Thin Myelin Sheaths As the Hallmark of Remyelination Persist Over Time And

Thin myelin sheaths as the hallmark of remyelination PNAS PLUS persist over time and preserve axon function

Ian D. Duncana,1, Rachel L. Marika, Aimee T. Bromanb, and Moones Heidaria

aDepartment of Medical Sciences, School of Veterinary Medicine, University of Wisconsin–Madison, Madison, WI 53706; and bDepartment of Biostatistics and Medical Informatics, School of Medicine and Public Health, University of Wisconsin–Madison, Madison, WI 53792

Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved September 28, 2017 (received for review August 18, 2017)

The presence of thin myelin sheaths in the adult CNS is recognized as neural precursor cell-derived oligodendrocytes (OLs) were thicker a marker of remyelination, although the reason there is not a than those generated by oligodendrocyte progenitor cells (OPCs), recovery from demyelination to normal myelin sheath thickness and the g ratios of these fibers were normal (24). However, these remains unknown. Remyelination is the default pathway after myelin observations were made in the corpus callosum, where it has been loss in all mammalian species, in both naturally occurring and shown that g ratios can be unreliable measurements of remyeli- experimental disease. However, there remains uncertainty about nation (26), and this will require verification in other white matter whether these thin sheaths thicken with time and whether they tracts. In the study of Samanta et al. (25), remyelinated, small- remain viable for extended periods. We provide two lines of evidence diameter axons in the spinal cord were found to have normal g here that thin myelin sheaths may persist indefinitely in long-lived ratios. This, however, should be verified in all calibers of axons. We have previously shown that transplantation of OPCs into a animal models. In the first, we have followed thin myelin sheaths in a mature myelin mutant, the shaking pup, at 8 mo of age, resulted in model of delayed myelination during a period of 13 years that we predominantly thin myelin sheaths (27). This may be equivalent to propose results in the same myelin sheath deficiencies as seen in what is described in the present study; that is, delayed myelination remyelination; that is, thin myelin sheaths and short internodes. We of mature axons. In contrast, transplantation of OPCs into 2-wk- show that the myelin sheaths remain thin and stable on many axons old recipients led to the production of normal-thickness myelin throughout this period with no detrimental effects on axons. In a sheaths (27). Powers et al. (28) studied internode lengths and second model system, in which there is widespread demyelination myelin sheath thickness of remyelinated axons after spinal cord of the spinal cord and optic nerves, we also show that thinly injury. They showed that internode lengths of remyelinated axons NEUROSCIENCE remyelinated axons with short internodes persist for over the course were short early after injury, but elongated with time (6 mo), with of 2 y. These studies confirm the persistence and longevity of thin myelin sheath thickness also increasing to normal. Whether this myelin sheaths and the importance of remyelination to the long- relates only to this model (spinal cord injury) is unknown, yet term health and function of the CNS. unlikely. Taken in context, these studies suggest that the estima- tion of remyelination using thin myelin sheaths as evidence may axon | oligodendrocyte | remyelination be underestimated, yet the proof of this does not appear over- whelming at present, and longer-term and more complete studies emyelination of the CNS is the most robust endogenous form are needed. The reason or reasons that remyelinated axons have thin myelin of repair of the brain and spinal cord and a major thera- R sheaths have not been determined experimentally, and questions peutic target in demyelinating disease. It has been found to be remain about the long-term effectiveness of remyelination. For the default pathway in a wide variety of experimental demyelinating example, can thin myelin sheaths persist for long periods and disorders, with large and extensive areas of demyelination becoming continue to support normal conduction and function, preventing totally remyelinated in the recovery phase of each disorder (1). In axons from degeneration (29, 30)? If thin myelin is less than opti- human demyelinating diseases, and in particular in multiple scle- mum, is there a need to develop strategies that restore thin myelin rosis (MS), remyelination can be extensive (2–4), but in MS, it diminishes with aging and disease course (5, 6). Persistently Significance demyelinated axons are at risk for degeneration, and axon loss is likely the basis for patients developing secondary progressive MS The hallmark of remyelination in the CNS has been proposed to and permanent neurological deficits (7–10). Remyelination in the be the presence of thin myelin sheaths. This has been dem- CNS has been shown to restore nerve conduction (11), locomotor onstrated in multiple experimental models and in multiple function (12, 13), and global neurologic recovery (1). Importantly, – sclerosis. It is the only surrogate marker of remyelination, and remyelination protects axons from ongoing degeneration (14 17), therefore a crucial fingerprint of myelin repair. However, this and is therefore a crucial repair strategy. has been challenged recently in separate studies, in which, by The morphologic hallmark of remyelination in both human implication, the degree or presence of remyelinated axons has and experimental disease is proposed to be the presence of axons been underestimated. In this article, we provide evidence from with thin myelin sheaths that have a significant increase in the g ratio (axon diameter/total fiber diameter) (6, 18). Remyelinated two different models that thin myelin sheaths and short in- axons also have shortened internodal lengths. These features of ternodes persist almost indefinitely. Future attempts to pro- remyelinated axons have been described in many human and mote myelin repair in models of multiple sclerosis will be animal demyelinating disorders of any cause, in which there is crucially dependent on a definitive marker of repair. We pro- subsequent remyelination (19–21). Most important, short inter- pose that the thin myelin sheath remains the gold standard. nodes with thin myelin sheaths have also been reported in detail in MS tissue (22, 23). Although the prevailing view is that Author contributions: I.D.D. designed research; I.D.D., R.L.M., and M.H. performed re- remyelination of the adult CNS results only in thin myelin search; I.D.D. and A.T.B. analyzed data; and I.D.D. wrote the paper. sheaths (6), some recent studies have suggested that remyelina- The authors declare no conflict of interest. tion can lead to normal-thickness myelin with normal g ratios. This article is a PNAS Direct Submission. These studies have examined remyelination in the corpus cal- Published under the PNAS license. losum after cuprizone-induced demyelination (24, 25) and in the 1To whom correspondence should be addressed. Email: [email protected]. spinal cord in experimental autoimmune encephalomyelitis (25). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. It was proposed that remyelinated myelin sheaths induced by 1073/pnas.1714183114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1714183114 PNAS Early Edition | 1of7 Downloaded by guest on September 28, 2021 sheaths to their normal thickness (31)? These are difficult questions appeared identical to controls, and although rare degenerating to answer in MS, where it is impossible to determine how long axons were seen, this was similar to controls. There was no evi- axons have been remyelinated. In experimental rodent animal dence of abnormal axoplasm, as we have previously identified in models, their short lifespan limits the length of time remyelinated 1-μm sections in another canine myelin mutant, in which a wide axons can be followed. In an attempt to answer some of these array of axonal changes was documented (33). In addition, no questions, we studied two experimental models. In the first, for swollen axons or spheroids were present in either case in the more than a decade, we followed the fate of thinly myelinated cervical and thoracic spinal cord. axons in the spinal cord of a canine mutant, resulting from a de- Fig. 3 shows the distribution of g ratio measurements within velopmental delay in myelination, which we propose matches the each image, by subject and ventral column (VC) or lateral col- changesseeninremyelinatedfibers.Second,totestwhetherthinly umn (LC) locations. The number of axons measured at each remyelinated sheaths persist over time after demyelination, we position ranged from 249 to 505, and mean g ratio ranged from turned to a model in which this was studied in animals over the 0.62 to 0.84, with the variability having an SD of 0.07–0.10 (Table course of 2 y after myelin repair. S1). We tested the difference between mean affected and WT g Results ratio adjusting for VC or LC location, using random effects for the subject and 1 μm image factors, to account for potentially Genetic Disorder. The disorder presented here is a unique de- higher correlation of measurements for axons within the same velopmental disorder of myelination of the superficial tracts of the subject and/or the same 1-μm image (Table S2). g ratios mea- cervical and thoracic spinal cord caused by a mutation in the = FNIP2 sured in affected subjects were, on average, 0.126 (SE 0.027; gene (32). At 2 wk of age, affected dogs have a severe P < 0.0001) higher than in WT subjects. There was no difference tremor, resulting in difficulty in ambulation. We studied the tho- in the g ratio between VC and LC locations of the cord. racic spinal cord from a 2-wk-old affected dog and demonstrated a To determine whether these abnormally thin myelin sheaths also zone of hypomyelination of the superficial tracts of the lateral and had short internodes, a hallmark of remyelination, tissue from the ventral columns with almost a total lack of myelin (Fig. 1 C and D) VC of the cervical and thoracic cord of the male dog was em- compared with the same tracts in controls that were completely bedded for longitudinal sectioning. These sections enabled visual- myelinated (Fig. 1 A and B). The dorsolateral column appeared most deficient in myelin. On higher magnification of this area, the ization of myelinated axons from the subpial surface to those in the myelin deficiency was more obvious despite the presence of many more medial gray matter interface. Thinly myelinated short inter- glial cells and intact axons (Fig. 1D). Importantly, however, some nodes were only seen in the subpial area and not more medially, as larger-diameter axons in this zone were myelinated normally (Fig. would be expected from the transverse sections (Fig. 2). In addi- 1D, Inset). In contrast, in both of the affected 13-y-old dogs, these tion, some axons had adjacent, normal-thickness/thin myelin par- J areas appeared completely myelinated, but the outstanding fea- anodes (Fig. 2 ). This confirmed that this is a primary glial defect ture was the presence of many thinly myelinated axons of all ax- and not an abnormality of axons. Not all thinly myelinated inter- onal calibers in the lateral and ventral columns of the cervical and nodes were short, however, and on occasion, they were more than thoracic spinal cord (Fig. 2 and Fig. S1). These thinly myelinated 100 μm. It was also noted that nodal gaps were greater than normal axons were seen predominantly in the subpial zones of the lateral at many nodes of the thinly myelinated axons (Fig. 4). In the more and ventral columns, the site of original myelination delay (Fig. 1). medial, normally myelinated areas, nodes of Ranvier with normal This was also seen at both levels of the cord in the second dog, nodal gaps were seen on large-, medium-, and small-caliber axons, although less obviously than in the first case (Fig. S1). There were but it was impossible to measure internode length on these axons no other abnormalities in any of the spinal cord sections evalu- because of variation in fiber plane. However, those internodes that ated. There was no evidence of demyelination or myelin debris could be followed over some length were always longer than the that would suggest myelin was not stable. Myelinated fiber density thinly myelinated internodes seen in the subpial zone.

Fig. 1. Myelin deficiency in the subpial zone of the ventral and lateral tracts of the spinal cord in the mutant. Hemisections of the thoracic spinal cord from a 2-wk-old control dog (A and B) and age-matched affected dog (C and D). In the control, the spinal cord appears completely myelinated, and a higher-power image of the dorsolateral column (arrows) in B confirms this. In contrast, in the affected dog, there is a clear, pale subpial zone (C) that on higher power shows a marked myelin deficiency compared with the medial white matter (D). However, occasional large-diameter axons (arrow) are myelinated in this area (D, Inset). [Scale bars: 0.2 mm (A and C), 20 μm(B and D), and 10 μm(D, Inset).]

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Fig. 2. In the aged mutant, hypomyelination persists in the affected tracts, and internodes can be short. In the control dog, the spinal cord is normally myelinated (A), and this is confirmed on higher-power examination of subpial areas in the LCs (B)andVCs(C). The g ratio of myelinated axons in these areas was measured at two sites and the ratio noted A. In the first affected dog, the cord appears completely myelinated (D), yet on higher power, there are many thin myelin sheaths of all axon diameters in both the LC (E)andVC(F) in the subpial zone. This was confirmed by the g ratio measurements from these sites (D). High power of areas (*) in the control (G) and affected dog (H) show that these areas have a mix of normally myelinated (thick myelin sheaths) and hypomyelinated axons, as reflected by the gratiorange.(I) A short internode with a thin myelin sheath is seen between thicker internodes. (J) In the ventral column, a 7.6-μm-diameter axon has two short internodes, as marked by arrows. The node of Ranvier on the right has a paranode with a normal myelin sheath adjacent to the thin, short internode (g ratio, 0.83) (A). Arrows mark nodes of Ranvier. Internode lengths are detailed. [Scale bars: 0.5 mm (A and D), and 20 μm(B, C, E–J).]

FIDID. We have previously shown that irradiation of food at more thinly myelinated axons interspersed between mature myelin than 30 Kgray results in a widespread and severe demyelinating sheaths (Fig. 5A). These appeared to be stable lesions, as there was and remyelinating disorder in cats (1). Here we studied this phe- no myelin vacuolation and no foamy, lipid-filled macrophages, nomenon in the spinal cord of three cats that were examined over which are seen during and shortly after demyelination and the course of 16 mo to 2 y after neurologic recovery, and found remyelination. Longitudinal sections of the spinal cord showed

Duncan et al. PNAS Early Edition | 3of7 Downloaded by guest on September 28, 2021 cord of cats that had been demyelinated by focal lysolecithin in- jections (34). Some remyelinated axons can have longer internodes, close to the predicted diameter for the axon caliber (34), and this was also seen in some thinly myelinated axons in the present study. Axons in the medial aspect of the cord of varying diameters and normal-thickness myelin did not appear to have short internodes, although it was difficult to follow them for long distances. Given that neurologic function was restored in the affected dogs by 4–6 mo of age, and this is correlated with the myelination of the spinal cord superficial tracts (32), it can be concluded that the thin myelin sheaths restored conduction. It has been known for some time that endogenous remyelination of CNS axons restores secure conduction from previous conduction block of axons demyelinated by lysolecithin injection (11). Despite the neurologic normality and likely normal conduction in the thinly myelinated axons, we saw numerous cases of nodal gap widening on the thinly myelinated axons that may also result in conduction abnormalities. Nodal gap widening is common and a nonspecific finding in many neurologic disorders (35). Despite these findings of thin myelin sheaths and nodal widening, they appear not to have any negative effect on neurologic function. Fig. 3. Measurements of g ratios in the dorsolateral columns and VCs confirms Thus, although we propose that the thin myelin sheaths that the presence of abnormally thin myelin sheaths. Distribution of g ratios for persist in this mutant are the same as those seen after remyeli- randomly sampled axons within the 1-μm image of the cervical cord from af- nation of the adult CNS, the question remains, as with remye- fected and WT dogs. The suffix 1 and 2 in the LC and VC are two sections taken lination: Why do these myelin sheaths remain thin? Franklin and from those locations. The number of axons measured is shown below the im- Hinks (36) proposed that thin myelin sheaths in remyelination age location. The box represents the median and 25th and 75th percentiles of result from OLs unsheathing mature axons of mature diameter the distribution of the values. Whiskers extend out no more than 1.5× the and length. In contrast in normal development, early myelin interquartile range away from the box. Values outside of this range are plotted internodes adapt to changes in axon caliber and length by be- as points. coming thicker in diameter, and longer. We propose that the Franklin and Hinks hypothesis applies in the developmental delay in myelination in these mutants. Myelin sheath thickness in frequent thinly myelinated axons with short but variable internode B–E the peripheral nervous system is controlled by neuregulin III lengths (Fig. 5 ). In one of these animals, the optic nerves were signaling (37), whereas in the CNS, ERK1/ERK2 signaling, as dominated by thinly myelinated axons, and short internodes were found in both loss- and gain-of-function experiments, decreases also seen on longitudinal sections (Fig. 6). Two of the cats with feline or increases myelin sheath thickness, respectively (38, 39). The irradiated diet-induced demyelination (FIDID) were part of a clin- model proposed by Franklin and Hinks (36) to explain why ical trial in Madison, Wisconsin, and the other was a pet cat from remyelinated sheaths are thin could equally apply to the late-stage Sydney, Australia, confirming the ubiquitous nature of the disease developmental myelination documented here. As oligodendro- process and the long-term persistence of thinly myelinated axons. cytes are myelinating (or remyelinating) axons that are close to their maximum diameter and length, there is no need to undergo Discussion remodeling that is required in the early developing CNS. That is, We provide here compelling evidence for this persistence of thin the original association of the OL process with an axon is when the myelin sheaths over extended periods in two models, most notably axon reaches a critical diameter, although it is still to undergo in the canine genetic disorder. During the prolonged period of increases in its diameter and length. Indeed, Franklin and Hinks observation of the two affected dogs, from the time of recovery (36) suggest that remyelination in the adult CNS resulting from from their neurologic deficit at 6 mo, to their termination 13 y later, oligodendrocytes derived from adult progenitor cells represents four possibilities were predicted regarding the fate of the thinly “delayed” myelination, similar to what occurs in the Weimaraner myelinated axons in the spinal cord. First, they could remain mutant. The similarities between myelination and repair have led hypomyelinated, as we have previously demonstrated in mutant to the supposition that remyelination is simply a recapitulation of dogs up to 2 y of age (32). Second, the thin myelin sheaths thick- development, and although there are differences, these may be ened with time, eventually achieving normal thickness and g ratio as redundant (40). This has been confirmed recently in an in- a result of oligodendrocyte compensation and plasticity. Third, thin vestigation of the key transcription factor, myelin regulatory myelin was not stable and eventually degenerated, leading to demyelination, or fourth, was not sufficient to maintain axonal via- bility with subsequent axon degeneration. We demonstrate here that the first outcome, that is, the persistence of hypomyelination with no other pathologic changes, was the outcome. As thin myelin sheaths in the adult CNS are thought to indicate remyelination, do the results in this genetic model match the changes seen after myelin restoration? As detailed here, many thinly myelinated axons were seen in the LCs and VCs after 13 y. These differences were confirmed by the g ratio measurements in these sites in the cervical and thoracic cord of both dogs. g ratios were, on average, greater in the affected dogs at all sites measured, and although the number of test subjects was small, the myelin sheath abnormalities were consistent in both dogs. In addition to thin myelin sheaths, as seen on transverse section, thinly myelinated Fig. 4. Widened nodal gaps seen on affected fibers. Although normal nodes axons in the subpial zone frequently had short internode lengths were common on axons with normal thickness myelin sheaths (A) and those (<100 μm). This is similar to that seen in remyelinated fibers in the with thin myelin sheaths (B), many examples of nodal widening were seen at CNS, as shown in study of single teased nerve fibers from the spinal nodes where adjacent myelin sheaths were thin (C and D). (Scale bars: 20 μm.)

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Fig. 5. Thin remyelinated sheaths persist in the spinal cord for more than a year after demyelination. (A) Area from the ventral column of the cervical spinal cord from a cat, 11/4 y after neurologic recovery from FIDID. Remyelinated axons are scattered between axons with normal myelin sheath thickness in an area of normal axon density. This is a nonactive lesion with no demyelinated axons and no lipid-filled macrophages. (B and C) Longitudinal sections from these areas show short internodes with thin myelin sheaths, all of which had g ratios >0.80. On some axons, multiple short internodes with thin myelin sheaths were seen (D). (D) The fiber seen continues in the panel below (E). Arrows mark nodes of Ranvier. (Scale bars: 20 μm.)

factor in experimental remyelination after lysolecithin-induced 95% of axons had thin myelin sheaths with only occasional re- demyelination of the mouse corpus callosum, and in MS le- sidual mature sheaths, primarily on large axons. The more ex- sions (41). Myelin regulatory factor expression, which was es- tensive remyelination seen in the optic nerves reflects the global sential for remyelination in both, broadly recapitulated the OL demyelination in the nerve compared with the spinal cord. expression seen during development (41). Are there alternative explanations for the finding of thinly my- Although we propose that the thin myelin sheaths result from elinated axons with short internodes in the mature CNS? The delayed myelination of the subpial axons, it could be suggested concept that myelin sheaths remain static throughout life has been that the causative mutation in FNIP2 has a negative effect on challenged by recent studies on oligodendrocyte dynamics in the myelination in addition to the proposed effect on migration and normal adult CNS, which showed continuous division of OPCs in differentiation of OLs and their progenitors (32). However, we the brain and shortening of internode lengths in some tracts (42). would argue against this, as some OLs that are found early (2 wk) This was not related to ongoing myelination of nonmyelinated in the subpial zone are able to myelinate axons normally (Fig. axons; instead, it was suggested that there is continuous myelin 1D), leading to the presence of scattered, thickly myelinated remodeling that results in shortened internode lengths. Could this axons with a normal g ratio at 13 y, interspersed with the late- be the reason for this finding in the two dogs examined here? This myelinated thin sheaths (Fig. 1H). There is no published evi- would seem to be unlikely, however, as the only areas with thin dence to indicate there is heterogeneity of OLs in the subpial myelin/short internodes were in the superficial tracts of the LCs zone of the spinal cord that could result in a difference in and VCs. Myelinated axons medial to these zones had normal myelinating capacity of a subset of OLs in mutations of FNIP2. myelin sheath thickness and no evidence of shortened internodes The persistence of thin, remyelinated sheaths after demyelination as far as could be determined in longitudinal sections. was also clearly seen in the FIDID model. In all three cases in Despite the fact that many axons in the spinal cord were only which tissue was collected over the course of 1–2 y after neurologic ensheathed by thin myelin sheaths, there was no evidence that this recovery, scattered thin myelin sheaths were seen in the spinal compromised axon health or resulted in degeneration (29). The cord VCs and LCs, with these fibers being interspersed between interactions between the axon and its myelin sheath/oligodendro- mature myelin sheaths. This pattern of fiber mixing is seen during cyte that are essential for axon survival are well recognized (43–46). acute disease; that is, scattered myelinated axons (with mature Although the primary source of support for the axon has been myelin sheaths) escape demyelination and persist adjacent to shown to the metabolic coupling between the oligodendrocyte and demyelinated and remyelinated axons. In the three recovered cats axon, involving importing glucose and lactate, the myelin sheath recorded here, however, there was no evidence of active myelin itself is also likely to be involved. In Plp1 knockout mice, there is breakdown, demyelinated axons, or macrophages; that is, these late-stage axonal degeneration (47), whereas mutation of the my- were nonactive areas of myelin damage and repair. The persis- elin gene Cnp1 likewise results in axon degeneration (48). In 19- tence of thin myelinated sheaths and short internodes confirms mo-old rumpshaker mice which have a mutation in the Plp1 gene, that these abnormalities had remained for more than 2 y. In one thin myelin sheaths persist, yet the genetically deficient myelin cat, this was even more dramatic in the optic nerves, where over results in a late-stage axonopathy (49). Here, however, we show

Duncan et al. PNAS Early Edition | 5of7 Downloaded by guest on September 28, 2021 model studied here is a disorder in the Weimaraner breed of dog that has a unique developmental deficiency in myelination of the superficial tracts of the cervical and thoracic spinal cord (32, 51). Affected dogs develop a severe tremor and ataxia at 12–14 d of age, which gradually diminish and are lost in most cases by 3–4 mo. The disorder is inherited as an autosomal recessive trait, and the mutation is in the folliculin-interacting protein 2 (FNIP2) (32). We have proposed that the mutation results in a failure of migration or differentiation of a subpopulation of oligodendrocytes (32). In the present study, we followed two homozygous affected littermates, a male and female, for more than 13 y. The male had a more severe and persistent tremor and ataxia than the female. In both dogs, however, no obvious tremor could be detected by 6 mo, and the male was no longer ataxic. Both dogs were neurologically normal for up to 13 y. Each was killed at around 13 1/2 y because of nonneurologic disease. Samples of the cervical and thoracic spinal cord were collected from the male immediately after death and immersion fixed in 2.5% glutaraldehyde. Tissue from the female dog was only available 3–4 h after death and immersion fixed, but the delay resulted in poorer fixation. For controls for the mature affected dogs, we used sections from the cervical and thoracic cord of 2-y-old normal dogs (WT; n = 2), as age-matched controls were not available. By 2 y of age, myelination is complete in the canine spinal cord, and myelin sheath thickness is repre- sentative of all mature canines (33). To illustrate the developmental defect on early myelination of the superficial tracts of the spinal cord and the change that occurs with time, we examined the thoracic spinal cord of a 2-wk-old affected Weimaraner with tremor and an age-matched control dog. Blocks from the cervical and thoracic cord were trimmed and processed for Fig. 6. The optic nerve contains predominantly remyelinated axons. (A)Inthe plastic embedding and preparation of 1-μm toluidine blue-stained sections. second cat, 2 y after recovery, the optic nerve contains predominantly thin, One-micrometer sections were then used to measure the g ratios of ∼250– remyelinated sheaths with only two mature, normal-thickness myelinated axons 500 myelinated axons in the LCs and VCs of both animals from the cervical (arrows) that were not demyelinated. (B) The nerve also contained short, thin cord at two adjacent sites, using ImageJ software (NIH). The mean difference internodes. (Scale bars: 20 μm.) between case and control dogs was analyzed using a linear mixed-effects model (52), with two levels of random effects: one for measurements from the same dog and one for measurements taken within the same 1-μm slide. that biochemically normal, yet thin, myelin sheaths appear to This model is considered a mixed-effect model with nested factors. support the long-term integrity of axons as the myelin fiber density The second model used to study the persistence of thinly myelinated axons was qualitatively normal (Fig. 2). was a unique feline disorder in which feeding irradiated food resulted in The tissue fixation precluded immunolabeling axons for am- profound and generalized demyelination and remyelination of the CNS (1). yloid precursor protein or for demonstrating any increase in The disorder has been named FIDID. Two cats were studied as part of the original study at University of Wisconsin–Madison (1) and were killed over neurofilament phosphorylation (50), both of which may have the course of 2 y after complete neurologic recovery. The third cat was part been used to indicate axonal disturbance. However, from ob- μ of an outbreak of clinical disease seen in Sydney, Australia (53). While on an servations of axonal pathology in 1- m sections from another irradiated diet, the cat developed severe neurologic disease (tetraparesis, canine myelin mutant (33), we believe we would have identified ataxia) but recovered to almost normal ambulation and was studied 14 mo most potential changes in the axoplasm in the present model and after recovery. In this animal, pieces of the cervical and thoracic spinal cord certainly noted if axons were swollen. Absence of any such ab- were immersion fixed in 2.5% glutaraldehyde after euthanasia, processed, normalities leads us to conclude that thin myelin sheaths can and embedded in plastic. Plastic blocks from spinal cord and optic nerve of support axon health and function for over the course of 13 y, and the first two cats were also studied. potentially indefinitely. Hence, the need to develop remyelinating strategies that restore myelin to its normal thickness to ACKNOWLEDGMENTS. We are grateful to Abigail Radcliff for her skilled protect axons and sustain function (31) is not required. preparation of this manuscript and to Dr. John Svaren and Dr. Ian Griffiths for our many discussions on this research and their review of the paper. We Materials and Methods are also grateful to Dr. Sara Levén for her careful collection of the spinal cord of the first case and to Dr. Georgina Child for the second cat tissue from All animals were handled and treated according to the guidelines and rec- Sydney. These studies were supported in part by National Multiple Sclerosis ommendations of the Research Animal Resources Center and the Animal Care Society Grant RG-1501-02876 and by a prior grant from the MS Hope for and Use Committee at the University of Wisconsin–Madison. The genetic a Cure Foundation.

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