Human Brain Mapping
Functio nal integrity in children with anoxic brain injury from drowning
Journal:For Human Peer Brain Mapping Review Manuscript ID HBM-16-0612.R3
Wiley - Manuscript type: Research Article
Date Submitted by the Author: 10-Jul-2017
Complete List of Authors: Ishaque, Mariam; UT Health Science Center San Antonio, Research Imaging Institute Manning, Janessa; Wayne State University Woolsey, Mary; UT Health Science Center San Antonio, Research Imaging Institute Franklin, Crystal; UT Health Science Center San Antonio, Research Imaging Institute Tullis, Elizabeth; UT Health Science Center San Antonio, Research Imaging Institute Beckmann, Christian; Donders Centre for Cognitive Neuroimaging, Fox, Peter; University of Texas Health Science Center, Research Imaging Institute; South Texas Veterans Health Care System; Shenzhen University
anoxic brain injury, hypoxic-ischemic encephalopathy, neural networks, Keywords: functional magnetic resonance imaging, independent components analysis, resting-state, fMRI, locked-in syndrome, minimally conscious state, ICA
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1 1 2 Functional integrity in children with anoxic brain injury from drowning 3 4 Mariam Ishaque 1, 2 *, Janessa H. Manning 3, Mary D. Woolsey 1, Crystal G. Franklin 1, Elizabeth W. 5 4 5,6,7 1,2,8,9 6 Tullis , Christian F. Beckmann , Peter T. Fox * 7 8 9 1. Research Imaging Institute, University of Texas Health Science Center at 10 San Antonio, San Antonio, Texas, USA 11 12 2. Department of Radiology, University of Texas Health Science Center at 13 San Antonio, San Antonio, Texas, USA 14 3. Merrill Palmer Skillman Institute, Wayne State University, Detroit, Michigan, USA 15 4. Conrad Smiles Fund 16 5. Department of Cognitive Neuroscience, Radboud University Medical Center, Donders Institute for 17 Brain, Cognition and Behaviour, Nijmegen, The Netherlands 18 6. Donders InstituteFor for Brain, CognitionPeer and Behaviou Reviewr, Donders Center for Cognitive Neuroimaging, 19 Radboud University, Nijmegen, The Netherlands 20 7. Centre for Functional MRI of the Brain, University of Oxford, Oxford, United Kingdom 21 8. South Texas Veterans Healthcare System, San Antonio, Texas, USA 22 9. Shenzhen University School of Medicine, Shenzhen, People's Republic of China 23 24 25 26 *Corresponding Authors : Mariam Ishaque, Peter T. Fox 27 Address : 7703 Floyd Curl Drive, San Antonio, TX 78229 28 29 Email : [email protected]; [email protected] 30 31 Phone : (469) 441 7065; (210) 567 8150 32 33 Fax : (210) 567 8103 34 35 36 Running Title : Functional integrity with pediatric drowning 37 38 Keywords : anoxic brain injury, locked in syndrome, minimally conscious state, functional magnetic 39 40 resonance imaging, hypoxic ischemic encephalopathy, independent components analysis, neural 41 42 networks, resting state, fMRI, rs fMRI, rs fcMRI 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 2 of 51
2 1 2 ABSTRACT 3 4 5 Drowning is a leading cause of accidental injury and death in young children. Anoxic brain injury (ABI) 6 7 is a common consequence of drowning and can cause severe neurological morbidity in survivors. 8 9 Assessment of functional status and prognostication in drowning victims can be extremely challenging, 10 11 12 both acutely and chronically. Structural neuroimaging modalities (CT and MRI) have been of limited 13 14 clinical value. Here, we tested the utility of resting state functional MRI (rs fMRI) for assessing brain 15 16 functional integrity in this population. Eleven children with chronic, spastic quadriplegia due to 17 18 For Peer Review 19 drowning induced ABI were investigated. All were comatose immediately after the injury and gradually 20 21 regained consciousness, but with varying ability to communicate their cognitive state. Eleven 22 23 24 neurotypical children matched for age and gender formed the control group. Resting state fMRI and co 25 26 registered T1 weighted anatomical MRI were acquired at night during drug aided sleep. Network 27 28 integrity was quantified by independent components analysis (ICA), at both group and per subject 29 30 31 levels. Functional status assessments based on in home observations were provided by families and 32 33 caregivers. Motor ICNs were grossly compromised in ABI patients both group wise and individually, 34 35 concordant with their prominent motor deficits. Striking preservations of perceptual and cognitive ICNs 36 37 38 were observed, and the degree of network preservation correlated (ρ = 0.74) with the per subject 39 40 functional status assessments. Collectively, our findings indicate that rs fMRI has promise for assessing 41 42 brain functional integrity in ABI and, potentially, in other disorders. Further, our observations suggest 43 44 45 that the severe motor deficits observed in this population can mask relatively intact perceptual and 46 47 cognitive capabilities. 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 3 of 51 Human Brain Mapping
3 1 2 INTRODUCTION 3 4 5 Drowning is the second most prevalent cause of unintentional injury death in children 1 to 4 years of age. 6 7 Nonfatal drowning (i.e., cardiopulmonary resuscitation is successful) is prevalent in this age group, with 8 9 an estimated 2 out of 3 drowned children surviving [Borse and Sleet, 2009; Kriel et al., 1994; Topjian et 10 11 12 al., 2012]. Neurological morbidity from anoxic brain injury (ABI) is a frequent outcome, as the brain is 13 14 exquisitely sensitive to oxygen deprivation [Topjian et al. 2012]. 15 16 17 18 For Peer Review 19 Functional assessment and prognostication are extremely challenging in this disorder, both acutely and 20 21 chronically. In the acute state, victims are typically comatose and may remain unresponsive for weeks. 22 23 24 Acutely, T1 and T2 weighted MRI are often normal or suggest diffuse swelling; chronically, diffuse 25 26 atrophy is the most frequently reported finding [Rafaat et al., 2008; Rabinsten and Resnick, 2009]. 27 28 When focal pathology is present, basal ganglia and cerebellar damage are the most commonly reported 29 30 31 and both are associated with poor outcomes [Rabinsten and Resnick, 2009]. In the chronic state, spastic 32 33 quadriparesis resembling severe cerebral palsy is the most common presentation. This entails loss of 34 35 self mobility, self feeding and verbal communication, with various movement disorders being reported 36 37 38 [Lu Emerson and Khot, 2010]. Children with anoxic brain damage are frequently deemed to be in a 39 40 minimally conscious or vegetative state, but these determinations may well be flawed, as 41 42 communication and task cooperation limitations often preclude reliable assessment of cognitive status 43 44 45 [Ibsen and Koch, 2002; Topjian et al., 2012; Childs et al., 1993; Levy et al., 1985]. 46 47 48 49 50 The neuropathology of pediatric, non fatal ABI is not well established. Post drowning anoxic injury has 51 52 been described as diffusely affecting grey matter more than white matter, reflecting the respective 53 54 metabolic demands of the two tissue types [Huang and Castillo, 2008; Rabinstein and Resnick, 2009]. 55 56 57 However, the possibility of a more selective injury must be considered in view of the predominant 58 59 motor system disability observed in survivors. Until recently, structural imaging methods had 60 John Wiley & Sons, Inc. Human Brain Mapping Page 4 of 51
4 1 2 contributed little to our understanding of the disorder. The limited utility of these methods stemmed 3 4 chiefly from the use of standard, clinical acquisition protocols which were not optimized for this 5 6 7 condition (the neuropathology of was unknown), compounded by a reliance on visual inspection for 8 9 image interpretation (i.e., on non quantitative methods) [Gutierrez et al., 2010; Howard et al., 2011; 10 11 Huang and Castillo, 2008; Topjian et al., 2012]. In the same patient cohort reported here, our group 12 13 14 recently reported quantitative analyses of two structural imaging modalities. Voxel based morphometry 15 16 (VBM) was applied to T1 weighted MRI to independently quantify grey and white matter tissue loss 17 18 [Ishaque et al., 2016]. GreyFor matter loss Peer was largely restrictedReview to the basal ganglia and thalamus; white 19 20 21 matter loss predominately affected the posterior limb of the internal capsule (PLIC). Tract based spatial 22 23 statistics (TBSS) was applied to diffusion weighted MRI to quantify fractional anisotropy and mean 24 25 26 diffusivity [Ishaque et al., 2017]. TBSS independently confirmed our VBM white matter findings, also 27 28 showing highly focal, deep subcortical lesions. Further, per subject motor function scores correlated 29 30 highly with both fractional anisotropy (Spearman’s ρ = 0.8) and mean diffusivity (ρ = 0.83) in the PLIC. 31 32 33 For both modalities, the lesion was largely confined to the lenticulostriate vascular distribution, 34 35 suggesting that this end arterial watershed zone may be uniquely susceptible in young children. A 36 37 similar distribution has been reported in perinatal asphyxia using diffusion tensor imaging (DTI) 38 39 40 [Barkovich et al., 2001] and magnetic resonance spectroscopy [Pu et al., 2000], lending further credence 41 42 to this hypothesis. Descending corticospinal and corticobulbar fibers (pyramidal tract) pass through the 43 44 PLIC, the cerebral peduncles (midbrain) and anterior pons en route to the pyramidal decussation 45 46 47 (medulla). Collectively, these observations suggest that pediatric drowning victims may be an at risk 48 49 population for a selective de efferentation syndrome, with disproportionate motor impairment and 50 51 52 relatively preserved perceptual and cognitive function. To test this hypothesis, we implemented rs fMRI 53 54 and quantitative behavioral assessments in the same pediatric ABI cohort in the present study. 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 5 of 51 Human Brain Mapping
5 1 2 In pediatric brain injury, traditional, task activation fMRI is extremely difficult to implement reliably 3 4 [Munson et al., 2006]. Resting state fMRI is a task free alternative that has been widely used in adult 5 6 7 populations unable to comply with in scanner tasks. Resting state fMRI, however, is notoriously 8 9 susceptible to movement artifacts [Power et al. 2014], which poses a substantial obstacle for pediatric 10 11 studies, in general, and for pediatric brain injury, in particular. Sleep has been successfully used in 12 13 14 pediatric populations to achieve in scanner immobility for anatomical MRI [Giedd et al., 1999], task 15 16 activation fMRI (using auditory stimuli) [Anderson et al., 2001; Redcay and Courchesne, 2008], and 17 18 during resting state fMRI For[Manning, CourchesnePeer and Review Fox, 2013]. In prior work from our laboratory, we 19 20 21 demonstrated that intrinsic connectivity networks (ICN's) persist during natural, nocturnal sleep in pre 22 23 school aged neurotypical children [Manning, Courchesne and Fox, 2013]. In that study, we applied 24 25 26 independent components analysis (ICA), a whole brain (i.e., not region of interest based), voxel wise, 27 28 data driven procedure that extracts functionally connected networks by temporal coherence of 29 30 spontaneous fluctuations in T2* signal [Beckmann and Smith, 2004; Beckmann et al., 2005]. ICN's are 31 32 33 known to persist during natural sleep [Fukunaga et al., 2006; Horovitz et al., 2008; Manning Courchesne 34 35 and Fox, 2013] and sedation [Peltier et al., 2005; Vincent et al., 2007]. Importantly, the canonical, 36 37 discrete ICNs extracted with ICA [Smith et al., 2009] correlate to more or less discrete behavioral 38 39 40 domains and task activation paradigms [Laird et al., 2009; Laird et al., 2011] facilitating correlations 41 42 with behavioral assessments. ICNs also predict individual differences in task activation fMRI effects 43 44 [Tavor et al., 2016], making them a suitable alternative in populations not able to comply with task 45 46 47 activation protocols. An acquisition, analysis and interpretation strategy combining the approaches of 48 49 Manning, Smith and Laird was applied here. 50 51 52 53 54 Quantitative behavioral assessments were obtained via structured interviews with families and 55 56 caregivers using Likert style questionnaires developed for this population and designed to correspond to 57 58 59 canonical ICA derived ICNs. Caregiver assessments were used because: 1) families have knowledge of 60 John Wiley & Sons, Inc. Human Brain Mapping Page 6 of 51
6 1 2 the patients’ premorbid personalities, habits and preferences which could help them recognize coherent 3 4 response patters; 2) families and care staff have extensive opportunities for observation and interaction 5 6 7 with patients; and, 3) caregivers are accessible to the research team and accepting of the time demands 8 9 involved. Additional motivation for this approach came from evidence that family and care staff are 10 11 most often the first to recognize the return of awareness in locked in syndrome and other disorders of 12 13 14 consciousness (Leon Carrion et al., 2002; Smith & Delargy, 2005; Owen, 2017). 15 16 17 18 For the data reported here,For eleven children Peer with chronic Review post drowning ABI and eleven neurotypical 19 20 21 controls matched for age and gender were imaged during nocturnal, drug aided sleep, acquiring both rs 22 23 fMRI (BOLD) and structural MRI (T1 weighted). ICA was implemented both group wise and per 24 25 26 subject at a dimensionality of 20. From these 20 networks, 11 canonical networks were identified: five 27 28 perceptual networks (Visual x 3, Auditory, Sensorimotor); two motor networks (Basal Ganglia, 29 30 Cerebellum); and, four cognitive networks (Default Mode, Executive Function, left and right 31 32 33 Frontoparietal). We hypothesized that (1) resting state fMRI would demonstrate group level differences 34 35 between these cohorts, that (2) resting state fMRI would demonstrate per patient abnormalities, that (3) 36 37 imaging based results would be concordant with behavioral results, and, (4) that motor network 38 39 40 abnormalities would greatly outweigh perceptual and cognitive network abnormalities. 41 42 43 44 45 MATERIALS AND METHODS 46 47 Participants. 48 49 50 ABI subjects were recruited by means of collaborations with the Nonfatal Drowning Registry 51 52 (nonfataldrowningresigstry.org), the Conrad Smiles Fund (www.conradsmiles.org) and Miracle Flights 53 54 55 (miracleflights.org). Families of drowning victims were made aware of the study via the Nonfatal 56 57 Drowning Registry, to which they had previously provided contact information. Interested families 58 59 initiated contact with the study project coordinator at the University of Texas Health Science Center, 60 John Wiley & Sons, Inc. Page 7 of 51 Human Brain Mapping
7 1 2 San Antonio for screening and enrollment. Ten of the recruited ABI victims resided in North America; 3 4 one ABI victim resided in Europe. The North American participants were widely geographically 5 6 7 dispersed, with most travelling thousands of miles (i.e. from the East and West Coasts) to participate. 8 9 Air travel costs were subsidized by Miracles Flights; lodging costs were subsidized by the Conrad 10 11 Smiles Fund. No other monetary incentives were provided. In all instances, the parents’ primary 12 13 14 motivation for participation was to better understand their child’s neurological and cognitive status. In 15 16 keeping with this expectation, parents were provided with a formal report of their child’s imaging and 17 18 behavioral results at the completionFor ofPeer the study. Available Review funding limited the scope of enrollment to 19 20 21 11 ABI victims and 11 controls. Controls were recruited chiefly from friends and family of patients. The 22 23 study’s protocol was approved by the University of Texas Health Science Center at San Antonio’s 24 25 26 Institutional Review Board, in compliance with the Code of Ethics of the World Medical Association 27 28 (Declaration of Helsinki). Informed consent was obtained from the participants’ parent(s). Per patient 29 30 descriptions are provided as Supplementary Material (SM) for the 10 patients with good quality imaging 31 32 33 data. In the following, we make reference to patient identification numbers provided in the SM. 34 35 36 37 Medical History. Medical history was taken at the time of scan acquisition and supplemented by video 38 39 40 conference structured interviews subsequent to scanning (described below in Quantitative Behavioral 41 42 Assessment). All ABI participants: had accidentally drowned in early childhood (age at injury: 17 48 43 44 months); had been pulseless and apneic at the scene; had been administered cardiopulmonary 45 46 47 resuscitation and cardioversion by emergency medical technicians en route to the hospital; had been 48 49 admitted to the hospital in a comatose state; had required intubation and mechanical ventilation; had 50 51 52 been treated for cerebral swelling (e.g., with steroids or hypothermia); and, had protracted hospital stays. 53 54 In the acute setting, most (7/10) were advised by the medical team to withdraw care but opted not to 55 56 follow this guidance. All transitioned from coma through a minimally conscious state to a state in which 57 58 59 the caregivers believed that the patients were conscious and aware of themselves and their environment. 60 John Wiley & Sons, Inc. Human Brain Mapping Page 8 of 51
8 1 2 At the time of study participation, all were at least 6 months post injury, were not mechanically 3 4 ventilated, and were being cared for in the parents’ home. All had normal sleep wake cycles and were 5 6 7 attentive to their environment. Nine had permanent gastric tubes for feeding, being unable to safely take 8 9 food by mouth. Most had difficulty swallowing saliva and required periodic suctioning. 10 11 12 13 14 [Table I] 15 16 17 18 For Peer Review 19 Neurological Assessment. Neurological examination was performed by a neurologist (PTF), at the 20 21 time of scan acquisition. All ABI participants had severe, spastic quadriplegia with involvement of neck 22 23 24 and facial musculature, indicative of a supranuclear, corticospinal tract lesion. All were awake, alert and 25 26 attentive to the environment but had little (3 patients) or no intelligible speech (7 patients). Using 27 28 published clinical diagnostic criteria [Plum and Posner, 1982; Bauer, Gerstenbrand and Rumpl, 1979; 29 30 31 Laureys, Owens and Schiff, 2004; Bruno et al., 2011] for Locked In Syndrome (LIS) and Minimally 32 33 Conscious State (MCS), these patients were clinically categorized as Classical LIS (2 patients), as 34 35 36 Incomplete LIS (5 patients) and as somewhere on the spectrum of MCS+ to Total LIS (3 patients). Two 37 38 patients (Patients 1 and 6) had Classic LIS, communicating effectively by eye movements. Five patients 39 40 (Patient 2, 3, 7, 8, 9) were clinically categorized as Incomplete LIS. Of these, two (Patients 2 and 9) 41 42 43 were clinically categorized as Incomplete LIS because they had some limb movement and 44 45 communicated by pointing or button pressing. Three were categorized as Incomplete LIS in that they 46 47 have some limited, effortful speech (Patients 3, 7, and 8); of these, two also had some limb movement 48 49 50 (Patients 3 and 7). Even in these Incomplete LIS patients, however, the predominant mode of 51 52 communication was eye gaze (Patients 2, 7, 8, 9) or non speech sounds (Patient 3), reflecting the 53 54 55 severity of their de efferentation. Two patients (Patients 7, 9) made use of eye gaze controlled 56 57 computer interfaces to communicate; one of these (Patient 9) read electronic books via an eye gaze 58 59 controlled system and communicating by spelling. Three patients (Patients 4, 5, and 10) were clinically 60 John Wiley & Sons, Inc. Page 9 of 51 Human Brain Mapping
9 1 2 classified as Total LIS or MCS+, being quadriplegic, aphonic, awake (sustained eye opening) and aware 3 4 of their environment (aware of touch, voice & other sounds, etc.) but being unable to communicate 5 6 7 effectively by eye movements or gesture. Per patient descriptions and clinical categorizations are 8 9 provided in Supplementary Material. 10 11 12 13 14 Quantitative Behavioral Assessment 15 16 Quantitative behavioral assessments were performed using both normed, published instruments designed 17 18 For Peer Review 19 for cerebral palsy (cerebralpalsy.org.au) and non normed, in house instruments designed for this patient 20 21 population. The four published instruments were: Gross Motor Function Classification System 22 23 (GMFCS) [Palisaono et al., 1997]; Manual Ability Classification System (MACS) [Eliasson et al., 2006; 24 25 26 Communication Function Classification System (CFCS) [Hidecker et al., 2011; and, Eating and 27 28 Drinking Ability Classification System (EDACS) [Sellers et al., 2014]. All measures are Likert type 29 30 scales rated 1 5. Assessments using these four instruments were completed during the patient’s visits to 31 32 33 the Research Imaging Institute at the University of Texas Health Science Center at San Antonio. These 34 35 instruments proved of limited value for several reasons. First, the severity of the motor deficits caused 36 37 38 floor effects on three of these measures (GMFCS, MACS, and EDACS). Specifically, ten patients 39 40 scored V (i.e., worst score) in GMFCS, MACS, and EDACS, despite having a discernible range in 41 42 abilities; one patient scored IV on each instrument. Second, the measures did not include assessments 43 44 45 for perceptual abilities (vision, audition, somesthesis), as these are not typically impaired in cerebral 46 47 palsy. As perceptual networks feature prominently in ICN analyses, quantitative assessments of 48 49 perceptual abilities were needed for the planned imaging:behavior correlation analysis. Third, the 50 51 52 communication assessment was not sufficiently fine grained. Fourth, there was no assessment of social 53 54 and emotional behaviors. To address these limitations, we expanded the range of the GMFCS, MACS 55 56 and EDACS and added additional, similarly configured assessments. 57 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 10 of 51
10 1 2 The in house assessment battery evaluated ten dimensions of behavior using Likert type (1 5 rating 3 4 scale, low – normal function) questions and two dimensions using binary response (No = 1 / Yes = 5) 5 6 7 questions, for a total of 12 behavioral categories. The Likert type instruments were: Gross Motor 8 9 Function, Fine Motor Function, Eating and Drinking Ability, Tactile Perception, Visual/Visuomotor 10 11 Function, Auditory Function, Receptive Language Function, Expressive Language Function, Overall 12 13 14 Communication, and Social Emotional Responsiveness. The first three were modifications of the 15 16 GMFCS, MACS and EDACS, respectively. The three language assessment instruments were modeled 17 18 on the CFCS. The three perceptualFor and Peer the social em Reviewotional instruments were new but were similarly 19 20 21 constructed. The two binary response instruments assessed Expression of Pleasure/Displeasure and 22 23 Anticipation of Future. 24 25 26 27 28 Assessment forms were distributed to families after successful completion of the image acquisition 29 30 sessions and prior to the release of any imaging results. Parents were instructed to score their child’s 31 32 33 functional abilities based on empirical observations and perceptions. Where an adequate response could 34 35 not be identified, explanations and notes were encouraged. Upon receipt of the completed behavioral 36 37 assessment forms, structured interviews were performed with each family either by videoconference or 38 39 40 in person, to review the scores, ensure scoring was consistently applied across families, and to collect 41 42 detailed information about their child’s functional status. The outline of the interview followed the 43 44 assessment form and reviewed the functional deficits of each child, requesting specific examples of 45 46 47 behaviors that informed the scoring. Three members of our team (MI, PTF, MDW) participated in each 48 49 interview, which were recorded and transcribed. The complete behavioral assessment battery, per 50 51 52 patient clinical summaries and per patient behavioral scores are provided in Supplementary Data. 53 54 55 56 Image Acquisition 57 58 59 60 John Wiley & Sons, Inc. Page 11 of 51 Human Brain Mapping
11 1 2 Participants were brought to the imaging suite by their parent(s) at or after their typical bedtime, 3 4 typically around 8:00–9:00 p.m. Children were mildly sleep deprived by limiting daytime naps. All 5 6 7 participants received 5 mg/kg of diphenhydramine HCl 12.5 mg/5 mL oral solution to aid in sleep 8 9 induction. The children were put to bed by their parent(s) in a child friendly bedroom adjacent to the 10 11 MRI suite. When judged by the parent(s) to be deeply asleep, they were carried to the MRI suite and 12 13 14 prepared for imaging. Preparations included placement of noise reduction earplugs, foam head restraints, 15 16 and blankets. Extra padding and sheets had already been added to the MRI table to increase comfort 17 18 through the sleep scanningFor sequence. Peer Review 19 20 21 22 23 T1 weighted, diffusion weighted, and functional (BOLD) MR data were obtained in a single scanning 24 25 26 session when possible. If all sequences could not be obtained in one night, the subject returned for repeat 27 28 scanning (2 patients were rescanned). We previously reported analyses of the T1 weighted [Ishaque et 29 30 al., 2016] and diffusion weighted [Ishaque et al., 2017] data from these sessions. The present study 31 32 33 reports the rs fMRI data. 34 35 36 37 MRI data were obtained on a 3T Siemens TIM Trio (Siemens Medical Solutions, Erlangen, Germany), 38 39 40 using a standard 12 channel head coil as the RF receiver and the integrated circularly polarized body 41 42 coil as the RF transmitter. T1 weighted images were acquired using the MPRAGE pulse sequence with 43 44 TR / TE = 2200 / 2.72 ms, flip angle = 13°, TI = 766 ms, and volumes = 208, 0.8 mm isotropic voxel 45 46 47 size. Functional (BOLD; T2* weighted) imaging used a gradient echo, echo planar sequence, acquiring 48 49 29 slices, with TR / TE = 3000 / 30 ms, flip angle = 90°, 2 x 2 x 4 mm spatial resolution, FOV = 200 50 51 52 mm, and acquisition time = 30 minutes. 53 54 55 56 Acquired images were visually inspected per subject for artifacts, non related pathology, and/or 57 58 59 evidence of motion. No images showed gross pathology other than variable degrees of atrophy. Three 60 John Wiley & Sons, Inc. Human Brain Mapping Page 12 of 51
12 1 2 ABI and two control subject datasets demonstrated gross signs of excessive motion. These were 3 4 preprocessed with FSL’s MCFLIRT to determine absolute displacement parameters and were found to 5 6 7 be beyond acceptable limits (> 2 mm) in the first instance (3.27 mm, 3.59 mm, 3.98 mm, 4.47mm, and 8 9 8.16 mm, respectively). These datasets were excluded from further consideration. Two of the ABI 10 11 subjects were successfully rescanned, ultimately yielding high quality datasets in 10 ABI and 9 control 12 13 14 participants. 15 16 17 18 For Peer Review 19 Image Preprocessing 20 21 Independent intrinsic connectivity networks were computed using the FSL (FMRIB Software Library, 22 23 www.fmrib.ox.ac.uk/fsl) MELODIC software package, as follows [Beckmann and Smith, 2004]. T1 24 25 26 weighted anatomical images were preprocessed using the brain extraction tool (BET) to remove skull 27 28 and non brain tissues [Smith, 2002]. FSL’s MELODIC was run on the T2* weighted BOLD images 29 30 using a high pass filter of 100 s. MCFLIRT motion correction, brain extraction, and spatial smoothing 31 32 33 of 5 mm FWHM were applied [Jenkinson et al., 2002]. Single session ICA with fixed dimensionality of 34 35 20 components was implemented on each subject’s resting state data. Manual denoising was performed 36 37 38 per subject (images were thresholded at z > 2.3) using FSL’s fsl_regfilt tool with the following noise 39 40 component criteria: ring patterns, large activation areas across white matter/grey matter boundaries, 41 42 checkerboarding, predominant posterior sagittal sinus signal, saw tooth patterns or large spikes in 43 44 45 component time series, and >50% of component time series power spectrum at frequencies > 1 Hz 46 47 [Kelly et al., 2010]. This was an "aggressive" filtering, removing all aspects of the components 48 49 identified as bad, not simply those orthogonal to good components. Global signal regression was not 50 51 52 performed: because the technique is controversial, because "GSR should be used with care when 53 54 comparing groups with different noise characteristics or varying neural network structures" [Fox and 55 56 Murphy, 2017], because GSR has not been shown to improve spatial ICA network segregation, and 57 58 59 because the FSL development team does not recommend its use. Prior to motion correction the 60 John Wiley & Sons, Inc. Page 13 of 51 Human Brain Mapping
13 1 2 absolution motion in the ABI group was 0.192 ± 0.070 mm and in the control group was 0.188 ± 3 4 0.83 mm. These were not significantly different (p = 0.907). Following motion correction, the 5 6 7 absolute motion in the ABI group was 0.026 ± 0.011 mm and in the control group was 0.018 ± 8 9 0.13 mm. These were not significantly different (p = 0.156). Following motion correction, the 10 11 averages of mean absolute displacement and mean relative displacement for all subjects (i.e., patients 12 13 14 and controls) were found to be 0.022 mm and 0.021 mm, respectively. At the individual level, all 15 16 subjects had mean absolute and relative displacement values ≤ 0.050 mm. 17 18 For Peer Review 19 20 21 Between-Group Image Analysis 22 23 Multi session temporal concatenation ICA with a fixed dimensionality of 20 components was 24 25 26 implemented on all subjects’ denoised data to yield group average ICNs [Beckmann et al., 2005]. T2* 27 28 BOLD images were registered to anatomical images with a normal search and 6 degrees of freedom. 29 30 Registered brains were linearly converted into standard Talairach space (full affine registration; 12 31 32 33 degrees of freedom) using a resampling resolution of 2 mm, with registration to the Colin27 brain 34 35 template (normalized to Talairach space). Accurate registration was visually verified for all subjects, and 36 37 the Colin27 template was found to be appropriate across the cohort’s age range. Thresholded ICNs (z > 38 39 40 3) were identified and behaviorally interpreted by reference to the 10 resting state networks (Cerebellum, 41 42 Sensorimotor, Visual 1, Visual 2, Visual 3, Auditory, Left Frontoparietal, Right Frontoparietal, Default 43 44 Mode, and Executive Function) extracted with ICA of resting state data and the BrainMap database, as 45 46 47 described by Smith et al. (i.e., by referencing the functional attributions they made to these ICNs) [Smith 48 49 et al., 2009]. The basal ganglia network was additionally identified and interpreted [Robinson et al., 50 51 52 2009]. Only the 11 components of interest were retained for further analysis. 53 54 55 56 The refined set of spatial maps from the group average analysis was used to generate subject specific 57 58 59 spatial maps and associated time series using dual regression in FSL [Beckmann et al., 2009; Filippini et 60 John Wiley & Sons, Inc. Human Brain Mapping Page 14 of 51
14 1 2 al., 2009]. For each subject, the group average set of spatial maps was regressed (as spatial regressors in 3 4 a multiple regression) into the subject's 4D space time dataset. This resulted in a set of subject specific 5 6 7 time series, one per group level spatial map. Those time series were regressed (as temporal regressors, 8 9 again in a multiple regression) into the same 4D dataset, resulting in a set of subject specific spatial 10 11 maps, one per group level spatial map. We tested for group differences per network using FSL's 12 13 14 randomise permutation testing tool (5000 permutations) and Bonferroni corrected for multiple 15 16 comparisons (for 11 networks and a two tailed t test). 17 18 For Peer Review 19 20 21 Within-Patient-Group Image Analysis 22 23 For per subject, within patient group analysis, study specific network templates were established from 24 25 26 the group ICA of control subjects’ data (z > 2.3) (see Fig. 5). These network component templates 27 28 served as the standard for identification and comparison of each subject’s individual ICA decomposition. 29 30 Included networks reflect the 10 ICNs described by Smith [et al., 2009] and the basal ganglia network. 31 32 33 34 35 Network preservation was investigated through network absence/presence and network volume 36 37 measures. In measuring network preservation, we assess the extent to which the ICNs are intact, or 38 39 40 uninjured. Network absence/presence was noted through visual inspection of the overlap of individual 41 42 component maps with network templates. Network identification was then validated by spatial cross 43 44 correlation of single subject networks with the control group derived network templates following the 45 46 47 methods described by Smith [et al., 2009]. This automated labeling technique did not change any 48 49 component identifications. The paired networks had a mean Pearson’s r = 0.43 (0.20:0.73). 50 51 52 53 54 Total network volumes and percent overlap of each network with the corresponding template network 55 56 were calculated per subject using Mango tools (www.ric.uthscsa.edu/mango/). Total network volume 57 58 59 was calculated as follows: (1) a single component was loaded onto the standard Talairach brain, (2) the 60 John Wiley & Sons, Inc. Page 15 of 51 Human Brain Mapping
15 1 2 Threshold to ROI function was used to create an ROI around the component, (3) creation of an accurate 3 4 ROI was confirmed by visual inspection, and (4) volume in voxels was calculated within the ROI using 5 6 7 the ROI Statistics → Volume functions. Network percent overlap was calculated as follows: (1) the two 8 9 components of interest were loaded onto a single standard Talairach brain (e.g., single patient Auditory 10 11 12 network and template Auditory network), (2) the Create Overlay Logicals tool was used to show areas 13 14 of overlap between the two component images, (3) the volume in voxels of the overlapping region(s) 15 16 was calculated using the Create stats of this region tool from within the Logicals interface, and (4) this 17 18 For Peer Review 19 volume was divided by the total network volume of the template network to determine a percentage of 20 21 overlap (e.g., (overlap volume of Auditory networks / volume of template Auditory network) * 100) in 22 23 Microsoft Excel. These imaging metrics characterized the relative extent and spatial position of the 24 25 26 identified ICNs, and they were further utilized in correlation analyses. 27 28 29 30 31 Imaging-Behavioral Correlation 32 33 Correlation between per subject imaging metrics (Network Percent Overlap) and behavioral scores (as 34 35 described above) in ABI patients was assessed after sub classifying ICNs and behavioral categories into 36 37 38 three sub groups: Motor, Sensory, and Cognition. The Motor network grouping was comprised of the 39 40 Basal Ganglia and Cerebellar networks; the Motor behavioral grouping included the Gross Motor 41 42 43 Function, Fine Motor Function, Eating & Drinking Ability, and Expressive Language Function 44 45 categories. The Sensory network grouping was comprised of the Sensorimotor, Visual 1, Visual 2, 46 47 Visual 3, and Auditory networks; the Sensory behavioral grouping included the Tactile Perception, 48 49 50 Visual/Visuomotor Function, and Auditory Function categories. The Cognition network grouping was 51 52 comprised of the Left Frontoparietal, Right Frontoparietal, Default Mode, and Executive Function 53 54 networks; the Cognition behavioral grouping included Receptive Language Function, Overall 55 56 57 Communication, Social Emotional Responsiveness, Expression of Pleasure/Displeasure, and 58 59 Anticipation of the Future categories. Network Percent Overlap values and behavioral scores were 60 John Wiley & Sons, Inc. Human Brain Mapping Page 16 of 51
16 1 2 averaged within the described groupings to provide a Motor, Sensory, and Cognition network 3 4 preservation value and a Motor, Sensory, and Cognition behavioral value per patient (3 behavioral 5 6 7 scores and 3 network values per patient). The R statistical environment (R Core Team 2017) and lme4 8 9 [Bates, Maechler, Bolker, & Walker, 2015] were used to perform a linear mixed effects model analysis 10 11 of the relationship between network percent overlap and behavioral scores. The mixed effect model 12 13 14 controlled for the variance associated with multiple measures from the same subject. In computing 15 16 Spearman’s rank correlation, the network preservation and behavioral scores within subject were 17 18 modeled as a fixed effect, Forand the random Peer intercept s whichReview are allowed to vary between subjects were 19 20 21 modeled as a random effect. 22 23 24 25 26 RESULTS 27 28 Group-level Analysis 29 30 31 Behavioral Assessment 32 33 Average behavioral scores for the anoxic brain injury patients were: .50 (± 0.97) for Gross Motor 34 35 36 Function; 1.90 (± 0.74) for Fine Motor Function; 2.50 (± 1.35) for Eating and Drinking Ability; 5.00 (± 37 38 0) for Tactile Perception; 4.30 (± 0.67) for Visual/Visuomotor Function; 4.80 (± 0.42) for Auditory 39 40 Function; 4.30 (± 0.95) for Receptive Language Function; 2.40 (± 0.70) for Expressive Language 41 42 43 Function; 3.00 (± 0.47) for Overall Communication; 4.50 (± 0.53) for Social Emotional Responsiveness; 44 45 5.00 (± 0) for Expression of Pleasure/Displeasure; and, 5.00 (± 0) for Anticipation of the Future (Fig. 1). 46 47 The lowest average scores thus involved motor functions (i.e., gross and fine motor function, eating and 48 49 50 drinking, and expressive language). Other sensory and higher order functions were found to be 51 52 relatively behaviorally intact across the patient group. All control subjects were scored at a 5.00 for all 53 54 55 categories, thus representing normal functioning. 56 57 58 59 60 John Wiley & Sons, Inc. Page 17 of 51 Human Brain Mapping
17 1 2 [Figure 1] 3 4 5 6 7 Network Analysis 8 9 We were able to identify and isolate all 11 networks of interest (Basal Ganglia, Cerebellum, 10 11 12 Sensorimotor, Visual 1, Visual 2, Visual 3, Auditory, Left Frontoparietal, Right Frontoparietal, Default 13 14 Mode, and Executive Function) from the initial ICA analysis implemented on all subjects’ resting state 15 16 fMRI data (Fig. 2, top row ). 17 18 For Peer Review 19 20 21 Regression of these ICNs to group specific maps demonstrated substantial preservation of the 22 23 24 Sensorimotor, Visual 1 3, Auditory, and Default Mode networks in the patient group. Less preservation 25 26 was observed in the Left Frontoparietal, Right Frontoparietal, and Executive Function networks. The 27 28 least preservation was observed in the Basal Ganglia and Cerebellar networks ( P < 0.05; Fig. 2, bottom 29 30 31 row; Fig. 3). 32 33 34 35 Significant between group differences were measured in the Basal Ganglia, Cerebellar, and Left 36 37 38 Frontoparietal networks ( P < 0.05, corrected; Fig. 2, top row; Table II). Peak areas of decreased ICN 39 40 connectivity in the ABI patient group were identified in bilateral caudate bodies (Basal Ganglia), 41 42 posterior cerebellar lobe (Cerebellum), and left inferior parietal lobule (Left Frontoparietal). No 43 44 45 significant areas of increased network connectivity were observed in the patient group relative to the 46 47 control group. 48 49 50 51 52 [Figure 2] 53 54 55 56 57 [Table II] 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 18 of 51
18 1 2 3 4 [Figure 3] 5 6 7 8 9 Subject-Level Analysis 10 11 12 Behavioral Assessment 13 14 As reported above for average behavioral scores, ABI patients were found to overall have low motor 15 16 function (Gross Motor, Fine Motor, Eating and Drinking, Expressive Language) and relatively higher 17 18 For Peer Review 19 emotive/cognitive (Receptive Language, Overall Communication, Social Emotional, Expression of 20 21 Pleasure/Displeasure, Anticipation of the Future) and sensory perception (Tactile, Visual/Visuomotor, 22 23 24 Auditory) function (Fig. 4). The greatest variation in behavioral function was measured in Eating and 25 26 Drinking Ability; there was no variation in the Tactile Perception, Expression of Pleasure/Displeasure, 27 28 or Anticipation of Future categories. 29 30 31 32 33 [Figure 4] 34 35 36 37 38 Network Analysis 39 40 41 Using the group wise ICNs from control subjects as per network normal templates (Fig. 5), per subject 42 43 ICNs were extracted and quantified for both groups. Figure 6 demonstrates the range of network 44 45 preservation (Absent/Low, Moderate, and High) for each ICN (by total volume) among ABI patients. In 46 47 48 terms of absence/presence of ICNs, the Cerebellar network was most frequently absent (5 times). The 49 50 Left Frontoparietal network had the second highest frequency of absence (2 times), and the Basal 51 52 Ganglia, Visual 1, Right Frontoparietal, and Executive Function networks were each unidentified once 53 54 55 (Fig. 7, top ). 56 57 58 59 60 John Wiley & Sons, Inc. Page 19 of 51 Human Brain Mapping
19 1 2 Among the identified ICNs in ABI patients, relative preservation of the Sensorimotor, Visual 1 3, 3 4 Auditory, and Default Mode networks was observed. Less preservation was seen in the Left 5 6 7 Frontoparietal, Right Frontoparietal, and Executive Function networks. The least preservation was 8 9 observed in the Basal Ganglia and Cerebellar networks (Fig. 7, middle; bottom ). The largest per patient 10 11 variation in network volume was measured in the Left Frontoparietal, Right Frontoparietal, and 12 13 14 Executive Function networks, and the smallest variation was measured in the Sensorimotor, Visual 2, 15 16 and Basal Ganglia networks (in order). 17 18 For Peer Review 19 20 21 [Figure 5] 22 23 24 25 26 [Figure 6] 27 28 29 30 31 [Figure 7] 32 33 34 35 36 Imaging-Behavioral Correlation 37 38 Per patient Network Percent Overlap values and behavioral scores were sub grouped into Motor, 39 40 41 Sensory, and Cognition categories, and an overall positive correlation was measured between the image 42 43 and behavioral metrics. Spearman’s correlation coefficient was = 0.74 (P = 0.0001, 95% confidence 44 45 interval = 0.51 – 0.87) (see Fig. 8). Clear separation of values from the Motor, Sensory, and Cognition 46 47 48 sub groups was observed. A low to high trend in network preservation percentages and behavioral 49 50 scores was discernible with Motor, Cognition, and Perceptual values (in that order). 51 52 53 54 55 [Figure 8] 56 57 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 20 of 51
20 1 2 DISCUSSION 3 4 5 6 7 We acquired long duration resting state fMRI data in children with drowning induced ABI and 8 9 10 neurotypical controls using a nocturnal, drug aided sleep fMRI protocol. Network integrity was 11 12 quantified by independent components analysis (ICA), at both group and per subject levels. 13 14 15 Quantitative behavioral assessments designed to correspond to canonical, ICA derived networks were 16 17 obtained from parents and caregivers via structured interviews. Motor ICNs (Basal Ganglia, 18 For Peer Review 19 Cerebellum) were grossly compromised in ABI patients both group wise and individually, concordant 20 21 22 with their prominent motor deficits. Striking preservations of perceptual (Visual, Auditory, 23 24 Sensorimotor) and cognitive ICNs (Default Mode, Frontoparietal, Executive Function) were observed, 25 26 and the degree of network preservation correlated ( = 0.74) with per subject functional status 27 28 29 assessments. Our hypotheses were validated as (1) resting state fMRI demonstrated group level 30 31 differences between patients and controls, (2) per patient abnormalities were successfully and 32 33 meaningfully elicited, (3) imaging and behavioral results demonstrated concordance, and, (4) motor 34 35 36 network abnormalities were much more severe than perceptual and cognitive network abnormalities. 37 38 Collectively, our findings indicate that the severe motor deficits observed in this population can mask 39 40 41 relatively intact perceptual and cognitive capabilities. 42 43 44 45 The 11 ICNs of interest (Basal Ganglia, Cerebellum, Sensorimotor, Visual 1, Visual 2, Visual 3, 46 47 48 Auditory, Left Frontoparietal, Right Frontoparietal, Default Mode, and Executive Function) were 49 50 distinguishable at group and individual subject levels with ICA. In both group wise and per patient 51 52 network analyses, greatest network preservation was observed in the Sensorimotor, Visual 1, Visual 2, 53 54 55 Visual 3, Auditory, and Default Mode networks. Less preservation was observed in the Left 56 57 Frontoparietal, Right Frontoparietal, and Executive Function networks. The least preservation was 58 59 60 John Wiley & Sons, Inc. Page 21 of 51 Human Brain Mapping
21 1 2 observed in the Basal Ganglia and Cerebellar networks. Compared to the control group, patients with 3 4 ABI showed decreased functional connectivity in the Basal Ganglia, Cerebellar, and Left Frontoparietal 5 6 7 networks. The Cerebellar network was most frequently absent (i.e., undetectable) in individual patients. 8 9 10 11 Behavioral assessments demonstrated that children with ABI had relatively intact function on the Tactile 12 13 14 Perception, Visual/Visuomotor Function, Auditory Function, Receptive Language Function, Overall 15 16 Communication, Social Emotional Responsiveness, Expression of Pleasure/Displeasure, and 17 18 Anticipation of Future instrument.For Less Peer behavioral integrityReview was observed in the Gross Motor Function, 19 20 21 Fine Motor Function, Eating and Drinking Ability, and Expressive Language Function categories. Per 22 23 subject image metrics and behavioral scores overall correlated strongly. Furthermore, Motor, Cognitive, 24 25 26 and Sensory sub groups separated out quite well (in order of increasing preservation) along the network 27 28 and behavioral preservation scale. High concordance was thus observed in the evaluation of functional 29 30 integrity by intrinsic connectivity network preservation and behavioral assessments, at both group and 31 32 33 per subject levels. 34 35 36 37 In all analyses, pathology was largely limited to the motor system (i.e., Basal Ganglia and Cerebellar 38 39 40 networks; Gross Motor Function, Fine Motor Function, Eating and Drinking Ability, and Expressive 41 42 Language Function behavioral categories). Striking relative preservation of sensory (i.e., Sensorimotor, 43 44 Visual 1 3, and Auditory networks; Tactile Perception, Visual/Visuomotor Function, and Auditory 45 46 47 Function behavioral instruments) and higher cognitive (i.e., Default Mode network; Receptive Language 48 49 Function, Overall Communication, Social Emotional Responsiveness, Expression of 50 51 52 Pleasure/Displeasure, and Anticipation of Future behavioral instruments) functions was evident. 53 54 55 56 Taken together, this pattern of functional impairment/integrity builds upon our previous structural grey 57 58 59 and white matter analyses demonstrating predominant motor system damage to suggest that children 60 John Wiley & Sons, Inc. Human Brain Mapping Page 22 of 51
22 1 2 with drowning related anoxic brain injury can suffer from a de efferentation syndrome with relative 3 4 preservation of perceptual and cognitive abilities. Structural and functional motor deficits appear to 5 6 7 preclude outward expression of relatively intact perceptual, cognitive, and emotional abilities. 8 9 Indications of this level of functional integrity were detected behaviorally, but only through a novel, 10 11 caregiver driven behavioral assessment strategy designed for this population. The discussion below will 12 13 14 expand on the correlates of these impactful findings. Additionally, further examination of the potential 15 16 clinical impact of our imaging protocol in per subject assessment and prognostication is warranted. 17 18 For Peer Review 19 20 21 Group-level Image Analysis 22 23 Significant decreases in functional connectivity in the ABI patient group were measured in the Basal 24 25 26 Ganglia, Cerebellar, and Left Frontoparietal networks. Peak decreased connectivity within the Basal 27 28 Ganglia network localized to bilateral caudate bodies. The basal ganglia consist of subcortical nuclei 29 30 involved in motor, associative, and limbic circuits. Their role in voluntary motor activity, control, and 31 32 33 coordination is well established, and the Basal Ganglia intrinsic connectivity network has been shown to 34 35 be altered in several movement disorders [Laird et al. 2011; Luo et al., 2012; Robinson et al., 2009; 36 37 38 Szewczyk Krolikowski et al., 2014]. Functional compromise of this network likely underlies the 39 40 substantial motor deficits routinely reported in this population. Injury may extend to the associative and 41 42 limbic functions of the basal ganglia as well. 43 44 45 46 47 A large region of decreased functional connectivity was identified in the Cerebellar network, with a 48 49 maximum in the posterior lobe. The cerebellum is implicated in several functions, but the most prevalent 50 51 52 descriptions of this network involve action execution and motor coordination [Laird et al., 2011; Smith 53 54 et al., 2009]. Functional compromise of the Cerebellar network likely contributes to substantial motor 55 56 impairments observed in the patient group. Interestingly, prominent structural damage to the cerebellum 57 58 59 was not observed in our VBM analysis [Ishaque et al., 2016]. 60 John Wiley & Sons, Inc. Page 23 of 51 Human Brain Mapping
23 1 2 3 4 Finally, a small region of hypoconnectivity in the Left Frontoparietal network, with a peak in the left 5 6 7 inferior parietal lobule, was identified. The Left Frontoparietal network is strongly implicated in 8 9 language comprehension and production [Laird et al., 2011; Rosazza and Minati, 2011; Smith et al., 10 11 2009]. Functional compromise of the left fronto-parietal network likely contributes to the 12 13 14 expressive language deficits observed in the patient group. 15 16 17 18 Aside from these regions ofFor decreased Peer functional co nnectivity,Review no significant between group differences 19 20 21 were identified in the Sensorimotor, Visual 1 3, Auditory, Right Frontoparietal, Default Mode, or 22 23 Executive Function networks. This suggests relatively intact sensory and higher cognitive function in the 24 25 26 patient group. This pattern of preservation was seen in the behavioral data as well, with motor system 27 28 behavioral functions being most affected (1.50, 1.90, 2.50, and 2.40 average scores for Gross Motor, 29 30 Fine Motor, Eating and Drinking, and Expressive Language functions, respectively). 31 32 33 34 35 Subject-level Image Analysis 36 37 38 Long scan duration (30 minutes) enabled the application of ICA to individual patient data, mediating 39 40 meaningful per subject, per network interpretation. This is promising when considering the potential 41 42 clinical impact of these methods. 43 44 45 46 47 The greatest compromise in functional connectivity was seen in the motor resting state networks (i.e., 48 49 50 Cerebellum and Basal Ganglia) across ABI patients. The Cerebellar network was undetectable in 5/10 51 52 patients (17.8% average preservation). In some MRI acquisitions, the cerebellum is not fully covered in 53 54 the scan due to a limited field of view (FOV) [Smith et al., 2009]. Limited FOV cannot explain our 55 56 57 observation as we confirmed both at acquisition and prior to processing that the cerebellum was within 58 59 the FOV. Furthermore, the Cerebellar ICN was detected in all control subjects, and between group 60 John Wiley & Sons, Inc. Human Brain Mapping Page 24 of 51
24 1 2 functional connectivity differences were also observed in the group level analysis. Thus, as suggested 3 4 above, functional compromise of the Cerebellar network likely contributes to the observed motor 5 6 7 deficits in this population. The extent of cerebellar impairment is nevertheless quite striking in 8 9 comparison to our VBM structural findings, in which a relatively small region of atrophy was identified 10 11 in the posterior lobe [Ishaque et al., 2016]. Cerebellar dysfunction may arise, at least in part, due to 12 13 14 functional deafferentation following the predominant subcortical injury to the basal ganglia and PLICs. 15 16 Cerebellar diaschisis, first reported by Baron and colleagues in 1981 [Baron et al., 1981] has been 17 18 described to manifest as reducedFor metabolism Peer and blo Reviewod flow in the cerebellum following, and 19 20 21 contralateral to, a supratentorial lesion. Interruption of excitatory cortico ponto cerebellar pathways— 22 23 including PLIC lesions—is thought to be the underlying mechanism of cerebellar dysfunction [Carrera 24 25 26 and Tononi, 2014]. Functional disconnection of the cerebellum may thus be a downstream effect of the 27 28 primary central subcortical insult. This would be consistent with the absence of substantial cerebellar 29 30 atrophy in the group wise morphometric analysis, in which the strongest and most consistent structural 31 32 33 differences are measured. Cerebellar ischemia could be further investigated using perfusion weighted 34 35 imaging methods [Petrella and Provenzale, 2012]. 36 37 38 39 40 The Basal Ganglia network was undetected in one patient. In patients where it was detected, it showed 41 42 great impairment as measured by total volume and percent overlap with the normal template (19.8% 43 44 average preservation). Again, this likely drives the resultant motor dysfunction. 45 46 47 48 49 Sensory networks (i.e., Sensorimotor, Visual 1 3, and Auditory) were notably found to be intact per 50 51 52 patient (58.0%, 56.3% (Visual 1 3 average), and 47.0% average preservation, respectively), suggesting 53 54 preserved tactile, visual, and auditory processing in ABI children. The Default Mode network was also 55 56 largely intact per patient (detected in 10/10 patients, 46.6% average preservation), suggesting preserved 57 58 59 introspective processes, self awareness, and social cognition (self vs. other awareness) [Broyd et al. 60 John Wiley & Sons, Inc. Page 25 of 51 Human Brain Mapping
25 1 2 2009; Rosazza and Minati, 2011; Smith et al. 2009]; this point will be expanded upon below. Other 3 4 higher order networks (i.e., Left Frontoparietal, Right Frontoparietal, and Executive Function) were 5 6 7 more variable in their preservation (22.6%, 23.2%, and 25.2% average preservation, respectively). 8 9 Nevertheless, striking preservation of these ICNs was indeed discovered in some patients (see Figs. 11, 10 11 12). Complex functions including language comprehension, working memory, spatial awareness, 12 13 14 attention allocation, perception of the future, and cognitive control may well be conserved to a level 15 16 previously unsuspected in this population [Beckmann et al. 2004; Laird et al. 2011; Rosazza and Minati, 17 18 2011; Smith et al. 2009]. ForFunctional connectivityPeer findings Review overall corresponded well with per subject 19 20 21 behavioral scores, conveying low motor function and relatively high perceptual and cognitive function. 22 23 The observed integrity of higher order networks may in part stem from the typical timeline of cerebral 24 25 26 network development and integration. Neural regions serving more “elementary” motor and sensory 27 28 functions are thought to mature earlier than those involved in complex, higher order cognitive processes 29 30 [de Bie et al. 2012]. Given the average age of accidental drowning, both within our cohort (2.4 year 31 32 33 average) and in the population at large (1 4 years), it is possible the relative sparing of cognitive 34 35 networks results from their comparative immaturity at the time of the anoxic insult. That is, motor 36 37 networks may sustain greater damage in pediatric drowning than other systems that are less developed in 38 39 40 young children. 41 42 43 44 45 Imaging-Behavioral Correlation 46 47 The caregiver-based, quantitative behavioral assessment strategy -- using structured interviews to 48 49 assess behavioral comprehensively and in a network-based manner -- worked well. Behavioral and 50 51 52 image derived metrics of network integrity were mutually predictive at a highly significant level ( = 53 54 0.74; p < 0.0001). The behavioral validity of the imaging results is thus plausible. Furthermore, a clear 55 56 57 trend in network preservation and behavioral integrity was visible among the Motor, Sensory, and 58 59 Cognition sub groups. Motor system impairment was discernible through the lowest network 60 John Wiley & Sons, Inc. Human Brain Mapping Page 26 of 51
26 1 2 preservation and behavioral function in the Motor sub group. This mirrored the compromised gross and 3 4 fine motor function, eating and drinking abilities, and the ability to produce speech. Sensory perception 5 6 7 was shown to be relatively intact with the highest network preservation and behavioral function in the 8 9 Sensory sub group. This mirrored the relatively preserved abilities to perceive tactile, visual, and 10 11 auditory stimuli. Similarly, higher order cognitive functions were shown to be moderately intact, with 12 13 14 intermediate network preservation and behavioral scores. These trends support the overall group wise 15 16 and per subject conclusions derived from imaging and behavioral data. We believe implementation of 17 18 the same analyses in a largerFor cohort wouldPeer provide additionalReview support for the observed imaging 19 20 21 behavioral correlation. 22 23 24 25 26 Compensatory Neural Mechanisms 27 28 In group and subject level analyses, prominent expansion in certain networks was detected in patients 29 30 relative to controls (see red regions in Fig. 7 and 11; Fig. 12). Network volume averages in individual 31 32 33 patients approximated or exceeded those of the corresponding control templates in the Sensorimotor, 34 35 Visual 1, Visual 2, Visual 3, Auditory, and Default Mode networks. Networks with greater overall 36 37 38 preservation at both group and individual level analyses were thus also found to have notable expansion 39 40 in connected volume. Although subject variability and registration effects may explain this to some 41 42 extent, the observation suggests that neural plasticity is being exhibited. Less damaged networks will 43 44 45 experience preferential use, encouraging expansion. Attenuation of inhibitory input from damaged 46 47 networks and pathways (dis inhibition) could also contribute to the observed expansion [Merzenich et 48 49 al., 2014]. Neuroplasticity is most readily demonstrable during early post natal years [Hensch, 2005; 50 51 52 Merabet and Pascual Leone, 2009; Mundkur, 2005; Rocha Ferreira and Hristova, 2016] (although brain 53 54 remodeling is now thought to be inducible at any age) [Merzenich et al., 2014]. The average age at 55 56 injury in our patient cohort (2.6 ± 1.1 years) falls within this critical period, supporting this 57 58 59 interpretation. Neuroplasticity has been implicated in recovery from several pediatric neurological 60 John Wiley & Sons, Inc. Page 27 of 51 Human Brain Mapping
27 1 2 disorders, including other hypoxic ischemic etiologies [Merabet and Pascual Leone, 2009; Mundkur, 3 4 2005; Rocha Ferreira and Hristova, 2016], lending further credence to this hypothesis. Accordingly, 5 6 7 expansion of perceptual and Default Mode ICNs may well reflect active (and ongoing) neuroplasticity. 8 9 Monitoring ICN reorganization longitudinally following anoxic injury in this population could provide 10 11 further evidence in this regard. 12 13 14 15 16 Significance of Default Mode Network Preservation 17 18 For Peer Review 19 As reported above, the Default Mode Network (DMN) was substantially preserved in each patient. The 20 21 DMN is a highly investigated intrinsic connectivity network that corresponds to the brain’s default, task 22 23 negative state [Raichle et al. 2001; Rosazza and Minati, 2011]. The DMN has been associated with 24 25 26 higher mental processes including introspection, social cognition, motivational drive, self referential 27 28 thought, and interoception, as well as less definitive concepts such as mind wandering and day 29 30 dreaming [Heine et al. 2012; Raichle, 2015]. Dysfunction of this network has been implicated in 31 32 33 neuropsychiatric disorders including but not limited to dementia, epilepsy, unipolar and bipolar mood 34 35 disorders, autism, schizophrenia, and attention deficit/hyperactivity disorder [Broyd et al. 2009; Rosazza 36 37 38 and Minati, 2011]. Moreover, it has been posited as the functional basis of neural processes subserving 39 40 consciousness [Vanhaudenhuyse et al. 2010]. Studies in patients with disorders of consciousness 41 42 revealed decreased glucose metabolism [Laureys, Owens and Schiff, 2004], and decreased structural and 43 44 45 functional connectivity of the DMN in patients with decreased levels of arousal and awareness 46 47 [Demertzi et al. 2014; Fernandez Espejo et al. 2012; Vanhaudenhuyse et al. 2010]. Although the 48 49 diagnostic value of DMN network preservation in assessing level of consciousness in patients who 50 51 52 cannot communicate their internal states is not yet firmly established, its preservation in our patient 53 54 cohort and its correspondence with caregiver's assessment of situational awareness support the 55 56 possibility of greater levels of awareness of self and others than have been previously expected or 57 58 59 reported. 60 John Wiley & Sons, Inc. Human Brain Mapping Page 28 of 51
28 1 2 3 4 A physiological explanation for the striking preservation of the DMN, particularly relative to the other 5 6 7 higher order cognitive networks, might be found in its high glycolytic index. The DMN has been shown 8 9 to use less oxygen per mole of glucose consumed than other cortical networks [Vaishnavi et al. 2010; 10 11 Vlassenko et al. 2010]. Oxygen deprivation from drowning, therefore, would likely cause less direct 12 13 14 injury to areas with a high glycolytic index (i.e., DMN regions) relative to those with a greater 15 16 dependency on oxygen. 17 18 For Peer Review 19 20 21 Post-Drowning Pediatric ABI as a Selective De-efferentation Syndrome 22 23 Our prior structural neuroimaging reports in this same patient cohort demonstrated grey and white 24 25 26 matter loss in the lenticulostriate arterial distribution, predominately affecting motor system components 27 28 (pyramidal tract and basal ganglia), with relative sparing elsewhere [Ishaque et al. 2016; Ishaque et al., 29 30 2017]. TBSS clearly demonstrated a lesion in the PLIC in which the degree of white matter damage 31 32 33 correlated with motor performance impairment. The present report extends this line of investigation by 34 35 applying rs fMRI and quantitative behavioral assessments to provide functional neuroimaging and 36 37 38 behavioral evidence of selective motor system impairment with relative preservation of perceptual and 39 40 cognitive function in children with post drowning ABI. Collectively, these observations suggest that the 41 42 substantial motor deficits in these children – all are quadriplegic and most (7/10) are aphonic – arising 43 44 45 from structural damage to the basal ganglia and posterior limb of the internal capsule, and from 46 47 functional compromise of the Basal Ganglia and Cerebellar networks, effectively prohibit full 48 49 expression of their level of awareness and cognition. Task activation fMRI [Monti et al. 2010] and rs 50 51 52 fMRI [Heine et al. 2012] have been successfully applied to demonstrate cognitive system preservation 53 54 in cryptic disorders of consciousness. Here, we extend this line of investigation to rs fMRI in children 55 56 with post drowning anoxic injury. 57 58 59 60 John Wiley & Sons, Inc. Page 29 of 51 Human Brain Mapping
29 1 2 Categorization of cryptic disorders of consciousness and communication have evolved substantially 3 4 since the initial description of Locked In Syndrome (LIS) by Plum and Posner [1966], to include: 5 6 7 various degrees of LIS (Classical, Incomplete, Total) [Bauer, Gerstenbrand and Rumpl, 1979]; 8 9 Minimally Conscious State (MCS) and various degrees thereof (MCS+ and MCS ); and, most recently, 10 11 Functional LIS [Bruno et al. 2009]. In proposing the diagnostic category Functional LIS, Bruno [et al. 12 13 14 2009] distinguished between clinical and para clinical testing, with para clinical tests including EEG, 15 16 evoked potentials, positron emission tomography, and functional MRI. Laureys, Owens and Schiff 17 18 [2004] reviewed the role ofFor imaging studiesPeer in supp ortingReview diagnosis in these disorders, placing specific 19 20 21 emphasis on preservation of the DMN. Here, we have used network analysis of sleep state fMRI and 22 23 quantitative caregiver behavioral assessments as para clinical tests to supplement clinical assessments 24 25 26 and confirm that post drowning ABI can cause a selective de efferentation syndrome ranging from 27 28 Incomplete and Classical LIS to Total LIS/MCS+. All patients in our cohort met Bruno (et al. 2009) 29 30 criteria for Functional LIS. 31 32 33 34 35 Ethical issues regarding the clinical management (e.g., acute care decisions pertaining to withdrawal of 36 37 life support) and treatment (e.g., pain and symptom control, stimulation, and overall handling) of these 38 39 40 patients must be considered. Aside from establishing residual function, the per subject network analysis 41 42 results could inform potential avenues of communication, guide therapeutic stimulation through intact 43 44 functions (e.g., reading to a child with a strong Auditory network; displaying visual stimuli when Visual 45 46 47 networks are intact), guide targeted therapy, and monitor effects of therapeutic interventions through 48 49 qualitative and quantitative network analysis. The results of the present investigation have been provided 50 51 52 to their families and clinicians and are already impacting care in this cohort. Importantly, where metrics 53 54 such as the duration of circulatory arrest and acute imaging findings fail to reliably predict outcomes, 55 56 ICN profiles per patient could help clinicians with assessment and prognostication in post drowning 57 58 59 ABI. 60 John Wiley & Sons, Inc. Human Brain Mapping Page 30 of 51
30 1 2 3 4 Epidemiology 5 6 7 Post drowning pediatric selective de efferentation may well be a common condition. The precise 8 9 prevalence, however, is difficult to estimate. Data on nonfatal drownings are difficult to attain, 10 11 12 especially in a standardized way [Topjian et al., 2012]. However, given the rates of pediatric drowning 13 14 (> 3,500/year in the U.S.) and survival (> 2/3 of drowning victims), there must be a large population of 15 16 children that survive drowning with clinically significant anoxic brain injury (> 50% of survivors require 17 18 For Peer Review 19 hospitalization) [Borse and Sleet, 2009; Centers for Disease Control and Prevention (CDC), 2012]. The 20 21 fraction of children with post drowning ABI suffering from an unrecognized selective de efferentation is 22 23 difficult to estimate, as the syndrome was not previously recognized. In our cohort, 10/10 patients had 24 25 26 severe impairment of motor function and relative preservation of perception and cognition, both 27 28 behaviorally and by functional imaging, suggesting that this is a common condition. 29 30 31 32 33 We must consider, however, that our findings are likely affected by a patient selection bias. Specifically, 34 35 parents of victims were motivated to participate by their own impressions of preserved awareness and 36 37 38 cognition in their children (although this was not an inclusion criteria). That is, their suspicion of 39 40 functional preservation likely incited them to seek further support and information, and thus to learn 41 42 about and participate in our study. To address these important limitations in epidemiological knowledge 43 44 45 of this condition, we are preparing to expand our cohort to study children post drowning acutely, sub 46 47 acutely, and chronically. We encourage other imaging research centers to use the methods herein 48 49 described to do the same. A concerted, collaborative effort will be needed to determine the prevalence of 50 51 52 this condition. 53 54 55 56 57 Methodological Considerations 58 59 60 John Wiley & Sons, Inc. Page 31 of 51 Human Brain Mapping
31 1 2 It is important to consider the translation of our image acquisition and analysis methods into the clinical 3 4 realm. We saw successful application of our protocol on a per subject basis, allowing for meaningful 5 6 7 interpretation and quantification of individual networks in individual patients. Resting state fMRI during 8 9 sleep is an ideal paradigm for patients unable to cooperate with traditional task based or resting state 10 11 fMRI scans. Patient contribution, motor response, or comprehension, are not required to probe whole 12 13 14 brain intrinsic activity. From an experimental logistics standpoint, great expertise in administering the 15 16 scan is not essential [Heine et al. 2012]. ICA is also a voxel wise, relatively automated network analysis 17 18 technique that is readily adaptableFor for Peerclinical use. InReview our analysis, we fixed the number of components 19 20 21 at 20 and otherwise used the software with default parameters. We thus believe the imaging procedure 22 23 here described to be a readily applicable clinical protocol for use in pediatric drowning, known and 24 25 26 suspected disorders of consciousness, and other traditionally difficult patient populations where 27 28 functional assessments are desirable. 29 30 31 32 33 In order to assess the clinical impact of these methods, we are preparing to apply them more acutely. 34 35 The optimal timing for implementation of sleep fMRI and ICA network analysis is unknown in pediatric 36 37 drowning. We hope to investigate their feasibility once patients become stable in the hospital subsequent 38 39 40 to the drowning incident. This will provide insight into how soon following the anoxic insult meaningful 41 42 intrinsic network connectivity can be extracted and analyzed. We additionally aim to determine whether 43 44 single or multimodal imaging will be optimal in the acute setting. Based on our previous study of white 45 46 47 matter pathology in this cohort [Ishaque et al., 2017] and that of Barkovich [et al., 2001] in peri natal 48 49 ischemia, analyses such as probabilistic tractography across the PLICs using diffusion weighted MR 50 51 52 data may be more valuable acutely . Future studies should also evaluate longitudinal changes in ICNs 53 54 and behavioral function in the current cohort. Such investigations could then be extended to address the 55 56 effects of various interventions on intrinsic connectivity. In chronic ABI patients, functional integrity 57 58 59 could be further examined with visual and auditory evoked potentials and event related potentials target 60 John Wiley & Sons, Inc. Human Brain Mapping Page 32 of 51
32 1 2 situational awareness (P300) and language comprehension (N400). Assessment of the potential use of 3 4 brain computer interfaces and other genres of communicative devices is also warranted and would 5 6 7 further explore our tentative determination that these children have a variant of locked in syndrome. 8 9 10 11 This study is not without limitations. The sample size of 10 patients is admittedly small. Nonetheless, 12 13 14 statistically significant results were found both group wise and per subject. As mentioned above, an 15 16 expanded cohort would further improve the imaging behavioral data correlation analysis and provide 17 18 insight into the prevalenceFor of this disorder. Peer With ICA Review decomposition, selection of the dimensionality, or 19 20 21 number of components sought from the data, can be arbitrary. We applied a relatively common 22 23 dimensionality of 20 components, but higher or lower model orders can influence results [Cole et al. 24 25 26 2010; Smith et al. 2009; Elseoud et al., 2011]. Finally, through probing the patient sample with 27 28 established, normal network architecture, we are assuming the functional network architecture is more 29 30 or less normal. In other words, when a patient network is spatially consistent with the corresponding 31 32 33 canonical template, we assume it to be subserving the typical behavioral functions of that canonical 34 35 network. This may not be the case. To rigorously confirm normal function, one would need to consider 36 37 task based fMRI investigation of specific ICN driven functions. 38 39 40 41 42 CONCLUSION 43 44 45 This study demonstrates reduced functional connectivity predominantly limited to motor intrinsic 46 47 connectivity networks with relative preservation of sensory and some higher order networks in pediatric 48 49 50 anoxic brain injury from drowning. This pattern of pathology was seen in group wise and subject level 51 52 analyses and was highly concordant with behavioral assessments. Our findings are also consistent with 53 54 our previously reported structural analyses of grey and white matter pathology, which demonstrated 55 56 57 focal, subcortical disruption predominantly affecting motor system nuclei and tracts. These observations 58 59 together suggest children with ABI from drowning suffer from a selective de efferentation syndrome, in 60 John Wiley & Sons, Inc. Page 33 of 51 Human Brain Mapping
33 1 2 which motor deficits largely underlie their inability to convey intact cognition and perception. Moreover, 3 4 our methods for resting state fMRI data acquisition and network analysis proved powerful and should be 5 6 7 considered for per subject, clinical applications. 8 9 10 11 12 ACKNOWLEDGMENTS 13 14 The project described was supported by the National Center for Advancing Translational Sciences, 15 16 National Institutes of Health (TL1 TR001119, M.I.), the National Institute for Mental Health (R01 17 18 For Peer Review 19 MH074457, P.T.F.), and the Kronkosky Charitable Foundation. The Nonfatal Drowning Registry 20 21 (nonfataldrowningregistry.org) assisted with participant recruitment. The Conrad Smiles Fund 22 23 24 (www.conradsmiles.org) publicized this study and provided funding for travel and logistical support. 25 26 Miracle Flights (miracleflights.org) supported airfare costs. The content is solely the responsibility of 27 28 the authors and does not necessarily represent the official views of the NIH. The authors thank Thomas 29 30 31 Vanasse (UT Health San Antonio) for his guidance in data analysis, and all participants and families for 32 33 their time and involvement in this study. 34 35 36 37 38 CONFLICTS OF INTEREST 39 40 41 Nothing to report. 42 43 44 45 REFERENCES 46 47 48 Anderson AW, Marois R, Colson ER, et al. (2001) Neonatal auditory activation detected by functional 49 magnetic resonance imaging. Magn Reson Imaging 19:1 5. 50 Barkovitch AJ, Westmark KD, Bedi HS, Partrige JC, Ferriero DM, Vigneron DB. (2001) Protocol 51 52 Spectroscopy and Diffusion Imaging on the First Day of Life after Perinatal Asphyxia: 53 Preliminary Report. Am J Neuroradio 22:1786 1794. 54 Baron JC, Bousser MG, Comar D, Castaigne P (1981): “Crossed cerebellar diaschisis” in human 55 supratentorial brain infarction. Trans Am Neurol Assoc 105:459–461. 56 Bates D, Maechler M, Bolker B, and Walker S (2015) Fitting Linear Mixed Effects Models Using Ime4. 57 Journal of Statistical Software 67(1) 1 48. 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 34 of 51
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38 1 2 TABLE LEGENDS 3 4 Table 1. Patient Characteristics. For the ABI patient cohort, the gender, age at injury (months), age 5 6 7 at behavioral and imaging assessment (years), results of MRI at hospital admission, acute care 8 9 recommendation and clinical categorization are shown. Clinical categorizations use the terminologies of 10 11 Bauer (et al., 1979) and Bruno (et al., 2009). 12 13 14 15 16 17 18 Table II . Between-groupFor Network Differences.Peer Locations, Review coordinates, and extents of peaks of 19 20 21 significant between group differences in intrinsic connectivity network (ICN) connectivity (from group 22 23 ICA and dual regression analysis) are shown. Significant differences were identified in three ICNs: 24 25 Basal Ganglia, Cerebellum, and Left Frontoparietal (Controls > Patients; P < 0.05, corrected for 26 27 28 multiple comparisons). 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 39 of 51 Human Brain Mapping
39 1 2 FIGURE LEGENDS 3 4 Figure 1. Average ABI Behavioral Data. Average behavioral scores and standard deviations for 5 6 7 anoxic brain injury (ABI) patients are shown for 12 behavioral categories. The first 10 categories were 8 9 assessed with a 1 5 scoring system (low – normal function); the last 2 categories were assessed with a 10 11 Yes/No response (Yes = 5, No = 1). See Supplementary Data for complete behavioral assessment form 12 13 14 contents. Note: all control subjects were scored at a 5 for all behavioral categories. 15 16 17 18 For Peer Review 19 Figure 2. Group Independent Components Analysis and Dual-Regression Results. 20 21 Top row: The 11 intrinsic connectivity networks (ICNs) of interest resulting from independent 22 23 components analysis (ICA) of all subjects’ data are shown on the top row of panels (A) – (K) in red 24 25 26 yellow white. The identified ICNs are classified as follows: (A) Basal ganglia; (B) Cerebellum; (C) 27 28 Sensorimotor; (D) Visual 1; (E) Visual 2; (F) Visual 3; (G) Auditory; (H) Left Frontoparietal; (I) Right 29 30 31 Frontoparietal; (J) Default Mode; and (K) Executive Function. These ICNs were used to compute group 32 33 contrasts and group specific maps per network. 34 35 36 37 38 Between group differences (Controls > Patients) are overlaid onto the original ICN maps ( top row ) in 39 40 green ( P < 0.05, corrected for multiple comparisons). NB: Significant between group differences were 41 42 observed in three ICNs: (A) Basal Ganglia, (B) Cerebellum, and (H) Left Frontoparietal. 43 44 45 46 47 Bottom row: Group specific maps for each ICN are shown on the bottom row of each panel. Control 48 49 group specific networks are displayed in blue; patient group specific networks are displayed in red; 50 51 52 overlap between control and patient group networks is displayed in pink (P < 0.05). 53 54 All images are overlaid onto the Talairach standard brain. 55 56 57 IC = independent component; CON = control; ABI = anoxic brain injury. 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 40 of 51
40 1 2 3 4 Figure 3. Top: The total volume (in voxels) of each group specific intrinsic connectivity network 5 6 7 (ICN) is shown for patient and control groups. Bottom : The percent overlap of each patient group ICN 8 9 with the corresponding control group ICN is shown. 10 11 12 CON = control; ABI = anoxic brain injury. 13 14 15 16 Figure 4. Per-Subject ABI Behavioral Data. Per subject behavioral scores (with averages and 17 18 For Peer Review 19 standard deviations) for ABI patients are shown for 12 behavioral categories. The first 10 categories 20 21 were assessed with a 1 – 5 scoring system (low – normal function); the last 2 categories were assessed 22 23 24 with a Yes/No response (Yes = 5, No = 1). See Supplementary Data for complete behavioral assessment 25 26 form contents. 27 28 29 30 31 Figure 5. Intrinsic Connectivity Network Templates. Study specific templates of 11 intrinsic 32 33 connectivity networks (ICNs) extracted from independent components analysis of controls subjects’ 34 35 36 resting state fMRI data are shown. The included networks reflect the 10 standard ICNs, as described by 37 38 Smith [et al., 2009], plus the basal ganglia network. Networks are overlaid onto the Talairach standard 39 40 brain. 41 42 43 44 45 Figure 6. ABI Per-Subject Network Preservation. The range of intrinsic connectivity network (ICN) 46 47 48 preservation among individual anoxic brain injury (ABI) patients is shown for the 11 ICNs of interest. 49 50 Each row illustrates the range of network preservation observed across ABI patients within a single 51 52 network. Each column illustrates the least preservation (left column), typical preservation (middle 53 54 55 column), and most preservation (right column) across all subjects. Note, no single row or column 56 57 represents an individual subject. ICN templates derived from the control group independent components 58 59 60 John Wiley & Sons, Inc. Page 41 of 51 Human Brain Mapping
41 1 2 analysis (Fig. 5) are displayed in blue; individual patient networks are displayed in red; overlap between 3 4 the template and patient networks is displayed in pink (z > 2.3). Absent/Low, Moderate, and High 5 6 7 levels of network preservation are depicted based on network volume. 8 9 All images are overlaid onto the Talairach standard brain. 10 11 CON = control; ABI = anoxic brain injury. 12 13 14 15 16 Figure 7 . Top : The frequency of network absence in individual patients’ independent components 17 18 For Peer Review 19 analyses (ICA) is shown per intrinsic connectivity network (ICN). Middle : The total volume in voxels of 20 21 each ICN is shown for individual patients. Total volume means for each ICN are shown for individual 22 23 patient and control volumes. The volumes of ICN templates derived from the control group’s ICA are 24 25 26 also displayed. Bottom : The percent overlap of each patient’s ICN with the corresponding template ICN 27 28 is shown, along with means and standard deviations. 29 30 31 32 33 Figure 8. Imaging-Behavioral Correlation . Individual patients’ network percent overlap values (with 34 35 the corresponding network templates) are shown plotted against individual patients’ behavioral scores, 36 37 38 both averaged within Motor, Sensory and Cognition sub groups. Each symbol represents an individual 39 40 patient (P1 P10), with 3 behavioral values and 3 network preservation values per patient. 41 42 Motor = Basal Ganglia and Cerebellar networks; Gross Motor Function, Fine Motor Function, Eating & 43 44 45 Drinking Ability, and Expressive Language Function behavioral categories. 46 47 Sensory = Sensorimotor, Visual 1, Visual 2, Visual 3, and Auditory networks; Tactile Perception, 48 49 50 Visual/Visuomotor Function, and Auditory Function behavioral categories. 51 52 Cognition = Left Frontoparietal, Right Frontoparietal, Default Mode, and Executive Function networks; 53 54 Receptive Language Function, Overall Communication, Social Emotional Responsiveness, Expression 55 56 57 of Pleasure/Displeasure, and Anticipation of the Future behavioral categories. 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 42 of 51
1 2 3 Table I: Patient Characteristics 4 5 6 7 Gender Age at Age at Acute Acute Care Clinical 8 Injury Study MRI Recommendation Categorization 9 10 (mo) (yr) 11 P1 Male 21 5 Global Withdraw care Classical LIS 12 injury 13 P2 Male 36 5 Unknown Withdraw care Incomplete LIS 14 P3 Male 48 6 Global Withdraw care Incomplete LIS 15 16 injury 17 P4 Female 17 5 Normal Withdraw care Complete 18 For Peer Review LIS/MCS+ 19 P5 Female 36 8 Diffuse Withdraw care Complete 20 Edema LIS/MCS+ 21 22 P6 Female 24 7 Normal Proceed with Classical LIS 23 treatment 24 P7 Male 20 4 Normal Proceed with Incomplete LIS 25 treatment 26 P8 Male 36 11 Global Withdraw care Incomplete LIS 27 28 injury 29 P9 Male 24 11 Diffuse Withdraw care Incomplete LIS 30 Edema 31 P10 Male 17 12 Normal Institutionalize Complete 32 33 LIS/MCS+ 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 43 of 51 Human Brain Mapping
1 2 3 4 5 6 7 8 Talairach coordinates Extent 9 Anatomical location Hemisphere of global maxima (voxels) 10 x y z 11 Control > ABI 12 13 14 Basal Ganglia 15 16 Caudate Body L -14 1 19 3885 17 18 For Peer Review 19 Caudate Body R 15 2 12 1273 20 21 Cerebellum 22 23 Posterior Lobe L -27 -71 -24 37901 24 25 26 Left Frontoparietal 27 28 Inferior Parietal Lobule L -36 -46 54 2503 29 30 31 32 Table II . Between-group Network Differences. Locations, coordinates, and extents of 33 34 peaks of significant between-group differences in intrinsic connectivity network (ICN) 35 36 connectivity (from group ICA and dual regression analysis) are shown. Significant 37 38 39 differences were identified in three ICNs: Basal Ganglia, Cerebellum, and Left 40 41 Frontoparietal (Controls > Patients; P < 0.05, corrected for multiple comparisons). 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 44 of 51
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 Figure 1. Average ABI Behavioral Data. Average behavioral scores and standard deviations for anoxic brain 28 injury (ABI) patients are shown for 12 behavioral categories. The first 10 categories were assessed with a 1 29 - 5 scoring system (low – normal function); the last 2 categories were assessed with a Yes/No response (Yes 30 = 5, No = 1). See Supplementary Data for complete behavioral assessment form contents. Note: all control 31 subjects were scored at a 5 for all behavioral categories.
32 454x268mm (300 x 300 DPI) 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 45 of 51 Human Brain Mapping
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 Figure 2. Group Independent Components Analysis and Dual-Regression Results. 31 Top row: The 11 intrinsic connectivity networks (ICNs) of interest resulting from independent components 32 analysis (ICA) of all subjects’ data are shown on the top row of panels (A) – (K) in red-yellow-white. The 33 identified ICNs are classified as follows: (A) Basal ganglia; (B) Cerebellum; (C) Sensorimotor; (D) Visual 1; 34 (E) Visual 2; (F) Visual 3; (G) Auditory; (H) Left Frontoparietal; (I) Right Frontoparietal; (J) Default Mode; 35 and (K) Executive Function. These ICNs were used to compute group contrasts and group-specific maps per 36 network. 37 Between-group differences (Controls > Patients) are overlaid onto the original ICN maps (top row) in green 38 (P < 0.05, corrected for multiple comparisons). NB: Significant between-group differences were observed in 39 three ICNs: (A) Basal Ganglia, (B) Cerebellum, and (H) Left Frontoparietal. 40 41 Bottom row: Group-specific maps for each ICN are shown on the bottom row of each panel. Control group- 42 specific networks are displayed in blue; patient group-specific networks are displayed in red; overlap 43 between control and patient group networks is displayed in pink (P < 0.05). 44 All images are overlaid onto the Talairach standard brain. 45 IC = independent component; CON = control; ABI = anoxic brain injury.
46 47 182x127mm (300 x 300 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 46 of 51
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Figure 3. Top: The total volume (in voxels) of each group-specific intrinsic connectivity network (ICN) is 41 shown for patient and control groups. Bottom: The percent overlap of each patient-group ICN with the 42 corresponding control-group ICN is shown. 43 CON = control; ABI = anoxic brain injury. 44 45 397x396mm (300 x 300 DPI) 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 47 of 51 Human Brain Mapping
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 Figure 4. Per-Subject ABI Behavioral Data. Per-subject behavioral scores (with averages and standard 26 deviations) for ABI patients are shown for 12 behavioral categories. The first 10 categories were assessed 27 with a 1 – 5 scoring system (low – normal function); the last 2 categories were assessed with a Yes/No 28 response (Yes = 5, No = 1). See Supplementary Data for complete behavioral assessment form contents. 29 30 152x81mm (300 x 300 DPI) 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 48 of 51
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 Figure 5. Intrinsic Connectivity Network Templates. Study-specific templates of 11 intrinsic connectivity 24 networks (ICNs) extracted from independent components analysis of controls subjects’ resting-state fMRI 25 data are shown. The included networks reflect the 10 standard ICNs, as described by Smith [et al., 2009], 26 plus the basal ganglia network. Networks are overlaid onto the Talairach standard brain. 27 28 152x73mm (300 x 300 DPI) 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 49 of 51 Human Brain Mapping
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 6. ABI Per-Subject Network Preservation. The range of intrinsic connectivity network (ICN) preservation among individual anoxic brain injury (ABI) patients is shown for the 11 ICNs of interest. Each 48 row illustrates the range of netwo rk preservation observed across ABI patients within a single network. Each 49 column illustrates the least preservation (left column), typical preservation (middle column), and most 50 preservation (right column) across all subjects. Note, no single row or column represents an individual 51 subject. ICN templates derived from the control-group independent components analysis (Fig. 5) are 52 displayed in blue; individual patient networks are displayed in red; overlap between the template and 53 patient networks is displayed in pink (z > 2.3). Absent/Low, Moderate, and High levels of network 54 preservation are depicted based on network volume. 55 All images are overlaid onto the Talairach standard brain. CON = control; ABI = anoxic brain injury. 56 57 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 50 of 51
1 2 3 123x228mm (300 x 300 DPI) 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 51 of 51 Human Brain Mapping
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 7. Top: The frequency of network absence in individual patients’ independent components analyses (ICA) is shown per intrinsic connectivity network (ICN). Middle: The total volume in voxels of each ICN is 48 shown for individual patients. Total volume means for each ICN are shown for individual patient and control 49 volumes. The volumes of ICN templates derived from the control group’s ICA are also displayed. Bottom: 50 The percent overlap of each patient’s ICN with the corresponding template ICN is shown, along with means 51 and standard deviations. 52 53 215x279mm (300 x 300 DPI) 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Brain Mapping Page 52 of 51
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 Figure 8. Imaging-Behavioral Correlation. Individual patients’ network percent overlap values (with the 21 corresponding network templates) are shown plotted against individual patients’ behavioral scores, both 22 averaged within Motor, Sensory and Cognition sub-groups. Each symbol represents an individual patient (P1-P10), with 3 behavioral values and 3 network-preservation values per patient. 23 Motor = Basal Ganglia and Cerebellar networks; Gross Motor Function, Fine Motor Function, Eating & 24 Drinking Ability, and Expressive Language Function behavioral categories. 25 Sensory = Sensorimotor, Visual 1, Visual 2, Visual 3, and Auditory networks; Tactile Perception, 26 Visual/Visuomotor Function, and Auditory Function behavioral categories. 27 Cognition = Left Frontoparietal, Right Frontoparietal, Default Mode, and Executive Function networks; 28 Receptive Language Function, Overall Communication, Social-Emotional Responsiveness, Expression of 29 Pleasure/Displeasure, and Anticipation of the Future behavioral categories. 30
31 620x227mm (72 x 72 DPI) 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc.