Increased cyclooxygenase-2 and nuclear factor-κB/p65 expression in mouse hippocampi after systemic administration of tetanus toxin

Abstract

Brain inflammation has a crucial role in various diseases of the central nervous system. The hippocampus in the mammalian brain exerts an important memory function, which is sensitive to various insults, including inflammation induced by exo/endotoxin stimuli. Tetanus toxin (TeT) is an exotoxin with the capacity for neuronal binding and internalization. The present study investigated changes in inflammatory mediators in the mouse hippocampus proper (CA1‑3 regions) and dentate gyrus (DG) after TeT treatment. The experimental mice were intraperitoneally injected with TeT at a low dosage (100 ng/kg), while the control mice were injected with the same volume of saline. At 6, 12 and 24 h after TeT treatment, changes in the hippocampal levels of inflammatory mediators cyclooxygenase‑2 (COX‑2) and nuclear factor kappa‑B (NF‑κB/p65) were assessed using immunohistochemical and western blot analysis. In the control group, moderate COX‑2 immunoreactivity was observed in the stratum pyramidal (SP) of the CA2‑3 region, while almost no expression was identified in the CA1 region and the DG. COX‑2 immunoreactivity was increased by TeT in the SP and granule cell layer (GCL) of the DG in a time‑dependent manner. At 24 h post‑treatment, COX‑2 immunoreactivity in the SP of the CA1 region and in the GCL of the DG was high, and COX‑2 immunoreactivity in the SP of the CA2/3 region was highest. Furthermore, the present study observed that NF‑κB/p65 immunoreactivity was obviously increased in the SP and GCL at 6, 12 and 24 h after TeT treatment. In conclusion, the present study demonstrated that systemic treatment with TeT significantly increased the expression of COX-2 and NF-κB/p65 in the mouse hippocampus, suggesting that increased COX‑2 and NF-κB/65 expression may be associated with inflammation in the brain induced by exotoxins.

Figure 1

Immunohistochemical analysis of NeuN in the hippocampi of (A–D) the control mice and (E–P) 100 ng/kg TeT-treated mice. Numbers of NeuN-immunoreactive cells in the TeT-treated groups were similar to those in the control-group. CA, cornu ammonis; DG, dentate gyrus; GCL, cell layer; ML, molecular later; PL, polymorphic layer; SO, stratum oriens; SP, stratum pyramidal; SR, stratum radiatum. Scale bar, 400 µm (A, E, I and M) or 50 µm (B–D, F, G, H, J–L, N–P). TeT, tetanus toxin; NeuN, neuronal nuclei.

Figure 2

Immunohistochemical detection of COX-2 in the hippocampi of (A–C) the control mice and (D–L) 100 ng/kg TeT-treated mice. At 24 h post-treatment, COX-2 immunoreactivity was distinctively increased in the SP (asterisks) and GCL (asterisk and arrows). TeT, tetanus toxin; COX, cyclooxygenase; SP, striatum pyramidal; GCL, granule cell layer; SO, stratum oriens; SR, stratum radiatum; ML, molecular later; PL, polymorphic layer. Scale bar, 50 µm.

Figure 3

Immunohistochemical detection of NF-κB/p65 in the hippocampi of (A–C) control mice and (D–L) 100 ng/kg TeT-treated mice. NF-κB/p65 immunoreactivity was apparently increased in the SP (asterisks) and GCL (asterisks) after TeT treatment. TeT, tetanus toxin; NF, nuclear factor; SP, striatum pyramidal; GCL, granule cell layer SO, stratum oriens; SR, stratum radiatum; ML, molecular later; PL, polymorphic layer. Scale bar, 50 µm.

Figure 4

Western blot analysis of COX-2 and NF-κB/p65 in the hippocampi of control- and 100 ng/kg TeT-treated mice (n=7 in each group). (A) Representative western blot. ROD of (B) COX-2 and (C) NF-κB/p65 normalized to β-actin. Values are expressed as the mean ± standard error of the mean. ^*P<0.05 vs. control group; ^#P<0.05 vs. preceding time-point. NF, nuclear factor; COX, cyclooxygenase; TeT, tetanus toxin; ROD, relative optical density.