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UP-BEAT (Upper Limb Baby Early Action–observation Training): protocol of two parallel randomised controlled trials of action–observation training for typically developing infants and infants with asymmetric brain lesions

Introduction Infants with asymmetric brain lesions are at high risk of developing congenital hemiplegia. Action–observation training (AOT) has been shown to effectively improve upper limb motor function in adults with chronic stroke. AOT is based on action observation, whereby new motor skills can be learnt by observing motor actions. This process is facilitated by the Mirror Neuron System, which matches observed and performed motor actions. This study aims to determine the efficacy of AOT in: (1) influencing the early development of reaching and grasping of typically developing infants and (2) improving the upper limb activity of infants with asymmetric brain lesions. Methods and analysis This study design comprises two parallel randomised sham-controlled trials (RCTs) in: (1) typically developing infants (cohort I) and (2) infants with asymmetric brain lesions (eg, arterial stroke, venous infarction, intraventricular haemorrhage or periventricular leukomalacia; cohort II). Cohort II will be identified through a neonatal ultrasound or neonatal MRI. A sham control will be used for both RCTs, taking into consideration that it would be unethical to give no intervention to an at-risk population. Based on a two-tailed t test of two independent means, with a significance (α) level of 0.05, 80% power, predicted effect size of 0.8 and a 90% retention rate, we require 20 participants in each group (total sample of 40) for cohort I. The sample size for cohort II was based on the assumption that the effect size of the proposed training would be similar to that found by Heathcock et al in preterm born infants (n=26) with a mean effect size of 2.4. Given the high effect size, the calculation returned a sample of only four participants per group, on a two-tailed t test, with a significance (α) level of 0.05 and 80% power. As cohort II will consist of two subgroups of lesion type (ie, arterial stroke and venous infarction), we have quadrupled the sample to include 16 participants in each group (total sample of 32). Infants will be randomised to receive either AOT or standard Toy Observation Training (TOT). Both interventions will be of 4 weeks’ duration, from the infant's 9th–13th post-term week of age. Three sessions of 5 min each will be performed each day for 6 days/week (total of 6 h over 28 days). Parents of the AOT group will repeatedly show the infant a grasping action on a set of three toys, presented in random order. Parents of the TOT group will show the infant the same set of three toys, in random order, without demonstrating the grasping action. At 14, 16 and 18 weeks, the quantity and quality of reaching and grasping will be measured using the Grasping and Reaching Assessment of Brisbane; symmetry of reaching and grasping will be measured using the Hand Assessment of Infants (HAI) and pressure of grasping for each hand with a customised pressure sensor. At 6 months’ corrected age, the primary outcome measures will be the HAI and Bayley Scales of Infant and Toddler Development (third edition; BSID III), to measure cognitive and motor development. At 8 months, HAI and EEG will be used to measure brain activity and cortical coherence. At 12 months, the primary outcome measures will again be HAI and BSID III. Dissemination This paper outlines the theoretical basis, study hypotheses and outcome measures for two parallel RCTs comparing the novel intervention Action–observation training with standard TOT in: (1) influencing the early development of reaching and grasping of typically developing infants and (2) improving the upper limb motor activity of infants with asymmetric brain lesions. Trial Registration ACTRN1261100991910. Web address of trial http://www.ANZCTR.org.au/ACTRN12611000991910.aspx


Background
Infants with asymmetric brain lesions (e.g. intraventricular haemorrhages, periventricular leukomalacia, arterial strokes and venous infarctions occurring on one side or more involved on one side of the brain) are at high risk of developing congenital hemiplegia by the end of their first year of life. The incidence of asymmetric brain lesions at birth is 1-2 of every 1000 newborns (1).
Congenital hemiplegia is the most common type of Cerebral Palsy (CP), with a prevalence of 1 in 1300 live births (1). The economic impact of CP is substantial. In 2007, the financial cost of CP was estimated at Aus$1.47 billion, with $124.1 million of that cost directly attributed to intervention costs (2). Approximately 43% of these costs are covered by the families of individuals with CP, with the remaining 57% by various levels of government (2).
The main focus of early intervention for infants with asymmetric brain lesions who may progress to classification of unilateral CP is very early and accurate detection of the brain lesion, followed by provision of an enriched environment and training to maximise upper limb function during critical periods of development. The challenge for clinicians and researchers is the limited number of tools available to identify the problem and measure progress, as well as a paucity of evidence for efficacy of very early upper limb rehabilitation.
There are, broadly speaking, two common clinical presentations of asymmetric brain lesions, early or delayed. Early presentation consists of perinatal onset of neurological symptoms, or seizures, or reduced movement at 24 to 48 hours post birth with verification on cranial ultrasound and/or Magnetic Resonance Imaging (MRI) of the presence of an asymmetric brain lesion. Specific imaging protocols may be needed for diagnosis in the early phases, such as diffusion MRI to identify an acute stroke in the first hours or days (3). In a delayed presentation, the infant may have an initially uncomplicated perinatal course and  7 may not show signs of stroke or asymmetric brain injury until three to seven months of age, when unilateral weakness and early hand preference start to manifest (4,5).
The current most predictive tools for early diagnosis of CP are a combination of brain magnetic resonance imaging (MRI) at term and a Prechtl's Assessment of General Movements (GMs) in the fidgety period (at 12 weeks post-term; 6). Specifically, GMs at one month and three months post-term age are highly associated with white matter abnormalities on MRI at term age (7). The GMs is a well validated and reliable tool, and is more sensitive at predicting CP than other motor assessments used in infancy (8,9). The GMs is also useful for prediction of minor motor difficulties (10). Neuromotor assessments (such as the GMs) utilised in the neonatal period (< 4 months post-term) have strong validity to detect CP in infants born preterm, when correlated with criterion assessments at 12 months corrected age (such as the BSID III; 8). Although abnormalities in general movements are likely to be evident during the early writhing period (<6-9 weeks post-term) and the fidgety period (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) weeks post-term), asymmetries are only visible during the fidgety period (11)(12)(13).
Asymmetries in fidgety GMs around 12 weeks post-term can be the first definitive clinical sign of hemiplegia (11)(12)(13).
Very early detection of hemiparesis frequently requires serial evaluation of subtle signs of interlimb differences or asymmetries in upper limb reaching (both spontaneous and purposeful), and grasp strength (14). Both bimanual and unimanual reaching with early strong hand preference at four to six months of age can be considered to be a strong sign of early hemiplegia (4). Studies of infants who have sustained an early perinatal stroke before four to seven months corrected age have suggested that until reach to grasp behaviours have emerged, an asymmetry may not be clearly evident and hemiparesis not confirmed (15,16).
Early intervention for infants at risk of developing congenital hemiplegia is considered to be very important, however, standard rehabilitation programs generally  8 commence after six months of age due to delayed detection. A further consideration regarding the timing of commencement of intervention is that important phases of brain reorganisation may have already occurred (17,18).
Current approaches to rehabilitation in congenital hemiplegia in infants focus on toy presentation and sensory stimulation of the limb to encourage spontaneous reaching and grasping, however, the challenge is to obtain active movement from the impaired limb. A new approach utilising action observation to stimulate the mirror neuron system offers another opportunity to stimulate the damaged motor cortex before the infant has achieved volitional reach and grasp.

Theoretical Framework
The Mirror Neuron System (MNS) is comprised of 'mirror neurons'; specialised neurons which fire when one observes another performing an action and when one executes the action, facilitating understanding of the action and subsequent imitation of that action (19)(20)(21)(22). Mirror neurons were discovered initially in the premotor area (F5) of macaque monkeys and have since been identified in the rostral area of the inferior parietal lobule (PF) and the ventral premotor cortex (20,23). Direct evidence for the MNS in humans is lacking and there have been no studies published which have recorded single neurons from the proposed MNS in humans.
There is a growing body of neurophysiological and brain-imaging studies providing indirect evidence for the existence of the MNS in humans (24). Transcranial Magnetic Stimulation (TMS) studies have concluded that the MNS exists in humans and it differs to the MNS in monkeys (25)(26)(27)(28)(29). Non-purposeful and intransitive actions activate mirror neurons in humans and not in monkeys (26,28,30). When humans observe actions, the temporal features of cortical excitability suggest that the MNS codes for the whole action as well as the  9 individual movements that comprise the action. In contrast, only the whole action is coded by the MNS in monkeys (24). These unique properties of the MNS in humans suggest that humans' capacity to imitate others' actions is related to the MNS.
Several studies have identified two cortical areas which correspond to motor function and are activated during action observation in humans; (i) the rostral area of the PF; and (ii) the lower area of the precental gyrus combined with the posterior area of the inferior frontal gyrus (IFG; 31,[32][33][34][35][36][37][38][39][40][41][42][43][44][45]. It has been suggested that activation of mirror neurons located in the IFG (otherwise known as Broca's area) in humans corresponds to activation of mirror neurons located in the PF in monkeys (24). In humans, the two mirror areas receive afferent input from the superior temporal sulcus (involved in processing motion), and send efferent input to the motor cortex (46,47).
Demonstration of the MNS soon after birth introduces a new perspective to the treatment of infants with congenital brain injury. Emerging evidence from the basic sciences in infant rhesus macaque monkeys suggests that the immature MNS can facilitate the imitative capabilities of infants; and that consistent demonstration of imitative skills and subsequent manual skills can predict later motor development (53,54).

Sensorimotor reorganisation after early brain injury
It is well known that brain injuries impacting on the sensorimotor (SM) system may manifest in varying degrees of functional impairment, the extent of which is related to the size and site of the lesion, as well as the type of adaptive reorganisation that follows. The main mechanism for a reconnection of the motor cortex to the spinal cord consists of This alternative mechanism results in the undamaged hemisphere directly controlling both upper limbs, which is a pattern of reorganisation unknown to adult pathology (i.e. contralesional reorganisation (55).
Emerging evidence in humans suggests that the pattern of SM reorganisation after early brain injury is determined during the first year of life, and possibly within the first few months (56). As children with reorganisation occurring in the damaged hemisphere (which results in the undamaged hemisphere directly controlling both upper limbs) have suboptimal upper limb motor activity, this pattern appears to be maladaptive (55). It has been suggested that the MNS may influence cortical reorganisation associated with upper limb impairment, and could potentially be a target of very early intervention (11).

Action Observation and Imitation
The process of observing an action (i.e. action observation) leads to activation of the MNS and stimulates the corticospinal system (motor pathways) prior to imitating the action (38,(57)(58)(59). When the motor cortex is damaged (e.g. congenital brain lesion), action observation and imitation may influence cortical reorganisation by directly restoring the damaged motor pathways or reinforcing other pathways that originally helped to perform motor actions, or both (60).
In animal and human adult studies, action observation appears to activate the MNS and enhance excitability of the SM cortex (61). These findings suggest that the effects of an The authors concluded that current evidence for very early upper limb interventions suggested small effects on unimanual capacity, bimanual coordination and self-care skills; however, there is limited data on the safety and the neural mechanisms underlying activity changes in response to these interventions. Further research is required to investigate the efficacy of upper limb interventions of this at-risk population at preschool age (<3 years) and address the lack of attention to safety implications for infants.

Proposed intervention and justification: why UP-BEAT?
Action observation therapy is a novel upper limb rehabilitation approach based on the recent discovery of mirror neurons (62). This approach has been shown to effectively  12 improve upper limb motor function in adult patients with chronic stroke (61). Action Observation Therapy is currently being investigated in a population of school-aged children (5-15 years) with unilateral CP (62).
Action Observation Therapy has not yet been investigated in a randomised clinical trial for a population of infants with congenital brain lesion. The efficacy, benefits and safety implications of this novel rehabilitation for this at-risk population are unknown. Ideally, such an intervention should begin soon after the brain injury has occurred. It is difficult, however, to achieve voluntary activation of the motor cortex during the first weeks of life, as voluntary reaching is absent or immature. The activation of the motor cortex related to action observation may represent a unique opportunity for therapeutic intervention in this early period of development. As soon as voluntary reaching can be reliably elicited, very early intervention should be supplemented with standard rehabilitative approaches aimed at encouraging symmetrical reach and grasp behaviours, as well as use of the limb in developing mobility.
In adults with stroke, action observation has been shown to effectively increase cortical excitability of the SM cortex and improve upper limb motor outcomes (61). Based on the hypothesis that the same activation can be elicited in infants, we predict that Action Observation Training will enhance the excitability of the SM cortex, accelerate the maturation of the corticospinal tract and the shaping of spinal motor circuits leading to better spontaneous use of the impaired upper limb. This could prevent the development of asymmetric reach and grasp in young infants with early asymmetric brain lesions.

Broad aim of proposed study
The broad aim of this study is to evaluate in two parallel randomised controlled trials with an identical sham control, whether the novel intervention Action Observation Training (AOT) is improving the upper limb motor activity of infants with asymmetric brain lesions (n = 32).

Methods
Two randomised controlled trials (RCTs) with an identical sham control will be conducted to evaluate the efficacy of AOT compared to standard TOT in: (a) typically developing infants with a gestational age between 38 and 41 weeks at time of recruitment (n = 40); and (b) infants with asymmetric brain lesions aged 0 to 9 post-term weeks at time of recruitment (n = 32).
This study will involve an at-risk population of infants (i.e. infants with asymmetric brain lesions). It would therefore be unethical to give no intervention to this population in the control arm. Standard TOT is the sham control intervention that will be used for both RCTs.
It is similar to standard therapy as it involves the parents presenting toys to infants and encouraging spontaneous visual exploration, without demonstrating how to play with the toys. It does not include the active AOT component of the intervention for the treatment group.
The specific hypotheses to be tested are: 1. Typically developing infants receiving AOT will have faster development of reaching and grasping in both upper limbs, compared with infants receiving standard TOT; 2. Infants with asymmetric brain lesions receiving AOT will have faster development and greater quality and quantity of reaching and grasping in both upper limbs, compared with infants receiving standard TOT; 3. For both infant cohorts, AOT will result in greater equalisation of corticomotor pathways and retention of cortical reorganisation, compared with standard TOT;  14 4. Individual differences among infants in quality of general movements and imitative behaviour will modulate the effects of training on their development of reaching and grasping.
These hypotheses will address the following specific aims:-1. To determine if typically developing infants undergoing AOT will develop reaching and grasping earlier than those undergoing standard TOT. AOT is a novel upper limb training program based on action observation. Evidence suggests that action observation can activate the motor cortex and reinforce the corticospinal network. We will determine through an RCT if a 4-week AOT program (from 9 to 13 post-term weeks' age) will influence the short-term outcomes of reaching and grasping, compared to a standard TOT program whereby action observation is replaced with toy observation (no grasping action demonstrated).

2.
To determine if infants with asymmetric brain lesions undergoing AOT will develop reaching and grasping earlier, and have greater quality and quantity of reaching and grasping, compared to those undergoing standard TOT. Recent studies suggest that an early therapeutic intervention in infants with asymmetric brain injury should aim to activate the impaired motor cortex. We will determine through an RCT if a 4-week AOT program (from 9 to 13 post-term weeks' age) will lead to cortical activation associated with action observation and influence the development of reaching and grasping in these infants and improve their short-and long-term outcomes. AOT will again be compared with standard TOT. If we can show that this novel, very early intervention can improve short-term and long-term upper limb motor activity in infants with asymmetric brain lesions, this will guide clinical practice and enable more efficient allocation of therapy resources in the future.  15 3. To determine if AOT will lead to greater equalisation of corticomotor pathways and cortical reorganisation. We will determine if the 4-week AOT program will result in modified cortical coherence related to action observation through an electroencephalogram (EEG). If we can show that this novel very early intervention can lead to greater equalisation of cortical motor pathways and retention of cortical motor reorganisation, this will guide clinical practice with implications for other patients (infants with bilateral/ symmetric brain injury, school-aged children with unilateral CP, children with stroke). An understanding of the nature and timing of the brain lesion may indicate which infants respond better.

4.
To determine if the individual differences among infants in quality of general movements and imitative behaviour will modulate the effects of training on their development of reaching and grasping. We will investigate these using standardised measures of spontaneous motility and imitation skills in both cohorts pre-and posttraining. If we find these correlations, we can explore the possibility of individually tailoring very early therapeutic interventions.
Assessments will be performed at 9, 12, 14, 16 and 18 weeks. Follow-up will be performed at 6 and 12 months following intervention, to determine retention of effects. The timing of assessments coincides with early critical periods of spontaneous general movements and early imitation behaviours (9 weeks); period of fidgety movements (12 weeks); early symmetrical reaching (14-16 weeks) and symmetrical reaching to the midline

Study sample and recruitment
Infants and their families will be recruited within a 50-200 km radius from The Royal Children's Hospital, Brisbane, Australia. The recruitment process will target major metropolitan health districts across southeast Queensland, with the expectation that the cohort I sample will be representative of typically developing infants and the cohort II sample will be representative of infants with asymmetric brain lesions from Queensland.

Inclusion criteria
Cohort I: Typically Developing Infants (TDI) will include infants: 1. With a gestational age at birth between 38 and 41 weeks; 2. Living within a 50 km radius of the Royal Children's Hospital, Brisbane.
Cohort II: Infants with asymmetric Brain Injury (ABI) will include infants: 1. With an asymmetric (one sided or more involved on one side) or unilateral (one sided) brain injury (e.g. preterm or term arterial stroke, grade III or IV intraventricular haemorrhage, periventricular leukomalacia) identified on neonatal ultrasound or MRI; 2. Aged 0 to 9 post-term weeks at time of recruitment; 3. Living within a 200 km radius of the Royal Children's Hospital, Brisbane.
A parallel clinical trial will admit infants with the same inclusion criteria into two parallel RCTs at the Stella Maris Scientific Institute in Pisa, Italy. Cohort I (TDI) will exclude infants: 1. With any post-natal medical complications (e.g. jaundice) requiring extended hospital admission or medical treatments.

Cohort II (ABI) will exclude infants:
3. With epileptic seizures unresponsive to treatment.

Sample size
Cohort I (TDI): Based on a 2-tailed t-test of 2 independent means, significance (alpha) level of 0.05, 80% power, predicted effect size of 0.8 and a 90% retention rate, we require 20 participants in each group (total sample of 40) for cohort I.
Cohort II (ABI): Sample size for cohort II was based on the assumption that the effect size of the proposed training will be similar to that found by Heathcock and colleagues (14) in a population of pre-term infants (n = 26) with a comparable training program, with a mean effect size of 2.4. Given the high effect size, the calculation returned a sample of only 4 participants per group, on a 2-tailed t-test, significance (alpha) level of 0.05 and 80% power.
As our cohort II population will consist of two sub-groups of lesion type (i.e. arterial stroke and venous infarction) and will be highly variable with the presence of asymmetric brain injury; we have quadrupled the sample. We require 16 participants in each group (total sample of 32) for cohort II.

Randomisation
The allocation sequence will be comprised of computer-generated random numbers in a blocked design. Infants will be randomised to receive either Action Observation Training  18 (AOT) or standard Toy Observation Training (TOT), from concealed envelopes opened by non-study personnel. Treatment allocation will be recorded on a piece of folded paper inside each envelope in random order (computer generated). The randomisation process will involve allocating a code to each infant, which consists of the letter 'B' or 'G' according to gender and a number based on date of birth (e.g. "B1", "B2" and "G1", "G2"). The infant's name and code will be written on the paper inside the envelope and sealed. The envelope will be marked that it has been allocated and the infant's code will be written on the front of the envelope. As each infant is entered, he/she will be allocated the next consecutive envelope, which will be opened by the non-study personnel who will read and record the treatment allocation from the paper inside the envelope. The randomisation envelopes will be held and administered by the therapist providing training of the interventions for the parents.

Blinding
The therapists who will be training the parents in the interventions and the parents will be informed of group allocation; the therapists conducting the assessments will be masked to group allocation; and study personnel who will be assessing the outcomes will also be masked to group allocation. Randomised group allocation will remain concealed to the therapists who conducted the assessments until all data for the entire sample has been analysed.

Study treatments
Both cohorts will receive the same dosage of three 5-minute sessions (15 minutes per day) for 6 days per week, for 4 weeks. The total dosage of intervention will be 6 hours, over a  19 period of 28 days. After baseline screening and randomisation, infants will receive either AOT or standard TOT. Parents will be trained by an occupational therapist for approximately 30 minutes, and will be directly observed performing the training activities with the infant during training.
Two follow-up phone calls regarding questions on how to perform the training will be addressed over the telephone with the therapist who trained the parents. Parents will be asked to video-record the sessions each day. Parents of the AOT group will repeatedly show the infant a grasping action on a set of three toys, presented in random order. Parents of the TOT group will show the infant the same set of three toys, also presented in random order, without the grasping action. The toys are mostly cylindrical in shape and vary in appearance, colour and patterns (i.e. cow, clown and musical instrument). Groups will be compared at 14, 16 and 18 weeks, as well as 6 months and 12 months following intervention.
To optimise comfort and convenience for their families, intervention training for parents, delivery of intervention and all assessments from 9 to 18 weeks will be performed in the infants' home environment. To optimise the infant's engagement in the interventions, parents will be advised to: (a) perform the training when the infant is calm and alert; (b) wiggle their fingers to engage the infant's attention prior to commencing the training; and (c) stop the training, allow the infant to play briefly with the toys if the infant becomes distracted or stops attending to the parent's hand and toy, before continuing the training.

Therapy protocols and delivery
Several occupational therapists will plan and conduct both intervention groups. These core therapists will be responsible for liaising with the parents to organise home visits to train the parents in their allocated interventions and for each set of assessments at 9, 12, 14, 16, 18 weeks corrected age (CA). The core therapists will also be responsible for organising the 6 An online diary that will only be accessible to the core therapists will be completed after each training session, follow-up phone call, home visit and follow-up assessment to summarise each activity for each infant. Any issues of concern such as difficulties with training and adverse events will be considered when data is analysed, as potential factors that may account for differences between cohorts. Video footage of each training and assessment session will be qualitatively and/or quantitatively analysed to assess treatment fidelity.
The core investigator team (RB, JZ, AG, KP, LF, MP) will meet regularly to review the progress of training and assessments for both cohorts, and will decide when any modifications to the protocol are required.

Outcome measures and procedures
At 14, 16 and 18 weeks post-term or CA, quantity and quality of reaching and grasping will be measured using the Grasping and Reaching Assessment of Brisbane (GRAB); symmetry of reaching and grasping will be measured using the Hand Assessment of Infants (HAI); and pressure of grasping for each hand with a customised pressure sensor. At six months, the primary outcome measures will be the HAI and the Bayley Scales of Infant and Toddler Development (third edition; BSID III), to measure cognitive and motor development. At eight months, the HAI will be used as well as electroencephalogram, to measure brain activity and cortical coherence. At 12 months, the primary outcome measures will again be the HAI and the BSID III. This is the primary outcome measure of the study, developed by the research team.

Grasping and Reaching Assessment of Brisbane (GRAB)
The GRAB will be performed at 14, 16 and 18 weeks post-term. Typically developing infants in Western cultures have been observed to acquire the important motor skills of reaching between three to five months of age (63,64) and grasping as early as 18 weeks (65)(66)(67). Prior to reach onset, infants have been observed to demonstrate pre-reaching movements. These movements provide infants with multimodal input about their upper limb function within their environment, and provide sensorimotor experiences that can help infants to learn how to control their upper limbs (63,(68)(69)(70).
Infants will be secured in a Baby Björn Babysitter Balance infant chair, allowing full range of motion of the arms. They will be presented with a toy at shoulder height at 75% of arm length for six trials of 30 seconds each, in the midline. Three different toys will be used in the various trials, in random order, to maintain the infant's interest in the task. A video camera will be placed to the midline at approximately 1.2 metres above the infant to ensure a full view of the infant, his/her upper limbs and the toy. The video-recordings will be edited into fragments in which the infant is manipulating the toys, and will be analysed by researchers who are masked to group allocations. The following variables will be assessed: (1) number of hand-toy contacts, (2) hand-toy contact type, (3) hand-toy contact duration with visual attention, (4) hand-toy contact duration without visual attention, and (5) number of bilateral interactions. As the GRAB has been developed by our team, we propose to establish validity and reliability (intra-rater, inter-rater and test-retest). See Figure 2 for a schematic drawing of the GRAB set up.

Pressure of Grasping (GP)
Infants will be secured in the same infant chair used in the GRAB. They will be presented with a small customised pressure sensor in the form of a cylindrically shaped toy that allows recording of differential positive pressure. A soft foam strap is attached to the pressure sensor  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  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   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   22 to secure the infants' hand. Pressure will be continuously sampled at a minimum rate of 20 Hz and stored on a PC compatible computer for further analysis. Each hand will be approached separately, using the pressure sensor. One trial for each hand will be performed, in a random order. The recording will begin as soon as the infant grasps the pressure sensor will continue for 120 seconds, unless the infant drops it. In that case, the trial will be repeated. The assessment will be video-recorded, synchronising the images with the activity of the pressure sensor. Grasp pressure will be assessed by the time series of positive hand pressure (expressed in Volts) corresponding to the selected video fragments will be analysed.

Hand Assessment of Infants (HAI)
The HAI is a new assessment tool which aims to quantify hand function from two to eight months post-term. It will be performed at 14, 16 and 18 weeks, and again at six months post-term age. The scale was developed at the Karolinska Institute of Stockholm (Sweden; Prof Eliasson and Prof Sundholm) in collaboration with the University of Pisa. It is currently at the phase of standardisation in a normal population. The assessment is based on a videorecorded play session, which should be completed in approximately ten minutes. Upper limb movements, reaching and grasping will be elicited by presenting the infants with toys. The toys are designed to promote exploration and handling and are presented in various places (e.g. both sides, midline, close to the baby and at a distance) on multiple occasions, both from the assessor's hand and, when possible, on the table. The scale consists of 40 items and includes both unimanual and bimanual tasks. Video recordings will be assessed by a researcher who is masked to group allocations.  23 The assessment of GMs based on Prechtl's method of observation is largely used as a diagnostic tool for neurological evaluation of the newborn and the young infant (71)(72)(73). The GMs has shown a high predictive value for neurodevelopmental outcome at 12 to 24 months for at-risk infants (e.g. brain lesion, CP, preterm); sensitivity is ≥ 92% and specificity is ≥ 82%, p < 0.01 (74). The GMs has greater sensitivity in predicting CP than other motor assessments used in infancy (74). It involves assessing the quality of spontaneous motility using a short video-recording. Video-recordings will be performed at 9, 12, 14, 16 and 18 weeks CA. Video recordings will be performed for five minutes and then one additional minute to focus on each hand. The video-camera will be positioned in the midline approximately one metre above the infant, at an angle of 45°. Infants will be recorded during while the infant is in a calm, alert state at inter-feeding time, in a supine position and clothed with wrists and ankles exposed. The analysis of the GMs will be performed by one of the certified GMs assessors participating to the study, masked to group allocations.

Assessment of Imitation (AI)
All infants will be tested for simple gestural and vocal imitation on two separate occasions, pre-and post-training, at nine and 12 weeks post-term age. This assessment will determine whether individual infants have a reliable imitative response, which may be important in interpreting the intervention results, as individual differences between infants are   24 action) and index finger pointing; and (c) two vocal gestures: "EEE," "OOO", as well as tongue clicks. The order of presentation for the gestures will be randomised across infants.
The assessment will be video-recorded.
A trained coder, masked to group allocations for the entire duration of the study, will score imitation from the videotapes. The coder will view footage of the infants' behaviour during the assessment, and record frequencies for each of the gestures listed above. These frequencies will be interpreted relative to the gestures that were modelled. Imitation is evident when infants' production of a gesture is significantly greater in response to a matching gesture, than to any other gesture.

Bayley Scales of Infant and Toddler Development (third edition; BSID III)
The BSID III (Bayley-III Clinical Use and Interpretation. Saint Louis, MO, USA: Elsevier Science & Technology, 2010) will be performed at six and 12 months corrected age.
These time points were chosen as: (a) reaching and grasping are expected to be established by 6 months of age; (b) bimanual manipulation is expected to be established by 12 months of age. The BSID III will be used to assess cognitive and motor development. It is a frequently used standardized developmental assessment throughout Australia; however its clinical utility in various populations of children has not yet been established (82). It will consist of a series of simple interactions with the infant and will take between 50 and 80 minutes to administer.

Electroencephalogram (EEG)
This test will be performed at eight months post-term and will last approximately 25 minutes. EEG is a standard method used in infants to measure brain activity and will be used in this study to explore possible brain functional correlates of motor development. EEG demonstrates mu rhythm suppression, which is considered to be a possible index of mirror neuron activity during observation and execution of hand actions (84,85). We will use a certified advanced system extensively used in infant testing, known as the Geodesic Sensor Net. It consists of a high-density net, which is applied in a few seconds.
We have tested modifications of mu rhythm using independent component analysis (ICA) of high-density EEG recordings, according to the paradigm used by Nyström and colleagues (86). ICA is a blind source separation technique that aims to find components that are most statistically independent of each other. The mu rhythm is expected to decompose into one or a few components from each subject and these are the only components useful for the analysis. We will have three different conditions for the infants to perform, from which mu rhythm activation will be estimated: (1) observe a static human model (baseline); (2) move his/her hand by reaching for and grasping an object (goal-directed action); and (3) move his/her hand by placing it on the table (non-goal-directed action). The difference in mu rhythm activation between the baseline and the two movement conditions will be used for the selection of EEG sources and the difference between the two movement conditions will be analysed.

Analyses
Analyses will be conducted on an intention-to-treat basis using STATA 11. Data from each outcome measure will be summarised for each treatment group and descriptive statistics (frequencies, means, medians, 95% confidence intervals) calculated dependent on data distribution. A significance level of 0.05 will be used. The effects of action observation training on development of reaching and grasping (Hypotheses 1 to 3) will be explored by a two-way repeated-measures analysis of variance (ANOVA) for parametric variables, including duration of hand-toy contact, maximum and minimum pressure, and pressure variance. Correction for multiple comparisons will be applied. The Kruskal-Wallis statistic will be used for non-parametric measures, including the number of hand-toy contact and the IHA score. To test the possible influence of GMs quality and imitative behaviour (Hypothesis 4), these will be considered as covariates in a multifactorial analysis. The results of EEG signal analysis will be compared between the two groups of each cohort using parametric tests. Post hoc analyses will be undertaken to investigate clinical characteristics of infants who have a greater response to either intervention.

Discussion
This paper outlines the background and design for two parallel randomised controlled trials with an identical sham control, comparing Action Observation Training (AOT) with standard Toy Observation Training (TOT) to: (a) influence the early development of reaching and grasping of typically developing infants, and (b) improve the upper limb motor activity of infants with asymmetric brain lesions. To our knowledge this study is the first to directly compare the two approaches for this population. Furthermore, we will be establishing validity and reliability for the newly developed outcome measures. AG, VB, FC and GT are responsible for recruitment, data collection and implementation of the partner investigation in Italy. AG is responsible for the design, implementation and data collection in Italy and analysis of the EEG component of the study for infants with asymmetric brain lesions. KP will be involved with the EEG data collection in Queensland.

Reported on page No
Title and abstract 1a Identification as a randomised trial in the title 1 1b Structured summary of trial design, methods, results, and conclusions (for specific guidance see CONSORT for abstracts) 4

Introduction
Background and objectives 2a Scientific background and explanation of rationale 7-16 2b Specific objectives or hypotheses 13-16

Trial design 3a
Description of trial design (such as parallel, factorial) including allocation ratio 2 3b Important changes to methods after trial commencement (such as eligibility criteria), with reasons 17-18 Participants 4a Eligibility criteria for participants 17-18 4b Settings and locations where the data were collected 17 Interventions 5 The interventions for each group with sufficient details to allow replication, including how and when they were actually administered

19-21
Outcomes 6a Completely defined pre-specified primary and secondary outcome measures, including how and when they were assessed

21-27 6b
Any changes to trial outcomes after the trial commenced, with reasons N/A Sample size 7a How sample size was determined 18 7b When applicable, explanation of any interim analyses and stopping guidelines n/A Randomisation: Sequence generation 8a Method used to generate the random allocation sequence 18 8b Type of randomisation; details of any restriction (such as blocking and block size) 18 Allocation concealment mechanism 9 Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned 18 Implementation 10 Who generated the random allocation sequence, who enrolled participants, and who assigned participants to interventions 18 Blinding 11a If done, who was blinded after assignment to interventions (for example, participants, care providers, those 18

BMJ Open
Key words: infants, early brain injury, asymmetric brain lesions, congenital hemiplegia, cerebral palsy, stroke, randomised controlled trial, upper limb rehabilitation, brain (re)organisation, mirror neuron system, mirror neurons, imitation, action observation, upper limb motor development, electroencephalogram. Infants will be randomised to receive either Action Observation (AOT) or standard Toy Observation Training (TOT). Both interventions will be of four weeks' duration, from the infant's 9 th to 13 th post-term week of age. Three sessions of five minutes will be performed each day for six days per week (total of six hours over 28 days). Parents of the AOT group will repeatedly show the infant a grasping action on a set of three toys, presented in random order. Parents of the TOT group will show the infant the same set of three toys, in random order, without demonstrating the grasping action. At 14, 16 and 18 weeks, quantity and quality of reaching and grasping will be measured using the Grasping and Reaching Assessment of Brisbane (GRAB); symmetry of reaching and grasping will be measured using

Background
Infants with asymmetric brain lesions (e.g. intraventricular haemorrhages, periventricular leukomalacia, arterial strokes and venous infarctions occurring on one side or more involved on one side of the brain) are at high risk of developing congenital hemiplegia by the end of their first year of life. The incidence of asymmetric brain lesions at birth is 1-2 of every 1000 newborns (1).
Congenital hemiplegia is the most common type of Cerebral Palsy (CP), with a prevalence of 1 in 1300 live births (1). The economic impact of CP is substantial. In 2007, the financial cost of CP was estimated at Aus$1.47 billion, with $124.1 million of that cost directly attributed to intervention costs (2). Approximately 43% of these costs are covered by the families of individuals with CP, with the remaining 57% by various levels of government (2).
The main focus of early intervention for infants with asymmetric brain lesions who may progress to classification of unilateral CP is very early and accurate detection of the  may not show signs of stroke or asymmetric brain injury until three to seven months of age, when unilateral weakness and early hand preference start to manifest (4,5).
The current most predictive tools for early diagnosis of CP are a combination of brain magnetic resonance imaging (MRI) at term and a Prechtl's Assessment of General Movements (GMs) in the fidgety period (at 12 weeks post-term; 6). Specifically, GMs at one month and three months post-term age are highly associated with white matter abnormalities on MRI at term age (7). The GMs is a well validated and reliable tool, and is more sensitive at predicting CP than other motor assessments used in infancy (8,9). The GMs is also useful for prediction of minor motor difficulties (10). Neuromotor assessments (such as the GMs) utilised in the neonatal period (< 4 months post-term) have strong validity to detect CP in infants born preterm, when correlated with criterion assessments at 12 months corrected age (such as the BSID III; 8). Although abnormalities in general movements are likely to be evident during the early writhing period (<6-9 weeks post-term) and the fidgety period (9-20 weeks post-term), asymmetries are only visible during the fidgety period (11)(12)(13).
Asymmetries in fidgety GMs around 12 weeks post-term can be the first definitive clinical sign of hemiplegia (11)(12)(13).
Very early detection of hemiparesis frequently requires serial evaluation of subtle signs of interlimb differences or asymmetries in upper limb reaching (both spontaneous and purposeful), and grasp strength (14). Both bimanual and unimanual reaching with early strong hand preference at four to six months of age can be considered to be a strong sign of early hemiplegia (4). Studies of infants who have sustained an early perinatal stroke before four to seven months corrected age have suggested that until reach to grasp behaviours have emerged, an asymmetry may not be clearly evident and hemiparesis not confirmed (15,16).
Early intervention for infants at risk of developing congenital hemiplegia is considered to be very important, however, standard rehabilitation programs generally commence after six months of age due to delayed detection. A further consideration regarding the timing of commencement of intervention is that important phases of brain reorganisation may have already occurred (17,18).
Current approaches to rehabilitation in congenital hemiplegia in infants focus on toy presentation and sensory stimulation of the limb to encourage spontaneous reaching and grasping, however, the challenge is to obtain active movement from the impaired limb. A new approach utilising action observation to stimulate the mirror neuron system offers another opportunity to stimulate the damaged motor cortex before the infant has achieved volitional reach and grasp.

Theoretical Framework
The There is a growing body of neurophysiological and brain-imaging studies providing indirect evidence for the existence of the MNS in humans (24). Transcranial Magnetic Stimulation (TMS) studies have concluded that the MNS exists in humans and it differs to the MNS in monkeys (25)(26)(27)(28)(29). Non-purposeful and intransitive actions activate mirror neurons in humans and not in monkeys (26,28,30). When humans observe actions, the temporal features of cortical excitability suggest that the MNS codes for the whole action as well as the   (24). In humans, the two mirror areas receive afferent input from the superior temporal sulcus (involved in processing motion), and send efferent input to the motor cortex (46,47).
Demonstration of the MNS soon after birth introduces a new perspective to the treatment of infants with congenital brain injury. Emerging evidence from the basic sciences in infant rhesus macaque monkeys suggests that the immature MNS can facilitate the imitative capabilities of infants; and that consistent demonstration of imitative skills and subsequent manual skills can predict later motor development (53,54).

Sensorimotor reorganisation after early brain injury
It is well known that brain injuries impacting on the sensorimotor (SM) system may manifest in varying degrees of functional impairment, the extent of which is related to the size and site of the lesion, as well as the type of adaptive reorganisation that follows. The main mechanism for a reconnection of the motor cortex to the spinal cord consists of  This alternative mechanism results in the undamaged hemisphere directly controlling both upper limbs, which is a pattern of reorganisation unknown to adult pathology (i.e. contralesional reorganisation (55).
Emerging evidence in humans suggests that the pattern of SM reorganisation after early brain injury is determined during the first year of life, and possibly within the first few months (56). As children with reorganisation occurring in the damaged hemisphere (which results in the undamaged hemisphere directly controlling both upper limbs) have suboptimal upper limb motor activity, this pattern appears to be maladaptive (55). It has been suggested that the MNS may influence cortical reorganisation associated with upper limb impairment, and could potentially be a target of very early intervention (11).

Action Observation and Imitation
The process of observing an action (i.e. action observation) leads to activation of the MNS and stimulates the corticospinal system (motor pathways) prior to imitating the action (38,(57)(58)(59). When the motor cortex is damaged (e.g. congenital brain lesion), action observation and imitation may influence cortical reorganisation by directly restoring the damaged motor pathways or reinforcing other pathways that originally helped to perform motor actions, or both (60).
In animal and human adult studies, action observation appears to activate the MNS and enhance excitability of the SM cortex (61). These findings suggest that the effects of an  The authors concluded that current evidence for very early upper limb interventions suggested small effects on unimanual capacity, bimanual coordination and self-care skills; however, there is limited data on the safety and the neural mechanisms underlying activity changes in response to these interventions. Further research is required to investigate the efficacy of upper limb interventions of this at-risk population at preschool age (<3 years) and address the lack of attention to safety implications for infants.

Proposed intervention and justification: why UP-BEAT?
Action observation therapy is a novel upper limb rehabilitation approach based on the recent discovery of mirror neurons (62). This approach has been shown to effectively    14 both term born and preterm infants a parallel healthy term born clinical trial is planned. As the provision of Action Observation Training and Toy Observation Training in the developmental period of 9-18 weeks post term are considered to be low risk and developmentally appropriate training approaches there is no risk but potentially some additional benefit for all infants. Inclusion of a cohort of healthy term born infants in parallel randomised comparison trial will provide a typically developing comparison of training approaches to our RCT of infants with asymmetric brain injury.

Broad aim of proposed study
The broad aim of this study is to evaluate in two parallel randomised controlled trials with an identical sham control, whether the novel intervention Action Observation Training (AOT) is more effective than standard Toy Observation Training (TOT) in: (i) influencing the early development of reaching and grasping of typically developing infants (n = 40), and ii) improving the upper limb motor activity of infants with asymmetric brain lesions (n = 32).

Methods
Two randomised controlled trials (RCTs) with an identical sham control will be conducted to evaluate the efficacy of AOT compared to standard TOT in: (a) typically developing infants with a gestational age between 38 and 41 weeks at time of recruitment (n = 40); and (b) infants with asymmetric brain lesions aged 0 to 9 post-term weeks at time of recruitment (n = 32).
This study will involve an at-risk population of infants (i.e. infants with asymmetric brain lesions). It would therefore be unethical to give no intervention to this population in the control arm. Standard TOT is the sham control intervention that will be used for both RCTs.
It is similar to standard therapy as it involves the parents presenting toys to infants and The specific hypotheses to be tested are:

2.
To determine if infants with asymmetric brain lesions undergoing AOT will develop reaching and grasping earlier, and have greater quality and quantity of reaching and  16 grasping, compared to those undergoing standard TOT. Recent studies suggest that an early therapeutic intervention in infants with asymmetric brain injury should aim to activate the impaired motor cortex. We will determine through an RCT if a 4-week AOT program (from 9 to 13 post-term weeks' age) will lead to cortical activation associated with action observation and influence the development of reaching and grasping in these infants and improve their short-and long-term outcomes. AOT will again be compared with standard TOT. If we can show that this novel, very early intervention can improve short-term and long-term upper limb motor activity in infants with asymmetric brain lesions, this will guide clinical practice and enable more efficient allocation of therapy resources in the future.

Study sample and recruitment
Infants and their families will be recruited within a 50-200 km radius from The Royal Children's Hospital, Brisbane, Australia. The recruitment process will target major metropolitan health districts across southeast Queensland, with the expectation that the cohort I sample will be representative of typically developing infants and the cohort II sample will be representative of infants with asymmetric brain lesions from Queensland. Cohort II: Infants with asymmetric Brain Injury (ABI) will include infants: 1. With an asymmetric (one sided or more involved on one side) or unilateral (one sided) brain injury (e.g. preterm or term arterial stroke, grade III or IV intraventricular haemorrhage, periventricular leukomalacia) identified on neonatal ultrasound or MRI; 2. Aged 0 to 9 post-term weeks at time of recruitment; 3. Living within a 200 km radius of the Royal Children's Hospital, Brisbane.
A parallel clinical trial will admit infants with the same inclusion criteria into two parallel RCTs at the Stella Maris Scientific Institute in Pisa, Italy.

Exclusion criteria
Cohort I (TDI) will exclude infants: 1. With any post-natal medical complications (e.g. jaundice) requiring extended hospital admission or medical treatments.

Cohort II (ABI) will exclude infants:
3. With epileptic seizures unresponsive to treatment. Cohort II (ABI): Sample size for cohort II was based on the assumption that the effect size of the proposed training will be similar to that found by Heathcock and colleagues (14) in a population of pre-term infants (n = 26) with a comparable training program, with a mean effect size of 2.4. Given the high effect size, the calculation returned a sample of only 4 participants per group, on a 2-tailed t-test, significance (alpha) level of 0.05 and 80% power.
As our cohort II population will consist of two sub-groups of lesion type (i.e. arterial stroke and venous infarction) and will be highly variable with the presence of asymmetric brain injury; we have quadrupled the sample. We require 16 participants in each group (total sample of 32) for cohort II.

Randomisation
The allocation sequence will be comprised of computer-generated random numbers in a blocked design. Infants will be randomised to receive either Action Observation Training (AOT) or standard Toy Observation Training (TOT), from concealed envelopes opened by non-study personnel. Treatment allocation will be recorded on a piece of folded paper inside each envelope in random order (computer generated). The randomisation process will involve allocating a code to each infant, which consists of the letter 'B' or 'G' according to gender and a number based on date of birth (e.g. "B1", "B2" and "G1", "G2"). The infant's name and code will be written on the paper inside the envelope and sealed. The envelope will be marked that it has been allocated and the infant's code will be written on the front of the envelope. As each infant is entered, he/she will be allocated the next consecutive envelope, which will be opened by the non-study personnel who will read and record the treatment  20 allocation from the paper inside the envelope. The randomisation envelopes will be held and administered by the therapist providing training of the interventions for the parents.

Blinding
The therapists who will be training the parents in the interventions and the parents will be informed of group allocation; the therapists conducting the assessments will be masked to group allocation; and study personnel who will be assessing the outcomes will also be masked to group allocation. Randomised group allocation will remain concealed to the therapists who conducted the assessments until all data for the entire sample has been analysed.

Study treatments
Both cohorts will receive the same dosage of three 5-minute sessions (15 minutes per day) for 6 days per week, for 4 weeks. The total dosage of intervention will be 6 hours, over a period of 28 days. After baseline screening and randomisation, infants will receive either AOT or standard TOT.
Parents will be trained by an occupational therapist for approximately 30 minutes, and will be directly observed performing the training activities with the infant during training.
Two follow-up phone calls regarding questions on how to perform the training will be addressed over the telephone with the therapist who trained the parents. Parents will be asked to video-record the sessions each day. Parents of the AOT group will repeatedly show the infant a grasping action on a set of three toys, presented in random order. Parents of the TOT group will show the infant the same set of three toys, also presented in random order, without the grasping action. The toys are mostly cylindrical in shape and vary in appearance, colour  21 and patterns (i.e. cow, clown and musical instrument). Groups will be compared at 14, 16 and 18 weeks, as well as 6 months and 12 months following intervention.
To optimise comfort and convenience for their families, intervention training for parents, delivery of intervention and all assessments from 9 to 18 weeks will be performed in

Therapy protocols and delivery
Several occupational therapists will plan and conduct both intervention groups. These core therapists will be responsible for liaising with the parents to organise home visits to train the parents in their allocated interventions and for each set of assessments at 9, 12, 14, 16, 18 weeks corrected age (CA). The core therapists will also be responsible for organising the 6 and 12 month follow-up assessments at the Royal Children's Hospital, Brisbane. Two other occupational therapists will provide training and follow-up phone calls for the parents.
An online diary that will only be accessible to the core therapists will be completed after each training session, follow-up phone call, home visit and follow-up assessment to summarise each activity for each infant. Any issues of concern such as difficulties with training and adverse events will be considered when data is analysed, as potential factors that may account for differences between cohorts. Video footage of each training and assessment session will be qualitatively and/or quantitatively analysed to assess treatment fidelity.
The core investigator team (RB, JZ, AG, KP, LF, MP) will meet regularly to review the progress of training and assessments for both cohorts, and will decide when any modifications to the protocol are required. The alternate training program (AOT, TOT) is will be recorded at the next home visit and accounted for in secondary analysis. Parents in either study will be free to discontinue the training and exit the study if they wish and there would be no impact on their access to additional medical and allied health services.

Outcome measures and procedures
At 14, 16 and 18 weeks post-term or CA, quantity and quality of reaching and grasping will be measured using the Grasping and Reaching Assessment of Brisbane (GRAB); symmetry of reaching and grasping will be measured using the Hand Assessment of Infants (HAI); and pressure of grasping for each hand with a customised pressure sensor. At six months, the primary outcome measures will be the HAI and the Bayley Scales of Infant and Toddler Development (third edition; BSID III), to measure cognitive and motor development. At eight months, the HAI will be used as well as electroencephalogram, to measure brain activity and cortical coherence. At 12 months, the primary outcome measures will again be the HAI and the BSID III.

Grasping and Reaching Assessment of Brisbane (GRAB)
This is the primary outcome measure of the study, developed by the research team. Infants will be secured in a Baby Björn Babysitter Balance infant chair, allowing full range of motion of the arms. They will be presented with a toy at shoulder height at 75% of arm length for six trials of 30 seconds each, in the midline. Three different toys will be used in the various trials, in random order, to maintain the infant's interest in the task. A video camera will be placed to the midline at approximately 1.2 metres above the infant to ensure a full view of the infant, his/her upper limbs and the toy. The video-recordings will be edited into fragments in which the infant is manipulating the toys, and will be analysed by researchers who are masked to group allocations. The following variables will be assessed: (1) number of hand-toy contacts, (2) hand-toy contact type, (3) hand-toy contact duration with visual attention, (4) hand-toy contact duration without visual attention, and (5) number of bilateral interactions. As the GRAB has been developed by our team, we propose to establish validity and reliability (intra-rater, inter-rater and test-retest). See Figure 2 for a schematic drawing of the GRAB set up.

Pressure of Grasping (GP)
Infants will be secured in the same infant chair used in the GRAB. They will be presented with a small customised pressure sensor in the form of a cylindrically shaped toy that allows recording of differential positive pressure. A soft foam strap is attached to the pressure sensor to secure the infants' hand. Pressure will be continuously sampled at a minimum rate of 20 Hz and stored on a PC compatible computer for further analysis. Each hand will be approached separately, using the pressure sensor. One trial for each hand will be performed, in a random order. The recording will begin as soon as the infant grasps the pressure sensor  24 will continue for 120 seconds, unless the infant drops it. In that case, the trial will be repeated. The assessment will be video-recorded, synchronising the images with the activity of the pressure sensor. Grasp pressure will be assessed by the time series of positive hand pressure (expressed in Volts) corresponding to the selected video fragments will be analysed.

Hand Assessment of Infants (HAI)
The HAI is a new assessment tool which aims to quantify hand function from two to eight months post-term. It will be performed at 14, 16

Prechtl's Assessment of General Movements (GMs)
The assessment of GMs based on Prechtl's method of observation is largely used as a diagnostic tool for neurological evaluation of the newborn and the young infant (71)(72)(73). The GMs has shown a high predictive value for neurodevelopmental outcome at 12 to 24 months for at-risk infants (e.g. brain lesion, CP, preterm); sensitivity is ≥ 92% and specificity is ≥ weeks CA. Video recordings will be performed for five minutes and then one additional minute to focus on each hand. The video-camera will be positioned in the midline approximately one metre above the infant, at an angle of 45°. Infants will be recorded during while the infant is in a calm, alert state at inter-feeding time, in a supine position and clothed with wrists and ankles exposed. The analysis of the GMs will be performed by one of the certified GMs assessors participating to the study, masked to group allocations.

Assessment of Imitation (AI)
All infants will be tested for simple gestural and vocal imitation on two separate occasions, pre-and post-training, at nine and 12 weeks post-term age. This assessment will determine whether individual infants have a reliable imitative response, which may be The assessment will be video-recorded.  26 A trained coder, masked to group allocations for the entire duration of the study, will score imitation from the videotapes. The coder will view footage of the infants' behaviour during the assessment, and record frequencies for each of the gestures listed above. These frequencies will be interpreted relative to the gestures that were modelled. Imitation is evident when infants' production of a gesture is significantly greater in response to a matching gesture, than to any other gesture.

Bayley Scales of Infant and Toddler Development (third edition; BSID III)
The

Electroencephalogram (EEG)
This test will be performed at eight months post-term and will last approximately 25 minutes. EEG is a standard method used in infants to measure brain activity and will be used in this study to explore possible brain functional correlates of motor development. EEG demonstrates mu rhythm suppression, which is considered to be a possible index of mirror neuron activity during observation and execution of hand actions (84,85). We will use a certified advanced system extensively used in infant testing, known as the Geodesic Sensor Net. It consists of a high-density net, which is applied in a few seconds.
We have tested modifications of mu rhythm using independent component analysis (ICA) of high-density EEG recordings, according to the paradigm used by Nyström and colleagues (86). ICA is a blind source separation technique that aims to find components that are most statistically independent of each other. The mu rhythm is expected to decompose into one or a few components from each subject and these are the only components useful for the analysis. We will have three different conditions for the infants to perform, from which mu rhythm activation will be estimated: (1) observe a static human model (baseline); (2) move his/her hand by reaching for and grasping an object (goal-directed action); and (3) move his/her hand by placing it on the table (non-goal-directed action). The difference in mu rhythm activation between the baseline and the two movement conditions will be used for the selection of EEG sources and the difference between the two movement conditions will be analysed.

Analyses
Analyses will be conducted on an intention-to-treat basis using STATA 11. Data from each outcome measure will be summarised for each treatment group and descriptive statistics including duration of hand-toy contact, maximum and minimum pressure, and pressure variance. Correction for multiple comparisons will be applied. The Kruskal-Wallis statistic will be used for non-parametric measures, including the number of hand-toy contact and the IHA score. To test the possible influence of GMs quality and imitative behaviour (Hypothesis 4), these will be considered as covariates in a multifactorial analysis. The results of EEG signal analysis will be compared between the two groups of each cohort using parametric tests. Post hoc analyses will be undertaken to investigate clinical characteristics of infants who have a greater response to either intervention.

Discussion
This paper outlines the background and design for two parallel randomised controlled trials with an identical sham control, comparing Action Observation Training (AOT) with standard Toy Observation Training (TOT) to: (a) influence the early development of reaching and grasping of typically developing infants, and (b) improve the upper limb motor activity of infants with asymmetric brain lesions. To our knowledge this study is the first to directly compare the two approaches for this population. Furthermore, we will be establishing validity and reliability for the newly developed outcome measures.

Abbreviations
AOT Action Observation Therapy ABI Asymmetric Brain Injury ANOVA Analysis of Variance AG, VB, FC and GT are responsible for recruitment, data collection and implementation of the partner investigation in Italy. AG is responsible for the design, implementation and data collection in Italy and analysis of the EEG component of the study for infants with asymmetric brain lesions. KP will be involved with the EEG data collection in Queensland.

Methods and Analysis:
This study design comprises two parallel randomised sham controlled trials (RCTs) in: (i) typically developing infants (cohort I); and (ii) infants with asymmetric brain lesions (e.g. arterial stroke, venous infarction, intraventricular haemorrhage or periventricular leukomalacia; cohort II). Cohort II will be identified through a neonatal ultrasound or neonatal Magnetic Resonance Imaging. A sham control will be used for both RCTs, in consideration that it would be unethical to give no intervention to an at-risk population. Based on a 2-tailed t-test of 2 independent means, significance (alpha) level of 0.05, 80% power, predicted effect size of 0.8 and a 90% retention rate, we require 20 participants in each group (total sample of 40) for cohort I. Sample size for cohort II was based on the assumption that the effect size of the proposed training will be similar to that  Infants will be randomised to receive either Action Observation (AOT) or standard Toy Observation Training (TOT). Both interventions will be of four weeks' duration, from the infant's 9 th to 13 th post-term week of age. Three sessions of five minutes will be performed each day for six days per week (total of six hours over 28 days). Parents of the AOT group will repeatedly show the infant a grasping action on a set of three toys, presented in random order. Parents of the TOT group will show the infant the same set of three toys, in random order, without demonstrating the grasping action. At 14, 16 and 18 weeks, quantity and quality of reaching and grasping will be measured using the Grasping and Reaching Assessment of Brisbane (GRAB); symmetry of reaching and grasping will be measured using the Hand Assessment of Infants (HAI); and pressure of grasping for each hand with a customised pressure sensor. At six months CA the primary outcome measures will be the HAI and the Bayley Scales of Infant and Toddler Development (third edition; BSID III), to measure cognitive and motor development. At eight months the HAI will be used as well as electroencephalogram, to measure brain activity and cortical coherence. At 12 months, the primary outcome measures will again be the HAI and the BSID III.   1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  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

Background
Infants with asymmetric brain lesions (e.g. intraventricular haemorrhages, periventricular leukomalacia, arterial strokes and venous infarctions occurring on one side or more involved on one side of the brain) are at high risk of developing congenital hemiplegia by the end of their first year of life. The incidence of asymmetric brain lesions at birth is 1-2 of every 1000 newborns (1).
Congenital hemiplegia is the most common type of Cerebral Palsy (CP), with a prevalence of 1 in 1300 live births (1). The economic impact of CP is substantial. In 2007, the financial cost of CP was estimated at Aus$1.47 billion, with $124.1 million of that cost directly attributed to intervention costs (2). Approximately 43% of these costs are covered by the families of individuals with CP, with the remaining 57% by various levels of government (2).
The current most predictive tools for early diagnosis of CP are a combination of brain magnetic resonance imaging (MRI) at term and a Prechtl's Assessment of General Movements (GMs) in the fidgety period (at 12 weeks post-term; 6). Specifically, GMs at one month and three months post-term age are highly associated with white matter abnormalities on MRI at term age (7). The GMs is a well validated and reliable tool, and is more sensitive at predicting CP than other motor assessments used in infancy (8,9). The GMs is also useful for prediction of minor motor difficulties (10). Neuromotor assessments (such as the GMs) utilised in the neonatal period (< 4 months post-term) have strong validity to detect CP in infants born preterm, when correlated with criterion assessments at 12 months corrected age (such as the BSID III; 8). Although abnormalities in general movements are likely to be evident during the early writhing period (<6-9 weeks post-term) and the fidgety period (9-20 weeks post-term), asymmetries are only visible during the fidgety period (11)(12)(13).
Asymmetries in fidgety GMs around 12 weeks post-term can be the first definitive clinical sign of hemiplegia (11)(12)(13).
Very early detection of hemiparesis frequently requires serial evaluation of subtle signs of interlimb differences or asymmetries in upper limb reaching (both spontaneous and purposeful), and grasp strength (14). Both bimanual and unimanual reaching with early strong hand preference at four to six months of age can be considered to be a strong sign of early hemiplegia (4). Studies of infants who have sustained an early perinatal stroke before four to seven months corrected age have suggested that until reach to grasp behaviours have emerged, an asymmetry may not be clearly evident and hemiparesis not confirmed (15,16).
Early intervention for infants at risk of developing congenital hemiplegia is considered to be very important, however, standard rehabilitation programs generally commence after six months of age due to delayed detection. A further consideration regarding the timing of commencement of intervention is that important phases of brain reorganisation may have already occurred (17,18).
Current approaches to rehabilitation in congenital hemiplegia in infants focus on toy presentation and sensory stimulation of the limb to encourage spontaneous reaching and grasping, however, the challenge is to obtain active movement from the impaired limb. A new approach utilising action observation to stimulate the mirror neuron system offers another opportunity to stimulate the damaged motor cortex before the infant has achieved volitional reach and grasp.
Demonstration of the MNS soon after birth introduces a new perspective to the treatment of infants with congenital brain injury. Emerging evidence from the basic sciences in infant rhesus macaque monkeys suggests that the immature MNS can facilitate the imitative capabilities of infants; and that consistent demonstration of imitative skills and subsequent manual skills can predict later motor development (53,54).

Sensorimotor reorganisation after early brain injury
It is well known that brain injuries impacting on the sensorimotor (SM) system may manifest in varying degrees of functional impairment, the extent of which is related to the size and site of the lesion, as well as the type of adaptive reorganisation that follows. The main mechanism for a reconnection of the motor cortex to the spinal cord consists of  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  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  This alternative mechanism results in the undamaged hemisphere directly controlling both upper limbs, which is a pattern of reorganisation unknown to adult pathology (i.e. contralesional reorganisation (55).
Emerging evidence in humans suggests that the pattern of SM reorganisation after early brain injury is determined during the first year of life, and possibly within the first few months (56). As children with reorganisation occurring in the damaged hemisphere (which results in the undamaged hemisphere directly controlling both upper limbs) have suboptimal upper limb motor activity, this pattern appears to be maladaptive (55). It has been suggested that the MNS may influence cortical reorganisation associated with upper limb impairment, and could potentially be a target of very early intervention (11).

Action Observation and Imitation
The process of observing an action (i.e. action observation) leads to activation of the MNS and stimulates the corticospinal system (motor pathways) prior to imitating the action (38,(57)(58)(59). When the motor cortex is damaged (e.g. congenital brain lesion), action observation and imitation may influence cortical reorganisation by directly restoring the damaged motor pathways or reinforcing other pathways that originally helped to perform motor actions, or both (60).
In animal and human adult studies, action observation appears to activate the MNS and enhance excitability of the SM cortex (61). These findings suggest that the effects of an The authors concluded that current evidence for very early upper limb interventions suggested small effects on unimanual capacity, bimanual coordination and self-care skills; however, there is limited data on the safety and the neural mechanisms underlying activity changes in response to these interventions. Further research is required to investigate the efficacy of upper limb interventions of this at-risk population at preschool age (<3 years) and address the lack of attention to safety implications for infants.

Proposed intervention and justification: why UP-BEAT?
Action observation therapy is a novel upper limb rehabilitation approach based on the recent discovery of mirror neurons (62). This approach has been shown to effectively    14 both term born and preterm infants a parallel healthy term born clinical trial is planned. As the provision of Action Observation Training and Toy Observation Training in the developmental period of 9-18 weeks post term are considered to be low risk and developmentally appropriate training approaches there is no risk but potentially some additional benefit for all infants. Inclusion of a cohort of healthy term born infants in parallel randomised comparison trial will provide a typically developing comparison of training approaches to our RCT of infants with asymmetric brain injury.

Broad aim of proposed study
The broad aim of this study is to evaluate in two parallel randomised controlled trials with an identical sham control, whether the novel intervention Action Observation Training (AOT) is more effective than standard Toy Observation Training (TOT) in: (i) influencing the early development of reaching and grasping of typically developing infants (n = 40), and ii) improving the upper limb motor activity of infants with asymmetric brain lesions (n = 32).

Methods
Two randomised controlled trials (RCTs) with an identical sham control will be conducted to evaluate the efficacy of AOT compared to standard TOT in: (a) typically developing infants with a gestational age between 38 and 41 weeks at time of recruitment (n = 40); and (b) infants with asymmetric brain lesions aged 0 to 9 post-term weeks at time of recruitment (n = 32).
This study will involve an at-risk population of infants (i.e. infants with asymmetric brain lesions). It would therefore be unethical to give no intervention to this population in the control arm. Standard TOT is the sham control intervention that will be used for both RCTs.
It is similar to standard therapy as it involves the parents presenting toys to infants and The specific hypotheses to be tested are:

2.
To determine if infants with asymmetric brain lesions undergoing AOT will develop reaching and grasping earlier, and have greater quality and quantity of reaching and  16 grasping, compared to those undergoing standard TOT. Recent studies suggest that an early therapeutic intervention in infants with asymmetric brain injury should aim to activate the impaired motor cortex. We will determine through an RCT if a 4-week AOT program (from 9 to 13 post-term weeks' age) will lead to cortical activation associated with action observation and influence the development of reaching and grasping in these infants and improve their short-and long-term outcomes. AOT will again be compared with standard TOT. If we can show that this novel, very early intervention can improve short-term and long-term upper limb motor activity in infants with asymmetric brain lesions, this will guide clinical practice and enable more efficient allocation of therapy resources in the future.

Study sample and recruitment
Infants and their families will be recruited within a 50-200 km radius from The Royal Children's Hospital, Brisbane, Australia. The recruitment process will target major metropolitan health districts across southeast Queensland, with the expectation that the cohort I sample will be representative of typically developing infants and the cohort II sample will be representative of infants with asymmetric brain lesions from Queensland. Cohort II: Infants with asymmetric Brain Injury (ABI) will include infants: 1. With an asymmetric (one sided or more involved on one side) or unilateral (one sided) brain injury (e.g. preterm or term arterial stroke, grade III or IV intraventricular haemorrhage, periventricular leukomalacia) identified on neonatal ultrasound or MRI; 2. Aged 0 to 9 post-term weeks at time of recruitment; 3. Living within a 200 km radius of the Royal Children's Hospital, Brisbane.
A parallel clinical trial will admit infants with the same inclusion criteria into two parallel RCTs at the Stella Maris Scientific Institute in Pisa, Italy.

Exclusion criteria
Cohort I (TDI) will exclude infants: 1. With any post-natal medical complications (e.g. jaundice) requiring extended hospital admission or medical treatments.

Cohort II (ABI) will exclude infants:
3. With epileptic seizures unresponsive to treatment. Cohort II (ABI): Sample size for cohort II was based on the assumption that the effect size of the proposed training will be similar to that found by Heathcock and colleagues (14) in a population of pre-term infants (n = 26) with a comparable training program, with a mean effect size of 2.4. Given the high effect size, the calculation returned a sample of only 4 participants per group, on a 2-tailed t-test, significance (alpha) level of 0.05 and 80% power.
As our cohort II population will consist of two sub-groups of lesion type (i.e. arterial stroke and venous infarction) and will be highly variable with the presence of asymmetric brain injury; we have quadrupled the sample. We require 16 participants in each group (total sample of 32) for cohort II.

Randomisation
The allocation sequence will be comprised of computer-generated random numbers in a blocked design. Infants will be randomised to receive either Action Observation Training (AOT) or standard Toy Observation Training (TOT), from concealed envelopes opened by non-study personnel. Treatment allocation will be recorded on a piece of folded paper inside each envelope in random order (computer generated). The randomisation process will involve allocating a code to each infant, which consists of the letter 'B' or 'G' according to gender and a number based on date of birth (e.g. "B1", "B2" and "G1", "G2"). The infant's name and code will be written on the paper inside the envelope and sealed. The envelope will be marked that it has been allocated and the infant's code will be written on the front of the envelope. As each infant is entered, he/she will be allocated the next consecutive envelope, which will be opened by the non-study personnel who will read and record the treatment  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  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 20 allocation from the paper inside the envelope. The randomisation envelopes will be held and administered by the therapist providing training of the interventions for the parents.

Blinding
The therapists who will be training the parents in the interventions and the parents will be informed of group allocation; the therapists conducting the assessments will be masked to group allocation; and study personnel who will be assessing the outcomes will also be masked to group allocation. Randomised group allocation will remain concealed to the therapists who conducted the assessments until all data for the entire sample has been analysed.

Study treatments
Both cohorts will receive the same dosage of three 5-minute sessions (15 minutes per day) for 6 days per week, for 4 weeks. The total dosage of intervention will be 6 hours, over a period of 28 days. After baseline screening and randomisation, infants will receive either AOT or standard TOT.
Parents will be trained by an occupational therapist for approximately 30 minutes, and will be directly observed performing the training activities with the infant during training.
Two follow-up phone calls regarding questions on how to perform the training will be addressed over the telephone with the therapist who trained the parents. Parents will be asked to video-record the sessions each day. Parents of the AOT group will repeatedly show the infant a grasping action on a set of three toys, presented in random order. Parents of the TOT group will show the infant the same set of three toys, also presented in random order, without the grasping action. The toys are mostly cylindrical in shape and vary in appearance, colour  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  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 21 and patterns (i.e. cow, clown and musical instrument). Groups will be compared at 14, 16 and 18 weeks, as well as 6 months and 12 months following intervention.
To optimise comfort and convenience for their families, intervention training for parents, delivery of intervention and all assessments from 9 to 18 weeks will be performed in the infants' home environment. To optimise the infant's engagement in the interventions, parents will be advised to: (a) perform the training when the infant is calm and alert; (b) wiggle their fingers to engage the infant's attention prior to commencing the training; and (c) stop the training, allow the infant to play briefly with the toys if the infant becomes distracted or stops attending to the parent's hand and toy, before continuing the training.

Therapy protocols and delivery
Several occupational therapists will plan and conduct both intervention groups. These core therapists will be responsible for liaising with the parents to organise home visits to train the parents in their allocated interventions and for each set of assessments at 9, 12, 14, 16, 18 weeks corrected age (CA). The core therapists will also be responsible for organising the 6 and 12 month follow-up assessments at the Royal Children's Hospital, Brisbane. Two other occupational therapists will provide training and follow-up phone calls for the parents.
An online diary that will only be accessible to the core therapists will be completed after each training session, follow-up phone call, home visit and follow-up assessment to summarise each activity for each infant. Any issues of concern such as difficulties with training and adverse events will be considered when data is analysed, as potential factors that may account for differences between cohorts. Video footage of each training and assessment session will be qualitatively and/or quantitatively analysed to assess treatment fidelity.
The core investigator team (RB, JZ, AG, KP, LF, MP) will meet regularly to review the progress of training and assessments for both cohorts, and will decide when any modifications to the protocol are required. The alternate training program (AOT, TOT) is  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  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  will be recorded at the next home visit and accounted for in secondary analysis. Parents in either study will be free to discontinue the training and exit the study if they wish and there would be no impact on their access to additional medical and allied health services.

Outcome measures and procedures
At 14, 16 and 18 weeks post-term or CA, quantity and quality of reaching and grasping will be measured using the Grasping and Reaching Assessment of Brisbane (GRAB); symmetry of reaching and grasping will be measured using the Hand Assessment of Infants (HAI); and pressure of grasping for each hand with a customised pressure sensor. At six months, the primary outcome measures will be the HAI and the Bayley Scales of Infant and Toddler Development (third edition; BSID III), to measure cognitive and motor development. At eight months, the HAI will be used as well as electroencephalogram, to measure brain activity and cortical coherence. At 12 months, the primary outcome measures will again be the HAI and the BSID III.

Grasping and Reaching Assessment of Brisbane (GRAB)
This is the primary outcome measure of the study, developed by the research team.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  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  Infants will be secured in a Baby Björn Babysitter Balance infant chair, allowing full range of motion of the arms. They will be presented with a toy at shoulder height at 75% of arm length for six trials of 30 seconds each, in the midline. Three different toys will be used in the various trials, in random order, to maintain the infant's interest in the task. A video camera will be placed to the midline at approximately 1.2 metres above the infant to ensure a full view of the infant, his/her upper limbs and the toy. The video-recordings will be edited into fragments in which the infant is manipulating the toys, and will be analysed by researchers who are masked to group allocations. The following variables will be assessed: (1) number of hand-toy contacts, (2) hand-toy contact type, (3) hand-toy contact duration with visual attention, (4) hand-toy contact duration without visual attention, and (5) number of bilateral interactions. As the GRAB has been developed by our team, we propose to establish validity and reliability (intra-rater, inter-rater and test-retest). See Figure 2 for a schematic drawing of the GRAB set up.

Pressure of Grasping (GP)
Infants will be secured in the same infant chair used in the GRAB. They will be presented with a small customised pressure sensor in the form of a cylindrically shaped toy that allows recording of differential positive pressure. A soft foam strap is attached to the pressure sensor to secure the infants' hand. Pressure will be continuously sampled at a minimum rate of 20 Hz and stored on a PC compatible computer for further analysis. Each hand will be approached separately, using the pressure sensor. One trial for each hand will be performed, in a random order. The recording will begin as soon as the infant grasps the pressure sensor  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  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 24 will continue for 120 seconds, unless the infant drops it. In that case, the trial will be repeated. The assessment will be video-recorded, synchronising the images with the activity of the pressure sensor. Grasp pressure will be assessed by the time series of positive hand pressure (expressed in Volts) corresponding to the selected video fragments will be analysed.

Hand Assessment of Infants (HAI)
The HAI is a new assessment tool which aims to quantify hand function from two to eight months post-term. It will be performed at 14, 16 and 18 weeks, and again at six months post-term age. The scale was developed at the Karolinska Institute of Stockholm (Sweden; Prof Eliasson and Prof Sundholm) in collaboration with the University of Pisa. It is currently at the phase of standardisation in a normal population. The assessment is based on a videorecorded play session, which should be completed in approximately ten minutes. Upper limb movements, reaching and grasping will be elicited by presenting the infants with toys. The toys are designed to promote exploration and handling and are presented in various places (e.g. both sides, midline, close to the baby and at a distance) on multiple occasions, both from the assessor's hand and, when possible, on the table. The scale consists of 40 items and includes both unimanual and bimanual tasks. Video recordings will be assessed by a researcher who is masked to group allocations.

Prechtl's Assessment of General Movements (GMs)
The assessment of GMs based on Prechtl's method of observation is largely used as a diagnostic tool for neurological evaluation of the newborn and the young infant (71)(72)(73). The GMs has shown a high predictive value for neurodevelopmental outcome at 12 to 24 months for at-risk infants (e.g. brain lesion, CP, preterm); sensitivity is ≥ 92% and specificity is ≥  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  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  weeks CA. Video recordings will be performed for five minutes and then one additional minute to focus on each hand. The video-camera will be positioned in the midline approximately one metre above the infant, at an angle of 45°. Infants will be recorded during while the infant is in a calm, alert state at inter-feeding time, in a supine position and clothed with wrists and ankles exposed. The analysis of the GMs will be performed by one of the certified GMs assessors participating to the study, masked to group allocations.

Assessment of Imitation (AI)
All infants will be tested for simple gestural and vocal imitation on two separate occasions, pre-and post-training, at nine and 12 weeks post-term age. This assessment will determine whether individual infants have a reliable imitative response, which may be The assessment will be video-recorded.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  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 26 A trained coder, masked to group allocations for the entire duration of the study, will score imitation from the videotapes. The coder will view footage of the infants' behaviour during the assessment, and record frequencies for each of the gestures listed above. These frequencies will be interpreted relative to the gestures that were modelled. Imitation is evident when infants' production of a gesture is significantly greater in response to a matching gesture, than to any other gesture.

Bayley Scales of Infant and Toddler Development (third edition; BSID III)
The BSID III (Bayley-III Clinical Use and Interpretation. Saint Louis, MO, USA: Elsevier Science & Technology, 2010) will be performed at six and 12 months corrected age.
These time points were chosen as: (a) reaching and grasping are expected to be established by 6 months of age; (b) bimanual manipulation is expected to be established by 12 months of age. The BSID III will be used to assess cognitive and motor development. It is a frequently used standardized developmental assessment throughout Australia; however its clinical utility in various populations of children has not yet been established (82). It will consist of a series of simple interactions with the infant and will take between 50 and 80 minutes to administer.

Electroencephalogram (EEG)
This test will be performed at eight months post-term and will last approximately 25 minutes. EEG is a standard method used in infants to measure brain activity and will be used in this study to explore possible brain functional correlates of motor development. EEG demonstrates mu rhythm suppression, which is considered to be a possible index of mirror neuron activity during observation and execution of hand actions (84,85). We will use a certified advanced system extensively used in infant testing, known as the Geodesic Sensor Net. It consists of a high-density net, which is applied in a few seconds.
We have tested modifications of mu rhythm using independent component analysis (ICA) of high-density EEG recordings, according to the paradigm used by Nyström and colleagues (86). ICA is a blind source separation technique that aims to find components that are most statistically independent of each other. The mu rhythm is expected to decompose into one or a few components from each subject and these are the only components useful for the analysis. We will have three different conditions for the infants to perform, from which mu rhythm activation will be estimated: (1) observe a static human model (baseline); (2) move his/her hand by reaching for and grasping an object (goal-directed action); and (3) move his/her hand by placing it on the table (non-goal-directed action). The difference in mu rhythm activation between the baseline and the two movement conditions will be used for the selection of EEG sources and the difference between the two movement conditions will be analysed.

Analyses
Analyses will be conducted on an intention-to-treat basis using STATA 11. Data from each outcome measure will be summarised for each treatment group and descriptive statistics including duration of hand-toy contact, maximum and minimum pressure, and pressure variance. Correction for multiple comparisons will be applied. The Kruskal-Wallis statistic will be used for non-parametric measures, including the number of hand-toy contact and the IHA score. To test the possible influence of GMs quality and imitative behaviour (Hypothesis 4), these will be considered as covariates in a multifactorial analysis. The results of EEG signal analysis will be compared between the two groups of each cohort using parametric tests. Post hoc analyses will be undertaken to investigate clinical characteristics of infants who have a greater response to either intervention.

Discussion
This paper outlines the background and design for two parallel randomised controlled trials with an identical sham control, comparing Action Observation Training (AOT) with standard Toy Observation Training (TOT) to: (a) influence the early development of reaching and grasping of typically developing infants, and (b) improve the upper limb motor activity of infants with asymmetric brain lesions. To our knowledge this study is the first to directly compare the two approaches for this population. Furthermore, we will be establishing validity and reliability for the newly developed outcome measures.

Introduction
Background and objectives 2a Scientific background and explanation of rationale 7-16 2b Specific objectives or hypotheses 13-16

Methods
Trial design 3a Description of trial design (such as parallel, factorial) including allocation ratio 2 3b Important changes to methods after trial commencement (such as eligibility criteria), with reasons 17-18 Participants 4a Eligibility criteria for participants 17-18 4b Settings and locations where the data were collected 17 Interventions 5 The interventions for each group with sufficient details to allow replication, including how and when they were actually administered

19-21
Outcomes 6a Completely defined pre-specified primary and secondary outcome measures, including how and when they were assessed 21-27 6b Any changes to trial outcomes after the trial commenced, with reasons N/A Sample size 7a How sample size was determined 18 7b When applicable, explanation of any interim analyses and stopping guidelines n/A Randomisation: Sequence generation 8a Method used to generate the random allocation sequence 18 8b Type of randomisation; details of any restriction (such as blocking and block size) 18 Allocation concealment mechanism 9 Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned 18 Implementation 10 Who generated the random allocation sequence, who enrolled participants, and who assigned participants to interventions 18 Blinding 11a If done, who was blinded after assignment to interventions (for example, participants, care providers, those 18  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  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 assessing outcomes) and how 11b If relevant, description of the similarity of interventions 19-20 Statistical methods 12a Statistical methods used to compare groups for primary and secondary outcomes 27-28 12b Methods for additional analyses, such as subgroup analyses and adjusted analyses 27-28

Results
Participant flow (a diagram is strongly recommended) 13a For each group, the numbers of participants who were randomly assigned, received intended treatment, and were analysed for the primary outcome Sources of funding and other support (such as supply of drugs), role of funders 30 *We strongly recommend reading this statement in conjunction with the CONSORT 2010 Explanation and Elaboration for important clarifications on all the items. If relevant, we also recommend reading CONSORT extensions for cluster randomised trials, non-inferiority and equivalence trials, non-pharmacological treatments, herbal interventions, and pragmatic trials.
Additional extensions are forthcoming: for those and for up to date references relevant to this checklist, see www.consort-statement.org.