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Focal Immune-Mediated White Matter Demyelination Reveals an Age-Associated Increase in Axonal Vulnerability and Decreased Remyelination Efficiency

  • David W. Hampton
    Correspondence
    Address reprint requests to David Hampton, Ph.D., Euan MacDonald Centre, Centre for Neuroregeneration, Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor's Building, 49 Little France Crescent, Edinburgh, EH16 4SB, United Kingdom
    Affiliations
    Euan MacDonald Centre for Motor Neurone Disease Research, Centre for Neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom
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  • Neill Innes
    Affiliations
    Euan MacDonald Centre for Motor Neurone Disease Research, Centre for Neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom
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  • Doron Merkler
    Affiliations
    Division of Clinical Pathology, Geneva University Hospital, Geneva, Switzerland

    Department of Neuropathology, University Medical Center, Georg August University, Göttingen, Germany

    Department of Pathology and Immunology, University Medical Center, Georg August University, Göttingen, Germany
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  • Chao Zhao
    Affiliations
    Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom
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  • Robin J.M. Franklin
    Affiliations
    Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom
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  • Siddharthan Chandran
    Affiliations
    Euan MacDonald Centre for Motor Neurone Disease Research, Centre for Neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom
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      In addition to being an established risk factor for neurodegenerative diseases, age is increasingly recognized as adversely influencing regeneration. Accumulating evidence also suggests that age plays important, although poorly understood, roles with respect to course and prognosis in the degenerative and untreatable later phase of multiple sclerosis. Two experimental models of multiple sclerosis have been particularly influential in modeling the different aspects of neuronal injury and regeneration: global experimental autoimmune encephalomyelitis and focal toxin-mediated injury. Against this background, we report a focal model of immune-mediated demyelinating injury that reliably generates targeted primary demyelination and axonal injury. A detailed pathologic characterization of this model, modified extensively from an earlier study, showed that aged adult animals exhibited increased vulnerability to axonal injury and reduced efficiency of remyelination compared with younger animals. More important, remyelination in aged animals was predominantly Schwann cell mediated, in contrast to the central oligodendrocyte-mediated remyelination that predominated in younger rodents. Together, these findings establish an experimental platform to further study the influence of age on injury and repair in a biologically relevant model of human demyelinating injury.
      It is increasingly recognized that age not only is a risk factor for neurodegenerative disorders but also adversely influences regenerative processes. This finding has major implications for the development of neuroprotective therapies. Multiple sclerosis, one of the most common forms of acquired neurologic disability in young adults, shows age-related phenomena regarding course and prognosis.
      • Trojano M.
      • Liguori M.
      • Bosco Zimatore G.
      • Bugarini R.
      • Avolio C.
      • Paolicelli D.
      • Giuliani F.
      • De Robertis F.
      • Marrosu M.G.
      • Livrea P.
      Age-related disability in multiple sclerosis.
      • Confavreux C.
      • Vukusic S.
      Age at disability milestones in multiple sclerosis.
      • Tremlett H.
      • Zhao Y.
      • Rieckmann P.
      • Hutchinson M.
      New perspectives in the natural history of multiple sclerosis.
      Although age is particularly associated with the neurodegenerative phase of multiple sclerosis, the underlying mechanisms are unknown. In turn, this reflects uncertainty about the precise interplay between the dominant pathologic abnormalities of inflammation and neuronal injury in multiple sclerosis.
      However, it is generally accepted that the early phase of disease is driven by inflammation manifest clinically as relapses, with neurodegeneration as the principal substrate of clinical progression. Remyelination is variable, influenced by age and inflammation, and, although neuroprotective, is ultimately limited, resulting in accumulating irreversible disability.
      • Patrikios P.
      • Stadelmann C.
      • Kutzelnigg A.
      • Rauschka H.
      • Schmidbauer M.
      • Laursen H.
      • Sorensen P.S.
      • Brück W.
      • Lucchinetti C.
      • Lassmann H.
      Remyelination is extensive in a subset of multiple sclerosis patients.
      • Prineas J.W.
      • Connell F.
      Remyelination in multiple sclerosis: remyelination in multiple sclerosis.
      • Franklin R.J.M.
      • Ffrench-Constant C.
      Remyelination in the CNS: from biology to therapy.
      • Goldschmidt T.
      • Antel J.
      • König F.B.
      • Brück W.
      • Kuhlmann T.
      Remyelination capacity of the MS brain decreases with disease chronicity.
      These features of the disease highlight the need for improved experimental models of immune-mediated demyelinating injury that allow the influence of age on neurodegenerative and regenerative processes to be studied.
      Experimental autoimmune encephalomyelitis (EAE), in its various guises, has provided a powerful model of immune-mediated injury that has proved influential in the study of multiple sclerosis. There are many refinements of EAE with important variables, including species, strain, sex, and method of induction.
      • Basso A.S.
      • Frenkel D.
      • Quintana F.J.
      • Costa-Pinto F.A.
      • Petrovic-Stojkovic S.
      • Puckett L.
      • Monsonego A.
      • Bar-Shir A.
      • Engel Y.
      • Gozin M.
      • Weiner H.L.
      Reversal of axonal loss and disability in a mouse model of progressive multiple sclerosis.
      • Constantinescu C.S.
      • Farooqi N.
      • O'Brien K.
      • Gran B.
      Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS).
      • Hassen G.W.
      • Feliberti J.
      • Kesner L.
      • Stracher A.
      • Mokhtarian F.
      Prevention of axonal injury using calpain inhibitor in chronic progressive experimental autoimmune encephalomyelitis.
      Inevitably, the inherent complexity of such models limit their value in addressing targeted questions that benefit from a focal model of injury. For example, focal injections of toxins such as ethidium bromide and lysolecithin have complemented EAE models and have enabled major insights into the biology of remyelination, including the negative impact of age.
      • Irvine K.A.
      • Blakemore W.F.
      Age increases axon loss associated with primary demyelination in cuprizone-induced demyelination in C57BL/6 mice.
      • Rist J.M.
      • Franklin R.J.M.
      Taking ageing into account in remyelination-based therapies for multiple sclerosis.
      • Sim F.J.
      • Zhao C.
      • Penderis J.
      • Franklin R.J.M.
      The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation.
      • Shen S.
      • Sandoval J.
      • Swiss V.A.
      • Li J.
      • Dupree J.
      • Franklin R.J.M.
      • Casaccia-Bonnefil P.
      Age-dependent epigenetic control of differentiation inhibitors is critical for remyelination efficiency.
      • Zhao C.
      • Li W.W.
      • Franklin R.J.M.
      Differences in the early inflammatory responses to toxin-induced demyelination are associated with the age-related decline in CNS remyelination.
      Notwithstanding the successes of toxin models, there is a clear need for additional focal models to allow study of the influence of age on immune-mediated rather than chemical injury. A model of focal EAE (fEAE) has been described in the spinal cord and cerebral cortex characterized by florid inflammation but with limited analysis of neuroaxonal status.
      • Kerschensteiner M.
      • Stadelmann C.
      • Buddeberg B.S.
      • Merkler D.
      • Bareyre F.M.
      • Anthony D.C.
      • Linington C.
      • Brück W.
      • Schwab M.E.
      Targeting experimental autoimmune encephalomyelitis lesions to a predetermined axonal tract system allows for refined behavioral testing in an animal model of multiple sclerosis.
      • Merkler D.
      • Ernsting T.
      • Kerschensteiner M.
      • Brück W.
      • Stadelmann C.
      A new focal EAE model of cortical demyelination: multiple sclerosis-like lesions with rapid resolution of inflammation and extensive remyelination.
      In this study, we adapted the fEAE protocol to generate an immune-mediated lesion in which primary demyelination (ie, loss of myelin from otherwise intact axons) is a prominent component and that has, thus, enabled assessment of the influence of age on neuroaxonal injury and remyelination.

      Materials and Methods

      Animals and Surgery

      All the procedures were performed in compliance with national and institutional guidelines (UK Animals Scientific Procedures Act 1986 and the University of Cambridge Animal Care Committee).

      Induction of fEAE

      Female Lewis rats (Charles River UK Ltd, Kent, UK, or Harlan Laboratories, Oxon, UK), young adults (3 to 4 months old) and old adults (15 to 18 months old), were inoculated in a similar manner as previously described.
      • Kerschensteiner M.
      • Stadelmann C.
      • Buddeberg B.S.
      • Merkler D.
      • Bareyre F.M.
      • Anthony D.C.
      • Linington C.
      • Brück W.
      • Schwab M.E.
      Targeting experimental autoimmune encephalomyelitis lesions to a predetermined axonal tract system allows for refined behavioral testing in an animal model of multiple sclerosis.
      • Merkler D.
      • Ernsting T.
      • Kerschensteiner M.
      • Brück W.
      • Stadelmann C.
      A new focal EAE model of cortical demyelination: multiple sclerosis-like lesions with rapid resolution of inflammation and extensive remyelination.
      Briefly, recombinant myelin-associated oligodendrocyte glycoprotein (rMOG), at various concentrations ranging from 50 to 75 μg of protein, as stipulated throughout the text, emulsified in incomplete Freund's adjuvant (IFA) (Sigma-Aldrich, Poole, UK) was injected s.c. at the base of the tail. Rats were monitored every day after injection to be certain that no clinically apparent disseminated EAE had been induced.
      After this successful inoculation, a focal injection of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) at various concentrations (75 to 250 ng/μL TNF-α and 50 to 200 U of IFN-γ) was administered 18 to 21 days after rMOG-IFA injection. All the surgical procedures were performed in aseptic conditions under general anesthesia (isoflurane and nitrous oxide). Briefly, the prominent eighth cervical vertebra (C8) was identified, a dorsal laminectomy at C7 was performed, and a pulled glass micropipette tip was lowered 0.8 mm from the dorsal surface into the dorsal funiculus (at an angle of 45°) and then was raised 0.1 mm so that infusions would occur around C7-C6. The pulled glass micropipette was attached to a Hamilton syringe via silicone tubing, and oil red O–colored mineral oil was used to aid injections rather than relying on air pressure alone over a long distance that is ineffective. By using an infusion pump connected to the Hamilton syringe, it was possible to ensure that 1 μL was injected over a 10-minute period to minimize the trauma associated with introduction of a volume of liquid. The needle tip was left in the tissue for an extra 5 minutes before slowly removing it and the animals being sutured and placed in a heated recovery box. All the animals received postoperative sodium chloride solution containing 5% glucose to aid rehydration and postoperative analgesic [Rimadyl at 5 mg/kg s.c. (Pfizer Animal Health, Tadworth, UK)] and antibiotic (Terramycin at 60 mg/kg s.c.; Pfizer Animal Health).

      Preparation of Semithin Resin Sections

      Animals were euthanized 7 or 28 days after fEAE injection by perfusion fixation (using phosphate-buffered solution as a prewash, followed by 4% gluteraldehyde in PBS), and the spinal cord was removed as described previously.
      • Hampton D.W.
      • Anderson J.
      • Pryce G.
      • Irvine K.A.
      • Giovannoni G.
      • Fawcett J.W.
      • Compston A.
      • Franklin R.J.M.
      • Baker D.
      • Chandran S.
      An experimental model of secondary progressive multiple sclerosis that shows regional variation in gliosis, remyelination, axonal and neuronal loss.
      Briefly, the cord was dissected out from C5-C7, and sections were postfixed (≥24 hours up to 14 days) in 4% gluteraldehyde before being washed in PBS before embedding. Tissue blocks were then placed in 2% osmium tetroxide (Oxkem Ltd, Reading, UK) and were finally processed into resin through ascending alcohols. Semithin sections of 1 μm were cut using 6- or 8-mm glass knives using a microtome (RM2065; Leica Microsystems GmbH, Wetzlar, Germany) or an ultracut microtome [Leica (formerly Reichert-Jung)] and were stained with toluidine blue (5% in a borax solution).

      Removal of Resin from Sections and Immunohistochemical Analysis

      Resin etching was performed using a modified method using the following technique. Briefly, the process involved cutting sections 1 μm thick onto Polysine slides as described previously.
      • Hampton D.W.
      • Anderson J.
      • Pryce G.
      • Irvine K.A.
      • Giovannoni G.
      • Fawcett J.W.
      • Compston A.
      • Franklin R.J.M.
      • Baker D.
      • Chandran S.
      An experimental model of secondary progressive multiple sclerosis that shows regional variation in gliosis, remyelination, axonal and neuronal loss.
      Slides were baked at 80°C for 30 minutes before being immersed for 20 minutes in a matured (≥5 weeks) sodium ethoxide (a saturated solution of sodium hydroxide in 100% ethanol):ethanol (50:50) solution in the dark. Slides were then washed 4 times in ethanol, each wash being a minimum of 5 minutes, before being quenched by immersion in a 3% hydrogen peroxide (in methanol) solution in the dark for a further 20 minutes. This is followed by three 10-minute washes in distilled water and then immersion in an 8% formic acid solution (using distilled water) for 10 minutes to dissolve the resin, followed by a further two 5-minute washes in water and then TX-PBS (PBS containing 0.2% Triton X-100) solution (Sigma-Aldrich) for 10 minutes.
      Diaminobenzidine (DAB) staining was then performed on the slides using the universal elite ABC kit (PK-6200) and DAB substrate kit (SK-4100) (Vector Laboratories, Burlingame, CA). Briefly, slides were blocked using 3% normal horse serum in 0.2% TX-PBS for 1 hour. Primary antibody, P0 (H-60; Santa Cruz Biotechnology, Santa Cruz, CA) (1:50), was then added in 1% normal horse serum containing 0.2% TX-PBS overnight at room temperature. Slides were washed in PBS (a minimum of three 10-minute washes), and biotinylated secondary antibody (Vector Laboratories) was added (1:200) with 1% normal horse serum in TX-PBS for 2 hours at room temperature. The sections were washed again (three 10-minute washes) in PBS before Vector A-B solution was made (30 minutes before use) and added (1:200 of solution A and B in PBS) for 1 hour, followed by three 10-minute PBS washes and two 15-minute washes in Tris-buffered nonsaline. DAB was then applied (made as per kit instructions) until the sections turned brown/black (depending on whether nickel had been added to the DAB solution), whereupon they were washed in Tris-buffered nonsaline to inactivate the DAB before being mounted using xylene.

      Analysis

      Semithin resin sections (n = 5, a minimum of 4 sections per animal) were analyzed in several ways. Initially, total white matter damage was calculated by measuring the area of damage contained in the dorsal funiculus and dividing this number by the total amount of white matter for that spinal cord section and multiplying by 100. All measurements were generated on toluidine blue–stained sections and using raw “zvi” images obtained using an Axio Scope and AxioVision software version 4.8.2 (Carl Zeiss Ltd, Herts, UK).
      Further detailed counts through the dorsal funiculus of the toluidine blue–stained spinal cords were performed to quantify normal myelin, demyelinated axons, and remyelinated axons. Remyelination by either oligodendrocytes or Schwann cells is distinguishable using well-established morphologic features, including central-type remyelination (identified by thin myelin sheaths) and peripheral-type remyelination (based on identification in semithin sections by its characteristic signet ring–like appearance),
      • Ibanez C.
      • Shields S.A.
      • El-Etr M.
      • Baulieu E.E.
      • Schumacher M.
      • Franklin R.J.M.
      Systemic progesterone administration results in a partial reversal of the age-associated decline in CNS remyelination following toxin-induced demyelination in male rats.
      in the area of the focal injection. Counts were generated using images taken at high magnification (×63) under oil immersion using an Axio Scope and AxioVision software version 4.8.2 before images were saved as TIFFs and counted using Photoshop C4 (Adobe Systems Inc, San Jose, CA). Counts were undertaken by two independent and blinded researchers in 25 × 25-μm squares, with 10 to 11 squares being counted per image, 6 to 10 images per animal (n = 4 to 7 per group). Raw data were then converted into percentages and plotted.
      P0 counts were generated by taking multiple images (4 per small animal) from the surface and at a depth of 100 μm in the dorsal funiculus under a ×63 oil immersion lens. Counts were performed in four 50 × 50-μm squares per image, and then all the counts were averaged and plotted along with their SEMs. All statistical analysis was generated using SigmaPlot version 11.0 (Systat Software Inc, San Jose, CA), and analysis of variance or the Student's t-test was used to determine significance, at a threshold of P < 0.05.

      Electron Microscopy

      For ultrastructural analysis, selected blocks of resin-embedded tissue were trimmed, and ultrathin sections were cut onto copper grids. The tissue was stained with lead citrate and uranyl acetate according to standard protocols before being examined by transmission electron microscopy using an H-600 electron microscope (Hitachi High-Technologies Europe GmbH, Krefeld, Germany).

      Results

      Establishing a Focal Model of Mixed Primary Demyelination and Axonal Injury

      To determine the influence of age on demyelination, remyelination, and axonal injury, we first established a reliable variant of the fEAE model in which there was clear evidence of primary demyelination (henceforth referred to as demyelination). Our initial studies using established protocols of rMOG followed by focal injection of cytokines
      • Kerschensteiner M.
      • Stadelmann C.
      • Buddeberg B.S.
      • Merkler D.
      • Bareyre F.M.
      • Anthony D.C.
      • Linington C.
      • Brück W.
      • Schwab M.E.
      Targeting experimental autoimmune encephalomyelitis lesions to a predetermined axonal tract system allows for refined behavioral testing in an animal model of multiple sclerosis.
      generated destructive lesions in which there was substantial axonal loss but with limited demyelination (data not shown). We, therefore, tested different concentrations of rMOG, TNF-α, and IFN-γ with a view to generating focal lesions characterized by less axonal loss and more demyelination using young adult Lewis rats.
      Initial qualitative studies used a fixed dose of rMOG (50 μg) and varied the concentrations of TNF-α (250, 150, or 75 ng/mL, referred to as simply “ng” from this point forward) and IFN-γ (150, 100, or 50 U; 1 U is equivalent to 0.1 μg/mL, concentrations/units) paired in the following combinations: 250 ng/150 U, 150 ng/100 U, and 75 ng/50 U. This analysis identified the lowest dose of TNF-α/IFN-γ (75 ng/50 U) as being optimal owing to excessive axonal loss measured by semithin quantification at the higher TNF-α/IFN-γ doses (data not shown) 28 days after cytokine injection. Before this titration, a pilot investigation 7 days after cytokine injection had shown clear evidence of demyelination on semithin and electron microscopic images (Figure 1), therefore validating demyelination before remyelination could be studied.
      Figure thumbnail gr1
      Figure 1Semithin and electron microscopic images 7 days after cytokine injection in a young rodent showing primary demyelination. A and B: Semithin images show clear examples of demyelinated surviving axons (arrows) 7 days after cytokine injection (A) and evidence of primary demyelination (B). C and D: More detailed examination shows examples of denuded surviving axons on day 7 at higher magnifications. Scale bars: 20 μm [A and B (scale bar in A is applicable to B)]; 5 μm (C and D).
      Having established the TNF-α/IFN-γ dosage, we next tested two further concentrations of rMOG (60 and 75 μg) to identify the optimal combination of rMOG and TNF-α/IFN-γ to achieve focal demyelination-remyelination with limited axonal loss 28 days after cytokine injection. This was evaluated using quantitative analysis of semithin resin sections examined by light microscopy and compared with age-matched controls. At 50 μg of rMOG and 75 ng/50 U of TNF-α/IFN-γ, a mean ± SEM total of 71.0% ± 9.3% of axons were seen to be surviving (where 100% is derived from age- and region-matched normal Lewis female rats). In these 71%, the mean ± SEM proportion of demyelinated axons was 31.8% ± 4.2%, of remyelinated axons was 53.4% ± 7.0%, and of unaffected axons with normal myelin was 14.8% ± 1.9%. This compared with a mean ± SEM of 56.6% ± 5.2% surviving axons at 60 μg of rMOG, of which 20.9% ± 1.9% were demyelinated, 55.5% ± 5.1% were remyelinated, and 23.6% ± 2.2% were normal. Finally, at 75 μg of rMOG, a mean ± SEM of 36.3% ± 3.7% of axons survived, of which 11.1% ± 1.3% were demyelinated, 42.8% ± 4.4% were remyelinated, and 46.1% ± 4.7% were normally myelinated. Subsequent studies, therefore, used rMOG at 60 μg followed by 75 ng/50 U of TNF-α/IFN-γ injected into the cervical spinal cord as this resulted in frank demyelination (evidenced also by the presence of remyelination) with some axonal loss.

      Aged Animals Display Greater Axonal Loss and Less Remyelination

      Having established a dosing regimen that produced a reliable model of focal demyelination in young adult animals, we next quantified and compared the extent of white matter abnormality in young adult (3 months) and aged old (15 months) rats (Figure 2). We first established that in the intact uninjured spinal cords of normal young and old adult Lewis rats, there were no differences between the percentage of the dorsal funiculus compared with total white matter when analyzed or total axons counted in the dorsal funiculus (mean ± SEM: 35.6 ± 2.2 in young compared with 36.6 ± 2.8 in old normally myelinated axons per 25 μm2). This finding showed that there was no possibility of preinjury age-associated changes accounting for axonal differences observed. Furthermore, to exclude any potential effect due to colony difference, we also examined aged Lewis rats from Harlan Laboratories and Charles River UK Ltd. These studies revealed no differences in quantitative outcomes, including total axonal loss and remyelination efficiencies, between Lewis rat colonies (data not shown).
      Figure thumbnail gr2
      Figure 2White matter damage in the dorsal funiculus is significantly more pronounced in old versus young adult fEAE rats. A–D: Representative images from the 1-μm semithin, toluidine blue–stained, C5-C6 region of Lewis rat spinal cords from normal rats (A and D) and young (B and E) and old (C and F) adult rats after rMOG injection and cytokine infusions, 28 days after spinal cord infusions. Images A–C show the whole spinal cord, and D–F are the boxed areas from A–C, being more focused on the dorsal funiculus, in the area where infusion of cytokines occurred. G: By measuring the area of white matter damaged compared with the total amount of white matter in the section allows quantification of the total white matter damage and shows that there is significantly (*P < 0.05 by analysis of variance) more disruption to the white matter in old Lewis rats after rMOG and cytokine injections compared with in young rats. Data are given as mean ± SEM. Scale bars: 1 mm [A–C (scale bar in A is applicable to B and C)]; 0.5 mm [D–F (scale bar in D is applicable to E and F)].
      After fEAE induction, however, an age-related increase in white matter damage was evident, with mean ± SEM loss of white matter in the area of focal injection of 9.0% ± 2.9% in young adult rats compared with 24.0% ± 2.4% in old rats (Figure 2) when the total area of damaged tissue compared with all white matter was calculated. Independent and blinded quantification of total axonal loss using toluidine semithin sections revealed significant progressive age-related axonal loss after fEAE induction using TNF-α and IFN-γ.
      Further subtype analysis was next undertaken to determine the influence of age on the myelination status of surviving axons after fEAE induction (Figure 3). Aged rodents had mean ± SEM total axonal loss of 74.1% ± 2.9% compared with normal animals, and young rodents displayed only a 51.8% ± 9.2% loss (P < 0.05) (Figure 3G). The mean ± SEM overall level of remyelination of the surviving axons, identified according to well-established morphologic criteria by either oligodendrocytes or Schwann cells (Figure 3), was significantly greater in young adult rats (64.6% ± 6.4%) than in old adult rats (50.2% ± 5.6%) (P < 0.05) (Figure 3H). Furthermore, the percentage of axons remaining demyelinated 28 days after cytokine injection also varied significantly, with the mean ± SEM percentage being lower in young adult rats (16.7% ± 6.4%) than in old adult rats (30.9% ± 3.5%) (P < 0.05) (Figure 3H).
      Figure thumbnail gr3
      Figure 3Demyelination, axonal damage, and remyelination (peripheral and central) in the dorsal funiculus is altered in old versus young fEAE animals. Images of toluidine blue–stained 1-μm-thick sections from the surface (A–C) and from deeper into the dorsal funiculus (100 to 150 μm) (D–F) of Lewis rats after the induction of fEAE. Representative images from normal (A and D), young (B and E), and old (C and F) rats, with arrows highlighting examples of normally myelinated axons (orange), peripherally Schwann cell remyelinated axons (green) as identified by the signet ring–like appearance of the myelin, which is slightly darker than central oligodendrocyte remyelination (yellow) and demyelinated axons (red). G: Quantification of the total number of surviving axons (denuded, remyelinated, and normally myelinated) in old and young fEAE animals compared with in undamaged age-matched controls shows significantly greater loss of axons in old fEAE animals compared with in young animals. H: In the surviving axons, there is a significant increase in remyelinated (peripheral or central) axons in young fEAE animals compared with in old animals, and in the remaining axons that are demyelinated 28 days after cytokine injection, there is a significantly greater proportion in old animals than in young animals. I: Furthermore, a significant increase in peripheral remyelination based on morphologic criteria was detected in old adult rats compared with in young rats. Data are given as mean ± SEM. Scale bars: 20 μm [A–F (scale bar in A is applicable to B–F)]. *P < 0.05, one-way analysis of variance.

      Peripheral Remyelination Predominates in Aged Animals

      Central nervous system (CNS) remyelination can be mediated by either oligodendrocytes (central type) or Schwann cells (peripheral type).
      • Sim F.J.
      • Zhao C.
      • Penderis J.
      • Franklin R.J.M.
      The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation.
      • Zawadzka M.
      • Rivers L.E.
      • Fancy S.P.
      • Zhao C.
      • Tripathi R.
      • Jamen F.
      • Young K.
      • Goncharevich A.
      • Pohl H.
      • Rizzi M.
      • Rowitch D.H.
      • Kessaris N.
      • Suter U.
      • Richardson W.D.
      • Franklin R.J.M.
      CNS-resident glial progenitor/stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination.
      Central-type remyelination, identified by thin myelin sheaths, was more abundant in young rats than in old rats (Figure 3). A more detailed analysis of semithin sections showed that in young adult rats, a mean ± SEM of 40.4% ± 8.8% of all remyelination seemed to be Schwann cell mediated and 59.6% ± 8.8% oligodendrocyte derived. Conversely, in old animals, peripheral-type remyelination was more abundant than in young adult rodents based on identification in semithin sections by its characteristic signet ring–like appearance (Figure 3), showing a mean ± SEM of 88.5% ± 4.7% Schwann cell remyelination in old adult rats compared with 11.1% ± 4.5% oligodendrocyte remyelination (Figure 3I).
      We further quantified the extent of peripheral-type remyelination by immunohistochemical staining to identify P0 (Figure 4), a myelin protein uniquely expressed in peripheral myelin in mammals. This analysis further showed a significant age-related bias: a mean ± SEM of 4.8 ± 3.1 P0+ myelin cells per 2500 μm2 in young adult rats (Figure 4, A, C, and E) compared with 17.7 ± 5.3 in old rats (P < 0.05) (Figure 4, B, D, and E). Consistent with previous reports, P0+ cells were most evident in areas of the lesion from which glial fibrillary acidic protein–positive astrocytes were absent (see Supplemental Figure S1 at http://ajp.amjpathol.org).
      • Zawadzka M.
      • Rivers L.E.
      • Fancy S.P.
      • Zhao C.
      • Tripathi R.
      • Jamen F.
      • Young K.
      • Goncharevich A.
      • Pohl H.
      • Rizzi M.
      • Rowitch D.H.
      • Kessaris N.
      • Suter U.
      • Richardson W.D.
      • Franklin R.J.M.
      CNS-resident glial progenitor/stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination.
      • Woodruff R.H.
      • Franklin R.J.M.
      Demyelination and remyelination of the caudal cerebellar peduncle of adult rats following stereotaxic injections of lysolecithin, ethidium bromide, and complement/anti-galactocerebroside: a comparative study.
      Finally, ultrastructural analyses confirmed the comparative abundance of peripheral remyelination in aged compared with young adult Lewis rats (Figure 5). Together, these findings reveal an age-dependent effect on the extent and type of remyelination after fEAE.
      Figure thumbnail gr4
      Figure 4Acid-etched P0 immunohistochemical analysis in the dorsal funiculus in old and young fEAE animals showing significantly more peripheral myelin in old animals. A–D: P0 immunohistochemical analysis after acid-etching removal of resin from semithin-cut sections of young (A and C) and old (B and D) fEAE animals. A and B are low magnification, showing the complete dorsal funiculus; the boxed areas are enlarged in C and D, respectively. P0 is known to identify peripherally generated myelin only, and quantification of averaged total cell counts shows a significantly larger number of P0+ “rings” counted in old fEAE animals compared with in young animals, although there was clearly less remyelination occurring in same-aged animals. E: Mean ± SEM P0 counts per 50 × 50-μm grid in old compared with young fEAE animals. *P < 0.05, one-way analysis of variance. Scale bars: 500 μm [A and B (scale bar in A is applicable to B)]; 50 μm [C and D (scale bar in C is applicable to D)].
      Figure thumbnail gr5
      Figure 5Representative electron microscopic images taken from old and young fEAE animals showing exclusive Schwann cell remyelination in old animals compared with a mixture of peripheral and central remyelination in young fEAE animals. Electron microscopic images from young (A–D) and old (E–H) fEAE Lewis rats. In the old rodents, only examples of demyelinated axons (E) and Schwann cell remyelinated axons (F–H) were evident. We show Schwann cells in their premyelinating stage (F), perfect Schwann cell remyelination (G), and further evidence that this is peripheral remyelination as shown by the presence of the basal lamina (H). In the young fEAE animals, we also found clear evidence of Schwann cell remyelination (A), along with evidence of basal lamina being present (not shown); however, there was also central oligodendrocyte remyelination (B–D). At lower magnification (B), a large patch of oligodendrocyte remyelination and some damaged-looking axons are seen; at higher magnification (C), there are clear examples of oligodendrocyte remyelination and an astrocyte process. On detailed examination of these areas, no presence of basal lamina (D) was found, being further proof of central oligodendrocyte remyelination. Scale bars: 5 μm (A–C and E–G); 500 nm (D and H).

      Discussion

      This study reveals two important influences of age on response to focal immune-mediated inflammatory demyelinating injury: first, the same method of lesion induction in young compared with aged adult animals reveals increased susceptibility to axonal injury in the aged group and, second, not only is the efficiency of remyelination decreased with age but there is also a significant increase in Schwann cell–type remyelination.
      An age-related influence on remyelination is well established from experimental studies using focal injection or systemic delivery of demyelinating toxins.
      • Shields S.A.
      • Gilson J.M.
      • Blakemore W.F.
      • Franklin R.J.M.
      Remyelination occurs as extensively but more slowly in old rats compared to young rats following gliotoxin-induced CNS demyelination.
      • Shen S.
      • Sandoval J.
      • Swiss V.A.
      • Li J.
      • Dupree J.
      • Franklin R.J.M.
      • Casaccia-Bonnefil P.
      Age-dependent epigenetic control of differentiation inhibitors is critical for remyelination efficiency.
      However, the relevance of this phenomenon to remyelination after immune-mediated inflammatory demyelination, a model that more closely models key pathologic aspects of MS, has not been established. To resolve this issue, previously reported protocols of focal inflammatory white matter injury
      • Kerschensteiner M.
      • Stadelmann C.
      • Buddeberg B.S.
      • Merkler D.
      • Bareyre F.M.
      • Anthony D.C.
      • Linington C.
      • Brück W.
      • Schwab M.E.
      Targeting experimental autoimmune encephalomyelitis lesions to a predetermined axonal tract system allows for refined behavioral testing in an animal model of multiple sclerosis.
      • Merkler D.
      • Ernsting T.
      • Kerschensteiner M.
      • Brück W.
      • Stadelmann C.
      A new focal EAE model of cortical demyelination: multiple sclerosis-like lesions with rapid resolution of inflammation and extensive remyelination.
      were modified to generate a reliable model of focal immune-mediated demyelinating injury, fEAE.
      Titration of rMOG and cytokines resulted in the generation of a consistent model of white matter injury characterized by extensive primary demyelination but also some axonal loss. This enabled us to demonstrate a means of demyelination induction closer to that occurring in some multiple sclerosis lesions and the age-associated effect on remyelination similar to that previously shown in toxin models of demyelination.
      • Sim F.J.
      • Zhao C.
      • Penderis J.
      • Franklin R.J.M.
      The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation.
      Age has also been reported to be a determinant of axonal injury after demyelination.
      • Irvine K.A.
      • Blakemore W.F.
      Age increases axon loss associated with primary demyelination in cuprizone-induced demyelination in C57BL/6 mice.
      This study used the cuprizone model of demyelination/remyelination. However, to induce demyelination in older rats, a higher dose of cuprizone was required, resulting in some uncertainty as to whether the increase in axonal loss in older animals was due to the higher dose of toxin or an intrinsic age-associated vulnerability with the axons. In the present study, we partially resolved this issue by using the same method of lesion induction in both age groups and demonstrate an age-associated increase in axonal vulnerability.
      The reason for increased axonal loss in older animals is unclear, although there are several possible explanations. Older axons may be intrinsically more susceptible to fEAE. This is consistent with accumulating evidence for global intrinsic cellular changes with aging that include accumulating nuclear and particularly mitochondria mutations with concomitant impaired DNA repair and cellular and organ deterioration.
      • Rist J.M.
      • Franklin R.J.M.
      Taking ageing into account in remyelination-based therapies for multiple sclerosis.
      • Hayflick L.
      How and Why We Age.
      • Clark W.
      A Means to an End: The Biological Basis of Aging and Death.
      • Weissman L.
      • de Souza-Pinto N.C.
      • Stevnsner T.
      • Bohr V.A.
      DNA repair, mitochondria, and neurodegeneration.
      Differences in the nature of the immune response attributable to age may also contribute. This is consistent with studies showing a reduced macrophage response in a rodent eye injury model and altered cytokine levels of IFN-γ and IL-1 in aged compared with young adult dogs.
      • Chung J.Y.
      • Choi J.H.
      • Lee C.H.
      • Yoo K.Y.
      • Won M.H.
      • Yoo D.Y.
      • Kim D.W.
      • Choi S.Y.
      • Youn H.Y.
      • Moon S.M.
      • Hwang I.K.
      Comparison of ionized calcium-binding adapter molecule 1-immunoreactive microglia in the spinal cord between young adult and aged dogs.
      • Luo J.M.
      • Geng Y.Q.
      • Zhi Y.
      • Zhang M.Z.
      • van Rooijen N.
      • Cui Q.
      Increased intrinsic neuronal vulnerability and decreased beneficial reaction of macrophages on axonal regeneration in aged rats.
      Previous studies using toxin-induced models of demyelination have reported increased and sustained cytokine levels after lysolecithin injection in older compared with younger adult animals
      • Zhao C.
      • Li W.W.
      • Franklin R.J.M.
      Differences in the early inflammatory responses to toxin-induced demyelination are associated with the age-related decline in CNS remyelination.
      and age-associated altered patterns of growth factor expression.
      • Hinks G.L.
      • Franklin R.J.M.
      Delayed changes in growth factor gene expression during slow remyelination in the CNS of aged rats.
      In addition, since remyelination is neuroprotective
      • Irvine K.A.
      • Blakemore W.F.
      Remyelination protects axons from demyelination-associated axon degeneration.
      and age is known to negatively influence regenerative responses, the reduced remyelination in older animals could itself result in increased axonal loss. The finding that age slows the rate rather than the extent of remyelination argues against this possibility and recommends potential analysis of older animals at later time points.
      • Shields S.A.
      • Gilson J.M.
      • Blakemore W.F.
      • Franklin R.J.M.
      Remyelination occurs as extensively but more slowly in old rats compared to young rats following gliotoxin-induced CNS demyelination.
      The present study does not directly address this possibility, but the results suggest that reduced remyelination per se is not solely responsible for the increased axonal loss observed in fEAE given that only 30.9% of surviving axons in the aged animals are demyelinated 4 weeks after fEAE. This contrasts with >50% demyelinated axons after toxin-induced demyelination 4 weeks after lesion induction.
      • Shields S.A.
      • Gilson J.M.
      • Blakemore W.F.
      • Franklin R.J.M.
      Remyelination occurs as extensively but more slowly in old rats compared to young rats following gliotoxin-induced CNS demyelination.
      A striking finding was the influence of age on remyelination type. Aged animals, aside from less remyelination, also had almost predominantly peripheral remyelination determined by ultrastructure and immunostaining. Peripheral-type remyelination of axons in the CNS by Schwann cells is a well-recognized phenomenon, being especially prevalent in traumatic spinal cord injury
      • Blight A.R.
      • Young W.
      Central axons in injured spinal cat spinal cord recover electrophysiological function following remyelination by Schwann cells.
      • Griffiths I.R.
      • McCulloch M.C.
      Nerve fibres in spinal cord impact injuries, part 1: changes in the myelin sheath during the initial 5 weeks.
      and occurring in genetic demyelination
      • Duncan I.D.
      • Hoffman R.L.
      Schwann cell invasion of the central nervous system of the myelin mutants.
      and in multiple experimental models of demyelination, including EAE
      • Raine C.S.
      • Traugtt U.
      • Stone S.H.
      Glial bridges and Schwann cell migration during chronic demyelination in the CNS.
      and especially those involving the use of demyelinating toxins.
      • Woodruff R.H.
      • Franklin R.J.M.
      Demyelination and remyelination of the caudal cerebellar peduncle of adult rats following stereotaxic injections of lysolecithin, ethidium bromide, and complement/anti-galactocerebroside: a comparative study.
      • Blakemore W.F.
      Remyelination by Schwann cells of axons demyelinated by intraspinal injection of 6-aminonicotinamide in the rat.
      • Dusart I.
      • Marty S.
      • Peschanski M.
      Demyelination, and remyelination by Schwann cells and oligodendrocytes after kainate-induced neuronal depletion in the central nervous system.
      • Felts P.A.
      • Woolston A.M.
      • Fernando H.B.
      • Asquith S.
      • Gregson N.A.
      • Mizzi O.J.
      • Smith K.J.
      Especially relevant to this study is the description of Schwann cell remyelination in multiple sclerosis,
      • Ghatak N.R.
      • Hirano A.
      • Doron Y.
      • Zimmerman H.M.
      Remyelination in multiple sclerosis with peripheral type myelin.
      • Itoyama Y.
      • Webster H.D.
      • Richardson Jr, E.P.
      • Trapp B.D.
      Schwann cell remyelination of demyelinated axons in spinal cord multiple sclerosis lesions.
      • Yamamoto T.
      • Kawamura J.
      • Hashimoto S.
      • Nakamura M.
      Extensive proliferation of peripheral type myelin in necrotic spinal cord lesions of multiple sclerosis.
      • Ikota H.
      • Iwasaki A.
      • Kawarai M.
      • Nakazato Y.
      Neuromyelitis optica with intraspinal expansion of Schwann cell remyelination.
      where, as in other situations, it is most often but not exclusively found in the spinal cord. Indeed, Schwann cell remyelination might be regarded as an expected form of spinal cord remyelination, which becomes more prominent with increasing age, as we and others
      • Shields S.A.
      • Gilson J.M.
      • Blakemore W.F.
      • Franklin R.J.M.
      Remyelination occurs as extensively but more slowly in old rats compared to young rats following gliotoxin-induced CNS demyelination.
      have shown.
      Until recently, it was thought that this type of CNS remyelination was due to Schwann cell lineage cells of peripheral nervous system origin immigrating into the CNS after a breach in the glia limitans, an explanation consistent with Schwann cell remyelination occurring in areas where astrocytes are absent and the preponderance of CNS Schwann cell remyelination is close to peripheral nervous system sources of Schwann cells, such as spinal roots or blood vessels. However, recent fate mapping studies in which CNS precursors and peripheral nervous system Schwann cells are labeled with green fluorescent protein such that they and their progeny can be identified have indicated somewhat unexpectedly that most CNS Schwann cells are derived from CNS intrinsic stem/progenitor cells, including when this type of remyelination occurs contiguously with spinal roots.
      • Zawadzka M.
      • Rivers L.E.
      • Fancy S.P.
      • Zhao C.
      • Tripathi R.
      • Jamen F.
      • Young K.
      • Goncharevich A.
      • Pohl H.
      • Rizzi M.
      • Rowitch D.H.
      • Kessaris N.
      • Suter U.
      • Richardson W.D.
      • Franklin R.J.M.
      CNS-resident glial progenitor/stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination.
      The association of CNS Schwann cells with the absence of glial fibrillary acidic protein–positive astrocytes observed in these experiments, therefore, suggests a potential mechanism in which the presence or absence of astrocytes determines the CNS or peripheral nervous system fate of CNS intrinsic stem/progenitor cells. Several studies have suggested that bone morphogenetic protein may play a role in this differentiation and that this pathway is inhibited by astrocyte-derived inhibitors of bone morphogenetic protein signaling.
      • Hampton D.W.
      • Asher R.A.
      • Kondo T.
      • Steeves J.D.
      • Ramer M.S.
      • Fawcett J.W.
      A potential role for bone morphogenetic protein signalling in glial cell fate determination following adult central nervous system injury in vivo.
      • Talbott J.F.
      • Cao Q.
      • Enzmann G.U.
      • Benton R.L.
      • Achim V.
      • Cheng X.X.
      • Mills M.D.
      • Rao M.S.
      • Whittemore S.R.
      Schwann cell-like differentiation by adult oligodendrocyte precursor cells following engraftment into the demyelinated spinal cord is BMP-dependent.
      In the context of the present study, the significantly predominant Schwann cell remyelination in older animals is likely to be related to increased astrocyte loss and/or the reduced ability of lost astrocytes to be regenerated.
      Taken together and in view of the increased recognition that progression or neurodegeneration in multiple sclerosis is an age-dependent process, a formal study of age and remyelination in multiple sclerosis tissue preferably from primary and secondary progressive samples would be of great value. The present findings of age-related increased vulnerability to injury and reduced regenerative response are of relevance not just to the degenerative phase of human demyelinating injury but also to other age-related neurodegenerative disorders in an aging population.

      Supplementary data

      • Supplemental Figure S1

        Acid-etched glial fibrillary acidic protein (GFAP) immunohistochemical (IHC) analysis in the dorsal funiculus in old versus young fEAE rats showing significantly fewer reactive astrocytes in old animals. GFAP IHC analysis after acid-etching removal of resin from semithin-cut sections of young (A) and old (B) fEAE animals. Images are low magnification, showing the complete dorsal funiculus from comparable sections to the P0 IHC analysis. A significant decrease in GFAP-positive reactive astrocytes and processes can be seen in the old example (B) compared with the young (A). Scale bars: 500 μm (A and B).

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