Injection Of Ciliary Neurotrophic Factor Research Articles

Endogenous and exogenous ciliary neurotrophic factor enhances forebrain neurogenesis in adult mice

Neurogenesis in the adult mammalian CNS occurs in the subventricular zone (SVZ) and dentate gyrus. The receptor for ciliary neurotrophic factor (CNTF), CNTFRα, is expressed in the adult subventricular zone. Because the in vitro effects of CNTF on neural precursors have been varied, including proliferation and differentiation into neurons or glia, we investigated its role in vivo. Injection of CNTF in the adult C57BL/6 mice forebrain increased the number of cells labeled with ip BrdU in both neurogenic regions. In the dentate gyrus, CNTF also appeared to enhance differentiation of precursors into neurons, i.e., increased the proportion of NeuN+/BrdU+ cells from ∼14 to ∼29%, but did not affect differentiation into astrocytes (GFAP+) or oligodendrocytes (CNPase+). In the SVZ, CNTF increased the proportion of GFAP+/BrdU+ cells from ∼1 to ∼2%. CNTF enhanced the distance of migration of new neurons into the granule cell layer. Intraventricular injection of neutralizing anti-CNTF antibodies reduced the number of BrdU-labeled cells in the SVZ. These results suggest that endogenous CNTF regulates adult neurogenesis by increasing proliferation of neural stem cells and/or precursors. Alternatively, CNTF could maintain cells longer in the S-phase, resulting in increased BrdU labeling. In the neurogenic region of the SVZ, CNTFRα was exclusively present in GFAP-positive process-bearing cells, suggesting that CNTF affects neurogenesis indirectly via neighboring astroglia. Alternatively, these cells may be part of the neural precursor lineage. The restricted expression of CNTF within the nervous system makes it a potential selective drug target for cell replacement strategies.

GFAP promoter drives Müller cell-specific expression in transgenic mice.

In an attempt to identify Müller cell-specific promoters and to better understand the gene regulatory mechanisms in retinal glial cells, the expression of the glial fibrillary acidic protein (GFAP) gene was studied in Müller cell cultures and in GFAP-enhanced green fluorescent protein (EGFP) transgenic mice. A transfection assay of GFAP-luciferase constructs carrying a series of nested deletions was performed in an established Müller cell line. For in vivo analysis, transgenic mice were generated by injecting a construct carrying a 2.5-kb, 5' fragment of the mouse GFAP gene linked to the EGFP gene. Isolated retinas from transgenic mice were screened for GFP expression. Subsequently, the identity of the GFP-expressing cells was established by immunostaining cryostat sections of the retina with antibodies against Müller cell antigenic markers. Induction of the transgene and the endogenous GFAP gene was examined by injecting ciliary neurotrophic factor (CNTF) into the eye. The DNA transfection data suggested that proximal 5' sequences of the GFAP gene are sufficient to direct high-level reporter expression in Müller cell cultures. In transgenic mice, GFP fluorescence appeared in radially oriented processes that spanned almost the entire thickness of the retina. Immunostaining with antibodies to cellular retinaldehyde-binding protein (CRALBP) and glutamine synthetase showed that the GFP-expressing cells were Müller cells. GFP-expressing Müller cells were observed in the retinas of both albino and pigmented transgenic mice. In eyes injected with CNTF, both GFP and GFAP levels were highly elevated. These observations suggest that the 2.5-kb, 5' GFAP sequence can direct inducible reporter gene expression in Müller cells. In addition to Müller cells, a few GFP-labeled astrocytes were present in the adult retina. In the developing retina, GFP-expressing astrocytes were first present at the optic nerve head, and as development progressed, the cells gradually moved toward the periphery of the retina and acquired their adult, stellate morphology. The present study shows that the 2.5-kb, 5' flanking region of the mouse GFAP gene can be used to express GFP, and possibly other genes, specifically in Müller cells in the mouse retina. Furthermore, expression of the transgene can be upregulated by intravitreal injection of CNTF.

Mitotic activation of proliferative cells in the inner nuclear layer of the mature fish retina: regulatory signals and molecular markers.

New neurons continuously differentiate within the otherwise mature retina of teleost fish, both under normal conditions and in response to injury. We investigated the effects of surgical injury and intraocular injection of neurotrophic factors on the mitotic rate of proliferative inner nuclear layer cells (PINC). PINC are continually born in the inner nuclear layer and then migrate to the outer nuclear layer (ONL). Surgical excision of a part of a retina activates PINC mitotic activity near and far from the lesion. In the injured eye, up-regulation of PINC cells is largest in the dorsonasal sector of the retina, regardless of the site of lesion. Up-regulation extends even to the unlesioned contralateral eye, where it occurs in the same dorsonasal sector. Intraocular injection of ciliary neurotrophic factor mimics the effect of injury on PINC in the treated eye but not on the untreated contralateral retina. We searched for the expression in PINC of Pax6, a transcription factor linked to retinal progenitor cells and found that less than 0.5% of all PINC cells express it. Importantly, the number of Pax6-expressing PINC does not change significantly in the retinas subjected to any of the experimental manipulations tested. Under normal conditions, the default fate of PINC cells is to migrate to the ONL and, likely, replenish the rod progenitor pool. PINC respond to injury (both surgical and light-dependent) by increasing their mitotic rate; this increase is long lived, but there are no changes in the expression level of Pax6. PINC probably are a heterogenous cell population that can be specified for ultimate, different purposes: creating rod precursors, creating founder cells, creating cone precursors. Several fates are recognized now, but others may also be possible.

Ciliary neurotrophic factor induces glial fibrillary acidic protein in retinal Müller cells through the JAK/STAT signal transduction pathway

Purpose. Intravitreal injection of ciliary neurotrophic factor (CNTF) is known to induce glial intermediate filament protein (GFAP) expression in retinal Müller cells. Because CNTF binding can activate multiple signaling kinases, we have examined the involvement of JAK/STAT pathway in GFAP induction in Müller cells. Methods. CNTF was injected intravitreally into mouse eyes. Immunocytochemistry and immunoblotting were used to study GFAP and STAT3-p (phosphorylated STAT3) levels either in mouse eyes, retinal explant cultures or in a Müller cell line, rMC-1. Results. In protein extracts of CNTF-injected eyes, retinal explants and the Müller cells, there was a substantial increase in STAT3-p level. Immunocytochemistry showed that STAT3-p was now present in many cell bodies in the INL and the GCL. To prove that CNTF acted via the JAK-STAT pathway, rMC-1 cells were transfected with a dominant-negative STAT3 mutant prior to treatment with CNTF. In the immunoblots of transfected cells, there was decrease in GFAP level. Conclusions. The results establish that CNTF can induce GFAP expression in retinal Müller cells through the JAK/STAT signaling pathway.

Leptin and ciliary neurotrophic factor enhance the formation of gap junctions between folliculo-stellate cells in castrated male rats.

We investigated the effects of the leptin and ciliary neurotrophic factor (CNTF) on gap junction formation between folliculo-stellate cells in the anterior pituitary glands of male rats. Thirty-day-old Wistar-Imamichi strain male rats were castrated, and 30 days later they received intraperitoneal injections of either human recombinant leptin or recombinant rat CNTF. They were divided into seven groups according to the injected materials: PBS as a control, either 0.04, 0.2 or 1.0 mg/kg leptin or either 0.004, 0.02, or 0.1 mg/kg CNTF. Five rats from each group were killed 1, 2, 3, 4 and 5 days after the injections, and the pituitary gland was removed from each rat. Then the specimens were prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions and calculated the rate of occurrence of gap junctions as the ratio of the number of gap junctions existing between folliculo-stellate cells per intersected follicle profile in electron photomicrographs. The administration of 1.0 mg/kg leptin and 0.1 mg/kg CNTF to castrated male rats increased the number of gap junctions between folliculo-stellate cells. These observations indicate that the formation of gap junctions within the anterior pituitary gland of male rats is under the influence of leptin and CNTF.

Ciliary Neurotrophic Factor Activates Spinal Cord Astrocytes, Stimulating Their Production and Release of Fibroblast Growth Factor-2, to Increase Motor Neuron Survival

At focal CNS injury sites, several cytokines accumulate, including ciliary neurotrophic factor (CNTF) and interleukin-1β (IL-1β). Additionally, the CNTF alpha receptor is induced on astrocytes, establishing an autocrine/paracrine loop. How astrocyte function is altered as a result of CNTF stimulation remains incompletely characterized. Here, we demonstrate that direct injection of CNTF into the spinal cord increases GFAP expression and astroglial size and that primary cultures of spinal cord astrocytes treated with CNTF, IL-1β, or leukemia inhibitory factor exhibit nuclear hypertrophy comparable to that observed in vivo. Using a coculture bioassay, we further demonstrate that CNTF treatment of astrocytes increases their ability to support ChAT+ ventral spinal cord neurons (presumably motor neurons) more than twofold compared with untreated astrocytes. Also, the complexity of neurites was significantly increased in neurons cultured with CNTF-treated astrocytes compared with untreated astrocytes. RT–PCR analysis demonstrated that CNTF increased levels of FGF-2 and nerve growth factor (NGF) mRNA and that IL-1β increased NGF and hepatocyte growth factor mRNA levels. Furthermore, both CNTF and IL-1β stimulated the release of FGF-2 from cultured spinal cord astrocytes. These findings demonstrate that cytokine-activated astrocytes better support CNS neuron survival via the production of neurotrophic molecules. We also show that CNTF synergizes with FGF-2, but not epidermal growth factor, to promote DNA synthesis in spinal cord astrocyte cultures. The significance of these findings is discussed by presenting a new model depicting the sequential activation of astrocytes by cytokines and growth factors in the context of CNS injury and repair.

Ciliary neurotrophic factor increases muscle fiber number in the developing levator ani muscle of female rats

In addition to its well characterized neurotrophic properties, ciliary neurotrophic factor (CNTF) also has myotrophic effects in several experimental paradigms. We have previously observed that the volume of the levator ani (LA) muscle is increased in female rats treated with CNTF during the perinatal period. In order to determine the cellular basis for the effect of CNTF on LA muscle volume, female rat pups were given daily perineal injections of CNTF or a control solution from postnatal day 1 through 6. Mean cross-sectional area of LA muscle fibers and LA fiber number were assessed on postnatal day 7. CNTF treatment increased LA muscle fiber number more than 300% while having no effect on LA fiber size. We conclude that CNTF prevents muscle fiber degeneration and/or increases myogenesis in the developing LA muscle.

Ciliary neurotrophic factor induces expression of the IGF type I receptor and FGF receptor 1 mRNAs in adult rat brain oligodendrocytes

Ciliary neurotrophic factor (CNTF) is produced and released in response to injury in the central nervous system (CNS). While CNTF initially was characterized as a trophic factor for neurons, more recent evidence supports roles for this factor in survival, proliferation, and maturation of oligodendrocyte lineage cells. Evidence is emerging to support the hypothesis that CNTF's actions may include enhancing other growth and trophic factors. Here we tested the hypothesis that CNTF can induce expression of receptors on oligodendrocytes for factors that are known to promote their generation, maturation, and survival. Specifically, we used an in vivo paradigm to test whether CNTF, when injected stereotactically into forebrain white matter of adult rats, could induce mRNA expression for the insulin-like growth factor (IGF) type I receptor (IGF-IR), fibroblast growth factor (FGF) receptor (FGFR)-1, FGFR3, and platelet-derived growth factor (PDGF) receptor-alpha (PDGFRα). We determined that CNTF injection increased expression of IGF-IR and FGFR1 mRNAs in adult white matter to 200–250% of control levels. Cellular analysis indicated that these receptor mRNAs were induced in interfascicular oligodendrocytes. In contrast, CNTF had no effect on levels of FGFR3 and PDGFRα mRNAs. These results suggest that CNTF enhances the sensitivity of oligodendrocytes to other mitogens and trophic factors via induction of their receptors. J. Neurosci. Res. 57:447–457, 1999. © 1999 Wiley-Liss, Inc.

Ciliary neurotrophic factor induces expression of the IGF type I receptor and FGF receptor 1 mRNAs in adult rat brain oligodendrocytes

Ciliary neurotrophic factor (CNTF) is produced and released in response to injury in the central nervous system (CNS). While CNTF initially was characterized as a trophic factor for neurons, more recent evidence supports roles for this factor in survival, proliferation, and maturation of oligodendrocyte lineage cells. Evidence is emerging to support the hypothesis that CNTF's actions may include enhancing other growth and trophic factors. Here we tested the hypothesis that CNTF can induce expression of receptors on oligodendrocytes for factors that are known to promote their generation, maturation, and survival. Specifically, we used an in vivo paradigm to test whether CNTF, when injected stereotactically into forebrain white matter of adult rats, could induce mRNA expression for the insulin-like growth factor (IGF) type I receptor (IGF-IR), fibroblast growth factor (FGF) receptor (FGFR)-1, FGFR3, and platelet-derived growth factor (PDGF) receptor-alpha (PDGFRalpha). We determined that CNTF injection increased expression of IGF-IR and FGFR1 mRNAs in adult white matter to 200-250% of control levels. Cellular analysis indicated that these receptor mRNAs were induced in interfascicular oligodendrocytes. In contrast, CNTF had no effect on levels of FGFR3 and PDGFRalpha mRNAs. These results suggest that CNTF enhances the sensitivity of oligodendrocytes to other mitogens and trophic factors via induction of their receptors.

Ciliary neurotrophic factor stimulates nuclear hypertrophy and increases the GFAP content of cultured astrocytes

Studies using transgenic mice that overexpress ciliary neurotrophic factor (CNTF), direct injection of CNTF into brain parenchyma, and ectopic expression of CNTF by an adenoviral vector have demonstrated that CNTF activates astrocytes. Paradoxically, studies to date have failed to show an effect of CNTF on the expression of GFAP by cultured astrocytes. Therefore, the goal of this study was to use nuclear hypertrophy and GFAP expression as indices of glial activation to compare the responsiveness of forebrain type 1 and type 2 astrocytes to CNTF. As reported by others, CNTF did not increase GFAP in type 1 astrocytes; however, it rapidly increased their nuclear size by 20%. Nuclear hypertrophy was apparent within 4 h after CNTF exposure and persisted for at least 48 h. In contrast, type 2 astrocyte GFAP increased 2-fold over the course of 48 h of CNTF treatment. During this same treatment period type 2 astroglial nuclei enlarged by 25%. We conclude that CNTF stimulates both type 1 and type 2 astrocytes directly. Together with our in vivo studies (Levison et al., 1996: Exp. Neurol. 141: 256), these data support the concept that CNTF is responsible for many of the progressive astroglial changes that appear after CNS injury and disease.

Regulatory effects of trophic factors on expression and distribution of CGRP and GAP-43 in rat motoneurons.

Trophic factors play important roles for the regulation of several motoneuron characteristics. In this report, we have studied the effects of different trophic factors on the regulation of two substances that have been linked with axon sprouting and regeneration in rat spinal motoneurons, i.e., calcitonin gene-related peptide (CGRP) and growth-associated protein-43 (GAP-43). The expression of both GAP-43 and CGRP is regulated developmentally and after different lesions. In the adult animal a sciatic nerve transection normally leads to an increased expression of both alpha-CGRP and GAP-43. Local administration of insulin-like growth factor-1 (IGF-1), IGF-2, or ciliary neurotrophic factor (CNTF) did not significantly change this response, whereas basic fibroblast growth factor (bFGF) attenuated the lesion-induced upregulation of alpha-CGRP mRNA. None of the studied factors had any influence on the upregulation of GAP-43 after lesion. In vitro, GAP-43 and alpha-CGRP mRNAs could be detected in enriched motoneuron cultures prepared from E14 rat embryos. The GAP-43 expression was upregulated by bFGF, IGF-1, IGF-2, and CNTF. CNTF also upregulated alpha-CGRP mRNA in cultured motoneurons, whereas bFGF had the opposite effect. IGF-1 and -2 did not significantly affect the alpha-CGRP mRNA levels. The decrease in GAP-43-immunopositive neuromuscular junctions (NMJs), seen during normal postnatal development, was attenuated after intramuscular injections of bFGF, CNTF, IGF-2, or CGRP 8-37, a CGRP antagonist. At the same time bFGF decreased and CNTF increased the number of CGRP-immunoreactive NMJs, whereas no difference from control was seen in IGF-2-treated muscles. These results show important regulatory effects of trophic factors on the expression of both GAP-43 and alpha-CGRP in motoneurons. However, it also is shown that the trophic factors used here influence the expression of these two substances in distinctively different patterns. This may have important implications for the understanding of trophic interactions in the spinal motor system and also should be considered in the evaluation of possible effects of CGRP and GAP-43 in motoneurons. The effect of a CGRP antagonist on GAP-43 in muscle indicates a role for CGRP in sprouting-related processes.

Adenovirus-mediated gene transfer of ciliary neurotrophic factor can prevent photoreceptor degeneration in the retinal degeneration (rd) mouse.

Mutations in the beta-subunit of the cGMP phosphodiesterase gene (betaPDE) can cause photoreceptor degeneration leading to blindness in humans, dogs, and mice. Since intravitreal administration of trophic factors has been shown to reduce photoreceptor death in the Royal College of Surgeons rat and the light overexposure models of photoreceptor degeneration, we have tested the possibility of rescuing photoreceptors in the rd mouse using ciliary neurotrophic factor (CNTF). Following intravitreal injection of an adenoviral vector encoding a ngf/cntf fusion gene into one eye of rd/rd mice, many strong CNTF-immunoreactive profiles are detected in various cell types of the injected eyes. Semiquantitative analysis of the corresponding retinae reveals that the photoreceptor-containing layer (outer nuclear layer, or ONL) retains significantly more rows of photoreceptor nuclei than that of eyes treated with a control (LacZ) vector, or untreated, for at least 18 days after vector administration (the longest survival time analyzed). A smaller, but significant, protective effect was also seen 9 days after intravitreal injection of recombinant CNTF. Because apoptotic cell death has been shown to be the common terminal fate of photoreceptors in the rd mouse and many other animal models of retinitis pigmentosa, these results suggest the possibility that administration of neurotrophic factors may prove beneficial in reducing photoreceptor loss in multiple forms of retinitis pigmentosa.

CNTF induces GFAP in a S-100α brain cell population: the pattern of CNTF-αR suggests an indirect mode of action

In a recent report, we demonstrated that intracerebral injections of the pleiotropic cytokine, ciliary neurotrophic factor (CNTF), into developing postnatal rats evoked a severe inflammatory response as determined by the appearance of reactive astrocytes and activated microglia. Considering the likely involvement of CNTF in the injury response, we felt it was important to further understand the role of CNTF in the developing rat CNS. In this study, we examined the responsiveness of other cell populations to intracerebral injections of CNTF. We report that CNTF increases glial fibrillary acidic protein (GFAP), while having no appreciable effect on the levels of other intermediate filaments including vimentin and neurofilament. Moreover, CNTF did not affect the expression of the mature oligodendrocyte gene, myelin basic protein. These results suggest that CNTF is highly specific in its regulation of GFAP. In our previous study, we showed CNTF to increase GFAP in a cell population that already exists in the CNS parenchyma. To determine the origin of the CNTF-induced reactive astrocytes, therefore, we have utilized a technique of combined in situ hybridization and immunocytochemistry. To examine the possibility that CNTF acts on oligodendrocyte precursors to give rise to reactive astrocytes, the platelet-derived growth factor alpha receptor (PDGF-αR) was utilized as a riboprobe in conjunction with an antibody to GFAP. Examination of CNTF-induced GFAP + astrocytes revealed no colocalization with PDGF-αR mRNA. In contrast, when we utilized an S100α antibody recognizing a calcium binding protein in immature astrocytes, we found colocalization of S100α and GFAP mRNA. These data suggest that CNTF induces an upregulation of GFAP in immature S100α + astrocytes. Examination of the CNTF-α receptor mRNA revealed no change in expression following CNTF treatment. Unexpectedly, however, the CNTF-induced astrogliotic response appears to be indirect since the CNTF-α receptor was solely expressed by neurons in the cytokine-treated animals.

Ciliary Neurotrophic Factor Induces Down-regulation of Its Receptor and Desensitization of Signal Transduction Pathways in Vivo: NON-EQUIVALENCE WITH PHARMACOLOGICAL ACTIVITY

Despite the widespread use of polypeptide growth factors as pharmacological agents, little is known about the extent to which these molecules regulate their cognate cell surface receptors and signal transduction pathways in vivo. We have addressed this issue with respect to the neurotrophic molecule ciliary neurotrophic factor (CNTF). Administration of CNTF in vivo resulted in modest decreases in levels of CNTFRalpha mRNA and protein in skeletal muscle. CNTF causes the rapid tyrosine phosphorylation of LIFRbeta and gp130 and the induction of the immediate-early gene, tis11; injection of CNTF 3-7 h after an initial exposure failed to re-stimulate these immediate-early responses, suggesting a biochemical desensitization to CNTF not accounted for by decreased receptor protein. To determine whether the desensitization of immediate-early responses caused by CNTF resulted in a functional desensitization, we compared the efficacy of multiple daily injections versus a single daily dose of CNTF in preventing the denervation-induced atrophy of skeletal muscle. Surprisingly, injections of CNTF every 6 h, which falls within the putative refractory period for biochemical responses, resulted in efficacy equal to or greater than injections once daily. These results suggest that although much of the CNTF signal transduction machinery is down-regulated with frequent CNTF dosing, biological signals continue to be recognized and interpreted by the cell.

Morphological effects of ciliary neurotrophic factor treatment during neuromuscular synapse elimination.

In adult skeletal muscles, exogenous ciliary neurotrophic factor (CNTF) induces axons and their nerve terminals to sprout. CNTF also regulates the amount of multiple innervation in developing skeletal muscles during synapse elimination, maintaining multiple innervation of muscle fibers. While CNTF may maintain multiple innervation by regulating developmental synapse elimination, it is also possible that CNTF induces the formation of new multiple innervation through a sprouting response. In this study I examined morphologically the effects of CNTF during synapse elimination in the extensor digitorum longus (EDL) muscle. Rat pups received injections of CNTF in one leg and vehicle in the other either early [postnatal day 7 (P7)-P13] or late (P14-P20) in development. The early treatment period corresponds to that time when the pattern of innervation in the EDL is converted from predominantly multiple to single innervation. The late treatment period is at the end of synapse elimination for the EDL but corresponds to the major period of synapse elimination in the levator ani (LA), allowing a comparison of effects on these two muscles from the same animals. On the day after the final injection, EDL muscles were dissected and stained with tetranitroblue tetrazolium and the resulting pattern of innervation was assessed. The present findings indicate that only the early CNTF treatment regulates the level of multiple innervation in the EDL. Moreover, the effect o early CNTF treatment was local, affecting multiple innervation only in the EDL from the CNTF-treated leg. CNTF injected during the late treatment period had no apparent effects on the EDL but had a potent effect on the pattern of innervation in the LA, significantly increasing the level of multiple innervation in this muscle. Thus, CNTF affected multiple innervation in these two muscles only if provided during the period when single innervation normally develops. There was no evidence to indicate that CNTF induced axons or their terminals to sprout during either treatment period. In conclusion, CNTF increases the level of multiple innervation, probably by regulating synapse elimination, and skeletal muscles themselves may be an important target site for CNTF action. Presumably, the sprouting response to CNTF found in adult muscle develops sometime after P21.

Both Basic Fibroblast Growth Factor and Ciliary Neurotrophic Factor Promote the Retention of Polyneuronal Innervation of Developing Skeletal Muscle Fibers

At birth, nearly all rat muscle fibers receive synaptic inputs from more than one motoneuron at a single end-plate site. By the end of the third postnatal week all but one of these inputs has been eliminated. During this loss of polyneuronal innervation, developing neuromuscular synapses compete with one another. Although the nature of this competition is not known, it is commonly assumed that it is mediated through differential activity of the competing inputs. One means by which such differential activity might be translated into a biological signal would be if the synapses compete in an activity-dependent manner for a scarce supply of neurotrophic molecules. A prediction of this hypothesis is that excess quantities of such trophic molecules will reduce competition and thereby slow down or abolish the normal loss of polyneuronal innervation. In newborn rats, the effects of injection of either basic fibroblast growth factor (bFGF) or ciliary neurotrophic factor (CNTF) on the outcome of neuromuscular synapse elimination were investigated. Daily injections of either bFGF or CNTF were made for 1 week into the lateral gastrocnemius muscle beginning at the postnatal age of 2 days. The amount of polyneuronal innervation of fibers in trophic molecule-injected muscles and saline-injected contralateral muscles was assayed using intracellular recording methods. For both bFGF- and CNTF-injected muscles, an increase in the percentage of polyneuronally innervated fibers relative to saline-injected muscles was noted. For bFGF-injected muscles, the amount of polyneuronal innervation remained at nearly 60% as late as the postnatal age of 14 days (P14). This is the amount of polyneuronal innervation found at age 6 days in normal animals. Nearly 40% of the fibers of CNTF-injected muscles remained polyneuronally innervated at age P14, the amount expected at age 9 days. These results indicate that both bFGF and CNTF exert powerful and long-lasting effects on developing neuromuscular synapses.

Cytokines regulate IGF binding proteins in the CNS

Growth factor induction is a major component of the response to central nervous system trauma. The insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) are among the molecules induced by injury that have demonstrated neuroprotective actions. Induction of IGFBPs 2, 3, 4 and 5 have been documented following injury and are hypothesized to function in transport or localization of the IGFs to injured cells. It is unclear what factors lead to induction of these molecules following trauma, however, several cytokines including ciliary neurotrophic factor (CNTF) and interleukin-1β (IL-1β) have been described as major injury signals and can induce aspects of reactive gliosis. To establish whether these cytokines also are responsible for inducing the IGFBPs following CNS injury, we injected CNTF or IL-1β intracerebrally into the neocortex of adult rats and measured changes in mRNA expression for the IGFBPs. IGFBP-2 mRNA showed a dramatic increase by 24–48 h following either CNTF or IL-1β injection as compared with the contralateral side injected with heat-inactivated cytokine. Neither CNTF nor IL-1β caused alterations in BP3 or BP5. Levels of BP4 and BP6 mRNAs also were unchanged following CNTF injection. These results suggest that IGFBP2 is uniquely regulated among the IGFBPs in the CNS and is induced by cytokines that signal CNS injury.

Systemic injections of ciliary neurotrophic factor induce sprouting by adult motor neurons.

The ability of ciliary neurotrophic factor (CNTF) to induce sprouting by undamaged adult motor neurons was studied in gluteal muscles of adult ICR mice. Low doses of CNTF (0.02 mg kg-1 day-1) only induced sprouting in gluteus muscles that were beneath the site of injection, whereas high doses of CNTF (0.4-1.2 mg kg-1 day-1) acted systemically to induce motor neuron sprouting. We found little difference between the type or quality of sprouting induced by CNTF compared with muscle paralysis. Both stimuli induced sprouts of the same length, although muscle paralysis tended to induce more sprouts per end-plate. Paralysis also induced more nodal sprouting than did CNTF, but both were weaker stimuli for nodal sprouting than was partial denervation. In addition to its effects on motor neuron sprouting, high doses of CNTF induced loss of up to 36% of the body weight of treated mice. The substantial wasting caused by CNTF indicates that the factor has potent cachectic activity.

Ciliary neurotrophic factor is an endogenous pyrogen.

Fever is initiated by the action of polypeptide cytokines called endogenous pyrogens, which are produced by the host during inflammation, trauma, or infection and which elevate the thermoregulatory set point in the hypothalamus. Ciliary neurotrophic factor (CNTF) supports the differentiation and survival of central and peripheral neurons. We describe the activity of CNTF as intrinsically pyrogenic in the rabbit. CNTF induced a monophasic fever which rose rapidly (within the first 12 min) following intravenous injection; CNTF fever was blocked by pretreatment with indomethacin. The fever induced by CNTF was not due to contaminating endotoxins. Increasing doses of CNTF resulted in prolongation of the fever, suggesting the subsequent induction of additional endogenous pyrogenic activity. After passive transfer of plasma obtained during CNTF-induced fever, endogenous pyrogen activity was not present in the circulation; CNTF also did not induce the endogenous pyrogens interleukin 1, tumor necrosis factor, or interleukin 6 in vitro. Nevertheless, a second endogenous pyrogen may originate within the central nervous system following the systemic injection of CNTF. Of the four endogenous pyrogens described to date (interleukin 1, tumor necrosis factor, interferon, and interleukin 6), CNTF, like interleukin 6, utilizes the cell-surface gp 130 signal-transduction apparatus.