Delayed transplantation of olfactory ensheathing cells for thoracic cord injury in adult rats

Yong TANG; Yanfeng WU; Yupeng SHI; Huiyong SHEN

ABSTRACT

BACKGROUND:

Spinal cord can regenerate after injury in certain microenvironment. Olfactory ensheathing cells (OECs)have the characteristics of astrocytes and Schwann cells and can accelerate the spinal cord axonal regeneration.

OBJECTIVE:

To make injured thoracic cord rat models and observe the effect of OECs on injured spinal cord axonal regeneration.

DESIGN:

Observational experiment.

SETTING:

Second Affiliated Hospital of Sun Yat-Sen University.MATERIALS The experiment was performed at the Second Affiliated Hospital of Sun Yat-Sen University from January 2001 to November 2002.Totally 20 adult SD male rats with the body mass of (380±20) g were provided by Experimental Animal Center of Sun Yat-Sen University (number of institution license SYXK2004-0020). There were DMEM culture solution with low glucose (L-DMEM, GibcoBRL), fetal calf serum (FCS) (Hyclone), myelin basic protein (MBP) (Sigma) and nerve growth factor receptor antibody (Sigma). They were divided into cell transplantation group and control group by the method of random digits table with 10 in each group.

METHODS:

The adult SD rats were anaesthetized and decapitated to obtain the whole olfactory bulb and then isolate olfactory nerve with a sterile operation. Thoracic cord injury models were established by modified Allen method. 10μL OECs suspension (2.5×1010 L-1) was injected into injured spinal cord of the cell transplantation group, whereas DMEM/F12 (11) culture solution of the same dose was injected in the control group. The influence of OECs on spinal cord axonal regeneration was observed by histological and immunohistochemical method 6 weeks after transplantation.MAIN OUTCOME

MEASURES:

①OECs were identified by nerve growth factor receptor antibody staining. ②Repair of myelin sheath was observed by MBP staining. ③Nerve axonal regeneration was observed by argentaffin staining.

RESULTS:

Two rats in the cell transplantation group and 3 rats in the control group died, so totally 15 rats were involved in the result analysis. ①In the cell transplantation group,injured spinal meninges were integrated,but spinal cord became thin as compared with the normal spinal cord. By hematoxylin-eosin (HE) staining, multipolar cells appeared in injured region and the cells were fused excessively with myeloid tissues. It proved that survival OECs were integrated well within the host. New axons were clustered in bundles and infiltrated by small round lymphocytes. By argentaffin staining, regenerated axons grew in tissues of injured region, which mostly accompanied with fascicular-arranged multipolar cells. In the control group, spinal cord became thin markedly and spinal meninges were integrated. No new axon appeared in the injured spinal cord by HE staining. No regenerated axon appeared by argentaffin staining, either. ②In the cell transplantation group, most multipolar cells were clustered in bundles. A mass of positive granules of nerve growth factor receptor antibody appeared in cytoplasm, which further verified that OECs still survived and integrated well within the host 6 weeks after transplantation. Linear MBP positive fibers appeared in the injured region by MBP staining,which indicated that myelin-like substance appeared and drew closely in both ends of injured region. At the same time,MBP positive substance also appeared in the multipolar cells, which illustrated that transplanted OECs could induce the occurrence of myelin-like substance. No regenerated axon was found in the control group.

CONCLUSION:

Delayed transplantation of OECs can survive and induce the occurrence of myelin-like substance in injured spinal cord of adult rats.