Our report builds on our previous pilot infant MRI study that received ethical approval from Institutional Review Board as a “no more than minimal risk” study. Specifically, we extended our previous T1-weighted brain MRI analysis of total CC volumes (11) and T2-weighted analysis of total forebrain volumes (12) to the current T1-weighted analysis of sub-regional volumes of both CC, as well as that of the forebrain. Since presented data is from the same infant study subjects, previously described methodological approach (11, 22) for (1) recruitment criteria and (2) MRI scanning process apply to this study as well. Briefly, informed written parental consent was obtained prior to subject participation, in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. The family of each subject received a $90 gift card for participation in the non-sedated research brain MRI scan.

All infant patients underwent non-sedated research brain MRI at a point in time just before the hospital discharge following completion of initial surgical repair, or during subsequent admissions for follow up management in the 1st year of life. Thus, subjects were scanned throughout the infancy, depending on the time of their recruitment. A “feed and wrap” approach was used on all infants undergoing a non-sedated research brain MRI scan after the completion of complex perioperative treatment (32–35). Corrected age at scan for all cohort subjects was calculated as follows: postnatal age (weeks) – [40 – GA at birth (weeks)]. Please, refer to our previous publication in regard to individual total CC [Figure 5 in (11)] and total forebrain volumes [Figure 3 in (12)] with respect to corrected GA at scan. Patients were scanned at late evenings or nights using a 3T TrioTim MRI system equipped with 32-channel receive-only head coil and body-transmission (Siemens Healthcare Inc., USA) as per previously described protocol (12, 22). Foam earplugs (Newmatic Medical, Birmingham, AL) and earmuffs (MRI-Safe Neonatal Noise Guards, Universal Medical, Norwood, MA) were used for noise protection. Smaller infants were further supported using beanbags (viz. gentle vacuum bag immobilizer), while infants older than 3 months were allowed to assume a more relaxed position (e.g., arms next to face). Despite undergoing a non-sedated research scan, due to complexity and severity of clinical status, a single physician (DB) continuously monitored all infants for a stable heart rate and oxygenation throughout the MRI acquisition. Structural T1-weighted images were acquired using a MPRAGE sequence (repetition time = 2.52 s; echo time = 1.74 ms; flip angle = 7°; field of view = 192 × 192 mm²; voxel size = 1 × 1 × 1 mm³; 144 sagittal slices). We collected 100% of T1-weighted images for all patients (n = 13/group), and 91% (n = 21/23) for term-born controls (Table 1). Regarding the latter, one infant had partial brain coverage that precluded analysis of total brain volume (n = 20 controls for forebrain analysis) but allowed for CC analysis (n = 21 controls). Although we noted ringing artifact due to motion only in 1/21 controls and 1/13 premature patients, it was mild enough not to require motion correction or obscure tissue segmentation.

Prior to any tissue segmentation, Freeview (v.2.0) from Freesurfer (the General Hospital Corporation, Boston, MA) was used to correct for any head tilt during MRI acquisition. In other words, preprocessing of T1-weighted images included alignment along the anterior commissure - posterior commissure (AC-PC) line [see Figure 2 in (22)]. A single rater with neuroanatomical expertise blindly performed tissue segmentation, which was subsequently checked by a senior researcher for internal consistency. Specifically, ITK-SNAP software (v.3.6.0; www.itksnap.org) (36) was used for total and sub-regional CC segmentation (Figure 1) and total brain and forebrain segmentation (Figure 2A–C) of T1-weighted images. We also performed surface area analysis, but since finals 2-D data results did not significantly differ from volumetric data, 2D results are not presented.

The ITK-SNAP volume estimation tool was used to obtain absolute volumes (cm³) of segmentation masks. Normalization was calculated as part of a whole to allow for better understanding as to how a particular sub-region of interest changes with respect to the whole (viz. total forebrain volume). Specifically, volumetric data of both CC and forebrain sub-regions were normalized as a % total forebrain volume (Figures 3, 4, respectively).

This study used original data from our previous pilot study (22). Since no prior information was available regarding brain findings in the selected cohort of infants with LGEA, we used a convenience sample size of 13 patients/group, based on the anticipated number of eligible infants at our institution and an estimated 50% enrollment rate. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS, v.23.0; IBM Corporation, Armonk, NY), and normal distribution of all continuous variables was confirmed using the Shapiro-Wilk test. To account for the potential confounding variable of having subjects scanned at various ages throughout the first year of life, comparisons of sub-regional volumes between the three groups were assessed using a general linear model (GLM) univariate analysis with corrected age at scan as a covariate and Bonferroni adjusted p-values. Statistical significance was assessed at the α <0.05 level after conservative Bonferroni correction that accounted for multiple comparisons. In this report, we present differences between three groups studied. A comprehensive statistical report for absolute and normalized group differences is also condensed in a table format (Tables 2, 3).

We previously reported disproportionately smaller total CC volume (as % of total brain volume) in both patient groups (11). Our novel analysis of total CC as a % of total forebrain volume matched these previous findings. Specifically, normalized total CC volumes showed significant group differences [F_(2,42) = 16.16, p < 0.001], with both term-born and preterm-born patients having smaller normalized total CC volumes compared to controls, with no differences between patient groups (graph not shown).

To evaluate sub-regional vulnerability of CC, we analyzed volumes of six CC sub-regions as illustrated in Figure 1A',B'. Statistical analysis of absolute values (cm³) between groups is summarized in Table 2. We normalized all six sub-regions of the CC as % total forebrain to see if any of the specific CC sub-regions in patients are smaller when compared to the forebrain as a whole. We report that normalized CC sub-regional volumes (as % forebrain volume; Figure 3) were significantly smaller in both patient groups compared to controls in all sub-regions except for the sub-region II (rostral body). For full statistical details, (see Table 2). As such, results implicate the rostral body, sub-region II of the CC to be least affected.

Our current T1-weighted analysis parallels the results of previously published T2-weighted data report (12) that showed proportionally smaller total forebrain (as % of total brain volume). Specifically, no group differences were observed in normalized volumes for total forebrain [F_(2,42) = 0.1, p = 0.90; graph not shown].

We extended the study to perform 3-D segmentation of seven forebrain sub-regions according to previous report in literature (24, 25), as illustrated in Figure 2C. Of the parcellated seven forebrain sub-regions, six are known to send homotopic projections via selective sub-regions of CC (26), as schematically color-coded in Figure 2C,D. In the absence of better methodology, presented results should be interpreted with caution due to linear parcellation methodological approach not fully aligned to functional connectivity (see section Study Limitations: Methodological Consideration). Statistical analysis of decreased absolute values (cm³) per sub-region in patients is summarized in Table 3. However, we only found group differences for normalized sub-regional volumes of the forebrain (as % total forebrain volume; Figure 4) for two forebrain sub-regions: parieto-occipital and subgenual sub-regions (term-born patients were different from both controls and premature patients). For full statistical details, (see Table 3). Future studies with more power and narrower clinical criteria for inclusion of premature patients should be considered.

Our current data of sub-regional CC size implicates globally smaller CC in premature and term-born patients. The uniformly decreased normalized CC volume with respect to the whole forebrain [with the exception of the sub-region II of the CC (viz. rostral body)], may implicate delayed and/or impaired maturation of white matter tracts in the setting of LGEA as a congenital anomaly and/or result of LGEA complex perioperative treatment. As such, CC volume analysis may serve as an early marker of brain (mal)adaptations of brain size in the selected group of infants born with LGEA undergoing complex perioperative critical care with Foker process (14–16). Our volumetric analysis showed disproportionally larger normalized volume in the rostral body (sub-region II of the CC) known to connect to the pre-motor cortex, indicating that this region may be least likely affected (schematics summary in Figure 5) in the context of globally smaller brain in selected cohort of infants (22). The anterior callosal fibers such as those projecting from the rostral body have been shown to interconnect to the frontal lobe and relate motor information (6). Larger normalized rostral body findings, and homotopic-like linkages between the rostral body and the premotor area, which was also shown to have potentially altered growth trajectories for the patient groups, may indicate possible protection in this area of the forebrain, an interesting and unexpected finding given the previously established delay in motor development in critically ill infants (46). Conversely, the more adversely affected posterior CC fibers connecting the parietal, temporal, and occipital lobes, the greater likelihood of (mal)adaptations in integration of sensory information (6) might be encountered. To our knowledge, no studies yet have evaluated the neurodevelopmental outcomes in either term- or preterm-born infants following complex perioperative critical care with Foker process for LGEA repair. As such, future clinical evaluations that include motor and sensory assessments should be used to correlate neuroanatomical findings to the neurodevelopmental implications.

Although no previous studies have investigated sub-regional CC development in the setting of complex perioperative critical care, previous work has shown that the more posterior sub-regions of the CC (viz. anterior midbody, posterior midbody, isthmus, and splenium; Figure 1) are likely to have smaller surface areas, decreased volume of interhemispheric fiber tracts, and reduced fractional anisotropies in very premature babies (41). This is consistent with our findings of decreased volumes in such posterior areas, and possible protective effects in the more anterior rostral body sub-region (sub-region II of CC). Importantly, as CC myelination occurs anteriorly to posteriorly (10), such findings may indicate the posterior sub-regions of the CC are more vulnerable to adverse early life events in the context of the complex perioperative critical care for LGEA repair.

Our current sub-regional analysis of normalized forebrain volumes failed to identify vulnerable sub-region of the forebrain in homotopic-like relation to CC sub-regions. As such, results indicate that uniformly decreased sub-regional forebrain volumes contribute to the global decrease in brain size (22) in the selected pilot cohort of infants following LGEA repair. Interestingly, a disproportionally smaller normalized volume is reported only for the subgenual sub-region of the forebrain (Figure 2C), which does not have any homotopic connections with the CC (Figure 5), and suggest that, possibly - limbic circuits may be adversely impacted following complex perioperative critical care for LGEA repair.

The disproportionately smaller CC sub-regional volumes may indicate hypo or incomplete myelination in term-born and premature patients. Whether this is due to decreased oligodendrocyte cells, decreased axonal density, or degradation of the myelin, remains unclear. It should be noted, that previous studies have linked prematurity to focal non-cystic white matter injuries in nearly half of premature newborns with MRI data, and much of this white matter vulnerability has been linked to late oligodendrocyte progenitor cells/ subplate cells (47). Whether this is true of the underlying pathology of term-born infants undergoing complex perioperative care warrants future studies. Previous literature has also reported term-born infants undergoing complex care, including those both with and without major cardiac anomalies, are more likely to have white matter lesions (cardiac and non-cardiac cases) and lower fractional anisotropy in the CC (cardiac cases) (48, 49). Whether the decreased CC size in our sample population of infants with LGEA reflects preexisting brain abnormalities, risk of abnormal development in the setting of perioperative critical care, or a double-hit etiology of brain injury both pre and peri-Foker process, as previously discussed (18), remains unknown. Our most recent preliminary data published as a small case report series (18) are in support of possible double-hit etiology of brain injury (pre-Foker and peri-Foker process) in both premature and term-born infants with LGEA. Future studies in this vulnerable infant population should also include diffusion tensor imaging (DTI) and tractography to shed light on the possible mechanisms affecting white matter microstructural integrity during infancy, a critical age for the white matter maturation and global brain growth.

The exact neurological effects and long-term neurobehavioral impact of complex perioperative care for LGEA repair remain unknown. Previous reports indicate that infants with non-cardiac congenital anomalies that undergo complex perioperative care are at an increased risk for abnormal brain development and poor long-term outcomes (9, 48). More broadly, as the largest white matter tract in the brain that serves to connect and facilitate cross-talk between the two hemispheres, abnormal development of the CC in infancy may lead to altered cognitive/executive function that persists into childhood and adolescence (50). Prematurity, white matter impairment, and decreased CC volume have also all been associated with poor executive functioning and language outcomes (51–53). Since functional implications of reported structural differences remains unknown in infants following complex perioperative critical care for LGEA repair, investigation into clinical correlations and long-term neurodevelopmental outcomes represents the next steps in clinical research investigation.