Neuroprotective effect of estrogen after chronic spinal cord injury in ovariectomized rats

NEURAL REGENERATION RESEARCH Volume 2, Issue 8, August 2007

Cite this article as: Neural Regen Res,2007,2(8),471-4

Basic Medicine

1HXURSURWHFWLYHHIIHFWRIHVWURJHQDIWHUFKURQLFVSLQDO  FRUGLQMXU\LQRYDULHFWRPL]HGUDWV  Yanhong Li1, Shuanke Wang2, Yayi Xia1, Jing Wang1, Wenjie Pan1, Yingbin Shi1, Mige Wang1 1Institute of Orthopeadics, Second Hospital of Lanzhou University, Lanzhou 730030, Gansu Province, China 2Department of Orthopaedics, Second Hospital of Lanzhou University, Lanzhou 730030, Gansu Province, China

Abstract BACKGROUND: At present, there is still lack of effective drugs for chronic spinal cord injury, whereas it is found recently that estrogen has a neuroprotective effect on brain and spinal cord injuries. OBJECTIVE: To observe the effect of estrogen on the apoptosis of nerve cells after gradual chronic spinal cord injury in ovariectomized rats. DESIGN: A randomized controlled animal trial. SETTING: Institute of Orthopaedics, the Second Hospital of Lanzhou University. MATERIALS: Sixty-five female Wistar rats of common degree, weighing 220250 g, were provided by the experimental animal center of Lanzhou University. The rats were randomly divided into sham-operated group (n =5), estrogen-treated group (n =30) and saline control group (n =30), and the latter two groups were observed at 1, 3, 7, 14, 28 and 60 days respectively, and 5 rats for each time point. METHODS: All the rats were treated with bilateral oophorectomy 2 weeks before the experiment. T10 vertebral lamina was revolved into using plastic screw. The spinal canal impingement was not induced initially. After that, the original incision was opened to expose the screw every 710 days. MAIN OUTCOME MEASURES: The apoptosis and Caspase-3 positive cells in the damaged spinal cord were detected using terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and Caspase-3 immunohistochemical staining at 1, 3, 7, 14, 28 and 60 days after chronic spinal cord injury respectively. RESULTS: Totally 65 rats were used, and the deleted ones during the experiment were supplemented by others. Changes of Caspase-3 expression after spinal cord injury: In the sham-operated group, only a small amount of Caspase-3 proteins were observed in the rat spinal cord, mainly located in motor neurons of spinal cord anterior horn. In the estrogen-treated group and saline control group, positive cells expressed occasionally at 1 day postoperatively, began to increase obviously at 7 days after injury, strongly expressed at 14 and 28 days, but decreased at 60 days, mainly located in the neurons of spinal cord gray matter anterior horn, and they expressed fewer in the motor neurons and white matter of ventral horn, and there were obvious differences between the estrogen-treated group and saline control group at 7, 14, 28 and 60 days (P < 0.05). CONCLUSION: Estrogen can reduce the apoptosis of nerve cells and promote the recovery of neurological function following gradual chronic spinal cord injury. Key Words: estrogen; chronic; spinal cord; neuroprotection

  ,1752'8&7,21 A great amount of in vivo and in vitro experiments have confirmed that estrogen is an effective neuroprotectant[1]. It has been reported that there is sex difference in the response and recovery of nerve injury[2,3]. Demographic investigation showed that after both symptomless cerebral infarction and traumatic brain injury, females are relatively insensitive to injury, and the recovery of neurological function is also better in females than in males[2,4]. Study indicates that exogenous

Yanhong Li , Studying for master's degree, Physician, Institute of Orthopeadics, Second Hospital of Lanzhou Lanzhou University, 730030, Gansu Province, China Li YH, Wang SK, Xia YY, Wang J, Pan WJ, Shi YB, Wang MG.Neuroprotective effect of estrogen after chronic spinal cord injury in ovariectomized rats. Neural Regen Res 2007;2(8):471-4 www.sjzsyj.com/Journal/ 0708/07-08-471.html

estrogen has certain protective effect on spinal cord injury[5], but the mechanism is still unclear. In this study, model of gradual spinal cord compression from posterior approach were established in rats to observe whether estrogen had neuroprotective effect on chronic spinal cord injury.

0$7(5,$/6$1'0(7+2'6 0DWHULDOV The experiment was carried out in the Institute of

(07-S-5-0471/SHM) Received: 2007-05-10;Accepted: 2007-06-20 Corresponding author: Yanhong Li, Studying for master's degree, Physician, Institute of Orthopeadics, Second Hospital 730030, Gansu Province, China of Lanzhou University, Lanzhou E-mail: [email protected]

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Li YH, et al. / Neural Regeneration Research,2007,2(8):471-4

Orthopaedics, the Second Hospital of Lanzhou University from March to August in 2006. The experiment was approved by the committee of ethics. Sixty-five female Wistar rats of common degree, weighing 220250 g, were provided by the experimental animal center of Lanzhou University (certificate number: 14-006). The rats were randomly divided into sham-operated group (n =5), estrogen-treated group (n =30) and saline control group (n =30), and the latter two groups were observed at 1, 3, 7, 14, 28 and 60 days respectively, and 5 rats for each time point. 0HWKRGV ,QWHUYHQWLRQV All the rats were treated with bilateral oophorectomy 2 weeks before the experiment. The rats were anesthetized with intraperitoneal injection of 20 g/L pentobarbital sodium (40 mg/kg), then a median incision was made, T10 spinous process was resected, vertebral lamina boring was performed, and T10 vertebral lamina was revolved into using plastic screw with diameter of 3 mm and pitch of 0.5 mm. The spinal canal impingement was not induced initially, then the skin was sutured. Aseptic operation was strictly required perioperatively. Model of chronic spinal cord compression was successfully established if there was no neurological dysfunction. After that, the original incision was opened to expose the screw every 710 days, the screw was revolved for 0.2 turn, and about 50% of the spinal cord was compressed till 60 days. 'UXJLQWHUYHQWLRQV Rats in the estrogen-treated group were treated with intraperitoneal injection of benovocylin (1 mg/kg) on the 4th and 7th days every week after model establishment, and those in the saline control group were given intraperitoneal injection of isovolume saline. Not any drug intervention was given to those in the sham-operated group. 0DWHULDOFROOHFWLRQIL[DWLRQDQGVHFWLRQSUHSDUDWLRQ The compressed spinal cord tissue was collected to detect the conditions of cell apoptosis of the injured spinal cord at 1, 3, 7, 14, 28 and 60 days after model establishment respectively. 'HWHUPLQDWLRQRI&DVSDVHH[SUHVVLRQ Rabbit-anti-rat Caspase-3 p20 subunit primary antibody was used, the instruction of S-ABC kit was followed, and the number of positive cells in each section was recorded under microscope. The cytoplasm of positive cells were stained as bubby at cytoplasm and the process root. The stained positive cells in high power visual field ( 400) were counted, and the positive rate was calculated. 7HUPLQDO GHR[\QXFOHRWLGDO WUDQVIHUDVHPHGLDWHG G873ELRWLQQLFNHQGODEHOLQJ781(/ SRVLWLYHFHOOV The above-mentioned sections were used, the disciplines for operation supplied by Boster Company were strictly 472

followed. TUNEL labeled positive cells were re-stained with haematine, thus the nucleus were brown and blue color. Each section was counted independently by two observers under light microscope ( 400), 5 unrepeated visual fields were randomly observed in the stained region, the number of positive cells in each visual sight and positive rate were calculated, and then the average value was calculated. 6WDWLVWLFDODQDO\VLV The data were statistically analyzed by the first, fourth and fifth authors, and the results were expressed as Mean SD. The SPSS 12.0 software was used. The one-way analysis of variance and two-two comparison among the groups were performed, the differences between two sample means were compared with the t test, and P < 0.05 was considered as significant difference.

5(68/76  4XDQWLWDWLYHDQDO\VLVRIWKHH[SHULPHQWDODQLPDOV Totally 65 female Wistar rats were used, the dead and completely paralytic ones during the model establishment were excluded, and supplemented by other animals. 3DWKRORJLFDOFKDQJHVDIWHUVSLQDOFRUGLQMXU\ In the sham-operated group, there was no obvious pathological change. In the saline control group, it was observed under microscope at 28 days after compression that there was obvious edema in spinal cord gray matter, loss of Nissl body, satellitism, obvious phagocytosis of neurons. The surrounding interspace of white matter axon was enlarged, the axonal fibers arranged in disorders, there was obvious degenerative edema, demyelination, obvious proliferation of glial cells in the white matter of the compressed site, vacuole-like changes. In the estrogen-treated group, it was observed under microscope at 28 days after compression that there was mild edema, swelling of neurons in the spinal cord gray matter, proliferation of some glial cells and astrocytes, the pathological changes were alleviated. &KDQJHVRI&DVSDVHH[SUHVVLRQDIWHUVSLQDOFRUGLQMXU\ In the sham-operated group, only a small amount of Caspase-3 proteins were observed in the rat spinal cord, mainly located in motor neurons of spinal cord anterior horn. In the estrogen-treated group and saline control group, positive cells expressed occasionally at 1 day postoperatively, began to increase obviously at 7 days after injury, strongly expressed at 14 and 28 days. But decreased at 60 days, mainly located in the neurons of spinal cord gray matter anterior horn, and they expressed fewer in the motor neurons and white matter of ventral horn, and there were obvious differences between the estrogen-treated group and saline control group (P < 0.05) (Table 1).

Li YH, et al. / Neural Regeneration Research,2007,2(8):471-4

Table 1 Comparison of the number of Caspase-3 positive cells between estrogen-treated group and saline control group (x s, n=5) t (after model establishment)/d Group 1 Estrogen-treated Saline control

3

10.77 3.70 11.48 3.28

12.24 14.32

7 2.05 1.95

14.13 17.43

1.85a 2.33

t (after model establishment)/d Group 14 Estrogen-treated Saline control

28 3.67a 3.10

17.49 22.63

20.47 26.48

60 3.00a 4.22

16.35 22.00

3.45a 2.67

a

P < 0.05, vs. the saline control group

781(/ODEHOLQJDQGFRXQWLQJUHVXOWV In the sham-operated group, only very a few scattered positively labeled glial cells were observed. In the estrogen-treated group and saline control group, the cells were occasionally stained at 1 day after the chronic spinal cord compression, the positive cells were increased at 3 and 7 days, mainly located in gray matter, then the positive cells were increased gradually, plenty of the positive cells were observed in gray matter and white matter at 14 days, reached the peak value at 28 days, then decreased at 60 days, the positive cells were mainly the motor neurons in spinal cord gray matter anterior horn, astrocytes, oligodendrocytes and microglia in white matter. The apoptotic rate was significantly different between the saline control group and estrogen-treated group at 7, 14, 28 and 60 days (P < 0.05) (Table 2). Table 2 Apoptotic rate in the estrogen-treated group and saline control group (x s,%) t (after model establishment)/d Group 1 Estrogen-treated Saline control

3

11.72 2.24 14.86 2.99

16.44 19.36

7 2.38 2.65

20.02 24.83

2.65 2.35a

t (after model establishment)/d Group 14 Estrogen-treated Saline control

22.46 28.85

28 3.87 3.52a

26.66 35.60

60 5.14 4.27a

20.61 30.10

4.63 5.29a

a

P < 0.05, vs. the saline control group

',6&866,21 Caspases are considered to be associated with the apoptosis of nerve cells after central nervous system injury and in

neurodegenerative diseases[6-8]. Caspases (cysteine protease) can directly hydrolyze and activate proteins closely associated with apoptotic characteristic changes such as DNA breakage, and it is also called death protease[9]. Recent evidences have demonstrated that after chronic spinal cord injury, Caspase-3 as a common performance molecule is involved in cell apoptosis after activation[10-12]. The Caspase-3 P20 subunit detected in our study can accurately react the apoptosis of neurons and glial cells. thus Caspase-3 activation is the final common pathway of apoptosis occurrence after various cellular injuries[13,14]. Central nervous system is considered as one of the important target organs for exogenous steroid hormone, and nerve cell itself can also synthetize estrogen[15]. During the individual development, these locally synthetized estrogens also play an important role in the occurrence and differentiation of neurons and synapse[16]. Plenty of in vivo and in vitro experiments showed that estrogen was an effective neuroprotectant[17,18]. Recent studies found that estrogen as a neuroprotectant has many functions, including anti-inflammation, agains the toxicity of excitatory amino acids, anti-apoptosis, etc. Estrogen can also protect many kinds of nerve cells from various damages[19]. Sribnick et al[20] found that there were more TUNEL positive cells in the damaged spinal cord of the animals treated with drug vector, while those were obviously decreased in the estrogen-treated group. In cerebral hippocampus, estrogen treatment reduces ischemia induced Caspase-3 activation and the release of cytochrome C in mitochondrion, and it is associated with the neuroprotection[21,22]. Our results showed that no Caspase-3 expression was observed in normal spinal cord tissue; In the estrogen-treated group and saline control group, the Caspase-3 expressions were fewer at 1 and 3 days after spinal cord compression, gradually increased at 7 and 14 days, reached peak value at 28 days, and decreased at 60 days, which were concordant with the changes of TUNEL positive cells at each time point, it was indicated that Caspase-3 was involved in the regulation of cell apoptosis in spinal cord. Certain apoptosis was observed in both the estrogen-treated group and saline control group, but the Caspase-3 expression at each time point was lower in the estrogen-treated group than in the saline control group, and the TUNEL detection also reflected that there were obviously fewer apoptotic cells at each time point in the estrogen-treated group than in the saline control group. The results of behavioral examination also showed that the functional recovery in the in the estrogen-treated group was significantly better than that in the saline control group. Yune et al[5] confirmed that estrogen promoted the recovery of neurological function through reducing apoptosis in rats with spinal cord injury. Therefore, administration of certain estrogen following acute nerve injury can protect and promote 473

Li YH, et al. / Neural Regeneration Research,2007,2(8):471-4

rehabilitation. It is concluded that estrogen can protect nerve cells, and the neuroprotective mechanism is partially mediated by inhibiting Caspase-3 apoptosis, that was to say, estrogen treatment can decrease various damages induced Caspase-3 activity, then reduce the apoptotic cell death[9,17,23]. Our results also suggested that exogenous estrogen could obviously inhibit apoptosis following chronic spinal cord injury. The neuroprotective effect of estrogen may also participate in the several links of chronic spinal cord injury, including the anti-inflammatory role of estrogen. Estrogen can inhibit in cerebrovessels the process that interleukin-1 induced cyclooxygenasa-2 to produce prostaglandin E[24], and the early anti-inflammatory mechanism mediated by neurotrophic factor receptor p75NTR[25], etc., it also can act against the secondary injury after acute central nervous system injury. In addition, estrogen also regulates the expressions of insulin-like growth factor-I (IGF-I) and brain-derived neurotrophic factor (BDNF), activates the cell cascade reaction including IGF-I receptor and BDNF receptor, then plays a protective role[26,27]. The neuroprotective effect of estrogen mainly has the following three ways: Effect of direct genome: The neurotrophic and neuroprotective effects are mediated by estrogen receptor alpha and beta, and through up-regulating downstream gene. It mainly refers to that estrogen of physiological concentration enters cytoplasm and binds with estrogen receptor [28, 29]. Effect of indirect genome: This effect is rapid, and it is the pathway of the second messenger mediated by activating membrane estrogen receptor. Anti-inflammatory and anti-oxidative effects directly mediated by estrogen.

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(Edited by Ao Q/Yin YL/Wang L)