Systematic Review Of Neuroimaging Studies Research Articles

Unveiling the hippocampal subfield changes across the Alzheimer's disease continuum: a systematic review of neuroimaging studies.

Studies exploring the hippocampal subfield atrophy in Alzheimer's disease (AD) have shown contradictory results. This review aims to disentangle such heterogeneity by investigating the dynamic changes of hippocampal subfields across the AD continuum. We systematically searched the PubMed and EMBASE databases for case-control studies. Selected studies included investigations of biomarker-based amyloid status and reported data on hippocampal subfield atrophy using advanced MRI techniques. Twelve studies were included. Despite high heterogeneity, a distinguishable pattern of vulnerability of hippocampal subfields can be recognized from the cognitively unimpaired phase to the dementia stage, shedding light on hippocampal changes with disease progression. Consistent findings revealed atrophy in the subiculum and presubiculum, along with a potential increase in volume in the cornu ammonis (CA) among the cognitively unimpaired group, a feature not observed in patients experiencing subjective cognitive decline. Atrophy in the subiculum, presubiculum, CA 1-4, and the dentate gyrus characterized the mild cognitive impairment stage, with a more pronounced severity in the progression to dementia.

Effects of systemic lupus erythematosus on the brain: a systematic review of structural MRI findings and their relationships with cognitive dysfunction

BackgroundCognitive dysfunction (CD) is highly prevalent in systemic lupus erythematosus (SLE), yet the underlying mechanisms are poorly understood. Neuroimaging utilising advanced MRI metrics may yield mechanistic insights. We conducted a...

The Association Between Chronic Tobacco Smoking and Brain Alterations in Schizophrenia: A Systematic Review of Magnetic Resonance Imaging Studies.

The high co-occurrence of tobacco smoking in patients with schizophrenia spectrum disorders (SSD) poses a serious health concern, linked to increased mortality and worse clinical outcomes. The mechanisms underlying this co-occurrence are not fully understood. Addressing the need for a comprehensive overview of the impact of tobacco use on SSD neurobiology, we conducted a systematic review of neuroimaging studies (including structural, functional, and neurochemical magnetic resonance imaging studies) that investigate the association between chronic tobacco smoking and brain alterations in patients with SSD. Eight structural and fourteen functional studies were included. Structural studies show widespread independent and additive reductions in gray matter in relation to smoking and SSD. The majority of functional studies suggest that smoking might be associated with improvements in connectivity deficits linked to SSD. However, the limited number of and high amount of cross-sectional studies, and high between-studies sample overlap prevent a conclusive determination of the nature and extent of the impact of smoking on brain functioning in patients with SSD. Overall, functional results imply a distinct neurobiological mechanism for tobacco addiction in patients with SSD, possibly attributed to differences at the nicotinic acetylcholine receptor level. Our findings highlight the need for more longitudinal and exposure-dependent studies to differentiate between inherent neurobiological differences and the (long-term) effects of smoking in SSD, and to unravel the complex interaction between smoking and schizophrenia at various disease stages. This could inform more effective strategies addressing smoking susceptibility in SSD, potentially improving clinical outcomes.

Effect of spaceflight experience on human brain structure, microstructure, and function: systematic review of neuroimaging studies.

Spaceflight-induced brain changes have been commonly reported in astronauts. The role of microgravity in the alteration of the brain structure, microstructure, and function can be tested with magnetic resonance imaging (MRI) techniques. Here, we aim to provide a comprehensive overview of Spaceflight studies exploring the potential role of brain alterations identified by MRI in astronauts. We conducted a search on PubMed, Web of Science, and Scopus to find neuroimaging correlates of spaceflight experience using MRI. A total of 20 studies (structural MRI n = 8, diffusion-based MRI n = 2, functional MRI n = 1, structural MRI and diffusion-weighted MRI n = 6, structural MRI and functional MRI n = 3) met our inclusion criteria. Overall, the studies showed that regardless of the MRI techniques, mission duration significantly impacts the human brain, prompting the inclusion of various brain regions as features in the analyses. After spaceflight, notable alterations were also observed in the superior occipital gyrus and the precentral gyrus which show alterations in connectivity and activation during spaceflight. The results provided highlight the alterations in brain structure after spaceflight, the unique patterns of brain remodeling, the challenges in drawing unified conclusions, and the impact of microgravity on intracranial cerebrospinal fluid volume.

Socio-cognitive, expertise-based and appearance-based accounts of the other-'race' effect in face perception: A label-based systematic review of neuroimaging results.

Two competing theories explain the other-'race' effect (ORE) either by greater perceptual expertise to same-'race' (SR) faces or by social categorization of other-'race' (OR) faces at the expense of individuation. To assess expertise and categorization contributions to the ORE, a promising-yet overlooked-approach is comparing activations for different other-'races'. We present a label-based systematic review of neuroimaging studies reporting increased activity in response to OR faces (African, Caucasian, or Asian) when compared with the SR of participants. Hypothetically, while common activations would reflect general aspects of OR perception, 'race'-preferential ones would represent effects of 'race'-specific visual appearance. We find that several studies report activation of occipito-temporal and midcingulate areas in response to faces across different other-'races', presumably due to high demand on the visual system and category processing. Another area reported in response to all OR faces, the caudate nucleus, suggests the involvement of socio-affective processes and behavioural regulation. Overall, our results support hybrid models-both expertise and social categorization contribute to the ORE, but they provide little evidence for reduced motivation to process OR faces. Additionally, we identify areas preferentially responding to specific OR faces, reflecting effects of visual appearance.

Unveiling the neural underpinnings of optimism: a systematic review.

Optimism is a personality trait strongly associated with physical and psychological well-being, with correlates in nonhuman species. Optimistic individuals hold positive expectancies for their future, have better physical and psychological health, recover faster after heart disease and other ailments, and cope more effectively with stress and anxiety. We performed a systematic review of neuroimaging studies focusing on neural correlates of optimism. A search identified 14 papers eligible for inclusion. Two key brain areas were linked to optimism: the anterior cingulate cortex (ACC), involved in imagining the future and processing of self-referential information; and the inferior frontal gyrus (IFG), involved in response inhibition and processing relevant cues. ACC activity was positively correlated with trait optimism and with the probability estimations of future positive events. Behavioral measures of optimistic tendencies investigated through the belief update task correlated positively with IFG activity. Elucidating the neural underpinnings of optimism may inform both the development of prevention and treatment strategies for several mental disorders negatively associated with optimism, such as depression, as well as help to foster new resilience promotion interventions targeting healthy, vulnerable, and mentally ill individuals.

Effects of Prenatal Methamphetamine Exposure on the Developing Human Brain: A Systematic Review of Neuroimaging Studies.

Methamphetamine (MA) can cross the placenta in pregnant women and cause placental abruption and developmental alterations in offspring. Previous studies have found prenatal MA exposure effects on the social and cognitive performance of children. Recent studies reported some alterations in structural and functional magnetic resonance imaging (MRI) of prenatal MA-exposed offspring. In this study, we aimed to investigate the effect of prenatal MA exposure on brain development using recently published structural, metabolic, and functional MRI studies. According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we searched PubMed and SCOPUS databases for articles that used each brain imaging modality in prenatal MA-exposed children. Seventeen studies were included in this study. We investigated brain imaging alterations using 17 articles with four different modalities, including structural MRI, diffusion tensor imaging (DTI), magnetic resonance spectroscopy (MRS), and functional MRI (fMRI). The participants’ age range was from infancy to 15 years. Our findings demonstrated that prenatal MA exposure is associated with macrostructural, microstructural, metabolic, and functional deficits in both cortical and subcortical areas. However, the most affected regions were the striatum, frontal lobe, thalamus and the limbic system, and white matter (WM) fibers connecting these regions. The findings from our study might have valuable implications for targeted treatment of neurocognitive and behavioral deficits in children with prenatal MA exposure. Even so, our results should be interpreted cautiously due to the heterogeneity of the included studies in terms of study populations and methods of analysis.

Anxiety in Parkinson’s Disease: A Systematic Review of Neuroimaging Studies

The mechanisms and neuronal networks associated with anxiety in Parkinson's disease (PD) are incompletely understood. One of the best tools for investigating both component function and neuronal networks associated with psychiatric symptoms is functional MRI (fMRI). Unlike structural scans, functional scans, whether task-based or resting-state, are more likely to be clinically relevant and sensitive to changes related to treatment. The investigators provide a comprehensive review of and present results for imaging studies of anxiety in PD. A systematic review of the literature on fMRI and anxiety in PD was conducted, and the quality of all included studies was simultaneously assessed. Eighteen studies were included: 15 studies assessed anxiety directly, and three evaluated emotional processing. Imaging methodology and behavioral assessments varied across studies, preventing direct comparison of results in most cases. There was a convergence in findings across methods, implicating involvement of the amygdala, caudate, and putamen in association with anxiety in PD. For both task-based activation and resting-state connectivity, dopamine medication status was associated with differences in activation and behavioral function. Although there is little consensus in the current fMRI literature studying anxiety in PD, these results suggest an overlap between structures classically involved in the brain's fear circuit (particularly the amygdala) and the alterations in the nigro-striatal system (e.g., the caudate and putamen and on-off dopamine findings) related to PD and its dopaminergic treatments.

Structural and functional brain abnormalities in postherpetic neuralgia: A systematic review of neuroimaging studies

In recent decades, an increasing number of neuroimaging studies utilizing magnetic resonance imaging (MRI) have explored the differential effects of postherpetic neuralgia (PHN) on brain structure and function. We systematically reviewed and integrated the findings from relevant neuroimaging studies in PHN patients. A total of 15 studies with 16 datasets were ultimately included in the present study, which were categorized by the different neuroimaging modalities. The results revealed that PHN was closely associated with structural/microstructural and functional abnormalities of the brain mainly located in the ‘pain matrix’, including the thalamus, insula, parahippocampus, amygdala, dorsolateral prefrontal cortex, precentral gyrus and inferior parietal lobe, as well as other regions, such as the precuneus, lentiform nucleus and brainstem. Furthermore, a disruption of multiple networks, including the default-mode network, salience network and limbic system, may contribute to the neurophysiological mechanisms underlying PHN. The findings indicate that the cerebral abnormalities of PHN were not restricted to the pain matrix but extended to other regions, profoundly affecting the regulation and moderation of pain processing in PHN. Future prospective and longitudinal neuroimaging studies with larger samples will elucidate the progressive trajectory of neural changes in the pathophysiological process of PHN.

Functional Near-Infrared Spectroscopy in the Study of Speech and Language Impairment Across the Life Span: A Systematic Review.

Purpose Functional brain imaging is playing an increasingly important role in the diagnosis and treatment of communication disorders, yet many populations and settings are incompatible with functional magnetic resonance imaging and other commonly used techniques. We conducted a systematic review of neuroimaging studies using functional near-infrared spectroscopy (fNIRS) with individuals with speech or language impairment across the life span. We aimed to answer the following question: To what extent has fNIRS been used to investigate the neural correlates of speech-language impairment? Method This systematic review was preregistered with PROSPERO, the international prospective register of systematic reviews (CRD42019136464). We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol for preferred reporting items for systematic reviews. The database searches were conducted between February and March of 2019 with the following search terms: (a) fNIRS or functional near-infrared spectroscopy or NIRS or near-infrared spectroscopy, (b) speech or language, and (c) disorder or impairment or delay. Results We found 34 fNIRS studies that involved individuals with speech or language impairment across nine categories: (a) autism spectrum disorders; (b) developmental speech and language disorders; (c) cochlear implantation and deafness; (d) dementia, dementia of the Alzheimer's type, and mild cognitive impairment; (e) locked-in syndrome; (f) neurologic speech disorders/dysarthria; (g) stroke/aphasia; (h) stuttering; and (i) traumatic brain injury. Conclusions Though it is not without inherent challenges, fNIRS may have advantages over other neuroimaging techniques in the areas of speech and language impairment. fNIRS has clinical applications that may lead to improved early and differential diagnosis, increase our understanding of response to treatment, improve neuroprosthetic functioning, and advance neurofeedback.

Central Nervous System Reorganization and Pain After Spinal Cord Injury: Possible Targets for Physical Therapy-A Systematic Review of Neuroimaging Studies.

Pain is one of the main symptoms associated with spinal cord injury (SCI) and can be associated with changes to the central nervous system (CNS). This article provides an overview of the evidence relating to CNS changes (structural and functional) associated with pain in SCIs. A systematic review was performed, according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations, on PubMed, Embase, and Web of Science in March 2018. Studies were selected if they concerned changes in the CNS of patients with SCI, regardless of the type of imagery. Data were extracted by 2 blinded reviewers. There is moderate evidence for impaired electroencephalographic function and metabolic abnormalities in the anterior cingulate in patients experiencing pain. There is preliminary evidence that patients with pain have morphological and functional changes to the somatosensory cortex and alterations to thalamic metabolism. There are conflicting data regarding the relationships between lesion characteristics and pain. In contrast, patients without pain can display protective neuroplasticity. Further studies are required to elucidate fully the relationships between pain and neuroplasticity in patients with SCIs. However, current evidence might support the use of physical therapist treatments targeting CNS plasticity in patients with SCI pain.

Brain structural effects of treatments for depression and biomarkers of response: a systematic review of neuroimaging studies.

Antidepressive pharmacotherapy (AD), electroconvulsive therapy (ECT) and cognitive behavioural therapy (CBT) are effective treatments for major depressive disorder. With our review, we aim to provide a systematic overview of neuroimaging studies that investigate the effects of AD, ECT and CBT on brain grey matter volume (GMV) and biomarkers associated with response. After a systematic database research on PubMed, we included 50 studies using magnetic resonance imaging and investigating (1) changes in GMV, (2) pre-treatment GMV biomarkers associated with response, or (3) the accuracy of predictions of response to AD, ECT or CBT based on baseline GMV data. The strongest evidence for brain structural changes was found for ECT, showing volume increases within the temporal lobe and subcortical structures - such as the hippocampus-amygdala complex, anterior cingulate cortex (ACC) and striatum. For AD, the evidence is heterogeneous as only 4 of 11 studies reported significant changes in GMV. The results are not sufficient in order to draw conclusions about the structural brain effects of CBT. The findings show consistently that higher pre-treatment ACC volume is associated with response to AD, ECT and CBT. An association of higher pre-treatment hippocampal volume and response has only been reported for AD. Machine learning approaches based on pre-treatment whole brain patterns reach accuracies of 64-90% for predictions of AD or ECT response on the individual patient level. The findings underline the potential of brain biomarkers for the implementation in clinical practice as an additive feature within the process of treatment selection.

Structural and Functional Alterations in Betel-Quid Chewers: A Systematic Review of Neuroimaging Findings.

Background: A number of neuroimaging studies have investigated structural, metabolic, and functional connectivity changes in betel quid (BQ) chewers. We present a systematic review of neuroimaging studies with emphasis on key brain systems affected by BQ chewing to bring a better understanding on the neuro mechanisms involved in BQD.Methods: All BQ neuroimaging studies were identified by searching PubMed, EMBASE, and Google scholar for English articles published until March 2018 using the key words: Betel-quid, resting state, functional MRI, structural MRI, diffusion tensor imaging (DTI), and betel quid dependence basing on the PRISMA criteria. We also sought unpublished studies, and the rest were obtained from reference lists of the retrieved articles. All neuroimaging studies investigating brain structural, and functional alterations related to BQ chewing and BQ dependence were included. Our systematic review registration number is CRD42018092669.Results: A review of 12 studies showed that several systems in the brain of BQ chewers exhibited structural, metabolic, and functional alterations. BQ chewing was associated with alterations in the reward [areas in the midbrain, and prefrontal cortex (PFC)], impulsivity (anterior cingulate cortex, PFC) and cognitive (PFC, the default mode, frontotemporal, frontoparietal, occipital/temporal, occipital/parietal, temporal/limbic networks, hippocampal/hypothalamus, and the cerebellum) systems in the brain. BQ duration and severity of betel quid dependence were associated with majority of alterations in BQ chewers.Conclusion: Betel quid chewing is associated with brain alterations in structure, metabolism and function in the cognitive, reward, and impulsivity circuits which are greatly influenced by duration and severity of betel quid dependence.

Ventral Striatal Reactivity in Compulsive Sexual Behaviors.

Compulsive Sexual Behaviors (CSB) are a reason to seek treatment. Given this reality, the number of studies on CSB has increased substantially in the last decade and the World Health Organization (WHO) included CSB in its proposal for the upcoming ICD-11. Sixty percent of the neuroimaging studies on CSB published since 2014 aimed to examine similarities and differences between brain mechanisms underlying CSB, gambling disorder, and substance use disorders. One of the crucial brain circuits involved in addiction is the reward system involving the ventral striatum (including nucleus accumbens). There are two distinct theories describing ventral striatal activity in addictions: Incentive Salience Theory (IST) and Reward Deficiency Syndrome (RDS). IST describes increased ventral striatal activations during the anticipation of addiction-related reward, while RDS describes decreased ventral striatal reactivity both during the anticipation of the reward and during the reward processing. Here, we aim to investigate how the findings on ventral striatal reactivity in CSB support each of these two addiction frameworks. For this purpose, we conducted a systematic review of neuroimaging studies on CSB available in Pubmed, EBSCO, and Google Scholar between 2005 and 2018. We found nine relevant research papers. Only four of these studies directly investigated processing of erotic cues and/or rewards and reported findings related to ventral striatum activations. Three of these studies indicate increased ventral striatal reactivity for erotic stimuli, which is consistent with IST and does not support predictions based on RDS. Therefore, the current state of this data suggest that CSB is related to increased ventral striatal reactivity during the anticipation of erotic stimuli.

A systematic review of neuroimaging in delirium: predictors, correlates and consequences.

Neuroimaging advances our understanding of delirium pathophysiology and its consequences. A previous systematic review identified 12 studies (total participants N=764, delirium cases N=194; years 1989-2007) and found associations with white matter hyperintensities (WMH) and cerebral atrophy. Our objectives were to perform an updated systematic review of neuroimaging studies in delirium published since January 2006 and summarise the available literature on predictors, correlates or outcomes. Studies were identified by keyword and MeSH-based electronic searches of EMBASE, MEDLINE and PsycINFO combining terms for neuroimaging, brain structure and delirium. We included neuroimaging studies of delirium in adults using validated delirium assessment methods. Thirty-two studies (total N=3187, delirium N=1086) met the inclusion criteria. Imaging included magnetic resonance imaging (MRI; N=9), computed tomography (N=4), diffusion tensor imaging (N=3), transcranial Doppler (N=5), near infrared spectroscopy (N=5), functional-MRI (N=2), single photon emission computed tomography (N=1), proton MRI spectroscopy (N=1), arterial spin-labelling MRI (N=1) and 2-13 fluoro-2-deoxyglucose positron emission tomography (N=1). Despite heterogeneity in study design, delirium was associated with WMH, lower brain volume, atrophy, dysconnectivity, impaired cerebral autoregulation, reduced blood flow and cerebral oxygenation and glucose hypometabolism. There was evidence of long-term brain changes following intensive care unit delirium. Neuroimaging is now used more widely in delirium research due to advances in technology. However, imaging delirious patients presents challenges leading to methodological limitations and restricted generalisability. The findings that atrophy and WMH burden predict delirium replicates findings from the original review, while advanced techniques have identified other substrates and mechanisms that warrant further investigation.

Commentary: Critical considerations for studying low-functioning autism.

Jack and Pelphrey provide a systematic review of neuroimaging studies in understudied populations within the autistic spectrum, focussing specifically on those with minimal verbal ability, intellectual disability and developmental regression. Despite accounting for nearly a third of the autistic spectrum, the number of studies focussing on these populations is extremely low. This review highlights a critical need for further neuroimaging research on these populations, and provides practical suggestions for overcoming the challenges posed by it. In this commentary, I discuss some of the theoretical questions that arise from the review, on the conceptualisation of the autistic spectrum as well as on optimising experimental design and analysis.

C9ORF72 and the FTD-ALS spectrum: A systematic review of neuroimaging studies.

ObjectiveTo perform a systematic review of the literature on the neuroimaging investigation of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) associated with C9ORF72 mutation.MethodsThe search was performed on PubMed and LILACS with the following terms: C9ORF72, MRI, SPECT, PET, ALS, FTD. No filters were added.ResultsTwenty articles were selected. Most studies found consistent involvement of frontotemporal regions in C9ORF72 carriers, including prefrontal cortex, and also cingulate, subcortical regions, especially the thalami, and posterior regions such as the parietal and occipital lobes. Functional connectivity was also explored and impaired sensorimotor connectivity in striatum and thalami was found in behavioral variant FTD C9ORF72 carriers. Some papers have reported an absence of significant abnormalities on brain imaging.ConclusionThe inclusion of patients at different stages of the disease, differences in neuroimaging methods across studies, and distinct clinical phenotypes associated with C9ORF72 may account for the heterogeneity of results.

An Updated Systematic Review of Neuroimaging Studies of Children and Adolescents with Perinatally Acquired HIV

A recent review of neuroimaging studies related to perinatally acquired human immunodeficiency virus (HIV) infection by Hoare and colleagues (Metabolic Brain Disease 29:221–229, 2014) included studies published between 1995 and 2012, with all but two studies being published in or before 2006. Although the review synthesized the extant research available at the time, the findings may not be relevant to the current population of children and adolescents who are HIV positive due to more recent advances in antiretroviral medication and new medication guidelines. As such, the purpose of this paper was to extend the findings of Hoare and colleagues by reviewing the imaging literature that has been published since 2012 and to compare the more recent studies to the earlier ones. The results were that 11 articles met criteria for inclusion in this review. The majority of studies were cross-sectional and analyzed MRI data.. Samples included children and adolescents of nearly all ages. Studies differed widely on the inclusion and reporting of clinical variables. Imaging was focused primarily on white matter, and white matter issues such as lesions, poor tract integrity, and reduced volume were found in samples of children and adolescents of various age ranges. Two studies compared the cognitive functioning of children with HIV to controls, and both found that children with HIV perform more poorly than their non-infected peers on various cognitive tasks. Based on the findings of this review, recommendations for future imaging research are made.

Pain in multiple sclerosis: a systematic review of neuroimaging studies.

IntroductionWhile pain in multiple sclerosis (MS) is common, in many cases the precise mechanisms are unclear. Neuroimaging studies could have a valuable role in investigating the aetiology of pain syndromes. The aim of this review was to synthesise and appraise the current literature on neuroimaging studies of pain syndromes in MS.MethodsWe systematically searched PubMed and Scopus from their inception dates to the 2nd of April 2013. Studies were selected by predefined inclusion and exclusion criteria. Methodological quality was appraised. Descriptive statistical analysis was conducted.ResultsWe identified 38 studies of variable methodology and quality. All studies but one used conventional structural magnetic resonance imaging, and the majority reported a positive association between location of demyelinating lesions and specific neuropathic pain syndromes. Most investigated headache and facial pain, with more common pain syndromes such as limb pain being relatively understudied. We identified a number of methodological concerns, which along with variable study design and reporting limit our ability to synthesise data. Higher quality studies were however less likely to report positive associations of lesion distribution to pain syndromes.ConclusionsFurther high quality hypothesis-driven neuroimaging studies of pain syndromes in MS are required to clarify pain mechanisms, particularly for the commonest pain syndromes.

Systematic review of neuroimaging studies in vertically transmitted HIV positive children and adolescents

One of the most serious consequences of vertical HIV-infection is its impact on the central nervous system (CNS). Although much work has been done to elucidate the complex mechanism of HIV associated neurotoxicity, several questions remain unanswered. The purpose of this review is to summarise what is already known in the field of neuroimaging in vertically acquired HIV, addressing three aims and to highlight possible future directions in using neuroimaging and neurocognitive testing to understand the spectrum of neurocognitive disorders in HIV positve children. Here we aim to address several clinically relevant questions in pediatric neuroHIV, using the current evidence base by conducting a systematic review. We aim to investigate what is known about the relationship between cognitive impairment and central nervous system damage in HIV as seen in neuroimaging studies, and to search for any evidence in the current literature which suggests a spectrum of neuocognitive disorders in vertically infected HIV. Secondly, we aim to enquire whether children with a clinical diagnosis of encephalopathy are clearly distinguishable from HIV positive children without encephalopathy on neuroimaging and neurocognitive testing. Finally aim to investigate what is known about the effect on the CNS of antiretroviral therapy in paediatric HIV. Three separate databases were searched and two investigators systematically evaluated the titles, abstracts, and keywords associated with each individual article to determine those that may have met the inclusion and exclusion criteria. Following this process 11 studies were included in the review. Thus there was limited available data to address the 3 questions posed.