Effect of recombinant human erythropoietin on neural cell apoptosis and related cytokine expression in rats with spinal cord injury

Jinguang WANG; Qixin ZHENG; Yuntao WANG; Ming ZHAO

ABSTRACT

BACKGROUND:

Beside direct trauma, a series of secondary pathological changes would occur in local injured spinal cord area during spinal cord injury. It has been reported that recombinant human erythropoietin (rhEPO)could inhibit cell apoptosis and inflammatory reaction, and possess neuroprotective role.

OBJECTIVE:

To explore the neuroprotective role of rhEPO in spinal cord injury by observing the nerve cell apoptosis and related cytokine expression in traumatic spinal cord.

DESIGN:

Randomized and controlled study.

SETTING:

Orthopedic Surgery Laboratory of Affiliated Cooperation Hospital of Tongji Medical College, and Department of Pathology of Tongji Medical College, Central China Science and Technology University.

PARTICIPANTS:

The study was conduced at Orthopedic Surgery Laboratory of Affiliated Cooperation Hospital of Tongji Medical College, Central China Science and Technology University and Department of Pathology of Tongji Medical College from September 2003 to May 2004. Thirty healthy female adult rats were randomly divided into 4 groups ① Six rats in blank control group only subjected to spinal cord exposure without injury. ②Eight rats in injury group were subjected to spinal cord injury without medication. ③ Eight rats in medication A group were treated with rhEPO.④ Eight rats in medication B group were treated with thEPO and βaeacine sodium.

METHODS:

① Animal model preparation Spinal cord injury model was established on 24 rats by using improved Allen method. ② Administration Rats in medication A group were treated with rhEPO in dosage of 300 U/(kg·d) at postoperative 1, 3, 5, 8, 11 days, while rats in medication B group were given additional β-aeacine sodium in dosage of 0.1 mg/kg,once a day for consecutive 11 days. Rats in blank control group and injury group were injected with the same volume physical saline from tail veins.③ Neurological function The neurological function was scored by the same examiner at 1 day and 12 days after model establishment. Behavioral observation Basing on improved Gale's neurological functional behavioral analysis, 0 score presented severer dysfunction and 6 scores represents normal. Slope test The gradient was determined by the grasping capability of rat, which could reflect the recovery of neurological function. ④ Pathological examination Rat was put to death at postoperative 12 days; traumatic spinal cord was made into slices for HE staining. ⑤ Expression of apoptosis cytokine bcl-2, bax and fas in nerve cells Specimen was collected for IHC stain and brown colored positive cells was counted for calculating positive expressing rate. ⑥ Apoptosis nerve cells In situ end-labeling techniques was used to calculate apoptosis index (the number of apoptosis nucleus/total number). All data were analyzed by using paired chi-test.MAIN OUTCOME

MEASURES:

① Neurological functional behavioral score and results of slope test. ② Histological observation of traumatic spinal cord. ③ Expression of apoptosis nerve cell cytokines. ④ Examination of apoptostic nerve cells.

RESULTS:

① Neurological functional behavioral score and results of slope test There was no significant difference between 1 day and 12 days in blank control group, while the gradient in traumatic group was significantly larger in 12 days than in 1 day (P ＜ 0.05). In both therapeutic A and B groups, the behavioral scores and slope gradient were found significantly larger at 12 days than 1 day (P ＜ 0.05) and that of traumatic group (P ＜ 0.05). ② Histological observation of traumatic spinal cord Traumatic spinal cord became slim with a majority of necrosis and glial cell hyperplasia in it; most of neurological tissues were found normal in medication A and B groups, with the neural structure of medication B group better than A group. ③Expression of nerve cell apoptosis cytokines bax and fas positive cells in traumatic group were more than medication group A and B [traumatic group of (25.75±3.37)% and (41.37±2.83)% vs medication group A of (19.87±3.56) and (26.00±3.29)% vs medication group B of (12.00±2.97)and (17.50±2.20)％, P ＜ 0.05]; bcl-2 was significantly lower in medication group A and group B [(9.75±1.83)％, (14.63±2.83)％, (21.63±5.34)％,P ＜ 0.05]. ④ Examination of apoptotic nerve cells Cell apeptosis index in traumatic group was significantly higher than medication group A and group B (50.75±5.39, 34.75±3.01, 24.00±3.46, P ＜ 0.05).

CONCLUSION:

Cell apoptosis is an important kind of neuronal death following spinal cord injury. rhEPO can inhibit nerve cell apoptosis and possess neuroprotective effect for traumatic spinal cord.