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Steroids and the reversal of age-associated changes in myelination and remyelination
Progress in Neurobiology 71 (2003) 49–56
Steroids and the reversal of age-associated changes in myelination and remyelination C. Ibanez b , S.A. Shields a , M. El-Etr b , E. Leonelli c , V. Magnaghi c , W.-W. Li a , F.J. Sim a , E.-E. Baulieu b , R.C. Melcangi c , M. Schumacher b , R.J.M. Franklin a,∗ a
Department of Clinical Veterinary Medicine and Cambridge Centre for Brain Repair, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK b INSERM U488, 80 rue du Général Leclerc, 94276 Le Kremlin Bicˆ etre-Cedex, France c Department of Endocrinology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milano, Italy Received 27 May 2003; accepted 9 September 2003
Abstract The myelin sheaths that surround all but the smallest diameter axons within the mammalian central nervous system (CNS) must maintain their structural integrity for many years. Like many tissues, however, this function is prone to the effects of ageing, and various structural anomalies become apparent in the aged CNS. Similarly, the regenerative process by which myelin sheaths, lost as a consequence of exposure to a demyelinating insult, are restored (remyelination) is also affected by age. As animals grow older, the efficiency of remyelination progressively declines. In this article, we review both phenomena and describe how both can be partially reversed by steroid hormones and their derivatives. © 2003 Elsevier Ltd. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ageing and CNS myelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CNS remyelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ageing and CNS remyelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The age-related decline in remyelination efficiency occurs due to an impairment of oligodendrocyte progenitor recruitment and differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Inflammation and the age-related decline in remyelination efficiency . . . . . . . . . . . . . . . . . . . 5. The effects of progesterone on CNS remyelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Progesterone does not impair efficient remyelination in young animals . . . . . . . . . . . . . . . . . 5.2. Systemic progesterone administration results in a small but significant increase in oligodendrocyte remyelination following demyelination in old adult male rats . . . . . . . . . . . . . . . . . 6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. 2. 3. 4.
Abbreviations: CNPase, 2 -3 -cyclic nucleotide-3-phosphodiesterase; CNS, central nervous system; DHP, dihydro-progesterone; IGF-I, insulin-like growth factor I; MBP, myelin-basic protein; MRI, magnetic resonance imaging; OPC, oligodendrocyte progenitor; PMP-22, peripheral myelin protein 22; PNS, peripheral nervous system; PROG, progesterone; PLP, proteolipid protein; THP, tetrahydro-progesterone ∗ Corresponding author. Tel.: +44-1223-337642; fax: +44-1223-337610. E-mail address: [email protected] (R.J.M. Franklin). 0301-0082/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.pneurobio.2003.09.002
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1. Introduction Myelin sheaths surround all but the smallest diameter axons within the mammalian central nervous system (CNS). Their function is to enable axons to conduct action potentials by saltatory conduction, a more rapid and efficient means of conduction than that which occurs in their absence. In the CNS, myelin is produced and maintained by oligoden-
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drocytes, a highly specialised cell type that can support between 1 and 40 separate myelin sheaths depending on the size of axon with which they associate. The vexed question of whether there is a turnover of oligodendrocytes has not been fully resolved and continues to provoke heated debate amongst those who believe that there is no turnover and those who cite evidence supporting their view that at least some replacement of oligodendrocytes and their myelin sheaths must occur as a normal physiological process. It is not the intention in this article to engage in this discussion but instead to discuss our own recent work on how myelin sheaths and the intrinsic ability to replace myelin sheaths lost as a consequence of pathological processes (remyelination) are affected by ageing, and how these effects can be modulated by steroid hormones.
2. Ageing and CNS myelin In common with all other tissues, myelin is subject to degenerative changes attributable to ageing. These have not been extensively studied but informative morphological analysis of white matter in the brain of aged old world monkeys has revealed a number of distinct changes that occur in ageing CNS myelin similar to those known to occur in ageing peripheral myelin. These include a significantly high incidence of: (1) splitting of the major dense line (the line formed by the apposition of the internal membrane surface during the extrusion of cytoplasm that occurs during the formation of the compacted myelin sheath) to create swellings containing electron dense oligodendrocyte cytoplasm; (2) splitting of the intraperiod line (the line formed by the apposition of the external membrane surfaces during myelination) to create fluid filled swellings; (3) formation of redundant myelin so that the sheath is too large for the enclosed axon; and (4) thickened sheaths that are almost completely split (Peters, 1996; Peters et al., 2000, 2001; Peters and Sethares, 2002). The age-associated changes in myelin sheaths may in part account for the cognitive decline that occurs in monkeys over a similar time frame (Peters and Sethares, 2002). However, it is not certain how closely changes in myelin sheath structure correlate with MRI and histologically detectable loss of white matter in ageing human brain (Guttmann et al., 1998; Tang et al., 1997). What are the molecular changes occurring with ageing that may account for the disturbances in the normal morphology of myelin sheaths with ageing? Recent observations on the effects of ageing on the constitutive levels of expression of key genes encoding for major protein constituents of the myelin sheath may provide clues. The maintenance of myelin sheath integrity involves the sustained expression of a range of genes specifically associated with the myelin sheath, foremost amongst which are the major myelin proteins myelin-basic protein (MBP) and proteolipid protein (PLP). In a recent study, we compared the constitutive levels of expression of mRNAs of both these myelin genes in the
Fig. 1. Relative optical densities of constitutive mRNA expression of the myelin protein genes, MBP and PLP, and the transcription factor Gtx (±S.E.M.) in the brain stem white matter of young and old adult rats (see Sim et al., 2000).
brain stem white matter of young adult rats (8–10 weeks) and older adult rats (>12 months) by quantitative in situ hybridisation based on densitometry of autoradiographs obtained following use of 35 S-labelled oligonucleotide probes (Sim et al., 2000). Although the distribution of expression of both MBP and PLP mRNA was the same between the two groups, there was a significant decrease in the constitutive levels of expression of both genes (Fig. 1). This difference was mirrored by lower levels of expression of Gtx, a homeodomain transcription factor associated with myelinating oligodendrocytes and potentially involved in the regulation of both MBP and PLP (Awatramani et al., 1997). The age-associated decline in expression of myelin protein mRNAs in the CNS resembles similar changes in myelin protein mRNA expression occurring in the peripheral nervous system (PNS) (Melcangi et al., 1998). In the PNS, changes in P0 mRNA expression can be reversed by the administration of various steroid derivatives. We have therefore addressed whether the same may be true for the myelin protein genes in the CNS. Aged (22–24 month-old) male rats have been treated with progesterone (PROG), dihydro-progesterone (DHP), and tetrahydro-progesterone (THP) for 1 month and the mid-brains from both control (vehicle only) and treatment groups cryostat-sectioned and subjected to in situ hybridisation with 35 S-labelled oligonucleotide probes against MBP, PLP and Gtx (Fig. 2A). The most striking change observed was the dramatic reduction in constitutive levels of all genes in the aged controls compared to our earlier data on constitutive expression in young and middle-aged adult rats, indicating that constitutive levels of expression continue to decline with increasing age. In the treatment groups, it was observed that both DHP and THP induced a slight but significant increase in MBP mRNA expression compared to controls, implying that the age-associated decline in levels of expression of this key myelin-associated gene is partially reversible by steroid
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Fig. 2. (A) Autoradiographs of brain stem/cerebellar sections following in situ hybridisation with 35 S-labelled oligonucleotide probes for MBP, PLP and Gtx from young adult rat, aged adult rat and aged adult rats treated with PROG (P), DHP and THP. Rats received eight subcutaneous injections of 1 mg of steroid, suspended in 250 l of sesame oil. Age-matched control rats were injected with 250 l of sesame oil only. Injections were administered every 4 days, and the animals were killed 24 h after the last treatment; (B) Relative optical densities of mRNA expression of the myelin protein genes, MBP and PLP, and the transcription factor Gtx (±S.E.M.). Significant increases in the levels of expression compared to control aged rats were obtained for aged rats treated with DHP and THP.
hormone treatment (Fig. 2B). An effect of these metabolites of PROG is perhaps not surprising, since oligodendrocytes possess both the capability to convert PROG into DHP, by the enzyme 5␣-reductase, and subsequently into THP by the enzyme 3␣-hydroxysteroid dehydrogenase (Melcangi et al., 1994; Gago et al., 2001). Whether these changes in levels of MBP expression relate to morphological changes such as fewer myelin sheaths with split major dense lines remains to be addressed.
3. CNS remyelination Remyelination, the process in which new myelin sheaths are restored to demyelinated axons, is one of the few spontaneous regenerative processes that occurs within the adult mammalian CNS (Franklin and Hinks, 1999). However, although this helpful regenerative process can occur even in demyelinating diseases in adult humans such as multiple sclerosis (MS), it is not an invariable consequence of demyelination, and there are many occasions where it fails to occur or is incomplete (Franklin, 2002). Failure of
remyelination in MS becomes increasingly pronounced as the disease progresses, leading to impaired conduction and probably contributing to the axonal atrophy that typifies the secondary progressive phase of the disease. Attempts to promote remyelination have frequently focused on enhancing or reactivating the inherent process, whose failure accounts for the persistence of demyelination. This approach has proved difficult to achieve, largely because of shortcomings in our understanding of the mechanisms by which remyelination proceeds. Using a variety of experimental models of demyelination, the sequence of cellular events involved in oligodendrocyte-mediated remyelination of the CNS have been identified. Firstly, in response to demyelination, oligodendrocyte progenitor cells (OPCs) present within the adult CNS are recruited into the demyelinated area (Gensert and Goldman, 1997; Franklin et al., 1997; Reynolds et al., 2001; Carroll et al., 1998; Levine and Reynolds, 1999; Redwine and Armstrong, 1998; Sim et al., 2002). This initial recruitment process involves OPC proliferation, and, to a lesser extent migration. The second phase of remyelination involves the differentiation of the recruited OPCs into mature
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remyelinating oligodendrocytes (Ludwin and Sternberger, 1984; Jordan et al., 1990; Woodruff and Franklin, 1998, 1999b; Sim et al., 2000).
4. Ageing and CNS remyelination In common with many regenerative processes in the body, ageing has a detrimental effect on remyelination (Franklin et al., 2002). Using a toxin model of demyelination, we found that demyelinated foci were fully remyelinated at 4 weeks in young adult rats (<3 months of age) but not until 9 weeks in older adult rats (>9 months of age) (Shields et al., 1999), indicating that although remyelination proceeds to completion in the older age-group, it does so more slowly. This age-related slowing of remyelination is reflected in a slowing of the rate of reappearance of transcripts for the major myelin proteins MBP and PLP, and their putative regulatory factor Gtx, within lesions in old animals compared to young (Sim et al., 2000) (Fig. 3). Our recent unpublished data indicate that the rate of remyelination continues to slow with increasing age. The decline in the efficiency of remyelination simply by virtue of the animal having become older has clear implications for the likelihood of recovery in a disease such as MS from which a patient may suffer for several decades. If ageing effects are indeed contributing to the disease progression in MS, then devising means of reversing the ageing trend represents a legitimate approach to MS therapy. 4.1. The age-related decline in remyelination efficiency occurs due to an impairment of oligodendrocyte progenitor recruitment and differentiation Why does the rate of remyelination slow with ageing? On the basis of the model of remyelination presented earlier, there are at least three possibilities: (1) the numbers of resident OPCs decline with age, leading to a reduction in the size of the pool of cells available for recruitment; (2) the rate of OPC recruitment decreases; and (3) the rate of
differentiation of recruited OPCs into remyelinating oligodendrocytes decreases. The first of these possibilities has been addressed by comparing the absolute numbers of OPCs in the white matter of young and old adult rats. No significant difference occurs between the two, in spite of the difference in the rate of remyelination (Sim et al., 2002), suggesting that the impairment of remyelination is not due to a decline in OPC availability but rather to an impairment of the factors involved in their recruitment and differentiation, or a change in their intrinsic ability to respond to these factors. The rate of OPC recruitment following demyelination in young and old adults has been established by following the OPC response to chemically-induced demyelination using two OPC markers: PDGF-␣R mRNA is an established OPC marker (Pringle et al., 1992), and MyT-1 is a zinc finger transcription factor expressed by OPCs in tissue culture but less often used as an OPC marker in histology (Armstrong et al., 1995; Wrathall et al., 1998). In both cases, the patterns of expression indicated a slower rate of OPC recruitment in the older animals (Sim et al., 2002). Thus, one of the contributory factors in the slowing of the rate of remyelination that occurs with ageing is the impairment of OPC recruitment. Finally, comparing the interval between equivalent levels of expression of MyT-1 transcripts, indicative of the OPC response, and Gtx and MBP transcripts, indicative of the appearance of differentiated oligodendrocytes, revealed that there is also a decrease in the rate at which recruited OPCs differentiate into remyelinating oligodendrocytes (Sim et al., 2002). Thus, in addition to the delay in OPC recruitment that occurs in the old animals, there was also a decrease in the rate at which the recruited cells differentiated, and together both these phenomena result in a decrease in the rate of remyelination. 4.2. Inflammation and the age-related decline in remyelination efficiency A major feature of the cellular environment of remyelination is the presence of macrophages in the latter,
Fig. 3. Scatter plot showing the results of a ranking analysis in older adult rats 5 weeks after lesion induction. The highest rank is given to the animal exhibiting the lowest proportion of demyelinated axons or the highest proportion of either oligodendrocyte or Schwann cell remyelination. The plot illustrates the control (Cont.) and progesterone-treated (PROG) animals ranked according to the degree of demyelination (D) and remyelination by either oligodendrocytes (O) or Schwann cells (SC). Mann–Whitney U-test results with corresponding P-values are reported on the graph.
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recruited in response to the presence of myelin debris generated by demyelination (Ousman and David, 2000). The macrophage has been identified as a major source of remyelination-associated growth factors during remyelination (Hinks and Franklin, 1999; Diemel et al., 1998). In old animals, the macrophage response following toxin-induced demyelination is delayed compared with young animals, suggesting that this response may be a key determinant of efficient remyelination. Indeed, a clear association exists between widespread remyelination and a robust macrophage presence, or conversely poor remyelination and a sparse macrophage presence (Ludwin, 1980; Graca and Blakemore, 1986). If the macrophage response was important for remyelination then reducing it during the rapid remyelination following demyelination in young animals should result in an impairment of remyelination. This has been tested using clodronate liposomes to deplete circulating monocytes and thereby reduce the monocyte-derived contribution to the macrophages within the lesion (van Rooijen, 1989). This led to a significant reduction in the extent of oligodendrocyte remyelination of lysolecithin lesions (Kotter et al., 2001), providing yet further evidence of a pro-remyelination role for the macrophage (Hamilton and Rome, 1994; Loughlin et al., 1997). Prima facie, the beneficial role of macrophages contradicts the more widely recognised role for macrophages in myelin pathology as mediators of the demyelinating process (Dijkstra et al., 1992). The functional roles of macrophages are undoubtedly diverse, and their role in demyelinating disease is likely to be similarly so. There is little doubt that in immune-mediated demyelination, where macrophages are activated by lymphocytes, their predominant effects are harmful to the myelin sheath (Cuzner and Norton, 1996; Merrill and Benveniste, 1996; Merrill and Scolding, 1999). However, in toxin-induced demyelination, macrophages are recruited as a consequence of demyelination and are not likely to be involved in its mediation. The macrophages are therefore present as part of the regenerative process. How is the macrophage response beneficial? One of the obvious functions of macrophages in demyelinating disease is the removal of myelin debris. Whether this function is directly beneficial for remyelination has never been formally tested. Another way in which macrophages might be beneficial is by producing molecules crucially involved in the remyelination process. These may be growth factors that have direct effects on the cells of the oligodendrocyte lineage, or they may be molecules that activate other cells within the remyelination environment, such as astrocytes, to produce pro-remyelination factors.
5. The effects of progesterone on CNS remyelination Progesterone has beneficial effects on remyelination in the PNS, manifested as an increase in the myelin sheath thickness during the remyelination of regenerating ax-
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ons after cryolesion of the sciatic nerve (Koenig et al., 1995). Moreover, it has been recently demonstrated that PROG and its metabolites DHP and THP are able to reduce ageing-associated morphological abnormalities of myelin and ageing-associated myelin fibre loss in the sciatic nerve (Azcoitia et al., 2003). These results are consistent with in vitro studies showing that progesterone can enhance the rate of myelin formation by Schwann cells co-cultured with dorsal root ganglia ( Koenig et al., 1995; Chan et al., 1998). The mechanisms of this effect are unclear, but may be due to a progesterone-mediated increase in the expression of peripheral myelin specific genes, such as P0, MBP and PMP22 (Desarnaud et al., 1998), and transcription factors involved in PNS myelination such as Krox-20 (Guennoun et al., 2001). In the context of CNS myelination, progesterone regulates the expression of MBP and 2 -3 -cyclic nucleotide-3-phosphodiesterase (CNPase) genes by mature oligodendrocytes (Verdi and Campagnoni, 1990; Jung-Testas et al., 1996), which also express the receptor for this steroid (Jung-Testas and Baulieu, 1998). It is possible therefore that progesterone might also have pro-remyelination effects following CNS demyelination. However, since progesterone can also have anti-inflammatory actions (Ganter et al., 1992; Drew and Chavis, 2000), the consequence of its administration following demyelination might be to impair remyelination. We have addressed this issue by establishing whether the effects of progesterone are on balance favourable, and therefore of potential therapeutic value, or unfavourable to CNS remyelination using a toxin-model of demyelination (Woodruff and Franklin, 1999a). We have used young adult animals, in which remyelination occurs very efficiently, to verify that there are no adverse effects of progesterone on remyelination, and used older animals, in which remyelination occurs slowly, to ascertain whether progesterone has a pro-remyelination effect (Ibanez et al., in press). 5.1. Progesterone does not impair efficient remyelination in young animals Using male rats, which have constant low levels of endogenous progesterone that contrast with the cyclic variations that occur in females, we examined the effects of sustained progesterone delivery, from slow release pellets positioned subcutaneously, on the remyelination that follows toxin-induced demyelination of brain stem white matter (Ibanez et al., in press). In both control and progesterone-treatment groups, extensive remyelination had occurred at 3 weeks after lesion induction, and the lesions were almost fully remyelinated by 6 weeks. The remyelination was by both oligodendrocytes and Schwann cells, with a distribution similar to that previously observed (Shields et al., 1999; Woodruff and Franklin, 1999a). An estimate was then made for each section of the proportion of axons that remained demyelinated or were remyelinated by either oligodendrocytes or Schwann cells. When analysed, there
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were no statistical differences in the extent of oligodendrocyte or Schwann cell remyelination between the control and the treated animals at either survival time, indicating that elevated levels of progesterone do not have a direct inhibitory effect on CNS remyelination. 5.2. Systemic progesterone administration results in a small but significant increase in oligodendrocyte remyelination following demyelination in old adult male rats We next addressed whether progesterone might have a pro-remyelination effect following CNS demyelination using old animals in which remyelination proceeds slowly (Ibanez et al., in press). The extent of remyelination was considerably less in the older animals at both 3 and 5 weeks after lesion induction compared to that seen in the young animals at either 3 or 6 weeks survival. Moreover, a notable feature of the lesions in older animals was the persistence of myelin debris that had not been cleared by phagocytes and remained within the extracellular space. Estimation of remyelination at 5 weeks revealed that although the oligodendrocyte remyelination in the treatment group was not extensive at approximately 20% of the axons within the lesion, it nevertheless constituted a doubling of the proportion of oligodendrocyte remyelinated axons when compared to the control group. When sections were ranked blindly according to the degree of demyelination, oligodendrocyte remyelination and Schwann cell remyelination, we found that progesterone caused a small but significant decrease in the number of axons remaining demyelinated that was attributable to a corresponding small but significant increase in oligodendrocyte remyelination (Fig. 3). The approximate doubling in the amount of oligodendrocyte remyelination in the progesterone-treated group can be viewed as a considerable improvement pointing to a therapeutic potential for progesterone. On the other hand, the lesion at 5 weeks only had 20% remyelination in the treatment group in old animals, and that compares poorly with the near complete remyelination that occurs in only 3 weeks in the young animals. This would suggest that there remains considerable scope to make the remyelination process in old animals occur more efficiently. Nevertheless, together with other pro-remyelination factors, progesterone may form part of a remyelination-enhancing protocol. We therefore regard our results as encouraging given that the old animal remyelination model has proven remarkably refractory to other potentially pro-remyelinating strategies such as the direct delivery of the remyelination-associated growth factor IGF-I (O’Leary et al., 2002).
6. Summary The process of myelin sheath formation that occurs during developmental myelination and when myelin needs to be replaced following injury in adulthood is a complex biolog-
ical process mediated by a diversity of signalling pathways. It is clear that the stability of myelin once formed and the efficiency with which it can be repaired are both affected by the ageing process. Clearly, a more detailed understanding of the cellular and molecular basis of ageing as it affects CNS myelin is required before significant steps can be made towards their amelioration. The studies described in this article indicate that steroid hormones and their derivatives have beneficial effects on the ageing decline in both the maintenance of myelin integrity and in partially reversing the decline in the efficiency of myelin repair. The exciting tasks for the next years will be to establish the extent to which this potential can be fully harnessed and provide the basis for novel therapeutic approaches to white matter disease.
Acknowledgements The authors’ work described in this article has been supported by grants from Research into Ageing and the Commission of the European Communities, specific RTD programme “Quality of Life and Management of Living Resources”, QLK6-CT-2000-00179. The support given by the Myelin Project to the laboratories of MS and RJMF is also gratefully acknowledged. References Armstrong, R.C., Kim, J.G., Hudson, L.D., 1995. Expression of myelin transcription factor I (MyTI), a “zinc-finger” DNA-binding protein, in developing oligodendrocytes. Glia 14, 303–321. Awatramani, R., Scherer, S., Grinspan, J., Collarini, E., Skoff, R., OqHagan, D., Garbern, J., Kamholz, J., 1997. Evidence that the homeodomain protein Gtx is involved in the regulation of oligodendrocyte myelination. J. Neurosci. 17, 6657–6668. Azcoitia, I., Leonelli, E., Magnaghi, V., Veiga, S., Garcia-Segura, L.M., Melcangi, R.C., 2003. Progesterone and its derivatives dihydroprogesterone and teatrahydroprogesterone reduce myelin fiber morphological abnormalities and myelin fiber loss in the sciatic nerve of aged rats. Neurobiol. Ageing 24, 853–860. Carroll, W.M., Jennings, A.R., Ironside, L.J., 1998. Identification of the adult resting progenitor cell by autoradiographic tracking of oligodendrocyte precursors in experimental CNS demyelination. Brain 121, 293–302. Chan, J.R., Phillips, L.J., Glaser, M., 1998. Glucocorticoids and progestins signal the initiation and enhance the rate of myelin formation. Proc. Natl. Acad. Sci. U.S.A. 95, 10459–10464. Cuzner, M.L., Norton, W.T., 1996. Biochemistry of demyelination. Brain Pathol. 6, 231–242. Desarnaud, F., Do Thi, A.N., Brown, A.M., Lemke, G., Suter, U., Baulieu, E.E., Schumacher, M., 1998. Progesterone stimulates the activity of the promoters of peripheral myelin protein-22 and protein zero genes in Schwann cells. J. Neurochem. 71, 1765–1768. Diemel, L.T., Copelman, C.A., Cuzner, M.L., 1998. Macrophages in CNS remyelination: friend or foe? Neurochem. Res. 23, 341–347. Dijkstra, C.D., De Groot, C.J., Huitinga, I., 1992. The role of macrophages in demyelination. J. Neuroimmunol. 40, 183–188. Drew, P.D., Chavis, J.A., 2000. Female sex steroids: effects upon microglial cell activation. J. Neuroimmunol. 111, 77–85. Franklin, R.J.M., 2002. Why does remyelination fail in multiple sclerosis? Nat. Rev. Neurosci. 3, 705–714.
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Melcangi, R.C., Celotti, F., Martini, L., 1994. Progesterone 5-alpha-reduction in neuronal and in different types of glial cell cultures: type 1 and 2 astrocytes and oligodendrocytes. Brain Res. 639, 202–206. Melcangi, R.C., Magnaghi, V., Cavarretta, I., Martini, L., Piva, F., 1998. Age-induced decrease of glycoprotein Po and myelin basic protein gene expression in the rat sciatic nerve. Repair by steroid derivatives. Neuroscience 85, 569–578. Merrill, J.E., Benveniste, E.N., 1996. Cytokines in inflammatory brain lesions: helpful and harmful. Trends Neurosci. 19, 331–338. Merrill, J.E., Scolding, N.J., 1999. Mechanisms of damage to myelin and oligodendrocytes and their relevance to disease. Neuropathol. Appl. Neurobiol. 25, 435–458. O’Leary, M.T., Hinks, G.L., Charlton, H.M., Franklin, R.J.M., 2002. Increasing local levels of IGF-I mRNA expression using adenoviral vectors does not alter oligodendrocyte remyelination in the CNS of aged rats. Mol. Cell Neurosci. 19, 32–42. Ousman, S.S., David, S., 2000. Lysophosphatidylcholine induces rapid recruitment and activation of macrophages in the adult mouse spinal cord. Glia 30, 92–104. Peters, A., 1996. Age-related changes in oligodendrocytes in monkey cerebral cortex. J. Comp. Neurol. 371, 153–163. Peters, A., Sethares, C., 2002. Ageing and the myelinated fibers in prefrontal cortex and corpus callosum of the monkey. J. Comp. Neurol. 442, 277–291. Peters, A., Moss, M.B., Sethares, C., 2000. Effects of ageing on myelinated nerve fibers in monkey primary visual cortex. J. Comp. Neurol. 419, 364–376. Peters, A., Sethares, C., Killiany, R.J., 2001. Effects of age on the thickness of myelin sheaths in monkey primary visual cortex. J. Comp. Neurol. 435, 241–248. Pringle, N.P., Mudhar, H.S., Collarini, E.J., Richardson, W.D., 1992. PDGF receptors in the rat CNS: during late neurogenesis, PDGF alpha-receptor expression appears to be restricted to glial cells of the oligodendrocyte lineage. Development 115, 535–551. Redwine, J.M., Armstrong, R.C., 1998. In vivo proliferation of oligodendrocyte progenitors expressing PDGF-alphaR during early remyelination. J. Neurobiol. 37, 413–428. Reynolds, R., Cenci di Bello, I., Dawson, M., Levine, J., 2001. The response of adult oligodendrocyte progenitors to demyelination in EAE. Prog. Brain Res. 132, 165–174. Shields, S.A., Gilson, J.M., Blakemore, W.F., Franklin, R.J.M., 1999. Remyelination occurs as extensively but more slowly in old rats compared to young rats following gliotoxin-induced CNS demyelination. Glia 28, 77–83. Sim, F.J., Hinks, G.L., Franklin, R.J.M., 2000. The re-expression of the homeodomain transcription factor Gtx during remyelination of experimentally-induced demyelinating lesions in young and old rat brain. Neuroscience 100, 131–139. Sim, F.J., Zhao, C., Penderis, J., Franklin, R.J.M., 2002. The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation. J. Neurosci. 22, 2451–2459. Tang, Y., Nyengaard, J.R., Pakkenberg, B., Gundersen, H.J., 1997. Age-induced white matter changes in the human brain: a stereological investigation. Neurobiol. Ageing 18, 609–615. van Rooijen, N., 1989. The liposome-mediated macrophage ‘suicide’ technique. J. Immunol. Methods 124, 1–6. Verdi, J.M., Campagnoni, A.T., 1990. Translational regulation by steroids. Identification of a steroid modulatory element in the 5 -untranslated region of the myelin basic protein messenger RNA. J. Biol. Chem. 265, 20314–20320. Woodruff, R.H., Franklin, R.J.M., 1998. The expression of myelin basic protein exon 1 and exon 2 during myelination of the neonatal rat spinal cord—an in situ hybridisation study. J. Neurocytol. 27, 683– 693.
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Woodruff, R.H., Franklin, R.J.M., 1999a. Demyelination remyelination of the caudal cerebellar peduncle of adult following stereotaxic injections of lysolecithin, ethidium bromide complement/anti-galactocerebroside—a comparative study. Glia 216–228.
and rats and 25,
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Steroids and the reversal of age-associated changes in myelination and remyelination C. Ibanez b , S.A. Shields a , M. El-Etr b , E. Leonelli c , V. Magnaghi c , W.-W. Li a , F.J. Sim a , E.-E. Baulieu b , R.C. Melcangi c , M. Schumacher b , R.J.M. Franklin a,∗ a
Department of Clinical Veterinary Medicine and Cambridge Centre for Brain Repair, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK b INSERM U488, 80 rue du Général Leclerc, 94276 Le Kremlin Bicˆ etre-Cedex, France c Department of Endocrinology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milano, Italy Received 27 May 2003; accepted 9 September 2003
Abstract The myelin sheaths that surround all but the smallest diameter axons within the mammalian central nervous system (CNS) must maintain their structural integrity for many years. Like many tissues, however, this function is prone to the effects of ageing, and various structural anomalies become apparent in the aged CNS. Similarly, the regenerative process by which myelin sheaths, lost as a consequence of exposure to a demyelinating insult, are restored (remyelination) is also affected by age. As animals grow older, the efficiency of remyelination progressively declines. In this article, we review both phenomena and describe how both can be partially reversed by steroid hormones and their derivatives. © 2003 Elsevier Ltd. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ageing and CNS myelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CNS remyelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ageing and CNS remyelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The age-related decline in remyelination efficiency occurs due to an impairment of oligodendrocyte progenitor recruitment and differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Inflammation and the age-related decline in remyelination efficiency . . . . . . . . . . . . . . . . . . . 5. The effects of progesterone on CNS remyelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Progesterone does not impair efficient remyelination in young animals . . . . . . . . . . . . . . . . . 5.2. Systemic progesterone administration results in a small but significant increase in oligodendrocyte remyelination following demyelination in old adult male rats . . . . . . . . . . . . . . . . . 6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. 2. 3. 4.
Abbreviations: CNPase, 2 -3 -cyclic nucleotide-3-phosphodiesterase; CNS, central nervous system; DHP, dihydro-progesterone; IGF-I, insulin-like growth factor I; MBP, myelin-basic protein; MRI, magnetic resonance imaging; OPC, oligodendrocyte progenitor; PMP-22, peripheral myelin protein 22; PNS, peripheral nervous system; PROG, progesterone; PLP, proteolipid protein; THP, tetrahydro-progesterone ∗ Corresponding author. Tel.: +44-1223-337642; fax: +44-1223-337610. E-mail address: [email protected] (R.J.M. Franklin). 0301-0082/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.pneurobio.2003.09.002
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1. Introduction Myelin sheaths surround all but the smallest diameter axons within the mammalian central nervous system (CNS). Their function is to enable axons to conduct action potentials by saltatory conduction, a more rapid and efficient means of conduction than that which occurs in their absence. In the CNS, myelin is produced and maintained by oligoden-
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drocytes, a highly specialised cell type that can support between 1 and 40 separate myelin sheaths depending on the size of axon with which they associate. The vexed question of whether there is a turnover of oligodendrocytes has not been fully resolved and continues to provoke heated debate amongst those who believe that there is no turnover and those who cite evidence supporting their view that at least some replacement of oligodendrocytes and their myelin sheaths must occur as a normal physiological process. It is not the intention in this article to engage in this discussion but instead to discuss our own recent work on how myelin sheaths and the intrinsic ability to replace myelin sheaths lost as a consequence of pathological processes (remyelination) are affected by ageing, and how these effects can be modulated by steroid hormones.
2. Ageing and CNS myelin In common with all other tissues, myelin is subject to degenerative changes attributable to ageing. These have not been extensively studied but informative morphological analysis of white matter in the brain of aged old world monkeys has revealed a number of distinct changes that occur in ageing CNS myelin similar to those known to occur in ageing peripheral myelin. These include a significantly high incidence of: (1) splitting of the major dense line (the line formed by the apposition of the internal membrane surface during the extrusion of cytoplasm that occurs during the formation of the compacted myelin sheath) to create swellings containing electron dense oligodendrocyte cytoplasm; (2) splitting of the intraperiod line (the line formed by the apposition of the external membrane surfaces during myelination) to create fluid filled swellings; (3) formation of redundant myelin so that the sheath is too large for the enclosed axon; and (4) thickened sheaths that are almost completely split (Peters, 1996; Peters et al., 2000, 2001; Peters and Sethares, 2002). The age-associated changes in myelin sheaths may in part account for the cognitive decline that occurs in monkeys over a similar time frame (Peters and Sethares, 2002). However, it is not certain how closely changes in myelin sheath structure correlate with MRI and histologically detectable loss of white matter in ageing human brain (Guttmann et al., 1998; Tang et al., 1997). What are the molecular changes occurring with ageing that may account for the disturbances in the normal morphology of myelin sheaths with ageing? Recent observations on the effects of ageing on the constitutive levels of expression of key genes encoding for major protein constituents of the myelin sheath may provide clues. The maintenance of myelin sheath integrity involves the sustained expression of a range of genes specifically associated with the myelin sheath, foremost amongst which are the major myelin proteins myelin-basic protein (MBP) and proteolipid protein (PLP). In a recent study, we compared the constitutive levels of expression of mRNAs of both these myelin genes in the
Fig. 1. Relative optical densities of constitutive mRNA expression of the myelin protein genes, MBP and PLP, and the transcription factor Gtx (±S.E.M.) in the brain stem white matter of young and old adult rats (see Sim et al., 2000).
brain stem white matter of young adult rats (8–10 weeks) and older adult rats (>12 months) by quantitative in situ hybridisation based on densitometry of autoradiographs obtained following use of 35 S-labelled oligonucleotide probes (Sim et al., 2000). Although the distribution of expression of both MBP and PLP mRNA was the same between the two groups, there was a significant decrease in the constitutive levels of expression of both genes (Fig. 1). This difference was mirrored by lower levels of expression of Gtx, a homeodomain transcription factor associated with myelinating oligodendrocytes and potentially involved in the regulation of both MBP and PLP (Awatramani et al., 1997). The age-associated decline in expression of myelin protein mRNAs in the CNS resembles similar changes in myelin protein mRNA expression occurring in the peripheral nervous system (PNS) (Melcangi et al., 1998). In the PNS, changes in P0 mRNA expression can be reversed by the administration of various steroid derivatives. We have therefore addressed whether the same may be true for the myelin protein genes in the CNS. Aged (22–24 month-old) male rats have been treated with progesterone (PROG), dihydro-progesterone (DHP), and tetrahydro-progesterone (THP) for 1 month and the mid-brains from both control (vehicle only) and treatment groups cryostat-sectioned and subjected to in situ hybridisation with 35 S-labelled oligonucleotide probes against MBP, PLP and Gtx (Fig. 2A). The most striking change observed was the dramatic reduction in constitutive levels of all genes in the aged controls compared to our earlier data on constitutive expression in young and middle-aged adult rats, indicating that constitutive levels of expression continue to decline with increasing age. In the treatment groups, it was observed that both DHP and THP induced a slight but significant increase in MBP mRNA expression compared to controls, implying that the age-associated decline in levels of expression of this key myelin-associated gene is partially reversible by steroid
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Fig. 2. (A) Autoradiographs of brain stem/cerebellar sections following in situ hybridisation with 35 S-labelled oligonucleotide probes for MBP, PLP and Gtx from young adult rat, aged adult rat and aged adult rats treated with PROG (P), DHP and THP. Rats received eight subcutaneous injections of 1 mg of steroid, suspended in 250 l of sesame oil. Age-matched control rats were injected with 250 l of sesame oil only. Injections were administered every 4 days, and the animals were killed 24 h after the last treatment; (B) Relative optical densities of mRNA expression of the myelin protein genes, MBP and PLP, and the transcription factor Gtx (±S.E.M.). Significant increases in the levels of expression compared to control aged rats were obtained for aged rats treated with DHP and THP.
hormone treatment (Fig. 2B). An effect of these metabolites of PROG is perhaps not surprising, since oligodendrocytes possess both the capability to convert PROG into DHP, by the enzyme 5␣-reductase, and subsequently into THP by the enzyme 3␣-hydroxysteroid dehydrogenase (Melcangi et al., 1994; Gago et al., 2001). Whether these changes in levels of MBP expression relate to morphological changes such as fewer myelin sheaths with split major dense lines remains to be addressed.
3. CNS remyelination Remyelination, the process in which new myelin sheaths are restored to demyelinated axons, is one of the few spontaneous regenerative processes that occurs within the adult mammalian CNS (Franklin and Hinks, 1999). However, although this helpful regenerative process can occur even in demyelinating diseases in adult humans such as multiple sclerosis (MS), it is not an invariable consequence of demyelination, and there are many occasions where it fails to occur or is incomplete (Franklin, 2002). Failure of
remyelination in MS becomes increasingly pronounced as the disease progresses, leading to impaired conduction and probably contributing to the axonal atrophy that typifies the secondary progressive phase of the disease. Attempts to promote remyelination have frequently focused on enhancing or reactivating the inherent process, whose failure accounts for the persistence of demyelination. This approach has proved difficult to achieve, largely because of shortcomings in our understanding of the mechanisms by which remyelination proceeds. Using a variety of experimental models of demyelination, the sequence of cellular events involved in oligodendrocyte-mediated remyelination of the CNS have been identified. Firstly, in response to demyelination, oligodendrocyte progenitor cells (OPCs) present within the adult CNS are recruited into the demyelinated area (Gensert and Goldman, 1997; Franklin et al., 1997; Reynolds et al., 2001; Carroll et al., 1998; Levine and Reynolds, 1999; Redwine and Armstrong, 1998; Sim et al., 2002). This initial recruitment process involves OPC proliferation, and, to a lesser extent migration. The second phase of remyelination involves the differentiation of the recruited OPCs into mature
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remyelinating oligodendrocytes (Ludwin and Sternberger, 1984; Jordan et al., 1990; Woodruff and Franklin, 1998, 1999b; Sim et al., 2000).
4. Ageing and CNS remyelination In common with many regenerative processes in the body, ageing has a detrimental effect on remyelination (Franklin et al., 2002). Using a toxin model of demyelination, we found that demyelinated foci were fully remyelinated at 4 weeks in young adult rats (<3 months of age) but not until 9 weeks in older adult rats (>9 months of age) (Shields et al., 1999), indicating that although remyelination proceeds to completion in the older age-group, it does so more slowly. This age-related slowing of remyelination is reflected in a slowing of the rate of reappearance of transcripts for the major myelin proteins MBP and PLP, and their putative regulatory factor Gtx, within lesions in old animals compared to young (Sim et al., 2000) (Fig. 3). Our recent unpublished data indicate that the rate of remyelination continues to slow with increasing age. The decline in the efficiency of remyelination simply by virtue of the animal having become older has clear implications for the likelihood of recovery in a disease such as MS from which a patient may suffer for several decades. If ageing effects are indeed contributing to the disease progression in MS, then devising means of reversing the ageing trend represents a legitimate approach to MS therapy. 4.1. The age-related decline in remyelination efficiency occurs due to an impairment of oligodendrocyte progenitor recruitment and differentiation Why does the rate of remyelination slow with ageing? On the basis of the model of remyelination presented earlier, there are at least three possibilities: (1) the numbers of resident OPCs decline with age, leading to a reduction in the size of the pool of cells available for recruitment; (2) the rate of OPC recruitment decreases; and (3) the rate of
differentiation of recruited OPCs into remyelinating oligodendrocytes decreases. The first of these possibilities has been addressed by comparing the absolute numbers of OPCs in the white matter of young and old adult rats. No significant difference occurs between the two, in spite of the difference in the rate of remyelination (Sim et al., 2002), suggesting that the impairment of remyelination is not due to a decline in OPC availability but rather to an impairment of the factors involved in their recruitment and differentiation, or a change in their intrinsic ability to respond to these factors. The rate of OPC recruitment following demyelination in young and old adults has been established by following the OPC response to chemically-induced demyelination using two OPC markers: PDGF-␣R mRNA is an established OPC marker (Pringle et al., 1992), and MyT-1 is a zinc finger transcription factor expressed by OPCs in tissue culture but less often used as an OPC marker in histology (Armstrong et al., 1995; Wrathall et al., 1998). In both cases, the patterns of expression indicated a slower rate of OPC recruitment in the older animals (Sim et al., 2002). Thus, one of the contributory factors in the slowing of the rate of remyelination that occurs with ageing is the impairment of OPC recruitment. Finally, comparing the interval between equivalent levels of expression of MyT-1 transcripts, indicative of the OPC response, and Gtx and MBP transcripts, indicative of the appearance of differentiated oligodendrocytes, revealed that there is also a decrease in the rate at which recruited OPCs differentiate into remyelinating oligodendrocytes (Sim et al., 2002). Thus, in addition to the delay in OPC recruitment that occurs in the old animals, there was also a decrease in the rate at which the recruited cells differentiated, and together both these phenomena result in a decrease in the rate of remyelination. 4.2. Inflammation and the age-related decline in remyelination efficiency A major feature of the cellular environment of remyelination is the presence of macrophages in the latter,
Fig. 3. Scatter plot showing the results of a ranking analysis in older adult rats 5 weeks after lesion induction. The highest rank is given to the animal exhibiting the lowest proportion of demyelinated axons or the highest proportion of either oligodendrocyte or Schwann cell remyelination. The plot illustrates the control (Cont.) and progesterone-treated (PROG) animals ranked according to the degree of demyelination (D) and remyelination by either oligodendrocytes (O) or Schwann cells (SC). Mann–Whitney U-test results with corresponding P-values are reported on the graph.
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recruited in response to the presence of myelin debris generated by demyelination (Ousman and David, 2000). The macrophage has been identified as a major source of remyelination-associated growth factors during remyelination (Hinks and Franklin, 1999; Diemel et al., 1998). In old animals, the macrophage response following toxin-induced demyelination is delayed compared with young animals, suggesting that this response may be a key determinant of efficient remyelination. Indeed, a clear association exists between widespread remyelination and a robust macrophage presence, or conversely poor remyelination and a sparse macrophage presence (Ludwin, 1980; Graca and Blakemore, 1986). If the macrophage response was important for remyelination then reducing it during the rapid remyelination following demyelination in young animals should result in an impairment of remyelination. This has been tested using clodronate liposomes to deplete circulating monocytes and thereby reduce the monocyte-derived contribution to the macrophages within the lesion (van Rooijen, 1989). This led to a significant reduction in the extent of oligodendrocyte remyelination of lysolecithin lesions (Kotter et al., 2001), providing yet further evidence of a pro-remyelination role for the macrophage (Hamilton and Rome, 1994; Loughlin et al., 1997). Prima facie, the beneficial role of macrophages contradicts the more widely recognised role for macrophages in myelin pathology as mediators of the demyelinating process (Dijkstra et al., 1992). The functional roles of macrophages are undoubtedly diverse, and their role in demyelinating disease is likely to be similarly so. There is little doubt that in immune-mediated demyelination, where macrophages are activated by lymphocytes, their predominant effects are harmful to the myelin sheath (Cuzner and Norton, 1996; Merrill and Benveniste, 1996; Merrill and Scolding, 1999). However, in toxin-induced demyelination, macrophages are recruited as a consequence of demyelination and are not likely to be involved in its mediation. The macrophages are therefore present as part of the regenerative process. How is the macrophage response beneficial? One of the obvious functions of macrophages in demyelinating disease is the removal of myelin debris. Whether this function is directly beneficial for remyelination has never been formally tested. Another way in which macrophages might be beneficial is by producing molecules crucially involved in the remyelination process. These may be growth factors that have direct effects on the cells of the oligodendrocyte lineage, or they may be molecules that activate other cells within the remyelination environment, such as astrocytes, to produce pro-remyelination factors.
5. The effects of progesterone on CNS remyelination Progesterone has beneficial effects on remyelination in the PNS, manifested as an increase in the myelin sheath thickness during the remyelination of regenerating ax-
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ons after cryolesion of the sciatic nerve (Koenig et al., 1995). Moreover, it has been recently demonstrated that PROG and its metabolites DHP and THP are able to reduce ageing-associated morphological abnormalities of myelin and ageing-associated myelin fibre loss in the sciatic nerve (Azcoitia et al., 2003). These results are consistent with in vitro studies showing that progesterone can enhance the rate of myelin formation by Schwann cells co-cultured with dorsal root ganglia ( Koenig et al., 1995; Chan et al., 1998). The mechanisms of this effect are unclear, but may be due to a progesterone-mediated increase in the expression of peripheral myelin specific genes, such as P0, MBP and PMP22 (Desarnaud et al., 1998), and transcription factors involved in PNS myelination such as Krox-20 (Guennoun et al., 2001). In the context of CNS myelination, progesterone regulates the expression of MBP and 2 -3 -cyclic nucleotide-3-phosphodiesterase (CNPase) genes by mature oligodendrocytes (Verdi and Campagnoni, 1990; Jung-Testas et al., 1996), which also express the receptor for this steroid (Jung-Testas and Baulieu, 1998). It is possible therefore that progesterone might also have pro-remyelination effects following CNS demyelination. However, since progesterone can also have anti-inflammatory actions (Ganter et al., 1992; Drew and Chavis, 2000), the consequence of its administration following demyelination might be to impair remyelination. We have addressed this issue by establishing whether the effects of progesterone are on balance favourable, and therefore of potential therapeutic value, or unfavourable to CNS remyelination using a toxin-model of demyelination (Woodruff and Franklin, 1999a). We have used young adult animals, in which remyelination occurs very efficiently, to verify that there are no adverse effects of progesterone on remyelination, and used older animals, in which remyelination occurs slowly, to ascertain whether progesterone has a pro-remyelination effect (Ibanez et al., in press). 5.1. Progesterone does not impair efficient remyelination in young animals Using male rats, which have constant low levels of endogenous progesterone that contrast with the cyclic variations that occur in females, we examined the effects of sustained progesterone delivery, from slow release pellets positioned subcutaneously, on the remyelination that follows toxin-induced demyelination of brain stem white matter (Ibanez et al., in press). In both control and progesterone-treatment groups, extensive remyelination had occurred at 3 weeks after lesion induction, and the lesions were almost fully remyelinated by 6 weeks. The remyelination was by both oligodendrocytes and Schwann cells, with a distribution similar to that previously observed (Shields et al., 1999; Woodruff and Franklin, 1999a). An estimate was then made for each section of the proportion of axons that remained demyelinated or were remyelinated by either oligodendrocytes or Schwann cells. When analysed, there
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were no statistical differences in the extent of oligodendrocyte or Schwann cell remyelination between the control and the treated animals at either survival time, indicating that elevated levels of progesterone do not have a direct inhibitory effect on CNS remyelination. 5.2. Systemic progesterone administration results in a small but significant increase in oligodendrocyte remyelination following demyelination in old adult male rats We next addressed whether progesterone might have a pro-remyelination effect following CNS demyelination using old animals in which remyelination proceeds slowly (Ibanez et al., in press). The extent of remyelination was considerably less in the older animals at both 3 and 5 weeks after lesion induction compared to that seen in the young animals at either 3 or 6 weeks survival. Moreover, a notable feature of the lesions in older animals was the persistence of myelin debris that had not been cleared by phagocytes and remained within the extracellular space. Estimation of remyelination at 5 weeks revealed that although the oligodendrocyte remyelination in the treatment group was not extensive at approximately 20% of the axons within the lesion, it nevertheless constituted a doubling of the proportion of oligodendrocyte remyelinated axons when compared to the control group. When sections were ranked blindly according to the degree of demyelination, oligodendrocyte remyelination and Schwann cell remyelination, we found that progesterone caused a small but significant decrease in the number of axons remaining demyelinated that was attributable to a corresponding small but significant increase in oligodendrocyte remyelination (Fig. 3). The approximate doubling in the amount of oligodendrocyte remyelination in the progesterone-treated group can be viewed as a considerable improvement pointing to a therapeutic potential for progesterone. On the other hand, the lesion at 5 weeks only had 20% remyelination in the treatment group in old animals, and that compares poorly with the near complete remyelination that occurs in only 3 weeks in the young animals. This would suggest that there remains considerable scope to make the remyelination process in old animals occur more efficiently. Nevertheless, together with other pro-remyelination factors, progesterone may form part of a remyelination-enhancing protocol. We therefore regard our results as encouraging given that the old animal remyelination model has proven remarkably refractory to other potentially pro-remyelinating strategies such as the direct delivery of the remyelination-associated growth factor IGF-I (O’Leary et al., 2002).
6. Summary The process of myelin sheath formation that occurs during developmental myelination and when myelin needs to be replaced following injury in adulthood is a complex biolog-
ical process mediated by a diversity of signalling pathways. It is clear that the stability of myelin once formed and the efficiency with which it can be repaired are both affected by the ageing process. Clearly, a more detailed understanding of the cellular and molecular basis of ageing as it affects CNS myelin is required before significant steps can be made towards their amelioration. The studies described in this article indicate that steroid hormones and their derivatives have beneficial effects on the ageing decline in both the maintenance of myelin integrity and in partially reversing the decline in the efficiency of myelin repair. The exciting tasks for the next years will be to establish the extent to which this potential can be fully harnessed and provide the basis for novel therapeutic approaches to white matter disease.
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