This study found disrupted ACC functional connectivity in cirrhotic patients without overt HE which further deteriorated with the increasing severity of HE. Disrupted functional connectivity in the ACC in cirrhotic patients correlated with cognitive dysfunctions measured by NCT-A and DST. To the best of our knowledge, this is the first report on disrupted ACC functional connectivity in cirrhotic patients without overt HE mapped by resting state fMRI.
In this study, the ACC functional connectivity patterns mapped in healthy controls, non-HE patients, and MHE patients were in line with those reported in the previous study 
. The ACC functional connectivity positively correlated with the DMN, AN, and brain regions that process different aspects of emotionally salient stimuli, the thalamus, caudate, putamen and all other cingulate subregions. The ACC functional connectivity negatively correlated with the SMN, CN, VN. All subjects had the similar functional connectivity patterns of the ACC, however, cirrhotic patients had decreased tendency of functional connectivity of the ACC. This suggests that our findings on the ACC functional connectivity are reliable.
In this study, we found the ACC functional connectivity was predominantly decreased from non-HE to MHE, especially in the right MCC, bilateral STG/MTG, and right putamen. The MCC is activated during a variety of cognitive tasks including conflict monitoring, error detection, response selection, and attention control 
. FMRI studies showed the MCC positively connected with the DLPFC which showed decreased functional connectivity in this study and our previous DMN study 
. The anatomic studies demonstrated the reciprocal connections between these two brain regions 
. The MCC also negatively correlated with NCT-A scores. NCT-A test for psychomotor speed 
has been considered as the most sensitive diagnostic test for the detection of MHE. This indicated that MCC functional connectivity abnormality could reflect neurocognitive deficits in MHE patients. We found that bilateral STG/MTG functional connectivities were significantly decreased in MHE patients compared with non-HE patients. Recently, Montoliu et al. 
found a focal thinning of STG in MHE patients compared with non-HE patients and controls using a cortical-based analysis technique. STG contains the primary auditory cortex, which is involved in auditory processing, including language, but also has been implicated as a critical structure in social cognition 
. Previous studies showed that cognitive auditory components with neural sources out of the primary auditory cortex were helpful for diagnosis of MHE 
. Interestingly, the STG functional connectivity was increased in non-HE patients compared with controls, while it was decreased in MHE patients compared with non-HE patients and controls, indicating STG can be a specific brain region in the development of MHE. The non-HE patients may require more functional connectivity of STG to maintain cognitive balance, but it was further decreased with the increasing severity of HE. The negative correlation of STG functional connectivity with NCT-A scores also supported the role of STG in cognitive dysfunction in cirrhotic patients. However, the precise role of STG in the diagnosis of MHE remains unclear, and further investigation is needed in the future. We observed that DST scores positively correlated with bilateral ACC and right putamen. The putamen is one of the structures that comprise the basal ganglia, which is connected to the substantia nigra and globus pallidus through various pathways. The main function of the putamen is to regulate movements and influence various types of learning 
. In addition, the thalamus and insular functional connectivity were decreased in non-HE and MHE patients compared with controls. The disrupted functional connectivity of these cortical and subcortical networks, including the insula, thalamus and putamen may have an adverse effect on cognitive function in MHE patients. The above-mentioned brain areas are composed of anterior attention system proposed by Posner and Peterson 
; these decreased functional connectivities of the above-mentioned brain areas can better interpret neurocognitive abnormality in MHE patients.
Although no difference was found between MHE and non-HE patients in cirrhotic patients compared with healthy controls, we also observed increased negative functional connectivity between the ACC seed and MOG, lingual gyrus and temporo-occipital junction, and the components of posterior attention system. This latter observation indicates that the increased negative functional connectivity between the ACC seed and these brain areas is not specific for the development of MHE. Our findings were supported by one previous behavioral study which concluded that the anterior attention system is more sensitive than the posterior attention system to the early stages of hepatic encephalopathy 
. The above-mentioned brain areas composing of visual spatial information processing network are involved in attention information adjustment and orienting 
. It can also be interpreted as a compensatory mechanism for abnormal anterior attention system in MHE patients because the posterior attention system is thought to operate upon the ventral pathway during tasks requiring detailed processing of objects 
. However, further investigations are needed for the role of increased negative functional connectivity between the ACC seed and MOG, lingual gyrus, and temporo-occipital cortex in cirrhotic patients in the development of OHE.
Our study has several limitations. First, our study is confined to analyze the pregenual ACC rather than all subregions of the ACC or whole brain functional network in cirrhotic patients without overt HE. The effects of HE may be more global. Other additional networks or whole brain network should be performed in future. For example, we observed functional connectivity of DMN was impaired in cirrhotic patients 
. Second, although we did observe the functional connectivity changes of the ACC during the development of MHE, the functional connectivity changes of the ACC in overt HE was not further investigated because of severity of overt HE. Third, we choose bilateral pregenual ACC as a seed region rather than left or right subregions of ACC in this study because one previous study demonstrated that the subregions of the ACC had similar functional connectivity. Additionally, side difference of the ACC in cirrhotic patients did not perform although one previous study demonstrated no asymmetry was observed in healthy controls 
. Fourth, our study results could not exclude the interference of structural changes in the ACC. Future study linking structural and functional networks of the ACC is warranted to confirm the finding of this study.
Notwithstanding these limitations, our preliminary experimental study demonstrates that disrupted ACC functional connectivity existed in cirrhotic patients without overt HE. This further deteriorated with the increasing severity of HE and correlated cognitive dysfunction in cirrhotic patients. The disrupted ACC functional connectivity in cirrhotic patients appears imbalance of predominately decreased functional connectivity of anterior attention system and posterior attention system. This study shed on light for understanding neurocognitive basis of attention deficits in cirrhotic patients.