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, 26 (4), 28-38

Functional Antagonism of Sphingosine-1-Phosphate Receptor 1 Prevents Harmaline-Induced Ultrastructural Alterations and Caspase-3 Mediated Apoptosis

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Functional Antagonism of Sphingosine-1-Phosphate Receptor 1 Prevents Harmaline-Induced Ultrastructural Alterations and Caspase-3 Mediated Apoptosis

Narjes Dahmardeh et al. Malays J Med Sci.

Abstract

Background: There is a meaningful necessity for a targeted therapy of essential tremor (ET), as medications have not been developed specifically for ET. For nearly a century, many drugs have been applied in the treatment of tremor but the drug treatment of ET remains still unknown. Some potential therapeutic factors such fingolimod (FTY720) can be effectively used to treat ET in animals. In the present research, the effect of FTY720, the immunomodulatory sphingosine 1-phosphate (S1P) analog, on degeneration of cerebellar and olivary neurons induced by harmaline in male rats was investigated.

Methods: The animals were allotted into control dimethyl sulfoxide (DMSO), saline + harmaline [30 mg/kg, intraperitoneally, (i.p.)], harmaline + FTY720 (1 mg/kg, i.p, 1 h and 24 h before harmaline injection) groups (n = 10). The cerebellum and inferior olive nucleus (ION) were studied for neuronal degeneration using immunohistochemistry (IHC) and ultrastructural study by transmission electron microscopy (TEM) techniques.

Results: Harmaline caused neuronal cell loss, caspase-3 mediated apoptosis, astrocytosis and ultrastructural changes in cerebellar Purkinje cells and inferior olive neurons. FTY720 exhibited neuroprotective effects on cerebellar Purkinje cells and inferior olivary neurons.

Conclusion: These results suggest that FTY720 has potential efficacy for prevention of ET neurodegeneration and astrocytosis induced by harmaline in male rats.

Keywords: apoptosis; essential tremor; fingolimod; harmaline; rats.

Conflict of interest statement

Conflict of Interest None.

Figures

Figure 1
Figure 1
Immunohistochemical analysis of GFAP+ cells in the cerebellum of rats (right panel) and ION (left panel). DMSO (A), harmaline (B), FTY720/1h (C) and FTY720/24 h (D). Scale bar: 10 μm. Bottom panel, the bar graph shows the quantitative analysis of GFAP+ cells in the cerebellum and ION of rats in different groups. Data are presented as means S.E.M. In the cerebellum * was considered for P < 0.045 compared with DMSO group and # was for P < 0.006 compared with harmaline group. In ION * was considered for P < 0.001 compared with the DMSO group and # was considered for P < 0.001 compared with harmaline (one-way ANOVA with Tukey’s post-hoc test for all comparisons).
Figure 2
Figure 2
Upper panel, immunohistochemical analysis of caspase-3+ cells in the cerebellum. DMSO (A), harmaline (B), FTY720/1h (C) and FTY720/24 h (D). Scale bar: 10 μm. White arrow shows normal cell (neuron) and black arrow shows caspase-3+ neuron. Bottom panel, the bar graph shows the number of caspase-3+ neurons was increased in harmaline group and pretreatment with FTY attenuated caspase-3+ cells. ** P < 0.001 compared with DMSO group. # P < 0.031 compared with harmaline group (one-way ANOVA with Tukey’s post-hoc test for all comparisons).
Figure 3
Figure 3
Upper panel, immunohistochemical analysis of caspase-3+ in inferior olive of the rat. DMSO (A), harmaline (B), FTY720/1h (C) and FTY720/24h (D). Scale bar: 10 μm. White arrow shows normal cell (neuron) and black arrow shows apoptotic neuron. Bottom panel, bar graph show the number of caspase-3+ neuron was increased in harmaline group and pretreatment with FTY attenuated caspase-3+ cells. * P < 0.001 compared with DMSO group. # P < 0.008 compared with harmaline group (one-way ANOVA with Tukey’s post-hoc test for all comparisons).
Figure 4
Figure 4
Upper panel, an electron micrograph of the ultrastructure of Purkinje cells in the cerebellum of rats. Bottom panel, an electron micrograph of the ultrastructure of inferior olive neurons of rats. The prominent nucleolus (black arrow), intact nucleolemma and cell membrane (yellow and white arrows) were visible in part A. Note ultrastructural alterations including chromatin aggregation (dark red arrow) nuclear deformity and apoptotic bodies (narrow red arrow) in part B. Control (DMSO) (A), harmaline (B), FTY720/1h (C) and FTY720/24h (D). Scale bar: 1.5 μm.

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