Thermomineral water promotes axonal sprouting but does not reduce glial scar formation in a mouse model of spinal cord injury

Dubravka Aleksić, Milan Aksić, Nevena Divac, Vidosava Radonjić, Branislav Filipović, Igor Jakovčevski, Dubravka Aleksić, Milan Aksić, Nevena Divac, Vidosava Radonjić, Branislav Filipović, Igor Jakovčevski

Abstract

Thermomineral water from the Atomic Spa Gornja Trepča has been used for a century in the treatment of neurologic disease. The thermomineral water contains microelements, including lithium and magnesium, which show neural regeneration-promoting effects after central nervous system injury. In this study, we investigated the effects of oral intake of thermomineral water from the Atomic Spa Gornja Trepča on nerve regeneration in a 3-month-old mouse model of spinal cord injury. The mice receiving oral intake of thermomineral water showed better locomotor recovery than those without administration of thermomineral water at 8 and 12 weeks after lower thoracic spinal cord compression. At 12 weeks after injury, sprouting of catecholaminergic axons was better in mice that drank thermomineral water than in those without administration of thermomineral water, but there was no difference in glial reaction to injury between mice with and without administration of thermomineral water. These findings suggest that thermomineral water can promote the nerve regeneration but cannot reduce glial scar formation in a mouse model of spinal cord injury.

Keywords: astrocyte scar; catecholaminergic innervations; lithium; locomotor recovery; magnesium; microglia; nerve regeneration; neuroprotection; spinal cord injury; thermomineral water.

Conflict of interest statement

Conflicts of interest: None declared.

Figures

Figure 1
Figure 1
Time course of functional recovery after compressive spinal cord injury in mice from the experimental (EXP) and control (CON) groups. The values are shown as the mean ± SEM (n = 8 mice/group) of the open field locomotion score (Basso Mouse Scale (BMS)) at 3 days (d) and at 1, 2, 4, 8 and 12 weeks (w) after injury. Asterisks indicate significant differences between the groups (P < 0.05; two-way analysis of variance followed by Tukey's post-hoc test).
Figure 2
Figure 2
Sprouting of tyrosine hydroxylase (TH)-expressing axons caudal to the lesion site 12 weeks after injury. (A, B) Representative photomicrographs of immunofluorescent staning for TH (red) in the spinal cord of mice from the control (A) and experimental (B) groups. Scale bar: 100 μm. (C) Number of TH+ axons in the spinal cord crossing the line drawn 250 μm caudal to the lesion site (white lines in A, B) in mice from the experimental (EXP) and control (CON) groups (n = 8 mice/group). Values are shown as the mean ± SEM. Asterisk indicates significant difference between the groups (P = 0.026; two-tailed t-test).
Figure 3
Figure 3
NeuN+ neurons rostral and caudal to the lesion site at 12 weeks after spinal cord injury. (A, B) Representative photomicrographs of immunofluorescent staning for NeuN (red) in the lumbar spinal cord of mice from the control (A) and experimental (B) groups. Scale bar: 100 μm. (C) Density of NeuN+ cells in the spinal cord rostral and caudal from the injury site in mice from the experimental (EXP) and control (CON) groups (n = 8 mice/group). Values are shown as the mean ± SEM. There were no significant differences between the experimental and control groups (P = 0.50 and 0.62 for rostral and caudal, respectively; two-tailed t-test). NeuN: Neuronal nuclear antigen.
Figure 4
Figure 4
Glial fibrillary acidic protein (GFAP) expression around the lesion site at 12 weeks after spinal cord injury. (A, B) Representative photomicrographs of the GFAP-immunostained (red) injured spinal cords of mice from the control (A) and experimental (B) groups. White squares depict areas in which the measurements were performed. Scale bar: 200 μm. (C) Percentage of GFAP-immunoreactive area normalized to the total image area (shown as the mean ± SEM). There was no significant difference between the experimental and control groups (P = 0.77, two-tailed t-test; n = 8 mice/group). CON: Control group; EXP: experimental group
Figure 5
Figure 5
Iba-1+ microglia within the lesion site at 12 weeks after spinal cord injury. (A, B) Representative photomicrographs of the Iba-1-immunostained (red) lesion sites of spinal cords of mice from the control (A) and experimental (B) groups. White squares depict areas in which the measurements were performed. (C) Percentage of Iba-1-immunoreactive area normalized to the total image area (shown as the mean ± SEM). There was no significant difference between the experimental (EXP) and control (CON)groups (P = 0.30, two-tailed t-test; n = 8 mice/group). Scale bar: 200 μm. Iba-1: Ionized calcium-binding adapter molecule 1.

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