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. 2005 Apr 6;2(1):10.
doi: 10.1186/1742-2094-2-10.

Anti-inflammatory therapy by ibudilast, a phosphodiesterase inhibitor, in demyelination of twitcher, a genetic demyelination model

Kuriko Kagitani-Shimono  1 , Ikuko Mohri, Yasushi Fujitani, Kinuko Suzuki, Keiichi Ozono, Yoshihiro Urade, Masako Taniike
Affiliations

Anti-inflammatory therapy by ibudilast, a phosphodiesterase inhibitor, in demyelination of twitcher, a genetic demyelination model

Kuriko Kagitani-Shimono et al. J Neuroinflammation. .

Abstract

BACKGROUND: Twitcher mouse (twi/twi) is an authentic murine model of Krabbe's disease. Accumulation of psychosine, resulting in apoptosis of oligodendrocytes and subsequent demyelination, is a cardinal event to the pathogenesis of this disease. Moreover, recruitment of inflammatory cells plays a significant role in the pathological process in the twi/twi central and peripheral nervous systems. In this study, we investigated the 1) the relationship between tumor necrosis factor-alpha (TNFalpha), pro-inflammatory cytokine, and the progression of this disease and 2) effect of the anti-inflammatory therapy by ibudilast, a phosphodiesterase inhibitor. METHODS: We quantified the expression level of TNFalpha and TNF-receptor mRNA in twi/twi using semi-quantitative RT-PCR. The relationship between TNFalpha expression, apoptosis of oligodendrocytes and demyelination was studied with immunohistochemistry and TUNEL method. We then treated twi/twi with a daily intraperitoneal injection of ibudilast (10 mg/kg), which suppress TNFalpha production in the brain. RESULTS: We found that TNFalpha-immunoreactive microglia/macrophages appeared in the twi/twi brain and that the mRNA levels of TNFalpha and TNF-receptor 1 was increased with the progression of demyelination. The distribution profile of TNFalpha-immunoreactive microglia/macrophages overlapped that of TUNEL-positive oligodendrocytes in the twi/twi brain. When twi/twi was treated with ibudilast from PND30, the number of oligodendrocytes undergoing apoptosis was markedly reduced and demyelination was milder. Obvious improvement of clinical symptom was noted in two of five. The failure of constant clinical improvement by ibudilast may result from hepatotoxicity and/or the inhibition of proliferation of NG2-positive oligodendrocyte precursors. CONCLUSION: We conclude that anti-inflammatory therapy by a phosphodiesterase inhibitor can be considered as a novel alternative therapy for Krabbe's disease.

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Figures

Figure 1
Figure 1
TNFα and its receptors increased as demyelination proceeded. A-B: Quantification of mRNA for TNFα (A) and its receptors (B). The copies of mRNA for TNFα have increased in twi/twi (■) after PND 30, especially in the cerebellum, when compared with those in +/+ (▴). Those for TNFR1 in the cerebellum have increased in twi/twi after PND 30. The copies of mRNA for TNFR2 have increased in twi/twi only after PND 40, when compared with those for +/+, but the difference was not significant (B). Bar represents mean ± SE. * p < 0.01. C-F: TNFα immunostaining in the cerebellum. There are no TNFα-positive cells in the cerebellum of twi/twi mice at PND 20 (C). Immunoreactive cells for TNFα are progressively increased in number in the twi/twi cerebellar white matter between PND 30 (D) and PND 40 (E). In contrast, there are no TNFα positive cells in +/+ brains at any ages examined (F). Tw and W represent twi/twi and wild-type mice, respectively. The data represent mean ± SE. IG: internal granular layer, CWM: cerebellar white matter. Scale bar = 50 μm.
Figure 2
Figure 2
TNFα is expressed in activated microglia/macrophages in the regions where many apoptotic OLs are recognized with severe demyelination. A: Double labeling of TNFα and RCA-1 of the twi/twi cerebrum at PND 40. Arrows indicate microglia/macrophages, which are double positive for TNFα and RCA-1. B-J : In twi/twi at PND 40, there are many TNFα-positive cells (B, E) as well as many TUNEL-positive cells (C, F) in the CWM and sp5, where severe demyelination was present as judged from the results of MBP immunostaining (D, G). These apoptotic cells are immunostained with pi-GST, identified to be OLs (inset in C). In the corpus callosum (cc), there are only a few TNFα-positive cells (H) and TUNEL-positive cells (I), where demyelination was milder than in the cerebellum (J). Asterisks and double asterisks represent the same region in the serial sections. Scale bars = 50 μm (B-J), 10 μm (inset in "C").
Figure 3
Figure 3
A: Two twi/twi at PND 44, one ibudilast-treated and other vehicle-treated from PND 30. The ibudilast-treated twi/twi is much bigger and can walk faster and reach the feedbox, in spite of mild paralysis and spasticity in lower limbs. In contrast, the vehicle-treated twi/twi can no longer walk nor feed itself. In addition, the ibudilast-treated twi/twi has much milder tremor than the vehicle-treated twi/twi. B: The change of body weight (g) of ibudilast- and vehicle-treated twi/twi. Both twi/twi treated with ibudilast or vehicle from PND 15 (●: ibudilast-treated twi/twi, ○: vehicle-treated twi/twi) showed less weight gain compared with those treated from PND 30 (■: ibudilast-treated twi/twi, □: vehicle-treated twi/twi), and no prolongation of the life span. However, ibudilast-treated twi/twi showed less body weight loss than vehicle-treated twi/twi. N = 3 and 2 in ibudilast- and vehicle-treated twi/twi from PND 15. The ibudilast-treated twi/twi from PND 30 were bigger and showed milder clinical detrerioration. N = 5 and 4 in ibudilast- and vehicle-treated twi/twi from PND 30. The data represent mean ± SE.
Figure 4
Figure 4
Suppression of TNFformula image mRNA expression is accompanied by inhibition of apoptosis and subsequent milder demyelination in ibudilast-treated twi/twi at PND45. A, B, E, F, I, J: CWM, C, D, G, H, K, L: sp5. A-D: In situ hybridization of TNFα mRNA in vehicle-treated twi/twi (A, C) and ibudilast-treated twi/twi (B, D). Whereas vehicle-treated twi/twi show abundant signals in CWM (A) and sp5 (C), TNFα mRNA signals are remarkably reduced in the ibudilast-treated twi/twi (B, D). Inset in "A" shows TNF-α mRNA-positive microglia. E-H: TUNEL staining of vehicle-treated twi/twi (E, G) and ibudilast-treated twi/twi (F, H). Ibudilast-treated twi/twi shows fewer TUNEL-positive cells than are seen in vehicle-treated twi/twi. Arrowheads indicate TUNEL-positive cells. I-L: LFB-PAS staining of vehicle-treated twi/twi (I, K) and ibudilast-treated twi/twi (J, L). In the ibudilast-treated twi/twi, CWM and sp5 show much milder demyelination than in vehicle-treated twi/twi. Scale bar = 100 μm (I-L), 50 μm (A-H), 10 μm (inset in "A").
Figure 5
Figure 5
Ibudilast-treated twi/twi show pathological improvement. Population of TUNEL-positive cells and neuropathological scores of LFB-PAS in ibudilast- (closed-boxed; N = 4) or vehicle-treated (hatched; N = 3) twi/twi. In CWM, 8 n, and sp5 of the ibudilast-treated twi/twi, the number of TUNEL-positive cells is decreased to half of those in the vehicle-treated twi/twi. They also recognized significantly milder demyelination in LFB-PAS stain. 8 n: the 8th nerve. *p < 0.01, **p < 0.05. The error bars represented standard deviations.
Figure 6
Figure 6
Ibudilast surpresses proliferation of NG2-positive OL progenitors. A: Vehicle-treated twi/twi shows many NG2-positive OL progenitors. B: Ibudilast-treated twi/twi shows decreased number of NG2-positive OL progenitors. Allows: NG2-positive OL progenitors labeled with Alexa 488. Scale bar = 50 μm
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