Effects of Ceftriaxone on Oxidative Stress and Inflammation in a Rat Model of Chronic Cerebral Hypoperfusion

Abstract

Ceftriaxone (CTX) exerts a neuroprotective effect by decreasing glutamate excitotoxicity. We further studied the underlying mechanisms and effects of CTX early post-treatment on behavior in a cerebral hypoperfusion rats. The rats' common carotid arteries (2VO) were permanently ligated. CTX was treated after ischemia. Biochemical studies were performed to assess antioxidative stress and inflammation. Behavioral and histological studies were then tested on the ninth week after vessel ligation. The 2VO rats showed learning and memory deficits as well as working memory impairments without any motor weakness. The treatment with CTX was found to attenuate white matter damage, MDA production, and interleukin 1 beta and tumor necrosis factor alpha production, mainly in the hippocampal area. Moreover, CTX treatment could increase the expression of glia and the glial glutamate transporters, and the neuronal glutamate transporter. Taken together, our data indicate the neuroprotective mechanisms of CTX involving the upregulation of glutamate transporters' expression. This increased expression contributes to a reduction in glutamate excitotoxicity and oxidative stress as well as pro-inflammatory cytokine production, thus resulting in the protection of neurons and tissue from further damage. The present study highlights the mechanism of the effect of CTX treatment and of the underlying ischemia-induced neuronal damage.

Keywords: ceftriaxone; inflammation; oxidative stress; white matter damage.

Figure 1

Experimental protocol of chronic cerebral hypoperfusion in rats. Arrow shows different sacrifice days. Dark block shows the duration of early post-treatment with ceftriaxone. Opened block shows the period of behavioral testing.

Figure 2

The Morris water maze test for rats in the 8th week. In the acquisition trial, escape latencies of sham-operated rats and 2VO rats were compared (a) and matching to the sample test was performed to test working memory (b). Prehensile traction test was performed and reported as motor scores (c). Data are expressed as mean ± SEM (n = 8). n.s. represents not statistically significant, * p < 0.05 vs. sham-operated group.

Figure 2

The Morris water maze test for rats in the 8th week. In the acquisition trial, escape latencies of sham-operated rats and 2VO rats were compared (a) and matching to the sample test was performed to test working memory (b). Prehensile traction test was performed and reported as motor scores (c). Data are expressed as mean ± SEM (n = 8). n.s. represents not statistically significant, * p < 0.05 vs. sham-operated group.

Figure 3

Special staining of Luxol fast blue (LFB) for the detection of white matter (WM) changes in the corpus callosum after ischemic induction and its protection by CTX treatment. Grading score of white matter changes in sham-operated rats, 2VO vehicle-treated rats, and 2VO CTX-treated rats (a). Representative images show white matter changes among groups (b). Nerve fiber disarrangement and vacuole formation were observed (arrowed). * p < 0.05 compared with sham-operated group; # p < 0.05 compared with 2VO-Veh-treated group. (n = 5).

Figure 4

Malondialdehyde (MDA) level of rat brain tissues in the hippocampus (a), cortex (b), and striatum (c). MDA production significantly increased from ischemia and was attenuated by CTX treatment in the hippocampus. Data are expressed as mean ± SEM (n = 5). * p < 0.05 compared with sham-operated group.

Figure 5

Immunohistochemistry of glial markers and glutamate transporters in the hippocampal sections. CTX treatment attenuated the reduction in the number of astrocytes and microglia by increasing the expression of GFAP (a) and OX42 (b), respectively. CTX treatment activated the expression of glutamate transporters in both glial cells and neuron cells by increasing the expression of EAAT1 (c), EAAT2 (d), and EAAT3 (e). *, **, and **** p < 0.05, 0.01, and 0.0001 Veh-treated group compared with sham-operated group. ##, ###, and #### p < 0.01, 0.001, and 0.0001 CTX-treated group compared with 2VO Veh-treated group. $$ and $$$$ p < 0.01 and 0.0001 CTX-treated group compared with sham-operated group (n = 5).

Figure 6

ELISA results of expression of inflammatory cytokines IL-1β (a) and TNFα (b) in the hippocampus. The expression of the inflammatory cytokines IL-1β (a) and TNFα (b) was increased in 2VO rats, and CTX-treated rats showed significantly decreased expression of IL-1β (a) and TNFα (b) in the first week after cerebral hypoperfusion. ** and *** p < 0.01 and 0.001 compared with sham-operated group; # and ## p < 0.05 and 0.01 compared with 2VO Veh-treated group (n = 5).