Content Of Ciliary Neurotrophic Factor Research Articles

Subconjunctival Bevacizumab Injection Impairs Corneal Innervations and Epithelial Wound Healing in Mice.

To investigate the effects of subconjunctival bevacizumab injection on the corneal nerve, sensitivity, and epithelial wound healing in mice. Adult C57BL/6 mice were treated with subconjunctival injection of 1, 2, 5, or 25 mg/mL bevacizumab. The corneal nerve was observed with whole-mount anti-β3-tubulin fluorescence staining. Corneal sensitivity was measured with a Cochet-Bonnet esthesiometer. The protein levels of pigment epithelium-derived factor (PEDF), nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) were measured by ELISA. The corneal epithelial wound-healing rate was evaluated by fluorescein staining. The recovery of impaired mouse corneal innervations and epithelial wound-healing rate following bevacizumab injection was evaluated with the co-injection of PEDF, NGF, or CNTF. Subconjunctival bevacizumab injection caused apparent corneal nerve degeneration, attenuated corneal sensitivity, and delayed corneal epithelial wound healing and nerve regeneration in normal mice, which was more significant with increased concentration and times of the bevacizumab injection. However, the corneal nerve and sensitivity gradually improved and recovered in mice with a single injection of 1 to 5 mg/mL bevacizumab. Moreover, the bevacizumab injection significantly decreased the corneal PEDF, NGF, and CNTF content, whereas exogenous PEDF, NGF, or CNTF supplement attenuated impairment of the corneal nerve, sensitivity, and epithelial wound healing after subconjunctival bevacizumab injection. Subconjunctival bevacizumab injection impairs corneal innervations, epithelial wound healing, and nerve regeneration in normal mice, which may be caused by the reduction of neurotrophic factor content in the cornea.

Соотношения нейротрофических факторов в слезной жидкости и влаге передней камеры у больных с возрастной катарактой

Purpose . To study a possibility of evaluation of neurotrophic factors content in the aqueous humor (AH) based on a determination of their level in lacrimal fluid (LF) on the examples of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in patients with age-related cataract. Material and methods . We studied the content of CNTF and BDNF in the LF, AH, and the blood serum (BS) in 55 patients (55 eyes) with agerelated cataract. Collection of stimulated LF was performed with a pipette on the day preceding surgery; the AH and BS were sampled during the phacoemulsification of a cataract. The CNTF and BDNF concentration was measured using an enzyme immunoassay. Results. After the exclusion of patients with outlying (>M+3σ) values for LF, AH or their ratios, the mean concentration of CNTF in patients was 38.6±17.6 pg/mL in the LF, 55.8±25.0 pg/mL in the AH, and 5.76±5.39 pg/mL in the BS, the concentrations ratio AH / LF was 1.50±0.51. The mean concentration of BDNF in patients was 120.8±42.8 pg/mL in the LF, 49.3±20.8 pg/mL in the AH, and 22270±7040 pg/mL in the BS, the concentrations ratio AH / LF was 0.41±0.13. The concentration of each neurotrophic factor in the AH was correlated with its content in the LF. Pearson’s correlation coefficient was 0.68 (P<0.000) for CNTF and 0.66 (P<0.000) for BDNF. Conclusion. Significant correlations found between AH and LF levels for both CNTF and BDNF confirm the possibility to use the LF concentration of these neurotrophic factors for an approximate evaluation of their levels in the AH.

Ciliary neurotrophic factor in patients with age-related cataract

We studied the content of ciliary neurotrophic factor (CNTF) in the lacrimal fluid, the aqueous humor, and the blood serum in 40 patients (40 eyes) with age-related cataracts. Collection of stimulated lacrimal fluid was performed by a pipette on the day preceding surgery; the aqueous humor and the blood were sampled during the phacoemulsification of a cataract. CNTF was measured using an enzyme immunoassay. On average, the concentration of CNTF in patients was 43.0 ± 21.2 pg/mL in the lacrimal fluid, 58.7 ± 26.4 pg/mL in the aqueous humor, and 4.4 ± 2.6 pg/mL in the blood serum. The concentration of CNTF in the aqueous humor correlated well with its content in the lacrimal fluid (Pearson’s correlation coefficient was 0.66, P < 0.000) and exceeded it on average by a factor of 1.4 ± 0.5. The data we obtained may be used for an approximate evaluation of the CNTF concentration in the aqueous humor on the basis of an analysis of the lacrimal fluid.

Effect of ammonia, glutamine, and serum on calcineurin, p38MAPK-diP, GADD153/CHOP10, and CNTF in primary rat astrocyte cultures

Primary astrocyte cultures were subjected to different experimental schedules using several concentrations of ammonia (1, 3, and 5 mM ammonium chloride), serum (2.5%, 5%, and 12%), and glutamine (0.5, 1, and 3 mM) to analyze the involvement of calcineurin (CaN) in hyperammonemia and its relation with p38MAPK-diP and ciliary neurotrophic factor (CNTF). We demonstrated that exposure to ammonia affects CaN content, and confirmed the ammonia-induced reduction of CNTF expression; however, the involvement of CaN and p38MAPK-diP in CNTF reduction could not be confirmed. On the contrary, an inverse relationship between CaN and p38MAPK-diP contents was clearly demonstrated. GADD153/CHOP10 content was always higher under hyperammonemic conditions as well as under glutamine exposure, probably due to the osmotic stress provoked by glutamine accumulation, which was induced after exposure to ammonia. Statistical analysis demonstrated significant interactions of ammonia and serum for CaN, GADD153/CHOP10 and CNTF contents. The exposure to glutamine also induced changes in GADD153/CHOP10 and CaN; however, CNTF content was not affected. In conclusion, CaN content was affected by exposure to ammonia and glutamine; the serum content of the culture medium had a strong influence on the astroglial response to ammonium chloride, and glutamine exposure only reproduced some of the ammonia effects.

Immobilization of Ciliary Neurotrophic Factor on Model Biomaterials by Plasma Graft Polymerization

Acrylic acid was grafted on the surface of polypropylene (PP) film via plasma‐induced technology. It was shown by FT‐IR that there were carboxyl groups on the surface of the modified PP film. The grafting ratio was analyzed quantitatively through a dyeing process. The effects of the technology parameters of the plasma‐treatment and graft polymerization on the grafting ratio, such as power, discharge time, concentration of monomer and active species density, reaction temperature and time, were characterized. Moreover, the content of ciliary neurotrophic factor (CNTF) that was anchored on the surface of the modified PP film was measured through enzyme‐linked immunosorbent assay (ELISA). The results show that the amount of CNTF immobilized on the surface of the modified PP film increase with increasing content of carboxyl groups, showing that the existence of carboxyl groups on the surface of the PP film is beneficial to the immobilization of CNTF.

Quinolinic acid lesions of the caudate putamen in the rat lead to a local increase of ciliary neurotrophic factor.

When applied prior to excitotoxic lesions, ciliary neurotrophic factor (CNTF) has been shown to be neuroprotective. However, data concerning the endogenous CNTF content of the intact rat striatum are rare and have not until now been available for the quinolinic acid (QA)-lesioned striatum. Therefore, we investigated the CNTF content in the QA-lesioned rat striatum for at least 1 month using immunohistochemistry and Western blot analysis. In lesioned striata a neuronal loss was observed by Nissl staining and by a reduction of NeuN-immunoreactive cells, whereas increased glial fibrillary acidic protein immunoreactivity showed a gliotic reaction. With CNTF immunohistochemistry we found that in the QA-lesioned striatum CNTF was increased over time, whereas it was not detectable in intact and sham-lesioned striata. CNTF-immunoreactive cells had the morphology of protoplasmatic astrocytes. Furthermore, quantitative Western blotting demonstrated that the content of CNTF protein from striatal lysates containing 1 mg of whole protein 1 month after QA lesioning (2.76 +/- 1.71 ng) was significantly increased (P < 0.05, U-test) compared with sham-lesioned hemispheres (0.68 +/- 0.25 ng) and intact controls (0.55 +/- 0.25 ng). We conclude that CNTF content is correlated with glial scar formation and suggest that our results may be of relevance to cell grafting strategies for the treatment of Huntington's disease.

Ciliary neurotrophic factor in the olfactory bulb of rats and mice

Ciliary neurotrophic factor (CNTF) is primarily regarded as an astrocytic lesion factor, promoting neuronal survival and influencing plasticity processes in deafferented areas of the CNS. Postnatal loss of neurons in CNTF-deficient mice indicates a function of the factor also under physiological conditions. In the olfactory bulb, where neurogenesis, axo- and synaptogenesis continue throughout life, CNTF content is constitutively high. The cellular localization of CNTF in the rat olfactory bulb is not fully resolved, and species differences between mouse and rat are not yet characterized. In the present study, four different CNTF antibodies and double immunolabeling with specific markers for glial and neuronal cells were used to study the cellular localization of CNTF in rat and mouse olfactory bulb. Specificity of the detection was checked with tissue from CNTF-deficient mice, and investigations were complemented by immunolocalization of reporter protein in mice synthesizing β-galactosidase under control of the CNTF promoter (CNTF lacZ-knock-in mice). In both species, CNTF localized to ensheathing cell nuclei, cell bodies and axon-enveloping processes. Additionally, individual axons of olfactory neurons were CNTF immunoreactive. Both CNTF protein content and immunoreaction intensity were lower in mice than in rats. Scattered lightly CNTF-reactive cells were found in the granular and external plexiform layers in rats. Some CNTF-positive cells were associated with immunoreactivity for the polysialylated form of the neural cell adhesion molecule, which is expressed by maturing interneurons derived from the rostral migratory stream. In CNTF lacZ-knock-in mice, β-galactosidase reactivity was found in ensheathing cells of the olfactory nerve layer, and in cells of the glomerular, external plexiform and granular layers. The study proves that CNTF is localized in glial and neuronal structures in the rodent olfactory bulb. Results in mice provide a basis for investigations concerning the effects of a lack of the factor in CNTF-deficient mice.

Therapeutic Effects of Aldose Reductase Inhibitor on Experimental Diabetic Neuropathy through Synthesis/Secretion of Nerve Growth Factor

We investigated alterations in nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF) contents during treatment with epalrestat, an aldose reductase inhibitor (ARI), on streptozotocin (STZ)-induced diabetic neuropathy in rats. Diabetic rats showed a statistically significant reduction in H-wave-related sensory nerve conduction velocity (HSNCV) and in NGF content in sciatic nerves during the experiment of 8 weeks. No reduction in the CNTF content in sciatic nerves was seen in the diabetic rats. The epalrestat treatment, which started 4 weeks after STZ injection, resulted in a significantly greater NGF content and faster HSNCV than those in untreated diabetic rats. But no statistically significant alterations of motor nerve conduction velocity (MNCV) or CNTF content were seen during the treatment. ARI showed the stimulating effect for NGF synthesis/secretion in rat Schwann cell culturein vitro.These findings suggest that decreased levels of NGF in diabetic sciatic nerves may be involved in the pathogenesis of diabetic neuropathy in these rats and further show that epalrestat treatment can be useful for the treatment of diabetic neuropathy through NGF-induction in Schwann cells and/or inhibition of the polyol pathway.

Increase of ciliary neurotrophic factor (CNTF)in the ischemic rat· brain as determined by a sensitive enzyme-linked immunoassay

To study the distribution of ciliary neurotrophic factor (CNTF) in the rat brain under physiological as well as pathological conditions, a sensitive sandwich enzyme linked immunoassay (EIA) for rat CNTF was developed using rabbit anti-CNTF antibody. This method detected as little as 10 pg ml-1 CNTF with a good linearity and accuracy. Analysis of various regions of rat brain and peripheral nerves showed a high content of CNTF in the sciatic nerves, spinal cord, optic nerves, and olfactory bulb. Transient focal ischemia of rat brain caused by middle cerebral artery occlusion significantly and specifically increased CNTF levels in the cerebral cortex and hippocampus regions in the ischemic hemisphere. [Neural Res 1997; 19: 593-598]

Distribution of and age-related changes in ciliary neurotrophic factor protein in rat tissues.

We developed a sensitive enzyme-linked immunosolvent assay for measuring ciliary neurotrophic factor (CNTF) and examined age-related changes in the CNTF contents of a variety of rat tissues during postnatal development. CNTF contents were substantially higher in the sciatic nerve and spinal cord than in the other tissues tested, the kidney coming third. In all the tissues except the sciatic nerve (90 ng/g), the CNTF content was less than 1 ng/g at 1 week of age, then gradually increased. It was highest at 5 weeks of age in the sciatic nerve (3171 ng/g), spinal cord (118 ng/g), and kidney (36.8 ng/g), after which it slowly decreased. In contrast, the maximum in the brain stem (9 ng/g) and cerebellum (3.6 ng/g) was at 8 weeks of age, whereas in skeletal muscle it was at 2 weeks of age (14.6 ng/g). These findings indicate that CNTF functions in the postnatal development of the rat.

Microencapsulated Ciliary Neurotrophic Factor: Physical Properties and Biological Activities

Controlled drug release in the CNS and PNS is still an obstacle to the treatment of neurodegenerative disorders. We have prepared a variety of microspheres containing either ciliary neurotrophic factor (CNTF) or genetically engineered cells able to synthesize and release this cytokine. CNTF is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. However, when given in therapeutically effective doses by systemic injections, it produces numerous adverse side effects. In order to minimize these effects we have microencapsulated it in biopolymers (chitosans, alginates, and copolymers in various proportions to achieve different kinetic properties). Size distribution profiles were determined by an image analysis system and surface characteristics were assessed by electron microscopy. The total content of CNTF as well as the amounts released per day were determined by ELISA andin vitrobioassays. The results from the release kinetics demonstrate that long-term secretion (up to 24 days) of CNTF is achieved by combining chitosan with copolymerized lactic and glycolic acid, whereas microspheres made of alginate provided only relatively short-term release (2–12 days). Neuron survival and neurite outgrowth in cultures of ciliary ganglia were supported by microencapsulated CNTF, indicating biological stability of CNTF. Genetically engineered human kidney cells 293 continued synthesizing CNTF within spheres and the released amounts of CNTF in the culture medium were comparable to the amounts secreted from monolayers (1 ng/ml of supernatant from confluent cultures) or even higher. These studies provide a basis for future testing of CNTF in encapsulated preparations using animal models of neurodegenerative disorders.

Changes in ciliary neurotrophic factor content in the rat brain after continuous intracerebroventricular infusion of β-amyloid(1–40) protein

We have previously shown that the continuous intracerebroventricular infusion of β-amyloid(1–40) protein results in memory impairments in rats, associated with a reduction of choline acetyltransferase activity in the frontal cortex and hippocampus. In the present study, we examined whether the infusion of β-amyloid(1–40) protein affected the content of ciliary neurotrophic factor (CNTF) in the rat brain. The β-amyloid(1–40) infusion increased CNTF content in the frontal cortex, hippocampus, and the cerebellum, but decreased its content in the brain stem. These results suggest that accumulation of β-amyloid(1–40) in the brain may affect CNTF production in vivo.