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(The FASEB Journal. 1999;13:2083-2090.)
© 1999 FASEB

Epidermal growth factor as a local mediator of the neurotrophic action of vitamin B12 (cobalamin) in the rat central nervous system

GIUSEPPE SCALABRINO*1, GABRIELLA NICOLINI{dagger}, FRANCESCA R. BUCCELLATO*, MADDALENA PERACCHI§, GIOVANNI TREDICI{dagger}, ALFREDO MANFRIDI and GIULIO PRAVETTONI{ddagger}

Institutes of
* General Pathology,
{dagger} Human Anatomy,
§ Medical Sciences, and
Human Physiology II, Faculty of Medicine, University of Milan, Milano, Italy; and
{ddagger} Gife Laboratory, Lugano, Switzerland

1Correspondence: Institute of General Pathology, University of Milan, Via Mangiagalli, 31, I-20133 Milan, Italy. E-mail: Giuseppe.Scalabrino{at}unimi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have recently demonstrated that the myelinolytic lesions in the spinal cord (SC) of rats made deficient in vitamin B12 (cobalamin) (Cbl) through total gastrectomy (TG) are tumor necrosis factor-{alpha} (TNF-{alpha})-mediated. We investigate whether or not permanent Cbl deficiency, induced in the rat either through TG or by chronic feeding of a Cbl-deficient diet, might modify the levels of three physiological neurotrophic factors—epidermal growth factor (EGF), vasoactive intestinal peptide (VIP), and somatostatin (SS)—in the cerebrospinal fluid (CSF) of these rats. We also investigated the ability of the central nervous system (CNS) in these Cbl-deficient rats to synthesize EGF mRNA and of the SC to take up labeled Cbl in vivo. Cbl-deficient rats, however the vitamin deficiency is induced, show a selective decrease in EGF CSF levels and an absence of EGF mRNA in neurons and glia in various CNS areas. In contrast, radiolabeled Cbl is almost exclusively taken up by the SC white matter, but to a much higher degree in totally gastrectomized (TGX) rats. Chronic administration of Cbl to TGX rats restores to normal both the EGF CSF level and EGF mRNA expression in the various CNS areas examined. This in vivo study presents the first evidence that the neurotrophic action of Cbl in the CNS of TGX rats is mediated by stimulation of the EGF synthesis in the CNS itself. It thus appears that Cbl inversely regulates the expression of EGF and TNF-{alpha} genes in the CNS of TGX rats.—Scalabrino, G., Nicolini, G., Buccellato, F. R., Peracchi, M., Tredici, G., Manfridi, A., Pravettoni, G. Epidermal growth factor as a local mediator of the neurotrophic action of vitamin B12 (cobalamin) in the rat central nervous system.


Key Words: cerebrospinal fluid • epidermal growth factor • subacute combined degeneration • totally gastrectomized rat


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN MAMMALIAN CELLS, deficiency of vitamin B12 [hereafter referred to as cobalamin (Cbl)] leads to the impairment of two enzymes, methionine synthase (EC 2.1.1.13) and methylmalonyl-CoA mutase (EC 5.4.99.2), and therefore to the accumulation of the metabolites homocysteine and methylmalonic acid immediately upstream of these reactions (1 , 2) . Hitherto, theories (reviewed in ref 1 2 3 4 ) advanced to explain the pathogenesis of neuropathy due to Cbl deficiency, classically termed subacute combined degeneration (SCD) (1 2 3 4) , have postulated a causal relationship between the impairment of either or both of the two Cbl-dependent reactions and the lesions of SCD. On the basis of the clinical and/or experimental data, however, this causal relationship is not established. Thus, children who develop marked methylmalonic acidemia due to inherited disorders other than Cbl deficiency do not show SCD (5) , and many experimental findings (reviewed in ref 6 ) do not support the hypothesis that Cbl deficiency neuropathy might reflect an impairment of methionine synthesis (7) . Furthermore, we have recently shown that the accumulation of methylmalonic acid and/or homocysteine, elicited by permanent Cbl deficiency in the serum and in spinal cord (SC) of totally gastrectomized (TGX) rats, does not play any etiologic role in the SCD-like lesions in these rats (8 , 9) . Total gastrectomy (TG) provides a surgical paradigm of pernicious anemia in that both surgery and autoimmune gastritis remove gastric intrinsic factor crucial for Cbl absorption (10 , 11) .

In the nervous system of our TGX rats, we observed widespread spongy vacuolation in the SC white matter (10) due to intramyelin and interstitial edema (12) , a marked astrogliosis (11) , and polyneuropathy (13) . We have subsequently demonstrated that Cbl deficiency central neuropathy of TGX rats, which is a purely myelinolytic disease (14) , is not caused directly by the withdrawal of the vitamin itself, but reflects enhanced production of the biologically active form of tumor necrosis factor {alpha} (TNF-{alpha}) (15) . The result in the first instance proves that Cbl regulates TNF-{alpha} synthesis in the mammalian central nervous system (CNS), albeit through an as yet unknown mechanism. Further support for the hypothesis that TNF-{alpha} has myelinolytic properties is that increased TNF-{alpha} synthesis has been shown to mediate the formation of intramyelin edema and vacuolation in the CNS of mice with experimental Creutzfeldt-Jakob disease (16) . In recent years, experimental evidence has accumulated that an imbalance in the temporal expression and production of the CNS cytokines, including TNF-{alpha}, contributes to a cascade of damage resulting in specific patterns of neurodegeneration (17 18 19) . In contrast, little is known about a possible role of neurotrophic factors in the pathogenesis of human neurodegenerative diseases (20) , although the clinical relevance of these molecules to therapy of this kind of disease has been canvassed (21 22 23) .

To explain the pathogenesis of SCD on the basis of our new findings (15) , we hypothesized that a deficiency of Cbl in rat CNS could lead to an increased production of neurotoxic agent(s) combined with a decreased production of neurotrophic agent(s). Cbl should thus induce an up-regulation of production of neurotrophic agent(s) and a down-regulation of the production of neurotoxic agent(s). If so, Cbl in the mammalian CNS may play a regulatory role independent of its coenzyme functions, and the effect of Cbl on the CNS may occur through shifting of the physiological equilibrium between neurotrophic and neurotoxic agents in favor of the former. We have already demonstrated (15) that Cbl inhibits the synthesis of TNF-{alpha}, a physiological, putatively neurotoxic agent, though it remains to be demonstrated whether Cbl stimulates the production of physiological neurotrophic agent(s) in the CNS.

To this end, in the current study we determined the cerebrospinal fluid (CSF) levels of several physiological neurotrophic agents and neurohormones, i.e., epidermal growth factor (EGF) (24 25 26 27) , vasoactive intestinal peptide (VIP) (28 29 30 31 32 33) , and somatostatin (SS) (32 33 34 35 36 37 38) , which are all produced in the CNS cells (24 25 26 27 28 29 30 31 32 33 34 35 36 37 38) . Animals were made Cbl deficient (Cbl-D) by TG or by chronic feeding of a Cbl-D diet. We also investigated EGF mRNA expression by in situ hybridization on sections of different CNS areas in these Cbl-D rats. The crucial experiment, however, was to see whether or not their levels would return to normal when the Cbl deficiency was corrected; this was done by injecting Cbl chronically into TGX rats and redetermining the above parameters in TGX-, and Cbl-treated rats. Last, since evidence will be presented (see Results) that the EGF synthesis is equivalently abrogated in both neurons and glia of the SC in prolonged Cbl deficiency, we attempted to settle the controversy as to whether Cbl deficiency primarily affects SC neuroglia or neurons (39) . We thus administered radiolabeled Cbl to TGX rats in vivo to investigate the uptake of the vitamin in the SC by histoautoradiography.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
[Cyano-14C] cyano-Cbl (specific activity 2.07 GBq/mmol) was from Amersham Life Science Ltd. (Little Chalfont, Bucks, U.K.), liquid emulsion (K5, in gel form) was from Ilford Nuclear Research (Mobberley, Ches., U.K.), and developer and fixer liquids were from Kodak (Eastman Kodak Co., Rochester, N.Y.).

Animal models and in vivo treatments
Adult male outbred Sprague-Dawley rats (Charles River Italia, Calco, Italy) [average body weight (b.w.), 250 g at the beginning of the experiment] were used. Experimental SCD was induced by TG, which was performed as previously reported (10) . Laparotomized (LPT) rats served as controls. The perioperative mortality rate and the mean change in b.w. of TGX rats during the investigation period were as reported (10) . A group of rats was chronically fed a Cbl-D diet, the composition of which has been reported (10) . To minimize the possible diurnal changes in CSF levels of neurohormones (40) and/or growth factors, the rats were always killed between noon and 14:00. When given, Cbl was injected subcutaneously into TGX rats weekly over the first two postoperative months (mo) (scheme a) (8) or over the third and the fourth mo after TG (scheme b) (8) , i.e., after SC abnormalities had already appeared (10) , or for the six postoperative mo (scheme c), at the dose previously reported (10) . In another set of experiments, radiolabeled cyano-Cbl (0.02 mCi/100 g b.w.) was administered by a single intraperitoneal injection to three 2mo-TGX rats and three 2mo-LPT rats, and the animals were killed 20 h after injection. At the time of death, all rats were the same age. Procedures involving animals and their care conformed to institutional guidelines, in compliance with national and international laws and policies (EEC Council Directive 86/609, 03 L 358, Dec. 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication no. 86–23, 1985).

Histological processing
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 (12) . SC segments were carefully dissected out from the cervical7–8, thoracic10–11, and lumbar2–4 cord. The SC segments were then embedded in paraffin and sectioned coronally at 10 µm, as described previously (12) . All procedures were carefully done in RNase-free conditions.

In situ hybridization
The EcoRI-BamHI fragment of human EGF cDNA (corresponding to nucleotides 113-1126 from ATG) (kindly provided by Dr. Klagsbrum, Children's Hospital, Boston, Mass.) was cut out from a lambda gt10 vector (ATCC, 59956) and cloned into pBluescript II SK± (Stratagene, La Jolla, Calif.). The antisense and sense probes were transcribed with T3 RNA polymerase, after linearization of the vector by BglII digestion, generating a 571 base probe. Single-strand antisense and sense RNA probes labeled with digoxigenin were made by an in vitro transcription system in the presence of digoxigenin-labeled uridine-triphosphate nonradioactive labeling kit for RNA detection (B 246 Boehringer, Mannheim, Germany) according to the manufacturer's instructions. Paraffin-embedded SC sections of unoperated, LPT, TGX, or rats receiving a Cbl-D diet were mounted on 3-aminopropyltriethoxisilane pretreated glass slides and baked at 37°C overnight. The SC sections were defatted and rehydrated in descending alcohols, treated with 0.2 M HCl, and washed twice with 2x saline-sodium citrate (SSC) buffer and once with phosphate-buffered saline. Samples were incubated with proteinase K (4 µg/ml) at 50°C for 30 min in a humid tray, rinsed twice in phosphate-buffered saline, once in 2x SSC buffer, once in 0.25% acetic acid anhydride/0.1 M triethanolamine, once in 2x SSC buffer, and then incubated for 1 h in a humid tray at room temperature with a prehybridization solution containing 10% dextran sulfate, 3x SSC, 1x Denhardt's reagent, salmon sperm DNA (0.1 mg/ml), yeast tRNA (0.1 mg/ml), poly(A)-polyC (0.01 mg/ml), Na-pyrophosphate 1 mg/ml, and 40% deionized formamide. SC sections were then incubated for at least 16 h at 45°C in a fresh hybridization solution containing 0.5 ng/µl of the labeled sense or antisense riboprobe. After hybridization, SC sections were washed twice at 50°C in 1:1 4x SSC/formamide, twice in 2x SSC buffer and twice in 1x SSC buffer. The remaining washes, twice at 0.5x SSC buffer and once in buffer A (NaCl 1.5M, Tris-HCl 100 mM, MgCl2 4 mM, Tween-20 0.05%, pH 7.5), were performed at room temperature. SC sections were then incubated in a humid tray at room temperature in buffer A with 1% normal swine serum and 0.3 Triton X-100 for 30 min, followed by a further incubation in the same buffer with a 1:400 dilution of anti-digoxigenin-alkaline phosphatase for 2 h. After a few washes in buffer A and buffer C (NaCl 0.1M, Tris-base 0.1M, and MgCl2 0.01M, pH 9.5), SC sections were incubated in buffer C with 1 mM levamisole, 0.02% 5-bromo-4-chloro-3-indolyl-phosphate, and 0.04% nitro-blue-tetrazolium-chloride in a humid tray wrapped in aluminum foil at room temperature. Reaction was stopped in buffer C. Immunostaining with sense cRNA probe was used as a negative control. Given the high expression of EGF mRNA in mammalian kidney (41) , histological sections from kidneys of unoperated rats were run in parallel with the SC sections and served as positive controls (not shown). No hybridization was seen with an EGF sense probe (not shown).

Light microscopy autoradiographs
The method used for preparing light microscopy autoradiographs from paraffin-embedded SC sections was as reported in ref 42 , with slight modifications. The slides were air-dried; SC sections were dipped in K5 emulsion (1:1 with water) and exposed at 4°C for 1 month before being developed with Kodak D19 developing solution and Kodak GBX fixer at room temperature for 5 min.

Assay of EGF, VIP, and SS in CSF
Rats were anesthetized with chloral hydrate (350 mg/kg b.w.) given intraperitoneally and samples (200–250 µl each) of CSF were drawn from the cisterna magna using a glass capillary with a tip of ~300 µm. Careful surgery prevented blood contamination. The samples were then transferred into prechilled polypropylene tubes containing aprotinin (Trasylol; Bayer, Leverkusen, Germany) (500 K IU/ml) for EGF assay, and aprotinin and ethylenediaminetetraacetic acid (1 mg/ml) for VIP and SS assays. For EGF assay, the samples were stored at -20°C until assayed; for VIP and SS assays, they were stored at -80°C. When assayed, the samples were no older than 2 months. EGF was measured by radioimmunoassay, using a commercially available kit for mouse EGF (DRG, Marburg, Germany). Because of high interspecies homology (43) , the antiserum used in the EGF assay had 100% cross-reactivity with rat EGF. Since VIP (44) and SS (45) have the same structure in the human and rat, VIP and SS were measured by radioimmunoassay using commercially available kits (Peninsula Laboratories, Inc., Belmont, Calif.), as described in detail (46) . Before being assayed for VIP and SS, the samples were extracted on Sep Pak C18 cartridges (46) . The antiserum used in the SS assay had 100% cross-reactivity with the two main forms of SS, i.e., SS-14 and SS-28 (46) . For the SS assay, the 95% confidence detection limit was 0.9 fmol/tube and the intra- and interassay coefficients of variation were 7.1% and 9.5%, respectively. For VIP assay, the 95% confidence detection limit was 0.4 fmol/tube and the intra- and interassay coefficients of variation were 7.9% and 9.6%, respectively. Samples were always determined in duplicate.

Assay of Cbl and folate in CSF
Samples (~200 µl each) from four to five rats for each experimental group were collected as described above, pooled, evaporated until dry in a vortex evaporator (Buchler Instruments, New York, N.Y.), and subsequently taken up in 0.2 ml of saline. When assayed, the samples were no older than 1 mo. Cbl level was determined by radioimmunoassay (8) and the folate level was determined as previously reported (10) . No changes in CSF folate levels were observed either in TGX rats or in rats chronically fed a Cbl-D diet (results not reported).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Table 1 shows that both TG and chronic feeding of a Cbl-D diet reduce the CSF content of Cbl to trace level, although the time required in rats on a Cbl-D diet is longer than that required for TGX rats. Table 1 also shows that the postoperative administration of Cbl to TGX rats for 2 mo significantly raises CSF Cbl levels above those of Cbl-D rats, whether begun immediately after TG (scheme a) or 2 mo later (scheme b), although the CSF Cbl level of TGX-, Cbl-treated rats remains almost 50% that of control rats.


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  		Table 1. Long-term experiments with TGX rats and with rats maintained on a Cbl-deficient diet. Effect on CSF level of Cbla

Data presented in Table 2 show that the EGF level in CSF of 2mo-TGX rats is significantly lower than that of controls, with no further decrease observed on lengthening the postoperative period of observation to 6 mo after TG (again see Table 2 ). Unlike EGF, the CSF levels of VIP and SS are unchanged in 2mo-TGX rats, demonstrating a specific link between decreased EGF content and the permanent Cbl-D status of TGX rats. This notion is further supported by the restoration of normal EGF levels in CSF of TGX rats injected postoperatively with Cbl, when Cbl has been given according to schemes a and b. A dramatic decrease in EGF levels was observed in CSF of rats fed a Cbl-D diet, albeit after 9 mo of treatment (see Table 2 ). The minimal experimental time required for decreasing EGF CSF levels is far longer for rats on a Cbl-D diet than for TGX rats, although the decrease by itself is significantly greater in the former (again Table 2 ).


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  		Table 2. Long-term experiments with TGX rats and with rats maintained on a Cbl-deficient diet. Effects on CSF levels of EGF, VIP, and SSa

Figure 1C shows the absence of EGF mRNA in SC cells of TGX rats from 6 mo after TG, in striking contrast to the strongly positive SC cells (both neurons and glial cells) of 6mo-LPT rats (Fig. 1A , B). Similarly, no EGF mRNA signal is seen in SC cells from rats maintained on a Cbl-D diet after 9 mo of treatment (Fig. 1F ), i.e., for a period longer than that required for TGX rats to achieve the same result. Permanent Cbl deficiency is followed by the disappearance of EGF mRNA signal in other CNS areas, such as hippocampus (not shown) and the frontal cortex, both of 6mo-TGX rats (Fig. 1H ) and of rats fed a Cbl-D diet for 9 mo (not shown), in striking contrast with the strongly positive EGF mRNA signal in the frontal cortex (see Fig. 1G ) and hippocampus (not shown) in controls. Chronic treatment of TGX rats with Cbl over the first 6 postoperative mo (scheme c) maintained the EGF mRNA levels in SC cells (Fig. 1D, E ) and other CNS areas tested (not shown).



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  		Figure 1. Photomicrographs demonstrating the detection of EGF mRNA in different portions of the rat CNS by in situ hybridization (A–H; CNS sections were hybridized with antisense EGF riboprobe, as described in Materials and Methods) and the in vivo uptake of radioactive Cbl in the rat SC (I–J). A) Typical coronal hemisection of a lumbar segment of the SC of a 6mo-LPT rat. Note the intense positive staining for EGF mRNA in both neurons and glial cells (x100). No differences in the intensity for EGF mRNA between SC of LPT rats and SC of unoperated rats were observed (not shown). B) Detail of panel A at higher magnification (x400). C) Typical coronal section of a lumbar segment of the SC of a 6mo-TGX rat. Note the absence of EGF mRNA signal in any SC cell type (x100). D) Typical coronal hemisection of a lumbar segment of the SC of a 6mo-TGX- and Cbl-treated rat (Cbl was given according to scheme c, as described in Materials and Methods). Note the restoration to normal of positivity for EGF mRNA in neurons and glia (x100). E) Detail of panel D at higher magnification (x400). F) Typical coronal section of a thoracic segment of the SC of a rat fed a Cbl-D diet for 9 mo. Note the absence of EGF mRNA signal in any SC cell type (x100). G) Typical section of the frontal cortex of the brain of a 6mo-LPT rat. Note the intense staining for EGF mRNA in the different cell types (x170). H) Typical section of the frontal cortex of the brain of a 6mo-TGX rat. Note the total disappearance of EGF mRNA signal in any cortical cell type (x170). I) Dark-field photomicrograph of a typical coronal hemisection of a thoracic segment of the SC of a 2mo-LPT rat injected with radioactive Cbl, as described in Materials and Methods. Note the scanty accumulation of labeled Cbl only in the white matter (x80). J) Dark-field photomicrograph of a typical coronal hemisection of a lumbar segment of the SC of a 2mo-TGX rat injected with radioactive Cbl, as in panel I. Note the intense accumulation of radioactive Cbl, mainly in the glia and fibers of the white matter (x80).

From the comparative analysis of Fig. 1I, J , it is evident that 1) radiolabeled Cbl is taken up almost exclusively in SC white matter of both controls and 2mo-TGX rats, and 2) the accumulation of radioactive Cbl in the SC white matter in 2mo-TGX rats is far greater than that in control SC white matter. On the flat map of the adult rat CNS, note that the SC is proportionally the most developed area (48) and is the CNS area most severely affected by chronic Cbl deficiency (10 , 12) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates for the first time that Cbl selectively regulates the synthesis of a physiological neurotrophic agent—namely, EGF (24 25 26 27) —in the rat CNS. The main findings of this study can be summarized: 1) the CSF level of EGF is significantly and permanently reduced in TGX rats and in rats chronically fed a Cbl-D diet; 2) the CSF levels of two physiological neurotrophic agents other than EGF, i.e., VIP (28 29 30 31 32 33) and SS (32 33 34 35 36 37 38) , are unmodified in TGX rats; 3) EGF mRNA in different CNS areas (i.e., SC, hippocampus, and frontal cortex) of Cbl-D rats falls to below detection level six mo after TG and nine mo on a Cbl-D diet; 4) both EGF CSF levels and those of EGF mRNA in the CNS of TGX rats are restored to normal after chronic postoperative treatment with Cbl. It is not surprising that more time is required for rats fed a Cbl-D diet than for TGX rats. Similar temporal differences between the two experimental groups of Cbl-D rats have been observed previously, when other biological parameters [such as serum levels of Cbl (10) and methylmalonic acid (9) , SC Cbl level (11) , degree of the severity of spongy vacuolation in SC white matter (10 , 11) , intramyelin and interstitial edema (12) , and the maximal nerve conduction velocity (13 ] have been determined.

Again, from the results reported here, three important conclusions can be drawn. 1) The finding that the EGF in CSF of TGX rats is still present when the EGF-mRNA signal has completely disappeared strongly suggests that EGF in the CSF of TGX rats considerably after TG derives from the plasma (24) . 2) It is conceivable that the cessation of EGF synthesis in the SC of Cbl-D rats contributes to the development of the severe myelinolytic lesions observed (12) . It remains to be elucidated the extent to which these myelinolytic lesions are the result of increased TNF-{alpha} synthesis (15) and decreased EGF synthesis, both induced by a permanent Cbl deficiency in the CNS. To the best of our knowledge, nothing is known about the possible role of the EGF on structure and/or architecture of myelin in the mammalian CNS, although the positive effects of the EGF on proliferation and/or differentiation of neurons and/or glia are well known (24 , 25 , 27 , 49 50 51) . 3) The temporal trend of the decrease of the EGF CSF level in TGX rats is very similar to that of the severity of SC spongy vacuolation, in which no worsening had been observed 2 mo after TG (8 , 11) .

The current study, together with its predecessor (15) , demonstrates that the neurotrophic effect of Cbl in the CNS of TGX rats is twofold: a stimulation of EGF synthesis accompanied by a decrease in TNF-{alpha} production (15) . Conversely, the neurotoxic effect of permanent Cbl deficiency in TGX rats reflects the enhanced production of the biologically active form of TNF-{alpha} (15) accompanied by reduced EGF synthesis. It has recently been reported (52) that in some human tumor cell lines, activation of the EGF receptor by EGF resulted in a reduction of the cytotoxic effect of TNF-{alpha}; conversely, inhibition of the EGF receptor led to increased toxicity of these cells by TNF-{alpha}. It is thus tempting to speculate that a similar biological antagonism between the EGF and TNF-{alpha} might also occur in the mammalian CNS and that the balance between them is ruled by Cbl availability.

We have shown that permanent Cbl deficiency, however induced, affects both neurons and neuroglia to a similar degree in terms of their ability to synthesize EGF mRNA. However, labeled Cbl has been shown to be taken up mainly by glial cells and fibers of the SC white matter, to a higher degree in TGX than in control rats. Although this result has to be considered with caution, it supports the possibility that Cbl deficiency in the rat CNS preferentially strikes glial cells, consistent with the massive astroglial reaction in the SC of TGX rats (11) and with the damage of human astrocytes cultured in Cbl-D medium (53) . This interpretation also fits with the absence of any features of morphological damage observable by light or electron microscopy in neurons in different CNS areas in TGX rats at any postoperative time examined (10 , 12) . Nevertheless, it should be noted that the cessation of EGF synthesis in CNS neurons of our TGX rats is, to the best of our knowledge, the first report showing that neurons, too, are biochemically affected by permanent Cbl deficiency.

Finally, our finding that the postoperative administration of Cbl to TGX rats restores EGF synthesis in the CNS to normal means that Cbl regulates EGF gene expression both in neurons and in glia, though the level at which this regulation occurs and the signaling pathways involved are unknown at this time. This feature of Cbl action—that it requires mediation by growth factor(s) to achieve one of its key biological effects—resembles that of vitamin D3, which inhibits cell proliferation and induces cell differentiation in vitro by enhancing the production of transforming growth factor-ß1 (54 , 55) . Although very little is known about the molecular mechanism(s) set in the brain by Cbl at the cellular level to produce its neurotrophic effects, Cbl, with a chemical structure quite different from that of classical hormones, appears to act in a hormone-like manner in modulating the expression of both EGF and TNF-{alpha} genes in the mammalian CNS. However, if we are evaluating the time required to observe such putative hormone-like effects of Cbl on the two CNS genes of TGX rats, we can conclude that the time (2 mo) to prevent the myelinolytic effect due to increased TNF-{alpha} synthesis is the same as that required, for example, to observe increased collagen synthesis in chronic experimental hypermineralocorticoid states developing cardiac fibrosis (56 , 57) . In contrast, to the best of our knowledge, the time (6 mo) to reverse the cessation of EGF synthesis exceeds that required for most of the known effects of the traditional hormones. It also should be borne in mind that our TGX rats develop severe Cbl deficiency shortly after TG (10) . The cellular and/or extracellular mechanisms mediating the temporal difference in response to Cbl between the expression of TNF-{alpha} and EGF genes in rat CNS await exploration.


   ACKNOWLEDGMENTS
 
The author (G.S.) is deeply indebted to Prof. J. W. Funder (Melbourne, Australia) for critical reading of the manuscript and helpful discussions. G.S. dedicates this study to Prof. V. Herbert (New York City) and Prof. P. Mantegazza (President of the University of Milan), who have both helped him greatly, albeit in different ways, over the past 10 years in accomplishing this and previous studies of Cbl-D neuropathy; even more important, they have believed in him since his research on Cbl-D neuropathy began. The help of Dr. M. Francolini (Department of Pharmacology, University of Milan) in the histoautoradiographic part of this study is gratefully acknowledged. G.S. also wants to acknowledge the help of Miss S. Bonato in preparing the manuscript. This work is 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
 
Received for publication March 12, 1999. Accepted for publication May 19, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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