The Combination of Adipose-derived Schwann-like Cells and Acellular Nerve Allografts Promotes Sciatic Nerve Regeneration and Repair through the JAK2/STAT3 Signaling Pathway in Rats.

Schwann cells (SCs) combined with acellular nerve allografts (ANAs) effectively promote the regeneration and repair of peripheral nerves, but the exact mechanism has not been fully elucidated. However, the disadvantages of SCs include their limited source and slow rate of expansion in vitro. Previous studies have found that adipose-derived stem cells have the ability to differentiate into Schwann-like cells. Therefore, we speculated that Schwann-like cells combined with ANAs could profoundly facilitate nerve regeneration and repair. The aim of the present study was to investigate the cellular and molecular mechanisms of regeneration and repair. In this study, tissue-engineered nerves were first constructed by adipose-derived Schwann-like cells and ANAs to bridge missing sciatic nerves. Then, the rats were randomly divided into five groups (n = 12 per group): a Control group; a Model group; an ADSC group; an SC-L group; and a DMEM group. Twelve weeks postsurgery, behavioral function tests and molecular biological techniques were used to evaluate the function of regenerated nerves and the relevant molecular mechanisms after sciatic nerve injury (SNI). The results showed that adipose-derived Schwann-like cells combined with ANAs markedly promoted sciatic nerve regeneration and repair. These findings also demonstrated that the expression of neurotrophic factors (NFs) was increased, and the expression of Janus activated kinase2 (JAK2)/P-JAK2, signal transducer and activator of transcription-3 (STAT3)/P-STAT3 was decreased in the spinal cord after SNI. Therefore, these results suggested that highly expressed NFs in the spinal cord could promote nerve regeneration and repair by inhibiting activation of the JAK2/STAT3 signaling pathway.

All-trans retinoic acid upregulates the expression of ciliary neurotrophic factor in retinal pigment epithelial cells.

Retinopathy of prematurity, a leading cause of visual impairment in low birth-weight infants, remains a crucial therapeutic challenge. Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that promotes rod and cone photoreceptor survival and cone outer segment regeneration in the degenerating retina. Ciliary neurotrophic factor expression is regulated by many factors such as all-trans retinoic acid (ATRA). In this study, we found that ATRA increased CNTF expression in mouse retinal pigment epithelial (RPE) cells in a dose- and time-dependent manner, and PKA signaling pathway is necessary for ATRA-induced CNTF upregulation. Furthermore, we showed that ATRA promoted CNTF expression through CREB binding to its promoter region. In addition, CNTF levels were decreased in serum of retinopathy of prematurity children and in retinal tissue of oxygen-induced retinopathy mice. In mouse RPE cells cultured with high oxygen, CNTF expression and secretion were decreased, but could be recovered after treatment with ATRA. In conclusion, our data suggest that ATRA administration upregulates CNTF expression in RPE cells.

Sodium Benzoate, a Metabolite of Cinnamon and a Food Additive, Upregulates Ciliary Neurotrophic Factor in Astrocytes and Oligodendrocytes.

Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that plays an important role in multiple sclerosis (MS). However, mechanisms by which CNTF expression could be increased in the brain are poorly understood. Recently we have discovered anti-inflammatory and immunomodulatory activities of sodium benzoate (NaB), a metabolite of cinnamon and a widely-used food additive. Here, we delineate that NaB is also capable of increasing the mRNA and protein expression of CNTF in primary mouse astrocytes and oligodendrocytes and primary human astrocytes. Accordingly, oral administration of NaB and cinnamon led to the upregulation of astroglial and oligodendroglial CNTF in vivo in mouse brain. Induction of experimental allergic encephalomyelitis, an animal model of MS, reduced the level of CNTF in the brain, which was restored by oral administration of cinnamon. While investigating underlying mechanisms, we observed that NaB induced the activation of protein kinase A (PKA) and H-89, an inhibitor of PKA, abrogated NaB-induced expression of CNTF. The activation of cAMP response element binding (CREB) protein by NaB, the recruitment of CREB and CREB-binding protein to the CNTF promoter by NaB and the abrogation of NaB-induced expression of CNTF in astrocytes by siRNA knockdown of CREB suggest that NaB increases the expression of CNTF via the activation of CREB. These results highlight a novel myelinogenic property of NaB and cinnamon, which may be of benefit for MS and other demyelinating disorders.

Human ciliary neurotrophic factor-overexpressing stable bone marrow stromal cells in the treatment of a rat model of traumatic spinal cord injury.

BACKGROUND AIMS: Traumatic injury to the central nervous system (CNS) often causes motor dysfunctions. However, because of the CNS complexity and variability in the clinical presentations, efforts to repair damaged CNS tissue and restoring its functions are particularly demanding. On the other hand, recent progress in the regenerative therapy field have led to novel approaches for the treatment of traumatic CNS injury and renewed hopes to overcome the obstacles. It appears that the balance between neurite re-growth-inhibiting and neurite re-growth-inducing molecules determines the axonal re-growth fate. Neurotrophic factors can tilt this balance and indeed promote cell survival and axonal re-growth over neurodegeneration. One of the promising neurotrophic factors in this field is ciliary neurotrophic factor (CNTF). METHODS: We transfected rat bone marrow stromal cells with a mammalian expression vector-inserted human CNTF gene through the use of a non-viral method to prepare human CNTF-overexpressing stem cells under ex vivo conditions. We transplanted these modified cells to the rat model of spinal cord traumatic injury to explore functional recovery after contusion induction. RESULTS: Our data from immunocytochemistry and behavioral tests showed that such cells can act as a powerful potential approach to treat traumatic CNS injuries because these modified cells improved the behavioral test scores in the rat model of spinal cord injury. CONCLUSIONS: CNTF-overexpressing bone marrow stromal cells can ameliorate spinal cord traumatic injury and can be used in the treatment of traumatic CNS injuries in the near future.

P2X7 receptor inhibition increases CNTF in the subventricular zone, but not neurogenesis or neuroprotection after stroke in adult mice.

Increasing endogenous ciliary neurotrophic factor (CNTF) expression with a pharmacological agent might be beneficial after stroke as CNTF both promotes neurogenesis and, separately, is neuroprotective. P2X7 purinergic receptor inhibition is neuroprotective in rats and increases CNTF release in rat CMT1A Schwann cells. We, first, investigated the role of P2X7 in regulating CNTF and neurogenesis in adult mouse subventricular zone (SVZ). CNTF expression was increased by daily intravenous injections of the P2X7 antagonist Brilliant Blue G (BBG) in naïve C57BL/6 or Balb/c mice over 3 days. Despite the ∼40-60 % increase or decrease in CNTF with BBG or the agonist BzATP, respectively, the number of proliferated BrdU+SVZ nuclei did not change. BBG failed to increase FGF2, which is involved in CNTF-regulated neurogenesis, but induced IL-6, LIF, and EGF, which are known to reduce SVZ proliferation. Injections of IL-6 next to the SVZ induced CNTF and FGF2, but not proliferation, suggesting that IL-6 counteracts their neurogenesis-inducing effects. Following ischemic injury of the striatum by middle cerebral artery occlusion (MCAO), a 3-day BBG treatment increased CNTF in the medial penumbra containing the SVZ. BBG also induced CNTF and LIF, which are known to be protective following stroke, in the whole striatum after MCAO, but not GDNF or BDNF. However, BBG treatment did not reduce the lesion area or apoptosis in the penumbra. Even so, this study shows that P2X7 can be targeted with systemic drug treatments to differentially regulate neurotrophic factors in the brain following stroke.

Loss of neuron-astroglial interaction rapidly induces protective CNTF expression after stroke in mice.

Ciliary neurotrophic factor (CNTF) is a potent neural cytokine with very low expression in the CNS, predominantly by astrocytes. CNTF increases rapidly and greatly following traumatic or ischemic injury. Understanding the underlying mechanisms would help to design pharmacological treatments to increase endogenous CNTF levels for neuroprotection. Here, we show that astroglial CNTF expression in the adult mouse striatum is increased twofold within 1 h and increases up to >30-fold over 2 weeks following a focal stroke caused by a transient middle cerebral artery occlusion (MCAO). Selective neuronal loss caused by intrastriatal injection of quinolinic acid resulted in a comparable increase. Cocultured neurons reduced CNTF expression in astrocytes, which was prevented by light trypsinization. RGD (arginine-glycine-aspartic acid) blocking peptides induced CNTF expression, which was dependent on transcription. Astroglial CNTF expression was not affected by diffusible neuronal molecules or by neurotransmitters. The transient ischemia does not seem to directly increase CNTF, as intrastriatal injection of an ischemic solution or exposure of naive mice or cultured cells to severe hypoxia had minimal effects. Inflammatory mechanisms were probably also not involved, as intrastriatal injection of proinflammatory cytokines (IFNγ, IL6) in naive mice had no or small effects, and anti-inflammatory treatments did not diminish the increase in CNTF after MCAO. CNTF-/- mice had more extensive tissue loss and similar astrocyte activation after MCAO than their wild-type littermates. These data suggest that contact-mediated integrin signaling between neurons and astrocytes normally represses CNTF expression and that neuronal dysfunction causes a rapid protective response by the CNS.

Adult rat mesenchymal stem cells delay denervated muscle atrophy.

To evaluate the function of rat mesenchymal stem cells (rMSCs) on denervated gastrocnemius muscles and to address the role of ciliary neurotrophic factor (CNTF) in rMSCs, denervated Wistar rats were separately injected with culture media (sham control), CNTF protein, 2.5 × 10(5) siCNTF-treated rMSCs, 2.5 × 10(5) GFP-transfected rMSCs, or 2.5 × 10(5) untreated rMSCs. Muscle function was assessed at different time points post-surgery. Tibial nerve and gastrocnemius muscle samples were taken at 4, 8, and 12 weeks for histochemistry, and neuromuscular junction repair was also examined by electron microscopy. Fluorescence immunocytochemistry on tissue sections confirmed neurotrophin expression in rMSCs but with little evidence of neuronal differentiation. The engraftment of rMSCs significantly preserved the function of denervated gastrocnemius muscle based both on evaluation of muscle function and direct examination of muscle tissue. Further, the density and depth of the junctional folds were visibly reduced 12 weeks after surgery and transplantation, especially in control group. Knockdown of CNTF expression in rMSCs failed to block muscle preservation, although administration of CNTF protein alone inhibited muscle atrophy, which indicating that delivery of rMSCs could preserve gastrocnemius muscle function following denervation and post-junctional mechanisms involved in the repairing capability of rMSCs.

Increased expression of IL-6 family members in tendon pathology.

OBJECTIVES: Histological examination of pathological tendon generally does not reveal signs of inflammation. However, the inflammatory cytokine IL-6 has been shown to be expressed in ruptured rotator cuff tendon. The aim of this study was to investigate the expression of IL-6 family members in painful posterior tibialis tendon (PTT) and in painful and ruptured Achilles tendon (AT) compared with normal tendon. METHODS: AT samples were obtained from cadavers (normal) or from patients undergoing surgical procedures to treat chronic painful tendinopathy or ruptured tendon. PTT samples were obtained from patients undergoing surgery for other reasons (normal) and from patients with PTT dysfunction (painful). Total RNA was extracted and mRNA expression was analysed by quantitative real-time PCR. RESULTS: Collagen type I α-chain I (COL1A1) expression was increased in both painful PTT and AT compared with normal. Ciliary neurotrophic factor levels were increased in painful PTT only. In the painful AT, cyclooxygenase-2 (COX2) and IL-6 expression increased compared with normal. In the ruptured AT, levels of VEGF A, COX2, oncostatin-M, leukaemia inhibitory factor and IL-6 expression were higher compared with both normal and painful AT. IL-6R expression decreased in both painful and ruptured AT compared with normal. CONCLUSION: Painful AT and PTT show different expression patterns, indicating a substantial difference between those two tendinopathies. Inflammatory markers are up-regulated in painful and particularly in ruptured AT, pointing towards a role of inflammation not only in rupture healing, but also in Achilles tendinopathy.

Non-virally modified human mesenchymal stem cells produce ciliary neurotrophic factor in biodegradable fibrin-based 3D scaffolds.

We report the adaptation of dendrimer-based nonviral expression system for ciliary neurotrophic factor (CNTF) overproduction in human mesenchymal stem cells (hMSCs) embedded into fibrin-based three-dimensional (3D) matrix. Time-restricted neurotrophin expression enables autologous adult stem cells for additional trophic support and increases their therapeutic potential in neuroregeneration applications. Polyamidoamine (PAMAM)-NH(2) dendrimers of fourth generation effectively provided virus-free delivery and expression of CNTF-internal ribosome entry site-green fluorescent protein cassette with a transfection efficiency in hMSCs over 11%. CNTF levels in transfected cultures were 10-fold higher as compared with the control cells. Dendrimer-driven CNTF expression also persisted in hMSCs embedded into fibrin-based 3D matrix, an emerging vehicle for cell delivery or bioartificial organ formation. Nonviral modification of autologous adult stem cells with use of dendrimers is a novel tool perspective in terms of biosafety and technological availability.

[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.

Aging exacerbates intracerebral hemorrhage-induced brain injury.

Aging may be an important factor affecting brain injury by intracerebral hemorrhage (ICH). In the present study, we investigated the responses of glial cells and monocytes to intracerebral hemorrhage in normal and aged rats. ICH was induced by microinjecting autologous whole blood (15 microL) into the striatum of young (4 month old) and aged (24 month old) Sprague-Dawley rats. Age-dependent relations of brain tissue damage with glial and macrophageal responses were evaluated. Three days after ICH, activated microglia/macrophages with OX42-positive processes and swollen cytoplasm were more abundantly distributed around and inside the hemorrhagic lesions. These were more dramatic in aged versus the young rats. Western blot and immunohistochemistry analyses showed that the expression of interleukin-1beta protein after ICH was greater in aged rats, whereas the expression of GFAP and ciliary neurotrophic factor protein after ICH was significantly lower in aged rats. These results suggest that ICH causes more severe brain injury in aged rats most likely due to overactivation of microglia/macrophages and concomitant repression of reactive astrocytes.

Cytokine-induced SOCS expression is inhibited by cAMP analogue: impact on regeneration in injured retina.

Injured adult retinal ganglion cells (RGCs) regrow axons into peripheral nerve (PN) grafted onto cut optic nerve. Survival and regeneration of RGCs is increased by intraocular injections of ciliary neurotrophic factor (CNTF) and axonal regeneration is further enhanced by co-injection of a cyclic AMP analogue (CPT-cAMP). Based on these data, and because cytokine signaling is negatively regulated by suppressor of cytokine signaling (SOCS) proteins, we set out to determine whether CNTF injections increase retinal SOCS expression and whether any changes are attenuated by co-injection with CPT-cAMP. Using quantitative PCR we found increased SOCS1, SOCS2 and SOCS3 mRNA levels at various times after a single CNTF injection. Expression remained high for many days. SOCS protein levels were also increased. In situ hybridization revealed that RGCs express SOCS3 mRNA, and SOCS expression in cultured RGCs was increased by CNTF. Co-injection of CPT-cAMP reduced CNTF induced expression of SOCS1 and SOCS3 mRNA and decreased SOCS3 protein expression. CNTF injection also transiently increased retinal leukemia inhibitory factor (LIF) expression, an effect that was also moderated by CPT-cAMP. We propose that, along with known reparative effects of elevated cAMP on neurons, reducing SOCS upregulation may be an additional way in which cyclic nucleotides augment cytokine-induced regenerative responses in the injured CNS.

Sustained striatal ciliary neurotrophic factor expression negatively affects behavior and gene expression in normal and R6/1 mice.

Huntington's disease (HD) is a neurodegenerative disorder caused by an elongation of CAG repeats in the HD gene, which encodes a mutant copy of huntingtin with an expanded polyglutatmine repeat. Individuals who are affected by the disease suffer from motor, cognitive, and emotional impairments. Levels of certain striatal-enriched mRNAs decrease in both HD patients and transgenic HD mice prior to the development of motor symptoms and neuronal cell death. Ciliary neurotrophic factor (CNTF) has been shown to protect neurons against chemically induced toxic insults in vitro and in vivo. To test the hypothesis that CNTF might protect neurons from the negative effects of the mutant huntingtin protein in vivo, CNTF was continuously expressed following transduction of the striatum by recombinant adeno-associated viral vectors (rAAV2). Wild-type and R6/1 HD transgenic (R6/1) mice that received bilateral or unilateral intrastriatal injections of rAAV2-CNTF experienced weight loss. The CNTF-treated R6/1 HD transgenic mice experienced motor impairments at an earlier age than expected compared with age-matched control R6/1 HD transgenic animals. CNTF also caused abnormal behavior in WT mice. In addition to behavioral impairments, in situ hybridization showed that, in both WT and R6/1 mice, CNTF expression caused a significant decrease in the levels of striatal-enriched transcripts. Overall, continuous expression of striatal CNTF at the dose mediated by the expression cassette used in this study was detrimental to HD and wild-type mice.

JAK-STAT signaling pathway mediates astrogliosis in brains of scrapie-infected mice.

Scrapie is characterized histologically, in part, by astrogliosis in brain and spinal cord. However, the mechanisms of astrogliosis in brain injury occurring during prion infection are not well understood. In this study, we investigated the expression levels and cellular localization of Janus kinase (JAK) -signal transducers and activators of transcription (STAT) signaling molecules and growth factors such as leukemia inhibitory factor (LIF) and ciliary neurotropic factor (CNTF) by western blot analysis and immunohistochemistry. We found that expression levels of LIF and CNTF were increased in scrapie-infected brains and phosphorylated (p)-JAK2, p-STAT1 (Ser727 and Tyr701), p-STAT3 (Tyr705), and glial fibrillary acidic protein were expressed strongly in scrapie-infected brains. Moreover, we found that p-STAT1 and p-STAT3 were found mainly in the nucleus in scrapie-infected brains. Immunohistochemically, p-STAT1 was colocalized with LIF and CNTF and p-JAK2 in many reactive astrocytes in scrapie-infected brains. In contrast, immunostaining for p-STAT3 was found in comparatively few astrocytes in limited regions; p-STAT3 staining merged with p-JAK2 in hippocampus sections of scrapie-infected brains. Taken together, our results suggest that activation of JAK2-STAT1 signaling pathway occurred in reactive astrocytes in hippocampus of scrapie-infected brains.

Effect of ammonia on ciliary neurotrophic factor mRNA and protein expression and its upstream signalling pathway in cultured rat astroglial cells: possible implication of c-fos, Sp1 and p38MAPK.

Ciliary neurotrophic factor (CNTF) may be implicated in the pathogenetic mechanisms of hepatic encephalopathy. We tested this hypothesis by treating confluent primary cultures of rat astroglial cells with ammonium chloride for various periods and analysing the effect of ammonia on the signalling pathway that regulates CNTF mRNA and protein expression. Ammonia treatment induced a dose- and time-dependent reduction in CNTF mRNA and protein expression. Surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry analysis of CNTF in the culture medium demonstrated that ammonia also induced a significant decrease in CNTF release. In addition, ammonia affected Sp1 and c-fos, transcription factors that regulate CNTF mRNA and protein expression, which showed partial dephosphorylation and significantly lower mRNA and protein levels. Total content of p38MAPK (for which Sp1 and c-fos are substrates) was unaffected by ammonia, although the diphosphorylated (active) form was significantly reduced after ammonia exposure.

Impaired wound healing after cerebral hypoxia-ischemia in the diabetic mouse.

Impaired peripheral wound healing is a hallmark of diabetics pathology and has been attributed to compromised macrophage activation. Stroke is another component of diabetic pathology, with increased tissue infarction and worsened recovery although the mechanisms remain unresolved. In this study, we investigated whether a compromised glial/macrophage response might contribute to cerebral hypoxic-ischemic (H/I) brain damage in diabetic (db/db), relative to their normoglycemic db/+ mice. Hypoxia-ischemia was induced in 8-week-old male db/db and db/+ mice by the ligation of right common carotid artery followed by systemic hypoxia (8% O2: 92% N2) for 17 mins. Mice were killed at specific intervals of reperfusion/recovery and the brains analyzed by in situ hybridization or total RNA isolation. In situ hybridization using bfl-1 (microglia) and glial fibrillary acidic protein (GFAP) (astrocytes) revealed expression of both bfl-1 and GFAP in the ipsilateral hemisphere at 4 h in the db/+ mice, which was delayed and minimal in the db/db mice. RNase protection assays showed a robust increase in expression of the proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha), interleukin-1 IL-1alpha, and IL-1beta mRNA in the db/+ mice at 6 to 8 h of reperfusion peaking at 8 to 12 h; in db/db mice expression was markedly delayed and diminished. Real-time-polymerase chain reaction (RT-PCR) confirmed the reduced and delayed expression TNFalpha, IL-1alpha, IL-1beta, and the growth factors insulin-like growth factor-1 and ciliary neurotrophic factor in the db/db mice; enzyme-linked immunosorbent assays confirmed the reduced and delayed translation of IL-1beta protein. These findings suggest that a compromised inflammatory response may underlie the greater infarct associated with diabetic db/db mice compared with their nondiabetic littermates following a hypoxic/ischemic insult.

Differential expression of suppressors of cytokine signaling-1 and -3 and related cytokines in central nervous system during remitting versus non-remitting forms of experimental autoimmune encephalomyelitis.

SJL mice exhibit a relapsing-remitting course of experimental autoimmune encephalomyelitis (EAE), whereas C57BL/6 (B6) mice display a more chronic course without complete remissions. Suppressor of cytokine signaling (SOCS)-1 and SOCS-3 are members of a family of inducible intracellular proteins that negatively regulate cytokine signaling in cells of hematopoietic origin and may influence the Th1 to Th2 balance. SOCS-1 and SOCS-3 are induced by cytokines that are known to be up-regulated during EAE, including IFN-gamma (IFN-g) and IL-6, respectively. To test the hypothesis that the level of induction of SOCS-1 and SOCS-3 correlates with the course of EAE, mRNA levels were compared in spinal cords of SJL and B6 mice during discrete stages of disease. SOCS-1 and SOCS-3 were elevated throughout active disease in both strains. At peak EAE, SOCS-1 was higher and SOCS-3 was lower in B6 cords compared with SJL cords. This correlated with greater expression of the Th1 cytokine, IFN-g, and less of the Th2 cytokine, IL-10, in B6 cords relative to SJL cords during onset and peak disease. SOCS-3 inducers in the IL-6 family were expressed differentially between the strains. IL-6 and leukemia inhibitory factor were higher at onset in B6 cords whereas ciliary neurotrophic factor was increased in SJL cords during peak disease. Expression of fibroblast growth factor-2, which may be involved in remyelination, was higher in SJL cords at peak. Comparison of these models suggests that cytokine autoregulatory mechanisms involving SOCS may play a role in determining the course of EAE.

Ciliary neurotrophic factor protects retinal ganglion cells from axotomy-induced apoptosis via modulation of retinal glia in vivo.

Adenoviral-mediated transfer of ciliary neurotrophic factor (CNTF) to the retina rescued retinal ganglion cells (RGCs) from axotomy-induced apoptosis, presumably via activation of the high affinity CNTF receptor alpha (CNTFRalpha) expressed on RGCs. CNTF can also activate astrocytes, via its low affinity leukemia inhibitory receptor beta expressed on mature astrocytes, suggesting that CNTF may also protect injured neurons indirectly by modulating glia. Adenoviral-mediated overexpression of CNTF in normal and axotomized rat retinas was examined to determine if it could increase the expression of several glial markers previously demonstrated to have a neuroprotective function in the injured brain and retina. Using Western blotting, the expression of glial fibrillary acid protein (GFAP), glutamate/aspartate transporter-1 (GLAST-1), glutamine synthetase (GS), and connexin 43 (Cx43) was examined 7 days after intravitreal injections of Ad.CNTF or control Ad.LacZ. Compared to controls, intravitreal injection of Ad.CNTF led to significant changes in the expression of CNTFRalpha, pSTAT(3), GFAP, GLAST, GS, and Cx43 in normal and axotomized retinas. Taken together, these results suggest that the neuroprotective effects of CNTF may result from a shift of retinal glia cells to a more neuroprotective phenotype. Moreover, the modulation of astrocytes may buffer high concentrations of glutamate that have been shown to contribute to the death of RGCs after optic nerve transection.

Protection of photoreceptor cells from phototoxicity by transplanted retinal pigment epithelial cells expressing different neurotrophic factors.

Transplantation of cells or tissues and the intravitreal injection of neurotrophic factors are two methods that have been used to treat retinal diseases. The purpose of this study was to examine the effects of combining both methods: the transplantation of retinal pigment epithelial (RPE) cells expressing different neurotrophic factors. The neutrophic factors were Axokine, brain derived-neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). The enhanced green fluorescence protein (eGFP) gene was used as a reporter gene. These genes were transduced into RPE cells by lipofection, selected by antibiotics, and transplanted into the subretinal space of 108 rats. The rats were examined at 1 week and 3 months after the transplantation to determine whether the transduced cells were present, were expressing the protein, and were able to protect photoreceptors against phototoxicity. The survival of the transplanted cells was monitored by the presence of eGFP. The degree of protection was determined by the thickness of the outer nuclear layer. Our results showed that the degree of photoreceptor protection was different for the different types of neurotrophic factors at 1 week. After 3 months, the number of surviving transplanted cell was markedly reduced, and protection was observed only with the BDNF-transduced RPE cells. A significant degree of rescue was also observed by BDNF-transduced RPE cells in the nontransplanted area of the retina at both the early and late times. Lymphocytic infiltration was not detected in the vitreous, retina, and choroid at any time. We conclude that the transplantation of BDNF-transduced RPE cells can reduce the photoreceptor damage induced by phototoxicity in the transplanted area and weakly in the nontransplanted area.

Long-term lentiviral-mediated expression of ciliary neurotrophic factor in the striatum of Huntington's disease transgenic mice.

Ciliary neurotrophic factor (CNTF) has been shown to prevent behavioral deficits and striatal degeneration in neurotoxic models of Huntington's disease (HD), but its effect in a genetic model has not been evaluated. Lentiviral vectors expressing the human CNTF or LacZ reporter gene were therefore injected in the striatum of wild-type (WT) and transgenic mice expressing full-length huntingtin with 72 CAG repeats (YAC72). Behavioral analysis showed increased locomotor activity in 5- to 6-month-old YAC72-LacZ mice compared to WT-LacZ animals. Interestingly, CNTF expression reduced the activity levels of YAC72 mice compared to control animals. In both WT and YAC72 mice, CNTF expression was demonstrated in striatal punches, up to a year after lentiviral injection. Stereological analysis revealed that the number of LacZ and DARPP-32-positive neurons were decreased in YAC72-LacZ mice compared to WT-LacZ animals. Assessment of the benefit of CNTF expression in the YAC72 mice was, however, complicated by a down-regulation of DARPP-32 and to a lesser extent of NeuN in all mice treated with CNTF. The expression of the neuronal marker NADPH-d was unaffected by CNTF, but expression of the astrocytic marker glial fibrillary acidic protein (GFAP) was increased. Finally, a reduction of the number of striatal dark cells was observed in YAC mice treated with CNTF compared to LacZ. These data indicate that sustained striatal expression of CNTF can be achieved with lentiviruses. Further studies are, however, needed to investigate the intracellular signaling pathways mediating the long-term effects of CNTF expression on dopamine signaling, glial cell activation and how these changes may affect HD pathology.