JV was a 48-year-old right-handed female at the time of injury, with 16 years of education. She is a native speaker of Tagalog, and was a fluent speaker of English as a second language. She sustained a moderate-severe traumatic brain injury (TBI) secondary to a fall from a ladder onto a concrete floor. A CT scan performed on the day of the injury indicated left frontal and temporal subdural hematomas (Glasgow Coma Scale (GCS) score not available), which were evacuated in an urgent craniotomy. JV was in a coma for five to six weeks. Unfortunately, she sustained a second TBI three months later, as a result of another fall while in hospital, at which time her language symptoms worsened. MRI at the time of the second TBI showed evidence of a new subarachnoid hemorrhage in the left medial temporal sulcus, in addition to underlying encephalomalacia in the left frontal and left temporal lobes associated with the initial hematomas (see Figure 1). The results of neuropsychological assessment conducted 25 months after the second injury and language assessment conducted 36 months after the second injury (immediately before therapy) show moderate-severe nonfluent aphasia, characterized by limited spontaneous speech output, stereotypical utterances, moderate comprehension deficits, and good use of gestures and facial expressions to support communication. The patient also presented with moderate apraxia of speech (based on tasks for assessing apraxia of speech), characterized by audible groping, sound distortions and substitutions, articulatory self-corrections, difficulty with words of increasing length, delayed response initiation, and slow rate of speech. She had normal visuoperceptual and hearing abilities. She appeared to respond well to musical stimulation, including melodic and rhythmic cueing, during informal diagnostic testing. Detailed language and neuropsychological assessment findings are presented in Tables 1 and 2.
The control TBI patient, GB, was a right-handed female, native speaker of English, with 13 years of education who was 54 years of age at the time of injury. She had sustained a moderate to severe TBI secondary to a motor vehicle accident. Her GCS at the scene was 5 and declined to 3 at the time of admission to the emergency department. A CT scan performed on the day of injury indicated a left temporal subarachnoid hemorrhage as well as an intraventricular hemorrhage. GB was in a coma for approximately 1 week following the accident. Neuropsychological assessment at 28 months postinjury did not indicate any persistent language deficits. Although the control patient is a native English speaker, while the treated patient speaks English as a second language, we do not expect this to affect the results because we are not comparing between them on language performance or on a language task-related functional imaging. Ethics approval was granted for the study, and both participants had signed an informed consent.
Thirty-six months after her second injury, as part of the current study, JV started receiving a melody-based treatment, which was a modified version of MIT. Therapy was developed jointly by a speech language pathologist and music therapist and administered in English, remotely via Skype  by the accredited music therapist (coauthor Cheryl Jones), specialized in Neurologic Music Therapy. All treatment stimuli were trained using the musical elements of melody and rhythm. A unique melody, distinct in shape and rhythmic pattern, was assigned to each target phrase and served as a timing template to support word retrieval and the fluency of word production. This is in contrast to the standard MIT protocol, which uses only two to three tones for all phrases. Rhythmic cues served to support oral motor timing impairments and word fluency.
Each target phrase, and its associated unique melody, was presented following the standard MIT hierarchy (e.g.,): the clinician, accompanied by an electric keyboard, first played and hummed the melody and then sang the target words. The participant then sung the phrase in unison with the therapist for a number of repetitions, with the therapist gradually omitting words until the participant was singing independently. These steps were repeated until the patient could sing the target phrase without support. The participant's son or daughter, present at every session, tapped her left hand, as per the MIT protocol.
Melody served as the primary cue to support word production within the target phrase, and the unique melody was played up to five times, as required, allowing the participant to gradually fill in the melody with the target words. Initially, the participant required maximal melodic cueing (i.e., 5 cues), but this need decreased (i.e., to 1-2 cues) over the course of treatment. Rhythm served as the secondary cue, whereby the rhythm of the target phrase was tapped on her left arm in order to help the participant overcome hesitations or sustained pauses during a phrase. Additional cues included modelling of oral-motor placement and providing the first word in the target phrase. The need for these cues, however, significantly reduced as treatment progressed. In addition to the hierarchical progression of cues, the treatment was designed to enhance generalization, by gradually decreasing the structure of the target phrases, thereby increasing the linguistic difficulty. Namely, the treatment involved progressing through 5 steps: steps 1 to 3 primarily involved repetition; step 4, sentence completion; and step 5, phrase production (in response to target questions). Novel responses were encouraged in steps 4 and 5.
The primary goal of steps 1 to 3 was to use melodic and rhythmic cues to encourage word retrieval and fluency in sentences of increasing length and complexity. The participant was required to produce target phrases with and without melodic intonation. The primary goal of step 4 (sentence completion task) was to encourage generalization beyond trained phrases, by asking the participant to generate novel words at the end of each rehearsed target phrase. In step 5, the primary goal of treatment was for JV to produce fully self-generated phrases, using two tones to produce nonrehearsed responses to target questions. Untreated items were also created and matched to all treatment stimuli in terms of number of syllables, syntactic complexity, and relevance to the participant's life and interests. These items were not treated and served for testing before, immediately after, and eight weeks following treatment (see outcome measures).
Treatment stimuli were created based on an "interest" inventory completed by JV and her family, whereby the participant listed her primary hobbies and interests, important members of her family and friend groups, details from her past, and her speech-related goals. Treatment stimuli were phrases on a continuum of difficulty in terms of syllable length and syntactic complexity. Shorter, simpler (e.g., imperative and wh-question) phrases were used initially, followed by longer, declarative present- and past-tense phrases (based on HELPSS hierarchy,). The target phrases were categorized into five distinct treatment steps: (1) 2-4 syllables (e.g., "How are you?"); (2) 5-7 syllables (e.g., "Please do the dishes"); (3) 8-10 syllables (e.g., "I drink coffee every morning"); (4) sentence completion cues (e.g., "For dinner I will make..."); and (5) question probes (e.g., "What did you do yesterday?"). There were seven target phrases in steps 1 to 3, nine target phrases in step 4, and 10 target questions in step 5.
JV started receiving treatment 36 months after her second injury and received treatment three days a week for 16 weeks, for a total of 48 sessions. Each session lasted approximately 30 minutes. At the beginning of each session, three previously learned phrases were rehearsed, to encourage maintenance and continued practice of all treated items. Progression through steps 1 to 5 occurred if either (a) the participant produced 80% of the target phrases with 80% accuracy on two consecutive sessions or (b) the participant completed 9 sessions of therapy within a single step. Target accuracy was measured as the proportion of syllables correctly produced within the target phrase (for steps 1 to 3) and the appropriateness of the responses given to sentence completion and question probes (for steps 4 and 5). Each melody and phrase was notated and emailed to the participant, who was instructed to practice the target phrases between sessions for 30 minutes, three times per week. The participant was also required to sing personally significant songs for ten minutes a day, five days a week, in an attempt to reinforce word retrieval through familiar melody and word associations. These activities were monitored by the patient using a "homework log." These logs indicated very good compliance of the patient with the homework.
Both treated and untreated items were administered pretherapy, immediately posttherapy, and at eight weeks posttherapy by a registered speech-language pathologist who was not involved in the treatment (coauthor Tijana Simic). The primary outcome measure for steps 1 to 3 was the ability to repeat target phrases accurately (measured as the proportion of correctly produced syllables in each phrase). In steps 4 and 5, the primary outcome measures were the raw number of syllables produced in appropriate responses to sentence completion cues (step 4) and questions (step 5). Syllables produced in inappropriate responses (e.g., that did not fit the context of the cue or question probe) were discarded and not included in the raw counts. All outcome measures were taken using spoken, not melodic, cues.
Whole head MR scans were acquired on a General Electric (GE) Signa-Echospeed 1.5 Tesla high-definition scanner, located at Toronto General Hospital:University Health Network, using an eight-channel head coil. The high-resolution 1 mm isotropic T1-weighted, three-dimensional radio-frequency spoiled-gradient recalled-echo (SPGR) images were acquired in the axial plane utilizing a 25 cm field of view (FOV) (TR/TE/TI = 12/5/300 ms), flip angle = 20 , slice thickness = 1 mm no gap, 160 slices, and matrix 256 x 256. Resting-state BOLD fMRI data were acquired with the following imaging parameters: TR/TE = 2000/40 ms, flip angle = 85 , FOV = 22, slice thickness 5 mm with no gap, 32 slices with 4800 images, and matrix 64 x 64. Resting-state data were collected at three time points. For JV, this was at 25 months, 35 months, and 39 months after the second injury, with treatment occurring between 36 and 39 months postinjury. Resting-state data for the control patient GB was collected at 28 months, 32 months, and 36 months postinjury, with no speech and language treatment. Additional sequences include DTI and axial fast spin-echo PD/T2-weighted images, which are not presented here. All sequences were obtained with a 22 cm FOV. The entire scanning session lasted approximately 55 min.
The preprocessing of resting-state data was performed using SPM12 (Wellcome Department of Imaging Neuroscience, London, UK; http://www.fil.ion.ucl.ac.uk/spm). The preprocessing steps included removal of the first five scans, slice-timing correction, rigid-body motion correction and unwarping, spatial normalization to the Montreal Neurological Institute (MNI) space, and smoothing with an 8 mm FWHM Gaussian kernel.
Functional connectivity analysis was performed using a region of interest (ROI) to ROI approach within CONN's functional connectivity toolbox (Whitfield-Gabrieli and Nieto-Castanon 2012; http://www.nitrc.org/projects/conn). We selected anatomical ROIs in bilateral frontal areas: inferior frontal gyrus (IFG) pars opercularis (Operc), pars triangularis (Tri), and pars orbitalis (Orb); precentral gyrus (PreC), insula, and supplementary motor area (SMA). Subregions within left IFG (opercularis, triangularis, and orbitalis), known to be involved in language production, were previously used as seed regions for the language network in resting-state studies. Their homologous regions in the right IFG were shown to be involved in melody-based treatments, in structural-imaging and brain stimulation studies. The left precentral gyrus, insula, and SMA are typically involved in motor sequence planning for articulation and initiation of speech, and the right SMA was also shown to be involved in melody-based treatments. A mask was created to encompass these ROIs using the automated anatomical labeling (AAL) atlas.
Sources of physiological noise (based on white matter and cerebrospinal fluid segmentation) and movement covariates (motion correction and scrubbing using Artifact Detection Tools; ART http://www.nitrc.org/projects/artifact_detect) were regressed out of the data. Data was also bandpass filtered to retain low-level frequencies (0.009 < f < 0.08 Hz). Semipartial correlations were then computed using the residual data across all ROIs, for each time point (T1-baseline, T2-pretreatment, and T3-posttreatment) and participant (treated versus control patient). In semipartial correlations, the effects of all other regions on the target region alone are held constant while computing the correlation between the source and the target region. Therefore, semipartial correlation between two regions is not symmetric and depends on which region is the target. The correlations were then transformed into z-scores using Fisher's r to z transformation.
Similar to the approach taken by Sandberg et al., difference matrices were computed to determine the level of increase and/or decrease in functional connectivity across time points. A difference matrix was calculated by subtracting post- minus pretreatment functional connectivity to identify changes during the "treatment period" (or its equivalent in the control patient; T3 - T2) and by subtracting pretreatment minus baseline functional connectivity to identify changes during the "baseline period" (T2 - T1). Significant differences in correlational changes across time points (treatment versus baseline period; separately for each participant; p < 0.05 (FDR corrected for 66 comparisons)) were determined using Fisher's test.