30 . Consistent with a prior study on GHD 16 , we also found significantly decreased ReHo in the right PreCG in Figure 1. The one-sample t-test results of the healthy control group shown as a Kendall's coefficient concordance (KCC) map (false discovery rate [FDR] corrected, p < 0.05). The figure was presented using the free DPABI software. Red areas indicate a positive ReHo. The number above each image refer to the z-plane coordinates of the Montreal Neurological Institute space. ReHo regional homogeneity, L left, R right. children with GHD. The PreCG is located in the premotor cortex and is associated with the somatic sensorimotor network [31][32][33] . Rech et al. found that the white matter underneath the premotor cortex could immediately elicit a unilateral negative motor response (UNMR) when stimulated 34 . Meanwhile, Schucht et al. suggested that the neural network causing UNMRs may be related to motor control 35 . Thus, we speculate that the GHD group's increased ReHo in the left PUT and decreased ReHo in the right PreCG may be associated with motor control dysfunction, which may reflect the pathophysiological mechanism of impulsive and hyperactive characteristics of behavior in children with GHD 5,36,37 .
In the current study, we also observed decreased ReHo in children with GHD in bilateral ORBsup, a region that is located in the medial orbitofrontal cortex (mOFC). The ORBsup has been shown to play a vital role in choosing the right response and suppressing the wrong response 38,39 . In a previous study, patients with  . Statistically significant differences in regional homogeneity (ReHo) between children with GHD and healthy controls (p < 0.05, AlphaSim corrected). The figure was presented using the free DPABI software. Cold and hot colors in the color bar indicate decreased and increased ReHo, respectively, in children with GHD. The number above each image refer to the z-plane coordinates of the Montreal Neurological Institute space. GHD growth hormone deficiency, L left, R right. www.nature.com/scientificreports/ ORBsup lesions were found to have behavioral regulation failures in the absence of external stimuli 38 . Decreased mOFC activity can be detected when patients with anxiety and depression fail to inhibit inappropriate anxiety responses [40][41][42] . In addition, decreased ReHo was also observed in the SFGmed, located in the medial prefrontal cortex (MPFC). The MPFC is one of the most important functional areas in the default mode network 43 , and is considered to participate in emotional processing, such as monitoring anxiety and other psychological states [44][45][46][47] .
The significantly decreased ReHo in the MPFC of anxiety patients was linked to a decrease in the ability to regulate emotion 13 . In the present study, the decreased ReHo of the ORBsup and SFGmed in the GHD group may be associated with inactivation of emotional regulation, which may underlie the anxiety and depression observed in children with GHD 5,36,37,41 . Several limitations of this study need to be addressed. Firstly, the number of subjects included in the study was small, especially in the HC group. Therefore, the generalizability of this study to the whole GHD population is limited. However, since both gender and age were matched between the two groups, we believe this constraint did not affect the robustness of our results. Secondly, participants in this study were not assessed by clinical cognitive and behavioral scales. It is valuable to evaluate the relationship of neuroimaging results with clinical scales. Therefore, in future studies, assessments of relevant scales should be included.

Methods
Participants and ethical considerations. All subjects were recruited from the pediatric clinic of the Zhejiang Provincial People's Hospital from October 2016 to March 2018. All participants' parents gave written informed consent for their inclusion in the study, and this study was approved by the Medical Ethics Committee of Zhejiang Provincial People's Hospital and adhered to the tenets of the Declaration of Helsinki. All subjects were right-handed and prepubescent. Participants were excluded if they met the following criteria: (1) head movements were obviously too large during MRI scans; (2) abnormalities identified on MRI associated with GHD; (3) had congenital multiple pituitary hormone deficiency (MPHD) or thyroid axis hormone level abnormalities; (4) chronic liver, kidney, or skeletal system diseases; (5) chromosomal abnormalities or special facial/ abnormal signs with diagnostic significance; (6) hyperthermia during the scanning; (7) past medical history of seizures, benign masses or cancers, or psychological disease/psychosis; (8) family history of mental retardation or congenital heart disease, or (9) contraindications to MRI (such as metal implants or claustrophobia).
Growth hormone (GH) stimulation tests were performed in participants with short stature. Because of the limitations of GH stimulation tests, GHD can only be diagnosed when the results of two or more drug stimulation tests are abnormal 1,48 . Seven children with short stature were excluded due to their peak GH value, which was higher than 10 μg/L, during the GH stimulation test. Accordingly, the first group was composed of 26 children with GHD (11 males, mean age: 8.2 ± 1.9 years), in whom both arginine and clonidine GH stimulation tests showed a partial or complete lack of GH (< l0 μg/L) 1 . Furthermore, 15 age-and sex-matched HCs formed the second group (7 males, mean age: 8.6 ± 1.7 years), who had normal height and normal growth variation. There were no statistically significant differences in age (p = 0.3823, two-tailed Mann-Whitney U test) or sex (p > 0.9999, two-tailed Fisher's exact test) between the two groups.
Imaging data acquisition. fMRI scans were performed with a 3.0-T MRI scanner (Discovery MR750; GE Healthcare, Milwaukee, WI) prior to GH stimulation tests in all participants during the resting state to avoid the effects of stimulating drugs. The subjects were instructed to lie as still as possible but not fall asleep. Two foam pads were used on the head to reduce head movement, and earplugs were worn to reduce external noise. The MRI equipment and data parameters used in all MR imaging examinations were consistent with our previous study 49 . The specific parameter settings were as follows: repetition time = 2000 ms, echo time = 30 ms, flip angle = 90°, field of view = 220 mm, matrix size = 64 × 64, voxel size = 3.44 × 3.44 × 3.2 mm 3 , no slice gap and 44 axial slices. The first 10 volumes of all acquired rs-fMRI data image sequences were excluded in order to ensure that the subjects were stable in the resting state. Table 1. Regions of increased/decreased ReHo in children with GHD. ReHo regional homogeneity, GHD growth hormone deficiency, MNI (x,y,z) coordinates of the peak voxel in the MNI space, t the statistical value of peak voxel, with a positive t-value indicating an increased ReHo and a negative t-value indicating a decreased ReHo in patients with GHD, BA Brodmann's area, L left, R right, PUT putamen, PreCG precentral gyrus, ORBsup superior frontal gyrus, orbital part, ORBsupmed superior frontal gyrus, medial orbital, SFGmed superior frontal gyrus, medial. www.nature.com/scientificreports/ Data preprocessing. The rs-fMRI data processing was performed using DPABI software (Version V4.0_190305, http://rfmri .org/dpabi ) 50 , followed by temporal difference and head-motion corrections on the remaining 200 volumes. No participants were excluded for translation or rotation parameters exceeding 3.0 mm or 3.0° of head motion. Previous studies have reported that rs-fMRI findings is sensitive to head motion effects 51,52 . Here, we also computed the frame-wise displacement (FD) index 51 , and found no significant differences (p = 0.7175) in head movement between children with GHD (0.0936 ± 0.0632) and HCs (0.1009 ± 0.0584) using two-sample t-tests. The echo-planar imaging (EPI) template was used to spatially normalize the image into the Montreal Neurological Institute (MNI) space and resampled to 3 × 3 × 3 mm 3 voxels. Subsequently, to remove the effects of head motion and other possible sources of artifacts, we regressed the constant, linear trend, and head motion parameters (Friston 24-parameter model) 53 as covariates 54 . The time series signals of the cerebrospinal fluid and white matter were eliminated by using the prior template in SPM8 software (Statistical Parametric Mapping 8, http://www.fil.ion.ucl.ac.uk/spm/). Finally, a band-pass filter (0.01 < f < 0.08 Hz) was used to remove the effects of noise, such as respiratory rhythm.

ReHo analysis.
For the preprocessed rs-fMRI data, the time series of specific voxels were extracted. We defined the 27 nearest voxels as a cluster and calculated their ReHo. The Kendall's coefficient concordance (KCC) values measure the consistency of activities between adjacent voxels and are assigned to a given voxel 17 , according to the following Eq. (1): where W was the KCC among given voxels, ranging from 0 to 1; R i was the sum rank of the ith time point; R = ((n + 1)K)/2 was the mean of the R i ' s; K was the number of time series within a measured cluster (here K = 27); and n was the number of ranks (here n = 200). The individual KCC map was generated with the free DPABI software (Version V4.0_190305, http://rfmri .org/dpabi ) 50 . Finally, spatial smoothing was performed in KCC maps with a full width at half maximum (FWHM) of 4 mm to decrease spatial noise.

Statistical analysis.
Second-level analyses of KCC maps were carried out using the SPM 8 software package. The brain regions with a KCC value greater than 1 were detected by performing one-sample t-tests within the two groups. A mask was used, according to the automated anatomical labeling (AAL) templates 55 , which matched the size of the spatially normalized voxels (3 × 3 × 3 mm 3 ). The multiple comparisons were performed using the false discovery rate (FDR) criterion (p < 0.05) 56 .
The one-sample t-tests results from the two groups were combined to get a new map. By binarizing the map, a combined explicit mask was obtained. Then, two-sample t-tests were performed within this mask to compare the ReHo between two groups. The results of the two-sample t-tests were corrected by using the AlphaSim program in the RESTplus software (Resting-State fMRI Data Analysis Toolkit plus V1.24, http://www.restf mri.net/forum / RESTp lus) 24 , as determined by a Monte Carlo simulation to calculate the probability of false-positive detection 13 . The AlphaSim correction was confined within the combined mask of one-sample t-tests results from the two groups, and the size of smooth kernel was set at 4 mm. The threshold of the t-map was set at a combined height threshold of p < 0.05 and a minimum cluster size of 56 voxels, and this corresponded to a corrected p < 0.05.

Conclusions
In the current study, we performed fMRI and ReHo calculations in children with GHD and HCs in the resting state. Our findings of increased ReHo in the left PUT and decreased ReHo in the right PreCG may suggest a dysfunction of inhibitory control in children with GHD. In addition, the decreased ReHo was observed in the bilateral ORBsup and left SFGmed, reflecting a possible inactivation of the inhibition of inappropriate anxiety and depression in children with GHD. In summary, the present findings confirmed that the activity homogeneity in specific brain regions is altered in children with GHD, providing imaging evidence to elucidate potential pathophysiological mechanisms of abnormal cognition and behavior in children with GHD.