Synergistic amelioration between Ligusticum striatum DC and borneol against cerebral ischemia by promoting astrocytes-mediated neurogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ligusticum chuanxiong Hort (LCH), with the accepted name of Ligusticum striatum DC in "The Plant List" database, is a widely used ethnomedicine in treating ischemic stroke, and borneol (BO) is usually prescribed with LCH for better therapy. Our previous study confirmed their synergistic effect on neurogenesis against cerebral ischemia. However, the underlying mechanism is still unclear. AIM OF THE STUDY: More and more evidence indicated that astrocytes (ACs) might be involved in the modulation of neurogenesis via polarization reaction. The study was designed to explore the synergic mechanism between LCH and BO in promoting astrocyte-mediated neurogenesis. MATERIALS AND METHODS: After primary cultures and identifications of ACs and neural stem cells (NSCs), the oxygen-glucose deprivation (OGD) model and the concentrations of LCH and BO were optimized. After the OGD-injured ACs were treated by LCH, BO, and their combination, the conditioned mediums were used to culture the OGD-injured NSCs. The proliferation, migration, and differentiation of NSCs were assessed, and the secretions of BDNF, CNTF, and VEGF from ACs were measured. Then the expressions of C3 and PTX3 were detected. Moreover, the mice were performed a global cerebral ischemia/reperfusion model and treated with LCH and (or) BO. After the assessments of Nissl staining, the expressions of Nestin, DCX, GFAP, C3, PTX3, p65 and p-p65 were probed. RESULTS: The most appropriate duration of OGD for the injury of both NSCs and ACs was 6 h, and the optimized concentrations of LCH and BO were 1.30 µg/mL and 0.03 µg/mL, respectively. The moderate OGD environment induced NSCs proliferation, migration, astrogenesis, and neurogenesis, increased the secretions of CNTF and VEGF from ACs, and upregulated the expressions of C3 and PTX3. For the ACs, LCH further increased the secretions of BDNF and CNTF, enhanced PTX3 expression, and reduced C3 expression. Additionally, the conditioned medium from LCH-treated ACs further enhanced NSC proliferation, migration, and neurogenesis. The in vivo study showed that LCH markedly enhanced the Nissl score and neurogenesis, and decreased astrogenesis which was accompanied by downregulations of C3, p-p65, and p-p65/p65 and upregulation of PTX3. BO not only decreased the expression of C3 in ACs both in vitro and in vivo but also downregulated p-p65 and p-p65/p65 in vivo. Additionally, BO promoted the therapeutic effect of LCH for most indices. CONCLUSION: A certain degree of OGD might induce ACs to stimulate the proliferation, astrogenesis, and neurogenesis of NSCs. LCH and BO exhibited a marked synergy in promoting ACs-mediated neurogenesis and reducing astrogenesis, in which LCH played a dominant role and BO boosted the effect of LCH. The mechanism of LCH might be involved in switching the polarization of ACs from A1 to A2, while BO preferred to inhibit the formation of A1 phenotype via downregulating NF-κB pathway.

CNTF Prevents Development of Outer Retinal Neovascularization Through Upregulation of CxCl10.

Purpose: Ciliary neurotrophic factor (CNTF) is a well-characterized neurotrophic factor currently in clinical trials for the treatment of macular telangiectasia type II. Our previous work showed that CNTF-induced STAT3 signaling is a potent inhibitor of pathologic preretinal neovascular tuft formation in the mouse model of oxygen-induced retinopathy. In this study, we investigated the effect of CNTF on outer retinal and choroidal angiogenesis and the mechanisms that underpin the observed decrease in outer retinal neovascularization following CNTF treatment. Methods: In the Vldlr-/- and laser-CNV mouse models, mice received a one-time injection (on postnatal day [P] 12 in the Vldlr-/- model and 1 day after laser in the Choroidal Neovascularization (CNV) model) of recombinant CNTF or CxCl10, and the extent of neovascular lesions was assessed 6 days posttreatment. STAT3 downstream targets affected by CNTF treatment were identified using quantitative PCR analysis. A proteome array was used to compare media conditioned by CNTF-treated and control-treated primary Müller cells to screen for CNTF-induced changes in secreted angiogenic factors. Results: Intravitreal treatment with recombinant CNTF led to significant reduction in neovascularization in the Vldlr-/- and laser-CNV mouse models. Treatment effect in the Vldlr-/- was long-lasting but time sensitive, requiring intravitreal treatment before P19. Mechanistic workup in vitro as well as in vivo confirmed significant activation of the STAT3-signaling pathway in Müller cells in response to CNTF treatment and upregulation of CxCl10. Intravitreal injections of recombinant CxCl10 significantly reduced outer retinal neovascularization in vivo in both the Vldlr-/- and laser-CNV mouse models. Conclusions: CNTF treatment indirectly affects outer retinal and choroidal neovascularization by inducing CxCl10 secretion from retinal Müller cells.

Application of CNTF or FGF-2 increases the number of M2-like macrophages after optic nerve injury in adult Rana pipiens.

We have previously shown that a single application of the growth factors ciliary neurotrophic factor (CNTF) or fibroblast growth factor 2 (FGF-2) to the crushed optic nerve of the frog, Rana pipiens, increases the numbers and elongation rate of regenerating retinal ganglion cell axons. Here we investigate the effects of these factors on the numbers and types of macrophages that invade the regeneration zone. In control PBS-treated nerves, many macrophages are present 100 µm distal to the crush site at 1 week after injury; their numbers halve by 2 weeks. A single application of CNTF at the time of injury triples the numbers of macrophages at 1 week, with this increase compared to control being maintained at 2 weeks. Application of FGF-2 is equally effective at 1 week, but the macrophage numbers have fallen to control levels at 2 weeks. Immunostaining with a pan-macrophage marker, ED1, and a marker for M2-like macrophages, Arg-1, showed that the proportion of the putative M2 phenotype remained at approximately 80% with all treatments. Electron microscopy of the macrophages at 1 week shows strong phagocytic activity with all treatments, with many vacuoles containing axon fragments and membrane debris. At 2 weeks with PBS or FGF-2 treatment the remaining macrophages are less phagocytically active, containing mainly lipid inclusions. With CNTF treatment, at 2 weeks many of the more numerous macrophages are still phagocytosing axonal debris, although they also contain lipid inclusions. We conclude that the increase in macrophage influx seen after growth factor application is beneficial for the regenerating axons, probably due to more extensive removal of degenerating distal axons, but also perhaps to secretion of growth-promoting substances.

Alleviative effect of ciliary neurotrophic factor analogue on high fat-induced hepatic steatosis is partially independent of the central regulation.

Ciliary neurotrophic factor (CNTF) analogues were reported to ameliorate fatty liver in db/db or high-fat diet-fed mice. It is generally thought that CNTF exerts its actions centrally. The aim of this study was to investigate whether peripheral effects of CNTF analogues are involved in the therapeutic effect on high fat-induced hepatic steatosis. The rat model of fatty liver was induced by a high-fat diet (HFD) for 12 weeks. In the next 2 weeks, rats were fed the HFD along with subcutaneous injection of vehicle or mutant recombinant human CNTF (rhmCNTF 0.05-0.2 mg/kg per day). Steatotic HepG2 cells were induced by 50% fetal bovine serum (FBS) for 48 hours, and then treated with rhmCNTF for 24 hours. The results showed that after rhmCNTF treatment, hepatic triglyceride (TG) accumulation was attenuated both in vivo and in vitro. RhmCNTF increased protein expression of CPT-1 and PPARα, and decreased SREBP-1c, FAS and SCD-1 in steatotic HepG2 cells. But the production of nitric oxide and 8-isoPGF2α in steatotic HepG2 cells was not affected by rhmCNTF. These results suggest that rhmCNTF has a peripheral effect that alleviates fat-induced hepatic steatosis.

In vitro assessment of TAT - Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration.

In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in the future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury.

Sustained Neural Stem Cell-Based Intraocular Delivery of CNTF Attenuates Photoreceptor Loss in the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis.

A sustained intraocular administration of neurotrophic factors is among the strategies aimed at establishing treatments for currently untreatable degenerative retinal disorders. In the present study we have analyzed the neuroprotective effects of a continuous neural stem (NS) cell-based intraocular delivery of ciliary neurotrophic factor (CNTF) on photoreceptor cells in the nclf mouse, an animal model of the neurodegenerative lysosomal storage disorder variant late infantile neuronal ceroid lipofuscinosis (vLINCL). To this aim, we genetically modified adherently cultivated NS cells with a polycistronic lentiviral vector encoding a secretable variant of CNTF together with a Venus reporter gene (CNTF-NS cells). NS cells for control experiments (control-NS cells) were modified with a vector encoding the reporter gene tdTomato. Clonal CNTF-NS and control-NS cell lines were established using fluorescent activated cell sorting and intravitreally grafted into 14 days old nclf mice at the onset of retinal degeneration. The grafted cells preferentially differentiated into astrocytes that were attached to the posterior side of the lenses and the vitreal side of the retinas and stably expressed the transgenes for at least six weeks, the latest post-transplantation time point analyzed. Integration of donor cells into host retinas, ongoing proliferation of grafted cells or adverse effects of the donor cells on the morphology of the host eyes were not observed. Quantitative analyses of host retinas two, four and six weeks after cell transplantation revealed the presence of significantly more photoreceptor cells in eyes with grafted CNTF-NS cells than in eyes with grafted control-NS cells. This is the first demonstration that a continuous intraocular administration of a neurotrophic factor attenuates retinal degeneration in an animal model of neuronal ceroid lipofuscinosis.

Ciliary neurotrophic factor reduces the proliferation and promotes the differentiation of TH- MYCN transformed sympathoadrenal progenitors.

Neuroblastoma is a childhood cancer caused by the transformation of sympathoadrenal progenitors. By following the formation of tumors in homozygous TH-MYCN mice, an established mouse model of neuroblastoma, we were able to capture transformed cells prior to the formation of large, vascularized tumors in order to determine the responsiveness of cells to neurotrophic factors. We discovered that the ciliary neurotrophic factor (CNTF) receptor is abundantly expressed in tumor cells from these mice. Furthermore, CNTF - but not nerve growth factor, brain-derived nerve growth factor, neurotrophin 3, or glial cell line-derived neurotrophic factor - promoted neuronal differentiation and withdrawal from the cell cycle. Thus, the transformation of sympathoadrenal progenitors by MYCN overexpression differentially affects responsiveness to neurotrophic molecules.

An experimental rat model of sporadic Alzheimer's disease and rescue of cognitive impairment with a neurotrophic peptide.

Alzheimer's disease (AD) is multifactorial and, to date, no single cause of the sporadic form of this disease, which accounts for over 99% of the cases, has been established. In AD brain, protein phosphatase-2A (PP2A) activity is known to be compromised due to the cleavage and translocation of its potent endogenous inhibitor, I2PP2A, from the neuronal nucleus to the cytoplasm. Here, we show that adeno-associated virus vector-induced expression of the N-terminal I2NTF and C-terminal I2CTF halves of I2PP2A , also called SET, in brain reproduced key features of AD in Wistar rats. The I2NTF-CTF rats showed a decrease in brain PP2A activity, abnormal hyperphosphorylation and aggregation of tau, a loss of neuronal plasticity and impairment in spatial reference and working memories. To test whether early pharmacologic intervention with a neurotrophic molecule could rescue neurodegeneration and behavioral deficits, 2.5-month-old I2NTF-CTF rats and control littermates were treated for 40 days with Peptide 6, an 11-mer peptide corresponding to an active region of the ciliary neurotrophic factor. Peripheral administration of Peptide 6 rescued neurodegeneration and cognitive deficit in I2NTF-CTF animals by increasing dentate gyrus neurogenesis and mRNA level of brain derived neurotrophic factor. Moreover, Peptide 6-treated I2NTF-CTF rats showed a significant increase in dendritic and synaptic density as reflected by increased expression of synapsin I, synaptophysin and MAP2, especially in the pyramidal neurons of CA1 and CA3 of the hippocampus.

Regional comparison of the neurogenic effects of CNTF-derived peptides and cerebrolysin in AßPP transgenic mice.

Adult neurogenesis, the production of new neurons in certain brain regions, is known to decrease with age and the loss of neurogenic potential has been implicated in Alzheimer's disease (AD), a leading cause of dementia in the elderly. Cerebrolysin (CBL) has been shown to increase neurogenesis in models of stroke and AD. CBL is composed of small peptides with activity similar to neurotrophic factors including ciliary neurotrophic factor (CNTF), which may mediate its neurogenic effects. This study compares the effects of CBL and two peptides with corresponding to an active region of CNTF (Peptide 6 and 6A) across neurogenic brain regions in amyloid-ß protein precursor (AßPP) transgenic (tg) mice. Both CBL and Peptides 6 and 6A were able to increase the numbers of neuroblasts (DCX+ cells) and BrdU+ cells in a regionally specific manner across the subventricular zone, olfactory bulb, and hippocampus. The increased generation of new cells and cell survival in animals treated with Peptides 6 and 6A was accompanied by an increase in PCNA+ cells. In contrast, AßPP tg mice treated with CBL displayed reduced levels of TUNEL staining, while levels of PCNA were unaltered. Collectively these results demonstrate that while CBL and Peptides 6 and 6A all potentiate neurogenesis in the AßPP tg mice, their relative modes of action may differ with CBL associated with reduced apoptosis and Peptides 6 and 6A working by augmenting cell proliferation. These results are consistent with a potential therapeutic relevance for Peptides 6 and 6A in AD and other disorders characterized by neurogenic deficits.

[Bone marrow stromal cells transfected with ciliary neurotrophic factor gene ameliorates the symptoms and inflammation in C57BL/6 mice with experimental allergic encephalomyelitis].

OBJECTIVE: To investigate the anti-inflammatory effect of bone marrow stromal cells (MSCs) transfected with recombinant adenovirus-mediated ciliary neurotrophic factor (CNTF) gene in C57BL/6 mice with experimental allergic encephalomyelitis (EAE). METHODS: An adenovirus vector containing CNTF gene Ad-CNTF-IRES-GFP was constructed and transfected in the MSCs (MSC-CNTF). After examination of CNTF expression, the transfected cells were transplanted in C57BL/6 mice with MOG 35-55-induced EAE, which were monitored for the changes in the symptoms scores. The levels of tumor necrosis factor-alpha (TNF-alpha), inteferon-gamma (IFN-gamma), interleukin-12P35 (IL-12P35), and IL-10 in the peripheral blood of the mice were detected, and the number of MSC-CNTF cells in the spleen and spinal cord was counted. CD3+ T cell infiltration and TNF-alpha and IFN-gamma expressions in the lesions were also observed after the cell transplantation. RESULTS: CNTF gene transfection resulted in significantly increased CNTF expression in the MSCs. The mice receiving MSC-CNTF transplantation exhibited significantly improved symptoms with shortened disease course and lessened disease severity. The cell transplantation also resulted in significantly decreased peripheral blood TNF-alpha levels, ameliorated CD3+T cell infiltrations and lowered TNF-alpha expression in the lesions, while the levels of IFN-gamma underwent no significant changes. CONCLUSION: Transplantation of CNTF gene-transfected MSCs results in decreased peripheral blood TNF-alpha and IFN-gamma levels and reduced inflammatory cells, CD3-positive cells and TNF-alpha expression in the lesion of EAE, therefore providing better effect than MSCs in relieving the symptoms of EAE in mice.

Chapter 10: Conduit luminal additives for peripheral nerve repair.

The use of nerve conduits as an alternative for nerve grafting has a long experimental and clinical history. Luminal additives, factors introduced into these nerve conduits, were later developed to enhance the nerve regeneration through conduits. This chapter generalizes the types of additives used, and the reported performance of luminal additives in conduits to present a preference list for the most effective additives to use over specific distances of nerve defect.

Ciliary neurotrophic factor fused to a protein transduction domain retains full neuroprotective activity in the absence of cytokine-like side effects.

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. CNTF prevents the degeneration of motor neurons after axotomy and in mouse mutant progressive motor neuronopathy, which has encouraged trials of CNTF for human motor neuron disease. Given systemically, however, CNTF causes severe side effects, including cachexia and a marked immune response, which has limited its clinical application. The present work describes a novel approach for administering recombinant human CNTF (rhCNTF) while conserving neurotrophic activity and avoiding deleterious side effects. rhCNTF was fused to a protein transduction domain derived from the human immunodeficiency virus-1 TAT (transactivator) protein. The resulting fusion protein (TAT-CNTF) crosses the plasma membrane within minutes and displays a nuclear localization. TAT-CNTF was equipotent to rhCNTF in supporting the survival of cultured chicken embryo dorsal root ganglion neurons. Local or subcutaneous administration of TAT-CNTF, like rhCNTF rescued motor neurons from death in neonatal rats subjected to sciatic nerve transection. In contrast to subcutaneous rhCNTF, which caused a 20-30% decrease in body weight in neonatal rats between postnatal days 2 and 7 together with a considerable fat mobilization in brown adipose tissue, TAT-CNTF lacked such side effects. Together, these results indicate that rhCNTF fused with the protein transduction domain/TAT retains neurotrophic activity in the absence of CNTFs cytokine-like side effects and may be a promising candidate for the treatment of motor neuron and other neurodegenerative diseases.

Overexpression of CNTF in Mesenchymal Stem Cells reduces demyelination and induces clinical recovery in experimental autoimmune encephalomyelitis mice.

Human Mesenchymal Stem Cells (MSCs) were previously reported to ameliorate neuronal functional deficits in the MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) mice by inducing T cell anergy. Human Ciliary neurotrophic factor (CNTF) recently was found to promote myelogenesis and reduce inflammation in CNTF-deficient EAE mice. We ectopically overexpressed CNTF in human MSCs to investigate its potential role in promoting remyelination and improving functional recovery in EAE mice. MSCs transfected by Ad-CNTF-IRES-EGFP (MSC-CNTF) were injected intravenously into EAE mice 10 days after the immunization. Neurological functional tests were scored daily by grading clinical signs (score 0-6). Immunofluorescence microscopy was used to detect MSC-CNTF in spinal cord. Expression of NG2, CNTF, and cleaved caspase-3 was measured by immunohistochemistry. CNTF expression was also analyzed by Western blot. Myelin was detected by Solochrome Cyanin staining. Our results found that CNTF concentration in MSC-CNTF cells was 20-fold higher than that in either MSC or Ad-EGFP-transfected MSCs (MSC-EGFP) in vitro. Mice receiving MSC-CNTF cells showed remarkable neuronal functional recovery: the cumulative clinical scores were significantly decreased, and the disease onset was statistically delayed. Mice receiving MSC-CNTF cells showed reduced TNF-alpha, IFN-gamma and increased the level of cytokine IL-10 in peripheral blood and a large number of MSC-CNTF cells were detected in the spleen, but were not detected in other organs such as lung, liver and kidney. In the lesions of these mice, 1) the number of cleaved caspase3-positive cells was significantly reduced; 2) MSC-CNTF- and NG2-positive cells were significantly increased; and 3) the expression of CNTF was dramatically increased. In addition, demyelination was significantly reduced in MSC-CNTF mice. These data indicated that MSC-CNTF may improve functional recovery in EAE mice, possibly by exerting their immunoregulatory activity, inhibiting inflammation, homing MSC-CNTF cells to the lesions, elevating CNTF expression, reducing demyelination, and stimulating oligodendrogenesis.

Emerging concepts in the medical and surgical treatment of obesity.

The relentless rise in the prevalence of obesity predicts an exponential increase in the incidence of obesity-related complications. Medical and surgical treatments are necessary to prevent and treat obese co-morbidities, thereby avoiding disability and premature death. Interventions for obesity should be evaluated not by weight loss alone but against the new incidence in obesity-related co-morbidities, their remission or improvement. In combination with lifestyle measures, currently available pharmacological therapies -- rimonabant, orlistat and sibutramine -- achieve 5-10% weight loss, although a return to baseline is the norm after cessation of medication. All these agents demonstrate approximately 0.5% reduction in HbA1c in diabetic subjects; orlistat also reduces the new incidence of type 2 diabetes. Modest improvement in lipid profiles and reduced calculated cardiovascular risk is observed, but data on improvement of other co-morbidities are sparse. In contrast, surgical procedures that restrict food ingestion and/or curtail the absorptive surface area of the gut consistently achieve substantial weight loss, typically 20-35%, effect resolution of co-morbid conditions and improve quality of life. Although mortality is low, complications and hospitalisation are not uncommon after bariatric surgery. Intriguingly, surgical patients experience a reduction in appetite and report changes in food preference. Accentuation of the normal gastrointestinal hormonal response to food intake and possible changes in vagal afferent signalling are proposed to induce satiety. Increased understanding of body weight homeostasis and appetite regulation has provided an impressive list of potential targets for drug development, with the promise that single or combination therapy may ultimately challenge the supremacy of bariatric surgery.

gp130 receptor ligands as potential therapeutic targets for obesity.

Obesity and its related cluster of pathophysiologic conditions including insulin resistance, glucose intolerance, dyslipidemia, and hypertension are recognized as growing threats to world health. It is now estimated that 10% of the world's population is overweight or obese. As a result, new therapeutic options for the treatment of obesity are clearly warranted. Recent research has focused on the role that gp130 receptor ligands may play as potential therapeutic targets in obesity. One cytokine in particular, ciliary neurotrophic factor (CNTF), acts both centrally and peripherally and mimics the biologic actions of the appetite control hormone leptin, but unlike leptin, CNTF appears to be effective in obesity and as such may have therapeutic potential. In addition, CNTF suppresses inflammatory signaling cascades associated with lipid accumulation in liver and skeletal muscle. This review examines the potential role of gp130 receptor ligands as part of a therapeutic strategy to treat obesity.

CNTF+BDNF treatment and neuroprotective pathways in the rd1 mouse retina.

The rd1 mouse is a relevant model for studying the mechanisms of photoreceptor degeneration in retinitis pigmentosa. Treatment with ciliary neurotrophic factor (CNTF) in combination with brain derived neurotrophic factor (BDNF) is known to rescue photoreceptors in cultured rd1 retinal explants. To shed light on the underlying mechanisms, we studied the effects of 9 days (starting at postnatal day 2) in vitro CNTF+BDNF treatment on the endogenous production of CNTF, BDNF, fibroblast growth factor 2 (FGF2), or the activation of extracellular signal-regulated kinase (ERK), Akt and cAMP-response-element-binding protein (CREB) in retinal explants. In rd1 explants, CNTF+BDNF decreased the number of TUNEL-positive photoreceptors. The treatment also increased endogenous rd1 levels of CNTF and BDNF, but lowered the level of FGF2 expression in rd1 explants. When wild-type explants were treated, endogenous CNTF was similarly increased, while BDNF and FGF2 levels remained unaffected. In addition, treatment of rd1 retinas strongly increased the phosphorylation of ERK, Akt and CREB. In treated wild-type explants, the same parameters were either unchanged (ERK) or decreased (Akt and CREB). The results suggest a role for Akt, ERK and CREB in conveying the neuroprotective effect of CNTF+BDNF treatment in rd1 retinal explants.

CNTF induces dose-dependent alterations in retinal morphology in normal and rcd-1 canine retina.

Ciliary neurotrophic factor (CNTF) provides morphologic preservation of rods in several animal models of retinitis pigmentosa (RP). However, CNTF may alter photoreceptor morphology and rod photoreceptor differentiation in vitro, as well as affecting normal retinal electrophysiology. In addition, the capacity of CNTF to support other cell types affected secondarily in RP (cones and ganglion cells) is unclear. The purposes of this study were to examine the effects of CNTF upon a canine model of RP, the rod-cone degeneration (rcd-1) dog. Archival tissue from a previous study assessing the capacity of CNTF to rescue photoreceptors in rcd-1 dogs was used. One eye was treated for 7 weeks before being explanted. The contralateral eye was untreated. A total of 23 rcd-1 dogs and seven control dogs (four untreated and three CNTF-treated) were used. Morphometric data describing outer and inner nuclear layer thickness, inner retinal thickness, cones and ganglion cells were collected at nine evenly spaced points along each retina and analysed using a mixed effects model. Immunohistochemistry was performed on a subset of 11 dogs for expression of rhodopsin, human cone arrestin (hCAR) and recoverin. CNTF protected the outer nuclear layer and increased inner retinal thickness in a dose-dependent manner (both were maximal at CNTF doses of 1-6 ng day-1). Significant cone loss or reduction of inner nuclear layer width in rcd-1 did not occur in this model, therefore we were unable to assess the protective effect of CNTF upon these parameters. CNTF did not afford significant ganglion cell protection. CNTF induced morphologic changes in rods and ganglion cells, as well as reducing expression of hCAR and rhodopsin, but not recoverin. The dose of CNTF which provided optimal outer nuclear layer protection also resulted in several other effects, including altered ganglion cell morphology, increased thickness of the entire retina, and reduced expression of some phototransduction proteins. These changes were more marked in rcd-1 retinas than in wild-type retinas. This implies that the consequences of CNTF treatment may be substantially influenced by the cellular context into which it is introduced.

Ciliary neurotrophic factor protects retinal ganglion cells from secondary cell death during acute autoimmune optic neuritis in rats.

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS which leads to demyelination, axonal destruction and neuronal loss in the early stages. Available therapies mainly target the inflammatory component of the disease but fail to prevent neurodegeneration. To investigate the effect of ciliary neurotrophic factor (CNTF) on the survival of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve, we used a rat model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. Optic neuritis in this model was diagnosed by recording visual evoked potentials, and RGC function was monitored by measuring electroretinograms. This study demonstrates that CNTF has a neuroprotective effect on affected RGCs during acute optic neuritis. Furthermore, we demonstrate that CNTF exerts its neuroprotective effect through activation of the Janus kinase/signal transducer and activator of transcription pathway, mitogen activated protein kinases and a shift in the Bcl-2 family of proteins towards the anti-apoptotic side. In summary, our results demonstrate that CNTF can serve as an effective neuroprotective treatment in a rat model of MS that especially reflects the neurodegenerative aspects of this disease.