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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: J Neuropathol Exp Neurol. 2010 Jul;69(7):704–716. doi: 10.1097/NEN.0b013e3181e3de90

Rostro-Caudal Analysis of Corpus Callosum Demyelination and Axon Damage Across Disease Stages Refines Diffusion Tensor Imaging Correlations with Pathological Features

Mingqiang Xie 1,*, Jennifer E Tobin 2,*, Matthew D Budde 1,†, Chin-I Chen 1,†, Kathryn Trinkaus 3, Anne H Cross 4, Dennis P McDaniel 5, Sheng-Kwei Song 1, Regina C Armstrong 1,§
PMCID: PMC2901930  NIHMSID: NIHMS213393  PMID: 20535036

Abstract

Non-invasive assessment of the progression of axon damage is important for evaluating disease progression and developing neuroprotective interventions in multiple sclerosis (MS) patients. We examined the cellular responses correlated with diffusion tensor imaging (DTI)-derived axial (λ||) and radial (λ⊥) diffusivity values throughout acute (4 weeks) and chronic (12 weeks) stages of demyelination and after 6 weeks of recovery using the cuprizone demyelination of the corpus callosum model in C57BL/6 and Thy1-YFP-16 mice. The rostro-caudal progression of pathologic alterations in the corpus callosum enabled spatially and temporally defined correlations of pathological features with DTI measurements. During acute demyelination, microglial/macrophage activation was most extensive and axons exhibited swellings, neurofilament dephosphorylation, and reduced diameters. Axial diffusivity values decreased in the acute phase but did not correlate with axonal atrophy during chronic demyelination. In contrast, radial diffusivity increased with the progression of demyelination but did not correlate with myelin loss or astrogliosis. Unlike other animals models with progressive neurodegeneration and axon loss, the acute axon damage did not progress to discontinuity or loss of axons even after a period of chronic demyelination. Correlations of reversible axon pathology, demyelination, microglia/macrophage activation, and astrogliosis with regional axial and radial diffusivity measurements will facilitate the clinical application of DTI in MS patients.

Keywords: Astrogliosis, Axon damage, Corpus callosum, Cuprizone, Demyelination, Diffusion tensor imaging, Microglia activation

INTRODUCTION

The pathologic features of the most common human demyelinating disease, multiple sclerosis (MS), include demyelination, axonal damage and inflammation in the acute stages and glial scar formation in chronic stages. MS patients have variable disease courses; relapsing-remitting disease generally correlates with inflammation whereas progressive disease more closely parallels axonal degeneration (1, 2). The complexity and variability of MS has fueled interest in the improvement of non-invasive imaging techniques for the evaluation of patient disease courses and therapeutic responses. Demyelinated lesion areas are typically identified by T2-weighted magnetic resonance (MR) imaging (MRI), with active inflammation and blood-brain barrier leakage detected as gadolinium-enhancement. Acute axonal damage has been observed in recent MS lesions by immunohistochemistry (IHC) for β-amyloid precursor protein (βAPP) and neurofilament dephosphorylation (3, 4). Acute axonal damage in neurodegenerative diseases can occur before clinical disease manifestations and may be reversible (5). However, MRI parameters of demyelination and inflammation have not matched well with evidence of axonal or neurodegeneration, such as MRI-assessed brain atrophy (6, 7). Therefore, additional non-invasive techniques are needed for the detection of acute axonal damage. In particular, there may be a window for stratifying therapeutic options to prevent axon degeneration in the first year after MS onset (3, 8).

Diffusion tensor imaging (DTI) has shown promise for the non-invasive detection of white matter injury (9, 10). Decreased axial diffusivity (λ||) describes water diffusion parallel to white matter fibers and indicates axonal injury in mouse traumatic brain injury models (11, 12), retinal ischemia with optic nerve degeneration (13–15), spinal cord injury (16–18), experimental autoimmune encephalomyelitis (EAE) (19–22), and corpus callosum (CC) demyelination following cuprizone treatment (23–25). Furthermore, λ|| has been used to detect axonal damage in patients with Alzheimer disease (26), and traumatic brain injury (27), and in postmortem specimens from MS patients (28). The DTI measurement of radial diffusivity (λ⊥) increases with experimental demyelination following cuprizone treatment (23–25) and secondary demyelination resulting from retinal ischemia (13, 15). The sensitivity of λ⊥ to detect demyelination may, however, be reduced in the presence of axonal injury (24).

Although axial and radial diffusivity measurements can be correlated with axon damage and demyelination, respectively, the potential impact of astrogliosis and inflammatory responses, including microglia/macrophage activation on these parameters, has not been investigated; different degrees or stages of axonal damage could impact λ||. For example, acute disease with axonal swellings and varicosities from disrupted axonal transport might alter axial diffusivity to a different extent than when there is chronic demyelination (which is typically associated with reduced axon diameter or atrophy). Similarly, λ⊥ values have not yet been evaluated for distinguishing active vs. chronic demyelination or for detecting regions of remyelination.

Here, we correlated λ|| and λ⊥ diffusivity measurements with quantitative analysis of relevant pathological features in the cuprizone neurotoxicant model of demyelination of the CC in mice (25, 29). Cuprizone demyelination involves oligodendrocyte apoptosis and microglial activation in the absence of lymphocytic infiltration, i.e. similar to the features reported in type III and IV MS lesions (30). DTI measurements of λ|| and λ⊥ were examined with parallel quantitative analysis of myelination, axon damage, gliosis, and microglia/macrophage activation in rostro-caudal regions of the CC over the acute demyelination, chronic demyelination, and remyelination stages of the model.

MATERIALS AND METHODS

Mice

All experimental procedures using mice were approved by the Washington University Animal Studies Committee and/or the USUHS Institutional Animal Care and Use Committee. Male C57BL/6 and Thy1-YFP-16 mice on the C57BL/6 background (both from The Jackson Laboratory, Bar Harbor, Maine) were fed ad libitum a diet of 0.2% (w/w) cuprizone (oxalic bis-(cyclohexylidenehydrazide); Sigma-Aldrich, Milwaukee, WI) mixed into milled chow pellets (Harlan Teklad, Indianapolis, IN) beginning at 8 weeks of age.

Diffusion Tensor Imaging

C57BL/6 mice (n = 5) underwent longitudinal DTI at time points throughout specific stages of the cuprizone disease progression. Specific time points for correlation with histological analysis were selected based on our previous longitudinal DTI analysis of cuprizone-treated C57BL/6 mice with scans every 2 weeks (24) as follows: 8 weeks of age before beginning cuprizone (0 weeks, pre-treatment controls); after 4 weeks of cuprizone (acute demyelination); after 12 weeks of cuprizone (chronic demyelination); and after 12 weeks of cuprizone plus 6 weeks of recovery on normal chow (12+6 weeks, recovery). As a non-treated control for aging during the prolonged cuprizone treatment time course, C57BL/6 mice (n = 5) fed with normal chow were imaged at 18 weeks of age.

Thy1-YFP-16 mice (n = 3) underwent serial DTI at 8 weeks of age before beginning cuprizone (0 weeks), after 4 weeks of cuprizone (4 weeks), and after 10 weeks of cuprizone (10 weeks). A set of age-matched Thy1-YFP-16 mice (n = 3) underwent DTI at corresponding time points as non-treated controls that were fed normal chow. Another cohort of Thy1-YFP-16 mice was used for cross-sectional analysis with DTI followed immediately by perfusion for histological analysis. Thy1-YFP-16 mice in this cohort were examined from time points corresponding to before beginning cuprizone (0 weeks, n = 5), after cuprizone treatment for 4 weeks (n = 5), or after cuprizone for 10 weeks (n = 5, including 2 mice from the longitudinally imaged cohort of YFP mice). For comparison to the C57BL/6 mice, the Thy1-YFP-16 mice were analyzed for the chronic condition after 10 weeks of cuprizone because the changes observed in the C57BL/6 mice were already able confirmed at this time point, as in our previous study (24).

The mice were anesthetized with a mixture of O2 and 4.5 % isoflurane, immobilized in a custom MR-compatible stereotaxic device, and placed on a pad of circulating warm water. Anesthesia was maintained at 1.0% to 1.5 % isoflurane in O2 and body temperature was maintained at 37°C throughout the DTI examination. A surface coil (1.5 cm o.d.) was placed above the head and served as the receiver. The mouse was positioned inside of a 9 cm i.d. Helmholtz transmit coil. The entire preparation was positioned in the center of a 4.7 Tesla Oxford Instruments magnet equipped with a 15 cm i.d. actively shielded Oxford gradient coil (20 G/cm, 200 μs rise time). The magnet, gradient coil, and Techron gradient power supply were interfaced with a Varian UNITY-INOVA console controlled by a Sun Microsystems Blade 1500 workstation.

Data were acquired using a spin-echo diffusion weighted imaging sequence. Acquisition parameters were repetition time (TR) = 1.5 s, echo time (TE) = 50 ms, Δ = 25 ms, δ = 8 ms, number of average = 4, slice thickness = 0.5 mm, field-of-view = 3 × 3 cm2, and data matrix = 256 × 256 (zero filled to 512 × 512). Diffusion sensitizing gradients were applied along 6 directions: [Gx,Gy,Gz] = [1,1,0], [1,0,1], [0,1,1], [−1,1,0], [0,−1,1], and [1,0,−1]. Two b-values (0 and 0.768 ms/μm2) were used. DTI data were acquired within 3 hours.

DTI Analysis

The eigenvalues (λ1, λ2, and λ3) of the diffusion tensor were derived by matrix diagonalization. On a pixel-by-pixel basis, axial (λ||) and radial diffusivity (λ⊥) were derived using software written in Matlab (MathWorks, Natick, MA), defined as previously reported (24).

Color-coded relative anisotropy maps were composed by associating each of the color channels with the components of the eigenvectors corresponding to λ||. The color reflects the primary axis of diffusion: red, left-right; green, superior-inferior; blue, anterior-posterior. Region of interest (ROI) was manually defined as the CC from the midline to under the peak of the cingulum on the color-coded relative anisotropy maps from 0.5 mm slices within the rostral (Fig. 1A), middle (Fig. 1B) and caudal (Fig. 1C) CC corresponding to anatomical positions centered at+0.75, −0.25 and −1.75 mm of bregma, respectively, using the point at which the anterior commissure crossed the midline as a means to register alignments (31). ROIs were applied to the corresponding λ|| and λ⊥ maps for subsequent analysis.

Figure 1.

Figure 1

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Diffusion tensor imaging (DTI) across rostro-caudal levels of the corpus callosum (CC) over the course of cuprizone treatment and recovery. (A–C) Color coded maps of relative anisotropy derived from DTI reflect the fiber orientation (red, medial-lateral; green, superior-inferior; blue, anterior-posterior) within white matter tracts in coronal MRI slices centered at anatomical positions within the CC to represent rostral (A; Bregma +0.75), middle (B; Bregma −0.25 mm), and caudal (C; Bregma −1.75) levels. The ROI analyzed within the CC extended from the midline to under the peak of the cingulum (outlined area). Example is after 4 weeks of cuprizone. (D, E) Quantification of axial (D) and radial (E) diffusivity values among C57BL/6 mice. One cohort of 5 mice was followed in a longitudinal series from prior to the start of treatment (pre-treatment; 8 weeks of age) and again at 4 and 12 weeks of cuprizone and after 12 weeks of cuprizone followed by a 6-week recovery period on normal chow (12 weeks cuprizone + 6 weeks). A second control cohort of 5 mice was scanned as a non-treated group at 18 weeks of age (18 weeks no cuprizone). Both axial and radial diffusivity values show a rostro-caudal pattern that is most pronounced in the caudal CC. Relative to pre-treatment controls, axial values are significantly decreased after 4 weeks of cuprizone (ap < 0.0001, bp = 0.0011, cp < 0.0001, for rostral, middle, and caudal CC, respectively) but overall exhibit a quadratic trend (p < 0.0001) with subsequent recovery to control levels. Radial diffusivity remains at pre-treatment levels through 4 weeks and then increases significantly by the 12-week time point (dp < 0.0001, ep = 0.004, and fp < 0.0001, for rostral, middle, and caudal, respectively) and remains elevated during the subsequent 6-week recovery period (gp = 0.0012, hp = 0.011, ip < 0.0001, for rostral, middle, and caudal, respectively). Pre-treatment and non-treated control cohorts exhibited similar values for each measurement. Scale bar = 1 mm.

Tissue Preparation, Electron Microscopy, and Immunohistochemistry in C57BL/6 Mice

Mice were perfused with 2% paraformaldehyde/3% acrolein for processing of alternate sections for electron microscopy (EM) and IHC from the same brain. Brains were dissected and cut on a vibratome into 40-μm sagittal serial sections. Parasagittal sections were processed for EM by post-fixing in 2% OsO4 in 0.1M phosphate buffer for 1 hour, followed by dehydration in a graduated series of ethanol. Dehydrated sections were then embedded in epoxy resin and a ROI for analysis was selected within each CC region (rostral, middle, and caudal). Thin sections (approximately 70 nm) were post-stained for 15 minutes in 2% aqueous uranyl acetate and 5 minutes in Reynolds lead citrate. Samples were examined in a Philips CM100 transmission electron microscope and images were recorded onto 4 × 5 cm photographic negatives developed according to the manufacturer’s instructions. Negatives were scanned at 1200 dpi and imported into MetaMorph (Molecular Devices) for quantitative analysis, including axon diameter and myelin thickness. A minimum of 100 axons was measured for each ROI in each mouse. Small diameter axons (<0.3 μm), which are typically within the unmyelinated population, were excluded from analysis in order to more specifically follow the changes during demyelination within the myelinated fiber population (32).

Alternate parasagittal floating 40-mm sections were double-immunostained for myelin oligodendrocyte glycoprotein (MOG) using monoclonal antibody 8-81C5 (mouse monoclonal IgG, donated by Dr. Minetta Gardinier, University of Iowa, Iowa City, IA) and total neurofilament (NF200, Chemicon, Billerica, MA) (33). Adjacent sections were double-immunostained for total (NF200; rabbit pan-neurofilament antibody) and non-phosphorylated neurofilaments (SMI-32 mouse monoclonal IgG; Covance, Princeton, NJ) to evaluate axonal integrity (4, 34). MOG and SMI-32 immunolabeling were detected with Cy3-conjugated donkey anti-mouse IgG F(ab′)2 and NF200 was detected using donkey anti-rabbit IgG F(ab′)2 conjugated with FITC (Jackson Immunoresearch, West Grove, PA).

Immunofluorescence images were acquired on an Olympus IX-70 microscope for low magnification (4x) analysis using a Spot 2 CCD digital camera (Diagnostic Instruments). High magnification (63x objective) images were captured using a Zeiss PASCAL 5 Laser Scanning confocal microscope. Morphometric studies of immunofluorescence were performed using MetaMorph software (Molecular Devices). The pathology of cuprizone toxicity differs along the rostro-caudal extent of the CC (25), therefore regions of the CC were examined separately for quantitative analysis corresponding to Bregma as approximately +0.25 to +0.75 mm (rostral), −0.5 to −1.0 mm (middle) and −1.5 to −2.0 mm (caudal) (31). NF200+ and SMI-32+ cross-sectional profiles were counted on high magnification confocal images that were imported into Adobe Photoshop 7. Axonal damage was estimated as the percentage of SMI-32+ profiles among all neurofilament+ profiles counted (summation of profiles detected with NF200, SMI-32, or labeled with both antibodies). The total density of axons was approximated by dividing the number of all neurofilament+ profiles by the area of each ROI.

Tissue Preparation and Immunohistochemistry in Thy1-YFP-16 Mice

At the conclusion of DTI examinations, Thy1-YFP-16 mice were perfused with 4% paraformaldehyde. Coronal brain sections (15 mm) were mounted onto slides and immunostained for either NG2 (rabbit polyclonal antibody, Chemicon), carbonic anhydrase II (rabbit polyclonal, Abcam, Cambridge, MA), glial fibrillary acidic protein (GFAP; mouse monoclonal IgG, Sigma Chemical Co., St. Louis, MO), or CD11b (rat monoclonal IgG, Abcam). Immunoreactivity (IR) was detected using goat anti-mouse, rat, or rabbit IgG conjugated with Cy3 (Jackson Immunoresearch) and cover-slipped using the Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) to visualize DNA (Vector Laboratories, Burlingame, CA). Immunofluorescence was examined with a Nikon Eclipse 80i fluorescence microscope, and images were acquired with a CCD digital camera using MetaMorph software (Molecular Devices).

To quantify axonal damage, infiltration of microglia/macrophages, and astrogliosis in Thy1-YFP-16 mice, 2 digital pictures were taken in the midline of the CC from sections for each mouse. Each image (1392 ×1040 pixels) was divided into 30 square subsections, and every subsection that contained the CC was randomized and then scored independently. Custom ImageJ macros were used to blind 2 raters to the group, animal, and section from which each subsection score was derived. A single score for each section was computed by averaging the scored subsections. Axonal injury was scored by modification of a published scale (35), where 0 = no injured axons, 1 = beaded axons, and 2 = beaded axons and loss of YFP fluorescence. The extent of microglia/macrophage infiltration and astrogliosis was scored in a similar fashion using the following scale: 0 = little to no CD11b or GFAP-IR; 1 = mild IR; 2 = moderate IR; 3 = severe IR (36). For each condition, 3 sections each were scored from 5 mice.

Statistical Analysis

Average DTI values, DAPI staining, YFP axonal damage, and immunostaining for microglia/macrophages and astrogliosis were compared across cuprizone time points of 0, 4 and 10 weeks using rank-based nonparametric tests. Average DTI values were also compared between cuprizone-treated and age-matched non-treated controls, at 4 and 10 weeks in Thy1-YFP-16 mice. A Kruskal-Wallis test was used to determine whether any differences exist among the experimental groups. Because the global test indicated that differences did exist, this was followed by pair-wise comparisons between experimental groups to identify the times at which differences were observed. A significance level of 0.05 was used for the overall test. Values are expressed as the mean ± SEM. DTI cohorts included 3 Thy1-YFP-16 mice or 5 C57BL/6 mice per condition examined; IHC analysis of Thy1-YFP-16 mice included 5 mice per condition or time point examined.

Quantification of neurofilament profiles detected by immunostaining and axons or myelinated fibers examined by EM was assessed using one-way analysis-of-variance with a Newman-Keuls post hoc test. Double immunofluorescence quantification of the proportion of SMI-32 profiles among those detected with NF200 was assessed using Chi square analysis. Values are expressed as the mean ± standard error of the mean. EM and IHC studies of C57BL/6 mice included 3 to 5 mice per condition or time point examined.

RESULTS

Radial (λ⊥) and Axial (λ||) Diffusivity Values Across the CC Rostro-Caudal Distribution in C57BL/6 Mice

We carried out a detailed analysis of axial and radial diffusivity at rostral, middle, and caudal sites in the CC and throughout cuprizone treatment stages (Fig. 1). Axial diffusivity values were significantly decreased overall and within each of the 3 regions after 4 weeks of treatment. The λ|| decrease relative to the pre-treatment imaging was more pronounced in the caudal than the rostral CC (34.6% vs. 21.5%) (Fig. 1D). Radial diffusivity values were unchanged after 4 weeks of treatment and were increased overall and within each of the 3 regions after 12 weeks. Relative to pre-treatment values, imaging of rostro-caudal slices throughout the CC demonstrated a gradient of increased λ⊥ diffusivity after 12 weeks that was more pronounced in the caudal than in the rostral CC (41.3% vs. 31.3%) (Fig. 1E).

Myelination Status Across the CC Rostro-Caudal Distribution in C57BL/6 Mice

Demyelination throughout the CC following cuprizone administration was evaluated by immunostaining for MOG (Fig. 2) and by EM (Fig. 3). Following 4 weeks of treatment, there was a dramatic rostro-caudal gradient of demyelination (Fig. 2A–C). During acute treatment, demyelinated areas have extensive infiltration and amplification of cells that distort the normal tissue structure and increase the area of the CC (Fig. 2D, E). Overall, there was a significant increase of CC area at 4 weeks of cuprizone treatment (50.6%; p < 0.001) whereas the CC area at 12 weeks of cuprizone (with or without a subsequent recovery period) was not significantly different from pre-treatment values (Fig. 2F).

Figure 2.

Figure 2

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Immunohistochemical analysis of myelination across rostro-caudal levels of the corpus callosum (CC) in C57BL/6 mice over the course of cuprizone treatment and recovery. (A–C) Confocal images from within sagittal brain sections immunostained for myelin oligodendrocyte glycoprotein (MOG; red; myelin marker) and NF200 (green; pan-neurofilament marker) in rostral (A), middle (B), and caudal (C) regions of the CC demonstrate a gradient in the extent of demyelination at 4 weeks of treatment. (D, E) Lower power images of coronal brain sections in the caudal region of the CC with myelin immunostained for MOG and nuclei labeled with DAPI. The normal CC area (D) expands with infiltration and amplification of cells during initial demyelination (E). Midline for each coronal section is aligned along the image border between D and E. (F) CC areas measured in low power sagittal sections of the entire CC immunostained for MOG. There was a significant increase in CC area at 4 weeks relative to all other time points (***p < 0.001). Scale bars: A = 25 μm, D = 250 μm.

Figure 3.

Figure 3

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Ultrastructural analysis of myelin and axonal parameters throughout rostro-caudal regions of the corpus callosum (CC) over the course of cuprizone treatment in C57BL/6 mice. (A–D) Electron micrographs illustrate demyelination and subsequent remyelination during cuprizone treatment and recovery: non-treated (0 weeks) controls (A), 4 weeks (B), 12 weeks (C), and 12+6 weeks (D) of cuprizone. There is a swollen axon with disorganized cytoskeletal elements and vacuolar material at 4 weeks (center of panel B). (E) Percentages of myelinated axons in rostral, middle, and caudal regions of the CC. All regions exhibited significant decreases in percent of myelinated axons after 4 weeks of treatment; this persisted through the recovery period (*p < 0.05, **p < 0.01, or ***p < 0.001 relative to non-treated (0 weeks) controls). In the rostral CC there was a significant increase in the percent of myelinated axons during the recovery period (##p < 0.01, relative to 12 weeks cuprizone). (F) There were significant decreases in axonal diameter vs. non-treated (0 weeks) controls after 4 weeks of cuprizone, which persisted through 12 weeks of cuprizone (**p < 0.01 for rostral, *p < 0.05 for middle and caudal). After 12 weeks of cuprizone and a 6-week period on normal chow there was a significant recovery in axon diameter (#p < 0.05 for rostral and caudal vs. 12 weeks cuprizone) approaching the pre-treatment values. A–D: scale bar = 1 μm.

Ultrastructural analysis showed significant differences in the proportions of myelinated fibers in each of the 3 CC regions after 4 weeks of cuprizone (Fig. 3B), with the greatest extent of demyelination in the caudal region (Fig. 3E). Demyelination throughout the CC was most extensive after 12 weeks of cuprizone (Fig. 3C, E). Following 12 weeks of cuprizone, partial remyelination occurred during the 6-week recovery period but the proportion of myelinated axons was still significantly below pre-treatment levels in all regions (Fig. 3C, E).

Axon diameter values were reduced after 4 weeks of cuprizone, even in the rostral CC where axons are not as frequently demyelinated as in caudal regions (Fig. 2A–C and 3E). Axonal atrophy persisted at 12 weeks of cuprizone with a significant return toward normal diameters in the rostral and caudal CC during the 6-week recovery period (Fig. 3F). During the recovery period, myelin thickness was consistently reduced for a given diameter axon (Table), an indication of remyelination. The ratio of axon diameter to myelinated fiber diameter, called the g-ratio, is not as clear an indicator of remyelination during the recovery period when axon diameters remain below values in non-treated mice (Table). The complexity of myelin thickness variation with axon diameter is further illustrated on a fiber population basis in Supplemental Figure 1.

Table.

Axon and Myelin Parameters Over the Course of Cuprizone Treatment

0 week control 4 weeks cuprizone 12 weeks cuprizone 12 weeks cuprizone, 6 weeks recovery
axon diameter (μm)
 Rostral 0.70 ± 0.034 0.55 ± 0.0055** 0.54 ± 0.0038** 0.64 ± 0.027#
 Middle 0.72 ± 0.053 0.55 ± 0.016* 0.52 ± 0.0052* 0.61 ± 0.045
 Caudal 0.68 ± 0.058 0.51 ± 0.029* 0.47 ± 0.023* 0.67 ± 0.026#
myelin thickness (μm)
 Rostral 0.093 ± 0.0028 0.099 ± 0.0027 0.11 ± 0.0050 0.085 ± 0.0041#
 Middle 0.093 ± 0.0023 0.11 ± 0.0066 0.099 ± 0.0031 0.076 ± 0.0041*#
 Caudal 0.092 ± 0.0036 0.11 ± 0.0085 0.091 ± 0.0024 0.082 ± 0.0011
g-ratio
 Rostral 0.80 ± 0.0031 0.74 ± 0.0038*** 0.76 ± 0.0057** 0.80 ± 0.0042###
 Middle 0.80 ± 0.0051 0.73 ± 0.0067*** 0.77 ± 0.0043** 0.82 ± 0.0049* ###
 Caudal 0.79 ± 0.010 0.74 ± 0.016* 0.77 ± 0.010 0.82 ± 0.00088#
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Statistical significance was evaluated using one-way ANOVA with a Newman-Keuls post hoc test within each anatomical region relative to 0 week control (*p < 0.05, **p < 0.01, ***p < 0.001) and for the 12 weeks cuprizone with 6 weeks recovery relative to the 12 weeks cuprizone (#p < 0.05, ### p < 0.001). Values are mean ± SEM. Axons less than 0.3 μm cannot be distinguished as unmyelinated or demyelinated axons and are not included in the axon diameter and subsequent measurements.

Immunohistochemical Evaluation of Axonal Damage and Loss

Accumulation of βAPP, an indication of impaired axonal transport, significantly increased after 3 and 5 weeks of cuprizone treatment but was greatly reduced after chronic demyelination for 12 weeks (23). To evaluate axonal alterations further, we immunostained for SMI-32 to detect dephosphorylated neurofilaments as an indicator of axonal damage along with the pan-neurofilament marker NF200 to detect the total population of axons (Fig. 4). At 4 weeks of cuprizone, the proportion of axons immunolabeled with SMI-32 was significantly increased only in the caudal CC (Fig. 4D). By 12 weeks of cuprizone treatment, the proportion of axons immunolabeled for SMI-32 was increased across all CC regions (Fig. 4D), in contrast to the λ|| values (Fig. 1D). The pattern of SMI-32 differences correlated with the axon diameter measurements (Fig. 3F).

Figure 4.

Figure 4

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Immunohistochemical analysis of axonal damage in C57Bl/6 mice. (A–C) Double immunolabeling with NF200 (green; pan-neurofilament marker) and SMI-32 (red; non-phosphorylated neurofilament epitope) in mice treated with cuprizone for 0 weeks (A), 4 weeks (B) or 12 weeks (C) and imaged using confocal microscopy of the caudal region of the corpus callosum (CC). (D) There is significant increase in the percent of SMI-32 immunolabeled axons among the total axon population after 4 weeks in the caudal CC and in all regions after 12 weeks of cuprizone vs. non-treated (0 weeks) controls (**p < 0.01 and ***p < 0.001). (E) There was no significant difference in the density of neurofilament profiles (NF200 only + SMI-32 only + double-labeled profiles) observed throughout cuprizone treatment. Scale bar = 20 μm.

We estimated the total density of axons based on NF200 pan-neurofilament immunolabeling; there were no significant differences in the densities of total neurofilament profiles throughout cuprizone treatment in any CC region or overall (Fig. 4E). Furthermore, the CC areas were not different between non-treated (0 weeks) and 12 weeks cuprizone mice (Fig. 2F), also suggesting that the total axon numbers were similar overall.

Cuprizone Administration in Thy1-YFP-16 Mice

To examine axonal structure over the cuprizone course, we used coronal sections of Thy1-YFP-16 mice. These mice have chimeric expression of YFP in neurons (35, 37) that permits fluorescence detection of the YFP distribution in longitudinal profiles of axons; this analysis can reveal structural changes such as varicosities and swellings that occur with impaired axonal transport. Thy1-YFP-16 mice exhibited loss of oligodendrocytes in the CC with 4 weeks of cuprizone (Supplemental Fig. 2). Coronal sections were used to characterize YFP axon distribution in the caudal CC region (Fig. 5), i.e. the area with the most extensive pathology following cuprizone treatment (Figs. 2–4). In control mice, YFP fluorescence was uniformly distributed along longitudinal axonal profiles with little or no examples of swelling (Fig. 5A, D). After 4 weeks of cuprizone, axons frequently showed regions of swelling and sites of apparent loss of YFP giving a beaded appearance in longitudinal profiles (Fig. 5B, E). With cuprizone treatment continued through 10 weeks (Fig. 5C, F), the YFP distribution appeared to have partially recovered with less prominent axonal varicosities. Analysis with a relative scoring system confirmed that YFP varicosities at 4 weeks of cuprizone were significantly greater than in the non-treated group or the 10 weeks cuprizone group (Fig. 5G). Compared to the 4-week time point, the incidence of varicosities was reduced approximately 65% at 10 weeks, indicating partial recovery of axonal integrity; however, values were still significantly above the levels estimated for non-treated mice (p < 0.0001).

Figure 5.

Figure 5

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Axonal varicosities detected by yellow fluorescent protein (YFP) distribution in Thy1-YFP-16 mice. (A–F) Coronal cryostat sections through the caudal corpus callosum to show YFP distribution longitudinally along axons. YFP is distributed uniformly along axons prior to the start of cuprizone (A, boxed area enlarged in D). At 4 weeks of cuprizone treatment the YFP distribution along axons is non-uniform, exhibiting swellings and varicosities or beading consistent with impaired axonal transport (B, boxed area enlarged in E). At 10 weeks of treatment, YFP is more distributed throughout axons and beading appears less marked than at the 4-week time point (C, boxed area enlarged in F). (G) The extent of acute axonal damage indicated by non-uniform YFP distribution was quantified as 0 = no beading or regions of YFP loss, 1 = beading, and 2 = beading and regions of YFP loss (see methods). YFP distribution is significantly different between the 0-, 4- and 10-week time points (G, p < 0.0001). Scale bars: A–C = 240 μm, D–F = 50 μm.

Axial and Radial Diffusivity Measurements in Thy1-YFP-16 Mice

Overall patterns of radial and axial diffusivity changes from pre-treatment through acute and continuous cuprizone administration stages was similar in both Thy1-YFP-16 (Fig. 6) and C57BL/6 (Fig. 1) mice. Axial diffusivity values were decreased after 4 weeks with recovery at 10 weeks of cuprizone (Fig. 6A). Radial diffusivity values were not altered after 4 weeks but then increased significantly by 10 weeks of cuprizone treatment (Fig. 6B). These similar findings between C57BL/6 and Thy1-YFP-16 mice indicate consistent patterns of λ|| and λ⊥ diffusivity measurements and the pathological analyses in each mouse strain.

Figure 6.

Figure 6

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Quantification of axial (A) and radial (B) diffusivity values in the corpus callosum (CC) of Thy1-YFP-16 mice. A cohort of 3 mice was followed in a longitudinal series from prior to the start of cuprizone treatment (0 weeks) and at 4 and 10 weeks of cuprizone treatment. Relative to controls (0 weeks), axial values are significantly decreased after 4 weeks of cuprizone (ap = 0.0006 for rostral and bp = 0.010 for caudal CC) but overall exhibit a quadratic trend (p < 0.0001) with subsequent recovery to control levels at 10 weeks (p = 0.87). In contrast, radial diffusivity is not changed until the 10 weeks cuprizone time point and is significantly increased compared to controls (cp = 0.0036, dp = 0.0001, and ep = 0.0003, for rostral, middle, and caudal, respectively).

Cell Populations Within the Corpus Callosum During Cuprizone Treatment in Thy1-YFP-16 Mice

A strong microglia/macrophage response has been reported during early cuprizone treatment (38) after which only a low level of activation appears to continue during prolonged feeding (39). To correlate the potential impact of the increased cell density during acute demyelination to DTI values and axonal damage, we examined patterns of microglia/macrophage activation with cuprizone treatment relative to axonal damage indicated by YFP distribution in the Thy1-YFP 16 mice. Using immunolabeling for CD11b to monitor microglia/macrophage responses in Thy1-YFP-16 mice, we found that compared to non-treated mice (Fig. 7A, D, G), the densities of CD11b+ cells was very high at 4 weeks of cuprizone treatment; DAPI labeling of nuclei was included to demonstrate the corresponding increase in cell density at this time point (Fig. 7B, E, H). By 10 weeks of cuprizone treatment the overall cell density and CD11b-IR were similar to non-treated mice but nuclear staining revealed a lack of organization that is typical in lesion areas (Fig. 7C, F, I). Quantitative analysis (Fig. 7J) with a relative scoring system of CD11b-IR demonstrated that pre-treatment values were increased 403% by 4 weeks of cuprizone but recovered to only a 60.5% increase over pre-treatment levels at 10 weeks of treatment. This pattern of CD11b-IR was similar to the pattern of change in axon damage in the Thy1-YFP-16 mice and, indeed, areas with YFP fluorescence patterns indicative of axon damage corresponded to areas of strong CD11b-IR (Fig. 7K–M).

Figure 7.

Figure 7

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Microglia/macrophage activation in the corpus callosum (CC) of Thy1-YFP-16 mice during acute and chronic demyelination. (A–I) DAPI nuclear stain (A–F) and CD11b immunolabeling (G–I) of microglia and macrophages in coronal CC sections of mice without (A, D, G), after 4 weeks (B, E, H), and after 10 weeks (C, F, I) of cuprizone treatment. Without cuprizone, DAPI-stained nuclei are mainly aligned in rows along axons, characteristic of interfascicular oligodendrocytes (A, boxed area enlarged in D) and few cells immunolabeled by CD11b (G). After 4 weeks, DAPI staining illustrates extensive cellular infiltration (B, boxed area enlarged in E) and increased CD11b immunolabeling (H). After 10 weeks, DAPI-stained nuclei exhibit a relatively normal density but are not aligned in rows (C, boxed area enlarged in F) and CD11b immunolabeling has normalized. (J) Microglia/macrophage activation and accumulation were quantified by scoring sections on a scale of 0 to 3, with 3 as the greatest extent of CD11b-immunoreactivity (IR). CD11b-IR was significantly increased in mice fed cuprizone for 4 weeks (***p = 0.0001) or 10 weeks (***p = 0.0001) vs. non-treated control mice (0 weeks). (K–M) Coronal CC sections from Thy1-YFP-16 mice treated with cuprizone for 4 weeks with axonal YFP fluorescence (K), CD11b immunolabeling (L), and the merged image (M) to show colocalization of high CD11B-IR in areas of vesiculated axons. Scale bars: 240 μm (A–C, K–M); 50 μm (D–I).

Astrogliosis has been reported during early cuprizone treatment in C57BL/6 mice (38). We used GFAP-IR to estimate the astrocyte response during cuprizone treatment for 4 and 10 weeks Semiquantitative analysis with a relative scoring system demonstrated an 80% increase in GFAP-IR in the caudal CC at 4 weeks with a further increase of 123% by 10 weeks of cuprizone, relative to pre-treatment (0 weeks) levels in adult Thy1-YFP-16 mice at 8 weeks of age (Fig. 8). This pattern of progressive changes across both 4 and 10 weeks of cuprizone is not consistent with the pattern observed for λ|| and λ⊥ diffusivity measures across these time points.

Figure 8.

Figure 8

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Astroglial response in the corpus callosum (CC) of Thy1-YFP-16 mice during acute and chronic demyelination. (A–F) Immunolabeling for glial fibrillary acidic protein (GFAP) in coronal CC sections to identify astrocytes in mice without (A, boxed area enlarged in D), after 4 weeks (B, boxed area enlarged in E), and after 10 weeks (C, with boxed area enlarged in F) of cuprizone treatment. (G) Astrogliosis was quantified using a scoring system from 0 to 3, with 3 as the greatest extent of GFAP-immunoreactivity (IR). Astrogliosis increases from the non-treated (0 weeks) control mice to the 4-week group (***p = 0.0005) and continues to increase from 4 to 10 weeks of (**p = 0.0064) of cuprizone treatment. Scale bars: A–C = 240 μm, D–F = 50 μm.

DISCUSSION

This study extends and refines the interpretation of DTI parameters relative to pathological features over the progression of demyelinating disease in the cuprizone model. Our analysis indicates that DTI-derived axial diffusivity values are reduced during the initial stages of demyelination in CC regions that are characterized by non-uniform axonal swellings, beads or varicosities along axon segments, loss of YFP within axons, increased numbers of SMI-32+ axons without loss of total number of axons, and microglia/macrophage activation. Axial diffusivity was not reduced during chronic demyelination in which axonal atrophy was prominent. Radial diffusivity values generally increased in chronically demyelinated CC regions but in areas with extensive axonal swelling and beading and marked inflammatory cell infiltration (e.g. the caudal CC at 4 weeks of cuprizone), radial diffusivity did not increase, possibly because of decreased intra-axonal water diffusivity after injury and/or the increased restriction arising from the infiltrating cells. These discrete differences in DTI signal relative to the onset and progression of white matter pathology are important for the application of DTI for the non-invasive assessment of the complex pathological features of human demyelinating diseases.

Our evaluation of axon pathology by IHC of Thy1-YFP transgenic reporter mice (40) and quantitative EM indicated that axonal swellings and varicosities in the early demyelination stage did not lead to significant axon loss over the course of chronic demyelination. The axonal swellings could be continuous with non-fragmented regions of axons and appeared to resolve between 4 and 10 weeks of cuprizone treatment. This observation is consistent with studies of Thy1-YFP mice with EAE in which inflammatory lesions exhibited axonal injury and loss of YFP while resolution of inflammation corresponded with return of YFP in axons (41). When the demyelination period was longer, continued axonal pathology was detected by IHC for dephosphorylated neurofilaments and by reduced axon diameters measured by EM However, axon densities were similar to those prior to cuprizone treatment and a lack of reduction of CC area between pre-treatment and chronic cuprizone treatment conditions also indicates a lack of significant axon loss. In MS, however, white matter atrophy is considered to be an indicator of axon loss (42). The ability of axons to remain viable following demyelination is supported in a recent example of non-immune demyelinating disease in cats (43).

Standard MR imaging of MS patients can monitor disease progression based on the number and size of lesion areas and the presence of gadolinium-enhancing lesions but cannot detect axonopathy; therefore, imaging frequently does not correlate well with neurological impairment. A combination of measurement of T2-weighted, T1-weighted, and magnetization transfer ratio has been suggested to distinguish axonal swelling and axonal loss in chronic MS (44). In DTI studies of mice with EAE, relative anisotropy was not sufficient to distinguish axonal pathology whereas axial diffusivity detected corresponding axonal damage (22). In a study of 4 weeks of cuprizone treatment of mice, reduced axial diffusivity corresponded with optical density area measurements in which neurofilament immunostaining was decreased and CD11b-IR was increased in the caudal CC (25). Because cellular infiltration increases the area of the CC at this time point, however, decreased optical density of neurofilament IHC may reflect spread in the area occupied by the axons rather than actual loss of axons. Therefore, the current findings are important for characterization of the directional diffusivity signals in analyses of axonal pathology throughout acute and chronic stages of demyelination. Specifically, axial diffusivity can distinguish regions of axonal swellings and varicosities, without there being a significant loss of axons, along with microglia/macrophage activation and accumulation (Figs. 6–8). Our present results show, however, that axial diffusivity does not reflect axonal atrophy (Figs. 1–4; Table).

The underlying mechanisms of DTI parameter changes responding to CNS injury have been difficult to resolve. Water diffusion in the CNS is affected by numerous cellular elements, including membranes, organelles, and cytoskeletal components, although lipid membranes appear to be the most prominent barriers to the motion of water molecules (45). These CNS components respond to injury in a highly complex and tightly interwoven manner. The resulting pathologies can occur with different severities and at different stages of the disease process, all of which contribute to the diffusion measurements in unique ways. We found that increased astrogliosis did not parallel the trend of change in λ|| (Fig. 8), suggesting that astrogliosis does not correspond with the observed decrease in λ|| after 4 wkof cuprizone, whereas microglia/macrophage infiltration coincided with acute axonal damage (Fig. 7). Thus, both acute axonal damage and microglia/macrophages may contribute to the observed decrease in λ||. A multiparametric analysis of mice with EAE revealed that λ|| was highly correlated with axonal damage, while increased cellularity also affected λ||, but to a lesser degree (22). DTI and histological techniques also have vastly different spatial resolutions and technical limitations may prevent direct correlations between these modalities (46). Continued studies to advance this correlation are important since histological determinants can usually provide more sensitive and direct examination of pathological features while DTI measures have strong advantages for non-invasive and longitudinal structural studies that can be performed in vivo.

Detection of axonal abnormalities in MS patients can be complicated by the presence of an inflammatory response in active lesions and scar formation in chronic inactive lesions. During the first year after the onset of MS, axon damage has been correlated with accumulation of CD-8+ cytotoxic T cells and activated microglia/macrophages (3). As in the present study and in EAE (40), CD11b-IR was colocalized in areas of axonal damage detected by dephosphorylated neurofilaments and loss of YFP in axons. βAPP accumulation is most extensive during the initial phase of CC demyelination with 3 to 6 weeks of cuprizone when microglial accumulation is most prominent (23, 39), but Lindner et al reported that SMI-32 immunostaining was not present after 4 weeks of cuprizone and only began to appear after 8 weeks (39). This observation has been interpreted as indicating 2 patterns of axonal injury, i.e. acute damage vs. the slow degeneration, occurring in the cuprizone model (39). In the present study, however, dissociation of axonal transport disruption and neurofilament dephosphorylation was not observed as there was SMI-32-IR in axonal swellings after 4 weeks of cuprizone (Fig. 4). Furthermore, we found reduced axonal diameter by EM (Fig. 3), which is consistent with neurofilament dephosphorylation (47, 48). Therefore, SMI-32-IR is present during both the acute axon damage associated with swellings and varicosities and during the chronic axon atrophy exhibiting relatively uniform reduction of axon diameter.

Evidence from several labs using similar methods for the cuprizone model indicates an ability of axons to recover from damage rather than progressing to discontinuity during either acute or chronic stages of demyelination. After 5 weeks of cuprizone, dystrophic axons appear to continue after removal of cuprizone from the diet for 1 or 2 weeks and comprised approximately 5% of the axon populations sampled (49). Reduction in axon diameter following the acute demyelination episode appears to be largely reversible upon return to normal chow and remyelination (32). Over the chronic cuprizone time course, loss of a small percentage of axons within the CC may occur and is not inconsistent with the current data. The restoration of YFP distribution along axons after the initial demyelination phase suggests that there is a resolution of the swellings and varicosities that might otherwise be expected to precede axon end bulb formation and transection (Fig. 5). Because our EM preparations were optimized for comparison of neurofilament IHC and ultrastructural analysis of axon diameter and myelin status in proximate sections from the same brain to better correlate these parameters for interpretation of our DTI, resolution of ultrastructural detail of axonal swellings following acute demyelination was not sufficient to determine whether observed accumulations of distended vacuolar structure and other variable electron dense elements were transient changes or likely to result in axonal disconnection (49). Similar to the analysis of acute cuprizone demyelination (32), we found reduced axon diameters throughout 12 weeks of treatment with a significant increase after a recovery period of 6 weeks (Table; Fig. 3F). Moreover, after chronic demyelination the remyelination is delayed and incomplete yet still serves as a significant indicator of axon viability (Fig. 3).

The mechanism for reversible axonal damage we observed is not clear. Cuprizone challenge causes copper deficiency, leading to dysfunction of oxidative phosphorylation and oligodendrocyte death which results in demyelination (29). The model was initially established in weanling mice with white matter pathology targeting the superior cerebellar peduncles (50, 51) and was subsequently adapted in 8- to 10-week-old mice to generate more extensive demyelination, particularly in the CC, and less toxicity (29, 32, 38, 49, 52, 53). The initial stage of demyelination results in a large cellular infiltrate consisting mainly of activated microglia/macrophages which function, in part, beneficially to clear apoptotic oligodendrocytes and myelin debris, facilitating spontaneous remyelination at 6 weeks of treatment (54). The pro-inflammatory cytokines secreted by activated microglia/macrophages may mediate axonal damage (55). Microglia/macrophages may similarly function both pathogenically and protectively in MS (56). In addition, oligodendrocyte progenitor cells proliferate to increase during acute cuprizone demyelination in both C57BL/6 mice (33) in Thy1-YFP-16 mice (Supplemental Fig. 2). These progenitors differentiate into oligodendrocytes so that partial remyelination begins even while mice are being fed cuprizone, progressing further during the return to normal chow ([32] and Fig. 3). Ongoing attempts at remyelination during the chronic cuprizone treatment may contribute to the preservation of atrophic axons. This rostro-caudal pattern of cuprizone induced CC demyelination was originally reported after 4 or 5 weeks of treatment (25, 49). The current study demonstrates that rostro-caudal differences are evident throughout the course of acute and chronic demyelination, with more extensive changes in the caudal CC (Figs 2, 3).

In summary, we demonstrate reversible axonal damage following severe acute swelling and robust infiltration of microglia/macrophages that may provide insight into mechanisms of early axon damage and the factors that mediate progression to discontinuity vs. continued viability. The reversibility of axon damage in this model may relate to a lack of cytotoxic T-cells and/or the relatively focal extent of injury (38, 57). The reversibility of demyelination in this model is also a factor since remyelination occurs in the presence of continued cuprizone treatment and demyelination (32). The non-invasive evaluation of axonal damage and demyelination afforded by axial and radial diffusivity measures in the discrete regions analyzed in the mouse CC should enhance the use of DTI parameters in MS and other conditions. In particular, the present results may translate to the development of a non-invasive means of detecting early, potentially reversible, acute axonal damage with regional specificity that affords a crucial window for effective therapy of MS patients.

Supplementary Material

Acknowledgments

Sources of support: National Institutes of Health Grants NS47592 (SKS), NS54194 (SKS) and NS39293 (RCA) and National Multiple Sclerosis Society Grants RG3515 (RCA), RG 3670A3/2 (SKS)

References

  • 1.Bruck W. Inflammatory demyelination is not central to the pathogenesis of multiple sclerosis. J Neurol. 2005;252 (Suppl 5):v10–5. doi: 10.1007/s00415-005-5003-6. [DOI] [PubMed] [Google Scholar]
  • 2.Wilkins A, Scolding N. Protecting axons in multiple sclerosis. Mult Scler. 2008;14:1013–25. doi: 10.1177/1352458508091370. [DOI] [PubMed] [Google Scholar]
  • 3.Kuhlmann T, Lingfeld G, Bitsch A, et al. Acute axonal damage in multiple sclerosis is most extensive in early disease stages and decreases over time. Brain. 2002;125:2202–12. doi: 10.1093/brain/awf235. [DOI] [PubMed] [Google Scholar]
  • 4.Trapp BD, Peterson J, Ransohoff RM, et al. Axonal transection in the lesions of multiple sclerosis. N Engl J Med. 1998;338:278–85. doi: 10.1056/NEJM199801293380502. [DOI] [PubMed] [Google Scholar]
  • 5.Pun S, Santos AF, Saxena S, et al. Selective vulnerability and pruning of phasic motoneuron axons in motoneuron disease alleviated by CNTF. Nat Neurosci. 2006;9:408–19. doi: 10.1038/nn1653. [DOI] [PubMed] [Google Scholar]
  • 6.Barkhof F, Filippi M. MRI--the perfect surrogate marker for multiple sclerosis? Nat Rev Neurol. 2009;5:182–3. doi: 10.1038/nrneurol.2009.31. [DOI] [PubMed] [Google Scholar]
  • 7.Inglese M, Benedetti B, Filippi M. The relation between MRI measures of inflammation and neurodegeneration in multiple sclerosis. J Neurol Sci. 2005;233:15–9. doi: 10.1016/j.jns.2005.03.001. [DOI] [PubMed] [Google Scholar]
  • 8.Dutta R, Trapp BD. Pathogenesis of axonal and neuronal damage in multiple sclerosis. Neurology. 2007;68:S22–31. doi: 10.1212/01.wnl.0000275229.13012.32. discussion S43–54. [DOI] [PubMed] [Google Scholar]
  • 9.Alexander AL, Lee JE, Lazar M, et al. Diffusion tensor imaging of the corpus callosum in Autism. Neuroimage. 2007;34:61–73. doi: 10.1016/j.neuroimage.2006.08.032. [DOI] [PubMed] [Google Scholar]
  • 10.Mori S, Zhang J. Principles of diffusion tensor imaging and its applications to basic neuroscience research. Neuron. 2006;51:527–39. doi: 10.1016/j.neuron.2006.08.012. [DOI] [PubMed] [Google Scholar]
  • 11.Mac Donald CL, Dikranian K, Bayly P, et al. Diffusion tensor imaging reliably detects experimental traumatic axonal injury and indicates approximate time of injury. J Neurosci. 2007;27:11869–76. doi: 10.1523/JNEUROSCI.3647-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Mac Donald CL, Dikranian K, Song SK, et al. Detection of traumatic axonal injury with diffusion tensor imaging in a mouse model of traumatic brain injury. Exp Neurol. 2007;205:116–31. doi: 10.1016/j.expneurol.2007.01.035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Song SK, Sun SW, Ju WK, et al. Diffusion tensor imaging detects and differentiates axon and myelin degeneration in mouse optic nerve after retinal ischemia. Neuroimage. 2003;20:1714–22. doi: 10.1016/j.neuroimage.2003.07.005. [DOI] [PubMed] [Google Scholar]
  • 14.Sun SW, Liang HF, Cross AH, et al. Evolving Wallerian degeneration after transient retinal ischemia in mice characterized by diffusion tensor imaging. Neuroimage. 2008;40:1–10. doi: 10.1016/j.neuroimage.2007.11.049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Sun SW, Liang HF, Le TQ, et al. Differential sensitivity of in vivo and ex vivo diffusion tensor imaging to evolving optic nerve injury in mice with retinal ischemia. Neuroimage. 2006;32:1195–204. doi: 10.1016/j.neuroimage.2006.04.212. [DOI] [PubMed] [Google Scholar]
  • 16.DeBoy CA, Zhang J, Dike S, et al. High resolution diffusion tensor imaging of axonal damage in focal inflammatory and demyelinating lesions in rat spinal cord. Brain. 2007;130:2199–210. doi: 10.1093/brain/awm122. [DOI] [PubMed] [Google Scholar]
  • 17.Kim JH, Loy DN, Liang HF, et al. Noninvasive diffusion tensor imaging of evolving white matter pathology in a mouse model of acute spinal cord injury. Magn Reson Med. 2007;58:253–60. doi: 10.1002/mrm.21316. [DOI] [PubMed] [Google Scholar]
  • 18.Loy DN, Kim JH, Xie M, et al. Diffusion tensor imaging predicts hyperacute spinal cord injury severity. J Neurotrauma. 2007;24:979–90. doi: 10.1089/neu.2006.0253. [DOI] [PubMed] [Google Scholar]
  • 19.Budde MD, Kim JH, Liang HF, et al. Toward accurate diagnosis of white matter pathology using diffusion tensor imaging. Magn Reson Med. 2007;57:688–95. doi: 10.1002/mrm.21200. [DOI] [PubMed] [Google Scholar]
  • 20.Wu Q, Butzkueven H, Gresle M, et al. MR diffusion changes correlate with ultra-structurally defined axonal degeneration in murine optic nerve. Neuroimage. 2007;37:1138–47. doi: 10.1016/j.neuroimage.2007.06.029. [DOI] [PubMed] [Google Scholar]
  • 21.Kim JH, Budde MD, Liang HF, et al. Detecting axon damage in spinal cord from a mouse model of multiple sclerosis. Neurobiol Dis. 2006;21:626–32. doi: 10.1016/j.nbd.2005.09.009. [DOI] [PubMed] [Google Scholar]
  • 22.Budde MD, Xie M, Cross AH, et al. Axial diffusivity is the primary correlate of axonal injury in the experimental autoimmune encephalomyelitis spinal cord: A quantitative pixelwise analysis. J Neurosci. 2009;29:2805–13. doi: 10.1523/JNEUROSCI.4605-08.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Song SK, Yoshino J, Le TQ, et al. Demyelination increases radial diffusivity in corpus callosum of mouse brain. Neuroimage. 2005;26:132–40. doi: 10.1016/j.neuroimage.2005.01.028. [DOI] [PubMed] [Google Scholar]
  • 24.Sun SW, Liang HF, Trinkaus K, et al. Noninvasive detection of cuprizone induced axonal damage and demyelination in the mouse corpus callosum. Magn Reson Med. 2006;55:302–8. doi: 10.1002/mrm.20774. [DOI] [PubMed] [Google Scholar]
  • 25.Wu QZ, Yang Q, Cate HS, et al. MRI identification of the rostral-caudal pattern of pathology within the corpus callosum in the cuprizone mouse model. J Magn Reson Imaging. 2008;27:446–53. doi: 10.1002/jmri.21111. [DOI] [PubMed] [Google Scholar]
  • 26.Huang J, Friedland RP, Auchus AP. Diffusion tensor imaging of normal-appearing white matter in mild cognitive impairment and early Alzheimer disease: Preliminary evidence of axonal degeneration in the temporal lobe. AJNR Am J Neuroradiol. 2007;28:1943–8. doi: 10.3174/ajnr.A0700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Kraus MF, Susmaras T, Caughlin BP, et al. White matter integrity and cognition in chronic traumatic brain injury: a diffusion tensor imaging study. Brain. 2007;130:2508–19. doi: 10.1093/brain/awm216. [DOI] [PubMed] [Google Scholar]
  • 28.Schmierer K, Wheeler-Kingshott CA, Boulby PA, et al. Diffusion tensor imaging of post mortem multiple sclerosis brain. Neuroimage. 2007;35:467–77. doi: 10.1016/j.neuroimage.2006.12.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Matsushima GK, Morell P. The neurotoxicant, cuprizone, as a model to study demyelination and remyelination in the central nervous system. Brain Pathol. 2001;11:107–16. doi: 10.1111/j.1750-3639.2001.tb00385.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Lucchinetti C, Bruck W, Parisi J, et al. A quantitative analysis of oligodendrocytes in multiple sclerosis lesions. A study of 113 cases. Brain. 1999;122:2279–95. doi: 10.1093/brain/122.12.2279. [DOI] [PubMed] [Google Scholar]
  • 31.Franklin KBJ, Paxinos G. The mouse brain in stereotaxic coordinates. San Diego, CA: Academic Press; 1997. [Google Scholar]
  • 32.Mason JL, Langaman C, Morell P, et al. Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre. Neuropathol Appl Neurobiol. 2001;27:50–8. doi: 10.1046/j.0305-1846.2001.00301.x. [DOI] [PubMed] [Google Scholar]
  • 33.Armstrong RC, Le TQ, Flint NC, et al. Endogenous cell repair of chronic demyelination. J Neuropathol Exp Neurol. 2006;65:245–56. doi: 10.1097/01.jnen.0000205142.08716.7e. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Sternberger LA, Sternberger NH. Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proc Natl Acad Sci U S A. 1983;80:6126–30. doi: 10.1073/pnas.80.19.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.McCarran WJ, Goldberg MP. White matter axon vulnerability to AMPA/kainate receptor-mediated ischemic injury is developmentally regulated. J Neurosci. 2007;27:4220–9. doi: 10.1523/JNEUROSCI.5542-06.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Plant SR, Arnett HA, Ting JP. Astroglial-derived lymphotoxin-alpha exacerbates inflammation and demyelination, but not remyelination. Glia. 2005;49:1–14. doi: 10.1002/glia.20089. [DOI] [PubMed] [Google Scholar]
  • 37.Feng G, Mellor RH, Bernstein M, et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron. 2000;28:41–51. doi: 10.1016/s0896-6273(00)00084-2. [DOI] [PubMed] [Google Scholar]
  • 38.Hiremath MM, Saito Y, Knapp GW, et al. Microglial/macrophage accumulation during cuprizone-induced demyelination in C57BL/6 mice. J Neuroimmunol. 1998;92:38–49. doi: 10.1016/s0165-5728(98)00168-4. [DOI] [PubMed] [Google Scholar]
  • 39.Lindner M, Fokuhl J, Linsmeier F, et al. Chronic toxic demyelination in the central nervous system leads to axonal damage despite remyelination. Neurosci Lett. 2009;453:120–5. doi: 10.1016/j.neulet.2009.02.004. [DOI] [PubMed] [Google Scholar]
  • 40.Bannerman PG, Hahn A. Enhanced visualization of axonopathy in EAE using thy1-YFP transgenic mice. J Neurol Sci. 2007;260:23–32. doi: 10.1016/j.jns.2007.03.020. [DOI] [PubMed] [Google Scholar]
  • 41.Shriver LP, Dittel BN. T-cell-mediated disruption of the neuronal microtubule network: correlation with early reversible axonal dysfunction in acute experimental autoimmune encephalomyelitis. Am J Pathol. 2006;169:999–1011. doi: 10.2353/ajpath.2006.050791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Miller DH, Barkhof F, Frank JA, et al. Measurement of atrophy in multiple sclerosis: Pathological basis, methodological aspects and clinical relevance. Brain. 2002;125:1676–95. doi: 10.1093/brain/awf177. [DOI] [PubMed] [Google Scholar]
  • 43.Duncan ID, Brower A, Kondo Y, et al. Extensive remyelination of the CNS leads to functional recovery. Proc Natl Acad Sci U S A. 2009;106:6832–6. doi: 10.1073/pnas.0812500106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Fisher E, Chang A, Fox RJ, et al. Imaging correlates of axonal swelling in chronic multiple sclerosis brains. Ann Neurol. 2007;62:219–28. doi: 10.1002/ana.21113. [DOI] [PubMed] [Google Scholar]
  • 45.Norris DG. The effects of microscopic tissue parameters on the diffusion weighted magnetic resonance imaging experiment. NMR in biomedicine. 2001;14:77–93. doi: 10.1002/nbm.682. [DOI] [PubMed] [Google Scholar]
  • 46.Sen PN, Basser PJ. A model for diffusion in white matter in the brain. Biophys J. 2005;89:2927–38. doi: 10.1529/biophysj.105.063016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Hoffman PN, Thompson GW, Griffin JW, et al. Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers. J Cell Biol. 1985;101:1332–40. doi: 10.1083/jcb.101.4.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Nixon RA, Paskevich PA, Sihag RK, et al. Phosphorylation on carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neurons in vivo: influences on regional neurofilament accumulation, interneurofilament spacing, and axon caliber. J Cell Biol. 1994;126:1031–46. doi: 10.1083/jcb.126.4.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Stidworthy MF, Genoud S, Suter U, et al. Quantifying the early stages of remyelination following cuprizone-induced demyelination. Brain Pathol. 2003;13:329–39. doi: 10.1111/j.1750-3639.2003.tb00032.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Blakemore WF. Remyelination of the superior cerebellar peduncle in the mouse following demyelination induced by feeding cuprizone. J Neurol Sci. 1973;20:73–83. doi: 10.1016/0022-510x(73)90119-6. [DOI] [PubMed] [Google Scholar]
  • 51.Ludwin SK. Central nervous system demyelination and remyelination in the mouse An ultrastructural study of cuprizone toxicity. Lab Invest. 1978;78:597–612. [PubMed] [Google Scholar]
  • 52.Hibbits N, Pannu R, Wu TJ, et al. Cuprizone demyelination of the corpus callosum in mice correlates with altered social interaction and impaired sensorimotor coordination. ASN Neuro. 2009;1:153–64. doi: 10.1042/AN20090032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Irvine KA, Blakemore WF. Age increases axon loss associated with primary demyelination in cuprizone-induced demyelination in C57BL/6 mice. J Neuroimmunol. 2006;175:69–76. doi: 10.1016/j.jneuroim.2006.03.002. [DOI] [PubMed] [Google Scholar]
  • 54.Streit WJ, Walter SA, Pennell NA. Reactive microgliosis. Prog Neurobiol. 1999;57:563–81. doi: 10.1016/s0301-0082(98)00069-0. [DOI] [PubMed] [Google Scholar]
  • 55.Pasquini LA, Calatayud CA, Bertone Una AL, et al. The neurotoxic effect of cuprizone on oligodendrocytes depends on the presence of pro-inflammatory cytokines secreted by microglia. Neurochem Res. 2007;32:279–92. doi: 10.1007/s11064-006-9165-0. [DOI] [PubMed] [Google Scholar]
  • 56.Heppner FL, Greter M, Marino D, et al. Experimental autoimmune encephalomyelitis repressed by microglial paralysis. Nat Med. 2005;11:146–52. doi: 10.1038/nm1177. [DOI] [PubMed] [Google Scholar]
  • 57.Crawford DK, Mangiardi M, Xia X, et al. Functional recovery of callosal axons following demyelination: A critical window. Neuroscience. 2009;164:1407–21. doi: 10.1016/j.neuroscience.2009.09.069. [DOI] [PubMed] [Google Scholar]

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