Neuroradiology DOI 10.1007/s00234-014-1380-9
PAEDIATRIC NEURORADIOLOGY
Magnetic resonance spectroscopy markers of axons and astrogliosis in relation to specific features of white matter injury in preterm infants Jessica L. Wisnowski & Vincent J. Schmithorst & Tena Rosser & Lisa Paquette & Marvin D. Nelson & Robin L. Haynes & Michael J. Painter & Stefan Blüml & Ashok Panigrahy
Received: 27 February 2014 / Accepted: 8 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Introduction Punctate white matter lesions (pWMLs) and diffuse excessive high signal intensity (DEHSI) are commonly observed signal abnormalities on MRI scans of high-risk preterm infants near term-equivalent age. To establish whether these features are indicative abnormalities in axonal development or astroglia, we compared pWMLs and DEHSI to markers of axons and astrogliosis, derived from magnetic resonance spectroscopy (MRS). Methods Data from 108 preterm infants (gestational age at birth 31.0 weeks±4.3; age at scan 41.2 weeks±6.0) who underwent MR examinations under clinical indications were included in this study. Linear regression analyses were used to test the effects of pWMLs and DEHSI on N-acetyl-aspartate (NAA) and myoinositol concentrations, respectively.
Results Across the full sample, pWMLs were associated with a reduction in NAA whereas moderate to severe DEHSI altered the normal age-dependent changes in myoinositol such that myoinositol levels were lower at younger ages with no change during the perinatal period. Subgroup analyses indicated that the above associations were driven by the subgroup of neonates with both pWMLs and moderate to severe DEHSI. Conclusion Overall, these findings suggest that pWMLs in conjunction with moderate/severe DEHSI may signify a population of infants at risk for long-term adverse neurodevelopmental outcome due to white matter injury and associated axonopathy. The loss of normal age-associated changes in myoinositol further suggests disrupted astroglial function and/or osmotic dysregulation.
Electronic supplementary material The online version of this article (doi:10.1007/s00234-014-1380-9) contains supplementary material, which is available to authorized users. J. L. Wisnowski : M. D. Nelson : S. Blüml : A. Panigrahy Department of Radiology, Children’s Hospital Los Angeles, Los Angeles, CA, USA J. L. Wisnowski : V. J. Schmithorst : A. Panigrahy (*) Department of Pediatric Radiology, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA 15224, USA e-mail: [email protected] T. Rosser Department of Pediatrics, Division of Neurology, Children’s Hospital Los Angeles, Los Angeles, CA, USA L. Paquette Department of Pediatrics, Division of Neonatology, Children’s Hospital Los Angeles, Los Angeles, CA, USA
R. L. Haynes Department of Pathology, Boston Children’s Hospital, Boston, MA, USA
M. J. Painter Department of Pediatrics, Division of Neurology, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh, Pittsburgh, PA, USA
S. Blüml Rudi Schulte Research Institute, Santa Barbara, CA, USA
Neuroradiology
Keywords Preterm birth . Diffuse excessive high signal intensity (DEHSI) . Magnetic Resonance Spectroscopy . N-acetyl-aspartate . Myoinositol
Introduction Neuroimaging studies have demonstrated that the majority of preterm infants have white matter abnormalities, including signal abnormalities, volume loss, cystic abnormalities, enlarged ventricles, thinning of the corpus callosum, and delayed myelination near term equivalency [1–3]. Although many of the above are now considered risk factors for later neurodevelopmental impairments, the sensitivity and specificity of these conventional imaging patterns for predicting future outcomes, particularly neurocognitive functioning, remains limited [2–6]. Moreover, even less is known about what these conventional imaging biomarkers signify at a cellular/ molecular level, a critical gap in knowledge at a time when human clinical trials targeting different strategies for neuroprotection in preterm infants are emerging. We used multimodal in vivo MR imaging to better characterize the two signal abnormalities most widely used to signify white matter injury in preterm infants in the neonatal period: punctate white matter lesions (pWMLs) and diffuse excessive high signal intensity (DEHSI). Thus far, the limited radiological-pathological data available have suggested that pWMLs, visualized as focal areas of high signal on T1weighted MRI, indicate focal necroses with lipid-laden macrophages or possibly foci of activated microglia [6, 7], which in human neuropathologic studies has been associated with axonopathy [8, 9]. In contrast, DEHSI, visualized on T2weighted MRI as diffuse regions of high signal, may reflect white matter gliosis, [1, 10, 11] which has been associated with arrested preoligodendrocyte maturation [12]. Importantly, although neuropathological studies demonstrate that focal necroses and diffuse white matter gliosis may frequently co-occur, there is not yet consensus as to whether they should be considered independently or combined—as in a single spectrum of white matter injury [8, 11, 13]. Similarly, there has been no consensus in neuroimaging studies as to whether pWMLs and DEHSI should be considered independently or combined to yield an overall white matter injury score [2, 14, 15], and such variability across studies may also explain the variability in the extent to which neonatal MRI is predictive of later neurodevelopmental disability [3–5, 14–16]. Previous research by our group has suggested pWMLs and DEHSI are associated with distinct patterns of metabolic alteration in the parietal white matter [17]. However, in that study, analyses pertaining to pWMLs and DEHSI were conducted in parallel and no direct statistical comparison was made between pWMLs and DEHSI.
To delineate the associations between signal abnormalities on conventional MRI and cellular/molecular aspects of white matter injury in vivo in preterm infants, we compared pWMLs and DEHSI to markers of neuronal/axonal integrity and astrogliosis derived from MR spectroscopy (MRS). Nacetyl-aspartate (NAA) is synthesized in the mitochondria of neurons and axons [18, 19] and is reliably depleted in the setting of traumatic brain injury [20], stroke [21], and other hypoxic-ischemic brain injuries [22] and, accordingly, is used as a marker of axonal integrity/axonopathy. In contrast, myoinositol, an osmolite expressed in high intracellular concentration in astroglia, is consistently elevated in the setting of multiple sclerosis and other neuroinflammatory CNS conditions [23–25] as well as in tumors characterized by a high fraction of glial cells [26], and thus is often used as a marker for astroglia. We first tested the hypothesis that the presence of pWMLs would be associated with a decrease in NAA, reflecting axonopathy, independent of DEHSI. In contrast, we hypothesized that increasing DEHSI severity would be associated with an elevation in myoinositol, reflecting astrogliosis, independent of pWMLs. Additionally, because pWMLs and DEHSI were considered distinct entities exerting independent effects on axons and astroglia, we also tested whether different subgroups defined by pWMLs and DEHSI (i.e., pWMLs without DEHSI, pWMLs with DEHSI) were associated with different effects on NAA and myoinositol, suggesting that pWMLs and DEHSI may be recombined to define distinct patterns of white matter injury (WMI).
Materials and methods Case selection Data from 108 preterm neonates (mean gestational age at birth 31.0 weeks±4.3; range 23–36 weeks; mean postconceptional age at scan 41.2 weeks±6.0; range 25.7– 60.7 weeks) were included in this study. Details regarding case selection are available in [17]. Briefly, all preterm infants who underwent clinically indicated MRIs were screened prospectively as part of ongoing longitudinal studies of neurodevelopment in neonates with prematurity. All available cases were included in this study provided: (1) the imaging study had been completed on an infant born before 37 gestational weeks of age; (2) the infant was not older than 60 weeks postconceptional age (PCA; calculated as the interval between the mother’s last menstrual period and birth plus postnatal age) at the time of the MRI; (3) there was no evidence of cerebral abnormality other than pWMLs or DEHSI (i.e., large vessel acute or chronic infarction, parenchymal hemorrhage, infection, tumor, or cerebral malformation); and (4) there was no clinical or laboratory evidence of liver failure, hyperbilirubinemia (requiring exchange transfusion), or
Neuroradiology
underlying inborn error of metabolism. Prior results from this cohort have been published in [17]. MR data acquisition MRI studies were acquired under clinical indications (most often to assess brain injury following preterm birth) on a GE 1.5 T (Signa LX, GE Healthcare, Milwaukee, WI) MR System using a customized neonatal transmit-receive head coil. Some studies were conducted using an MR compatible incubator; however, most were conducted with the neonate wrapped in a blanket and secured with appropriate physiological monitoring equipment. Per clinical protocol, most infants were sedated with choral hydrate. Ear protection was achieved using foam ear plugs in conjunction with MiniMuffs (Natus Medical Inc., San Carlos, CA). Conventional imaging studies were acquired with the MRS studies and included a 3D coronal SPGR sequence (echo time (TE)=6 ms; repetition time (TR)=25 ms, FOV= 18 cm; matrix=256×160; slice thickness 1.5 mm, spacing 0 mm) or axial and sagittal T1-weighted FLAIR sequences (TE=7.4, TR=2100; TI=750; FOV=20 cm; Matrix=256× 160), axial T2-weighted FSE sequence (TE=85 ms, TR= 5000 ms, FOV=20 cm, matrix=320×160 or 256×128), and a diffusion-weighted sequence (TE=80; TR=10000; FOV= 22 cm; Matrix=128×128; slice thickness=4.5 mm, spacing 0 mm). 1 H spectra were acquired from a single voxel (approximately 3 cm3) placed in the parietal white matter dorsolateral to the trigone of the lateral ventricle in the left hemisphere using a point resolved spectroscopy (PRESS) sequence with a short TE of 35 milliseconds (ms), a TR of 1.5 s, 128 signal averages, and a total acquisition time for each spectrum of approximately 5 min, including scanner adjustments. The parietal white matter location was selected because (1) the parietal white matter is known to be a region of vulnerability in preterm infants and (2) developing axons from numerous thalamocortical and corticocortical association pathways traverse that region [27, 28]. Determination of pWMLs and DEHSI based on conventional MR images Conventional MRI scans (T1-, T2-, and diffusionweighted sequences) for all studies were independently reviewed by two investigators (AP and JLW) and scored for the presence of both pWMLs (defined as punctate T1hyperintense lesions in the periventricular white matter and corona radiata; Fig. 1) and DEHSI (defined as high signal on T2-weighted MR images in the cerebral white matter) and scored on a four-point scale: 0/within normal limits, 1/mildly increased, 2/moderately increased, and 3/severely increased (Fig. 2) (see also Online Resource). Metabolites analyzed and data processing We focused our analyses on two key metabolites: NAA and myoinositol. Absolute concentration for each metabolite was
quantitated from the MRS spectra using LCModel software (Stephen Provencher Inc., Oakville, Ontario, Canada, LCModel Version 6.1-4 F). In accordance with prior publications [17, 29, 30], metabolite concentrations were corrected for the varying fractions of cerebrospinal fluid and tissue water content in the parietal white matter region of interest. For absolute quantitation, the signal from unsuppressed water was used as internal concentration reference. MR spectra of low quality were removed by limiting the sample empirically to spectra with a linewidth (measure of field homogeneity) of
PAEDIATRIC NEURORADIOLOGY
Magnetic resonance spectroscopy markers of axons and astrogliosis in relation to specific features of white matter injury in preterm infants Jessica L. Wisnowski & Vincent J. Schmithorst & Tena Rosser & Lisa Paquette & Marvin D. Nelson & Robin L. Haynes & Michael J. Painter & Stefan Blüml & Ashok Panigrahy
Received: 27 February 2014 / Accepted: 8 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Introduction Punctate white matter lesions (pWMLs) and diffuse excessive high signal intensity (DEHSI) are commonly observed signal abnormalities on MRI scans of high-risk preterm infants near term-equivalent age. To establish whether these features are indicative abnormalities in axonal development or astroglia, we compared pWMLs and DEHSI to markers of axons and astrogliosis, derived from magnetic resonance spectroscopy (MRS). Methods Data from 108 preterm infants (gestational age at birth 31.0 weeks±4.3; age at scan 41.2 weeks±6.0) who underwent MR examinations under clinical indications were included in this study. Linear regression analyses were used to test the effects of pWMLs and DEHSI on N-acetyl-aspartate (NAA) and myoinositol concentrations, respectively.
Results Across the full sample, pWMLs were associated with a reduction in NAA whereas moderate to severe DEHSI altered the normal age-dependent changes in myoinositol such that myoinositol levels were lower at younger ages with no change during the perinatal period. Subgroup analyses indicated that the above associations were driven by the subgroup of neonates with both pWMLs and moderate to severe DEHSI. Conclusion Overall, these findings suggest that pWMLs in conjunction with moderate/severe DEHSI may signify a population of infants at risk for long-term adverse neurodevelopmental outcome due to white matter injury and associated axonopathy. The loss of normal age-associated changes in myoinositol further suggests disrupted astroglial function and/or osmotic dysregulation.
Electronic supplementary material The online version of this article (doi:10.1007/s00234-014-1380-9) contains supplementary material, which is available to authorized users. J. L. Wisnowski : M. D. Nelson : S. Blüml : A. Panigrahy Department of Radiology, Children’s Hospital Los Angeles, Los Angeles, CA, USA J. L. Wisnowski : V. J. Schmithorst : A. Panigrahy (*) Department of Pediatric Radiology, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA 15224, USA e-mail: [email protected] T. Rosser Department of Pediatrics, Division of Neurology, Children’s Hospital Los Angeles, Los Angeles, CA, USA L. Paquette Department of Pediatrics, Division of Neonatology, Children’s Hospital Los Angeles, Los Angeles, CA, USA
R. L. Haynes Department of Pathology, Boston Children’s Hospital, Boston, MA, USA
M. J. Painter Department of Pediatrics, Division of Neurology, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh, Pittsburgh, PA, USA
S. Blüml Rudi Schulte Research Institute, Santa Barbara, CA, USA
Neuroradiology
Keywords Preterm birth . Diffuse excessive high signal intensity (DEHSI) . Magnetic Resonance Spectroscopy . N-acetyl-aspartate . Myoinositol
Introduction Neuroimaging studies have demonstrated that the majority of preterm infants have white matter abnormalities, including signal abnormalities, volume loss, cystic abnormalities, enlarged ventricles, thinning of the corpus callosum, and delayed myelination near term equivalency [1–3]. Although many of the above are now considered risk factors for later neurodevelopmental impairments, the sensitivity and specificity of these conventional imaging patterns for predicting future outcomes, particularly neurocognitive functioning, remains limited [2–6]. Moreover, even less is known about what these conventional imaging biomarkers signify at a cellular/ molecular level, a critical gap in knowledge at a time when human clinical trials targeting different strategies for neuroprotection in preterm infants are emerging. We used multimodal in vivo MR imaging to better characterize the two signal abnormalities most widely used to signify white matter injury in preterm infants in the neonatal period: punctate white matter lesions (pWMLs) and diffuse excessive high signal intensity (DEHSI). Thus far, the limited radiological-pathological data available have suggested that pWMLs, visualized as focal areas of high signal on T1weighted MRI, indicate focal necroses with lipid-laden macrophages or possibly foci of activated microglia [6, 7], which in human neuropathologic studies has been associated with axonopathy [8, 9]. In contrast, DEHSI, visualized on T2weighted MRI as diffuse regions of high signal, may reflect white matter gliosis, [1, 10, 11] which has been associated with arrested preoligodendrocyte maturation [12]. Importantly, although neuropathological studies demonstrate that focal necroses and diffuse white matter gliosis may frequently co-occur, there is not yet consensus as to whether they should be considered independently or combined—as in a single spectrum of white matter injury [8, 11, 13]. Similarly, there has been no consensus in neuroimaging studies as to whether pWMLs and DEHSI should be considered independently or combined to yield an overall white matter injury score [2, 14, 15], and such variability across studies may also explain the variability in the extent to which neonatal MRI is predictive of later neurodevelopmental disability [3–5, 14–16]. Previous research by our group has suggested pWMLs and DEHSI are associated with distinct patterns of metabolic alteration in the parietal white matter [17]. However, in that study, analyses pertaining to pWMLs and DEHSI were conducted in parallel and no direct statistical comparison was made between pWMLs and DEHSI.
To delineate the associations between signal abnormalities on conventional MRI and cellular/molecular aspects of white matter injury in vivo in preterm infants, we compared pWMLs and DEHSI to markers of neuronal/axonal integrity and astrogliosis derived from MR spectroscopy (MRS). Nacetyl-aspartate (NAA) is synthesized in the mitochondria of neurons and axons [18, 19] and is reliably depleted in the setting of traumatic brain injury [20], stroke [21], and other hypoxic-ischemic brain injuries [22] and, accordingly, is used as a marker of axonal integrity/axonopathy. In contrast, myoinositol, an osmolite expressed in high intracellular concentration in astroglia, is consistently elevated in the setting of multiple sclerosis and other neuroinflammatory CNS conditions [23–25] as well as in tumors characterized by a high fraction of glial cells [26], and thus is often used as a marker for astroglia. We first tested the hypothesis that the presence of pWMLs would be associated with a decrease in NAA, reflecting axonopathy, independent of DEHSI. In contrast, we hypothesized that increasing DEHSI severity would be associated with an elevation in myoinositol, reflecting astrogliosis, independent of pWMLs. Additionally, because pWMLs and DEHSI were considered distinct entities exerting independent effects on axons and astroglia, we also tested whether different subgroups defined by pWMLs and DEHSI (i.e., pWMLs without DEHSI, pWMLs with DEHSI) were associated with different effects on NAA and myoinositol, suggesting that pWMLs and DEHSI may be recombined to define distinct patterns of white matter injury (WMI).
Materials and methods Case selection Data from 108 preterm neonates (mean gestational age at birth 31.0 weeks±4.3; range 23–36 weeks; mean postconceptional age at scan 41.2 weeks±6.0; range 25.7– 60.7 weeks) were included in this study. Details regarding case selection are available in [17]. Briefly, all preterm infants who underwent clinically indicated MRIs were screened prospectively as part of ongoing longitudinal studies of neurodevelopment in neonates with prematurity. All available cases were included in this study provided: (1) the imaging study had been completed on an infant born before 37 gestational weeks of age; (2) the infant was not older than 60 weeks postconceptional age (PCA; calculated as the interval between the mother’s last menstrual period and birth plus postnatal age) at the time of the MRI; (3) there was no evidence of cerebral abnormality other than pWMLs or DEHSI (i.e., large vessel acute or chronic infarction, parenchymal hemorrhage, infection, tumor, or cerebral malformation); and (4) there was no clinical or laboratory evidence of liver failure, hyperbilirubinemia (requiring exchange transfusion), or
Neuroradiology
underlying inborn error of metabolism. Prior results from this cohort have been published in [17]. MR data acquisition MRI studies were acquired under clinical indications (most often to assess brain injury following preterm birth) on a GE 1.5 T (Signa LX, GE Healthcare, Milwaukee, WI) MR System using a customized neonatal transmit-receive head coil. Some studies were conducted using an MR compatible incubator; however, most were conducted with the neonate wrapped in a blanket and secured with appropriate physiological monitoring equipment. Per clinical protocol, most infants were sedated with choral hydrate. Ear protection was achieved using foam ear plugs in conjunction with MiniMuffs (Natus Medical Inc., San Carlos, CA). Conventional imaging studies were acquired with the MRS studies and included a 3D coronal SPGR sequence (echo time (TE)=6 ms; repetition time (TR)=25 ms, FOV= 18 cm; matrix=256×160; slice thickness 1.5 mm, spacing 0 mm) or axial and sagittal T1-weighted FLAIR sequences (TE=7.4, TR=2100; TI=750; FOV=20 cm; Matrix=256× 160), axial T2-weighted FSE sequence (TE=85 ms, TR= 5000 ms, FOV=20 cm, matrix=320×160 or 256×128), and a diffusion-weighted sequence (TE=80; TR=10000; FOV= 22 cm; Matrix=128×128; slice thickness=4.5 mm, spacing 0 mm). 1 H spectra were acquired from a single voxel (approximately 3 cm3) placed in the parietal white matter dorsolateral to the trigone of the lateral ventricle in the left hemisphere using a point resolved spectroscopy (PRESS) sequence with a short TE of 35 milliseconds (ms), a TR of 1.5 s, 128 signal averages, and a total acquisition time for each spectrum of approximately 5 min, including scanner adjustments. The parietal white matter location was selected because (1) the parietal white matter is known to be a region of vulnerability in preterm infants and (2) developing axons from numerous thalamocortical and corticocortical association pathways traverse that region [27, 28]. Determination of pWMLs and DEHSI based on conventional MR images Conventional MRI scans (T1-, T2-, and diffusionweighted sequences) for all studies were independently reviewed by two investigators (AP and JLW) and scored for the presence of both pWMLs (defined as punctate T1hyperintense lesions in the periventricular white matter and corona radiata; Fig. 1) and DEHSI (defined as high signal on T2-weighted MR images in the cerebral white matter) and scored on a four-point scale: 0/within normal limits, 1/mildly increased, 2/moderately increased, and 3/severely increased (Fig. 2) (see also Online Resource). Metabolites analyzed and data processing We focused our analyses on two key metabolites: NAA and myoinositol. Absolute concentration for each metabolite was
quantitated from the MRS spectra using LCModel software (Stephen Provencher Inc., Oakville, Ontario, Canada, LCModel Version 6.1-4 F). In accordance with prior publications [17, 29, 30], metabolite concentrations were corrected for the varying fractions of cerebrospinal fluid and tissue water content in the parietal white matter region of interest. For absolute quantitation, the signal from unsuppressed water was used as internal concentration reference. MR spectra of low quality were removed by limiting the sample empirically to spectra with a linewidth (measure of field homogeneity) of
