Remyelination after cuprizone-induced demyelination in the rat is stimulated by apotransferrin

doi:10.1016/j.expneurol.2005.12.027

Experimental Neurology

Volume 198, Issue 2, April 2006, Pages 519-529

https://doi.org/10.1016/j.expneurol.2005.12.027 Get rights and content

Abstract

Twenty-one-day-old Wistar rats were fed a diet containing 0.6% cuprizone for 2 weeks. Studies carried out after withdrawal of cuprizone showed histological evidences of marked demyelination in the corpus callosum. Biochemical studies of isolated myelin showed a marked decrease in myelin proteins, phospholipids, and galactocerebrosides as well as a marked decrease in myelin yield. Treatment of these animals with a single intracranial injection of 350 ng of apotransferrin at the time of withdrawal of cuprizone induced a marked increase in myelin deposition resulting in a significantly improved remyelination, evaluated by histological, immunocytochemical, and biochemical parameters, in comparison to what was observed in spontaneous recovery. Immunocytochemical studies of cryotome sections to analyze developmental parameters of the oligodendroglial cell population at the time of termination of cuprizone and at different times thereafter showed that in the untreated animals, there was a marked increase in the number of NG2-BrdU-positive precursor cells together with a marked decrease in MBP expression at the peak of cuprizone-induced demyelination. As expected, the amount of precursor cells decreased markedly during spontaneous remyelination and was accompanied by an increase in MBP reactivity. In the apotransferrin-treated animals, these phenomena occurred much faster, and remyelination was much more efficient than in the untreated controls. The results of this study suggest that apotransferrin is a very active promyelinating agent which could be important for the treatment of certain demyelinating conditions.

Introduction

In previous studies, we have demonstrated that a single intracranial injection (ICI) of apotransferrin (aTf) in 3-day-old rats produces an increase in various myelin constituents and in the expression of their mRNAs (Escobar Cabrera et al., 1994, Escobar Cabrera et al., 1997, Escobar Cabrera et al., 2000). Using both light and electron microscopy, we also showed that the ICI of aTf in 3-day-old rats results in a significantly increased deposition of myelin, especially in areas close to the lateral ventricles (Marta et al., 2003). The in vivo effects of aTf were reproduced in primary cultures of oligodendroglial cells (OLGcs) (Paez et al., 2002) and in cultures of two OLGc lines, N19 and N20.1 (Paez et al., 2004). Some of the results of our studies have been confirmed by works from another research group (Espinosa de los Monteros et al., 1989, Espinosa de los Monteros et al., 1999) who have also shown that Tf regulates the transcription of the MBP gene, and that its action synergizes with IGF-1 to enhance myelinogenesis in the md rats (Espinosa-Jeffrey et al., 2002). Transgenic mice overexpressing the human Tf gene (Saleh et al., 2003) showed an increase in their myelin components very similar to that found in rats ICI with aTf.

The addition of cuprizone (bis-cyclohexanone oxalydihydrazone, CPZ) to the diet of young adult mice produces a massive demyelination in different areas of the CNS and particularly in the corpus callosum (CC) (Suzuki and Kikkawa, 1969, Ludwin, 1978, Matsushima and Morell, 2001). Demyelination is closely followed by recruitment of microglia and by phagoctytosis of the disrupted myelin membrane. If the administration of CPZ is terminated, an almost complete remyelination takes place in a matter of weeks (Blakemore, 1974, Ludwin, 1978, Ludwin, 1994, Armstrong et al., 2002).

In view of the findings of our laboratories related to the pro-myelinating effects of aTf that we mentioned above, our interest was to find out if treatment with this glycoprotein could be used to improve recovery and remyelination in animals suffering from CPZ-induced demyelination. In the present study, we show for the first time, and contrary to what has been reported up to now, that CPZ can be effectively used to induce demyelination in Wistar rats. We also show that treatment of these animals with a single ICI of aTf, at the time of withdrawal of CPZ, induces a marked increase in myelin deposition resulting in a significantly improved remyelination in comparison to that observed in spontaneous recovery. The results obtained in this study suggest that aTf should be seriously considered as a remyelinating factor of possible application in the clinical treatment of certain demyelinating conditions.

Section snippets

Materials

Cuprizone (CPZ), rat apotransferrin (aTf), bovine serum albumin, paraformaldehyde, Triton X-100, 5′ bromo-2′ deoxyuridine (BrdU), and anti-BrdU monoclonal antibody were purchased from Sigma Chemical Co. (St. Louis, MO). The recently developed fluorescent BrainStain Kit (Molecular Probes, Cat #34650) was used for the selective staining of myelin, neurons, and nuclei. Anti-MBP antibody was a generous gift from Dr. A.T. Campagnoni (UCLA); anti-NG2 and anti-CD11b antibodies were from Chemicon Int.

Results

In preliminary studies, we analyzed the effects of CPZ fed to 21-day-old rats at different doses and during 1, 2, and 3 weeks. We found that CPZ administered in the diet during 2 weeks at a dose of 0.6% was effective in producing clear biochemical and histological evidences of demyelination in young rats. This protocol was adopted as our standard procedure. The results of these studies are presented in Table 1 which shows that feeding of CPZ up to 35 days of age (CPZ₃₅ group) produces a

Discussion

Cuprizone intoxication has been described as a useful model to study demyelination and remyelination phenomena (Ludwin, 1978, Matsushima and Morell, 2001). Demyelination and damage to OLGcs without damage to other cell types in the CNS have been shown to be the consequence of CPZ feeding to mice (Blakemore, 1973, Cammer and Zhang, 1993, Komoly et al., 1987). If the administration of CPZ is terminated, an almost complete and spontaneous remyelination takes place in a matter of weeks (Blakemore,

Acknowledgment

This work has been supported by Grant 092 from the University of Buenos Aires.

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Remyelination after cuprizone-induced demyelination in the rat is stimulated by apotransferrin

Abstract

Introduction

Section snippets

Materials

Results

Discussion

Acknowledgment

J. Neurol. Sci.

J. Neurol. Sci.

Exp. Mol. Pathol.

J. Neurosci. Methods

J. Biol. Chem.

J. Neurol. Sci.

J. Neurol. Sci.

Am. J. Pathol.

Absence of fibroblast growth factor 2 promotes oligodendroglial repopulation of demyelinated white matter

J. Neurosci.

TNFα promotes proliferation of oligodendrocyte progenitors and remyelination

Nat. Neurosci.

Remyelination of the superior cerebellar peduncle in old mice following demyelination induced by cuprizone

J. Neurol. Sci.

Atypical localization of the oligodendrocytic isoform (PI) of glutathion-S-transferase in astrocytes during cuprizone intoxication

J. Neurosci. Res.

Microdetermination of phosphorus

Anal. Chem.

Single intracerebral injection of apotransferrin in young rats induces increased myelination

Dev. Neurosci.

Single intracranial injection of apotransferrin in young rats increases the expression of specific myelin protein mRNA

J. Neurosci. Res.

The cytoskeletal components of the myelin fraction are affected by a single intracranial injection of apotransferrin in young rats

Neurochem. Res.

Does transferrin have a special role in the nervous system

J. Neurosci. Res.