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(The FASEB Journal. 1999;13:297-304.)
© 1999 FASEB

RESEARCH COMMUNICATION

Myelinolytic lesions in spinal cord of cobalamin-deficient rats are TNF-{alpha}-mediated

FRANCESCA R. BUCCELLATOa , MARIAROSARIA MILOSOb , MASSIMILIANO BRAGAd , GABRIELLA NICOLINIb , ALBERTO MORABITOc , GIULIO PRAVETTONIe , GIOVANNI TREDICIb and GIUSEPPE SCALABRINOa , 1


a Institutes of General Pathology,

b Human Anatomy,

c Biometrics and Medical Statistics,

d 2nd Department of Neurology, Faculty of Medicine, University of Milan, Milano, Italy; and

e Gife Laboratory, Lugano, Switzerland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Repeated intracerebroventricular (i.c.v.)microinjection of tumor necrosis factor-{alpha} (TNF-{alpha}) into normal rats causes intramyelin and interstitial edema in the white matter of the spinal cord (SC). This response is identical to that observed in the SC white matter of rats made cobalamin (Cbl) deficient by total gastrectomy (TG). Immunoblot analysis showed that: 1) the level of the biologically active form of the TNF-{alpha} protein (17 kDa) is higher in the SC of totally gastrectomized (TGX) rats 2 months after TG, i.e., at the postoperative time when edema is observed; 2) SC levels of TNF-{alpha} protein (17 kDa) in 2-mo-TGX-, Cbl-treated rats are reduced to control. Repeated i.c.v. microinjections of anti-TNF-{alpha} antibodies, transforming growth factor-ß1 (TGF-ß1) or interleukin-6 (IL-6) into TGX rats, begun shortly after TG, substantially reduced both intramyelin and interstitial edema in the SC white matter. This study provides the first evidence that the hallmark myelin damage of Cbl-deficient central neuropathy, which is a pure myelinolytic disease, is not caused directly by the withdrawal of the vitamin itself, but reflects enhanced production of the biologically active form of TNF-{alpha} by SC cells. This study thus supports the view that TGF-ß1 and IL-6 may act as neuroprotective agents in Cbl deficiency central neuropathy.—Buccellato, F. R., Miloso, M., Braga, M., Nicolini, G., Morabito, A., Pravettoni, G., Tredici, G., Scalabrino, G. Myelinolytic lesions in spinal cord of cobalamin-deficient rats are TNF-{alpha}-mediated.


Key Words: myelinolysis • subacute combined degeneration • tumor necrosis factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EQUIVALENT ANIMAL MODELS of human neurodegenerative diseases are needed to enable detailed analysis of the pathogenetic mechanism(s) involved and for the development and evaluation of new therapeutic strategies. The totally gastrectomized (TGX)2 rat, on becoming permanently cobalamin(Cbl) deficient, reproduces all the key neuropathological features of human Cbl-deficient neuropathy [classically known as subacute combined degeneration (SCD)] (1 , 2 ) in both the central (CNS) (1 , 2 ) and peripheral nervous systems (3) . The pathogenesis of SCD is largely unknown and still controversial; it remains unclear, for example, whether SCD reflects impairment of myelin synthesis or destruction of the existing myelin (1 , 2 , 4 ). To explain the pathogenesis of this neuropathy in mammalians, many theories have arisen from experimental investigations and have recently been reviewed (1 , 2 , 4 ).

In mammalian cells there are only two known Cbl-dependent enzymes. L-Methylmalonyl-coenzyme A (CoA) mutase (EC 5.4.99.2) requires adenosyl-Cbl and catalyzes the conversion of L- methylmalonyl-CoA to succinyl-CoA (4) . Methionine synthase (EC 2.1.1.13) requires methyl-Cbl and catalyzes the simultaneous conversion of N5-methyltetrahydrofolate to tetrahydrofolate and of homocysteine (HCYS) to methionine (4) . The metabolites methylmalonic acid (MMA) and HCYS accumulate when these two enzymatic reactions are impaired by Cbl deficiency (4) . In a previous paper (5) we described in reproducing in TGX rats the typical triad of biochemical abnormalities (i.e., decreased Cbl level and increased concentrations of MMA and HCYS) in serum components characteristic of humans with pernicious anemia and other disorders associated with Cbl deficiency (4) . We have previously shown (5) that the severity of the neuropathological features of experimental SCD in the spinal cord (SC) in TGX rats does not correlate with the accumulation in their sera and/or tissues of MMA and HCYS. These studies thus demonstrated that accumulation of putative neurotoxic metabolites such as MMA and HCYS is unlikely to be the cause of SCD in the SC of TGX rats (5) . Furthermore, we have demonstrated no substantial increase in the severity of SCD-like lesions in the SC white matter of 2-month(mo) -TGX rats, as the time after total gastrectomy (TG) lengthened (2 , 5 ).

It has been well established that various cytokines are released in large amounts from different CNS cell types, mainly from glial cells 6-11) , although the physiological role of these molecules in the mammalian CNS is only just beginning to be understood. Much attention has recently focused on the role of cytokines, especially of tumor necrosis factor-{alpha} (TNF-{alpha}) produced by CNS cells 12-23) , in the pathogenesis of neurological disease, especially those characterized by demyelination, since cytokines are rapidly produced in the mammalian CNS by experimental or clinical injury, ischemia, infection, or inflammation 12-23) . On the other hand, some cytokines [especially interleukin-6 (IL-6), transforming growth factor-ß1 (TGF-ß1), and interleukin-10], all of which are produced in the CNS cells mainly by glial cells (7 , 10-12 ), have recently been claimed to be neurotrophic and, therefore, potentially therapeutic in some neurological disease states 24-32) . Nevertheless, the key unanswered question is whether cytokines in the CNS serve to protect both neurons and glial cells, when either or both are injured, and stimulate the repair process or, conversely, whether they play a leading role in the pathophysiology of neuronal damage and/or death (33) .

In the present study we hypothesized that TNF-{alpha} may be involved in the pathogenesis of the SCD-like lesions, chiefly intramyelin and interstitial edema (34) in the SC of TGX rats and, conversely, that IL-6 and TGF-ß1 may act as neuroprotective molecules in the same situation. We therefore administered TNF-{alpha} to normal rats by repeated intracerebroventricular (i.c.v.) microinjection in order to investigate its morphologic effects on SC myelin and compare them with the SCD-like lesions in the SC of 2-mo-TGX rats. In addition, we have examined SCs of 2-mo-TGX rats and of 2-mo-TGX-, Cbl-treated rats for the presence of TNF-{alpha} protein by immunoblot analysis. Third, we administered anti-TNF-{alpha} antibodies, IL-6, or TGF-ß1 into TGX rats by repeated i.c.v. microinjection in order to evaluate whether each of these treatments, begun shortly after TG, might ameliorate the SCD-like lesions in SC white matter. We have used both optical and electron microscopy to gauge the extent of intramyelin and interstitial edema in SC sections from rats treated as described above in order to establish whether the injected material acted as a neuroprotective or neurotoxic agent, and have quantified the in vivo effects of the above treatments by morphometry.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals, induction of the Cbl-deficient central neuropathy (SCD), and i.c.v. drug administrations
Adult male albino non-inbred Sprague-Dawley rats (Charles River, Caleo, Italy) weighing 250 g at the beginning of the experiment were used, housed as previously described (1 , 2 ). To induce experimental SCD, TG was performed as previously reported (1 , 2 ). Laparotomy was performed as described previously, as was postoperative treatment (1 , 2 ). Cbl was injected subcutaneously into some TGX rats weekly over the first two postoperative months at the dosage previously reported (2) . To perform the i.c.v. microinjections, a polyethylene guide cannula was stereotaxically placed (1 mm posterior and 4 mm lateral from bregma) in the left lateral ventricle of all rats under ketamine anesthesia, in TGX rats 1 wk after TG. Cannulae were attached to the skull with dental acrylic. Correct placement of cannulae was checked by the drinking response elicited by injection of 200–400 µg of angiotensin II, dissolved in 3–4 µl of sterile pyrogen-free saline, and only rats with positive responses to angiotensin II were used. Control normal rats and control TGX rats were given i.c.v. microinjections of sterile pyrogen-free saline; other normal rats received i.c.v. microinjections of rmTNF-{alpha} (Calbiochem, San Diego, Calif.), h-TGF-ß1 (PeproTech, Inc., Rocky Hill, N.J.), rmIL-6 (Calbiochem), or rabbit anti-mouse TNF-{alpha} antibodies (Genzyme, Boston, Mass.). TGX rats were given i.c.v. h-TGF-ß1, rmIL-6, or rabbit anti-mouse TNF-{alpha} antibodies. All in vivo treatments included two i.c.v. microinjections weekly for 7 wk after attachment of the cannula, performed with a 10 µl Hamilton syringe and a volume ranging between 5 and 7 µl. The dose per injection was: 0.5 µg TNF-{alpha}, 0.75 µg IL-6, 1 µg TGF-ß1, and 7 µl anti-TNF-{alpha} antibodies (10 µl of antibody neutralize 1000 units of TNF-{alpha} bioactivity in the standard L929 cytotoxicity assay). Killing time was always fixed at 48 h after the last i.c.v. microinjection. At death, all rats were the same age. Procedures involving animals and their care conformed with institutional guidelines, in compliance with national and international laws and policies (EEC Council Directive 86/609, 0J L 358, 1, Dec. 12 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication no. 86-23, 1985).

Histological processing, staining, and morphometric analysis
Rats were anesthetized with ketamine and perfused via the heart with 1% paraformaldehyde and 1.25% glutaraldehyde in 0.12 M phosphate buffer solution, pH 7.4, as described in detail elsewhere (34) . SC segments were carefully dissected out from the cervical (C)7 and C8, thoracic (Th)10 and Th11, and lumbar (L)5 and L6 cord. From these segments, thin slices (0.5 mm) were osmified, dehydrated, and embedded in epoxy resin. Semithin sections (1 µm) were stained with toluidine blue for light microscopy; ultrathin sections were stained with uranyl acetate and lead citrate and observed in a CM 10 Philips electron microscope (34) . For each SC, four serially 10 µm-spaced semithin sections from each of the three above-mentioned SC levels were analyzed morphometrically (stereology-based measurements) (35) . With a camera lucida, a grid of 389 regularly spaced points was superimposed onto the microscopic field with a fixed magnification (objective lens: 63x). For each SC section, two samples of each funiculus (anterior, lateral, and posterior) were analyzed morphometrically. Therefore, the total number of the points (observations) for each white matter of each SC was 9336. Points falling on pathological findings (i.e., interstitial edema, intramyelin edema, myelin splitting) and on apparently normal structures in the SC white matter were counted. All examinations of the semithin SC sections and all electron microscopic examinations of SC sections were made blind, i.e., without knowledge of to which experimental group the SC sections belonged.

In situ nick-end labeling
Sections of SCs from 2-mo-TGX rats and from TNF-{alpha}-treated rats were processed for apoptosis recognition. The sections were cut from the different SC segments (C, Th, L), as described above, and examined by the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining (36) .

Immunoblot analysis for TNF-{alpha} protein
Total proteins were extracted from the SC of control rats and 2-mo-TGX rats by a single-step method (Tripure isolation reagent, Boehringer Mannheim, Germany). The protein pellets obtained were sonicated in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X100, 1.5 mM MgCl2, 5 mM EGTA, 4 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 10 mM sodium orthovanadate, 20 mM sodium pyrophosphate) with a microultrasonic cell disrupter (Kontes, Vineland, N.J.). Total proteins were measured by the Bio-Rad method. An aliquot of total lysate (150 µg) was mixed with 5x Laemmli buffer, boiled, run on a 15% sodium dodecyl sulfate-polyacrylamide gel, and transferred to nitrocellulose membrane (Schleicher & Schuell, Keene, N.H.). The membrane was blocked for 1 h at room temperature (RT) in Blotto A (10 mM Tris HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20, 5% non-fat milk) and then incubated for 1 h at RT with primary antibody anti-TNF-{alpha} 1 µg/ml in Blotto A (TNF-{alpha} goat polyclonal antibody) (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). After three washes with triphosphate-buffered saline (TBS) (10 mM Tris HCl pH 7.5, 150 mM NaCl), 0.05% Tween-20, the membrane was incubated for 45 min at RT with secondary antibody (horseradish peroxidase-conjugated anti-goat IgG, 1:3000) (Amersham Pharmacia, Amersham, U.K.) diluted 1:3000 in Blotto A. The membrane was then washed three times with TBS, 0.05% Tween-20 and once in TBS buffer. The immunoreactive proteins were visualized on an ECL chemiluminescence system (Amersham Pharmacia) and changes in TNF-{alpha} expression were assessed by densitometric analysis of autoluminographs. Additional details are reported elsewhere (37) .

Studies of blood components
Red cell count, hemoglobin concentration, and serum Cbl level were determined in all 2-mo-TGX rats immediately before killing, as described previously (1 , 2 , 4 ). Anemia and very low serum levels of Cbl were observed in 2-mo-TGX rats whether untreated or treated with the drugs. Changes in serum folate levels were never observed in TGX rats (1 , 5 ). The Cbl level in sera of TGX rats given Cbl postoperatively was 30% lower than normal values (2 , 5 ).

Cbl concentration in SC
SC Cbl concentration was determined as previously described (2) . The Cbl content of the SCs from the 2-mo-TGX rats, untreated or treated with the drugs, was greatly reduced (2) . Chronic in vivo administration of Cbl to TGX rats over the first 2 months after TG was effective in restoring a normal SC Cbl content in TGX rats (2) .

Statistical analyses
The Shapiro-Wilk test (38) was used to check the normality of the distribution of our morphometric data. The result of this test was statistically significant at the 5% level, providing statistical evidence against a normal distribution of the morphometric data. These data were then transformed by the Box-Cox transformation (39) . Thereafter, we evaluated the difference between each pair of means of the different treatments using the Student's t test for unpaired data at the 0.05 level of statistical significance. P levels are reported in Table 1 .Analysis of variance (ANOVA) for a two-factor (i.e., TG and TGF-ß1 or TG and anti-TNF-{alpha} antibodies) experimental design was performed in order to evaluate the presence of an interaction. An {alpha}-level of 0.05 was taken as the limit of statistical significance.


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  		Table 1. Morphometry of the pathological structures in SC white matter in rats treated with different drugs (see Materials and Methods)a


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphometry of cytokine-induced effects on SC white matter
The SC white matter of intact rats, saline-injected rats, TNF-{alpha}-treated rats, 2-mo-TGX rats, TGX- and saline-injected rats, and TGX rats treated with TGF-ß1, IL-6, or anti-TNF-{alpha} antibodies is shown at the structural and ultrastructural levels in Fig. 1 and Fig. 2 . I.c.v. microinjection of sterile pyrogen-free saline into normal rats caused no pathological changes in the SC white matter (Fig. 1A ).This parallels the morphometrical analysis (Table 1) , which shows no statistical difference between the SC white matter from normal untreated rats and that from normal saline-injected rats. Figure 1B shows the hallmark myelin alterations in the SC white matter from 2-mo-TGX rats, intramyelin and interstitial edema, as described previously in detail (34) . The SC white matter of a typical TNF-{alpha}-treated rat (Fig. 1C ) shows similar intramyelin and interstitial edema and is morphologically identical to the SC white matter of 2-mo-TGX rats (Fig. 1B ). Repeated i.c.v. administration of TNF-{alpha} affects both large and small fibers of the SC white matter, with splitting of the myelin sheath and a reduction in its thickness (Fig. 1C ). The statistical analysis of morphometrical data (Table 1) shows a highly significant increase in the number of lesions in SC white matter of TNF-{alpha}-treated rats compared with either intact or normal saline-injected rats. The neuropathological lesions clearly imply edema at both the interstitial and intramyelin levels in the SC white matter of TNF-{alpha}-treated rats.



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  		Figure 1. Semithin sections of white matter of a typical SC from a normal saline-injected rat (A), a 2-mo-TGX rat (B), a TNF-{alpha}-treated rat (C), a 2-mo-TGX rat treated with anti-TNF-{alpha} antibodies (D), a 2-mo-TGX-, TGF-ß1-treated rat (E), and a 2-mo-TGX-, IL-6-treated rat (F). The areas of interstitial edema are evident, as indicated by asterisks (B, C). Scale bar, 5 µm.



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  		Figure 2. Electron micrographs of myelinated fibers in the white matter of a typical SC from a TNF-{alpha}-treated rat (A), a 2-mo-TGX rat treated with anti-TNF-{alpha} antibodies (B), a 2-mo-TGX-, TGF-ß1-treated rat (C), and a 2-mo-TGX-, IL-6-treated rat (D). Note the intramyelin edema (A) (indicated by asterisks) and the normal appearance of the myelin sheaths (B–D). Scale bars, 0.5 µm.

Figure 1D–F shows areas of white matter in the SC of typical 2-mo-TGX rats treated with anti-TNF-{alpha} antibodies, TGF-ß1, or IL-6. Chronic i.c.v. microinjection of sterile pyrogen-free saline into TGX rats did not modify their appearance (see Table 1 for morphometric analysis). Chronic i.c.v. treatment with anti-TNF-{alpha} antibodies (Fig. 1D ) or TGF-ß1 (Fig. 1E ) substantially reduced the severity of the hallmark neuropathological lesions in the SC white matter of 2-mo-TGX rats. Statistical analysis of the morphometric data similarly shows a significant reduction in the number of lesions in the SC white matter in both 2-mo-TGX-, TGF-ß1-treated rats, and 2-mo-TGX, anti-TNF-{alpha}-antibody-treated rats compared with untreated 2-mo-TGX rats (P <0.003 for TGF-ß1-treated rats; P <0.04 for anti-TNF-{alpha}-antibody-treated rats) (see Table 1 ). Although improvement in edema in the SC white matter of 2-mo-TGX-, IL-6-treated rats is morphologically evident, as shown in Fig. 1F , and seems similar to that observed in the SC white matter of TGX rats treated with TGF-ß1 or anti-TNF-{alpha} antibodies, statistical analysis of the morphometric data shows no statistically significant difference (Table 1) . ANOVA does not show any statistically significant interaction between the two experimental factors (TG and TGF-ß1 or TG and anti-TNF-{alpha} antibodies) (results not reported). It is almost unnecessary to say that SCs from normal rats treated by i.c.v. microinjection of anti-TNF-{alpha} antibodies, TGF-ß1, or IL-6 show no pathological change (figures not shown); Table 1 contains morphometric analysis of these data.

No histological signs of apoptosis by the TUNEL method (36) were observed in any SC region of TNF-{alpha}-treated rats or of 2-mo-TGX rats (figures not shown), despite reports of apoptosis from elsewhere in the CNS of TNF-{alpha}-treated rats (18 , 40-42 ) and in cultured Cbl-deprived neoplastic cells (43) .

Ultrastructural effects of cytokines on SC white matter
Plates A–D of Fig. 2 show typical ultrastructural findings in the SC of TNF-{alpha}-treated rats (panel A) and of TGX rats treated with anti-TNF-{alpha} antibodies (panel B), TGF-ß1 (panel C), or IL-6 (panel D). SC white matter of TNF-{alpha}-treated rats shows overt splitting of the myelin lamellae (Fig. 2A ) identical to that previously described for SC white matter of 2-mo-TGX rats (34) . Chronic i.c.v. treatment of TGX rats with anti-TNF-{alpha} antibodies (Fig. 2B ), TGF-ß1 (Fig. 2C ), or IL-6 (Fig. 2D ) substantially reduced both types of edema observed in the SC white matter of 2-mo-TGX rats (34) .

We never observed any structural or ultrastructural feature of an inflammatory reaction or of oligodendrocyte death in SCs from TNF-{alpha}-treated rats (figures not shown), which differs from findings (14 , 44-48 ).

Immunoblot analysis for TNF-{alpha} protein
Immunoblot analysis shows that the TNF-{alpha} protein in its mature form (17 kDa) (46 , 49 , 50 ) is present in the SC of both control and 2-mo-TGX rats (see Fig. 3 ).On densitometric analysis, TNF-{alpha} levels in Cbl-deficient SCs from 2-mo-TGX rats are 2.6–2.8 higher than control. Chronic postoperative Cbl treatment of TGX rats substantially reduced SC levels of TNF-{alpha} protein almost to control levels (Fig. 3) .



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  		Figure 3. Levels of the biologically active form of TNF-{alpha} protein (17 kDa) (46, 49, 50) in typical SCs of two control (2-mo-laparotomized) rats (lanes 1 and 2), two 2-mo-TGX rats (lanes 3 and 4), and two 2-mo TGX- and Cbl-treated rats (lanes 5 and 6). The levels of TNF-{alpha} protein were determined by immunoblot analysis. No difference was observed between the SCs of control unoperated rats (not shown) and the SCs from 2-mo-laparotomized rats (lanes 1 and 2), as determined by densitometry. The control rats, whether unoperated or 2-mo-laparotomized, were of the same age as the 2-mo-TGX rats. The data are from one representative experiment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This in vivo study presents the first evidence that: 1) the biologically active 17 kDa form of TNF-{alpha} (46 , 49 , 50 ) is overexpressed in Cbl-deficient SC of 2-mo-TGX rats; 2) a chronic postoperative Cbl treatment to TGX rats substantially reduces the SC level of the biologically active form of TNF-{alpha} near to that of normal; 3) a chronic i.c.v. treatment with TNF-{alpha} reproduces the hallmark structural and ultrastructural lesions (intramyelin and interstitial edema) that we (34) and others (51 , 52 ) observed previously in animals with experimentally induced SCD; and 4) chronic i.c.v. treatment with different agents—one of which (i.e., the anti-TNF-{alpha} antibodies) antagonizes TNF-{alpha} and the others (i.e., TGF-ß1 and IL-6) substantially reduce its cellular production (15 , 27 , 53-55 )—largely prevents SCD-like lesions in the SC white matter of 2-mo-TGX rats. These results clearly provide both direct and indirect evidence that TNF-{alpha} is involved in the pathogenesis of Cbl-deficient central neuropathy, which is typically characterized by myelinolytic damage (1 , 34 , 56 ) without inflammation or demyelination (1 , 2 , 34 , 51 , 52 ). Different drugs that are directly or indirectly anti-TNF-{alpha} have been shown to be neuroprotective in experimental SCD of TGX rats. The deleterious effect on myelin structures of TNF-{alpha} in the rat SC, without simultaneous damage to the myelin-producing cells, agrees with the results of other investigators (17 , 57 , 58 ); the lack of histological evidence for apoptosis or ultrastructural evidence for complete demyelination (1 , 2 , 34 ) in TNF-{alpha}-treated or 2-mo-TGX rats also supports this interpretation. In the current debate as to whether TNF-{alpha} is neuroprotective 59-62) or has a pathological role in several neurological disorders 62-69) , our data clearly support a toxic effect of this cytokine on SC myelin structures in the rat. Our results are also in keeping with those of others showing that blockade of TNF-{alpha} activity inhibits in different ways the development of the experimental autoimmune encephalomyelitis (20 , 70-73 ). In addition, in vivo administration of TGF-ß1 into mice with experimental allergic encephalomyelitis is successful in prevention and/or improvement of the clinical course of the disease (32 , 74 , 75 ).

Our results also support the view that TGF-ß1 and IL-6 may function as endogenous neuroprotectants. TGF-ß1 and IL -6, traditionally viewed as pivotal modulators of immune and/or inflammatory responses 24-32) , have neurotrophic properties in a myelinolytic disease, i.e., Cbl-deficient central neuropathy. This activity appears to be independent of the modulation of immune and/or inflammatory processes 24-32) , since neither inflammatory nor immune-mediated processes appear to be required for Cbl-deficient central neuropathy (1 , 2 , 34 , 56 ). We cannot say with certainty whether the neuroprotective effect of TGF-ß1 we observe is due to the direct action of this cytokine or to an increase in IL-6 production by the CNS cells in response to TGF-ß1 (11 , 27 ). Similarly, what cannot be excluded at this time is whether the improvement of the SCD-like lesions by IL-6 may reflect increased release of adrenocorticotropic hormone, elicited by the cytokine and elevating glucocorticoids (9 , 76 , 77 ). Whatever the mechanism(s) of the neuroprotective effect of IL-6 might be, the present studies confirm the neurotrophic activity of IL-6 in vivo, in agreement with other authors (24 , 25 , 29 ), although neurological diseases have also been found in mice overexpressing IL-6 (23 , 78 ).

In conclusion, these data support the hypothesis that altered expression of cytokines in the CNS of TGX rats may play a crucial role in the pathogenesis of Cbl-deficient central neuropathy, although the sequence of molecular events that trigger this response awaits investigation. It is thus possible that abnormal production of TNF-{alpha}, TGF-ß1, and/or IL-6 is induced in the rat SC by permanent Cbl deficiency and is responsible for the onset of the neuropathological lesions typical of Cbl-deficient central neuropathy. In keeping with this is the fact that morphological improvement of the SCD-like lesions in TGX rats is seen after chronic i.c.v. treatments with TGF-ß1 or IL-6 or with anti-TNF-{alpha} antibodies, improvement of which is identical from a structural and ultrastructural point of view to that observed in SC white matter from 2-mo-TGX rats after chronic postoperative Cbl treatment (34) . Furthermore, it is well known that one of the major hypotheses advanced to explain the pathogenesis of SCD envisions a decrease in methionine synthase activity (4 , 79 ). It is also known that the ability of TNF-{alpha} to induce type II nitric oxide (NO) synthase in CNS (80) results in an increased NO concentration. NO has been shown to be implicated in the pathogenesis of several CNS diseases (80) and to be able to specifically inhibit the methionine synthase activity (4 , 81 ). Thus, it is conceivable that the increase in TNF-{alpha} in SCs of 2-mo-TGX rats might ultimately produce a decreased concentration of methionine and a methylation deficiency via increased NO concentrations. However, against this hypothesis lie the following: 1) the content of S-adenosyl-L-methionine of SCs of 2-mo-TGX rats is significantly increased (1) ; and 2) serum levels of methionine are significantly increased in 4-mo-TGX- and in 6-mo-TGX-rats (J. Lindenbaum et al., unpublished results). This last result apparently does not agree with the report (82) showing decreased methionine levels in plasma of nitrous oxide-treated pigs.


   ACKNOWLEDGMENTS
 
G.S. is deeply indebted to Prof. J. W. Funder (Melbourne, Australia) for critical reading of the manuscript and helpful discussions. G.S. expresses his deep gratitude to Prof. P. Mantegazza (President of the University of Milan) for his continued interest in this research. G.S. also wishes to thank the Hoechst Foundation (Milan) for the financial support given for part of this research. This work was supported in part by grants from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST 40% and 60%, Rome, Italy) and from C.N.R. Center for Research in Cell Pathology (Institute of General Pathology, University of Milan).


   FOOTNOTES
 
1 Correspondence: Institute of General Pathology, University of Milan, Via Mangiagalli 31, 20133 Milano, Italy. E-mail: scalb{at}imiucca.csi.unimi.it

2 Abbreviations: ANOVA, analysis of variance; C, cervical; Cbl, cobalamin; CNS, central nervous system; HCYS, homocysteine; i.c.v., intracerebroventricular; IL-6, interleukin-6; L, lumbar; MMA, L-methylmalonic acid; mo, month; NO, nitric oxide; NS, not significant; R, rat; RT, room temperature; SC, spinal cord; SCD, subacute combined degeneration; TBS, triphosphate-buffered saline; TG, total gastrectomy; TGF-ß1, transforming growth factor-ß1; TGX, totally gastrectomized; Th, thoracic; TNF-{alpha}, tumor necrosis factor-{alpha}; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick-end labeling.

Received for publication July 27, 1998. Revision received October 2, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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G. SCALABRINO, G. NICOLINI, F. R. BUCCELLATO, M. PERACCHI, G. TREDICI, A. MANFRIDI, and G. PRAVETTONI
Epidermal growth factor as a local mediator of the neurotrophic action of vitamin B12 (cobalamin) in the rat central nervous system
FASEB J, November 1, 1999; 13(14): 2083 - 2090.
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