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Anatomy and Pathology/Oncology  |   September 2012
Myelin Sheath Decompaction, Axon Swelling, and Functional Loss during Chronic Secondary Degeneration in Rat Optic Nerve
Author Affiliations & Notes
  • Sophie C. Payne
    From Experimental and Regenerative Neurosciences, the
    School of Animal Biology and Western Australian Institute of Medical Research, and the
  • Carole A. Bartlett
    From Experimental and Regenerative Neurosciences, the
    School of Animal Biology and Western Australian Institute of Medical Research, and the
  • Alan R. Harvey
    From Experimental and Regenerative Neurosciences, the
    School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, Western Australia, Australia.
  • Sarah A. Dunlop
    From Experimental and Regenerative Neurosciences, the
    School of Animal Biology and Western Australian Institute of Medical Research, and the
  • Melinda Fitzgerald
    From Experimental and Regenerative Neurosciences, the
    School of Animal Biology and Western Australian Institute of Medical Research, and the
  • Corresponding author: Melinda Fitzgerald, Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, Crawley, 6009, WA, Australia; lindy.fitzgerald@uwa.edu.au
Investigative Ophthalmology & Visual Science September 2012, Vol.53, 6093-6101. doi:10.1167/iovs.12-10080
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      Sophie C. Payne, Carole A. Bartlett, Alan R. Harvey, Sarah A. Dunlop, Melinda Fitzgerald; Myelin Sheath Decompaction, Axon Swelling, and Functional Loss during Chronic Secondary Degeneration in Rat Optic Nerve. Invest. Ophthalmol. Vis. Sci. 2012;53(10):6093-6101. doi: 10.1167/iovs.12-10080.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To examine chronic changes occurring at 6 months following partial optic nerve (ON) transection, assessing optic axons, myelin, and visual function.

Methods.: Dorsal ON axons were transected, leaving ventral optic axons vulnerable to secondary degeneration. At 3 and 6 months following partial transection, toluidine-blue stained sections were used to assess dimensions of the ON injury site. Transmission electron microscopy (TEM) images of ventral ON were used to quantify numbers, diameter, area, and myelin thickness of optic axons. Immunohistochemistry and fluoromyelin staining were used to assess semiquantitatively myelin protein, lipids in ventral ON, and retinal ganglion cells (RGCs) in midventral retina. Visuomotor function was assessed using optokinetic nystagmus.

Results.: Following partial ON transection, optic axons and function remained disrupted at 6 months. Although ventral ON swelling observed at 3 months (P ≤ 0.05) receded to normal by 6 months, ultrastructurally, myelinated axons remained swollen (P ≥ 0.05), and myelin thickness increased (P ≤ 0.05) due to loosening of lamellae and an increase in the number of intraperiodic lines. Axons with decompacted myelin persisted and were distinguished as having large axonal calibers and thicker myelin sheaths. Nevertheless, progressive loss of myelin lipid staining with fluoromyelin was seen at 6 months. Despite no further loss of ventral optic axons between 3 and 6 months (P ≥ 0.05), visuomotor function progressively declined at 6 months following partial transection (P ≤ 0.05).

Conclusions.: Continued decompaction of myelin, altered myelin structure, and swelling of myelinated axons are persistent features of the chronic phases of secondary degeneration and likely contribute to progressive loss of visual function.

Introduction
Partial or incomplete injury to the central nervous system (CNS) is a debilitating medical condition in which neural tissue directly damaged by the lesion (primary injury) is lost via apoptosis. 1,2 Neurons and glia adjacent to the lesion initially remain intact and functional but are vulnerable to a cascade of metabolic events initiated by the primary injury, causing widespread secondary loss (secondary degeneration) and progressive decline in function. 38 Similarly, optic neuropathies such as glaucoma involve primary injury of retinal ganglion cell (RGC) axons and secondary degeneration of neural tissue with gradual loss of vision. 912 The chronic progression of secondary degeneration in the visual system can be studied using a well established model of partial optic nerve (ON) transection. 35,13 Dorsal optic axons are transected while neighboring ventral optic axons adjacent to the primary injury area are spatially separated from the lesion and remain intact but vulnerable to secondary degeneration. Previously, we have shown disruptions and abnormalities in ON morphology, axon caliber, and myelin integrity at 3 months following partial transection. However, it is likely the condition of the ON, axons, and myelin progressively worsens and becomes even more disrupted during chronic secondary degeneration. Characterizing ongoing changes associated with secondary damage in neurotrauma 6,14,15 and glaucoma 1618 is necessary in designing therapeutic strategies to improve long-term functional outcome and quality of life. 
Secondary degeneration involves a cascade of self-propagating events that continually affect neurons and glia, including oligodendrocytes and astrocytes, long after the time of the initial partial injury. 9,19,20 High levels of extracellular glutamate, released from the primary loss of neural tissue, affect nearby intact but vulnerable tissue causing excitotoxicity, intra-axonal calcium overload, mitochondrial dysfunction and energy failure, oxidative stress and production of free radical species, lipid peroxidation, cleavage of cytoskeletal proteins, axon swelling, and eventual secondary death via necrosis and/or apoptosis. 13,19,21,22 Secondary loss of neural tissue in turn releases more glutamate into the extracellular space, fuelling additional excitotoxicity in nearby tissue and furthering the cascade of secondary events. 19 Thus, due to the self-propagating nature of secondary degeneration, there is prolonged vulnerability of neurons and glia, which are perpetually affected long after the initial primary injury. Myelinating oligodendrocytes that form insulating myelin sheaths around axons are particularly susceptible to secondary degeneration. 23 Myelin has a high lipid (proteolipid/galactolipid) content and forms compact layers of lamellae held together with proteins that are vulnerable to disruption by reactive oxidative species and lipid peroxidation arising during secondary degeneration. 24 Given the prolonged nature of secondary degeneration and the vulnerability of myelin to secondary events, myelin loss and abnormalities may continue to worsen during chronic secondary degeneration. 
Previous studies describing secondary degeneration have focused extensively on the acute (1 day–12 weeks) changes to, and responses of, neurons and oligodendrocytes/myelin following partial CNS injury. 5,2527 Such time points represent a narrow range of assessment within the lifespan of the animal. Patients may live with glaucoma-induced blindness for decades, 28 while patients receiving a partial CNS injury experience life-long cognitive, motor, and sensory deficits. 2933 However, relatively few in vivo studies have investigated the responses of neurons and oligodendrocytes (or myelin) during chronic phases of secondary degeneration following partial CNS injury. Of the few studies that extended their investigation to, or beyond, 6 months after injury, demyelination was found to be a persistent feature, 34,35 intact neurons failed to conduct nerve impulses following spinal cord contusion, 35 and varying degrees of remyelination were observed. 34,36 Here we characterize chronic changes occurring at 6 months following partial ON transection and assess disruptions and abnormalities in ON morphology, myelin lipids, and visuomotor function. At the ultrastructural level, we characterize changes in numbers, caliber, and myelin integrity of myelinated axons and axons with decompacted myelin. 
Materials and Methods
Animals and Surgical Procedures
Procedures were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by The University of Western Australia Animal Ethics Committee. Female piebald-virol-glaxo (PVG) hooded rats (150–200 g; Animal Resources Centre, Perth, Australia) were kept under standard animal house conditions with ad libitum access to food and water. As described previously, the partial ON transection was made by incising the dorsal aspect of the right ON to a depth of 200 μm at 1 mm behind the eye, while avoiding damage to major blood vessels. 4,5,13 Normal, age matched, unoperated ONs were used as controls since sham operated ONs were no different from normal with respect to myelinated axon density, immune and oxidative stress response, RGC density, and visual behavior. 5  
Transmission Electron Microscopy (TEM) Tissue Preparation and Analysis
Following transcardial perfusion (4% paraformaldehyde 0.1 M phosphate buffer; pH 7.2–7.4), ONs of animals (normal n = 5; 3 months n = 3; 6 months n = 6) were sectioned for light microscopy (1 μm) and TEM (100 nm) as described previously. 37 Fewer animals were assessed at 3 months as we have already demonstrated axon and myelin abnormalities at this time, 37 and the focus of the current study is 6-month outcomes. Light microscope images (20x magnification) were taken of toluidine blue stained semithin ON sections to identify the section containing the injury site and to quantify ON dimensions and cross-sectional ventral ON area using image analysis software (ImageJ; public domain). The dorsal aspect of the ON was defined as the area where the lesion was located, and the line between dorsal and ventral ON was drawn on an image of the injury site at the location where the arc of the ON was at its widest when the dorsal lesion was uppermost in the image. Ventral ON was defined as the area below this line; the demarcation assumed approximately uniform tissue reorganisation across the ON and referred only to the area in the immediate vicinity of the injury site and not proximal or distal to it in the longitudinal dimension. Ten 1500×-magnification TEM (JEM2100; Jeol, Tokyo, Japan) images were digitally generated by an 11-Megapixel digital camera (Orius; Gatan, Pleasanton, CA) from the ventral ON at the level of the injury site, as described in detail previously, 37 covering a total field of view (FOV) area of 2193 μm2 (approximately 2% of ventral ON area; 800–1100 axons/animal). Percentage area of major reactive tissue processes, minimum axon diameter, myelin sheath thickness, and g-ratio (axon diameter/axon plus myelin diameter) of normally myelinated axons 23 and axon diameter and area of axons with decompacted myelin were determined using image analysis software (ImageJ). Minimum axon diameter was used to avoid artificial skewing of data due to axons projecting through the ON at an oblique angle. Axons were separated into three categories according to the condition of the myelin sheath, as described previously. 37 Briefly, at the FOV in which analysis was conducted, “myelinated axons” were observed as having compact, electron-dense myelin. When increasing the magnification to view individual myelin layers, some myelinated axons were observed to be mildly delaminated. However if 85% of the axon was ensheathed in compact myelin, such axons were still classified as “myelinated.” Some axons had delaminated myelin in which over 15% of myelin had split and formed distinct multiple layers of lamellae, and these were classified as having “decompacted myelin.” Axons with no myelin were classified as “unmyelinated.” For a subset of axons assessed at higher magnification (15,000×, number of axons = approximately 130/time point, normal and 6 months), the number of intraperiodic lines and the myelin thickness of axons with normal, loosened, or decompacted myelin were also determined (ImageJ). 
Immunohistochemistry and Fluoromyelin Staining
Serial transverse cryosections (12 μm) of ONs (normal n = 5–6; 3 months n = 6; 6 months n = 7) and sagittal sections (20 μm) of the eye cup and retina were cut along the dorsal-ventral axis and were collected onto microscope slides (Superfrost Plus; Thermo Fisher Scientific, Vic, Australia) and stored at −80°C. Immunohistochemical procedures using goat anti-myelin basic protein (MBP) antibody (1:150; Abcam, Cambridge, UK); rabbit anti-βIII-tubulin (1:500; Covance, Princeton, NJ)—a specific marker for RGCs, 5,13,38 secondary antibody AlexaFluor 546 (1:400; Molecular Probes, Grand Island, NY), and fluorescent myelin staining (1:300; FluoroMyelin; Invitrogen, Carlsbad, CA) were performed as described previously. 37 Slides were coverslipped using an antifade reagent (ProLong Gold; Invitrogen) and viewed using fluorescence microscopy (TE300; Nikon Instruments, Melville, NY) and imaging software (Metamorph; Molecular Devices, Sunnyvale, CA). For ONs, one FOV per animal was generated at the injury site in ventral ON (40× magnification; FOV area: 0.0354 mm2). Semiquantification of the intensities of MBP immunohistochemistry and fluoromyelin staining was determined using ImageJ software to generate arbitrary units with no thresholds set, while ensuring uniformity of processing, imaging, and exposure to light. All images for analyses of intensities at the different time points were taken at the same time, and any differences in background were due to fluorescence flaring from strongly immunopositive or stained tissue. For retinae, βIII-tubulin immunopositive RGCs were quantified in midventral retina in sections closest to the ON head (3 retinal sections/animal; 100× magnification, FOV 0.011 mm2) using the optical dissector technique to avoid double counting of RGCs in adjacent sections. 39  
Visuomotor Response: Optokinetic Nystagmus
Optokinetic nystagmus was examined in accordance to previous studies. 5,40 In brief, animals (normal n = 8; 3 months n = 9; 6 months n = 15) with left eyes (uninjured side) sutured shut, were placed inside a Perspex cylinder within a rotating optokinetic drum fitted with an alternating black and white striped stimulus pattern (0.13 cycles per degree). Following 2 minutes of acclimatization, the drum was rotated (1.4 rotations/minute) anticlockwise for 2 minutes and responses recorded. After 15 to 20 minutes break, a second acclimatization and trial was conducted. Smooth pursuit and fast reset responses were quantified and analyzed from the second trial and expressed as mean responses per unit time that the animal was attending to the task. Light levels were standardized to 900 to 1100 lux. 
Statistical Analysis
All data were normally distributed with equal variances, and differences between groups (normal, 3, and 6 months) were examined using one-way ANOVA and Bonferroni/Dunn post hoc test (P ≤ 0.05). 
Results
Ventral ON Morphology and Reactive Tissue during Chronic Secondary Degeneration
Following partial transection, ON diameter (Figs. 1A–C), measured mediolaterally across the nerve, was unchanged from normal at 3 months (P > 0.05) but decreased by 6 months (P ≤ 0.05; Fig. 1G). ON ventral radius (Figs. 1A–C), measured from the midpoint of the diameter to the most ventral point of the ON, increased at 3 months (P ≤ 0.05) but was significantly decreased by 6 months (P ≤ 0.05) to a length not different to normal (Fig. 1H). Ventral ON area significantly increased by 26% (P ≤ 0.05) at 3 months but returned to normal by 6 months (P > 0.05; Fig. 1I). Similarly, total ON area, with the area of reactive tissue excluded, significantly increased at 3 months (P 0.05; 0.417 ± 0.055 mm2) when compared to normal (0.256 ± 0.017 mm2) but returned to values not different from normal at 6 months (P ≥ 0.05; 0.269 ± 0.015 mm2). The percentage of ventral ON consisting of major processes of reactive tissue (Figs. 1D–F; area indicated within dotted red lines) increased 2-fold from 11.6% to 25% at 3 months, (P ≤ 0.05), remaining higher than normal at 6 months (P ≤ 0.05; 16.4%; Fig. 1J). Although ON swelling receded at 6 months, at the ultrastructural level we observed novel secondary changes in axon caliber and myelin sheath at 6 months (see below). 
Figure 1. 
 
Changes in ventral ON at 3 and 6 months following partial transection. Transverse toluidine-blue stained semithin sections (AC) were used to measure ON diameter, ventral radius (black line), and ventral ON area (below the black line). TEM images (DF) were used to quantify major reactive processes (outlined in red). Data for graphs (G, F) are expressed as mean ± SE with major reactive processes defined as the mean percentage of the FOV ± SE (J). Differences between experimental groups are indicated by *(P < 0.05). Scale bars AC: 100 μm, DF: 2 μm.
Figure 1. 
 
Changes in ventral ON at 3 and 6 months following partial transection. Transverse toluidine-blue stained semithin sections (AC) were used to measure ON diameter, ventral radius (black line), and ventral ON area (below the black line). TEM images (DF) were used to quantify major reactive processes (outlined in red). Data for graphs (G, F) are expressed as mean ± SE with major reactive processes defined as the mean percentage of the FOV ± SE (J). Differences between experimental groups are indicated by *(P < 0.05). Scale bars AC: 100 μm, DF: 2 μm.
Number, Diameter, and Myelin Thickness of Myelinated Axons during Chronic Secondary Degeneration
Absolute numbers of axons in ventral ON significantly decreased at 3 months by 30.7% (P ≤ 0.05), compared to normal, with no further loss at 6 months (P > 0.05, Fig. 2G). The pattern of axon loss in ventral ON was reflected in the pattern of RGC loss in midventral retina (β-III-tubulin+ve cell bodies). RGC density significantly decreased from normal (2243 ± 69.48 RGCs/mm2) by 32.5% at 3 months (P ≤ 0.05; 1515 ± 129 RGCs/mm2) with no further significant loss by 6 months (P > 0.05; 1239 ± 165 RGCs/mm2). 
Figure 2. 
 
Representative TEM images and quantification of the number, diameter, myelin thickness, and g-ratio of myelinated axons, vulnerable to secondary degeneration in normal ONs and at 3 and 6 months following partial transection. Myelinated axons from normal ON (AC) are indicated by >, and at 6 months following injury (DE) indicated by >>. Data for graphs (DJ, L) are expressed as total numbers of myelinated axons in ventral ON (G), mean ± SE, or frequency of axons with small (<0.4 μm) or large (>0.9 μm) diameters (L). Differences between experimental groups are indicated by *(P < 0.05). Scatter graph of axon diameter versus myelin thickness (K) shows mean values of each FOV (100–160 axons/FOV; 10 FOV/animal). Scale bars A , DF: 200 nm, B, C: 100 nm.
Figure 2. 
 
Representative TEM images and quantification of the number, diameter, myelin thickness, and g-ratio of myelinated axons, vulnerable to secondary degeneration in normal ONs and at 3 and 6 months following partial transection. Myelinated axons from normal ON (AC) are indicated by >, and at 6 months following injury (DE) indicated by >>. Data for graphs (DJ, L) are expressed as total numbers of myelinated axons in ventral ON (G), mean ± SE, or frequency of axons with small (<0.4 μm) or large (>0.9 μm) diameters (L). Differences between experimental groups are indicated by *(P < 0.05). Scatter graph of axon diameter versus myelin thickness (K) shows mean values of each FOV (100–160 axons/FOV; 10 FOV/animal). Scale bars A , DF: 200 nm, B, C: 100 nm.
Myelinated axons with compact layers of myelin lamellae were observed in the ON of normal animals (Figs. 2A–C, indicated by >). Although at 6 months after injury such myelinated axons were present, a maximum of 15% of the myelin sheath surrounding many of these axons was loosened, with slightly disrupted layers of myelin lamellae widespread at 6 months after injury (Figs. 2D–F, indicated by >>). Out of the total axon population in normal ventral ON, axons with ostensibly normal myelin represented 92% of the total axon population (Fig. 2G), which decreased to represent 81.3% of the total population at 3 months, and 77% of the population at 6 months (Fig. 2G). Axon diameter, myelin thickness, and g-ratio were assessed in axons classified as myelinated in ventral ON. Following partial transection, axon diameter increased at 3 months (P < 0.05) and remained larger than normal at 6 months (P < 0.05), with no difference between these time points (P > 0.05, Fig. 2H). 
A novel finding of this study was that myelin sheath thickness significantly increased at 6 months (P < 0.05, Fig. 2I), compared to normal and 3 months' thickness values. The increase partly reflected the loosening of myelin lamellae, as shown in Figs. 2D–F, as the thickness of the myelin sheath in areas of loosened or delaminated myelin, assessed in a subset of axons at high magnification, was higher (0.293 ± 0.027 μm) than myelin thickness in areas of compact myelin at 6 months after injury (0.203 ± 0.016 μm, P ≤ 0.05). Furthermore, the difference in thickness between compact and loosened myelin in normal ON was 0.014 ± 0.0005 μm, and this substantially increased at 6 months to 0.051 ± 0.005 μm. Assessment of the number of intraperiodic lines in the myelin sheath of myelinated axons revealed a significant increase at 6 months compared to ON of normal animals (normal: 7.04 ± 0.19; 6 months: 10.73 ± 1.29, P ≤ 0.05), regardless of myelin loosening. There was no change in the thickness of individual myelin lamellae between normal and 6 months (normal: 0.011 ± 0.0007 μm; 6 months: 0.014 ± 0.004 μm, P > 0.05). Consequently, the increase in myelin thickness compensated for axon swelling and resulted in the g-ratio of myelinated axons returning to normal by 6 months following partial transection (P > 0.05, Fig. 2J). A scatter graph of axon diameter and myelin thickness highlights the chronic changes in ventral optic axon diameters and myelin thickness between normal, 3, and 6-month groups (Fig. 2K). Furthermore, the proportion of axons with small diameters (<0.4 μm) decreased at 6 months (P ≤ 0.05) while the proportion of axons with larger diameters (>0.9 μm) increased at this time point (P ≤ 0.05, Fig. 2L). 
Number, Area, and Diameter of Axons with Decompacted Myelin during Chronic Secondary Degeneration
Axons with decompacted myelin (distinct from the myelinated axons described above with greater than a 15% proportion of the myelin sheath surrounding the axon decompacted) were seen in normal ventral ON (Figs. 3A, 3B) and at 3 months (Fig. 3C) and 6 months (Fig. 3D) following partial transection (indicated by >>). The degree of decompaction of myelin surrounding individual axons was similar within all groups (Figs. 3A–D, indicated by >>), although the frequency of axons with decompacted myelin increased during secondary degeneration. In normal ON 1.5% of axons were unmyelinated (1020 ± 182 axons), 5.5% of axons had decompacted myelin (3962 ± 528 axons; Fig. 3F), and 1% of axons were lightly or excessively myelinated (data not shown). The number of unmyelinated axons remained similar to normal following partial transection (P > 0.05); 3 months: 2032 ± 646 axons; 6 months: 2146 ± 386 axons). However, the number of axons with decompacted myelin doubled from normal at 3 months (P ≤ 0.05; 12.3% of the total axon population) and remained elevated at 6 months (P > 0.05; 15.4% of the total axon population; Fig. 3E), with no significant difference between these time points (P > 0.05). 
Figure 3. 
 
Representative TEM images and quantification of axons with decompacted myelin, vulnerable to secondary degeneration at 3 and 6 months following partial transection. Axons with decompacted myelin in normal ON (AB), and at 3 months (C) and 6 months (D) are indicated with arrows (>>). Data for graphs (EF) are expressed as mean ± SE. Differences between experimental groups are indicated by *(P ≤ 0.05). Relative frequency distribution of axon diameters (GI) is expressed as counts within 0.1 μm intervals, total of 27 intervals, and cumulative frequency (J) up to 1. Scale bar = AC: 200 nm, D: 100 nm.
Figure 3. 
 
Representative TEM images and quantification of axons with decompacted myelin, vulnerable to secondary degeneration at 3 and 6 months following partial transection. Axons with decompacted myelin in normal ON (AB), and at 3 months (C) and 6 months (D) are indicated with arrows (>>). Data for graphs (EF) are expressed as mean ± SE. Differences between experimental groups are indicated by *(P ≤ 0.05). Relative frequency distribution of axon diameters (GI) is expressed as counts within 0.1 μm intervals, total of 27 intervals, and cumulative frequency (J) up to 1. Scale bar = AC: 200 nm, D: 100 nm.
Contrary to the persistent swelling of myelinated axons during secondary degeneration, the mean diameter of unmyelinated axons remained unchanged and similar to normal after injury (normal: 0.478 ± 0.028 μm, 3 months: 0.538 ± 0.03 μm, 6 months: 0.59 ± 0.064 μm; P > 0.05). The cross-sectional area and diameter of axons with decompacted myelin were compared to the dimensions of myelinated axons. The diameter and area of axons with decompacted myelin (Figs. 3F–I) were consistently far larger than those of myelinated axons in normal ventral ON and at 3 and 6 months following injury (Fig. 3F; P < 0.05). Furthermore, at 6 months, in axons with decompacted myelin, the myelin thickness in specific areas of the myelin sheath that was compact was 0.287 ± 0.053 μm, equivalent to the thickness of loosened myelinated axons (0.293 ± 0.027 μm) and not significantly different from the compact myelin thickness of myelinated axons (0.203 ± 0.016 μm) despite increased axon area (Fig. 3F). In areas that were decompacted, myelin thickness was 0.773 ± 0.015 μm, significantly higher than the myelin thickness of compact or loosened myelin in myelinated axons at 6 months, as described above (P ≤ 0.05). The number of intraperiodic lines in the myelin sheath of axons with decompacted myelin was not further increased from the numbers in normally myelinated axons at 6 months (axons with decompacted myelin: 13.87 ± 2.46; normally myelinated axons: 10.73 ± 1.29; P > 0.05). The relative frequency distribution (numbers of axons with diameters within 0.1 μm intervals across 30 intervals; Figs. 3G–I) of the diameters of axons with decompacted myelin illustrates that there was no change in the distribution of these axon diameters following injury, also supported by the cumulative frequency (Fig. 3J). Thus, while myelinated axons swell as secondary degeneration progresses, axons with decompacted myelin have a comparatively larger axon diameter, regardless of injury. 
Myelin Loss and Functional Deficits during Chronic Secondary Degeneration
Following partial transection, fluoromyelin staining intensity in ventral ON decreased from normal at 3 months (P < 0.05) and further decreased at 6 months (P < 0.05; Figs. 4A–C, 4G). MBP immuno-intensity in ventral ON also decreased at 3 months (P ≤ 0.05) with no further loss at 6 months (P ≥ 0.05; Figs. 4D–F, 4H). Visual function loss at 6 months following partial ON transection largely reflected loss and decompaction of myelin. The number of smooth pursuit responses made by normal animals was 12 ± 1.3 responses per minute, and this decreased to 8.4 ± 0.6 responses per minute at 3 months (P ≤ 0.05), with a trend to a further decrease at 6 months (P > 0.05; 6.3 ± 0.6 responses/minute). The number of fast reset responses made by normal animals was 6.7 ± 1.2 responses per minute. Following partial transection, these responses remained unchanged at 3 months (P > 0.05; 4.7 ± 0.5 responses/minute) but were significantly less than normal at 6 months (P ≤ 0.05; 3.6 ± 0.5 responses/minute). 
Figure 4. 
 
Representative images and semiquantification of the intensity of fluoromyelin staining (AC, G) and immuno-intensity of MBP (DF, H) in ventral ON, vulnerable to secondary degeneration (FOV: 0.0354 mm), at 3 and 6 months following partial transection. Data were expressed as mean immunohistochemical and staining intensities ± SE and differences between experimental groups indicated by *(P ≤ 0.05). Scale bar = 50 μm.
Figure 4. 
 
Representative images and semiquantification of the intensity of fluoromyelin staining (AC, G) and immuno-intensity of MBP (DF, H) in ventral ON, vulnerable to secondary degeneration (FOV: 0.0354 mm), at 3 and 6 months following partial transection. Data were expressed as mean immunohistochemical and staining intensities ± SE and differences between experimental groups indicated by *(P ≤ 0.05). Scale bar = 50 μm.
Discussion
Following partial injury to ON, secondary degeneration causes disruptions and abnormalities to axon caliber, myelin sheath integrity, and visual function that persist for at least 6 months after injury. Ventral ON swelling resolves after 6 months, presumably due to a decrease in amounts of reactive tissue. Ultrastructurally, myelinated axon swelling and prevalence of large caliber axons with decompacted myelin persist as an abnormal feature sustained throughout chronic secondary degeneration. A novel finding of this study was an increase in the thickness of myelin surrounding myelinated axons at 6 months due, in part, to the loosening of lamellae in many ostensibly normally myelinated axons and also to an increase in the number of intraperiodic lines in the myelin sheath in the chronic phase of secondary degeneration, indicating possible additional wrapping of oligodendrocytes around myelinated axons at 6 months. Further loss of myelin lipid staining was seen at 6 months in the absence of further axon and RGC loss. Taken together, following 6 months of secondary degeneration, myelinated axon swelling, the continued prevalence of axons with decompacted myelin, and disruption of myelin sheath integrity are likely factors that contribute to the progressive loss of visuomotor function following partial ON transection. Furthermore, the lack of significant spontaneous and effective remyelination, as would be indicated by thinner myelin sheaths and restoration of vision, indicates that the chronic phase following partial ON transection may be a valuable model for demyelinating disease. 
Swelling of myelinated axons was a persistent feature seen in the chronic phase of secondary degeneration. Furthermore, we showed that 12% to 16% of the population of axons have decompacted myelin and large axon calibers, compared to all other axon types, that is, unmyelinated and myelinated axons. A growing body of evidence suggests that in neurodegenerative diseases and CNS pathologies the swelling of myelinated axons and later axon degeneration, are due to the loss of oligodendroglia-axon support. 41,42 In proteolipid protein (PLP)–deficient mutant mice, swelling of myelinated axons occurs due to an impairment of axon transport, suggesting that the loss of oligodendroglia-axon function leads to axon swelling. 42 Similarly, ultrastructural analysis of C-type natriuretic peptide (CNP)–deficient mice indicated that although the myelin sheath of axons appeared compact and unaffected, axons became swollen as the animal aged. 43 Furthermore, myelinated axons in the optic nerve of mutant mice lacking the myelin membrane proteolipids PLP and its splice isoform DM20 developed axonal swellings due to impaired transport in approximately 11% of optic axons, 42 a similar proportion of large caliber axons with decompacted myelin to that reported here. Loss of oligodendrocyte support of myelinated axons disrupts mitochondrial functioning and reduces adenosine tri-phosphate (ATP) production, causing a focal transport blockage, axon swelling, and eventually Wallerian degeneration. 41,44 The mechanisms of axon swelling induced by a breakdown in oligodendrocyte–axon support are not well understood, but may involve posttranslational modification of neurofilament (an axoskeletal protein), in which increases in neurofilament phosphorylation create larger spacing between filaments, thus producing a larger axon caliber. 41,45,46 Therefore, increases in axon caliber during chronic secondary degeneration may be a function of disruption to the complex oligodendrocyte–axon relationship. 
The increase in myelin thickness of swollen myelinated axons during chronic secondary degeneration is a novel finding of this study. One explanation for the increase in myelin thickness is the disruption of protein structural bonds between individual myelin layers (double intraperiodic line, Figs. 2D, 2F) and mild delamination of lamellae, observed when examining individual myelin lamellae at high magnification. The noxious environment of the ON extracellular matrix during secondary degeneration includes high levels of glutamate 47,48 that lead to excitotoxicity, the release and build-up of nitric oxide, free radicals, and pro-inflammatory cytokines such as TNF-α and interferon-γ released from microglia/macrophages as well as lipid peroxidation. 21,22,49 Multiple layers of negatively charged inner membrane of myelin lamellae are intimately held together (double intraperiodic line) by adhesion molecules, such as the hydrophobic PLP, MBP, and myelin-associated glycoprotein (MAG), to form a compact myelin sheath. 24,50 The stability of the myelin sheath may be affected during secondary degeneration by high levels of radical species including nitric oxide and hydroxyl radicals. These may disrupt both PLP via lipid peroxidation and myelin proteins residing between the external repulsive surfaces of myelin lamellae, causing a weakening or loosening of lamellae and the increase in myelin thickness. 5153 Our demonstrated loss of MBP immunoreactivity indicates that loss of this adhesion molecule contributes to myelin decompaction during the chronic phase of secondary degeneration. Importantly, we also observed an increased number of intraperiodic lines in the chronic phase of secondary degeneration, indicating that oligodendrocytes may be wrapping additional layers around myelinated axons in an attempt to repair, perhaps as a response to axon swelling, similar to the increased myelination of larger caliber axons seen in development. 54 This is a phenomenon not previously described in the CNS, to our knowledge. Nevertheless, visual function is not improved, indicating that the increased number of intraperiodic lines is not indicative of successful myelin repair or remyelination. Indeed, remyelination is normally considered to involve thinner myelin sheaths 23,25 rather than the thicker myelin sheaths observed here. Analysis of proliferation and maturation of specific oligodendrocyte precursor cell subpopulations into newly generated myelinating oligodendrocytes, as well as assessments of internode lengths after injury will be required to address this issue more fully. 
Decompaction of myelin was a persistent, key feature of chronic secondary degeneration and was also observed in normal ON but to a considerably lesser extent. Decompaction in normal ON may be associated with turnover of oligodendrocytes in the mature nerve. 55 Interestingly, although reactive oxygen species generated during secondary degeneration may contribute to the disruption and break down of the intraperiodic lines and, therefore, decompaction of myelin, 52 only 12% to 15% of the total axon population were affected to the extent of large scale myelin decompaction, suggesting that some axons and myelin are more susceptible to decompaction than others. Oligodendrocytes that myelinate larger axons, identified as being oligodendrocyte marker Rip+ve/carbonic anhydrase II–ve, are distinct from oligodendrocytes that myelinate multiple smaller caliber axons, indentified as being Rip+ve/carbonic anhydrase II+ve, suggesting that subpopulations of oligodendrocytes myelinate axons of different sizes. 56 Our data suggest that oligodendrocyte subpopulations that myelinate larger caliber axons may be more susceptible to oxidative damage and resultant decompaction than other subpopulations. 
CNS myelin is rich in lipids, such as phospholipids and galactocerebrosides, which are highly vulnerable to peroxidation. Lipid peroxidation is a known pathophysiological event occurring during secondary degeneration following spinal cord injury. 57,58 The cascade of toxic metabolic events occurring during secondary degeneration is self-perpetuating in that the failure of mitochondrial functioning and antioxidant mechanisms results in the buildup of reactive radical species that react with cellular membranes, including myelin, producing more free radicals. 59 During the chronic phase of secondary degeneration, we detected a progressive loss of myelin lipid staining between 3 to 6 months after partial ON transection, despite no further loss of axon and RGC cell bodies. One interpretation of these data is that the lipid content of individual myelin sheaths decreases with oxidative damage, which may also lead to delamination of myelin at 6 months (Figs. 2D–F). However, the degree of lipid lost, as detected by fluoromyelin staining, was perhaps greater than the observed myelin disruption as assessed by TEM, leading us to suggest that peroxidation of lipids in myelin vulnerable to secondary degeneration may render them less able to be detected by fluoromyelin staining. Biochemical studies assessing oxidized lipid species will be required to understand the nature and mechanism of lipid loss and myelin disruption. Alternatively, many of the myelinated axons had a small degree of loosening of the myelin sheath at 6 months, which may result in reduced fluoromyelin staining. Lipid peroxidation was also detected in human multiple sclerosis brains and might be a contributor to disturbed fast axonal transport. 60 Thus, the continued disruption and loss of myelin lipids during chronic secondary degeneration may contribute to the additional loss of visuomotor function at this late stage, despite no further loss of axons. 
Acknowledgments
We thank Michael Archer for technical assistance with electron microscopy. The authors thank the facilities, scientific, and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, a facility funded by the University, State, and Commonwealth Governments. 
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Footnotes
 Supported by the Neurotrauma Research Program (Western Australia) and National Health and Medical Research Council, Australia (NHMRC) Grant 572550 (MF) and from NHMRC Grant APP1002347 (SAD).
Footnotes
 Disclosure: S.C. Payne, None; C.A. Bartlett, None; A.R. Harvey, None; S.A. Dunlop, None; M. Fitzgerald, None
Figure 1. 
 
Changes in ventral ON at 3 and 6 months following partial transection. Transverse toluidine-blue stained semithin sections (AC) were used to measure ON diameter, ventral radius (black line), and ventral ON area (below the black line). TEM images (DF) were used to quantify major reactive processes (outlined in red). Data for graphs (G, F) are expressed as mean ± SE with major reactive processes defined as the mean percentage of the FOV ± SE (J). Differences between experimental groups are indicated by *(P < 0.05). Scale bars AC: 100 μm, DF: 2 μm.
Figure 1. 
 
Changes in ventral ON at 3 and 6 months following partial transection. Transverse toluidine-blue stained semithin sections (AC) were used to measure ON diameter, ventral radius (black line), and ventral ON area (below the black line). TEM images (DF) were used to quantify major reactive processes (outlined in red). Data for graphs (G, F) are expressed as mean ± SE with major reactive processes defined as the mean percentage of the FOV ± SE (J). Differences between experimental groups are indicated by *(P < 0.05). Scale bars AC: 100 μm, DF: 2 μm.
Figure 2. 
 
Representative TEM images and quantification of the number, diameter, myelin thickness, and g-ratio of myelinated axons, vulnerable to secondary degeneration in normal ONs and at 3 and 6 months following partial transection. Myelinated axons from normal ON (AC) are indicated by >, and at 6 months following injury (DE) indicated by >>. Data for graphs (DJ, L) are expressed as total numbers of myelinated axons in ventral ON (G), mean ± SE, or frequency of axons with small (<0.4 μm) or large (>0.9 μm) diameters (L). Differences between experimental groups are indicated by *(P < 0.05). Scatter graph of axon diameter versus myelin thickness (K) shows mean values of each FOV (100–160 axons/FOV; 10 FOV/animal). Scale bars A , DF: 200 nm, B, C: 100 nm.
Figure 2. 
 
Representative TEM images and quantification of the number, diameter, myelin thickness, and g-ratio of myelinated axons, vulnerable to secondary degeneration in normal ONs and at 3 and 6 months following partial transection. Myelinated axons from normal ON (AC) are indicated by >, and at 6 months following injury (DE) indicated by >>. Data for graphs (DJ, L) are expressed as total numbers of myelinated axons in ventral ON (G), mean ± SE, or frequency of axons with small (<0.4 μm) or large (>0.9 μm) diameters (L). Differences between experimental groups are indicated by *(P < 0.05). Scatter graph of axon diameter versus myelin thickness (K) shows mean values of each FOV (100–160 axons/FOV; 10 FOV/animal). Scale bars A , DF: 200 nm, B, C: 100 nm.
Figure 3. 
 
Representative TEM images and quantification of axons with decompacted myelin, vulnerable to secondary degeneration at 3 and 6 months following partial transection. Axons with decompacted myelin in normal ON (AB), and at 3 months (C) and 6 months (D) are indicated with arrows (>>). Data for graphs (EF) are expressed as mean ± SE. Differences between experimental groups are indicated by *(P ≤ 0.05). Relative frequency distribution of axon diameters (GI) is expressed as counts within 0.1 μm intervals, total of 27 intervals, and cumulative frequency (J) up to 1. Scale bar = AC: 200 nm, D: 100 nm.
Figure 3. 
 
Representative TEM images and quantification of axons with decompacted myelin, vulnerable to secondary degeneration at 3 and 6 months following partial transection. Axons with decompacted myelin in normal ON (AB), and at 3 months (C) and 6 months (D) are indicated with arrows (>>). Data for graphs (EF) are expressed as mean ± SE. Differences between experimental groups are indicated by *(P ≤ 0.05). Relative frequency distribution of axon diameters (GI) is expressed as counts within 0.1 μm intervals, total of 27 intervals, and cumulative frequency (J) up to 1. Scale bar = AC: 200 nm, D: 100 nm.
Figure 4. 
 
Representative images and semiquantification of the intensity of fluoromyelin staining (AC, G) and immuno-intensity of MBP (DF, H) in ventral ON, vulnerable to secondary degeneration (FOV: 0.0354 mm), at 3 and 6 months following partial transection. Data were expressed as mean immunohistochemical and staining intensities ± SE and differences between experimental groups indicated by *(P ≤ 0.05). Scale bar = 50 μm.
Figure 4. 
 
Representative images and semiquantification of the intensity of fluoromyelin staining (AC, G) and immuno-intensity of MBP (DF, H) in ventral ON, vulnerable to secondary degeneration (FOV: 0.0354 mm), at 3 and 6 months following partial transection. Data were expressed as mean immunohistochemical and staining intensities ± SE and differences between experimental groups indicated by *(P ≤ 0.05). Scale bar = 50 μm.
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