Impaired Axonal Regeneration in Diabetes. Perspective on the Underlying Mechanism from In Vivo and In Vitro Experimental Studies.

Axonal regeneration after peripheral nerve injury is impaired in diabetes, but its precise mechanisms have not been elucidated. In this paper, we summarize the progress of research on altered axonal regeneration in animal models of diabetes and cultured nerve tissues exposed to hyperglycemia. Impaired nerve regeneration in animal diabetes can be attributed to dysfunction of neurons and Schwann cells, unfavorable stromal environment supportive of regenerating axons, and alterations of target tissues receptive to reinnervation. In particular, there are a number of factors such as enhanced activity of the negative regulators of axonal regeneration (e.g., phosphatase and tensin homolog deleted on chromosome 10 and Rho/Rho kinase), delayed Wallerian degeneration, alterations of the extracellular matrix components, enhanced binding of advanced glycation endproducts (AGEs) with the receptor for AGE, and delayed muscle reinnervation that can be obstacles to functional recovery after an axonal injury. It is also noteworthy that we and others have observed excessive neurite outgrowth from peripheral sensory ganglion explants from streptozotocin (STZ)-diabetic mice in culture and enhanced regeneration of small nerve fibers after sciatic nerve injury in STZ-induced diabetic rats. The excess of abortive neurite outgrowth may lead to misconnections of axons and target organs, which may interfere with appropriate target reinnervation and functional repair. Amelioration of perturbed nerve regeneration may be crucial for the future management of diabetic neuropathy.

Galectin-1 deficiency improves axonal swelling of motor neurones in SOD1(G93A) transgenic mice.

AIMS: Galectin-1, a member of the ß-galactoside-binding lectin family, accumulates in neurofilamentous lesions in the spinal cords of both sporadic and familial amyotrophic lateral sclerosis (ALS) patients with a superoxide dismutase 1 gene (SOD1) mutation (A4V). The aim of this study was to evaluate the roles of endogenous galectin-1 in the pathogenesis of ALS. METHODS: Expression of galectin-1 in the spinal cord of mutant SOD1 transgenic (SOD1(G93A) ) mice was examined by pathological analysis, real-time RT-PCR and Western blotting. The effects of galectin-1 deficiency were evaluated by cross-breeding SOD1(G93A) mice with galectin-1 null (Lgals1(-/-) ) mice. RESULTS: Before ALS-like symptoms developed in SOD1(G93A) /Lgals1(+/+) mice, strong galectin-1 immunoreactivity was observed in swollen motor axons and colocalized with aggregated neurofilaments. Electron microscopic observations revealed that the diameters of swollen motor axons in the spinal cord were significantly smaller in SOD1(G93A) /Lgals1(-/-) mice, and there was less accumulation of vacuoles compared with SOD1(G93A) /Lgals1(+/+) mice. In symptomatic SOD1(G93A) /Lgals1(+/+) mice, astrocytes surrounding motor axons expressed a high level of galectin-1. CONCLUSIONS: Galectin-1 accumulates in neurofilamentous lesions in SOD1(G93A) mice, as previously reported in humans with ALS. Galectin-1 accumulation in motor axons occurs before the development of ALS-like symptoms and is associated with early processes of axonal degeneration in SOD1(G93A) mice. In contrast, galectin-1 expressed in astrocytes may be involved in axonal degeneration during symptom presentation.

Characterization of galectin-1-positive cells in the mouse hippocampus.

Galectin-1 (gal-1) is one of several well-studied proteins from the galectin families. It is a 14.5 kDa glycoprotein with a single carbohydrate-binding domain. To examine the distribution and properties of gal-1 in the mouse hippocampus, we performed immunohistochemistry using an anti-gal-1 antibody. We found that most gal-1-positive cells showed both NeuN and ß-tubulin III (Tuj-1) immunoreactivity (NeuN: 93%, ß-tubulin III: 88%). Furthermore, we clarified that 77% of gal-1-positive cells expressed somatostatin, 79% of gal-1-positive cells expressed GAD67, 34% of gal-1-positive cells expressed parvalbumin, 5% of gal-1-positive cells expressed calretinin, 2% of gal-1-positive cells expressed calbindin, and 31% of gal-1-positive cells expressed neuropeptide Y in the mouse hippocampus. These results indicate that gal-1 is expressed in interneurons that also express ß-tubulin III and gal-1 may be a novel marker for interneuron subpopulations in the hippocampus.

GDNF promotes neurite outgrowth and upregulates galectin-1 through the RET/PI3K signaling in cultured adult rat dorsal root ganglion neurons.

Galectin-1 (GAL-1), a member of a family of ß-galactoside binding animal lectins, is predominantly expressed in isolectin B4 (IB4)-binding small non-peptidergic (glial cell line-derived neurotrophic factor (GDNF)-responsive) sensory neurons in the sections of adult rat dorsal root ganglia (DRG), but its functional role and the regulatory mechanisms of its expression in the peripheral nervous system remain unclear. In the present study, both recombinant nerve growth factor (NGF) and GDNF (50 ng/ml) promoted neurite outgrowth from cultured adult rat DRG neurons, whereas GDNF, but not NGF, significantly increased the number of IB4-binding neurons and the relative protein expression of GAL-1 in the neuron-enriched culture of DRG. The GAL-1 expression in immortalized adult rat Schwann cells IFRS1 and DRG neuron-IFRS1 cocultures was unaltered by treatment with GDNF, which suggests that GDNF/GAL-1 signaling axis is more related to neurite outgrowth, rather than neuron-Schwann cell interactions. The GDNF-induced neurite outgrowth and GAL-1 upregulation were attenuated by anti-GDNF family receptor (RET) antibody and phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor LY294002, suggesting that the neurite-outgrowth promoting activity of GDNF may be attributable, at least partially, to the upregulation of GAL-1 through RET-PI3K pathway. On the contrary, no significant differences were observed between GAL-1 knockout and wild-type mice in DRG neurite outgrowth in the presence or absence of GDNF. Considerable immunohistochemical colocalization of GAL-3 with GAL-1 in DRG sections and GDNF-induced upregulation of GAL-3 in cultured DRG neurons imply the functional redundancy between these galectins.

[One-year longitudinal change in parameters of myopic school children trained by a new accommodative training device--uncorrected visual acuity, refraction, axial length, accommodation, and pupil reaction].

PURPOSE: School children with myopia were trained using a visual stimulation device that generated an isolated blur stimulus on a visual target, with a constant retinal image size and constant brightness. Uncorrected visual acuity, cycloplegic refraction, axial length, dynamic accommodation and papillary reaction were measured to investigate the effectiveness of the training. SUBJECTS AND METHODS: There were 45 school children with myopia without any other ophthalmic diseases. The mean age of the children was 8.9 +/- 2.0 years (age range; 6-16)and the mean refraction was -1.56 +/- 0.58 D (mean +/- standard deviation). As a visual stimulus, a white ring on a black background with a constant ratio of visual target size to retinal image size, irrespective of the distance, was displayed on a liquid crystal display (LCD), and the LCD was quickly moved from a proximal to a distal position to produce an isolated blur stimulus. Training with this visual stimulus was carried out in the relaxation phase of accommodation. Uncorrected visual acuity, cycloplegic refraction, axial length, dynamic accommodation and pupillary reaction were investigated before training and every 3 months during the training. RESULTS: Of the 45 subjects, 42 (93%) could be trained for 3 consecutive months, 33 (73%) for 6 months, 23 (51%) for 9 months, and 21 (47%) for 12 months. The mean refraction decreased by 0.83 +/- 0.56 D (mean +/- standard deviation) and the mean axial length increased by 0.47 +/- 0.16 mm at 1 year, showing that the training bad some effect in improving the visual acuity. In the tests of the dynamic accommodative responses, the latency of the accommodative-phase decreased from 0.4 +/- 0.2 sec to 0.3 +/- 0.1 sec at 1 year, the gain of the accommodative-phase improved from 69.0 +/- 27.0% to 93.3 +/- 13.4%, the maximum speed of the accommodative-phase increased from 5.1 +/- 2.2 D/sec to 6.8 +/- 2.2 D/sec and the gain of the relaxation-phase significantly improved from 52.1 +/- 26.0% to 72.7 +/- 13.7% (corresponding t-test, p < 0.005). No significant changes were observed in the pupillary reaction. CONCLUSION: The training device was useful for improving the accommodative functions and accommodative excess, suggesting that it may be able to suppress the progression of low myopia, development of which is known to be strongly influenced by environmental factors.

Oxidized galectin-1 reduces lipopolysaccharide-induced increase of proinflammatory cytokine mRNA in cultured macrophages.

BACKGROUND: Periodontitis is prevalent in older humans. Limiting the inflammation associated with periodontitis may provide a therapy for this condition, because Gram-negative bacteria expressing lipopolysaccharide (LPS) have a key role in initiation of inflammation by activating macrophage functions. Because oxidized galectin-1 regulates macrophage functions in other systems, we sought to establish whether this galectin-1 mRNA is expressed in the oral cavity, and whether it could dampen LPS-induced macrophage activation in vitro. METHODS: Using the reverse transcriptase polymerase chain reaction (RT-PCR), we measured galectin-1 mRNA expression to clarify its localization to rat gingival tissues and studied the effect of Porphyromonas gingivalis challenge on galectin-1 expression. Next, we tested the effects of adding oxidized galectin-1 to cultured LPS-activated peritoneal macrophages on mRNA expression of proinflammatory factors by RT-PCR and real-time RT-PCR. RESULTS: We established that galectin-1 mRNA is expressed in gingival tissues and also showed that galectin-1 mRNA was significantly increased by challenge with P. gingivalis, indicating that galectin-1 may regulate oral inflammation. On the other hand, LPS 100 ng/mL in serum-containing medium induced macrophages to upregulate mRNA associated with a proinflammatory response, ie, interleukins 1ß and 6, and inducible nitric oxide synthase. We showed that application of 0.1-10 ng/mL of oxidized galectin-1 to LPS-treated macrophages reduced the intense LPS- induced increase by serum in proinflammatory mRNA expression in a concentration-dependent manner. Furthermore, application of oxidized galectin-1 10 ng/mL to LPS-treated macrophages in serum-free medium also showed a similar effect on LPS activity. CONCLUSION: Oxidized galectin-1 restricts the proinflammatory actions of LPS, and this protein could limit the negative effects of inflammation.

Training regimen involving cyclic induction of pupil constriction during far accommodation improves visual acuity in myopic children.

PURPOSE: Myopia in school-age children has become increasingly prevalent in industrialized countries, especially in Asia. A large population of school-age children still suffers from low visual acuity. We have developed a novel, safe and noninvasive training method to activate a pupillary constriction response during far accommodation that results in improved visual acuity. METHODS: Myopic children (n = 95) were treated for 3-minute sessions up to twice a week for 12-106 weeks. We stimulated quick cycles of near/far accommodation by displaying a visual object on a LCD screen and moving the screen in cycles from a near (25 cm) to a far (70 cm) point and back, while keeping the retinal projection size and brightness of the object constant. RESULTS: Mechanistically, we noted pupillary constriction upon far accommodation in trained myopic children, which was not seen in normal subjects or in untrained myopic children. Eighty five percent (52/61) of trained myopic right eyes with two sessions weekly experienced improved visual acuity (VA) by more than 0.1 logMAR units with an average improvement of 0.30 +/- 0.03 standard error of mean (SEM) logMAR units. With maintained training, most eyes' improved VA stayed almost constant, for more than 50 weeks in the case of 12 long trained subjects. CONCLUSIONS: This simple, short and safe accommodation training greatly improves the quality of vision in a large population suffering from refractive abnormalities.

The Galectin-1 level in serum as a novel marker for stress.

Galectin-1(Gal-1), a carbohydrate-binding protein with an affinity for beta-galactoside, is widely expressed in various normal and pathological tissues and it also plays an important role in regulating immune cell homeostasis and tumorigenesis. This study investigated the effects of restraint stress on serum Gal-1 by Western blot analyses and enzyme-linked immunosorbent assays. The Gal-1 levels of the restraint-stress group were significantly higher than those of the control group. However, this increase by stress was not obvious in adolescent rats. The pattern of these changes was similar to that of corticosterone. Furthermore, this Gal-1 increase in the serum was prevented by pre-treatment with a neurotoxin 6-hydroxydopamine (6-OHDA), which destroys the noradrenergic nerve terminals. However, a bilateral adrenalectomy (ADX) had no effect on the Gal-1 increase. These results suggest that Gal-1 is a candidate stress marker protein and that the stress-induced increase of Gal-1 in serum is regulated by the sympathetic nervous system under stress conditions.

A role for galectin-1 in the immune response to peripheral nerve injury.

Galectin-1 (Gal1) is a multi-functional protein that has key roles in organismal growth and survival. In the adult nervous system, Gal1 promotes axonal regeneration following peripheral nerve injury. Although the mechanism by which Gal1 promotes regeneration is unclear, previous reports suggested that Gal1 acts indirectly by activating macrophages. An appropriate response of macrophages is crucial for repair of injured nerves: these immune cells remove obstructive axon and myelin debris in the distal nerve. Here we establish a role for Gal1 in the accumulation of immune cells following peripheral axotomy. We used immunohistochemistry to visualize macrophages (F4/80) in wild-type (Lgals1(+/+)) and knockout (Lgals1(-/-)) mouse sciatic nerves following injury and/or manipulation of Gal1 levels. Density of F4/80 immunoreactivity, which peaks around 3 days post-injury, was decreased in Lgals1(+/+) nerves injected with Gal1 antibody. The typical injury-induced peak of macrophage/microglial density was delayed in the sciatic nerves and fifth lumbar dorsal root ganglia of Lgals1(-/-) mice relative to control mice. Injection of oxidized Gal1 into uninjured sciatic nerve promoted the accumulation of macrophages in Lgals1(+/+) nerves. Finally, we used transplants of sciatic nerve to uncover a compensatory mechanism in Lgals1(-/-) mice that allows for macrophage accumulation (albeit delayed and diminished) following axotomy. We conclude that Gal1 is necessary to direct the typical accumulation of macrophages in the injured peripheral nerve, and that Gal1 is sufficient to promote macrophage accumulation in the uninjured nerve of wild-type mice.

A carbohydrate-binding protein, Galectin-1, promotes proliferation of adult neural stem cells.

In the subventricular zone of the adult mammalian forebrain, neural stem cells (NSCs) reside and proliferate to generate young neurons. We screened factors that promoted the proliferation of NSCs in vitro by a recently developed proteomics technique, the ProteinChip system. In this screen, we identified a soluble carbohydrate-binding protein, Galectin-1, as a candidate. We show herein that Galectin-1 is expressed in a subset of slowly dividing subventricular zone astrocytes, which includes the NSCs. Based on results from intraventricular infusion experiments and phenotypic analyses of knockout mice, we demonstrate that Galectin-1 is an endogenous factor that promotes the proliferation of NSCs in the adult brain.

Cultured adult animal neurons and schwann cells give us new insights into diabetic neuropathy.

Since we introduced cultured dorsal root ganglia (DRG) neurons from streptozotocin (STZ)-induced diabetic mice as "an in vitro model to study diabetic neuropathy" (Sotelo et al., 1991), more than 30 papers have been devoted to the study of diabetic neuropathy with culture systems of neurons and Schwann cells derived from adult animals. So far, methods for dissociated cell culture of peripheral neurons (mainly DRG neurons) and Schwann cells, and for explant culture of peripheral ganglia and retinas have been applied to diabetic animals or patients. In addition to these diabetic cells, adult animal neurons and Schwann cells cultured under high glucose conditions and adult animal neurons exposed to diabetic serum have been utilized. The findings from these culture models clearly show that the exposure of mature neurons and Schwann cells to hyperglycemic conditions in vivo or in vitro can alter their biophysical and biochemical properties (e.g., cell viability, neurite outgrowth activity, polyol metabolism and electrophysiological features). Therefore, the cultured neurons and Schwann cells can be useful tools for investigating the precise mechanisms leading to diabetic neuropathy and the efficacy of therapeutic agents for the prevention and treatment of that condition.

Intravitreal macrophage activation enables cat retinal ganglion cells to regenerate injured axons into the mature optic nerve.

In mature mammals, retinal ganglion cells (RGCs) are generally unable to regenerate injured axons into the optic nerve. Here, we report that an intravitreal injection of either of two macrophage activators, oxidized galectin-1 or zymosan, strongly enhanced the regeneration of transected RGC axons beyond an optic nerve crush site in adult cats. Using WGA-HRP as an anterograde tracer, we found that injection of either macrophage activator caused many axons to grow into the distal optic nerve when evaluated 14 days later, with the strongest effects seen after injecting 100 ng of galectin-1. Elongation continued for at least another 2 weeks. Control eyes injected with saline contained very few labeled axons extending across the crush site. Elevation of intracellular cAMP levels using forskolin also enhanced regeneration beyond the crush site to some extent, but this treatment did not augment the effect of galectin-1 any further. These results indicate that RGCs of adult cats are capable of reverting to an active growth state and at least partially overcoming an inhibitory CNS environment as a result of intravitreal macrophage activation.

Impaired neurite outgrowth in the retina of a murine model of Sandhoff disease.

PURPOSE: To investigate the effects of lysosomal storage on the morphologic appearance and the neurite outgrowth capability of the retina in a mouse model of G(M2) gangliosidosis (Sandhoff disease). METHODS: Histopathologic appearances of retinas in Sandhoff (SD) mice at 3 and 4 months of age were examined by light and electron microscopy. Retinas of SD mice and wild-type (WT) mice at 1, 2, and 4 months of age were cultured in collagen gel in the presence or absence of brain-derived neurotrophic factor (BDNF), and neurite outgrowth was examined. RESULTS: Morphologic studies revealed accumulation of G(M2) ganglioside in the retinal ganglion cells of SD mice in a time-dependent manner. The number of neurites from the retinal explants after 7 and 10 days in culture were significantly lower in 2- and 4-month-old SD mice than in the age-matched WT mice. The application of BDNF significantly improved neurite outgrowth from the retina in both SD and WT mice at 2 months of age. At 4 months of age, BDNF was much less effective at stimulating neurite outgrowth in the retina of SD mice than in retina of WT mice. CONCLUSIONS: These results indicate that lysosomal storage of G(M2) ganglioside impairs the capability of neurite outgrowth in retinal ganglion cells in culture and that BDNF is effective at diminishing this impairment during the early stage of the disease.

Structural and functional studies of galectin-1: a novel axonal regeneration-promoting activity for oxidized galectin-1.

Recently, we discovered oxidized galectin-1 as a factor that regulates initial axonal growth in the peripheral nerve after axotomy. Galectin-1 is a member of the galectins, a family of animal lectins ranging from Caenorhabditis elegans to humans, which is defined by their affinity for beta-galactosides and by significant sequence similarity in the carbohydrate-binding site. Galectin-1 is a homodimer with a subunit molecular mass of 14.5 kDa, which contains six cysteine residues per subunit. The cysteine residues should be in a free state in order to maintain a molecular structure that is capable of showing lectin activity. However, our structural analysis revealed that the axonal regeneration-promoting factor exists as an oxidized form of galectin-1, containing three intramolecular disulfide bonds. The oxidized galectin-1 exhibited marked peripheral nerve regeneration-promoting activity, although it showed no lectin activity. It was also revealed that oxidized galectin-1 exists as a monomer in a physiological solution. Galectin-1 seems to have a variety of biological functions. These functions could vary according to the time at which a biological function is taking place, as well as the site in which a biological function is taking place. In addition, these functions could vary according to the structure of galectin-1 by which a particular biological function is taking place. Disulfide bond formation alters the structure of galectin-1, so as to confer the novel ability to promote axonal regeneration. Oxidized galectin-1 likely acts as an autocrine or paracrine factor to promote axonal regeneration, functioning more like a cytokine than as a lectin.

Oxidized galectin-1 is an essential factor for peripheral nerve regeneration.

Although many factors have been implicated in the regenerative response of peripheral axons to nerve injury, the signals that prompt neurons to extend processes in peripheral nerves after axotomy are not well-understood. As shown in the first chapter, oxidized recombinant human galectin-1 (rhGAL-1/Ox), which lacks lectin activity, promotes initial axonal growth in an in vitro peripheral nerve regeneration model at low concentrations (pg/ml). At a similarly low concentration, rhGAL-1/Ox has also been shown to be effective in enhancing axonal regeneration using in vivo experiments. Moreover, the application of functional anti-rhGAL-1 antibody strongly inhibited axonal regeneration in vivo as well as in vitro. Since galectin-1 (GAL-1) is expressed in the regenerating sciatic nerves as well as in both sensory and motoneurons, these results indicate that GAL-1, which is secreted into the extracellular space, is subsequently oxidized and then may regulate initial repair after axotomy. This possibility was confirmed by Western blot analysis, which revealed that both reduced and oxidized forms of GAL-1 are present in culture media of DRG neurons and immortalized adult mouse Schwann cells (IMS32). Externalized GAL-1/Ox has been found to stimulate macrophages to secrete an axonal regeneration-promoting factor. From these results, we propose that axonal regeneration occurs in axotomized peripheral nerves as a result of cytosolic reduced GAL-1 being released from Schwann cells and injured axons, which then becomes oxidized in the extracellular space. GAL-1/Ox in the extracellular space stimulates macrophages to secrete a factor that promotes axonal growth and Schwann cell migration, thus enhancing peripheral nerve regeneration and functional recovery. These results suggest that rhGAL-1/Ox may be a novel factor for functional restoration of injured peripheral nerves.

Regulation of the neuronal fate by DeltaFosB and its downstream target, galectin-1.

In mammals, the regulation of the cell fate to either proliferate, differentiate, arrest cell growth, or initiate programmed cell death is the most fundamental mechanism for maintaining normal cell function and tissue homeostasis. Under multiple signaling pathways, Jun and Fos family proteins are known to play important roles as components of an AP-1 (activator protein-1) complex, to regulate the transcription of various genes involved in cell proliferation, differentiation and programmed cell death. DeltaFosB, one of the AP-1 subunits encoded by alternatively spliced fosB mRNA, triggers one round of proliferation in quiescent rat embryo cell lines, followed by a different cell fate such as morphological alteration or delayed cell death. As one of the downstream targets of the DeltaFosB in rat3Y1 cell line, we identified rat galectin-1 and its novel variant, galectin-1beta, and demonstrated that the expression of galectin-1 is required for the proliferative activation of quiescent rat1A cells by DeltaFosB, thus indicating that galectin-1 is one of functional targets of DeltaFosB. The expression of DeltaFosB is highly inducible in the adult brain in response to various insults such as ischemic reperfusion injury, seizure induced by electric stimulation or cocaine administration. On the other hand, galectin-1 has also been shown to be involved in the regeneration of damaged axons in the peripheral nerve, as well as in neurite outgrowth or synaptic connectivity in the olfactory system during development. We herein propose that DeltaFosB together with galectin-1, may therefore mediate neuroprotection and neurogenesis in response to brain damage.

Oxidized galectin-1 advances the functional recovery after peripheral nerve injury.

Oxidized galectin-1 has been shown to promote axonal regeneration from transected-nerve sites in an in vitro dorsal root ganglion (DRG) explant model as well as in in vivo peripheral nerve axotomy models. The present study provides evidence that oxidized galectin-1 advances the restoration of nerve function after peripheral nerve injury. The sciatic nerve of adult rats was transected and the distal nerve was frozen after being sutured into a proximal site with four epineurial stitches. An osmotic pump delivered oxidized galectin-1 peripherally to the surgical site. Functional recovery was assessed by measurement of the degree of toe spread of the hind paw for 3 months after the sciatic nerve lesion. The recovery curves of toe spread in the test group showed a statistically significant improvement of functional recovery after day 21 by the application of oxidized recombinant human galectin-1 (rhGAL-1/Ox) compared to the control group. This functional recovery was supported by histological analysis performed by light microscopic examination. The regenerating myelinated fibers at the site 21 mm distal to the nerve-transected site were quantitatively examined at 100 days after the operation. The frequency distribution of myelinated fiber diameters showed that exogenous rhGAL-1/Ox increased the number and diameter of regenerating myelinated fibers; the number of medium-sized (6-11 microm in diameter) fibers increased significantly (P<0.05). These results indicate that oxidized galectin-1 promotes the restoration of nerve function after peripheral nerve injury. Thus, rhGAL-1/Ox may be a factor for functional restoration of injured peripheral nerves.

Long-term induction of beta-CGRP mRNA in rat lungs by allergic inflammation.

Calcitonin gene-related peptide (CGRP) is one of the major neuropeptides released from sensory nerve endings and neuroendocrine cells of the lung. Two CGRP isoforms, alpha-and beta-CGRP, have been identified in rats and humans, but no studies have attempted to reveal direct evidence of differences in action or location of these isoforms in allergic inflammation (AI). We investigated mRNA expressions of alpha-and beta-CGRP in lungs, nodose ganglia (NG), and dorsal root ganglia (DRG) of an animal model for AI of the airways, utilizing a model created by sensitizing Brown Norway (BN) rats with ovalbumin (OVA). By semiquantitative RT-PCR analysis, long-lasting enhanced expression of the beta-CGRP mRNA was shown in the lungs of the AI rats (14.5-fold enhancement at 6 hr, 8.1-fold at 24 hr, and 3.7-fold at 120 hr after OVA-challenge compared to the level in the lungs of phosphate-buffered saline (PBS)-challenged control rats). In contrast, the mRNA expression of the alpha-CGRP in AI lungs showed only a transient increase after OVA-challenge (2.7-fold at 6 hr) followed by a lower level of expression (0.5-fold at 48 hr and 0.6-fold at 120 hr). The mRNA expressions of both isoforms in NG, but not in DRG, were transiently up-regulated at 6 hr after antigen challenge. In situ RT-PCR in combination with immunohistochemical analysis revealed that beta-CGRP was expressed in neuroendocrine cells in clusters (termed neuroepithelial bodies [NEBs]) in AI lungs. These results indicate that the long-term induction of beta-CGRP in NEBs may play an important role in pulmonary AI such as bronchial asthma.

Oxidized galectin-1 stimulates macrophages to promote axonal regeneration in peripheral nerves after axotomy.

Various neurotrophic factors that promote axonal regeneration have been investigated in vivo, but the signals that prompt neurons to send out processes in peripheral nerves after axotomy are not well understood. Previously, we have shown oxidized galectin-1 (GAL-1/Ox) promotes initial axonal growth after axotomy in peripheral nerves. However, the mechanism by which GAL-1/Ox promotes axonal regeneration remains unclear and is the subject of the present study. To identify possible target cells of GAL-1/Ox, a fluorescently labeled recombinant human GAL-1/Ox (rhGAL-1/Ox) was incubated with DRG neurons, Schwann cells, and intraperitoneal macrophages from adult rats. Only the cell surfaces of intraperitoneal macrophages bound the rhGAL-1/Ox, suggesting that these cells possess a receptor for GAL-1/Ox. Experiments examining tyrosine phosphorylation revealed that rhGAL-1/Ox stimulated changes in signal transduction pathways in these macrophages. These changes caused macrophages to secrete an axonal growth-promoting factor. This was demonstrated when conditioned media of macrophages stimulated with rhGAL-1/Ox in 48 hr culture strongly enhanced axonal regeneration from transected-nerve sites of DRG explants. Furthermore, activated macrophage-conditioned media also improved Schwann cell migration from the transected-nerve sites. From these results, we propose that axonal regeneration occurs in axotomized peripheral nerves as a result of cytosolic reduced galectin-1 being released from Schwann cells and injured axons, which then becomes oxidized in the extracellular space. Oxidized galectin-1 then stimulates macrophages to secrete a factor that promotes axonal growth and Schwann cell migration, thus enhancing peripheral nerve regeneration.