Insulin-like growth factor-1 fails to enhance central nervous system myelin repair during autoimmune demyelination

Abstract

Previous studies have shown that insulin-like growth factor-1 (IGF-1) has beneficial effects, both clinically and histopathologically, on experimental autoimmune encephalomyelitis (EAE), although results vary depending on species and treatment regimen. The present study investigated whether IGF-1, delivered at different time points during the acute and chronic phases of adoptively transferred EAE in SJL mice, had the ability to affect or enhance myelin regeneration. Central nervous system tissue sampled at different stages of treatment was subjected to detailed neuropathological, immunocytochemical and molecular analysis. The results revealed some transient clinical amelioration and low level remyelination after IGF-1 administration during the acute phase of EAE. However, central nervous system tissue from acute phase treated animals sampled at chronic time points and from animals given IGF-1 during the chronic phase revealed no enhancing effect on remyelination in comparison to vehicle-treated controls. Examination of oligodendrocyte progenitor populations also revealed no differences between IGF-1- and vehicle-treated groups. At the cytokine level, the immunomodulatory molecules TGF-beta2 and TGF-beta3 displayed significant decreases that may have contributed to the transient nature of the effect of IGF-1 on EAE. Together with evidence from previous studies, it appears doubtful that IGF-1 is a good candidate for treatment in multiple sclerosis, for which EAE serves as a major model.

Figure 1.

Clinical charts of three experiments showing mice treated during the acute phase of EAE (A–C) and during the chronic phase (D–F). Arrows indicate period of treatment. In A and B, vehicle- and IGF-1-treated groups showed significantly different mean clinical scores at several time points. In A, using the Student t-test, IGF-1 provided protection at day 7 (P = 0.024), day 14 (P = 0.021), and day 17 (P = 0.041) and using the Mann-Whitney sum of rank analysis, on day 10 (P = 0.0l6). In B, the scores were significantly different on day 6 (P = 0.009, Mann-Whitney), and on day 7 (P = 0.022, t-test). Among the chronic phase groups, no significant differences were seen. y axis, clinical grade 1–4; x axis, days after transfer of cells.

Figure 2.

Pathology of lumbar spinal cord tissue from animals treated during the acute phase with IGF-1 and vehicle, sampled day 10 pt (A–D), and after 11 months pt (E–F). One-micrometer epoxy sections stained with toluidine blue. A: Vehicle-treated, 10 dpt, clinical score, grade 4. A large inflammatory demyelinated lesion is seen in an anterior column (×300). B: IGF-1-treated, 10 dpt, grade 1. Matching treated group for A. A less extensive inflammatory demyelinating lesion is seen in the anterior column. The asterisk indicates three fibers shown in detail in D (×300). C: Detail from A to show demyelinated axons (arrows) and macrophages containing myelin debris (×750). D: Same lesion as in B, higher magnification. The group of three fibers (shown in B) possess thin myelin sheaths suggestive of remyelination (asterisk). Demyelinated axons are also apparent (arrows) (×750). E: Vehicle-treated, 11 months pt, grade 3.5. A discrete zone of chronic demyelination and glial scarring in the subpial layer is seen. Note the glial scarring and demyelinated axons (arrows). A few remyelinated axons are present (asterisks) (×750). F: IGF-1-treated, 11 months pt, grade 2. In a matching animal, a lesion comparable in texture to E is seen. Demyelinated axons are shown at the arrows (×750).

Figure 3.

A: EM of a CNS lesion is shown from a mouse with EAE treated with IGF-1 during the acute phase and sampled day 10 pt (same field as shown in Figure 2, B and D ▶ ). The group of three thinly myelinated axons (asterisk) is seen to lie in the midst of a demyelinated, inflamed lesion. Vessel (V) above, demyelinated axons (a) (×3750). B: Detail of A illustrating the disproportionately thin myelin sheaths and oligodendroglial cytoplasmic tongues (arrows), features usually associated with ongoing myelination and remyelination (×16,000).

Figure 4.

Lumbar spinal cord tissue from mice with EAE treated with vehicle and IGF-1 during the chronic phase (41–55 dpt), and sampled day 60 (A and B), and 65–79 dpt, sampled day 93 (C–F). A: Vehicle-treated, grade 3. A gliotic demyelinated lesion is seen in the subpial zone. Some Wallerian degeneration and a few remyelinated fibers (arrows) are present. Inflammatory cells are located within the meningeal space above (×750). B: IGF-1-treated, grade 2, matching animal to A. A small lesion lies along the subpial margin. Note the extensive glial scarring, Wallerian degeneration, and a few infiltrating cells above (×750). C: Vehicle-treated, grade 2.5. A large chronically demyelinated lesion lies in an anterior column. Some inflammation is seen in the leptomeninges above (×300). D: IGF-1-treated, grade 3.5; matching animal to C. A lesion similar to the control is shown. Note the inflammation in the leptomeninges, above, and the widespread gliosis (×300). E: Detail of C to show glial scarring, chronically demyelinated axons (arrows) and Wallerian degeneration. Remyelinated axons are seen at the asterisks (×750). F: Detail of D to show scattered chronically demyelinated axons (arrows) and a few remyelinated fibers (asterisks) (×750).

Figure 5.

Lumbar spinal cord tissue from mice with EAE treated with IGF-1 or vehicle, immunoreacted for oligodendrocyte progenitors. No differences were observed between IGF-1 and control-treated groups at any time point. A: IGF-1 treatment during acute phase, sampled day 17 pt. Immunoreacted for IGF-1R. Several small rounded oligodendrocytes (arrows) show cytoplasmic staining (×750). B: Same animal as A, immunoreacted for PDGF-R7, a marker for oligodendrocyte progenitors. Several oligodendrocytes (arrows) display positive staining (×750). C: IGF-1 treatment, acute phase, sampled day 71 pt, immunoreacted for exon 2 MBP. Several oligodendrocytes (arrows) are intensely immunoreactive for this marker of immature oligodendrocytes (×750). D: Vehicle treatment, acute phase, sampled day 71 pt. Similar exon 2 MBP immunoreactivity is seen on two oligodendrocytes (×750). E: IGF-1 treatment, chronic phase (25–39 dpt), sampled day 52 pt, exon 2 MBP immunoreacted. A row of positively stained oligodendrocytes is seen (×750). F: Vehicle treatment, matching control for E, exon 2 MBP immunoreacted. Two oligodendrocytes stain intensely. Lesion activity is seen to the right (×750). G: Same animal as in A, CNPase immunoreacted. Note the intense staining of many oligodendrocytes (×750). H: Same animal as A, anti-MAG immunoreacted. Oligodendrocytes display moderate levels of immunoreactivity (×750).

Figure 6.

Multiprobe RPA analysis of cytokine mRNA expression in the spinal cord of IGF-1 (tx) and vehicle (v) treated mice measured at selected time points after treatment. The CNS was removed from saline-perfused animals and subjected to RPA analysis using the mCK-3b multiprobe template set (see Methods). Note that expression of TGF-β2 is decreased after treatment at one chronic time point. Also, TGF-β3 shows significant decreases after treatment. Figure 7 ▶ illustrates the quantitative analysis of these two cytokines, in addition to TNF-α, IFN-γ, IL-6, and MIF, none of which display significant changes.

Figure 7.

The gel shown in Figure 6 ▶ was phosphoimaged and the volumes of the protracted bands normalized to the bands for L32 and GAPDH. The data shown for TGF-β3 illustrates statistically significant differences in relative levels of mRNA between the vehicle- and IGF-1-treated animals at both time points, as is also the case for TGF-β2 at day 93 pt (asterisks).