Noradrenergic innervation of the rat spinal cord caudal to a complete spinal cord transection: effects of olfactory ensheathing glia

Abstract

Transplantation of olfactory bulb-derived olfactory ensheathing glia (OEG) combined with step training improves hindlimb locomotion in adult rats with a complete spinal cord transection. Spinal cord injury studies use the presence of noradrenergic (NA) axons caudal to the injury site as evidence of axonal regeneration and we previously found more NA axons just caudal to the transection in OEG- than media-injected spinal rats. We therefore hypothesized that OEG transplantation promotes descending coeruleospinal regeneration that contributes to the recovery of hindlimb locomotion. Now we report that NA axons are present throughout the caudal stump of both media- and OEG-injected spinal rats and they enter the spinal cord from the periphery via dorsal and ventral roots and along large penetrating blood vessels. These results indicate that the presence of NA fibers in the caudal spinal cord is not a reliable indicator of coeruleospinal regeneration. We then asked if NA axons appose cholinergic neurons associated with motor functions, i.e., central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more NA varicosities adjacent to central canal cluster cells, partition cells, and SMNs in the lumbar enlargement of OEG- than media-injected rats. As non-synaptic release of NA is common in the spinal cord, more associations between NA varicosities and motor-associated cholinergic neurons in the lumbar spinal cord may contribute to the improved treadmill stepping observed in OEG-injected spinal rats. This effect could be mediated through direct association with SMNs and/or indirectly via cholinergic interneurons.

Fig. 1

Noradrenergic (NA) axons in sagittal sections of lower thoracic spinal cord processed for dopamine β-hydroxylase (DBH) from an intact rat (a), and media- (b, d), and OEG-injected (c, e) rats 7 months after transection. (a–c) Many DBH-labeled axons are found around presumed sympathetic preganglionic neurons in lamina VII in an intact (a) rat. Only a few DBH immunopositive axons (arrowheads) are detected in lamina X along the central canal (cc; b) or at the edge of the white matter (c) in spinal rats. (d, e) DBH-positive axons (arrowheads) arborize extensively in the dorsal horn. (f) The density of DBH-labeled fibers is greater in OEG- than media-injected rats just below the transection site (data from Kubasak et al., 2008), but not significantly different in the remainder of the caudal spinal cord. Bars in (f) represent the mean ± SEM for 6 media-and 6 OEG-injected rats. Scale a–e = 50 µm.

Fig. 2

Noradrenergic (NA) axons enter the spinal cord in association with blood vessels via ventral roots and meninges regardless of transplantation status. (a) Lumbar spinal cord of an intact rat shows numerous dopamine β-hydroxylase (DBH)-positive axons (black; arrowheads) aligned along a penetrating blood vessel identified with platelet endothelial cell adhesion molecule (PECAM; brown). (b, c) DBH-positive axons (black; arrowheads) course along the wall of large ventralmedium arteries (VMA) and extend toward lamina X that contains ChAT-positive central canal cluster cells (amber brown; c is enlarged in inset). cc, central canal (d) A ventral root contains DBH-positive axons (arrowheads) in adult spinal rats. (e) DBH-labeled axons (arrowheads) often are detected along the meninges. Scale a = 25 µm, Scale d, e = 50 µm, Scale b, c = 100 µm.

Fig. 3

Noradrenergic (NA) axons labeled with dopamine β-hydroxylase (DBH) associate with blood vessels caudal to (a, b) and in the (c, d) injury site in adult spinal rats. (a, b) DBH-positive axons (black; arrowheads) in the caudal stump of spinal rats associate with large (a; 35 µm) and small (b; 10 µm) blood vessels identified with the platelet endothelial cell adhesion molecule (PECAM, brown). (c, d) DBH-positive axons (arrowheads) associate with PECAM-labeled blood vessels within the astroglial scar. Inset in c depicts the close association of NA axons along vessels. Scale a, c, d = 50 µm, Scale b = 25 µm.

Fig 4

Noradrenergic cells are present only at cervical levels while dopaminergic somata are distributed at cervical and sacral levels. (a, b) Rare NA (a; labeled with dopamine β-hydroxylase, DBH) and dopaminergic (b; labeled with tyrosine hydroxylase, TH) interneurons are found in the dorsal funiculi at upper cervical levels. (c, d) TH-positive only interneurons (c; arrows) at the sacral levels have smaller somata than those found at the cervical levels. Double-labeling experiments detected a TH-positive soma (d; amber brown, arrow) in the sacral spinal cord that was DBH-negative with DBH-labeled axons (black, arrowheads) coursing nearby. Scale a, b = 50 µm, Scale c, d = 25 µm.

Fig. 5

Noradrenergic (NA) axons labeled with dopamine β-hydroxylase (DBH) appose central canal cluster cells (a–c) and somatic motor neurons (f–h) in the lumbar spinal cord from intact, media-, and OEG-injected spinal rats. (a–c) Many DBH-positive fibers (black) course near central canal cluster cells (brown) in a sagittal section from an intact rat (a) with fewer fibers found in spinal rats (b, c; arrowheads). cc = central canal (d) OEG-injected rats contain significantly more appositions of DBH-positive fibers along central canal cluster cells than media-injected rats at lower lumbar (P = 0.006) but not at sacral (P = 0.09) levels. When data from both segments were combined, OEG-injected rats contained more DBH-positive varicosities apposing central canal cluster cells than media-injected rats (P = 0.003) (e) The number of DBH-labeled fibers apposing SMNs at the lumbar level was greater in OEG- than media-injected rats (P = 0.014). When data from all segmental levels were combined, OEG-injected rats contained more DBH-positive varicosities apposing SMNs than media-injected rats (P = 0.017). (f–h) Numerous NA axons appose SMNs in an intact rat (f). DBH-positive axons (arrowheads) associate with blood vessels that course within the white matter and then appose SMNs (brown) in a media-injected rat (g). Varicose NA axons are associated with SMNs in an OEG-injected spinal rat (h). Bars in (d, e) indicate mean ± SEM for 6 media-injected and 6 OEG-injected rats. *significant difference between media- and OEG-injected rats. Scale a–b, f–h = 50 µm, Scale c = 25 µm.

Fig. 6

Dopamine β-hydroxylase (DBH)-labeled axons appose cholinergic partition cells in sagittally sectioned lower lumbar spinal cord and parasympathetic preganglionic neurons (PPNs) in the sacral cord of an intact rat, and in media- and OEG-injected spinal rats. (a–c) Many DBH-labeled axons (black) course near partition cells (amber-brown) in intact rats (a). DBH-positive axons (arrowheads) also course near partition cells in the caudal stump of media- (b) and OEG-injected (c) spinal rats. (d) The density of DBH-labeled axons that appose partition cells in the lumbar spinal cord is significantly higher in OEG- than media-injected rats (P = 0.004). (e–g) DBH-labeled axons, presumably from the locus coeruleus, densely innervate cholinergic PPNs (brown) in intact rats (e). DBH-positive axons (arrowheads) course among clusters of PPN in media- (f) and OEG-injected (g) spinal rats. Area in (g) marked by double arrowhead is enlarged (inset) to illustrate the proximity of DBH-labeled axons to PPNs. Bars in (d), mean ± SEM for 6 media- and 6 OEG-injected rats. *significant difference between media- and OEG-injected rats. Scale a–c, e–f = 50 µm.