Neurotrophic factors and corneal nerve regeneration

The cornea has unique features that make it a useful model for regenerative medicine studies. It is an avascular, transparent, densely innervated tissue and any pathological changes can be easily detected by slit lamp examination. Corneal sensitivity is provided by the ophthalmic branch of the trigeminal nerve that elicits protective reflexes such as blinking and tearing and exerts trophic support by releasing neuromediators and growth factors. Corneal nerves are easily evaluated for both function and morphology using standard instruments such as corneal esthesiometer and in vivo confocal microscope. All local and systemic conditions that are associated with damage of the trigeminal nerve cause the development of neurotrophic keratitis, a rare degenerative disease. Neurotrophic keratitis is characterized by impairment of corneal sensitivity associated with development of persistent epithelial defects that may progress to corneal ulcer, melting and perforation. Current neurotrophic keratitis treatments aim at supporting corneal healing and preventing progression of corneal damage. Novel compounds able to stimulate corneal nerve recovery are in advanced development stage. Among them, nerve growth factor eye drops showed to be safe and effective in stimulating corneal healing and improving corneal sensitivity in patients with neurotrophic keratitis. Neurotrophic keratitis represents an useful model to evaluate in clinical practice novel neuro-regenerative drugs.

Expression of nerve growth factor mRNA in splenic lymphocytes of bronchial asthma rats and its influencing factors

BACKGROUND： Previous research has proved that nerve growth factor （NGF） participates in the onset of asthma by the induction of neurogenic inflammation. OBJECTIVE： To investigate the effect of interleukin-13 （IL-13） and interferon- γ; （IFN- γ ） on the expression of NGF mRNA in the splenic lymphocytes of bronchial asthma rats. DESIGN, TIME AND SETTING： The experiment, a completely randomized study based on cellular immunology, was performed in the Laboratory of Neurology in Chongqing Medical University and the Department of Clinical Pharmacy in College of Clinical Medicine, Chongqing Medical University （Chongqing, China） from January 2006 to April 2007. MATERIALS： Four adult male Wistar rats were used in this study. Rat IL-13, IFN- γ probe and the total RNA extraction kit were produced by Shanghai Sangon Biological Technology ＆ Services Co., Ltd （China）. The NGF ELISA kit was a product of Wuhan Boster Bioengineering Co., Ltd （China）. A Du-70 automatic UV spectrophotometer was produced by Beckman Company （USA）. METHODS： Rats were subjected to 1-mL intraperitoneal injections each containing 100 mg of ovalbumin, and were sensitized by using antigen solution, which was sensitized with 5×10^9 Bacillus pertussis and 100 mg aluminum hydroxide powder. Four rats were challenged with 1% ovalbumin using an ultrasonic nebulizer for 60 minutes to establish an asthmatic model. After rats were anesthetized, splenic lymphocytes were isolated and cultured in medium, which was supplemented with IL-13 or IFN- γ, for 0, 12, 24 or 48 hours. A parallel study was conducted with cultured splenic lymphocytes, which were divided into a control group, an IL-13 group and an IFN- γ group. Culture medium was added with different concentrations of IL-13 （10, 50, 100 U g/L） and IFN- γ; （1, 10, 50 u g/L）; 24 hours later, all samples were harvested. MAIN OUTCOME MEASURES： The expression levels of NGF mRNA were detected by reverse transcription-polymerase chain reaction. RESULTS： In the control group, the lymphocytes of the asthmatic model expressed NGF mRNA in a time-dependent manner. Alter lymphocytes were cultured with IL-13 at a final concentration of 50 u g/L for 12, 24 or 48 hours, expression of NGF mRNA was upregulated in a time-dependent manner to higher levels than the basal values at the same time points （P 〈 0.01 ）. IL- 13 upregulated the expression of NGF mRNA in a dose-dependent manner, and the NGF mRNA expression levels at middle and high concentrations of IL-3 were significanlly higher than the values at a low concentration of IL-13 （P 〈 0.05）. With increasing IFN- γ concentration, the expression of NGF mRNA was gradually downregulated. The low concentration group showed lower levels of NGF mRNA than the blank control group, while the middle and high concentration IFN- γ, groups showed lower levels than the low concentration group （P 〈 0.05）. CONCLUSION： In asthmatic rats, IL-13, a Th2 cytokine, upregulates the expression ofNGF mRNA, while IFN- γ, a Thl cytokine, downregulates NGF mRNA expression. The effects of both cytokines were time and dose dependent. Th 1/Th2 cytokine immune imbalance may indirectly induce airway neurogenic inflammation by regulating NGF mRNA expression.

Chitosan-based thermosensitive hydrogel with long-term release of murine nerve growth factor for neurotrophic keratopathy

Neurotrophic keratopathy is a persistent defect of the corneal epithelium,with or without stromal ulceration,due to corneal nerve deficiency caused by a variety of etiologies.The treatment options for neurotrophic keratopathy are limited.In this study,an ophthalmic solution was constructed from a chitosan-based thermosensitive hydrogel with long-term release of murine nerve growth factor(CTH-mNGF).Its effectiveness was evaluated in corneal denervation(CD)mice and patients with neurotrophic keratopathy.In the preclinical setting,CTH-mNGF was assessed in a murine corneal denervation model.CTH-mNGF was transparent,thermosensitive,and ensured sustained release of mNGF for over 20 hours on the ocular surface,maintaining the local mNGF concentration around 1300 pg/mL in vivo.Corneal denervation mice treated with CTH-mNGF for 10 days showed a significant increase in corneal nerve area and total corneal nerve length compared with non-treated and CTH treated mice.A subsequent clinical trial of CTH-mNGF was conducted in patients with stage 2 or 3 neurotrophic keratopathy.Patients received topical CTH-mNGF twice daily for 8 weeks.Fluorescein sodium images,Schirmer’s test,intraocular pressure,Cochet-Bonnet corneal perception test,and best corrected visual acuity were evaluated.In total,six patients(total of seven eyes)diagnosed with neurotrophic keratopathy were enrolled.After 8 weeks of CTH-mNGF treatment,all participants showed a decreased area of corneal epithelial defect,as stained by fluorescence.Overall,six out of seven eyes had fluorescence staining scores<5.Moreover,best corrected visual acuity,intraocular pressure,Schirmer’s test and Cochet-Bonnet corneal perception test results showed no significant improvement.An increase in corneal nerve density was observed by in vivo confocal microscopy after 8 weeks of CTH-mNGF treatment in three out of seven eyes.This study demonstrates that CTH-mNGF is transparent,thermosensitive,and has sustained-release properties.Its effectiveness in healing corneal epithelial defects in all eyes with neurotrophic keratopathy suggests CTH-mNGF has promising application prospects in the treatment of neurotrophic keratopathy,being convenient and cost effective.

Electrodeposition of chitosan/graphene oxide conduit to enhance peripheral nerve regeneration

Currently available commercial nerve guidance conduits have been applied in the repair of peripheral nerve defects.However,a conduit exhibiting good biocompatibility remains to be developed.In this work,a series of chitosan/graphene oxide(GO)films with concentrations of GO varying from 0-1 wt%(collectively referred to as CHGF-n)were prepared by an electrodeposition technique.The effects of CHGF-n on proliferation and adhesion abilities of Schwann cells were evaluated.The results showed that Schwann cells exhibited elongated spindle shapes and upregulated expression of nerve regeneration-related factors such as Krox20(a key myelination factor),Zeb2(essential for Schwann cell differentiation,myelination,and nerve repair),and transforming growth factorβ(a cytokine with regenerative functions).In addition,a nerve guidance conduit with a GO content of 0.25%(CHGFC-0.25)was implanted to repair a 10-mm sciatic nerve defect in rats.The results indicated improvements in sciatic functional index,electrophysiology,and sciatic nerve and gastrocnemius muscle histology compared with the CHGFC-0 group,and similar outcomes to the autograft group.In conclusion,we provide a candidate method for the repair of peripheral nerve defects using free-standing chitosan/GO nerve conduits produced by electrodeposition.

Effects of Nerve Growth Factor on NMDA Receptor 1 and Neuronal Nitric Oxide Production after Spinal Cord Injury in Rats

Objective:To explore the protective mechanisms of nerve growth factor (NGF) on spinal cord injury (SCI) and provide theoretical basis for its clinical application. Methods: The SCI of Wistar rats was done by Allens weight dropping way by a 10 g×2.5 cm impact on the posterior of spinal cord T 8. NGF (3 g/L, 20 μl) or normal saline was injected through catheter into subarachnoid space 2, 4, 8, 12 and 24 h after SCI. The expression of N-methyl-D-asparate receptor 1 (NMDAR 1) and neuronal constitutive nitric oxide synthase (ncNOS) mRNA in rat spinal cord was detected by in situ hybridization. Results: Abnormal expression of NMDAR 1 and ncNOS mRNA appeared in spinal ventral horn motorneuron in injured rats, as compared with that in control group. The expression of NMDAR 1 and ncNOS mRNA in NGF group was significantly lower than that in saline group (P<0.01). Conclusion: NGF can protect spinal cord against injury in vivo. One of the mechanisms is that NGF can prohibit NMDAR 1 and nitric oxide (NO) production after spinal cord injury.

Regulatory effect of nerve growth factor on release of substance P in cultured dorsal root ganglion neurons of rat

Objective To investigate the regulatory effects of nerve growth factor （NGF） on basal and capsaicin-induced release of neuropeptide substance P （SP） in primary cultured embryonic rat dorsal root ganglion （DRG） neurons. Methods DRGs were dissected from 15-day-old embryonic Wistar rats. DRG neurons were dissociated and cultured, and then exposed to different concentrations of NGF （10 ng/mL, 30 ng/mL, or 100 ng/mL） for 72 h. The neurons cultured in media without NGF served as control. RT-PCR were used for detecting the mRNAs of SP and vanilloid receptor 1 （VR1） in the DRG neurons. The SP basal and capsaicin （100 nmol/L）-induced release in the culture were measured by radioimmunoassay （RIA）. Results SP mRNA and VR1 mRNA expression increased in primary cultured DRG neurons in a dose-dependent manner of NGF. Both basal release and capsaicin-evoked release of SP increased in NGF-treated DRG neurons compared with in control group. The capsaicin-evoked release of SP also increased in a dose-dependent manner of NGF. Conclusion NGF may promote both basal release and capsaicin-evoked release of SP. NGF might increase the sensitivity of nociceptors by increasing the SP mRNA or VR1 mRNA.

Müller cells are activated in response to retinal outer nuclear layer degeneration in rats subjected to simulated weightlessness conditions

A microgravity environment has been shown to cause ocular damage and affect visual acuity,but the underlying mechanisms remain unclear.Therefore,we established an animal model of weightlessness via tail suspension to examine the pathological changes and molecular mechanisms of retinal damage under microgravity.After 4 weeks of tail suspension,there were no notable alterations in retinal function and morphology,while after 8 weeks of tail suspension,significant reductions in retinal function were observed,and the outer nuclear layer was thinner,with abundant apoptotic cells.To investigate the mechanism underlying the degenerative changes that occurred in the outer nuclear layer of the retina,proteomics was used to analyze differentially expressed proteins in rat retinas after 8 weeks of tail suspension.The results showed that the expression levels of fibroblast growth factor 2(also known as basic fibroblast growth factor)and glial fibrillary acidic protein,which are closely related to Müller cell activation,were significantly upregulated.In addition,Müller cell regeneration and Müller cell gliosis were observed after 4 and 8 weeks,respectively,of simulated weightlessness.These findings indicate that Müller cells play an important regulatory role in retinal outer nuclear layer degeneration during weightlessness.

Effect of resuscitation after selective cerebral ultraprofound hypothermia on expressions of nerve growth factor and glial cell line-derived neurotrophic factor in the brain of monkey

Objective To investigate the expression of nerve growth factor （NGF） and glial cell line-derived neurotrophic factor （GDNF） in monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood flow occlusion. Methods The monkeys were immediately removed brain after death in operation of group A （identical temperature perfusion group） and group B （ultraprofound hypothermia perfusion group）. Immunohistochemical technique was used to determine frontal cellular expression of NGF and GDNF. Statistics were analyzed by ANOVA analyses with significance level at P 〈 0.05. Results The expressions of NGF and GDNF in the group B were significantly higher than those in the group A （P 〈 0.05）. Conclusion NGF and GDNF increased significantly in the monkeys of resuscitation after selective cerebral ultraprofound hypothermia and blood flow occlusion. It may be a protective mechanism for neuron survival and neural function recovery.

Isolation and Evaluation of Nerve Growth Factor

Objective: to obtain the high purified and active nerve growth factor (NGF) from mouse submaxillary glands. Methods: NGF was prepared from mouse submaxillary glands by the way of elution with CM 52 column. The molecular weight and purification of NGF were detected by SDS-PAGE polyacrylamide gel electrophoresis. The biological activity of NGF was verified thorough culturing DRG. Results: About 14 kDa stained band was observed on SDS-PAGE and it promoted proliferation of dorsal root gang lia (DRG). Conclusion: Good quality of NGF could be obtained with these methods.

Nerve growth factor pretreatment against glutamate-induced hippocampal neuronal injury Action mechanism of phosphatase and tensin homologue deleted on chromosome 10

BACKGROUND： Nerve growth factor （NGF） attenuates glutamate-induced injury to hippocampal neurons, and the human tumor suppressor gene phosphatase and tensin homologue deleted on chromosome 10 （PTEN） promotes neuronal apoptosis. However, effects of PTEN in NGF-mediated neuroprotection against glutamate excitotoxicity remain poorly understood. OBJECTIVE： To investigate the relationship between NGF inhibition of glutamate-induced injury and PTEN. DESIGN, TIME AND SE＇I＇rlNG： The randomized, controlled, in vitro study was performed at the Department of Pathophysiology, Medical School of Nantong University, China from October 2007 to March 2008. MATERIALS： Glutamate, NGF, 4, 6-diamidino-2-phenyl-indolediacetate, 3-[4, 5-dimethylthiazol-2-yl]- 2, 5-diphenyl tetrazoliumbromide （M-I-F）, and lactate dehydrogenase kit （Sigma, USA）, fluorescence microscope and inverted phase contrast microscope （Olympus, Japan） were used in this study. METHODS： Hippocampal neurons were obtained from newborn （〈 24 hours） Sprague Dawley rats and cultured for 7 days. The control group was not treated with any intervention factor, the glutamate group was treated with glutamate （0.2 mmol/L）, and NGF groups were treated with NGF （10, 50, 100, and 200 μg/L, respectively） prior to glutamate treatment. MAIN OUTCOME MEASURES： The MTT and lactate dehydrogenase assays were applied to evaluate viability of hippocampal neurons. Morphological changes in hippocampal neurons were observed using an inverted phase-contrast microscope, and neuronal apoptosis was detected by 4, 6-diamidino-2- phenyl-indolediacetate staining. PTEN mRNA and protein expression were measured by reverse transcription-polymerase chain reaction and Western blot analysis, respectively. RESULTS： Glutamate （0.2 mmol/L） induced significantly decreased neuronal viability and greater lactate dehydrogenase efflux compared with the control group （P 〈 0.01）. However, compared with the glutamate group, cell viability significantly increased and lactate dehydrogenase efflux decreased in the NGF group with increasing NGF concentrations （P 〈 0.05 or P 〈 0.01）. The apoptotic ratio and PTEN mRNA and protein expression decreased in the NGF group compared with the glutamate group （P 〈 0.01）. CONCLUSION： Pretreatment with NGF exerted neuroprotective effects against glutamate-induced injury, partially through inhibition of PTEN expression and neuronal apoptosis.

Electroacupuncture and moxibustion promote regeneration of injured sciatic nerve through Schwann cell proliferation and nerve growth factor secretion

Using electroacupuncture and moxibustion to treat peripheral nerve injury is highly efficient with low side effects. However, the electroacupuncture-and moxibustion-based mechanisms underlying nerve repair are still unclear. Here, in vivo and in vitro experiments uncovered one mechanism through which electroacupuncture and moxibustion affect regeneration after peripheral nerve injury. We first established rat models of sciatic nerve injury using neurotomy. Rats were treated with electroacupuncture or moxibustion at acupoints Huantiao （GB30） and Zusanli （ST36）. Each treatment lasted 15 minutes, and treatments were given six times a week for 4 consecutive weeks. Behavioral testing was used to determine the sciatic functional index. We used electrophysiological detection to measure sciatic nerve conduction velocity and performed hematoxylin-eosin staining to determine any changes in the gastrocnemius muscle. We used immunohistochemistry to observe changes in the expression of S100—a specific marker for Schwann cells—and an enzyme-linked immunosorbent assay to detect serum level of nerve growth factor. Results showed that compared with the model-only group, sciatic functional index, recovery rate of conduction velocity, diameter recovery of the gastrocnemius muscle fibers, number of S100-immunoreactive cells,and level of nerve growth factor were greater in the electroacupuncture and moxibustion groups. The efficacy did not differ between treatment groups. The serum from treated rats was collected and used to stimulate Schwann cells cultured in vitro. Results showed that the viability of Schwann cells was much higher in the treatment groups than in the model group at 3 and 5 days after treatment. These findings indicate that electroacupuncture and moxibustion promoted nerve regeneration and functional recovery; its mechanism might be associated with the enhancement of Schwann cell proliferation and upregulation of nerve growth factor.

Tissue engineering for the repair of peripheral nerve injury

Peripheral nerve injury is a common clinical problem and affects the quality of life of patients. Traditional restoration methods are not satisfactory. Researchers increasingly focus on the field of tissue engineering. The three key points in establishing a tissue engineering material are the biological scaffold material, the seed cells and various growth factors. Understanding the type of nerve injury, the construction of scaffold and the process of repair are necessary to solve peripheral nerve injury and promote its regeneration. This review describes the categories of peripheral nerve injury, fundamental research of peripheral nervous tissue engineering and clinical research on peripheral nerve scaffold material, and paves a way for related research and the use of conduits in clinical practice.

Intranasal nerve growth factor bypasses the blood-brain barrier and affects spinal cord neurons in spinal cord injury

The purpose of this work was to investigate whether, by intranasal administration, the nerve growth factor bypasses the blood-brain barrier and turns over the spinal cord neurons and if such therapeutic approach could be of value in the treatment of spinal cord injury. Adult Sprague-Dawley rats with intact and injured spinal cord received daily intranasal nerve growth factor administration in both nostrils for 1 day or for 3 consecutive weeks. We found an in-creased content of nerve growth factor and enhanced expression of nerve growth factor receptor in the spinal cord 24 hours after a single intranasal administration of nerve growth factor in healthy rats, while daily treatment for 3 weeks in a model of spinal cord injury improved the deifcits in locomotor behaviour and increased spinal content of both nerve growth factor and nerve growth factor receptors. These outcomes suggest that the intranasal nerve growth factor bypasses blood-brain barrier and affects spinal cord neurons in spinal cord injury. They also suggest exploiting the possible therapeutic role of intranasally delivered nerve growth factor for the neuroprotection of damaged spinal nerve cells.

Efficacy and safety of nerve growth factor for the treatment of neurological diseases:a meta-analysis of 64 randomized controlled trials involving 6,297 patients

OBJECTIVE： China is the only country where nerve growth factor is approved for large-scale use as a clinical medicine. More than 10 years ago, in 2003, nerve growth factor injection was listed as a national drug. The goal of this article is to evaluate comprehensively the efficacy and safety of nerve growth factor for the treatment of neurological diseases. DATA RETRIEVAL： A computer-based retrieval was performed from six databases, including the Cochrane Library, PubMed, EMBASE, Sino Med, CNKI, and the VIP database, searching from the clinical establishment of nerve growth factor for treatment until December 31, 2013. The key words for the searches were ＂nerve growth factor, randomized controlled trials＂ in Chinese and in English. DATA SELECTION： Inclusion criteria： any study published in English or Chinese referring to randomized controlled trials of nerve growth factor; patients with neurological diseases such as peripheral nerve injury, central nerve injury, cranial neuropathy, and nervous system infections; patients older than 7 years; similar research methods and outcomes assessing symptoms; and measurement of nerve conduction velocities. The meta-analysis was conducted using Review Manager 5.2.3 software. MAIN OUTCOME MEASURES： The total effective rate, the incidence of adverse effects, and the nerve conduction velocity were recorded for each study. RESULTS： Sixty-four studies involving 6,297 patients with neurological diseases were included. The total effective rate in the group treated with nerve growth factor was significantly higher than that in the control group （P 〈 0.0001, RR： 1.35, 95%CI： 1.30-1.40）. The average nerve conduction velocity in the nerve growth factor group was significantly higher than that in the control group （P 〈 0.00001, MD. 4.59 m/s, 95%CI： 4.12-5.06）. The incidence of pain or sclero- ma at the injection site in the nerve growth factor group was also higher than that in the control group （P 〈 0.00001, RR： 6.30, 95%CI： 3.53-11.27）, but such adverse effects were mild. CONCLUSION： Nerve growth factor can significantly improve nerve function in patients with nervous system disease and is safe and effective.

Effect of electroacupuncture on glial fibrillary acidic protein and nerve growth factor in the hippocampus of rats with hyperlipidemia and middle cerebral artery thrombus

Electroacupuncture(EA)has been shown to reduce blood lipid level and improve cerebral ischemia in rats with hyperlipemia complicated by cerebral ischemia.However,there are few studies on the results and mechanism of the effect of EA in reducing blood lipid level or promoting neural repair after stroke in hyperlipidemic subjects.In this study,EA was applied to a rat model of hyperlipidemia and middle cerebral artery thrombosis and the condition of neurons and astrocytes after hippocampal injury was assessed.Except for the normal group,rats in other groups were fed a high-fat diet throughout the whole experiment.Hyperlipidemia models were established in rats fed a high-fat diet for 6 weeks.Middle cerebral artery thrombus models were induced by pasting 50%FeCl3 filter paper on the left middle cerebral artery for 20 minutes on day 50 as the model group.EA1 group rats received EA at bilateral ST40(Fenglong)for 7 days before the thrombosis.Rats in the EA1 and EA2 groups received EA at GV20(Baihui)and bilateral ST40 for 14 days after model establishment.Neuronal health was assessed by hematoxylin-eosin staining in the brain.Hyperlipidemia was assessed by biochemical methods that measured total cholesterol,triglyceride,low-density lipoprotein and high-density lipoprotein in blood sera.Behavioral analysis was used to confirm the establishment of the model.Immunohistochemical methods were used to detect the expression of glial fibrillary acidic protein and nerve growth factor in the hippocampal CA1 region.The results demonstrated that,compared with the model group,blood lipid levels significantly decreased,glial fibrillary acidic protein immunoreactivity was significantly weakened and nerve growth factor immunoreactivity was significantly enhanced in the EA1 and EA2 groups.The repair effect was superior in the EA1 group than in the EA2 group.These findings confirm that EA can reduce blood lipid,inhibit glial fibrillary acidic protein expression and promote nerve growth factor expression in the hippocampal CA1 region after hyperlipidemia and middle cerebral artery thrombosis.All experimental procedures and protocols were approved by the Animal Use and Management Committee of Beijing University of Chinese Medicine,China(approval No.BUCM-3-2018022802-1002)on April 12,2018.

Topical delivery of nerve growth factor for treatment of ocular and brain disorders

Neurotrophins are a family of proteins that support neuronal proliferation, survival, and differentiation in the central and peripheral nervous systems, and are regulators of neuronal plasticity. Nerve growth factor is one of the best-described neurotrophins and has advanced to clinical trials for treatment of ocular and brain diseases due to its trophic and regenerative properties. Prior trials over the past few decades have produced conflicting results, which have principally been ascribed to adverse effects of systemic nerve growth factor administration, together with poor penetrance of the blood-brain barrier that impairs drug delivery. Contrastingly, recent studies have revealed that topical ocular and intranasal nerve growth factor administration are safe and effective, suggesting that topical nerve growth factor delivery is a potential alternative to both systemic and invasive intracerebral delivery. The therapeutic effects of local nerve growth factor delivery have been extensively investigated for different ophthalmic diseases, including neurotrophic keratitis, glaucoma, retinitis pigmentosa, and dry eye disease. Further, promising pharmacologic effects were reported in an optic glioma model, which indicated that topically administered nerve growth factor diffused far beyond where it was topically applied. These findings support the therapeutic potential of delivering topical nerve growth factor preparations intranasally for acquired and degenerative brain disorders. Preliminary clinical findings in both traumatic and non-traumatic acquired brain injuries are encouraging, especially in pediatric patients, and clinical trials are ongoing. The present review will focus on the therapeutic effects of both ocular and intranasal nerve growth factor delivery for diseases of the brain and eye.

Nerve growth factor involvement in liver cirrhosis and hepatocellular carcinoma

AIM: To define NGF (nerve growth factor) and its high- affinity receptor trkANGF presence and distribution in fibrotic liver and in HCC, and to verify if NGF might have a role in fibrosis and HCC. METHODS: Intracellular distribution of NGF and trkANGF were assessed by immunohistochemistry and immuno- electron microscopy in liver specimens from HCC, cirrhosis or both. ELISA was used to measure circulating NGF levels. RESULTS: NGF and trkANGF were highly expressed in HCC tissue, mainly localized in hepatocytes, endothelial and some Kupffer cells. In the cirrhotic part of the liver they were also markedly expressed in bile ducts epithelial and spindle-shaped cells. Surprisingly, in cirrhotic tissue from patients without HCC, both NGF and trkANGF were negative. NGF serum levels in cirrhotic and/or HCC patient were up to 25-fold higher than in controls. CONCLUSION: NGF was only detected in liver tissue with HCC present. Intracellular distribution suggests paracrine and autocrine mechanisms of action. Better definition of mechanisms may allow for therapeutic and diagnostic/prognostic use of NGF.

Adenomyosis uterine innervation in mice correlates to nerve growth factor expression,inflammation,and vascularization

BACKGROUND： Studies have shown that abnormal innervation is an important factor impacting occurrence and development of pathological pain in endometriosis. OBJECTIVE： To observe uterine innervation of adenomyosis mice and to analyze the cause of innervation changes due to nerve growth factor （NGF） expression, inflammation, and vascularization. DESIGN, TIME AND SETTING： This randomized, controlled, animal experiment was performed at the Research Institute of Obstetrics and Gynecology Hospital, and Central Laboratory of Zhongshan Hospital, Fudan University from March to December 2008. MATERIALS： Tamoxifen was provided by Fudan Forward, China. Rabbit anti-mouse NGF was purchased from Santa Cruz Corporation, USA; rabbit anti-protein gene product 9.5 （PGP9.5） and rabbit anti-substance P （SP） were purchased from Chemicon, USA. METHODS： A total of 40 newborn ICR mice were randomly assigned to adenomyosis model and control groups, with 20 animals in each group. Mice in the adenomyosis model group were orally administrated 2.7 μmol/kg tamoxifen on days 2-5 after birth, while the controls were not treated. MAIN OUTCOME MEASURES： Both uteri from all mice were harvested at days 135-145 after birth Expressions of polyclonal PGP9.5 and SP were immunohistochemically detected to demonstrate pan- and sensory nerve fibers. Microvessel density was quantified in the endometrium and myometrium using immunochemical staining for polyclonal rabbit anti-CD31, which stained vessels. Gene expression for NGF, high-affinity tyrosine kinase receptor （trkA）, p75 neuretrophin receptor （p75NTR）, bradykinin receptor-1 （BKR-1）, and 2 （BKR-2）, as well as substance P receptor （neurokininl receptor, NK1-R）, were detected by reverse transcription-polymerase chain reaction. NGF-13 protein expression was detected by Western blot analysis. RESULTS： More nerve fibers were stained with PGP9.5 in the endometrium and myometrium, and with SP in the endometrium, in adenomyosis mice compared with controls （P 〈 0.01 and P 〈 0.05）. Microvessel density in the myometrium of adenomyosis mice was significantly greater than the controls （P 〈 0.01）. In the uterus of adenomyosis mice, mRNA expression of NGF and its two receptors （trkA and p75 NTR）, BKR-1, and NK1-R, as well as protein expression of NGF-β, were greater than the control mice （P 〈 0.01 or P 〈 0.05）. CONCLUSION： Uterine innervation in the adenomyosis mice was increased compared with the controls. Moreover, NGF expression, inflammation, and vascularization, which have been shown to be impact factors of innervation, were abnormal in the uteri of adenomyosis mice.

Dynamic expression of nerve growth factor and its receptor Trk A after subarachnoid hemorrhage in rat brain

Delayed ischemic neurologic deficit after subarachnoid hemorrhage results from loss of neural cells.Nerve growth factor and its receptor Trk A may promote regeneration of neural cells,but their expression after subarachnoid hemorrhage remains unclear.In the present study,a rat model of subarachnoid hemorrhage was established using two injections of autologous blood into the cistern magna.Immunohistochemical staining suggested that the expression of nerve growth factor and Trk A in the cerebral cortex and brainstem increased at 6 hours,peaked at 12 hours and decreased 1 day after induction of subarachnoid hemorrhage,whereas the expression in the hippocampus increased at 6 hours,peaked on day 1,and decreased 3 days later.Compared with those for the rats in the sham and saline groups,neurobehavioral scores decreased significantly 12 hours and 3 days after subarachnoid hemorrhage（P 〈 0.05）.These results suggest that the expression of nerve growth factor and its receptor Trk A is dynamically changed in the rat brain and may thus participate in neuronal survival and nerve regeneration after subarachnoid hemorrhage.

INTRANASAL DELIVERY OF NERVE GROWTH FACTOR TO PROTECT THE CENTRAL NERVOUS SYSTEM AGAINST ACUTE CEREBRAL INFARCTION

Objective To confirmed reliability and feasibility of intranasal nerve growth factor (NGF) bypassing the blood-brain barrier and its potential neuroprotective effects on acute cerebral ischemia. Methods (1) To assay NGF concentrations in different brain regions after middle cerebral artery occlusion (MCAO).Rats were randomly divided into intranasal (IN) NGF, intravenous (IV) NGF, and untreated group (n= 4). The concentra-tions of NGF of different brain regions in the three groups after MCAO were measured by ELISA. (2) To observe neuro-protective action of NGF on focal cerebral ischemic damage. Rats were randomly assigned to 4 groups: IN vehicle, IN NGF, IV vehicle, IV NGF (n= 8). Treatment was initiated 30 minutes after onset of MCAO and given again 24 hours later. Three neurologic behavioral tests were performed 24 and 48 hours following onset of MCAO. Corrected infarct volumes were determined 48 hours after onset of MCAO. Results The olfactory bulb in IN NGF group obtained the highest concentration (3252 pg/g) of NGF among all regions, followed by the hippocampus. The NGF concentrations in the olfactory bulb and hippocampus in IN NGF group were markedly higher than that in IV NGF and control groups. The infarct volume in IN NGF group was markedly reduced by 38.8% compared with IN vehicle group. IN NGF group vestibulum function markedly improved compared with IN vehicle group at 24 and 48 hours after onset of MCAO (P 24 h = 0.02 and P 48 h = 0.04, respectively). Conclusion Intranasal NGF could pass through the blood-brain barrier, reach the central nervous system, reduce infarct volume, and improve neurologic function in rats following MCAO. Intranasal delivery of NGF may be a promising treat-ment for stroke.