Influence of ketamine on astrocyte damage in spinal dorsal horn of rats induced by N-methyl-D-aspartic acid

Qing LI; Juying LIU; Qingshan ZHOU; Tao ZHU; Chengming QIN

ABSTRACT

BACKGROUND:

Ketamine is a kind of frequently used general venous anesthesia drug in clinic, and the medication in vein or epidural cavum has analgesic effect. It is N-methyl-D-aspartic acid (NMDA) receptor noncompetitive antagonist, which can inhibit toxic effect of excitatory amino acids.

OBJECTIVE:

To observe effect of ketamine on apoptosis of dorsal horn astrocytes of spinal cord of rats induced by NMDA receptor over activation and explore its possible mechanism of action.

DESIGN:

Randomized controlled observation.

SETTING:

Department of Anesthesiology, Taihe Hospital Affiliated to Yunyang Medical College.MATERIALS The experiment was conducted at Cell Biology Laboratory,Institute of Basic Medical Sciences, Yunyang Medical College between September 2003 and January 2005. Neonatal Wistar rats of two or three days were provided by Animal Experimental Center of Wuhan University.

METHODS:

Primary astrocytes in dorsal horn of T11-L6 spinal cord of Wistar rats were purified and cultured. Astrocytes were used in the experiment when its purity coefficient reached 98% assessed by gial fibrillary acidic protein. The cultured cells in 24-well plates were divided randomly into 6 groups (9 portions in each group) ①50 μL Hanks liquor was added into the control group. ②Amount of 100μmol/L was added into the NMDA group. ③Amount of 1 mmol/L was added into the ketamine group. ④100μmol/L NMDA + 0.1 mmol/L ketamine group. ⑤100 μmol/L NMDA + 0.5 mmol/L ketamine group. ⑥100μmol/L NMDA + 1 mmol/L ke tamine group. 1 mmol/L ketamine was clinical antalgic dosage. Activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) were examined after 24-hour culture. Content of Bcl-2 protein and change of morphology were observed with immunocytochemistry. Apoptosis of astrocytes was measured with flow cytometry. MAIN OUTCOME

MEASURES:

① Counterstain cell staining and changes of morphology of Bcl-2 protein with immunohistochemical method and hematoxylin-esoin staining (HE). ②Apoptosis of astrocytes was detected with flow cytometry. ③Content of MDA and activity of SOD.

RESULTS:

①Mean absorbance (A) of Bcl-2 as expression of Bcl-2 protein measured semiquantitatively It was lower in the 100μmoL/L NMDA group than the control group, which had significant difference [0.054±0.021,0.108±0.039, respectively, P＜0.01]. It was higher in the 100 μmol/L NMDA + 1 mmol/L ketamine group than the 100 μmol/L NMDA group,which had significant difference [0.148±0.045, 0.054±0.021, respectively,P ＜ 0.01]. ②Apoptosis of astrocytes detected with flow cytometry It was higher in the 100μmol/L NMDA group than the control group, which had significant difference [(25.26±6.13)%, (5.66±2.24)%, respectively, P＜0.01].It was lower in the 100μmol/L NMDA + 1 mmol/L ketamine group than in the 100μmol/L NMDA group, which had significant difference[(24.41±4.82)%, (25.26±6.13)%, respectively, P＜0.01]. ③Content of MDA and activity of SOD 100 μmol/L NMDA made the content of MDA in astrocytes obviously increase , while the activity of SOD markedly decrease. 1 mmol/L ketamine remarkably decreased the content of MDA, distinctly increased the activity of SOD. This effectiveness had evidently dosage-effect relationship in clinical antalgic dosage, which had obviously difference as compared with that of the NMDA group (P ＜ 0.01 ). The differences between the 1 mmol/L ketamine group and the control group as well as between the 100 μmol/L NMDA + 0.1 mmol/L ketamine group and the NMDA group had insignificant difference.

CONCLUSION:

NMDA receptor over activation can induce apoptosis of a great number of astrocytes in spinal dorsal horn of rats. Suitable ketamine dramatically inhibits apoptosis, and its mechanism can enhance the expressionof Bcl-2 protein of astrocytes, at the same time inhibit the production of free radical and reinforce the activity of SOD.