Effect of lithium chloride on neuronal apoptosis and expression of P53 and nuclear factor kappa B after forebrain ischemia in gerbils

Yanning QIAN; Qingming BIAN; Yuke TIAN

ABSTRACT

BACKGROUND:

Recently, lithium was reported shown neuroprotective effect against apoptosis induced by a variety of insults in vitro and in vitro,but the precise mechanisms underlying its neuroprotective effect remain unknown.

OBJECTIVE:

To observe the effect of lithium chloride on neuronal apoptosis and the expression of P53 or nuclear factor kappa B (NF-κB) protein in the CA1 region of the hippocampus after global ischemia in gerbils.

DESIGN:

A randomized controlled experimental research.

SETTING:

Department of Anatomy of Nanjing Medical University.MATERIALS Fifty-four healthy male gerbils weighing 50-70 g, clearing grade, were purchased from Experimental Animal Center of Zhejiang Province.

METHODS:

Totally 54 gerbils were randomly divided into three groups namely sham-operation group (SH group), ischemia-reperfusion group (IR group) and lithium chloride group (LI group), with 18 in each group. SH group, IR group and LI group were further divided into 3 subgroups respectively (SH1d, SH3d, SH7d; IR1d, IR3d, IR7d; LI1d, LI3d, LI7d), according to the time of reperfusion, with 6 gerbils in each. Gerbils in LI group were injected intraperitoneally with lithium chloride 3 mEq /kg, once a day for 7consecutive days before operation. Normal saline was used instead of lithium in SH group and IR group as vehicle control. Forebrain ischemia was induced at 24 hours after the last injection of lithium chloride. After gerbils being anesthetized, the bilateral common carotid arteries were blocked with micro aneurysm clips for 5 minutes, and the micro aneurysm clips were removed and the cerebral blood flow restored. Sham-operation animals were underwent the same operation except occlusion of bilateral common carotid arteries. Gerbils in each group were killed at every time points.4 μm coronal sections at 1.7-4.0 mm visual cross were cut at the level of the dorsal hippocampus. The apoptosis cells were assayed with in situ Cell Death Detection Kit, and assay of positive cell in cell apoptosis, P53 and positive NF-κB was performed with immunohistochemistry staining. The total number of TUNEL positive cells, P53 or NF-κB positive cells per image (area of 1 mm2) was counted.MAIN OUTCOME

MEASURES:

Neuronal apoptosis and expression of P53 or NF-κB protein in the CA1 region after cerebral ischemic reperfusion.apoptosis cell in cerebral hippocampus CA1 region No TUNEL positive cells were detected in SH group, a large majority of TUNEL positive cells were detected in the CA1 region in IR group on the 3rd day after reperfusion [(552.0±145.5, 142.4±103.5) pcs/mm2, t= 5.623, P ＜ 0.01], and TUNEL positive cells declined on the 7th day after reperfusion. The numbers of TUNEL positive cells in the CA1 region of LI3d, LI7d group were significantly lower than those of IR3d, IR7d group [(408.0±119.8, 156.0±108.2) pcs/mm2,CA1 region In IR group, the expression of P53 protein was increased on the 1st, 3rd and 7th day after reperfusion compared with that in SH group and cerebral hippocampus CA1 region No NF-κB protein was expressed in SH group. In IR group, the expression of NF-κB protein was increased on the 1st day after reperfusion (78.5±25.2)/mm2, significantly increased on the 3rd day after reperfusion (176.5±35.5)/mm2 and on the 7th day after reperfusion, the expression of NF-κB protein disappeared. There were no significant statistical difference between LI group and IR group on the 1st day after reperfusion. The expression of NF-κB protein in LI group was significantly lower than that in IR group on the 3rd day after reperfusion [(64.5±30.8)/mm2,t=5.824, P ＜ 0.01].

CONCLUSION:

Lithium chloride can significantly suppress neuronal apoptosis after global ischemia in gerbils. The down-regulation of expression of P53 or NF-κB protein is one of the mechanisms of the neuroprotective effect by lithium chloride.