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

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.

Oligodendroglial cell differentiation in rat brain is accelerated by the intracranial injection of apotransferrin.

In the present paper we first studied the brain distribution and the time and dose dependent effects of apotransferrin, after its intracranial injection into young rats and at different post-natal ages. Its action upon the transferrin receptor (TfR) and upon the expression of brain transferrin, as well as its effect on the proliferation and differentiation of oligodendroglial cells (OLGc) was one of the main objectives of our investigation. Total DNA and BrdU labeling, as an index of cellularity and proliferation, respectively, were the same in the control and experimental groups of rats. A significant increase in the MBP+ and CA II+ OLGc, and a decrease in the more immature (A2B5+) OLGc were found in the aTf injected rats. At 10 and 17 days of age, Tf-mRNA decreased to around 20% of the amount present in control animals. The TfR-mRNA in the animals receiving a single dose of aTf at 3 days of age showed an increase in its expression at 10 and 17 days of age, coincident with a higher immunoreactivity of the TfR itself of neurons, choroid plexus and brain capillaries in different brain areas. Although TfR+ OLGc were present up to 7 days of age in controls and in the Tf injected rats, no positive cells were observed at 17 days of age, even in the aTf injected rats. Our results give support to the hypothesis that aTf is an important factor necessary for the maturation of the OLGc, and that the effects that it produces in the OLGc-myelin unit after its intracranial injection in young rats are not due to an increase in proliferation, but to an accelerated differentiation of Tf-sensitive OLGc.

Myelin membranes isolated from rats intracranially injected with apotransferrin are more susceptible to in vitro peroxidation.

Purified myelin obtained from 17 day old rats intracranially injected with aTf at 3 days of age was submitted to in vitro peroxidation using Fe + ascorbic acid (FeA) or Cu + H2O2 (CuH), to investigate the susceptibility of this membrane to in vitro metal catalyzed peroxidation. There was an increase in thiobarbituric acid-reactive-substances (TBARS) (60%) and in protein-associated carbonyls (PAC) (20%) in the myelin from aTf injected rats in comparison to myelin from controls, indicating a higher susceptibility to peroxidation. Desferoxamine (DFX) injected simultaneously with aTf did not change the response of myelin to aTf. CNS myelin is highly vulnerable to oxidative stress, and its susceptibility to peroxidation increases in myelin isolated from aTf injected rats. This increased liability to peroxidation as well as the previously reported aTf-dependent increment in certain myelin proteins and lipids and in the expression of specific myelin mRNAS, does not appear to be due to an increased amount of iron bound to the injected aTf. The changes in composition that we have previously reported could result in an abnormal myelin, allowing the peroxidative system to act upon the membrane more easily than in normal circumstances.

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

Transferrin (Tf) is a possible regulator of oligodendrocyte development in vitro (Espinosa de los Monteros et al., 1989). At least two different mechanisms may account for the effects of Tf on myelin synthesis. It may act as a trophic factor and enhance the formation of new myelin sheaths. Tf may also induce the synthesis of myelin proteins in the central nervous system. We recently demonstrated that a single intracranial injection of apotransferrin (aTf) in young rats induces an increased myelination (Escobar Cabrera et al., 1994). In the present study, we investigated the in vivo effect of aTf on the expression of mRNAs of specific myelin genes. Three-day-old rats were injected intracranially with aTf and killed at different ages after injection. Total brain RNA was isolated, and the expression of different mRNAs was analyzed by Northern blot. The amount of mRNAs of myelin basic protein and of 2'-3' cyclic nucleotide 3'-phosphohydrolase were markedly increased in the experimental animals, whereas myelin proteolipid protein mRNA did not show differences relative to controls. These results indicate that in the animals treated with aTf, there is a differential effect on the expression of certain specific myelin protein genes. They also suggest that aTf might exert its action at the posttranscriptional level and/or by direct transcriptional regulation of the genes.

Single intracerebral injection of apotransferrin in young rats induces increased myelination.

Three-day-old rats were injected intracranially with 210 or 350 ng of apotransferrin (aTf) to study the possible neurotrophic effects of this iron transport protein. Treated animals and appropriate controls were injected with saline, denatured aTf or ovalbumin. Myelin was isolated from the brains and used to study its chemical composition and the protein electrophoretic pattern. Total myelin galactolipids and especially total phospholipids were significantly increased with reference to controls at both ages studied. A slight increase in total cholesterol was also observed. Total myelin proteins were markedly increased both at 10 and 17 days in comparison to controls. Chloroform:methanol-soluble proteins (proteolipids) were only slightly increased. The electrophoretic profile showed that the two main bands corresponding to myelin basic protein (MBP) were relatively increased in the treated animals. The enzymatic activity of 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) was significantly increased. The changes observed were dose- and age-dependent. The injection of aTf was effective only within a short developmental period since animals treated at 20 days of age showed no apparent changes in myelin composition. The actions of aTf injection persisted at least up to 60 days, since at this age the myelin obtained from injected animals still contained higher amounts of total proteins phospholipids and galactolipids in comparison to control animals. The action of aTf appears to be specific for myelin, since no significant effects were observed in membranes of a total brain homogenate. The effects are specific for the apoprotein injected since other proteins (ovalbumin) or heat-denatured aTf were ineffective.