Tsutomu Iseda

Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons specifically project axons to central and peripheral targets according to their sensory modality. The Runt-related genes Runx1 and Runx3 are expressed in DRG neuronal subpopulations, suggesting that they may regulate the trajectories of specific axons. Here we report that Runx3-deficient (Runx3−/−) mice displayed severe motor discoordination and that few DRG neurons synthesized the proprioceptive neuronal marker parvalbumin. Proprioceptive afferent axons failed to project to their targets in the spinal cord as well as those in the muscle. NT-3-responsive Runx3−/− DRG neurons showed less neurite outgrowth in vitro. However, we found no changes in the fate specification of Runx3−/− DRG neurons or in the number of DRG neurons that expressed trkC. Our data demonstrate that Runx3 is critical in regulating the axonal projections of a specific subpopulation of DRG neurons.

Tenascin-C regulates proliferation and migration of cultured astrocytes in a scratch wound assay

Tenascin-C (TNC), an extracellular matrix glycoprotein, is involved in tissue morphogenesis like embryogenesis, wound healing or tumorigenesis. Astrocytes are known to play major roles in wound healing in the CNS. To elucidate the roles of TNC in wound closure by astrocytes, we have examined the morphological changes of cultured astrocytes in a scratch wound assay and measured the content of soluble TNC released into the medium. We have also localized the expression of TNC mRNA, TNC, glial fibrillary acidic protein (GFAP), vimentin and integrin beta1. After wounding, glial cells rapidly released the largest TNC isoform and proliferated in the border zones. Subsequently, they became polarized with unidirectional processes and finally migrated toward the denuded area. The proliferating border zone cells and pre-migratory cells intensely expressed TNC mRNA, TNC-, vimentin-, GFAP- and integrin beta1-like immunoreactivity, while the migratory cells showed generally reduced expression except the front. Exogenous TNC enhanced cell proliferation and migration, while functional blocking with anti-TNC or anti-integrin beta1 antibody reduced both of them. These results suggest that mechanical injury induces boundary astrocytes to produce and release TNC that promotes cell proliferation and migration via integrin beta1 in an autocrine/paracrine fashion.

Spontaneous regeneration of the corticospinal tract after transection in young rats: a key role of reactive astrocytes in making favorable and unfavorable conditions for regeneration.

We demonstrated the occurrence of marked regeneration of the corticospinal tract (CST) after a single transection and failure of regeneration after a repeated transection in young rats. To provide convincing evidence for the complete transection and regeneration we used retrograde neuronal double labeling. Double-labeled neurons that took up the first tracer from the transection site and the second tracer from the injection site caudal to the transection site were observed in the sensorimotor cortex. The anterograde tracing method revealed various patterns of regeneration. In the most successful cases the vast majority of regenerated fibers descended in the normal tract and terminated normally whereas a trace amount of fibers coursed aberrantly. In the less successful cases fibers descended partly normally and partly aberrantly or totally aberrantly. To clarify the role of astrocytes in determining the success or failure of regeneration we compared expression of glial fibrillary acidic protein (GFAP), vimentin and neurofilament (NF) immunoreactivity (IR) in the lesion between single and repeated transections. In either transection, astrocytes disappeared from the CST near the lesion site as early as 3 h after lesioning. However, by 24 h after a single transection, immature astrocytes coexpressing GFAP- and vimentin-IR appeared in the former astrocyte-free area and NF-positive axons crossed the lesion. By contrast, after a repeated transection the astrocyte-free area spread and NF-positive axons never crossed the lesion. It appears likely that the major sign, and possibly cause of failure of regeneration is the prolonged disappearance of astrocytes in the lesioned tract area.

Runx3 is essential for the target-specific axon pathfinding of trkc-expressing dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons project their axons to specific target layers in the gray matter of the spinal cord, according to their sensory modality (Neuron 30 (2001), 707; Cell 101 (2000), 485; Neuron 31 (2001), 59; J. Comp. Neurol. 380 (1997), 215; Sensory Neurons, Oxford Univ. Press, New York, 1992, p. 131). Expression of runt-related Runx/AML genes (Mech. Dev. 109 (2001), 413) on subtypes of DRG neurons suggests their involvement in lamina-specific afferent differentiation and maturation. Here we show that Runx3-/- mice display severe limb ataxia and abnormal posture and that most of them die shortly after birth. They show that proprioceptive afferent axons fail to reach the ventral horn and have a smaller dorsal funiculus in their spinal cords. Despite the strong resemblance of this phenotype to that of knockout mice deficient in neurotrophin-3 (NT-3) (Cell 77 (1994), 503; Nature 369 (1994), 658) and its receptor, trkC, (Nature 368 (1994), 249), which show proprioceptive afferent loss through selective neuronal cell death, Runx3-/- mice maintain normal number of TrkC/trkC positive DRG neurons throughout development. Our results suggest that Runx3 controls the target-specific axon pathfinding of trkC-expressing DRG neurons in the spinal cord.

Simultaneous up-regulation of urokinase-type plasminogen activator (uPA) and uPA receptor by hepatocyte growth factor/scatter factor in human glioma cells.

Several lines of evidence indicate that hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, c-Met, may play an important role in progression of human glioma. In this study, effects of HGF/SF on urokinase- type plasminogen activator (uPA)-mediated proteolysis network were examined in c-Met-positive human glioma cell lines. Treatment of the glioma cells with various concentrations of HGF/SF resulted in an enhanced secretion of uPA proteins accompanying increased transcription of uPA mRNA in a dose dependent fashion. The levels of uPA receptor (uPAR) mRNAs were also elevated simultaneously upon HGF/SF stimulation, and the cell-surface associated uPA activity was also elevated by the treatment. Since concomitant expression of HGF and its receptor c-Met are frequently observed in malignant gliomas, these results suggest that HGF/SF participates in invasive process of malignant glioma cells not only by its motility-stimulating activity but also through enhanced degradation of the extracellular matrix induced by autocrine activation of uPA proteolysis network.

Appearance of Early Venous Filling During Intra-Arterial Reperfusion Therapy for Acute Middle Cerebral Artery Occlusion A Predictive Sign for Hemorrhagic Complications

Background and Purpose— The purpose of this study was to evaluate the correlation between appearance of angiographic early venous filling during intra-arterial reperfusion therapy and posttherapeutic hemorrhagic complications. Methods— For the past 7 years, 104 patients prospectively underwent superselective local angiography via a microcatheter before and during intra-arterial reperfusion therapy for acute middle cerebral artery occlusion to evaluate the presence or absence of early venous filling. In principle, reperfusion therapy was discontinued just after appearance of early venous filling for fear of hemorrhage. There were 2 types of early venous filling: early filling of the thalamostriate vein from the lenticulostriate arteries and that of the cortical vein from the cortical arteries. Results— Among these 104 patients, 31 (29.8%) had early venous filling: 19 had early filling of the thalamostriate vein, and the other 12 had early filling of the cortical vein. Eight of the 19 patients (42.1%) and 2 of the 12 patients (16.7%) had massive hematoma with neurological worsening, whereas only 1 of the 73 patients (1.4%) without early venous filling had massive hematoma. There was a significant correlation between early venous filling and massive hematoma in both the deep (P <0.0001) and superficial (P =0.0019) middle cerebral artery territories. The sensitivity and specificity of the presence of early venous filling as an indicator of parenchymal hematoma were 71% and 83%, respectively. None of the 31 ischemic areas with early venous filling could escape cerebral infarction. Conclusions— Appearance of early venous filling may indicate irreversible brain damage and may be a predictive sign for parenchymal hematoma.

Spontaneous regeneration of the corticospinal tract after transection in young rats: collagen type IV deposition and astrocytic scar in the lesion site are not the cause but the effect of failure of regeneration.

In young rats the corticospinal tract regenerated after a single transection of the spinal cord with a sharp blade, but regeneration failed if the transection was repeated to make a more traumatic injury. To identify cells and associated molecules that promote or impede regeneration, we compared expression of collagen type IV, glial fibrillary acidic protein (GFAP), and vimentin immunoreactivity (IR) at the lesion sites in combination with anterograde axonal tracing between animals with two types of transection. Axonal regeneration occurred as early as 18 hours after transection; regenerating axons penetrated vessel‐like structures with collagen type IV‐IR at the lesion site, while reactive astrocytes coexpressing GFAP‐ and vimentin‐IR appeared in the lesioned white matter. In contrast, when regeneration failed astrocytes were absent near the lesion. By 7 days sheet‐like structures with collagen type IV‐IR and astrocytic scar appeared in the lesioned white matter and persisted until the end of the observation period (31 days). On the basis of their spatiotemporal appearance, collagen type IV‐IR sheet‐like structures and the astrocytic scar follow, rather than cause, the failure of regeneration. The major sign, and perhaps cause, of failure of axonal regeneration is likely the prolonged disappearance of astrocytes around the lesion site in the early postinjury period. J. Comp. Neurol. 464:343–355, 2003.

Secretion of tenascin-C by cultured astrocytes: regulation of cell proliferation and process elongation.

Tenascin-C (TNC), an extracellular matrix glycoprotein, is involved in tissue morphogenesis like embryogenesis, wound healing or tumorigenesis. Quiescent astroglia in long-term primary cultures are known to show rapid morphological changes after subculture and serum deprivation/re-addition (SSDR). To elucidate roles of TNC in the morphogenetic processes of cultured astrocytes, we have revealed morphological changes in association with soluble TNC contents in the medium and expression of TNC mRNA, TNC, glial fibrillary acidic protein (GFAP) and integrin beta1, one of its cell surface receptors, in glial cells after SSDR. Soluble TNC in the medium rapidly increased in amount at 4 h when GFAP-positive cells expressed TNC mRNA, TNC and integrin beta1. Cellular proliferation and growth occurred in colonies expressing TNC mRNA, TNC and integrin beta1 during the first 24 h. During the next 24 h, process elongation and cell migration occurred in association with increased GFAP expression and re-elevation of soluble TNC in the medium. Cell bodies became flat and larger with increased GFAP and reduced TNC expression at 72 h, while cultures became confluent with reduced GFAP and TNC expression at 96 h after SSDR. Functional blocking with anti-TNC antibody reduced cell proliferation and induced morphological change from a process-bearing slender shape to a flat and wide shape presumably due to increased cell adhesion. These findings strongly support the idea that endogenous TNC produced and released by astrocytes in response to serum stimulation induces their proliferation and process elongation through a paracrine/autocrine mechanism.

Effects of an embryonic repair graft on recovery from spinal cord injury.

It is widely believed that mammalian CNS axons have little regenerative capacity because their environment is non-permissive to regrowth. This viewpoint is based, in large part, on the fact that in virtually all previous studies on regeneration following spinal cord injury, regenerated axonal projections have been few in number, quite short, and considered to be mostly aberrant. As a result, motor recovery has been very limited in both experimental preparations and the human. In this chapter, we describe use of a neonatal, spinally transected animal model in which selected spinal cord segments were carefully replaced with equivalent tissue from embryonic tissue of the same species. We demonstrate that the new spinal environment is indeed permissive, and reconstruction is possible of neural connections, which are similar to the pre-injury, normal projections. Moreover, the distribution and number of regenerated axons are closely related to the extent of functional motor recovery. Our results suggest that contrary to doctrinaire thought, the mammalian CNS possesses a remarkable capacity for regrowth. For this to be efficacious, however, regenerating axons must contact the inherent, pre-injury guidance system, whose cues were used for establishing appropriate neural connections in the developing animal, and are retained in the adult. It is argued that by use of these guidance cues, regenerating axons that traverse the site of a spinal cord injury, can project on to locate their pre-injury pathways and targets, and thereby restore function.

Intravenous low-dose native tissue plasminogen activator for distal embolism in the middle cerebral artery divisions or branches: a pilot study.

OBJECTIVE We prospectively evaluated the safety and efficacy of an intravenous infusion of low-dose native tissue plasminogen activator for distal embolisms in the middle cerebral artery divisions or branches. METHODS Twenty patients were selected according to the following computed tomographic and angiographic criteria and treated with intravenous infusion of 7.2 mg of tisokinase: 1) no early ischemic changes on the initial computed tomographic scan, and 2) embolic occlusion of the middle cerebral artery divisions or branches without the involvement of the lenticulostriate arteries. For comparison, the records of 12 patients from previous years who met the above inclusion criteria but underwent no thrombolytic therapy were reviewed retrospectively. The degree of neurological recovery was assessed using the National Institutes of Health Stroke Scale at 24 hours after admission. Major neurological improvement was defined as a decrease in the stroke score by 4 points or more. RESULTS There was no significant difference in stroke scores at the time of admission between the treatment group (mean +/- standard deviation, 12.8 +/- 2.8) and the untreated group (14.0 +/- 2.4). In the treatment group, major neurological improvement was seen in 17 (85%) of 20 patients, whereas in the untreated group only 5 (41.7%) of 12 patients showed major neurological improvement (P < 0.05). The mean score at 24 hours in the treatment group (3.6 +/- 3.5) was significantly lower than that in the untreated group (9.4 +/- 7.3) (P < 0.05). There was no hemorrhagic complication with neurological exacerbation in the treatment group. CONCLUSION Even with delayed initiation (>3 h after symptom onset), intravenous infusion of low-dose tisokinase may be safe and effective for small distal emboli in the middle cerebral artery divisions or branches, when early ischemic changes on computed tomographic scans and involvement of the lenticulostriate arteries are absent.