Hsiao-Fang Liang

Noninvasive detection of cuprizone induced axonal damage and demyelination in the mouse corpus callosum.

Previously, we tested the prediction that axonal damage results in decreased axial diffusivity (λ∥) while demyelination leads to increased radial diffusivity (λ⟂). Cuprizone treatment of C57BL/6 mice was a highly reproducible model of CNS white matter demyelination and remyelination affecting the corpus callosum (CC). In the present study, six C57BL/6 male mice were fed 0.2% cuprizone for 12 weeks followed by 12 weeks of recovery on normal chow. The control mice were fed normal chow and imaged in parallel. Biweekly in vivo DTI examinations showed transient decrease of λ∥ in CC at 2–6 weeks of cuprizone treatment. Immunostaining for nonphosphorylated neurofilaments demonstrated corresponding axonal damage at 4 weeks of treatment. Significant demyelination was evident from loss of Luxol fast blue staining at 6–12 weeks of cuprizone ingestion and was paralleled by increased λ⟂ values, followed by partial normalization during the remyelination phase. The sensitivity of λ⟂ to detect demyelination may be modulated in the presence of axonal damage during the early stage of demyelination at 4 weeks of cuprizone treatment. Our results suggest that λ∥ and λ⟂ may be useful in vivo surrogate markers of axonal and myelin damage in mouse CNS white matter. Magn Reson Med, 2006. Published 2006 Wiley‐Liss, Inc.

Toward accurate diagnosis of white matter pathology using diffusion tensor imaging

Diffusion tensor imaging (DTI) has been widely applied to investigate injuries in the central nervous system (CNS) white matter (WM). However, the underlying pathological correlates of diffusion changes have not been adequately determined. In this study the coregistration of histological sections to MR images and a pixel‐based receiver operating characteristic (ROC) analysis were used to compare the axial (λ∥) and radial (λ⟂) diffusivities derived from DTI and histological markers of axon (phosphorylated neurofilament, SMI‐31) and myelin (Luxol fast blue (LFB)) integrity, respectively, in two different patterns of injury to mouse spinal cord (SC) WM. In contusion SC injury (SCI), a decrease in λ∥ matched the pattern of axonal damage with high accuracy, but λ⟂ did not match the pattern of demyelination detected by LFB. In a mouse model of multiple sclerosis (MS), λ⟂ and λ∥ did not match the patterns of demyelination or axonal damage, respectively. However, a region of interest (ROI) analysis suggested that λ⟂‐detected demyelination paralleled that observed with LFB, and λ∥ decreased in both regions of axonal damage and normal‐appearing WM (NAWM) as visualized by SMI‐31. The results suggest that directional diffusivities may reveal abnormalities that are not obvious with SMI‐31 and LFB staining, depending on the type of injury. Magn Reson Med 57:688–695, 2007.

Radial diffusivity predicts demyelination in ex vivo multiple sclerosis spinal cords.

OBJECTIVE Correlation of diffusion tensor imaging (DTI) with histochemical staining for demyelination and axonal damage in multiple sclerosis (MS) ex vivo human cervical spinal cords. BACKGROUND In MS, demyelination, axonal degeneration, and inflammation contribute to disease pathogenesis to variable degrees. Based upon in vivo animal studies with acute injury and histopathologic correlation, we hypothesized that DTI can differentiate between axonal and myelin pathologies within humans. METHODS DTI was performed at 4.7 T on 9 MS and 5 normal control fixed cervical spinal cord blocks following autopsy. Sections were then stained for Luxol fast blue (LFB), Bielschowsky silver, and hematoxylin and eosin (H&E). Regions of interest (ROIs) were graded semi-quantitatively as normal myelination, mild (<50%) demyelination, or moderate-severe (>50%) demyelination. Corresponding axonal counts were manually determined on Bielschowsky silver. ROIs were mapped to co-registered DTI parameter slices. DTI parameters evaluated included standard quantitative assessments of apparent diffusion coefficient (ADC), relative anisotropy (RA), axial diffusivity and radial diffusivity. Statistical correlations were made between histochemical gradings and DTI parameters using linear mixed models. RESULTS Within ROIs in MS subjects, increased radial diffusivity distinguished worsening severities of demyelination. Relative anisotropy was decreased in the setting of moderate-severe demyelination compared to normal areas and areas of mild demyelination. Radial diffusivity, ADC, and RA became increasingly altered within quartiles of worsening axonal counts. Axial diffusivity did not correlate with axonal density (p=0.091). CONCLUSIONS Increased radial diffusivity can serve as a surrogate for demyelination. However, radial diffusivity was also altered with axon injury, suggesting that this measure is not pathologically specific within chronic human MS tissue. We propose that radial diffusivity can serve as a marker of overall tissue integrity within chronic MS lesions. This study provides pathologic foundation for on-going in vivo DTI studies in MS.

Detecting axon damage in spinal cord from a mouse model of multiple sclerosis

In the current study, the feasibility and reproducibility of in vivo diffusion tensor imaging (DTI) of the spinal cord in normal mice are illustrated followed by its application to mice with experimental allergic encephalomyelitis (EAE) to detect and differentiate axon and myelin damage. Axial diffusivity, describing water movement along the axonal fiber tract, in all regions of spinal cord white matter from EAE-affected C57BL/6 mice was significantly decreased compared to normal mice, whereas there was no statistically significant change in radial diffusivity, describing water movement across the fiber tract. Furthermore, a direct comparison between DTI and histology from a single mouse demonstrated a decrease in axial diffusivity that was supported by widespread staining of antibody against beta-amyloid precursor protein. Regionally elevated radial diffusivity corresponded with locally diminished Luxol fast blue staining in the same tissue from the EAE mouse cord. Our findings suggest that axonal damage is more widespread than myelin damage in the spinal cord white matter of mice with EAE and that in vivo DTI may provide a sensitive and specific measure of white matter injury.

Differential sensitivity of in vivo and ex vivo diffusion tensor imaging to evolving optic nerve injury in mice with retinal ischemia

Decreased axial (lambda(||)) and increased radial (lambda( perpendicular)) diffusivity have been shown to reflect axonal and myelin injury respectively. In the present study, evolving white matter injury within the optic nerves of mice with retinal ischemia was examined by in vivo and ex vivo measurements of lambda(||) and lambda( perpendicular). The results show that at 3 days after retinal ischemia, a 33% decrease in vivo and a 38% decrease ex vivo in lambda(||) without change in lambda( perpendicular) was observed in the injured optic nerve compared to the control, suggestive of axonal damage without myelin injury. At 14 days, both in vivo and ex vivo measured lambda( perpendicular) increased significantly to 220-240% of the control level in the injured optic nerve suggestive of myelin damage. In contrast, the axonal injury that was clearly detected in vivo as a significantly decreased lambda(||) (33% decrease) was not as clearly detected by ex vivo lambda(||) (17% decrease). The current findings suggest that ex vivo lambda( perpendicular) is comparable to in vivo lambda( perpendicular) in detecting myelin injury. However, the structural changes resulting from axonal damage causing the decreased in vivo lambda(||) may not be preserved ex vivo in the fixed tissues. Despite the accurate depiction of the pathology using lambda(||) and lambda( perpendicular) in vivo, the use of ex vivo lambda(||) to extrapolate the status of axonal injury in vivo would require further investigation.

Evolving Wallerian Degeneration after Transient Retinal Ischemia in Mice Characterized by Diffusion Tensor Imaging

Wallerian degeneration plays a significant role in many central nervous system (CNS) diseases. Tracking the progression of Wallerian degeneration may provide better understanding of the evolution of many CNS diseases. In this study, a 28-day longitudinal in vivo DTI of optic nerve (ON) and optic tract (OT) was conducted to evaluate the temporal and spatial evolution of Wallerian degeneration resulting from the transient retinal ischemia. At 3-28 days after ischemia, ipsilateral ON and contralateral OT showed significant reduction in axial diffusivity (32-40% and 21-29% respectively) suggestive of axonal damage. Both ON and OT showed significant increase in radial diffusivity, 200-290% and 58-65% respectively, at 9-28 days suggestive of myelin damage. Immunohistochemistry of phosphorylated neurofilament (pNF) and myelin basic protein (MBP) was performed to assess axonal and myelin integrities validating the DTI findings. Both DTI and immunohistochemistry detected that transient retinal ischemia caused more severe damage to ON than to OT. The current results suggest that axial and radial diffusivities are capable of reflecting the severity of axonal and myelin damage in mice as assessed using immunohistochemistry.

Noninvasive diffusion tensor imaging of evolving white matter pathology in a mouse model of acute spinal cord injury

We examined in vivo measurements of directional diffusivity derived from diffusion tensor imaging (DTI) to study the evolution of ventrolateral white matter (VWM) changes following contusive spinal cord injury (SCI) in C57BL/6 mice at 1, 3, 7, and 14 days postinjury. Relative anisotropy maps provided excellent gray matter (GM)/white matter (WM) contrast for characterization of evolving WM injury at all time points. Longitudinal DTI measurements clearly demonstrated rostral‐caudal injury asymmetry. Axial diffusivity provided a sensitive, noninvasive measure of axonal integrity within the injury epicenter and at remote levels. Quantitative measurements of axial and radial diffusivities in VWM showed a trend of acute primary axonal injury followed by delayed, subacute myelin damage at the impact site, with good histological correlation. Magn Reson Med 58:253–260, 2007.

Selective vulnerability of cerebral white matter in a murine model of multiple sclerosis detected using diffusion tensor imaging.

In this study, axial (lambda(parallel)) and radial (lambda(perpendicular)) diffusivities derived from diffusion tensor imaging (DTI) were used to evaluate white matter injury in brains of mice affected by experimental autoimmune encephalomyelitis (EAE). Sixteen female C57BL/6 mice were immunized with amino acids 35-55 of myelin oligodendrocyte glycoprotein (MOG(35-55)). Three months after immunization, optic nerve and tract were severely affected with 19% and 18% decrease in lambda(parallel) respectively, suggesting the presence of axonal injury. In addition, a 156% and 86% increase in lambda( perpendicular) was observed in optic nerve and tract respectively, suggestive of myelin injury. After in vivo DTI, mice were perfusion fixed and immunohistochemistry for the identification of myelin basic protein (MBP) and phosphorylated neurofilament (pNF) was performed to verify the presence of axonal and myelin injury. The present study demonstrated that the visual pathway is selectively affected in MOG(35-55) induced murine EAE and these injuries are non-invasively detectable using lambda(parallel) and lambda( perpendicular).

Quantitative Magnetization Transfer Measured Pool Size Ratio Reflects Optic Nerve Myelin Content in ex vivo Mice

Optic nerves from mice that have undergone retinal ischemia were examined using a newly implemented quantitative magnetization transfer (qMT) technique. Previously published results indicate that the optic nerve from retinal ischemia mice suffered significant axon degeneration without detectable myelin injury at 3 days after reperfusion. At this time point, we acquired ex vivo qMT parameters from both shiverer mice (which have nearly no myelin) and control mice that have undergone retinal ischemia, and these qMT measures were compared with diffusion tensor imaging (DTI) results. Our findings suggests that the qMT estimated ratio of the pool sizes of the macromolecular and free water protons reflected the different myelin contents in the optic nerves between the shiverer and control mice. This pool size ratio was specific to myelin content only and was not significantly affected by the presence of axon injury in mouse optic nerve 3 days after retinal ischemia. Magn Reson Med 61:364–371, 2009.

Noninvasive Topical Loading for Manganese-Enhanced MRI of the Mouse Visual System

PURPOSE To evaluate topical loading as an alternative to intravitreal injection for Mn(2+)-enhanced magnetic resonance imaging (MEMRI) of the visual system. METHODS Topical administration of 0.5 to 1.5 M MnCl(2) and intravitreal injections with 0.5 μL 100 mM and 2 μL 1 M MnCl(2) for mouse MEMRI were conducted, followed by immunohistochemistry. In another mouse group, two topical administrations of 1 M Mn(2+) were applied to the same animals 7 days apart, to evaluate the use of MEMRI in a time course study. Dynamic imaging was also conducted to reveal how Mn(2+) travels to the retina. MEMRI with topically loaded MnCl(2) was also conducted in eyes with retinal ischemia, to evaluate whether the enhancements required healthy neurons. RESULTS After 1 day, topical administration of 1 M and 1.5 M MnCl(2) rendered significant signal enhancement (up to 20%) in the superior colliculus (P < 0.05) that was equivalent to that of the 2-μL 1 M injection. Repeated exposure to Mn(2+) showed reproduced enhancement. Dynamic imaging showed significant enhancement in the iris, retina, and lens boundary, but not in the vitreous space. In retinal ischemic eyes, no enhancement of MEMRI was detected in the optic nerves. The immunohistochemistry of the optic nerve (1.5 mm anterior to the chiasm) and retina showed no injury 1 week after Mn(2+) topical administrations to each mouse. CONCLUSIONS The results demonstrated the feasibility of using topical administration of Mn(2+) for MEMRI. Topically loaded Mn(2+) did not diffuse into the vitreous space, but was it may have been absorbed into the iris to diffuse or travel via the capillary circulation to reach the retina.