In vivo measurement of ADC change due to intravascular susceptibility variation.

The apparent diffusion coefficient (ADC) of extravascular tissue water in rat brains was measured in response to step-wise injections of the superparamagnetic intravascular contrast agent AMI-227. These data were normalized and compared with measured changes in R2* and blood magnetic susceptibility. Linear regression showed that ADC changes 33%/ppm shift of intravascular susceptibility and 0.43% s(-1) change in R2*. These changes correspond to a predicted ADC change of approximately 6% for a change between fully oxygenated and fully deoxygenated blood. The source of these ADC changes was confirmed to be background gradients within the sample by the use of diffusion weighting with bipolar gradients of odd symmetry designed to cancel such background gradient effects on ADC. The results suggest that diffusion-weighted imaging is sensitive to blood-oxygenation and may provide a means of measuring changes in blood oxygen. They also provide estimates of the potential contribution of susceptibility changes to changes in ADC that occur, for example, in stroke and seizure.     

Rapid hyperpolarized 3He diffusion MRI of healthy and emphysematous human lungs using an optimized interleaved-spiral pulse sequence

PURPOSE: To develop and validate an interleaved-spiral diffusion pulse sequence capable of hyperpolarized (3)He MR imaging of the whole lung in less than 10 seconds. MATERIALS AND METHODS: Hyperpolarized (3)He diffusion measurements were performed in seven healthy volunteers and five patients with emphysema using an interleaved-spiral pulse sequence that provided 11 contiguous 15-mm thick coronal ADC maps, with an in-plane resolution of 3.9 mm, covering the whole lung in 5.5 seconds. The resulting means and SDs of ADC values were compared statistically to those from a gradient-echo pulse sequence with identical resolution and diffusion-weighting gradients that acquired five ADC maps in 10.5 seconds. RESULTS: High-quality diffusion-weighted interleaved-spiral images covering the whole lung were obtained, and showed no significant susceptibility-induced image degradation compared to corresponding gradient-echo images. On a subject-by-subject basis, the means and SDs of ADC values for the interleaved-spiral technique were not statistically different from those for the gradient-echo technique. The mean ADC values from the two techniques were highly correlated on a section-by-section basis (R = 0.99). CONCLUSION: The interleaved-spiral diffusion pulse sequence permits rapid acquisition of contiguous ADC maps covering the whole lung during a short breath-hold period, and provides ADC values that are statistically equivalent to those from standard gradient-echo techniques.     

"Squashing peanuts and smashing pumpkins": how noise distorts diffusion-weighted MR data.

New diffusion-weighted imaging (DWI) methods, including high-b, q-space, and high angular resolution MRI methods, attempt to extract information about non-Gaussian diffusion in tissue that is not provided by low-b-value (b approximately 1000 s mm(-2)) diffusion or diffusion tensor magnetic resonance imaging (DT-MRI). Additionally, DWI data with higher spatial resolution are being acquired to resolve fine anatomic structures, such as white matter fasciculi. Increasing diffusion-weighting or decreasing voxel size can reduce the signal-to-noise ratio so that some DWI signals are close to the background noise level. Here we report several new artifacts that can be explained by considering how background noise affects the peanut-shaped angular apparent diffusion coefficient (ADC) profile. These include an orientationally dependent deviation from Gaussian behavior of the ADC profile, an underestimation of indices of diffusion anisotropy, and a correlation between estimates of mean diffusivity and diffusion anisotropy. We also discuss how noise can cause increased gray/white matter DWI contrast at higher b values and an apparent elevation of diffusion anisotropy in acute ischemia. Importantly, all of these artifacts are negligible in the b-value range typically used in DT-MRI of brain (b approximately 1000 s mm(-2)). Finally, we demonstrate a strategy for ameliorating the rectified noise artifact in data collected at higher b values.     

Determination of Background Gradients with Diffusion MR Imaging

A new magnetic resonance (MR) imaging technique, opposite-polarity pulsed-field-gradient technique, with which the effects of background magnetic field gradients can be separated from the effects of diffusion, is described. It is based on the processing of two sets of diffusion-weighted images, the acquisition parameters of which differ only in the polarity of the applied diffusion pulses. The two effects can be separated because the cross term (b_c) of the gradient factor function is antisymmetric with respect to reversal of the sign of the applied diffusion pulses. The technique permits simultaneous measurement of the spatial distribution of both the diffusion constants and background magnetic field gradients, with the same spatial resolution as the parent images from which they were derived. The technique has been validated with a phantom in which the spatial distribution of susceptibility-induced background gradients is known, the results showing excellent agreement with theory. The technique was applied to two systems in which the spatial distribution of the background gradient is unknown. Sources of error in the measurement of background gradients and (unrestricted) diffusion constants are analyzed, including the effects of voxel size, partial volumes, and interactions between background and imaging gradients.     

Eddy current correction in diffusion-weighted imaging using pairs of images acquired with opposite diffusion gradient polarity.

In echo-planar-based diffusion-weighted imaging (DWI) and diffusion tensor imaging (DTI), the evaluation of diffusion parameters such as apparent diffusion coefficients and anisotropy indices is affected by image distortions that arise from residual eddy currents produced by the diffusion-sensitizing gradients. Correction methods that coregister diffusion-weighted and non-diffusion-weighted images suffer from the different contrast properties inherent in these image types. Here, a postprocessing correction scheme is introduced that makes use of the inverse characteristics of distortions generated by gradients with reversed polarity. In this approach, only diffusion-weighted images with identical contrast are included for correction. That is, non-diffusion-weighted images are not needed as a reference for registration. Furthermore, the acquisition of an additional dataset with moderate diffusion-weighting as suggested by Haselgrove and Moore (Magn Reson Med 1996;36:960-964) is not required. With phantom data it is shown that the theoretically expected symmetry of distortions is preserved in the images to a very high degree, demonstrating the practicality of the new method. Results from human brain images are also presented.     

Water diffusion heterogeneity index in the human brain is insensitive to the orientation of applied magnetic field gradients.

The alpha diffusion-weighted imaging (DWI) method was developed to study heterogeneous water diffusion in the human brain using magnetic resonance imaging (MRI). An advantage of this model is that it does not require an assumption about the shape of the intravoxel distribution of apparent diffusion rates, and it has a calculable relationship to this distribution. The alpha-DWI technique is useful for detecting microstructural tissue changes associated with brain tumor invasion, and may be useful for directing therapy to invading tumor cells. In previous work, alpha-DWI was performed with magnetic field gradients applied along a single direction in order to avoid artificially introducing a source of heterogeneity to the decay. However, it is known that restricted diffusion is anisotropic in the brain, and the alpha-DWI method must take this into account to be complete. In this work the relationship between the applied magnetic field gradients and the fitted stretched-exponential model parameters was studied in the human brain. It was found the distributed diffusion coefficient (DDC) varies with the direction of applied gradients, while the heterogeneity index alpha is relatively direction-insensitive. It is proposed that in clinical use, maps of alpha can be created using diffusion-weighting gradients applied in a single direction that reflect the tissue heterogeneity.     

CPMG measurements and ultrafast imaging in human lungs with hyperpolarized helium-3 at low field (0.1 T).

This work reports the use of single-shot spin echo sequences to achieve in vivo diffusion gas measurements and ultrafast imaging of human lungs, in vivo, with hyperpolarized (3)He at 0.1 T. The observed transverse relaxation time of (3)He lasted up to 10 s, which made it possible to use long Carr-Purcell-Meiboom-Gill echo trains. Preliminary NMR studies showed that the resolution of lung images acquired with hyperpolarized (3)He and single-shot sequences is limited to about 6 mm because of the diffusion of the gas in applied field gradients. Ultrafast images of human lungs in normal subjects, achieved in less than 0.4 s with the equivalent of only 130 micromol of fully polarized (3)He, are presented. Comparison with other studies shows that there is no SNR penalty by using low fields in the hyperpolarized case. Advantage was taken of the self diffusion-weighting of the rapid acquisition with relaxation enhancement (RARE) sequence to acquire apparent diffusion coefficient (ADC) images of the lungs. Time scales of seconds could be explored for the first time because there is no hindrance from T(*)(2) as with the usual approaches. At 0.1 T, 180 degrees RF pulses can be repeated every 10 ms without exceeding specific absorption rate limits, which would not be the case for higher fields. Moreover, at low field, susceptibility-induced phenomena are expected to be milder. This supports the idea that low-field imagers can be used for hyperpolarized noble gas MRI of lungs and may be preferred for ADC measurements.     

Visualization of the protective ability of a free radical trapping compound against rat C6 and F98 gliomas with diffusion tensor fiber tractography

PURPOSE: To apply fiber tractography to assess the effect of a possible antiglioma drug, phenyl N-tert-butyl nitrone (PBN), on glioma-affected neuronal fibers. The fiber tractography method was able to differentiate between different tumor types, such as the C6 and F98 rat glioma models. MATERIALS AND METHODS: C6 or F98 cells were intracranially injected into the cortex of male Fischer 344 rats. PBN treatment was initiated before or after cell implantation. Tumor growth was monitored with diffusion tensor imaging (DTI) and fiber tractography using diffusion-weighting gradients in 30 noncolinear directions. RESULTS: Although proton density-weighted (PDw) and T2-weighted (T2w) images did not show any difference between C6 and F98 gliomas without edema, the apparent diffusion coefficient (ADC) and fractional anisotropy (FA) maps were able to discriminate between these two tumor models. Fiber tractography was used to visualize C6 glioma-induced ischemia of tumor-surrounding tissues, whereas F98 glioma was found to infiltrate and penetrate into the corpus callosum (CC). During glioma growth, neuronal fibers were found to disappear at the border regions between the tumor and surrounding tissues. PBN treatment was shown to inhibit glioma growth with accompanying changes in the surrounding tissue. CONCLUSION: By noninvasively monitoring the degree of neuronal fiber integrity and connectivity with the use of neuronal fiber tractography, we were able to evaluate the protective effect of PBN against invasive glioma growth in rat brains. PBN provided protection of the neuronal fibers against tumor-induced ischemia and tumor invasion.     

Computing diffusion rates in T2-dark hematomas and areas of low T2 signal

BACKGROUND AND PURPOSE: It has been suggested that restricted diffusion is present within hematomas with intact red cell membranes; however, computing apparent diffusion coefficient (ADC) values in areas of low T2 signal can be problematic. Our purpose was to show the pitfalls of measuring diffusion within hematomas with intracellular blood products and to present a framework based on the properties of expected values for computing ADC values from regions with signal intensities close to that of the background noise (ie, T2-dark hematomas). METHODS: Twelve patients with intracranial hematomas who had undergone diffusion imaging were retrospectively identified during a 2-year period (four intracellular oxyhemoglobin, seven intracellular deoxyhemoglobin, one intracellular methemoglobin). Regions of interest were drawn on the hematomas, the contralateral white matter, and over the background. ADC values were computed using a variety of methods: 1) using expected values incorporating the variance of the background, 2) computing the mean of the regions of interest before taking the natural log, 3) masking negative values, and 4) masking the background at 0.5% increments from 0.5 to 5.5% and including the masked voxels (an intrinsically flawed method). Two-tailed Student's t test was performed between the white matter and the hematoma ADC values. RESULTS: There was no statistically significant difference between the hematomas and the white matter for methods 1 through 3 (P = .14, P = .23, and P = .83, respectively). Only method 4 revealed a statistically significant difference, beginning at 0.5% masking (P = .04) and becoming progressively more significant with increased masking (P = 4.14 x 10(-7) at 5.5% masking). The effect of masking was limited to the T2-dark hematomas. CONCLUSION: There is no restriction of diffusion for in vivo hematomas with intracellular blood products. The T2 blackout effect for T2-dark hematomas on diffusion-weighted images should not be interpreted as fast diffusion. The method of expected values can be used to obtain measurements for regions with signal intensities near the background noise. Using literature values for RBC self-diffusion, we computed lower limits of diffusion for hematomas with intracellular blood products to be 0.3 x 10(-3) mm2/s.     

Statistical model for diffusion attenuated MR signal.

A general statistical model that can describe a rather large number of experimental results related to the structure of the diffusion-attenuated MR signal in biological systems is introduced. The theoretical framework relies on a phenomenological model that introduces a distribution function for tissue apparent diffusion coefficients (ADC). It is shown that at least two parameters--the position of distribution maxima (ADC) and the distribution width (sigma)--are needed to describe the MR signal in most regions of a human brain. A substantial distribution width, on the order of 36% of the ADC, was found for practically all brain regions examined. This method of modeling the MR diffusion measurement allows determination of an intrinsic tissue-specific ADC for a given diffusion time independent of the strength of diffusion sensitizing gradients. The model accounts for the previously found biexponential behavior of the diffusion-attenuated MR signal in CNS.     

Analysis of partial volume effects in diffusion-tensor MRI.

The diffusion tensor is currently the accepted model of diffusion in biological tissues. The measured diffusion behavior may be more complex when two or more distinct tissues with different diffusion tensors occupy the same voxel. In this study, a partial volume model of MRI signal behavior for two diffusion-tensor compartments is presented. Simulations using this model demonstrate that the conventional single diffusion tensor model could lead to highly variable and inaccurate measurements of diffusion behavior. The differences between the single and two-tensor models depend on the orientations, fractions, and exchange between the two diffusion tensor compartments, as well as the diffusion-tensor encoding technique and diffusion-weighting that is used in the measurements. The current single compartment model's inaccuracies could cause diffusion-based characterization of cerebral ischemia and white matter connectivity to be incorrect. A diffusion-tensor MRI imaging experiment on a normal human brain revealed significant partial volume effects between oblique white matter regions when using very large voxels and large diffusion-weighting (b approximately 2.69 x 10(3) sec/mm(2)). However, the apparent partial volume effects in white matter decreased significantly when smaller voxel dimensions were used. For diffusion tensor studies obtained using typical diffusion-weighting values (b approximately 1 x 10(3) sec/mm(2)) partial volume effects are much more difficult to detect and resolve. More accurate measurements of multiple diffusion compartments may lead to improved confidence in diffusion measurements for clinical applications.     

A method for improving the performance of gradient systems for diffusion-weighted MRI.

The MR signal is sensitive to diffusion. This effect can be increased by the use of large, balanced bipolar gradients. The gradient systems of MR scanners are calibrated at installation and during regular servicing visits. Because the measured apparent diffusion constant (ADC) depends on the square of the amplitude of the diffusion sensitizing gradients, errors in the gradient calibration are exaggerated. If the error is varying among the different gradient axes, it will affect the estimated degree of anisotropy. To assess the gradient calibration accuracy in a whole-body MRI scanner, ADC values were calculated for a uniform water phantom along each gradient direction while monitoring the temperature. Knowledge of the temperature allows the expected diffusion constant of water to be calculated independent of the MRI measurement. It was found that the gradient axes (+/-x, +/-y, +/-z) were calibrated differently, resulting in offset ADC values. A method is presented to rescale the amplitude of each of the six principal gradient axes within the MR pulse sequence. The scaling factor is the square root of the ratio of the expected and observed diffusion constants. In addition, fiber tracking results in the human brain were noticeably affected by improving the gradient system calibration.     

Optimal acquisition orders of diffusion-weighted MRI measurements

PURPOSE: To propose a new method to optimize the ordering of gradient directions in diffusion-weighted MRI so that partial scans have the best spherical coverage. MATERIALS AND METHODS: Diffusion-weighted MRI often uses a spherical sampling scheme, which acquires images sequentially with diffusion-weighting gradients in unique directions distributed isotropically on the hemisphere. If not all of the measurements can be completed, the quality of diffusion tensors fitted to the partial scan is sensitive to the order of the gradient directions in the scanner protocol. If the directions are in a random order, then a partial scan may cover some parts of the hemisphere densely but other parts sparsely and thus provide poor spherical coverage. We compare the results of ordering with previously published methods for optimizing the acquisition in simulation. RESULTS: Results show that all methods produce similar results and all improve the accuracy of the estimated diffusion tensors significantly over unordered acquisitions. CONCLUSION: The new ordering method improves the spherical coverage of partial scans and has the advantage of maintaining the optimal coverage of the complete scan.     

Diffusion-weighted MR imaging of the normal human spinal cord in vivo

BACKGROUND AND PURPOSE: Diffusion-weighted imaging is a robust technique for evaluation of a variety of neurologic diseases affecting the brain, and might also have applications in the spinal cord. The purpose of this study was to determine the feasibility of obtaining in vivo diffusion-weighted images of the human spinal cord, to calculate normal apparent diffusion coefficient (ADC) values, and to assess cord anisotropy. METHODS: Fifteen healthy volunteers were imaged using a multi-shot, navigator-corrected, spin-echo, echo-planar pulse sequence. Axial images of the cervical spinal cord were obtained with diffusion gradients applied along three orthogonal axes (6 b values each), and ADC values were calculated for white and gray matter. RESULTS: With the diffusion gradients perpendicular to the orientation of the white matter tracts, spinal cord white matter was hyperintense to central gray matter at all b values. This was also the case at low b values with the diffusion gradients parallel to the white matter tracts; however, at higher b values, the relative signal intensity of gray and white matter reversed. With the diffusion gradients perpendicular to spinal cord, mean ADC values ranged from 0.40 to 0.57 x 10(-3) mm2/s for white and gray matter. With the diffusion gradients parallel to the white matter tracts, calculated ADC values were significantly higher. There was a statistically significant difference between the ADCs of white versus gray matter with all three gradient directions. Strong diffusional anisotropy was observed in spinal cord white matter. CONCLUSION: Small field-of-view diffusion-weighted images of the human spinal cord can be acquired in vivo with reasonable scan times. Diffusion within spinal cord white matter is highly anisotropic.     

弥散加权成像鉴别乳腺良恶性病变的价值初探

目的　探讨弥散加权成像（diffusionweightedimaging,DWI）的表面弥散系数（apparentdiffusioncoefficients,ADC）鉴别乳腺良恶性病变的价值。方法　健康志愿者10人,经手术病理证实的乳腺病变49例,其中恶性肿瘤26例,良性病变23例。DWI采用单次激发回波平面成像（echo-planarimaging,EPI）技术,14例取5个b值（b为扩散敏感度）,余者取2个b值,计算ADC值。以恶性肿瘤ADC值单侧上界95%容许区间为界限判断病灶的良恶性,诊断结果与动态增强比较。结果　除1例原位癌和1例小腺瘤外,DWI显示所有良恶性病变。恶性肿瘤组ADC值为(0.9608±0.2043)×10     

Quantifying the intra- and extravascular contributions to spin-echo fMRI at 3 T.

Functional MRI (fMRI) by means of spin-echo (SE) techniques provides an interesting alternative to gradient-echo methods because the contrast is based primarily on dynamic averaging associated with the blood oxygenation level-dependent (BOLD) effect. In this article the contributions from different brain compartments to BOLD signal changes in SE echo planar imaging (EPI) are investigated. To gain a better understanding of the underlying mechanisms that cause the fMRI contrast, two experiments are presented: First, the intravascular contribution is decomposed into two fractions with different regimes of flow by means of diffusion-weighting gradient schemes which are either flow-compensated, or will maximally dephase moving spins. Second, contributions from the intra- and extravascular space are selectively suppressed by combining flow-weighting with additional refocusing pulses. The results indicate two qualitatively different components of flowing blood which contribute to the BOLD contrast and a nearly equal share in functional signal from the intra- and extravascular compartments at TE approximately 80 ms and 3 T. Combining these results, there is evidence that at least one-half of the functional signal originates from the parenchyma in SE fMRI at 3 T. The authors suggest the use of flow-compensated diffusion weighting for SE fMRI to improve the sensitivity to the parenchyma.     

Analytical error propagation in diffusion anisotropy calculations

PURPOSE: To develop an analytical formalism describing how noise and selection of diffusion-weighting scheme propagate through the diffusion tensor imaging (DTI) computational chain into variances of the diffusion tensor elements, and errors in the relative anisotropy (RA) and fractional anisotropy (FA) indices. MATERIALS AND METHODS: Singular-value decomposition (SVD) was used to determine the tensor variances, with diffusion-weighting scheme and measurement noise incorporated into the design matrix. Anisotropy errors were then derived using propagation of error. To illustrate the applications of the model, 12 data sets were acquired from each human subject, over a range of b-values (500-2500 seconds/mm2) and diffusion-weighting gradient directions (N = 6-55). The mean RA and FA values and their respective errors were calculated within a region of interest (ROI) in the splenium. The RA and FA errors as a function of b-value and N were evaluated, and a number of diffusion-weighting schemes were assessed based on a new metric, sum of diffusion tensor variances. RESULTS: When the acquisition time was held constant, the sum of the diffusion tensor variances decreased as N increased. The same trend was also observed for several diffusion-weighting schemes with constant condition number when noise in the diffusion-weighted (DW) images was assumed unity. Errors in both FA and RA increased with b-value and decreased with N. The FA error in the splenium was approximately threefold smaller than RA error, irrespective of b-value or N. CONCLUSION: The condition number may not adequately characterize the noise sensitivity for a given diffusion-weighting scheme. Signal averaging may not be as effective as increasing N, especially when N is small (e.g., N < 13). Due to its smaller error, FA is preferred over RA for quantitative DTI applications.     

Apparent diffusion coefficient value of the hippocampus in patients with hippocampal sclerosis and in healthy volunteers

BACKGROUND AND PURPOSE: MR diffusion-weighted (DW) imaging with apparent diffusion coefficient (ADC) has had widespread use clinically in a variety of intracranial diseases; however, only a few studies report ADC changes in patients with hippocampal sclerosis. We sought to determine the ability of ADC to lateralize the epileptogenic lesion in patients with hippocampal sclerosis. METHODS: Nineteen healthy volunteers and 18 patients with intractable temporal lobe epilepsy whose MR imaging diagnosis was unilateral hippocampal sclerosis were examined prospectively with DW imaging and ADC mapping. DW images were obtained at 1.5 T with a spin-echo echo-planar sequence (6500/103 [TR/TE]) with variable diffusion gradients. ADCs were calculated from bilateral hippocampi. The ability of DW imaging and ADC to lateralize the lesion was evaluated visually and by comparing ADC values between healthy volunteers and patients with hippocampal sclerosis. RESULTS: In all patients, visual assessment of DW images failed to lateralize the lesion. However, the mean ADC value measured at the hippocampal area was significantly higher on the lesion side than on the contralateral side (P <.001). The overall correct lateralization rate of ADC was 100% (18 of 18 patients). Mean ADC in sclerotic hippocampi was also significantly higher than that in healthy volunteers. The normal-appearing hippocampus of the contralateral side in the patients had higher ADC values compared with those of healthy volunteers (P =.045). CONCLUSION: ADC can be used as a complementary tool in lateralizing the epileptogenic lesion in patients with hippocampal sclerosis, although the practical role of ADC value is yet to be determined in patients with inconclusive MR imaging findings.     

Diffusion tensor imaging and fiber tractography of C6 rat glioma

PURPOSE: To apply diffusion tensor images using 30 noncollinear directions for diffusion-weighted gradient schemes to characterize diffusion tensor imaging (DTI) features associated with C6 glioma-bearing rat brains, and ideally visualize fiber tractography datasets. MATERIALS AND METHODS: Fiber tractographies of normal male Fischer 344 rat brains were constructed from DTI datasets acquired with a 30 noncollinear diffusion gradient scheme. Cultured C6 cell were intracranially injected into the cortex of male Fischer 344 rats. The time course of the tumor growth was monitored with DTI and fiber tractography using diffusion-weighting gradients in 30 noncollinear directions. RESULTS: Fiber tractographies through the corpus callosum (CC) were easily visualized with the 30-direction gradient scheme, and the fiber trajectories of the motor cortex and striatum were well represented in normal rats. Fiber tractography indicated that the neuronal fibers of the CC were compressed or disappeared by growing C6 glioma, which affected surrounding brain tissue. CONCLUSION: We have demonstrated in this study that fiber tractography with the 30 noncollinear diffusion gradient scheme method can be used to help provide a better understanding regarding the influence of a tumor on the surrounding regions of normal brain tissue in vivo.     

Diffusion MRI findings in phenylketonuria

Two patients with phenylketonuria were studied who were under dietary control since infancy, and who were mentally normal. Diffusion MRI was obtained using a spin-echo, echo-planar sequence with a gradient strength of 30 mT/m at 1.5 T. A trace sequence (TR=5700 ms, and TE=139 ms) was used, acquired in 22 s. Heavily diffusion-weighted (b=1000 mm2/s) images, and the apparent diffusion coefficient (ADC) values from automatically generated ADC maps were studied. There were two different patterns in these two patients, restricted and increased diffusion patterns. Restricted diffusion pattern consisted of high-signal on b=1000 s/mm2 images with low ADC values ranging from 0.46 to 0.57x10(-3) mm2/s. Increased diffusion pattern consisted of normal b=1000 s/mm2 images with high ADC values ranging from 1.37 to 1.63x10(-3) mm2/s. It is likely that these values reflected presence of two different histopathological changes in phenylketonuria or reflected different stages of the same disease.