Differential expression of class 3 and 4 semaphorins and netrin in the lamprey spinal cord during regeneration

To explore the role of axon guidance molecules during regeneration in the lamprey spinal cord, we examined the expression of mRNAs for semaphorin 3 (Sema3), semaphorin 4 (Sema4), and netrin during regeneration by in situ hybridization. Control lampreys contained netrin-expressing neurons along the length of the spinal cord. After spinal transection, netrin expression was downregulated in neurons close (500 mum to 10 mm) to the transection at 2 and 4 weeks. A high level of Sema4 expression was found in the neurons of the gray matter and occasionally in the dorsal and the edge cells. Fourteen days after spinal cord transection Sema4 mRNA expression was absent from dorsal and edge cells but was still present in neurons of the gray matter. At 30 days the expression had declined to some extent in neurons and was absent in dorsal and edge cells. In control animals, Sema3 was expressed in neurons of the gray matter and in dorsal and edge cells. Two weeks after transection, Sema3 expression was upregulated near the lesion, but absent in dorsal cells. By 4 weeks a few neurons expressed Sema3 at 20 mm caudal to the transection but no expression was detected 1 mm from the transection. Isolectin I-B(4) labeling for microglia/macrophages showed that the number of Sema3-expressing microglia/macrophages increased dramatically at the injury site over time. The downregulation of netrin and upregulation of Sema3 near the transection suggests a possible role of netrin and semaphorins in restricting axonal regeneration in the injured spinal cord.     

Early development of the retina and pineal complex in the sea lamprey: comparative immunocytochemical study.

Lampreys have a complex life cycle, with largely differentiated larval and adult periods. Despite the considerable interest of lampreys for understanding vertebrate evolution, knowledge of the early development of their eye and pineal complex is very scarce. Here, the early immunocytochemical organization of the pineal complex and retina of the sea lamprey was studied by use of antibodies against proliferating cell nuclear antigen (PCNA), opsin, serotonin, and gamma-aminobutyric acid (GABA). Cell differentiation in the retina, pineal organ, and habenula begins in prolarvae, as shown by the appearance of PCNA-negative cells, whereas differentiation of the parapineal vesicle was delayed until the larval period. In medium-sized to large larvae, PCNA-immunoreactive (-ir) cells were numerous in regions of the lateral retina near the differentiated part of the larval retina (central retina). A late-proliferating region was observed in the right habenula. Opsin immunoreactivity appears in the pineal vesicle of early prolarvae and 3 or 4 days later in the retina. In the parapineal organ, opsin immunoreactivity was observed only in large larvae. In the pineal organ, serotonin immunoreactivity was first observed in late prolarvae in photoreceptive (photoneuroendocrine) cells, whereas only a few of these cells appeared in the parapineal organ of large larvae. No serotonin immunoreactivity was observed in the larval retina. GABA immunoreactivity appeared earlier in the retina than in the pineal complex. No GABA-ir perikaryon was observed in the retina of larval lampreys, although a few GABA-ir centrifugal fibers innervate the inner retina in late prolarvae. First GABA-ir ganglion cells occur in the pineal organ of 15-17 mm larvae, and their number increases during the larval period. The only GABA-ir structures observed in the parapineal ganglion of larvae were afferent fibers, which appeared rather late in development. The time sequence of development in these photoreceptive structures is rather different from that observed in teleosts and other vertebrates. This suggests that the unusual development of the three photoreceptive organs in lampreys reflects specialization for their different functions during the larval and adult periods.     

Semaphorins and their receptors in lamprey CNS: Cloning, phylogenetic analysis, and developmental changes during metamorphosis

The large, conserved semaphorin gene family encodes axon guidance molecules in both invertebrates and vertebrates. The primitive vertebrate lamprey diverged near the time of vertebrate origins and is useful for understanding the gene duplication events that led to the increased complexity of the vertebrate genome. We characterized the sequence and expression pattern of semaphorins and their receptors genes in the sea lamprey, Petromyzon marinus. We uncovered two members of the semaphorin family in sea lamprey. The first encodes a diffusible class 3 type semaphorin protein that is most similar to the human and mouse Sema3F (71% amino acid identity). The second encodes a transmembrane class 4 type semaphorin that is most similar to mouse Sema4D and human Sema4G, with 38% amino acid identity within the Sema domain. We also identified in lamprey two members of the semaphorin receptor family, lamprey Plexin A1 and Plexin A2. Phylogenetic analysis indicates that lamprey Sema3 and Sema4 represent precursor genes existing prior to the origin of the vertebrate Sema3A-G and Sema4A-G subfamilies. Therefore, the gene duplication event that gave rise to those subfamilies must have occurred after the divergence of jawed vertebrates from jawless fish. These semaphorins and plexins are expressed in unique and dynamic patterns in lamprey spinal cord and brain during development.     

Synergistic effects of bone marrow stromal cells and a Rho kinase (ROCK) inhibitor,Fasudil on axon regeneration in rat spinal cord injury

Transplanted bone marrow stromal cells (BMSC) promote functional recovery after spinal cord injury (SCI) through multiple mechanisms. A Rho kinase inhibitor, Fasudil also enhances axonal regeneration. This study was aimed to evaluate whether combination therapy of BMSC transplantation and Fasudil further enhances axonal regeneration and functional recovery in rats subjected to SCI. Fasudil or vehicle was injected for 2 weeks. BMSC or vehicle transplantation into the rostral site of SCI was performed at 7 days after injury. Neurological symptoms were assessed throughout the experiments. Fluoro‐Ruby was injected into the dorsal funiculus of the rostral site of SCI at 63 days after injury. The fate of the transplanted BMSC was examined using immunohistochemistry. BMSC transplantation significantly increased the number of Fluoro‐Ruby ‐labeled fibers of the dorsal corticospinal tracts at the caudal site of SCI, enhancing functional recovery of the hind limbs. Some of the engrafted BMSC were positive for Fluoro‐Ruby, neuronal specific nuclear protein and microtubule‐associated protein‐2, suggesting that they acquired neuronal phenotypes and built synaptic connection with the host's neural circuits. Fasudil treatment also improved axonal continuity, but did not promote functional recovery. Combination therapy dramatically increased the number of Fluoro‐Ruby‐labeled fibers of the dorsal corticospinal tracts at the caudal site of SCI, but did not further boost the therapeutic effects on locomotor function by BMSC transplantation. The findings suggest that BMSC transplantation and Fasudil provide synergistic effects on axon regeneration after SCI, although further studies would be necessary to further enhance functional recovery.     

Transplantation of glial-restricted precursor cells into the adult spinal cord: survival, glial-specific differentiation, and preferential migration in white matter

Glial-restricted precursor (GRP) cells are among a number of candidate cells for transplantation repair of CNS injury. The isolation and characterization of these cells in vitro have been described previously, but their in vivo properties are not well understood. We examined the fate and migration of grafted fetal GRP cells harvested from alkaline phosphatase-expressing transgenic rats into intact and injured spinal cord. Transplanted GRP cells survived for at least 6 weeks and differentiated along astrocytic and oligodendrocytic but not neuronal lineages. Cells grafted into the intact spinal cord exhibited robust migration along longitudinal white matter tracts and by 6 weeks migrated more than 15 mm. In contrast, migration of GRP cells in the gray matter was very limited. We then examined the phenotypic properties of proliferating endogenous precursors in response to injury by BrdU labeling. The predominant proliferating population seen after injury consisted of GRP-like cells with Nkx2.2/olig2 phenotype. Incorporation of BrdU by endogenous cells suggests that the environment provides proliferation signals and is permissive to glial precursor survival. To test if exogenous GRP cells would respond similarly, we transplanted GRP cells into a lateral funiculus injury. GRP cells survived and differentiated along glial lineages and migrated along white matter tracts in the injured spinal cord. Directed homing toward the lesion was not seen and there was no significant bias in differentiation between cells transplanted into injured and uninjured spinal cord. GRP cell transplants may therefore provide a cellular transplant that can respond to appropriate endogenous cues to produce therapeutic molecules and new glial cells after injury.     

Immunohistochemical demonstration of some putative neurotransmitters in the lamprey spinal cord and spinal ganglia: 5-hydroxytryptamine-, tachykinin-, and neuropeptide-Y-immunoreactive neurons and fibers

The distribution of some putative neurotransmitters was investigated in the spinal cord and spinal ganglia of the lamprey, a primitive vertebrate, by using immunohistochemical methods. In the spinal cord a midline row of 5-hydroxytryptamine (5-HT)-immunoreactive neurons was present immediately ventral to the central canal over the entire length of the spinal cord. The ventral processes of these neurons formed a dense ventromedial plexus of varicosities. In the dorsal, lateral, and ventral spinal axon columns, several longitudinal 5-HT fibers were present. After chronic spinal transections the distribution of 5-HT fibers was unchanged; it is therefore concluded that there was no substantial descending 5-HT contribution and that the spinal 5-HT neurons supplied the regional 5-HT innervation. The spinal 5-HT cells sent fibers into the dorsal and ventral roots; 5-HT cell bodies and fibers were also present in the spinal dorsal root ganglia, in their dorsal, ventral, and lateral nerve branches, and in the dorsal and ventral branches of the ventral roots. Neurons and fibers containing peptides of the tachykinin (TK) family (to which, amongst others, substance P belongs) were found in the spinal cord. TK neurons in the spinal cord supplied the local TK innervation, as well as TK fibers in the dorsal and ventral roots. Fibers have been found containing either TK, or 5-HT, or both compounds. Neurons containing neuropeptide-Y (NPY)-immunoreactive material were present in a medial column just dorsal to the central canal. The NPY neurons have longitudinal, mainly descending, fibers that provide the local NPY innervation of the lamprey spinal cord. The present results provide evidence for local spinal systems containing 5-HT, TK, 5-HT and TK, or NPY, but in contrast to mammals, these compounds do not seem to arise from supraspinal neurons.     

Innexins in the lobster stomatogastric nervous system: cloning, phylogenetic analysis, developmental changes and expression within adult identified dye and electrically coupled neurons

Gap junctions play a key role in the operation of neuronal networks by enabling direct electrical and metabolic communication between neurons. Suitable models to investigate their role in network operation and plasticity are invertebrate motor networks, which are built of comparatively few identified neurons, and can be examined throughout development; an excellent example is the lobster stomatogastric nervous system. In invertebrates, gap junctions are formed by proteins that belong to the innexin family. Here, we report the first molecular characterization of two crustacean innexins: the lobster Homarus gammarus innexin 1 (Hg-inx1) and 2 (Hg-inx2). Phylogenetic analysis reveals that innexin gene duplication occurred within the arthropod clade before the separation of insect and crustacean lineages. Using in situ hybridization, we find that each innexin is expressed within the adult and developing lobster stomatogastric nervous system and undergoes a marked down-regulation throughout development within the stomatogastric ganglion (STG).The number of innexin expressing neurons is significantly higher in the embryo than in the adult. By combining in situ hybridization, dye and electrical coupling experiments on identified neurons, we demonstrate that adult neurons that express at least one innexin are dye and electrically coupled with at least one other STG neuron. Finally, two STG neurons display no detectable amount of either innexin mRNAs but may express weak electrical coupling with other STG neurons, suggesting the existence of other forms of innexins. Altogether, we provide evidence that innexins are expressed within small neuronal networks built of dye and electrically coupled neurons and may be developmentally regulated.     

Olfactory ensheathing cell transplantation and inhibition of NogoA, NgR and RhoA expression in the damaged zone to ameliorate spinal cord injury

BACKGROUND:Olfactory ensheathing cell(OEC)transplantation promotes repair of spinal cord injury. Neural regeneration inhibits binding of the myelin protein Nogo to its receptor(NgR),activates downstream inhibitory signal RhoA, and leads to axonal degeneration.OBJECTIVE: To determine the relationship between OECs transplantation for spinal cord injury and NogoA, NgR, and RhoA protein expression in the damaged zone.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed from September 2006 to May 2007 at the Key Laboratory of Environment and Genes in Xi'an Jiaotong University School of Medicine, China.MATERIALS: OECs were harvested from healthy, adult, male, Sprague Dawley rats aged 6 months. Mouse anti-rat NogoA, NgR, and RhoA monoclonal antibodies were utilized for detection.METHODS: A total of 40 adult Sprague Dawley rats were randomly assigned to four groups: normal, model, OECs, and DF12, with 10 animals in each group. Transverse section spinal cord injury was established in the OECs and DF12 groups, followed by injection of 1 μL OECs suspension(1×108/mL)or equivalent DF12 medium at 1 mm above and below the injury site.MAIN OUTCOME MEASURES: Immunohistochemistry and Western blot were utilized to detect NogoA, NgR, and RhoA expression in the spinal cord injury lesions. Morphological changes were observed by argyrophilia staining, and lower extremity function of the animals was assessed using Basso, Beattie, and Bresnahan scores.RESULTS: Eight weeks following OECs transplantation, a significant increase in new axons was observed in the OECs group, and nerve fibers crossed the injury site to repair spinal cord injury.Qualitative and quantitative results from the OECs group were superior to the model and DF12 groups. At 8 weeks after transplantation, Basso, Beattie, and Bresnahan scores were significantly greater in the OECs group compared with the model and DF12 groups(P< 0.01), but expression of NogoA, NgR, and RhoA protein was significantly decreased compared with the model and DF12 groups(P< 0.05).CONCLUSION: OEC transplantation could inhibit NogoA, NgR, and RhoA expression in spinal cord injury lesions, thereby promoting repair of spinal cord injury.