Abstract

BACKGROUND: Many factors affect the outcome of olfactory ensheathing cell transplantation for obsolete spinal cord injury, such as the time of injury, segment and sex. The best time to do olfactory ensheathing cell transplantation is unknown up to now.
OBJECTIVE: To investigate the influence of time windows of olfactory ensheathing cell transplantation on the recovery of motion and sensation function in spinal cord injury patients.
DESIGN: Self-control observation.
SETTING: Department of Neurosurgery, Taian Disabled Soldier's Hospital.
PARTICIPANTS: 135 patients with spinal cord injury were enrolled at the Department of Spinal Cord Surgery, Taian Disabled Soldier's Hospital of Shandong Province from June 2004 to June 2007, including 121 males and 14 females, aged 7-59 years, averagely 36 years. Duration of spinal cord injury included 0-6 months in 21 cases, 7 months-2 years in 71 cases and over 2 years in 43 cases. These patients or their guardians signed an informed consent of the cell transplantation. The experimental procedures were accorded with the rules of Ministry of Health of China (No. 91-006) and approved by the Taian Disabled Soldier's Hospital of Shandong Province.
METHODS: ①Olfactory bulbs of aborted fetus were digested into single olfactory ensheathing cells, and then cultured for 7-15 days. Parturients signed the informed consent. This study was approved by the Hospital Ethical Committee. ②After general anesthesia, olfactory ensheathing cell suspension was implanted into the corresponding region by the multi-targeted injection with a microscope. According to the injury condition, targets generally located in upper or lower injured region and left or right normal spinal cord. The amount of targets depends on the size of the injured region. About 1 000 000 units of cells were injected into each target, about 50 μL of suspension, at 2×1010 L-1, 2-5 targets. ③American Spinal Injury Association impairment scale was used to assess the motion and sensation function in spinal cord injury patients before transplantation and 2-8 weeks after transplantation.
MAIN OUTCOME MEASURES: Scores on American Spinal Injury Association impairment scale.
RESULTS: 135 spinal cord injury patients were involved in the result analysis. Motion and sensation function was improved in spinal cord injury patients at different time windows compared with that before transplantation (P < 0.01). There was no significant difference in scores on motion and sensation function and the increased degree of the score at different time windows after transplantation (P > 0.05).
CONCLUSION: Olfactory ensheathing cell transplantation can promote the recovery of nerve function in spinal cord injury patients, without the difference in time windows.

INTRODUCTION

A series of studies showed that olfactory ensheathing cell transplantation could promote the neural regeneration of injured spinal cord and improve the nerve function of partial spinal cord[1-6]. Cell transplantation time is important to improve the nerve function of the injured spinal cord from the view of therapeutics. We investigate the influence of time windows of olfactory ensheathing cell transplantation on the recovery of spinal cord function.

SUBJECTS AND METHODS

Subjects
135 patients with spinal cord injury were enrolled at the Department of Spinal Cord Surgery, Taian Disabled Soldier's Hospital of Shandong Province from June 2004 to June 2007, including 121 males and 14 females, aged 7-59 years, averagely 36 years. Causes of injuries included trauma spinal injury in 130 cases, hurt by mistake in surgery in 4 cases and puncture with a knife in 1 case. Duration of spinal cord injury contained 0-6 months in 21 cases, 7 months-2 years in 71 cases and over 2 years in 43 cases. Inclusive criteria: ①These patients or their guardians knew the particularity and possible results of this clinical test; ②They voluntarily signed an informed consent of cell transplantation; ③The subjects were patients with complete or incomplete injury; ④All patients received postoperative rehabilitation only; ⑤The experimental procedures were accorded with the rules of Ministry of Health of China (No. 91-006) and approved by the Taian Disabled Soldier's Hospital of Shandong Province; ⑥Data of these cases are complete.

Methods
Cell culture
Olfactory bulb of aborted fetus was offered by the Taian Maternity and Child Care Hospital. This study was approved by the Hospital Ethical Committee. Parturients signed the informed consent. Olfactory bulb of aborted fetus was cut into small pieces, digested into single olfactory ensheathing cell, then cultured in D/F12 medium containing fetal bovine serum of 0.1 volume fraction for 3-4 days. Olfactory ensheathing cell culture medium was

purified with DMEM, successively cultured for 7-12 days, and then digested into single cell suspension, at 2×1010 L-1.

Transplantation method
After general anesthesia, injured spinal cord was exposed. Olfactory ensheathing cell suspension was implanted into the corresponding region by the multi-targeted injection with a microscope. According to the injury condition, targets generally located in upper or lower injured region and left or right normal spinal cord. The amount of targets depends on the size of the injured region. About 1 000 000 units of cells were injected into each target, about 50 μL of suspension, at 2×1010 L-1, about 2-5 targets.

Assessment on nerve function
American Spinal Injury Association impairment scale[7] was used to assess the motion and sensation function in spinal cord injury patients before the transplantation and 2-8 weeks after the transplantation. The highest score of motion function was 100 points. The highest score of sensation function was 112 points in light touch and 112 points in acupuncture.

Statistical analysis
Data were analyzed by Professor Cheng from Department of Statistics of Taishan Medical College with SPSS12.0 software. Measurement data were expressed by Mean±SD. Single factor analysis of variance was performed, but not t test. A value of P < 0.05 was considered as significant differences.

RESULTS

Quantitative analysis of the participants
135 patients with spinal cord injury were involved in the result analysis.

Influence of time windows on olfactory ensheathing cell transplantation in the treatment of spinal cord injury (Table 1)

Table 1 shows that motion and sensation function was improved in spinal cord injury patients at different time windows compared with that before the transplantation (P < 0.01). There was no significant difference in scores on motion and sensation function at different time windows after the transplantation (P > 0.05).

Increased degree of American Spinal Injury Association function score after olfactory ensheathing cell transplantation in patients with spinal cord injury in different time windows (Table 2)

DISCUSSION

Olfactory ensheathing cells as a special kind of glial cells process double characteristics of Schwann cells and astrocytes[8], immigrate into central nervous system from peripheral nervous system along with olfactory ensheathing nerve axon. Olfactory sensory neuron in sensory epithelium with renewal ability and axon can grow into olfactory bulb in center induced by olfactory ensheathing cells. Olfactory ensheathing cells cultured in vitro express many neurotrophic factors that can promote the growth, differentiation and neural regeneration of nerve cells, such as NGF，BDNF，N73/4 and GDNF. Thus, olfactory ensheathing cells are implanted into the injured spinal cord. Olfactory ensheathing cells can split, proliferate, inhibit the form of glial scar, prevent the activity of inhibitory factors of neural regeneration, wrap regenerated axons and form myelin sheath. Clinical and basic tests verified that olfactory ensheathing cell transplantation was one of the effective methods to treat spinal cord injury[9-15]. In this study, we found that dermal nutrient, spasm, bladder function (increased urine volume, reduced residual urine volume) and intestinal function (unobstructed defecation) were improved firstly, followed by motion and sensation function, generally within 2 weeks after transplantation. Vegetative nerve function is improved by repairing the reticular structure (reticulospinal tract and spinoreticular tract) of the injured spinal cord after olfactory ensheathing cell transplantation. This recovery rule met the evolutionary law of repairing ability from low classes to high classes. In other words, recovery order of injured spinal cord was from reticular structure (the lowest class of nerve structure) to Betz's cells (the highest class of neurons and conduction system such as pyramidal tract) after olfactory ensheathing cell transplantation. The optimal time windows of transplantation in spinal cord injury deserve further studies.
Many people believed that early transplantation was the optimal transplantation time, which could relieve secondary injury, improve the internal environment earlier and was beneficial for axonal growth[16]. A study found that 2-4 week transplantation was better than early transplantation[17]; a mass of inhibitory factors in early transplantation could prevent the implanted cell survival and axonal regeneration, and the transplantation could induce the spinal secondary injury. We found that motion and sensation function was improved after transplantation in spinal cord injury for 6 months, 6 months to 2 years and over 2 years. There was no significant difference in the three time windows. That is, there was no significant effect of time windows on olfactory ensheathing cell transplantation for treatment of spinal cord injury. Authors definitely believed that it is necessary to treat spinal cord injury in early phase by olfactory ensheathing cell transplantation, especially after the shock stage of the spinal cord when secondary injury of the spinal cord has stopped, and edema of the spinal cord has declined. Thus, olfactory ensheathing cells after transplantation can effectively prevent the formation of glial scar, promote the regeneration of nerve axon and the repair of myelin sheath. However, there is the possibility of natural recovery of the spinal cord in early transplantation (half a year), so the outcome of olfactory ensheathing cell transplantation is doubtable; there is no effective measure to make clear the differences between early natural recovery and post-transplanted recovery in spinal cord injury patients. We selected these cases of spinal cord injury over half a year in our study.
Kakulas[18] assigned the pathological process of spinal cord injury into early, middle and late phases. Early phase refers to acute phase, including changes in injury and secondary injury. Middle phase refers to persisted lesion phase, lasted for 4-6 weeks from the 72nd hour after injury, showing glial scar in spinal cord. Late phase refers to terminal phase, i.e. final pathologic change was found in 6 months. Self-repair of the spinal cord has been done, but some scholars believe that self-repair of some injured spinal cord lasts for 2 years. Thus, we divided spinal cord injury patients into 3 groups, shorter than 6 months, 6 months to 2 years, longer than 2 years.
Olfactory ensheathing cell transplantation was effective for treatment of spinal cord injury[19]. There were few reports on the optimal time of olfactory ensheathing cell transplantation. In our study, 135 patients with spinal cord injury were enrolled and divided into 3 groups. Clinical and statistical analysis demonstrated that spinal function of each patient was improved after transplantation, and there was no significant difference in function recovery in the 3 groups. That was, there was no effect of time windows on improving nerve function of each patient after olfactory ensheathing cell transplantation. Therefore, we believed that the earlier the olfactory ensheathing cell transplantation, the better the outcome was. In early phase, olfactory ensheathing cells can more effectively recover the injured spinal cord by repairing the function of neuron and axon.

REFERENCES

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移植时间对嗅鞘细胞移植治疗脊髓损伤效果的影响★

郑遵成，刘 超，高 瑞，张 林，魏树刚，张 坤，张 磊，匡乃峰，张立清，宋延军
山东省泰安荣军医院神经外科，山东省泰安市 271000
郑遵成★，男，1965年生，山东省济宁市人，汉族，1989年泰山医学院毕业，硕士，副主任医师，主要从事神经外科、脊柱脊髓损伤方面的研究。
通讯作者：刘 超，硕士，主治医师,山东省泰安荣军医院神经外科，山东省泰安市 271000
摘要
背景：嗅鞘细胞移植治疗陈旧性脊髓损伤的疗效可受多种影响因素如受伤的时间、节段、性别等的影响，而患者受伤后应在哪个时间段进

行嗅鞘细胞移植目前尚无定论。
目的：关注嗅鞘细胞移植时间窗的选择对脊髓损伤患者运动和感觉功能恢复的影响。
设计：自身前后对照观察。
单位：泰安荣军医院神经外科。
对象：选择2004-06/2007-06山东省泰安荣军医院脊柱脊髓外科收治的脊髓损伤患者135例，其中男121例，女14例；年龄7~59岁，平均36岁。脊髓损伤时间：0~6个月21例，7个月~2年71例，2年以上43例。纳入患者或其（未成年）父母了解这一临床试验的特殊性和可能结果；患者或其监护人同意接受细胞移植治疗并签自愿接受协议书；实验和治疗方案符合中国卫生部（91-006）文件规定，经山东省泰安荣军医院伦理委员会批准。
方法：①将流产胚胎的嗅球消化成单个嗅鞘细胞后培养7~15 d待用。产妇同意提供流产胚胎用于实验，此方案经过医院伦理委员会批准。②患者全身麻醉后，借助显微镜将已培养好的嗅鞘细胞悬液采用多靶点注射的方法分别移植到相应的区域内。靶点的选择据损伤情况而定，一般位于损伤区域的上下两端及左右侧正常脊髓处。据损伤区域的大小决定靶点的多少，每个靶点注射细胞量约100万单位，悬液约50 μL，含量大约2×1010 L-1,一般为2~5个靶点。③于移植前及移植后2~8周按照美国脊髓损伤学会制定的美国脊髓损伤学会标准评估患者运动和感觉功能。
主要观察指标：美国脊髓损伤学会标准评分。
结果：脊髓损伤患者135例均进入结果分析。不同移植时间窗脊髓损伤患者运动和感觉功能均较移植前有明显提高，差异有非常显著性意义（P < 0.01）；不同移植时间窗患者移植后运动和感觉功能分数及分数提高程度比较，差异无显著性意义（P > 0.05）。
结论：嗅鞘细胞移植可促进脊髓损伤患者的神经功能恢复，且不存在移植时间窗差异。
关键词：嗅鞘细胞移植;时间窗;脊髓损伤;多靶点移植
中图分类号：R394.2 文献标识码:A 文章编号：1673-8225(2008)03-00583-04
郑遵成，刘超，高瑞，张林，魏树刚，张坤，张磊，匡乃峰，张立清，宋延军.移植时间对嗅鞘细胞移植治疗脊髓损伤效果的影响[J].中国组织工程研究与临床康复，2008，12(3):583-586
[www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-583(ps).pdf]
(Edited by Wang JW/Qiu Y/Wang L)