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§
From the Departments of Pathology (Neuropathology),*
Neurology,
and
Neuroscience,§
Albert Einstein College of
Medicine, Bronx, New York; and the Laboratorio di
Neuropatologia,
University of Genova,
Genoa, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Another growth factor, insulin-like growth factor-1 (IGF-1), has been shown like GGF2, to promote oligodendrocyte development and stimulate myelin gene expression in vitro.5,6 Previous studies in vivo have shown that IGF-1 is capable of reducing clinical signs and lesion severity in the Lewis rat and SJL mouse models of acute and chronic relapsing EAE.7,8 Another laboratory has shown somewhat different results in the (PL x SJL/J) F1 mouse model of EAE,9 where a delay in disease onset was seen after treatment during the acute phase. However, once signs developed, the disease was more severe with higher doses of IGF-1, and treatment of chronic animals had no effect. Since CNS remyelination is a major ultimate goal of many therapeutic protocols in MS, the present study was designed to compare the long-term effects of IGF-1 delivered during both the acute and chronic phases of chronic relapsing EAE from the standpoint of myelin gene expression and repair. The findings have shown some transient clinical improvement and low-level remyelination during the acute phase, but no clinical improvement and no difference in the level of remyelination between controls and groups chronically treated with IGF-1, although some indication of an influence on immunoregulatory cytokines was seen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL/J mice (Jackson Laboratory, Bar Harbor, ME), were maintained in an NIH/AAALAC-approved facility. Donor mice were immunized at 5 to 12 weeks of age with an emulsion of 0.4 mg/ml bovine myelin basic protein (MBP; Sigma, St. Louis, MO), dissolved in PBS and emulsified with incomplete Freund’s adjuvant containing 60 µg of M. tuberculosis H37Ra (Difco Labs., Detroit, MI). Ten days later, lymph nodes were removed from these mice, the tissue homogenized into a single cell suspension, and the resultant cells cultured in the presence of 50 µg/ml of MBP for 3 days. These cells were then harvested, washed, counted and injected at a dose of 3 x 107 cells/0.2 ml into the tail vein of 5- to 6-week old naïve syngeneic recipients. Onset of signs in recipients occurred 6 to 10 days post-transfer (dpt) of cells. To prevent possible adverse physiological effects (hypoglycemia) in the IGF-1-treated groups, the diets of all animals were supplemented by the addition of sugar cubes to the regular mouse chow. Animals were graded according to a standard clinical index: 1 = limp tail; 2 = hind limb weakness; 3 = one limb plegic; 4 = plegia of 2 limbs; 5 = moribund or dead.4 A total of 99 mice sensitized for EAE were examined for this study (51 IGF-1-treated, 48 control).
Treatment with IGF-1
Recombinant human IGF-1 (rhIGF-1) was provided by Genentech (S. San Francisco, CA). The IGF-1 was suspended in a vehicle solution consisting of 10 mmol/L acetic acid, 0.1 mol/L NaCl, and 0.1% Tween, pH 5.4. To determine dose, vehicle and two doses of rhIGF-1 (100 µg and 200 µg) in vehicle were tested using osmotic pumps implanted under the skin over the animal’s back on day 3 post-transfer (pt) of cells and removed 14 days later. Twenty-four animals were tested, 8 in each group, under code. Animals given 100 µg of IGF-1 displayed greater amelioration of EAE than the 200 µg IGF-1 group. Based on the better clinical outcome, a dose of 100 µg of IGF-1 was selected. Animals were treated by subcutaneous (s.c.) injection of the selected dose of IGF-1 or vehicle, delivered twice daily for 14 days before onset of signs (3 to 17 dpt), and at different stages (beginning at 25 to 78 dpt) of the chronic phase of EAE. Control and IGF-1-treated groups were observed clinically and scored under code for effects on disease onset, severity, and relapse rate.
Neuropathology
For neuropathological analysis, representative animals were sampled at selected time points during and after treatment. Ether-anesthetized mice were perfused with 2.5% glutaraldehyde in phosphate buffer. CNS tissue was removed and thin slices taken from 10 levels of the neuraxis (optic nerve, cerebrum, cerebellum/brainstem, cervical, thoracic, lumbar and sacral cord, and spinal nerve roots. These were postfixed in 1% osmium tetroxide for 1 hour, dehydrated, and embedded in epoxy resin. One-micrometer epoxy sections were stained with toluidine blue and examined by light microscopy in a blinded fashion by two investigators. A score from 0 to 5 was determined for inflammation, demyelination, remyelination, and Wallerian degeneration, based on the degree of CNS involvement, according to established criteria.3,10 Remyelination was scored as follows: 1 = scattered thinly remyelinated fibers along the lesion margin; 2 = a narrow rim or small groups of remyelinated axons along the lesion margin; 3 = larger groups of remyelinating fibers; 4 = extensive remyelination of lesions; 5 = total remyelination. For electron microscopy (EM), thin sections were contrasted with uranyl acetate and lead citrate and examined in a Siemens 101.
Immunocytochemistry
Animals were perfused with PBS, the CNS removed, and slices from cerebral hemispheres, cerebellum, cervical, thoracic and lumbar cord embedded in OCT in a dry-ice bath. Frozen sections were fixed in acetone for 10 minutes and stained using the avidin-biotin-peroxidase complex technique (Vector Laboratories, Burlingame, CA). Overnight incubations were performed at 4°C with the following primary antibodies: rabbit anti-human IGF-1R (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 1:100 dilution; mouse anti-human IGF-1R (PharMingen, San Diego, CA) at 1:20; rabbit anti-human PDGF-R7 (a kind gift of C-H. Heldin of the Ludwig Institute for Cancer Research, Uppsala, Sweden) at 1:200; rabbit anti-rat NG2 (a kind gift of W. B. Stallcup, La Jolla, CA) at 1:100; anti-CNPase (Sigma) at 1:20; and rabbit anti-mouse MBP exon-2 (a kind gift of Dr. Carol Readhead, University of Southern California). Slides were scored in a blinded fashion by two observers on a scale of 0 to 4 based on the density of labeled cells.
Ribonuclease Protection Assay
For RNA preparation, anesthetized mice were perfused transcardially with ice-cold PBS and the spinal cord removed and snap-frozen in liquid nitrogen. Total RNA was extracted with tri-reagent (Molecular Research Center, Cincinnati, OH), as per manufacturer’s instructions using a Power Gen 125 homogenizer (Fisher Scientific), and adjusted to 5 µg/µl RNase-free H2O. The expression of various cytokine mRNAs was determined using a multiprobe RPA template set (Riboquant; PharMingen). A total of 20 µg total RNA from spinal cord was hybridized overnight to the [32P]UTP-labeled cRNA transcripts of the mCK-3b probe set using the RPA II Kit (Ambion, Austin, TX). Non-hybridized RNA was digested and the protected fragments were precipitated, dissolved in loading buffer and loaded on a denaturing 5% acrylamide gel. Bands were detected by autoradiography and quantified by phosphoimaging on a Storm 860 scanner using the ImageQuaNT 3.01 software package (Molecular Dynamics, San Francisco, CA). Results were calculated as a ratio of the volume of the band of interest to the sum of the bands for the housekeeping genes, L32 and GAPDH. Statistical significance was determined by Student’s t-test and ANOVA with a threshold value of P < 0.01.
Statistical Methods
The effect of treatment on clinical outcome was assessed by Student’s t-test and when the data were not normally distributed, the Mann-Whitney sum of ranks test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The clinical charts of six representative experimental groups are
shown in Figure 1
. There were no
significant differences in body weight between treated and control
groups.
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Mice treated with rhIGF-1 preceding and during the acute phase of
adoptively transferred EAE (2 x 100 µg rhIGF-1 per diem, s.c.
for 14 days beginning 3 dpt), displayed a less severe clinical course
commencing on the same day of onset as vehicle-treated animals (4–6
dpt) (Figure 1)
. In one experiment (Figure 1A)
, the clinical course of
the IGF-1-treated group with EAE (n = 5),
remained at least one clinical grade lower than the vehicle-treated
control group (n = 5), the differences being
significant at day 7 (P = 0.024), day 14
(P = 0.021), day 17 (P =
0.041) using the t-test, and on day 10
(P = 0.016) using the Mann-Whitney sum of ranks
test. However, in the subsequent experiment (Figure 1B)
, the clinical
scores for the two groups were different only at day 6
(P = 0.009, Mann-Whitney) and day 7
(P = 0.022, t-test). In the third
experiment (Figure 1C)
, there were no differences.
IGF-1 Treatment during the Chronic Phase
Three experiments were conducted during which mice with
established chronic EAE were administered rhIGF-1 at different time
points post-transfer (viz. days 25–39, days 41–55, and days 65–79
pt; Figure 1, D–F
). In the first experiment, the clinical course of
the IGF-1-treated group (n = 5), appeared to
improve posttreatment. In the second and third groups, the
IGF-1-treated animals (n = 3), maintained
courses similar to those of the vehicle-treated controls (Figure 1, D–F)
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Neuropathology
IGF-1 Treatment during the Acute Phase
At early time points during treatment, CNS lesions in
IGF-1-treated animals were less inflammatory and demyelinative than in
vehicle-treated controls, an observation correlating with the observed
decreased clinical severity (Table 1
and
Figure 1
). However, with time, as the clinical course of both groups
became similar, the histopathology was comparable to the extent that
the two groups could not be distinguished. In general, however, control
groups tended to show more Wallerian degeneration (axonal
pathology), than treated groups during early time points (Table 1)
.
Lesions were typically distributed around the subpial regions of the
lumbar spinal cord (Figure 2A)
.
Interestingly, while control animals sampled during the acute phase of
disease had inflammatory demyelinating lesions, similar to
IGF-1-treated groups, one animal from an IGF-1 group sampled on day 10
pt, displayed suggestions of low level remyelination (Figure 2, B and D)
, with several fibers possessing disproportionately thin myelin
sheaths around large diameter axons. These fibers existed in areas
showing ongoing inflammation and demyelination. These same areas were
examined by EM and remyelination was confirmed. The thin myelin sheaths
were associated with numerous outer loops of oligodendroglial
cytoplasm, (Figure 3,A and B)
, features
reminiscent of early myelination.11
Control-treated and
animals treated with IGF-1 during the acute phase and maintained
long-term (up to 11 months pt), displayed spinal cord lesions that were
indistinguishable from one another, being intensely gliotic with some
residual inflammatory activity (Figure 2, E and F)
. Such lesions
contained numerous chronically demyelinated axons and a few
remyelinated fibers toward the periphery. However, there was no
difference in the degree of remyelination in both groups.
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In all, five experiments were conducted in which mice that had
developed chronic EAE were treated with IGF-1 and compared with
vehicle-treated controls (Table 2)
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Except for a reduced amount of inflammation in one group,
IGF-1-injected animals treated from 25 to 39 dpt, the same group
showing a clinical difference (Figure 1D)
, there were no differences in
any of the pathological parameters examined (inflammation,
demyelination, remyelination and Wallerian degeneration) (Table 2)
.
Variation in pathology was noted in groups treated at different time
points post-transfer, although there were no apparent effects
attributable to IGF-1. For example, in one group given IGF-1 from 41 to
55 dpt and examined on day 60, both groups still displayed abundant
inflammation and Wallerian degeneration while the levels of
demyelination and remyelination were unremarkable (Figure 4, A and B)
. In another group treated from
65–79 dpt and sampled on day 93, more typical gliotic and demyelinated
lesions were seen (Figure 4, C–F)
. None of the mice treated with IGF-1
during the chronic phase of EAE displayed a level of CNS remyelination
significantly different from vehicle-treated controls.
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As part of our study on the possible effects of IGF-1 on
remyelination in this model, frozen sections immunoreacted for IGF-1R,
PDGF-R, and MBP exon 2 transcripts were examined from all treated and
control groups at different time points post transfer. In most cases,
IGF-1R staining was seen on oligodendrocytes although the level of
expression was low and similar between groups (Figure 5A)
. Occasionally, astrocytes displayed
immunoreactivity for IGF-1R. Staining for PDGF-R, a marker for
progenitor oligodendrocytes,12-14
was obtained in
sections from both acute and chronic IGF-1-treated and control groups
where scattered oligodendrocytes throughout the white matter displayed
immunoreactivity (Figure 5B)
. There was some increase in
immunoreactivity for PDGF-R toward the margins of lesions but there
were no differences in pattern at any time point between IGF-1 and
control-treated mice. With a polyclonal rabbit antibody raised against
mouse MBP exon 2 peptide, evidence for immature or remyelinating
oligodendrocytes,15
was sought in both IGF-1-treated and
control mice, at early and late time points. In all animals, a few MBP
exon 2-positive oligodendrocytes could be found at the margins of CNS
lesions and scattered throughout the spinal cord white matter (Figure 5, C–F)
. The numbers of MBP exon 2 positive cells were similar in
IGF-1 and control-treated mice. Oligodendrocyte phenotype was confirmed
with parallel sections stained for CNPase and MAG (Figure 4, G and H)
.
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Data obtained with the mCK-3b set for cytokines from spinal cord
mRNA from IGF-1-treated and control mice are shown (Figure 6)
. No differences were noted for the
expression of mRNA for TNF-
, IFN-
, IL-6, and MIF. LT and
TGF-ß1 levels were not detectable in any of the samples. However,
significant differences in the relative levels of mRNA were observed
for TGF-ß2 on day 93 pt of the chronic treatment group, and for
TGF-ß3 at both time points during acute treatment and the first time
point in the chronic treatment groups (Figure 7)
. The levels of these two cytokines
were much reduced in IGF-1-treated mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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That a previous9 and the present study on the IGF-1 paradigm in EAE has demonstrated transient beneficial effects suggests a common mechanistic pathway. Previous workers9 examined brain and spinal cord from experimental and control groups for adhesion molecule expression and found decreased ICAM-1 in IGF-1-treated mice. This, they suggested, may merely have reflected differences in inflammatory cell infiltration. On the other hand, it may have correlated with the delayed entry of cells into the CNS. Results from the same authors, using lymph node cells stimulated in vitro with MBP and/or IGF-1, revealed no significant differences in cytokine profile. From the present work, two cytokines displayed significant differences in expression in the CNS between experimental and control groups. These were TGF-ß2 and TGF-ß3 and the decreases in expression in the IGF-1-treated groups occurred at both acute and chronic time points. Whether this represents evidence for decreased immunomodulatory cytokine activity (at the mRNA level, at least), has relevance to the transient nature of the beneficial effect on disease severity, remains a possibility in need of further investigation. This is particularly apropos since TGF-ß may play an important role in oligodendrocyte differentiation,19 and is an effective therapeutic molecule in the treatment of acute and chronic relapsing EAE.20-22 More recently, TGF-ß2 has been reported to reduce demyelination in the Theiler’s virus model,23 further testifying to its beneficial effect on CNS myelin. These observations may be relevant to the present findings in that the decrease in TGF-ß2 and -ß3 may be the indirect effect of IGF-1 acting on cells of the immune system and the CNS, where astrocytes have been demonstrated to synthesize TGF-ß1, -ß2, and -ß3.24
The observation of "precocious" remyelination seen after treatment during the acute phase of EAE may be genuine and related to a stimulatory effect on oligodendrocytes by IGF-1, as has been shown at the level of myelin gene expression in previous work on rat EAE.16 However, it was encountered in a single animal only and may have been the result of a number of pleiotropic factors, as has been speculated in acute fulminant MS lesions where remyelination is also a feature,25,26 the result perhaps of soluble mediators produced by macrophages. Nevertheless, the appearances were consistent with remyelination (as opposed to incomplete demyelination), since it was associated with elaboration of multiple oligodendrocyte processes. On the down side, this appeared to be a transient repair phenomenon, as is believed to be the case in MS. Gene expression by oligodendrocytes examined from the standpoint of progenitor cells and myelin demonstrated a lack of increased involvement of myelinating cells in IGF-1-treated mice.
Taken in concert, under the conditions studied, IGF-1 had a marked transient beneficial effect on the early course of EAE in the mouse when administered before clinical onset, but no measurable clinical or histopathological effect, particularly at the level of remyelination, when delivered during chronic disease. Since the latter situation is that which parallels most closely the course of human MS, it is therefore doubtful that IGF-1 would be efficacious in the human disease. Thus, while manipulation of immune-mediated demyelinating disease with growth factors with known propensities to cause oligodendrocyte stimulation or proliferation provides a novel approach which avoids complications arising during immune-mediated therapy, careful screening of each growth factor in different models remains essential.
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Acknowledgements |
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Footnotes |
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Supported in part by National Multiple Sclerosis Society grant RG 1001-J-10; USPHS grants NS 08952, NS 11920 and NS 07098; a grant from Genentech Inc., S. San Francisco, CA; and the Wollowick Family Foundation.
Accepted for publication June 6, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  V. C. Russo, P. D. Gluckman, E. L. Feldman, and G. A. Werther The Insulin-Like Growth Factor System and Its Pleiotropic Functions in Brain Endocr. Rev., December 1, 2005; 26(7): 916 - 943. [Abstract] [Full Text] [PDF]
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 J. L. Mason, S. Xuan, I. Dragatsis, A. Efstratiadis, and J. E. Goldman Insulin-Like Growth Factor (IGF) Signaling through Type 1 IGF Receptor Plays an Important Role in Remyelination J. Neurosci., August 20, 2003; 23(20): 7710 - 7718. [Abstract] [Full Text] [PDF]
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