Involvement of vimentin- and BLBP-positive glial cells and their MMP expression in axonal regeneration after spinal cord transection in goldfish.

In goldfish, spinal cord injury triggers the formation of a fibrous scar at the injury site. Regenerating axons are able to penetrate the scar tissue, resulting in the recovery of motor function. Previous findings suggested that regenerating axons enter the scar through tubular structures surrounded by glial elements with laminin-positive basement membranes and that glial processes expressing glial fibrillary acidic protein (GFAP) are associated with axonal regeneration. How glia contribute to promoting axonal regeneration, however, is unknown. Here, we revealed that glial processes expressing vimentin or brain lipid-binding protein (BLBP) also enter the fibrous scar after spinal cord injury in goldfish. Vimentin-positive glial processes were more numerous than GFAP- or BLBP-positive glial processes in the scar tissue. Regenerating axons in the scar tissue were more closely associated with vimentin-positive glial processes than GFAP-positive glial processes. Vimentin-positive glial processes co-expressed matrix metalloproteinase (MMP)-14. Our findings suggest that vimentin-positive glial processes closely associate with regenerating axons through tubular structures entering the scar after spinal cord injury in goldfish. In intact spinal cord, ependymo-radial glial cell bodies express BLBP and their radial processes express vimentin, suggesting that vimentin-positive glial processes derive from migrating ependymo-radial glial cells. MMP-14 expressed in vimentin-positive glial cells and their processes might provide a beneficial environment for axonal regeneration.

Innervation and osteoclast distribution in the inferior pharyngeal jaw of the cichlid Nile tilapia (Oreochromis niloticus).

In addition to an oral jaw, cichlids have a pharyngeal jaw, which is used for crushing and processing captured prey. The teeth and morphology of the pharyngeal jaw bones adapt to changes in prey in response to changes in the growing environment. This study aimed to explore the possible involvement of the peripheral nervous system in remodeling the cichlid pharyngeal jaw by examining the innervation of the inferior pharyngeal jaw in the Nile tilapia, Oreochromis niloticus. Vagal innervation was identified in the Nile tilapia inferior pharyngeal jaw. Double staining with tartrate-resistant acid phosphatase and immunostaining with the neuronal markers, protein gene product 9.5, and acetylated tubulin, revealed that osteoclasts, which play an important role in remodeling, were distributed in the vicinity of the nerves and were in apposition with the nerve terminals. This contact between peripheral nerves and osteoclasts suggests that the peripheral nervous system may play a role in remodeling the inferior pharyngeal jaw in cichlids.

Distribution of 5HT receptors during the regeneration process after spinal cord transection in goldfish.

Spinal cord injury in teleosts leads to a fibrous scar, but axons sometimes spontaneously regenerate beyond the scar. In goldfish, regenerating axons enter the scar through tubular structures and enlargement of the tubular diameter is proportional to the increase in the number of regenerating axons. During the regeneration process, mast cells containing 5-hydroxytryptamine (5HT) are recruited to the injury site, and 5HT neurons are newly generated. Here, we investigated the distribution of 5HT receptors during this process to determine their role in remodeling the fibrous scar and tubular structures. At 2 weeks after spinal cord transection (SCT) in goldfish, expression of the 5HT2A and 5HT2C receptor subtypes was observed in the ependymo-radial glial cells lining the central canal of the spinal cord. 5HT2A was expressed at the luminal surface, suggesting that it is receptive to 5HT in the cerebrospinal fluid. 5HT2C, on the other hand, was expressed around the nuclei and in the radial processes protruding from the basal surface, suggesting that it is receptive to 5HT released from nearby nerve endings. 5HT2C was also expressed in the fibrous scar where mast cells containing 5HT were abundant. 5HT1B expression was coincident with the basement membrane bordering the fibrous scar and the surrounding nervous tissue, and with the basement membrane of the tubular structure through which axons pass during regeneration. Our findings suggest that multiple 5HT receptors are involved in remodeling the injured site during the regenerative process following SCT. Ependymo-radial glial cells expressing 5HT2A and 5HT2C are involved in neurogenesis and gliogenesis, which might contribute to remodeling the fibrous scar in coordination with 5HT-containing mast cells. Coincident expression of 5HT1B with the basement membrane might be involved in remodeling the tubular structures, thereby promoting axonal regeneration.

Expression of matrix metalloproteinases during axonal regeneration in the goldfish spinal cord.

Spinal cord injury in fish produces fibrous scar, but spontaneous axonal regeneration beyond the scar sometimes occurs. A previous study revealed that regenerating axons enter the scar through tubular structures with laminin, and that an increased number of axons within the tube is coincident with enlargement of the tube diameter and reduction of the fibrous scar area. The present study investigated the expression of matrix metalloproteinases (MMPs) that might play a role in the degradation of the extracellular matrix in fibrous scar tissue and in the remodeling of tubular structures. Spinal hemisection produced fibrous scar tissue in the lesion center, surrounded by nervous tissue. Two weeks after spinal lesioning, MMP-9 was expressed in some regenerating axons in the fibrous scar tissue. MMP-14 was expressed in the regenerating axons, as well as in glial processes in the fibrous scar tissue. MMP-2 was suggested to be expressed in mast cells in the fibrous scar. The mast cells were in contact with fibroblasts, and in close proximity to the basement membrane of tubular structures surrounding the regenerating axons. The present findings suggest that several MMPs are involved in axon regenerating processes following spinal cord injury in goldfish. MMP-9 and MMP-14 expressed in the regenerating axons might degrade extracellular matrix and support axonal growth deep into the fibrous scar tissue. MMP-14 expressed in glial cells and MMP-2 expressed in mast cells might also provide a beneficial environment for axonal regeneration, leading to successful motor recovery.

Chondroitin Sulfate Expression in Perineuronal Nets After Goldfish Spinal Cord Lesion.

Perineuronal nets (PNNs) surrounding neuronal cell bodies regulate neuronal plasticity during development, but their roles in regeneration are unclear. In the PNNs, chondroitin sulfate (CS) is assumed to be involved in inhibiting contact formation. Here, we examined CS expression in PNNs in the ventral horn of a goldfish hemisected spinal cord in which descending axons regenerate beyond the lesion to connect with distal spinal neurons. In intact fish, chondroitin sulfate A (CS-A)-positive PNNs accounted for 5.0% of HuC/D-immunoreactive neurons, and 48% of choline acetyltransferase (ChAT)-immunoreactive neurons. At 2, 4 and 8 weeks after spinal hemisection, CS-A-positive PNNs accounted for 8.4%-9.9% of HuC/D-immunoreactive neurons, and 50%-60% of ChAT-immunoreactive neurons, which was not significantly different from intact fish. Chondroitin sulfate C (CS-C)-positive PNNs accounted for 6.4% of HuC/D-immunoreactive neuron, and 67% of ChAT-immunoreactive neurons in intact fish. At 2, 4 and 8 weeks after spinal hemisection, CS-C-positive PNNs accounted for 7.9%, 5.5% and 4.3%, respectively, of HuC/D-immunoreactive neurons, and 65%, 52% and 42%, respectively, of ChAT-immunoreactive neurons, demonstrating a significant decrease at 4 and 8 weeks after spinal hemisection. Among ventral horn neurons that received descending axons labeled with tetramethylrhodamine dextran amine (RDA) applied at the level of the first spinal nerve, CS-A-positive PNNs accounted for 53% of HuC/D-immunoreactive neurons. At 2 and 4 weeks after spinal hemisection, CS-A-positive PNNs accounted for 57% and 56% of HuC/D-immunoreactive neurons, which was not significantly different from intact fish. CS-C-positive PNNs, accounted for 48% of HuC/D-immunoreactive neurons that received RDA-labeled axons. At 2 and 4 weeks after spinal hemisection, CS-C-positive PNNs significantly decreased to 22% of the HuC/D-immunoreactive neurons, and by 4 weeks after spinal hemisection they had returned to 47%. These findings suggest that CS expression is maintained in the PNNs after spinal cord lesion, and that the descending axons regenerate to preferentially terminate on neurons not covered with CS-C-positive PNNs. Therefore, CS-C in the PNNs possibly inhibits new contact with descending axons, and plasticity in the spinal neurons might be endowed by downregulation of CS-C in the PNNs in the regeneration process after spinal hemisection in goldfish.

Chondroitin sulfates do not impede axonal regeneration in goldfish spinal cord.

Chondroitin sulfate proteoglycans produced in glial scar tissue are a major inhibitory factor for axonal regeneration after central nervous system injury in mammals. The inhibition is largely due to chondroitin sulfates, whose effects differ according to the sulfation pattern. In contrast to mammals, fish nerves spontaneously regenerate beyond the scar tissue after spinal cord injury, although the mechanisms that allow for axons to pass through the scar are unclear. Here, we used immunohistochemistry to examine the expression of two chondroitin sulfates with different sulfation variants at the lesion site in goldfish spinal cord. The intact spinal cord was immunoreactive for both chondroitin sulfate-A (CS-A) and chondroitin sulfate-C (CS-C), and CS-A immunoreactivity overlapped extensively with glial processes positive for glial fibrillary acidic protein. At 1week after inducing the spinal lesion, CS-A immunoreactivity was observed in the cell bodies and extracellular matrix, as well as in glial processes surrounding the lesion center. At 2weeks after the spinal lesion, regenerating axons entering the lesion center overtook the CS-A abundant area. In contrast, at 1week after lesion induction, CS-C immunoreactivity was significantly decreased, and at 2weeks after lesion induction, CS-C immunoreactivity was observed along the regenerating axons entering the lesion center. The present findings suggest that after spinal cord injury in goldfish, chondroitin sulfate proteoglycans are deposited in the extracellular matrix at the lesion site but do not form an impenetrable barrier to the growth of regenerating axons.

A retrograde tracing study of compensatory corticospinal projections in rats with neonatal hemidecortication.

To examine the compensatory mechanisms in rats that underwent left decortication at postnatal day 7 (P7), we injected the retrograde tracers fluorescein isothiocyanate-cholera toxin B subunit (FITC-CTB) and Fast Blue (FB) into the right and left upper cervical spinal cord, respectively, at postoperative weeks 2, 3, 4, and 5 and counted the number of retrogradely labeled corticospinal neurons in the right cerebral cortex compared with that in normally developed rats. Significantly more ipsilaterally projecting neurons were labeled with FITC-CTB in the decorticated rats compared with normal rats at all time points examined. The number of labeled neurons was similar to that at P7 in normal rats. There were also some FITC-CTB and FB double-labeled neurons in both decorticated and normal rats. The number of double-labeled neurons in the decorticated rats increased each week and was significantly greater than that in normal rats at postoperative weeks 4 and 5. The present results suggest that the elimination of ipsilaterally projecting axons observed in normal rats was prevented in the decorticated rats, so that the cerebral cortex neurons on the unlesioned side projected corticospinal tracts to the ipsilateral spinal cord. Furthermore, the collaterals of the corticospinal tracts originating from the cerebral cortex on the unlesioned side also project to the ipsilateral spinal cord. These compensatory mechanisms might underlie the acquisition of motor function in these animals.

Regeneration of descending projections to the spinal motor neurons after spinal hemisection in the goldfish.

Following spinal transection, descending spinal projections from goldfish brainstem neurons spontaneously regenerate beyond the lesion site. The nucleus of the medial longitudinal fasciculus (nFLM), which has a critical role in swimming, also sends regenerated axons over a long distance to the ipsilateral spinal cord. To examine whether regenerated axons re-innervate the appropriate targets, we injected rhodamine dextran amine (RDA) into the nFLM of spinally transected goldfish and examined anterogradely labeled axons in the spinal cord. In intact controls, there were many RDA-labeled boutons or varicosities in the spinal cord in close apposition to both neurons positive for calcitonin gene-related peptide (CGRP), and those negative for CGRP. This suggests that the nFLM neurons project axons directly to the motoneurons and interneurons in the spinal cord. Four days after hemisection 1 mm caudal to the rostral end of the spinal cord, the number of RDA-labeled boutons in close apposition to the spinal neurons was significantly decreased on the side ipsilateral to the injection. Six to twelve weeks after spinal hemisection, regenerated axons ran through the repaired lesion site, and the number of RDA-labeled boutons or varicosities in close apposition to the ipsilateral spinal neurons had returned to the control level. These findings suggest that the midbrain-spinal pathway, critical for locomotion in fish, spontaneously regenerates beyond the lesion site to re-innervate the appropriately innervated targets after spinal lesion.