Re: article on demyelination

[Guest guest]

Thanks, yes your concern is valid. What the authors are trying to

show is that by transplanting myelin producing cells, one is able to

myelinate the axons of the retina that have never been myelinated

before. Thus suggesting that longstanding demyelinated axons in MS

can be repaired. Some excerpts from the article.

Article Text

A feature characteristic of chronic multiple sclerosis (MS) lesions

is the failure of remyelination (Prineas et al., [2002]). Loss of

myelin may be an important cause of axonal damage contributing to

the chronic progressive nature of this disease (Kornek et al.,

[2000]; Bjartmar et al., [2003]). Therapeutic strategies to enhance

remyelination by increasing the number of oligodendroglial cells

able to form myelin in the lesion, either by transplantation or by

stimulation of endogenous cells, may therefore be beneficial in MS.

These strategies obviously require that chronically demyelinated

axon segments remain competent for myelination. Axons are competent

for myelination during development, and also directly following

demyelination in both acute experimental models of white matter

injury (Bunge et al., [1961]; Lampert, [1965]; Blakemore, [1973];

Dal Canto and Barbano, [1984]), and in MS (Prineas and Connell,

[1979]). However, it is unknown whether axons that remain

unmyelinated after an episode of demyelination retain this

competence or lose it progressively with time. Any such loss would

clearly limit the efficacy of remyelination strategies based on

increasing cell numbers.

This important question can, in principle, be addressed by the use

of transplantation. If competence for myelination is lost

progressively, transplantation of myelin-forming cells into areas

containing axons without myelin sheaths will produce less

myelination the longer the time the axon has remained unmyelinated.

However, the lack of appropriate animal models that provide

consistent temporal and spatial patterns of chronic demyelination

means that this is a difficult prediction to test experimentally.

Transplantation of myelin-forming cells into the nerve fibre layer

(NFL) of the normal retina may represent an alternative approach to

this question. Axons in this layer remain unmyelinated throughout

life as a consequence of the inability of oligodendrocyte precursor

cells to migrate out of the optic nerve into the retina (Small et

al., [1987]; ffrench-Constant et al., [1988]; and Lund,

[1990]). Myelination of the NFL following transplantation of myelin-

forming cells has been described previously in developing rats and

mice (Huang et al., [1991]; Laeng et al., [1996]; Ader et al.,

[2000]). These studies indicate that the usually unmyelinated

portion of the retinal ganglion cell axon within the retina is

competent for myelination at the time when these axons are being

myelinated within the optic nerve. Thus, transplantation of cells

into the NFL of older adult animals represents an in vivo model in

which to ask whether those regions of the axons that remain

unmyelinated progressively lose the competence to support the

myelination process.

To establish the most effective method for delivery of cells into

the adult NFL, the retinae of adult female Fischer rats were

injected with fluorescent beads of 10m in diameter (Molecular

Probes, Eugene, OR). We compared a transvitreal approach, in which

the needle was inserted through the corneal-scleral junction and the

NFL approached through the vitreous, with a transscleral approach in

which the needle was inserted through the sclera and the NFL

approached through the outer retinal layers. Animals were sacrificed

3 days following transplantation, and cryostat sections were cut

from injected eyes. Nuclei of the retinal layers were visualised

using mounting media containing DAPI (Vector Laboratories,

Burlingame, CA), and sections examined under fluorescence

microscopy. Beads were more reliably delivered to the NFL with the

transvitreal approach than with the transscleral approach (Fig. 1).

In the latter approach, beads were often seen distant from the NFL

in deeper retinal layers or behind the sclera. All subsequent

transplants were therefore performed using the transvitreal

approach.

Figure 1. Retinal localisation of red fluorescent beads 3 days

after transvitreal (a, or transscleral (c,d) injection; 10-m

cryostat sections were mounted in DAPI-containing media, to

visualise nuclei of the retinal layers. With a transvitreal

injection (a), beads were more often located within the vicinity of

the nerve fibre layer (NFL) (indicated by the dashed line) (. A

transscleral approach © often resulted in beads located behind the

NFL (d). Scale bar = 100m.

[Normal View 29K | Magnified View 130K]

We next established that it is possible to myelinate the adult NFL

following transplantation of oligodendrocyte lineage cells. We

transplanted CG-4 cells, an oligodendrocyte progenitor cell (OPC)

line originally isolated from neonatal rat brains (Louis et al.,

[1992]). These cells have been shown previously to remyelinate axons

both in toxin models of demyelination (lin et al., [1995]) and

in spontaneously occurring myelin mutant rats (Tontsch et al.,

[1994]). For transplantation, cells grown and passaged in DMEM/Sato

medium supplemented with 30% B104 conditioned medium, as described

previously (Louis et al., [1992]) were resuspended at a density of

30,000 cells/l; 1 l of this suspension was injected into the NFL of

three young adult (8-week-old) Fischer rats. Potential rejection of

cells was averted by daily injections of Cyclosporin A (15 mg/kg,

subcutaneously) beginning on the day before transplantation. Animals

were sacrificed 4 weeks following transplantation by cardiac

injection of pentobarbital (Pentoject, Animalcare Ltd.), and eyes

were immersion-fixed in 4% glutaraldehyde for 24 h before being

processed into resin. Semi-thin sections were cut and analysed by

light microscopy, and selected sections were also cut for electron

microscopy. In two of the three transplanted animals, myelinated

axons could be identified within the NFL by light and electron

microscopy (Fig. 2a,.

Figure 2. Myelination of nerve fibre layer (NFL) axons following

transplantation of CG-4 cells (a, and primary oligodendrocyte

progenitor cells (OPCs) (c,d). Occasional myelinated axons were

observed following transplantation of CG-4 cells by both light

microscopy (a), and electron microscopy ( (transverse sections).

Extensive myelination was seen following transplantation of primary

oligodendrocyte precursor cells by light microscopy © (transverse

section) and by immunohistochemistry with an antibody against myelin

basic protein on flat-mounted retinae (d). Scale bars = 20m in a,c;

1 m in b; 200 m in d.

[Normal View 73K | Magnified View 296K]

We also examined myelination by primary (OPCs) transplanted into the

NFL of young adult Fischer rats. The goals of these experiments were

first, to show that primary cells as well as an immortalised cell

line will myelinate adult NFL axons, and second, to demonstrate the

feasibility of using cells derived from inbred rat strains so

obviating the need for posttransplant immunosuppression. OPCs were

obtained by mechanical dissociation from neonatal Fischer rat

cortical cultures, using techniques described previously (Milner and

ffrench-Constant, [1994]). For transplantation, OPCs were

resuspended in DMEM/Sato at 30,000 cells/l, and 1 l of this solution

injected. The animals were sacrificed 4 weeks (n = 5) and 8 weeks (n

= 5) following transplant, and the eyes were prepared for resin

sectioning as described above. Alternatively, in two of the 4-week

survival animals, retinae were dissected away from the sclera and

were flat-mounted; immunohistochemistry was performed with an

antibody against myelin basic protein. Extensive myelination of axon

fascicles in the NFL was seen both in resin sections (Fig. 2C) and

under fluorescent microscopy (Fig. 2D). Confocal microscopy of flat-

mounts revealed areas of myelin extending radially from the point of

injection close to the optic nerve head, following the path of axon

fascicles toward the retinal margin.

Having demonstrated the feasibility of cell transplantation to the

adult NFL as a model for studying myelination, we then asked whether

the extent of myelination varied dependent on the duration of time

the axons have been without a myelin sheath. In parallel with the

experiments above using OPCs transplanted into 2-month-old Fischer

rats, an identical population of primary OPCs was also transplanted

into the NFL of much older adult (12-18-month-old) Fischer rats. The

animals were sacrificed at either 4 weeks (n = 3) or 8 weeks (n = 4)

posttransplantation, and the eyes were processed into resin as

described above. Transplanted retinae were sectioned at 100-m

intervals throughout the myelinated area in both the young and old

age groups. Myelination of the NFL was observed in both young and

old animals at 4 and 8 weeks posttransplantation. In both age

groups, more myelin was seen at 8 weeks than at 4 weeks following

transplant. In all animals, the myelin formed a distinct patch,

which was maximal at the optic nerve head, where the NFL is thickest

and followed the fascicles radially toward the periphery. Near the

point of injection, almost every axon within a fascicle was

myelinated, although farther from the optic nerve head, the ratio of

myelinated to unmyelinated axons decreased.

The results were quantified using an Microcomputer Imaging Device

(Imaging Research Inc.) image analysis system, with a point grid

overlaid on the myelin containing sections, and the numbers of

points falling on myelinated axons counted. For each retina, a

volume of myelination was estimated using the Cavalieri method

( and , [1998]), with the estimated volume of myelin being

equal to the number of points counted × area associated with each

point × distance between sections. There was no significant

difference in the mean volume of myelination between the different

age groups at either 4 or 8 weeks posttransplantation (Fig. 3).

Figure 3. Mean volume of myelination following transplantation of

primary oligodendrocyte precursor cells (OPCs) into young adult (2-

month-old) and old adult (12-18-month-old) female Fischer rats at 4-

and 8-week survival times. In both age groups, more extensive

myelination occurred at 8 weeks survival. Statistical analysis of

the means was performed with the Mann-Whitney test. There was no

significant difference in the mean volume of myelination between the

two age groups at either survival time. Young adult: 4 week, n = 3,

8 week, n = 5; old adult: 4 week, n = 3; 8 week, n = 4.

[Normal View 5K | Magnified View 12K]

In this study, we present a novel in vivo model for the study of

myelination biology with which it is possible to study the

myelination of long-term unmyelinated axons. In an extension to the

work of others in neonatal animals (Huang et al., [1991]; Laeng et

al., [1996]; Ader et al., [2000]), we show that a transplant can be

delivered effectively to the NFL of the adult rat retina, and also

that widespread myelination of the adult NFL is observed following

transplantation of myelinating cells. These results indicate that,

despite remaining unmyelinated into adulthood, the axons of the

adult rat NFL remain capable of supporting myelination. Our results

also demonstrate no difference between mean myelin volume in the NFL

of young or old adult animals.

This indicates that the competence of a retinal ganglion cell axon

to be myelinated is independent of its age and shows that cell

surface or secreted axonal factors that promote myelination remain

throughout life, while factors that inhibit myelination are not

expressed with time. In support of this, we have found that

polysialylated-neuronal cell adhesion molecule (PSA-NCAM) and

jagged, two axonal cell surface molecules shown to inhibit

myelination (Oumesmar et al., [1995]; et al., [2000];

Givogri et al., [2002]) and implicated in the persistent

demyelination within MS lesions ( et al., [2002]; et

al., [2002]) are not expressed on the surface of adult NFL axons

(unpublished observations). It has been reported that chronic MS

lesions contain pre-myelinating oligodendrocytes (Chang et al.,

[2002]); our results suggest that the failure of these cells to

progress to a myelinating phenotype is not a consequence of the

length of time the axons have remained unmyelinated. Although the

extent to which the axolemma of an unmyelinated axon differs from

that of a chronically demyelinated axon is an important issue

requiring further investigation, we conclude from this work that

there are no changes in portions of axons remaining unmyelinated for

many months that would prevent effective remyelination. This

suggests that chronically demyelinated regions of axons such as

those in seen in MS are likely to remain competent to be

remyelinated, and that therapeutic strategies designed to promote

remyelination need not be hindered by an inevitable decrease in

chronically demyelinated axons to support myelination.

> Dear Yash,

> Are rat retina axons unmyelinated from birth? Is that their

natural State?

> If it is ,there could be a huge difference between their

behaviour and

> unmyelinated nerves of

> MS person that were naturally myelinated.Even though they set

off myelin

> growth in this experiment.

> Does this make sense to you?

> Alarm bells went off in my head to do with the experimental design

around

> this question.

> Regards

> Louise (OZ) B.Sc (Neurostuff , Genetics)