Lab Anim Res.  2013 Sep;29(3):148-155. 10.5625/lar.2013.29.3.148.

Systemic administration of low dosage of tetanus toxin decreases cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus

• Yan BC
• Kim IH
• Park JH
• Ahn JH
• Cho JH
• Chen BH
• Lee JC
• Choi JH
• Yoo KY
• Lee CH
• Cho JH
• Kim JD
• Won MH

• KMID: 2312107
• DOI: http://doi.org/10.5625/lar.2013.29.3.148

Abstract

In the present study, we investigated the effect of Tetaus toxin (TeT) on cell proliferation and neuroblast differentiation using specific markers: 5-bromo-2-deoxyuridine (BrdU) as an exogenous marker for cell proliferation, Ki-67 as an endogenous marker for cell proliferation and doublecortin (DCX) as a marker for neuroblasts in the mouse hippocampal dentate gyrus (DG) after TeT treatment. Mice were intraperitoneally administered 2.5 and 10 ng/kg TeT and sacrificed 15 days after the treatment. In both the TeT-treated groups, no neuronal death occurred in any layers of the DG using neuronal nuclei (NeuN, a neuron nuclei maker) and Fluoro-Jade B (F-J B, a high-affinity fluorescent marker for the localization of neuronal degeneration). In addition, no significant change in glial activation in both the 2.5 and 10 ng/kg TeT-treated-groups was found by GFAP (a marker for astrocytes) and Iba-1 (a marker for microglia) immunohistochemistry. However, in the 2.5 ng/kg TeT-treated-group, the mean number of BrdU, Ki-67 and DCX immunoreactive cells, respectively, were apparently decreased compared to the control group, and the mean number of each in the 10 ng/kg TeT-treated-group was much more decreased. In addition, processes of DCX-immunoreactive cells, which projected into the molecular layer, were short compared to those in the control group. In brief, our present results show that low dosage (10 ng/kg) TeT treatment apparently decreased cell proliferation and neuroblast differentiation in the mouse hippocampal DG without distinct gliosis as well as any loss of adult neurons.

Keyword

Exotoxin; neuronal damage; neurogenesis; sub-granular zone; granule cell

MeSH Terms

Adult
Animals
Bromodeoxyuridine
Cell Proliferation
Dentate Gyrus
Exotoxins
Fluoresceins
Gliosis
Humans
Immunohistochemistry
Mice
Neurogenesis
Neurons
Tetanus
Tetanus Toxin
Bromodeoxyuridine
Exotoxins
Fluoresceins
Tetanus Toxin

Figure

• 

  Figure 1 NeuN immunohistochemistry (A-C), F-J B histofluorescence (D-F), GFAP immunohistochemistry (G-I) and Iba-1 immunohistochemistry (J-L) in the mouse DG of the control-, 2.5 and 10 ng/kg TeT-treated-groups. The distribution of NeuN-immunoreactive cells in the TeT-treated-groups is similar to that in the control group. F-J B-positive cells are hardly detected in the DG of all the experimental groups. Microglia are slightly activated (arrows) in the 10 ng/kg TeT-treated-group. ML, molecular layer; GCL, granule cell layer; PL, polymorphic layer; SGZ, subgranular zone. Scale bar=100 µm.

• 

  Figure 2 BrdU immunohistochemistry in the mouse DG in the control- (A and D), 2.5 ng/kg TeT- (B and E) and 10 ng/kg TeT (D and F)-treated-groups. BrdU-positive (+) cells (arrows) are distinctively decreased in the 10 ng/kg TeT-treated-group. ML, molecular later; GCL, granule cell layer; PL, polymorphic layer; SGZ, subgranular zone. Scale bar=50 ìm. G: The mean number of BrdU+ cells per section in the control- and TeT-treated-groups (n=7 per group; F=62.14; *P<0.05: significantly different from the control group, #P<0.05, significantly different from the preceding group). The bars indicate the means±SEM.

• 

  Figure 3 Ki-67 immunohistochemistry in the mouse DG of the control- (A and D), 2.5 ng/kg TeT (B and E)- and 10 ng/kg TeT (C and F)-treated-groups. Ki-67-immunoreactive (+) cells (arrows) are apparently decreased in the 10 ng/kg TeT-treated-group. ML, molecular later; GCL, granule cell layer; PL, polymorphic layer; SGZ, subgranular zone. Scale bar=50 ìm. G: The mean number of Ki-67+ cells per section in the control- and TeT-treated-groups (n=7 per group; F=67.40; *P<0.05: significantly different from the control group, #P<0.05, significantly different from the preceding group). The bars indicate the means±SEM.

• 

  Figure 4 DCX immunohistochemistry in the mouse DG of the control- (A and D), 2.5 ng/kg TeT (B and E)- and 10 ng/kg TeT (C and F)-treated-groups. DCX-immunoreactive (+) cells (arrows) are significantly decreased in the 10 ng/kg TeT-treated-group. ML, molecular later; GCL, granule cell layer; PL, polymorphic layer; SGZ, subgranular zone. Scale bar=50 ìm. G: The mean number of DCX+ cells per section in the control- and TeT-treated-groups (n=7 per group; F=112.44; *P<0.05: significantly different from the control group, #P<0.05, significantly different from the preceding group +P<0.05, significantly different from the w/o tertiary dendrites group). The bars indicate the means±SEM.

• 

  Figure 5 Western blot analysis of DCX in the mouse DG derived from the control-, 2.5 and 10 ng/kg TeT-treated-groups (n=7 in each group). The relative optical density (ROD) of immunoblot bands are demonstrated as percent values (*P<0.05, significantly different from the control group, #P<0.05, significantly different from the preceding group). The bars indicate the means±SEM.

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