Transplanted olfactory ensheathing cells remyelinate and enhance axonal conduction in the demyelinated dorsal columns of the rat spinal cord

Abstract

Olfactory ensheathing cells (OECs), which have properties of both astrocytes and Schwann cells, can remyelinate axons with a Schwann cell-like pattern of myelin. In this study the pattern and extent of remyelination and the electrophysiological properties of dorsal column axons were characterized after transplantation of OECs into a demyelinated rat spinal cord lesion. Dorsal columns of adult rat spinal cords were demyelinated by x-ray irradiation and focal injections of ethidium bromide. Cell suspensions of acutely dissociated OECs from neonatal rats were injected into the lesion 6 d after x-ray irradiation. At 21-25 d after transplantation of OECs, the spinal cords were maintained in an in vitro recording chamber to study the conduction properties of the axons. The remyelinated axons displayed improved conduction velocity and frequency-response properties, and action potentials were conducted a greater distance into the lesion, suggesting that conduction block was overcome. Quantitative histological analysis revealed remyelinated axons near and remote from the cell injection site, indicating extensive migration of OECs within the lesion. These data support the conclusion that transplantation of neonatal OECs results in quantitatively extensive and functional remyelination of demyelinated dorsal column axons.

Fig. 1.

Illustration of regions of the dorsal column identified for subsequent analysis of myelinated axon density and size.A, Cross section of an EB-X-lesioned dorsal column with transplanted OECs. Areas A, B, andC indicate the dorsomedial, centromedial, and ventromedial regions of the dorsal column, respectively; within these areas the sizes (cross-sectional area) of myelinated axons were measured for one representative video field every millimeter along the length of the spinal cord segment that was studied. B, Photomicrograph shown in A, with the target (central region used for analysis) dorsal column areas, lesioned area, and “myelin-rich” and “myelin-poor” areas outlined. Myelinated, demyelinated, and remyelinated areas were identified at low magnification by the staining density. C–F, Diagrams showing the cross-sectional areas of the target dorsal column area, demyelinated area, and myelin-rich and myelin-poor areas within the lesion, respectively. These areas were used to calculate the percentage of the target areas lesioned as well as the estimated numbers of remyelinated axons at leach level of the cord.

Fig. 2.

A–C, Low-magnification light micrograph of cross sections through dorsal columns from unlesioned control rats (A), EB-X-lesioned rats (B), and animals with EB-X lesions, followed by the transplantation of 30,000 OECs (C). Micrographs B and C show areas near the centers of the lesioned areas. D–F, High magnification of images of the centers of the dorsal column shown in the panels directly above these. Few myelinated axons can be detected in the center of the lesioned dorsal column without transplanted OECs, but many can be observed in the lesion with transplanted OECs. Calibration in A refers toA–C and that in D toD–F.

Fig. 3.

Electron micrographs of representative regions of EB-X-lesioned area of the OEC transplanted dorsal column shown in Figure 2C. A, Peripheral edge of EB-X lesion. Note primarily nonmyelinated axons. B,C, Central areas of the lesion showing examples of remyelinated axons. Intercellular spacing is increased, and most axons appear either to be myelinated (B) or to be in some stage of myelin formation (C).Inset in C, High magnification of remyelinated axon showing multilayered membrane structure consistent with that of myelin surrounding the axon. A–C, 12,000× magnification; inset, 170,000× magnification.

Fig. 4.

Graphs showing areas of dorsal columns and EB-X-lesioned areas as well as the estimated numbers of remyelinated axons at successive points along the length of the spinal cord for a representative EB-X-lesioned dorsal column (A) and a lesioned dorsal column after 30,000 transplanted OECs (B). The data shown here are for the same lesions shown in Figure 2B, C.

Fig. 5.

Graphs illustrating the size (area) distribution of myelinated axons in the dorsomedial (A), centromedial (B), and ventromedial (C) of the dorsal columns (designated as areasA, B, and C in Fig.1B). Each graph shows the percentage of axons in each size category for unoperated controls (white bars) and OEC remyelinated dorsal columns (black bars). Representative fields were analyzed every millimeter along the dorsal column. A minimum of 250 control axons and 1200 OEC remyelinated axons were measured from at least three different experiments to produce each graph. The population distribution for each region is shifted toward larger axon sizes in the OEC remyelinated condition, but the effects were most marked in the ventromedial or corticospinal tract region of the dorsal column.

Fig. 6.

Field potential recordings from control (A), demyelinated (B), and transplanted (C) dorsal columns. Compound action potentials were recorded at four points at 1 mm increments (arrows) along the dorsal columns. Note the increase in latency for recordings obtained from the demyelinated axons and the decrease in latency for those obtained from the dorsal columns with OEC transplantation.

Fig. 7.

Summary data of conduction velocity for normal (n = 6), demyelinated (n = 7), and remyelinated (n = 6) dorsal column axons at 26°C (left) and 36°C (right). Conduction velocity of the demyelinated axons was reduced as compared with control at both temperatures (p < 0.00001, Student’s t test). Conduction velocity of the transplant group was increased as compared with the demyelinated axons but decreased as compared with the normal axons. Significance levels are indicated by asterisks (*p < 0.05; **p < 0.01; shown as comparisons of normal or demyelinated groups to the transplant group).

Fig. 8.

Amplitude decrement with conduction distance for normal (n = 6), demyelinated (n= 7), and transplanted (n = 6) dorsal columns at 26°C (A) and 36°C (B). Note that axons in the transplant group show less amplitude decrement with increasing conduction distance than axons in the EB-X lesion condition; p value notation and n values are the same as described in Figure 7.

Fig. 9.

Amplitude recovery for the second of two stimuli at varying interstimulus intervals for normal, demyelinated, and transplanted dorsal columns at 26°C (A) and 36°C (B). There is less amplitude decrement for the remyelinated axons than for the demyelinated axons.p value notation and n values are the same as described in Figure 7 and indicate the comparison between the normal or demyelinated condition to the transplant group.

Fig. 10.

Frequency–response properties of normal, demyelinated, and transplanted dorsal columns at 26°C (A) and 36°C (B). The demyelinated axons display considerable reduction in their ability to follow high-frequency stimulation, but the remyelinated axons are able to follow higher frequencies of stimulation. Comparisons of responses between normal or demyelinated groups are shown related to the transplant group. p value notation and nvalues are the same as described in Figure 7.