Glial cell line-derived neurotrophic factor (GDNF) has been shown to rescue developing motoneurons in vivo and in vitro from both naturally occurring and axotomyinduced cell death. To test whether GDNF has trophic effects on adult motoneurons, we used a mouse model of injuryinduced adult motoneuron degeneration. Injuring adult motoneuron axons at the exit point of the nerve from the spinal cord (avulsion) resulted in a 70% loss of motoneurons by 3 weeks following surgery and a complete loss by 6 weeks. Half of the loss was prevented by GDNF treatment. GDNF also induced an increase (hypertrophy) in the size of surviving motoneurons. These data provide strong evidence that the survival of injured adult mammalian motoneurons can be promoted by a known neurotrophic factor, suggesting the potential use of GDNF in therapeutic approaches to adultonset motoneuron diseases such as amyotrophic lateral sclerosis.Factors that promote motoneuron survival have potential as therapeutic agents for the treatment of human neurodegenerative diseases (1). It has been shown that several neurotrophic factors, including brain-derived neurotrophic factor (BDNF) (2-4), insulin-like growth factor (I.GF) (5), ciliary neurotrophic factor (6), and glial cell line-derived neurotrophic factor (GDNF) (7-10), can rescue developing motoneurons from both naturally occurring and axotomy-induced cell death. However, whether these trophic factors also play a role in adult motoneuron survival is not known. Because many motoneuron diseases, such as amyotrophic lateral sclerosis, have a late (i.e., adult) onset, it is important to determine whether neurotrophic factors are effective on injured adult motoneurons.Interactions between motoneurons and their target muscles have been extensively investigated. For example, it is known that transection of axons of motoneurons or removal of their target during embryonic and early postnatal development results in massive motoneuron cell loss, whereas axotomy of adult peripheral nerve induces little if any neuronal death (11)(12)(13)(14)(15)(16)(17)(18)(19). A plausible explanation for this difference is that trophic support derived from mature nonneuronal cells (e.g., Schwann cells) associated with the peripheral nerve maintains the survival of adult motoneurons.A different type of lesion, ventral root avulsion, which involves pulling the root out of the spinal cord, induces the death of virtually all motoneurons in the adult rat and provides a good model to examine the response of adult motoneurons to trophic factors (20,21). The expression of nitric oxide synthase (NOS), an enzyme for synthesis of the free radical nitric oxide (NO), can be induced in adult rat motoneurons following both spinal root avulsion (20,22) and cranial nerve axotomy (23), and it has been suggested that the cell death following these lesions may be induced by oxidative stress and reactive oxygen species such as [20][21][22].Although the target dependency of motoneuron survival is diminished in adult animals (15, 18), adult rat m...

We have examined the ability of different neurotrophic and growth factors to prevent axotomy-induced motoneuron cell death in the developing mouse spinal cord. After postnatal unilateral section of the mouse sciatic nerve, most motoneuron (MN) loss occurs in the lateral motor column of the fourth lumbar segment (L4). Significant axotomy-induced cell death occurred after surgery performed on or before postnatal day (PN) 5. In contrast, no significant cell loss was found when axotomy was performed after PN10. Axotomy on PN2 or PN5 resulted in a 44% loss of L4 motoneurons by 7 days, and a 66% loss of motoneurons by 10 days postsurgery. Implantation of gelfoam presoaked in various neurotrophic factors at the lesion site rescued axotomized motoneurons. Nerve growth factor (NGF), neurotrophin-4/5 (NT-4/5) and ciliary neurotrophic factor (CNTF) rescued 20%-30% of motoneurons, whereas brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and insulin-like growth factor 1 (IGF-1) rescued virtually all motoneurons from axotomy-induced death. By contrast, platelet-derived growth factor (PDGF)-AA, PDGF-AB, basic fibroblast growth factor (bFGF), and interleukin (IL-6) were ineffective on motoneuron survival following axotomy. NGF, BDNF, NT-3, IGF-1, and CNTF also prevented axotomy-induced atrophy of surviving motoneurons. These data show that mouse lumbar motoneurons continue to be vulnerable to axotomy up to about 1 week after birth and that a number of trophic agents, including the neurotrophins, CNTF, and IGF-1, can prevent the death of these neurons following axotomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevation of intracellular heat shock protein (Hsp)70 increases resistance of cells to many physical and metabolic insults. We tested the hypothesis that treatment with Hsc70 can also produce that effect, using the model of axotomy-induced neuronal death in the neonatal mouse. The sciatic nerve was sectioned and in some animals purified bovine brain Hsc70 was applied to the proximal end of the nerve immediately thereafter and again 3 days later. Seven days postaxotomy, the surviving sensory neurons of the lumbar dorsal root ganglion (DRG) and motoneurons of the lumbar ventral spinal cord were counted to assess cell death. Axotomy induced the death of approximately 33% of DRG neurons and 50% of motoneurons, when examined 7 days postinjury. Application of exogenous Hsc70 prevented axotomy-induced death of virtually all sensory neurons, but did not significantly alter motoneuron death. Thus, Hsc70 may prove to be useful in the repair of peripheral sensory nerve damage.

To investigate the mechanical mechanisms behind tumor cell arrest in the microvasculature, we injected fluorescently labeled human breast carcinoma cells or similarly sized rigid beads into the systemic circulation of a rat. Their arrest patterns in the microvasculature of mesentery were recorded and quantified. We found that 93% of rigid beads were arrested either at arteriole–capillary intersections or in capillaries. Only 3% were at the capillary–postcapillary venule intersections and in postcapillary venules. In contrast, most of the flexible tumor cells were either entrapped in capillaries or arrested at capillary or postcapillary venule–postcapillary venule intersections and in postcapillary venules. Only 12% of tumor cells were arrested at the arteriole–capillary intersections. The differential arrest and adhesion of tumor cells and microbeads in the microvasculature was confirmed by a χ2 test (p < 0.001). These results demonstrate that mechanical trapping was responsible for almost all the arrest of beads and half the arrest of tumor cells. Based on the measured geometry and blood flow velocities at the intersections, we also performed a numerical simulation using commercial software (ANSYS CFX 12.01) to depict the detailed distribution profiles of the velocity, shear rate, and vorticity at the intersections where tumor cells preferred to arrest and adhere. Simulation results reveal the presence of localized vorticity and shear rate regions at the turning points of the microvessel intersections, implying that hemodynamic factors play an important role in tumor cell arrest in the microcirculation. Our study helps elucidate long-debated issues related to the dominant factors in early-stage tumor hematogenous metastasis.

Microtubules are important components of the cellular cytoskeleton that play roles in various cellular processes such as vesicular transport and spindle formation during mitosis. They are formed by an ordered organization of α-tubulin and β-tubulin hetero-polymers. Altering microtubule polymerization has been known to be the mechanism of action for a number of therapeutically important drugs including taxanes and epothilones. Traditional cell-based assays for tubulin-interacting compounds rely on their indirect effects on cell cycle and/or cell proliferation. Direct monitoring of compound effects on microtubules is required to dissect detailed mechanisms of action in a cellular setting. Here we report a high-content assay platform to monitor tubulin polymerization status by directly measuring the acute effects of drug candidates on the cellular tubulin network with the capability to dissect the mechanisms of action. This high-content analysis distinguishes in a quantitative manner between compounds that act as tubulin stabilizers versus those that are tubulin destabilizers. In addition, using a multiplex approach, we expanded this analysis to simultaneously monitor physiological cellular responses and associated cellular phenotypes.

Experimental lesions have been used widely to induce motoneuron (MN) degeneration as a model to test the ability of different trophic molecules to prevent lesion-induced alterations. However, the morphological mechanisms of spinal MN death following different types of lesions is not clear at the present time. In this study, we have characterized the morphological characteristics of MN cell death by examining DNA fragmentation and the ultrastructural and light microscopic morphological features of MNs following different types of spinal nerve injury (i.e., axotomy and avulsion) in the developing and adult mouse. In neonatal mice, axotomy induced cell death as well as the atrophy of MNs that survived the injury. DNA fragmentation could be detected by using the terminal deoxynucleotidyl transferase (TUNEL) method during the cell death process following neonatal axotomy, whereas TUNEL labeling was not observed following either neonatal or adult avulsion. However, with the exception of TUNEL labeling, the morphological characteristics of MN death following neonatal axotomy and avulsion were similar, and both resembled most closely the form of programmed cell death termed cytoplasmic or type 3B, which exhibits similarities as well as differences with currently accepted definitions of apoptosis. By contrast, adult avulsion resulted in a type of degeneration that resembled necrosis more closely. However, even there, the morphology was mixed, showing characteristics of both apoptosis and necrosis. These results indicate that the mode of MN degeneration is complex and is related to developmental age and type of lesion.

Icariside II (ICS II), a phosphodiesterase 5 inhibitor (PDE 5-I), is a major ingredient of Epimedium brevicornum, with wide spectrum of neuroprotective properties. However, little is known about the potential beneficial effect of ICS II on neuronal cell proliferation, and its possible underlying mechanism remains still unclear. We hypothesized that the beneficial effect of ICS II on neuron-like highly differentiated rat pheochromocytoma (PC12) cell proliferation is correlated with the nitric oxide (NO) signaling pathway and its upstream of PI3K/AKT pathway. PC12 cells were treated with ICS II alone or together with L-NMMA, H89, KT-5823, and/or LY294002 (the inhibitor of NOS, PKA, PKG, PI3K, respectively). It was found that ICS II concentration-dependently promoted PC12 cells proliferation, and cell cycle analysis showed that the proportion of ICS II-treated PC12 cells in S phase was higher than that of control. Moreover, ICS II at the appropriate concentration (100 μM) not only increased nNOS expression, NO production, but also enhanced cGMP content and PKG activity. The addition of L-NMMA and KT-5 823 significantly inhibited the effects of ICS II on nNOS expression, NO production and PKG activity. Furthermore, LY294002 significantly decreased p-AKT level, NOS activity, NO production and nNOS expression, but it did not affect iNOS expression. These findings demonstrate that the beneficial effect of ICS II on neuronal cell proliferation, and its possible underlying mechanisms are, at least partly, through activating AKT/nNOS/NO/cGMP/PKG signaling pathway.

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