Ciliary Neurotrophic Factor Group Research Articles

Preparation of Neuropeptide Nanoparticle and Its Mechanism in Corneal Nerve Regeneration in Substance P-Neurokinin 1 Receptor Signaling Pathway

The multiple emulsion method was applied to prepare the 1.5 mL solution of ciliary neurotrophic factor (CNTF), and dichloromethane solution with 2.5% polylactic acid (PLA) was added into the CNTF solution during the colostrum process, and then, polyethylene glycol solution was added into the mixed solution under ultrasonic conditions to form multiple emulsion; finally, it was stirred magnetically and centrifuged to obtain CNTF nanoparticles. The transmission electron microscope (TEM) was applied to characterize the prepared nanoparticles that detected their drug loading rate, encapsulation rate, and their drug release in vitro and in vivo. In the demonstration of neuropeptide nanoparticles participating in corneal nerve regeneration in the substance p-neurokinin 1 receptor (SP-NK-1R) signaling pathway, it was to detect the gene expression in the trigeminal ganglion cells based on the different groups like normal group, advanced glycation end products (AGE)/Stattic group, and AGE + CNTF/Stattic + CNTF group. In the characterization experiment of CNTF nanoparticles, their surfaces were smooth, they evenly distributed, and the average drug loading rate reached 1.27% ± 0.032%; the nanoparticles were able to continuously release CNTF molecules within 30 hours; what’s more, CNTF group (low concentration to high concentration) and CNTF nanoparticles group (low concentration to high concentration) could both enhance the expression of mouse optic nerve cells. Compared to AGE group, the expressions of substance p-signal transducer and activator of transcription 3 (p-STAT3) in trigeminal ganglion cells increased after adding CNTF and CNTF nanoparticles, respectively (P < 0.05); and the expression of p-STAT3 increased remarkably after the addition of CNTF nanoparticles (P < 0.05), with an obvious increase on the growth of nerve axon. On the other hand, it had the same results as the above, compared with Stattic group.

Collagen scaffolds combined with collagen-binding ciliary neurotrophic factor facilitate facial nerve repair in mini-pigs.

The preclinical studies using animal models play a very important role in the evaluation of facial nerve regeneration. Good models need to recapitulate the distance and time for axons to regenerate in humans. Compared with the most used rodent animals, the structure of facial nerve in mini-pigs shares more similarities with humans in microanatomy. To evaluate the feasibility of repairing facial nerve defects by collagen scaffolds combined with ciliary neurotrophic factor (CNTF), 10-mm-long gaps were made in the buccal branch of mini-pigs' facial nerve. Three months after surgery, electrophysiological assessment and histological examination were performed to evaluate facial nerve regeneration. Immunohistochemistry and transmission electron microscope observation showed that collagen scaffolds with collagen binding (CBD)-CNTF could promote better axon regeneration, Schwann cell migration, and remyelination at the site of implant device than using scaffolds alone. Electrophysiological assessment also showed higher recovery rate in the CNTF group. In summary, combination of collagen scaffolds and CBD-CNTF showed promising effects on facial nerve regeneration in mini-pig models.

Effect of locally administered ciliary neurotrophic factor on the survival of transected and repaired adult sheep facial nerve.

to determine whether the administration of Ciliary Neurotrophic Factor (CNTF) at the site of repaired facial nerve enhances regeneration in the adult sheep model. Ten adult sheep were divided into 2 groups: control and study group (CNTF group). In the CNTF group, the buccal branch of the facial nerve was transected and then repaired by epineural sutures. CNTF was injected over the left depressor labii maxillaris muscle in the vicinity of the transected and repaired nerve for 28 days under local anesthesia. In the CNTF group, the sheep were again anesthetized after nine months and the site of facial nerve repair was exposed. Detailed electrophysiological, tension experiments and morphometric studies were carried out and then analyzed statistically. The skin CV min, refractory period, Jitter and tension parameters were marginally raised in the CNTF group than the control but the difference was statistically insignificant between the two groups. Morphometric indices also did not show any significant changes in the CNTF group. CNTF has no profound effect on neuronal regeneration of adult sheep animal model. CNTF; Neurtrophic factors; Sheep; Facial nerve; Regeneration.

In vivo visualisation of murine corneal nerve fibre regeneration in response to ciliary neurotrophic factor

The aim of this study was to examine the murine subbasal nerve fibre plexus (SNP) regeneration altered by surgical dissection. Investigations in the mouse model addressed the regeneration capabilities of the SNP, and the influence of local ciliary neurotrophic factor (CNTF) application on the regeneration process. In preliminary experiments, the healthy mouse cornea was monitored using in vivo confocal laser-scanning microscopy (CLSM) from the age of 8–52 weeks, to reveal and rule out the age-dependent changes in SNP. Nerve fibre density (NFD) was determined with the semi-automatic nerve tracing program NeuronJ. No quantitative or qualitative changes in NFD were detected in untreated animals over time; mean NFD in mice aged 8 weeks (28.30 ± 9.12 mm/mm2), 16 weeks (29.23 ± 7.28 mm/mm2), 30 weeks (26.31 ± 8.58 mm/mm2) and 52 weeks (26.34 ± 6.04 mm/mm2) showed no statistically significant differences between time points (p > 0.05). For regeneration studies a circular incision through corneal epithelium and anterior stroma of minimum 60 μm depth was generated with a custom-made guided trephine system to cut the subbasal corneal nerves in adult mice. The corneal nerve pattern was monitored and NFD was measured before and up to 8 weeks after surgery. Animals were divided in three groups each comprising 6 mice. The CNTF group received eye drops containing CNTF (25 ng/ml) 3 times daily for 3 weeks, whereas the control group received no further medication. In the sham group the same treatment schedule was applied as in CNTF group, using vehicle.The regenerating subbasal nerve fibres sprouted out of stromal nerves within the cut and additionally regrew over the scar rim from outside. They showed parallel orientation but were thinner than before incision. Whorl patterning was observed after 4 weeks. All three groups revealed a marked NFD reduction starting at one week after incision, followed by continuous recovery. After 8 weeks the NFD reached 23.5 ± 2.4 mm/mm2 (78% of baseline), 21.9 ± 1.6 mm/mm2 (73% of baseline) and 29.2 ± 3.4 mm/mm2 (93% of baseline) in the control, sham and CNTF group, respectively.By comparison with control and sham group, the CNTF group demonstrated significantly higher NFD at every observation time point.The mouse cornea provides a practicable animal model for in vivo CLSM monitoring of corneal nerve behaviour over time and following injury. Non-penetrating trephination generated a severe reduction in the NFD of the SNP, but murine corneas recovered to pre-injury NFD levels within 8 weeks. Local application of CNTF served merely to temporarily accelerate the recovery of NFD.

Chemically extracted acellular allogeneic nerve graft combined with ciliary neurotrophic factor promotes sciatic nerve repair.

A chemically extracted acellular allogeneic nerve graft can reduce postoperative immune rejection, similar to an autologous nerve graft, and can guide neural regeneration. However, it remains poorly understood whether a chemically extracted acellular allogeneic nerve graft combined with neurotrophic factors provides a good local environment for neural regeneration. This study investigated the repair of injured rat sciatic nerve using a chemically extracted acellular allogeneic nerve graft combined with ciliary neurotrophic factor. An autologous nerve anastomosis group and a chemical acellular allogeneic nerve bridging group were prepared as controls. At 8 weeks after repair, sciatic functional index, evoked potential amplitude of the soleus muscle, triceps wet weight recovery rate, total number of myelinated nerve fibers and myelin sheath thickness were measured. For these indices, values in the three groups showed the autologous nerve anastomosis group > chemically extracted acellular nerve graft + ciliary neurotrophic factor group > chemical acellular allogeneic nerve bridging group. These results suggest that chemically extracted acellular nerve grafts combined with ciliary neurotrophic factor can repair sciatic nerve defects, and that this repair is inferior to autologous nerve anastomosis, but superior to chemically extracted acellular allogeneic nerve bridging alone.

Synergetic effects of ciliary neurotrophic factor and olfactory ensheathing cells on optic nerve reparation

This study is to investigate the effect of the combination of the olfactory ensheathing cells (OEC) transplantation and intravitreous injection of ciliary neurotrophic factor (CNTF) on the retinal ganglia cells' (RGC) apoptosis and axonals' reparation and regeneration. In this study, the supraorbital margin exposure of the optic nerves was used to establish adult SD rats' optic nerve inhausted injury model as control group. Then the purified OECs were injected into the optic nerve sheaths, and CNTF was injected into the vitreous body simultaneously. The rats were divided into control group, CNTF group, OECs group, and OEC+CNTF combined group. At 4 weeks postoperatively, a cholera toxin B subunit (CTB) anterograde tracing technique and fluorescence (FG) biotinylated dextran amine (BDA) retrograde tracing technique were adopted to evaluate the survival of RGC and the regeneration of optic nerve axons. The number of survival neurons of the same vision field and the density of neurons were evaluated by analysis of variance. At the one and three quadrant distance between optic disc 2 mm spot, the number of the RGC in the control group was significantly (F = 633.38, P < 0.01) decreased compared with the normal group (46.00 ± 1.42, 34.80 ± 1.34, 25.00 ± 1.61, 15.40 ± 2.30). The survived RGC in OEC group was significantly more than that in CNTF group, while the combined treatment with CNTF and OEC had strongest repair effect. The neuron axon density showed a statistically significant difference in the average optical density value at distance between foramen opticum 2 mm spot (OEC+CNTF: 3.18 ± 0.26, OEC: 2.96 ± 0.28, CNTF: 2.83 ± 0.37, and control: 2.75 ± 0.12, respectively, F = 17.66, P < 0.01) . Optic nerves damage can be repaired and regenerated by the combined treatment with OEC and CNTF suggesting that CNTF and OEC have synergistic effect on the treatment of optic nerve injury and repair. Transplantation of OECs may genetically modify the secretion of human CNTF and promote optic nerve injury repair.

A rapid, novel model of culturing cranial nerve X-derived motoneurons for screening trophic factor outgrowth response

Objectives: After cranial nerve X (CN X) injury, vocal fold paralysis treatments currently face a myriad of obstacles in achieving non-synkinetic, functional reinnervation. Of particular therapeutic interest is the targeted administration of locally expressed biological neurotrophic factors (NFs). To date, a method to culture mature CN X motoneurons for NF responsiveness screening has not been described.Methods: We herein present a novel method for establishing mature murine CN X motoneuron cultures, and use the model to test CN X motoneuron outgrowth response to individual and paired ascending concentrations of selected neurotrophic factors [glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF)].Results: Findings demonstrated low concentration (5 ng/ml) CNTF to have the greatest positive effect on motoneuron outgrowth, beyond that of both indivual NF and paired NF combinations, based on total neurite outgrowth [mean total neurite outgrowth = 445·7±84·45 μm in the (5 ng/ml) CNTF group versus 179·7±13·63 μm in saline controls (P<0·01)]. Paired treatments with CNTF/GDNF, and CNTF/BDNF promoted motoneuron branching at a variety of concentrations beyond saline controls, and paired GDNF/BDNF had inhibitory effects on motoneuron branching.Discussion: Our described in vitro model of establishing mature CN X cultures allowed rapid screening for responsiveness to therapeutic NFs at a variety of concentrations and combinations. While the model ultimately may be used to investigate the molecular mechanisms of CN X motoneuron regeneration, the current study identified CNTF as a promising therapeutic candidate for the promotion of CN X outgrowth.

Protective effect of ciliary neurotrophic factor on retinal ganglion cell in acute ocular hypertension rat

Background Glaucoma associates with apoptosis of retinal ganglion cells ( RGCs).Research showed that ciliary neurotrophic factor (CNTF) can repair optic nerve trauma,but it has not reported whether CNTF has a protective effect on glaucomatous optic neuropathy. Objective This experimental study was to explore the protective effect of CNTF on RGCs in rat eyes with acute ocular hypertension. Methods Ocular acute hypertension models were induced in bilateral eyes of 24 clean Wistar rats by forced perfusion of a balanced salt solution into the anterior chamber.Two days before molding,0.5 μg recombinant human CNTF (5 μl) was injected intravitreously in the left eyes,and 5 mmol/L sodium phosphate solution (5 μl) was injected in the same way as the control group.Three other Wistar rats were used as the normal control group.The animals were sacrificed by excessive anesthesia and the retinal sections were prepared 1,3,7,14 days after molding.The morphology of the retina was examined and RGCs counting was performed by hematoxylin & eosin staining and observed under a light microscope.The expression of glutamic acid in RGCs was assessed by immunochemistry. Results Regular retinal structure with clearly defined cell layers were observed in normal control rats.Changes in vacuoles in RGCs were seen in the model control group.The number of degenerative RGCs decreased in the CNTF group.Compared with the model control group,number of normal RGCs increased from 1 day to 14 days after molding with a significant difference between the two groups (all P=0.000).Immunochemistry assay showed that the numbers of positive cells for glutamic acid were 5.50±1.04 and 6.00±1.41 for the average of 3 fields at 3 days or 7 days in the CNTF group,and those in the model control group were 9.00±2.91 and 10.83 ± 1.94,respectively,showing significant differences between them ( all P =0.000 ).However,no comparable differences were found in the numbers of positive cells for glutamic acid at 1 day and 14 days between the two groups( P=0.578,0.180). Conclusions CNTF down-regulates the expression of glutamic acid in RGCs and offers neuroprotection for RGCs in an acute glaucoma rat model. Key words: Ciliary neurotrophie factor; Ocular hypertention; Retinal ganglion cell; Glutamic acid

Synergistic effects of NGF, CNTF and GDNF on functional recovery following sciatic nerve injury in rats

To investigate the synergistic effects of nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and glia cell line-derived neurotrophic factor (GDNF) on survival and growth of sensory neurons and motoneurons, as well as on the functional recovery following sciatic nerve injury in rats. Experimental rats and neurons were randomized into 8 groups: NGF group, CNTF group, GDNF group, NGF+CNTF group, CNTF+GDNF group, NGF+GDNF group, NGF+CNTF+GDNF group and control group. Each group received local intramuscular injection of indicated NTFs according to the treatment protocol. The sciatic nerve function index (SFI), nerve conduction velocity and wet weight recovery rate of gastrocnemius muscle were tested to evaluate the functional recovery in vivo. A 2 (presence or absence of NGF) x 2 (presence or absence of CNTF) x 2 (presence or absence of GDNF) analysis of variance (ANOVA) was used to examine the main effects and interactions among NGF, CNTF and GDNF, and one-way ANOVA was calculated for multiple comparison. NGF and GDNF acted significantly on the survival of sensory neuron and motoneuron, respectively. CNTF was a dominant factor promoting cell body development, and GDNF had the most powerful effect on neurite outgrowth and elongation of sensory neurons and motoneurons. Combined administration of the three factors resulted in optimal functional recovery following sciatic nerve injury in rats. It is demonstrated that differential and complementary biological effects of various neurotrophic factors contribute to synergistic promotion of nervous function recovery.

The use of ciliary neurotrophic factor to promote recovery after peripheral nerve injury by delivering it at the site of the cell body

Despite a body of evidence showing that various neurotrophic factors support the survival of nerve cells and stimulate axonal outgrowth, doubt remains about their optimal site of application as well as the more compelling question of what clinical benefit, if any, they would confer. Ciliary neurotrophic factor (CNTF) supports the survival of motorneurons in vitro and in vivo. Direct delivery of CNTF to the cell bodies may help reduce the side effects and overcome the problem of rapid systemic clearance. The aim of this study was to establish whether nerve regeneration may be improved upon by the controlled addition of a specific humoral neurotrophic substance (CNTF) at the level of the cell body. Three groups of five adult sheep were used. The first group acted as normal controls. In the second and third groups, the median nerve was divided and repaired using an epineurial suture technique. In the second group, CNTF was supplied into the CSF at the level of C6 by an implanted osmotic pump. In the third group physiological saline was placed in the osmotic pump. The animals underwent comprehensive electrophysiological and isometric tension experiments at six months. All of the animals had reduced electrophysiological, morphometric and isometric tension indices after surgery compared to normal. The CNTF group had better results than the saline group in the following; (1) area and amplitude of the muscle action potential (2) the percentage of tetanus and muscle mass preserved after repair. These differences were only statistically significant for amplitude of the muscle action potential. No statistical difference was found in the morphological indices (fibre diameter, axon diameter, myelin thickness and internodal length) between the CNTF and saline groups. CNTF does not confer a functional benefit when applied at the level of the cell body.

The effects of ciliary neurotrophic factor on neurological function and glial activity following contusive spinal cord injury in the rats

Ciliary neurotrophic factor (CNTF) has been implicated in the pathophysiology of injury to the central nervous system. The rapid increase in CNTF production following spinal cord injury (SCI) in rats is thought to serve a role in the neuronal survival and functional recovery. In this study, 40 SD rats were divided into four groups: sham-operated group, saline-treated group, 5- and 10-μg CNTF group. Saline and CNTF were given through lumbar intrathecal catheter for 10 days after T10 segment of spinal cord were injured by modified Allen contusion method. Animals were behaviorally tested for 6 weeks using the Basso, Beattie, Bresnahan locomotor rating scale and inclined plane test. At the end of 6 week, rubrospinal neurons of five rats in each group were labeled by retrograde transport of the horseradish peroxidase (HRP) from the lesion site, and then the labeled red nucleus neuron (RN) numbers were counted. Additional rats were histologically assessed for tissue sparing and neuronal loss and reactive gliosis at the injury site and adjacent areas. Rats treated with CNTF regained greater improvements in hindlimb function than controls. The amount of spared tissue was significantly higher in CNTF-treated animals than in controls. After CNTF treatment, the number of HRP-labeled RN neurons were significantly increased. Astrocytes and microglia reactivity was more pronounced in CNTF-treated animals than in controls. These results indicate that intrathecal infusion of exogenous CNTF following SCI may significantly reduce tissue damage and protect the rubrospinal descending tracks and enhances functional recovery, and may also induce more gliosis.

Protective effects of ciliary neurotrophic factor on denervated skeletal muscle.

To study the effects of ciliary neurotrophic factor (CNTF) on denervated skeletal muscle atrophy and to find a new approach to ameliorate atrophy of denervated muscle, a model was established by cutting the right sciatic nerve in 36 Wistar mice, with the left side serving as control. Then they were divided into two groups randomly. CNTF (1 U/ml) 0.1 ml was injected into the right tibial muscle every day in experimental group, and saline was used into another group for comparison. The muscle wet weight, muscle total protein, Ca2+, physiological response and morphology were analyzed on the 7th, 14th and 28th day after operation. Our results showed that compared to control group, there was a significant increase in muscle wet weight, total protein, Ca2+, muscle fiber cross-section area in CNTF group (P < 0.05). CNTF could ameliorate the decrease of tetanic tension (PO), post-tetanic twitch potentiation (PTP), and the prolonged muscle relaxation time (RT) caused by denervation (P < 0.05). The motor end-plate areas 7 days and 14 days after denervation was similar (P > 0.05), but significantly larger 28 days after the denervation (P < 0.05). Our results suggest that CNTF exerts myotrophic effects by attenuating the morphological and functional changes associated with denervation of rat muscles and has protective effects on denervated muscle and motor end plate.

Effect of CNTF on ischaemic cell damage in rat hippocampus.

The neuroprotective effect of neurotrophic factors has been demonstrated in experimental cerebral ischaemia recently. These include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), and basic fibroblast growth factor (basic FGF). The neuroprotective effect of ciliary neurotrophic factor (CNTF), however, has not been studied so far. We have examined the neuroprotective effect of recombinant rat CNTF in a rat forebrain ischaemia model. A continuous infusion of CNTF was started 1 week before the induction of ischaemia and continued until 1 week after the ischaemia. Reversible forebrain ischaemia was induced by 7 minutes of bilateral carotid occlusion with hypotension. Neuronal cell death in the hippocampal CA1 sector was evaluated 1 week after the ischaemia. For the control group artificial CSF (cerebrospinal fluid) was infused instead of CNTF. Per cent neuronal cell death was 83.4 +/- 5.9% (mean +/- SEM, n = 5) in the control group, and 71.1 +/- 10.0% (mean +/- SEM, n = 5) in the CNTF group. Although percentage of neuronal cell death was lower in the CNTF group, the difference was not statistically significant. This result suggests that the protective effect of CNTF in the rat forebrain ischaemia model may be limited compared with other neurotrophic factors. It is considered that the number of neurons protected by CNTF may be small.