Nerve Growth Factor (NGF) as Partaker in the Modulation of UV-Response in Cultured Human Conjunctival Fibroblasts.

Corroborating data sustain the pleiotropic effect of nerve growth factor (NGF) in the protection of the visual system from dangerous stimuli, including ultraviolet (UV). Since UV exposure might promote ocular surface changes (conjunctival inflammation and matrix rearrangement), as previously reported from in vivo studies sustaining some protective NGF effects, in vitro cultures of human conjunctival fibroblasts (FBs) were developed and exposed to a single UV exposure over 15 min (0.277 W/m2), either alone or supplemented with NGF (1-10-100 ng/mL). Conditioned media and cell monolayers were collected and analyzed for protein release (ELISA, ELLA microfluidic) and transcript expression (real-time PCR). A specific "inflammatory to remodeling" pattern (IL8, VEGF, IL33, OPN, and CYR61) as well as a few epigenetic transcripts (known as modulator of cell differentiation and matrix-remodeling (DNMT3a, HDAC1, NRF2 and KEAP1)) were investigated in parallel. UV-exposed FBs (i), showed no proliferation or significant cytoskeleton rearrangement; (ii), displayed a trkANGFR/p75NTR phenotype; and (iii), synthesized/released IL8, VEGF-A, IL33, OPN, and CYR61, as compared to unexposed ones. NGF addition counteracted IL8, IL33, OPN, and CYR61 protein release merely at lower NGF concentrations but not VEGF. NGF supplementation did not affect DNMT3a or HDAC1 transcripts, while it significantly upregulated NRF2 at lowest NGF doses and did not change KEAP1 expression. Taken together, a single UV exposure activated conjunctival FBs to release pro-inflammatory/fibrogenic factors in association with epigenetic changes. The effects were selectively counteracted by NGF supplementation in a dose-dependent fashion, most probably accountable to the trkANGFR/p75NTR phenotype. Further in vitro studies are underway to better understand this additional NGF pleiotropic effect. Since UV-shield impairments represent a worldwide alert and UV radiation can slowly affect ocular surface homeostasis (photo-ageing, cataract) or might exacerbate ocular diseases with a preexisting fibrosis (pterygium, VKC), these findings on NGF modulation of UV-exposed FBs might provide additional information for protecting the ocular surface (homeostasis) from low-grade long-lasting UV insults.

The First and Last Time I Met Rita Levi-Montalcini: From the Insect Neurotrophic Factor to the Nerve Growth Factor Clinical Application : RLM and the Discovery of Two Neurotrophic Factors.

The neurotrophic factor nerve growth factor (NGF) has been discovered in the 1950s by Rita Levi-Montalcini, first in a neoplastic tissue and, later, in the mouse salivary gland (see 1A). Levi-Montalcini characterized its action in the sensory and sympathetic neurons (1B) and, a few years later, in central nervous, endocrine, and immune systems. Nerve growth factor plays its trophic role both during development and in adulthood, ensuring the maintenance of phenotypic and functional characteristic of several populations of neurons as well as immune cells. The aim of the present overview is to describe my personal scientific and human experiences working with Rita Levi-Montalcini for over 45 years, first at Washington University in St. Louis, Missouri, USA, searching (1) the invertebrate neurotrophic factor in the cockroaches and, later, at the Institute of Neurobiology of the National Research Council (CNR) in Rome studying (2) the role of NGF for various neuronal and non-neuronal functions; (3) the potential involvement of NGF in the pathobiology of human cutaneous, ocular, neurodegenerative, and cardiometabolic diseases; and finally (4) NGF potential clinical application.

The role of ethanethiol in deaths from acute poisoning by gas mixtures: A suicide case involving a decomposed corpse and a review of the literature.

The most common volatile substances used in suicide are liquefied petroleum gas mixtures, which consist of propane and butane gases mixed in different proportions. These substances are odourless and colourless. Some substances, such as ethanethiol, are added to liquefied petroleum gas mixtures to provide a garlic scent. The main causes of death in acute liquefied petroleum gas inhalation are cardiac arrest and asphyxia, but determining the manner of death is difficult. We present a case of a 30-year-old man found dead at home. On his head was a black plastic bag with a hole through which he had run a gas hose connected to a domestic liquefied petroleum gas cylinder tank. Toxicological analysis revealed butane and ethanethiol in his body. This study aims at understanding the lethal role of ethanethiol through the analysis of its chemical action and its influence on decomposition.

Nerve Growth Factor: Early Studies and Recent Clinical Trials.

Since its discovery, nerve growth factor (NGF) has long occupied a critical role in developmental and adult neurobiology for its many important regulatory functions on the survival, growth and differentiation of nerve cells in the peripheral and central nervous system. NGF is the first discovered member of a family of neurotrophic factors, collectively indicated as neurotrophins, (which include brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin 4/5). NGF was discovered for its action on the survival and differentiation of selected populations of peripheral neurons. Since then, an enormous number of basic and human studies were undertaken to explore the role of purified NGF to prevent the death of NGF-receptive cells. These studies revealed that NGF possesses important therapeutic properties, after topical administration, on human cutaneous pressure ulcer, corneal ulcers, glaucoma, retinal maculopathy, Retinitis Pigmentosa and in pediatric optic gliomas and brain traumas. The aim of this review is to present our previous, recent and ongoing clinical studies on the therapeutic properties of NGF.

NGF protects corneal, retinal, and cutaneous tissues/cells from phototoxic effect of UV exposure.

PURPOSE: Based on evidence that nerve growth factor (NGF) exerts healing action on damaged corneal, retinal, and cutaneous tissues, the present study sought to assess whether topical NGF application can prevent and/or protect epithelial cells from deleterious effects of ultraviolet (UV) radiation. METHODS: Eyes from 40 young-adult Sprague Dawley rats and cutaneous tissues from 36 adult nude mice were exposed to UVA/B lamp for 60 min, either alone or in the presence of murine NGF. Corneal, retinal, and cutaneous tissues were sampled/processed for morphological, immunohistochemical, and biomolecular analysis, and results were compared statistically. RESULTS: UV exposure affected both biochemical and molecular expression of NGF and trkANGFR in corneal, retinal, and cutaneous tissues while UV exposure coupled to NGF treatment enhanced NGF and trkANGFR expression as well as reduced cell death. CONCLUSIONS: Overall, the findings of this in vivo/ex vivo study show the NGF ability to reduce the potential UV damage. Although the mechanism underneath this effect needs further investigation, these observations prospect the development of a pharmacological NGF-based therapy devoted to maintain cell function when exposed to phototoxic UV radiation.

NGF/anti-VEGF combined exposure protects RCS retinal cells and photoreceptors that underwent a local worsening of inflammation.

PURPOSE: Our previous study highlighted the potential nerve growth factor (NGF) effect on damaged photoreceptors from a rat model of spontaneous Retinitis Pigmentosa (RP). Herein, we tested the combined NGF/anti-vascular endothelial growth factor (αVEGF) effect on cultured retinal cells isolated from Royal College of Surgeons (RCS) rats receiving an intravitreal VEGF injection (iv-VEGF) to exacerbate retinal inflammation/neovascularization. METHODS: RCS (n = 75) rats were equally grouped as untreated (n = 25), iv-saline (single saline intravitreal injection; n = 25) and iv-VEGF (single VEGF intravitreal injection; n = 25). Morphological and biochemical analysis or in vitro stimulations with the biomolecular investigation were carried out on explanted retinas. Isolated retinal cells were treated with NGF and αVEGF, either alone or in combination, for 6 days and cells were harvested for morphological and biomolecular analyses. RESULTS: Infiltrating inflammatory cells were detected in iv-VEGF exposed RCS retinas, indicative of exacerbated inflammation and neovascularization. In cell cultures, NGF/αVEGF significantly increased retinal cell survival as well as rhodopsin expression and neurite outgrowth in photoreceptors. Particularly, NGF/αVEGF upregulated Bcl-2 mRNA, downregulated Bax mRNA, upregulated trkANGFR mRNA and finally upregulated both NGF mRNA and protein. CONCLUSIONS: These data confirm and extend our previous findings on NGF-photoreceptor crosstalk, highlighting that the NGF/αVEGF combination might be an interesting approach for improving neuroprotection of RCS retinal cells and likewise photoreceptors in the presence of neovascularization. Further studies are required to translate this in vitro approach into clinical practice.

Nerve growth factor: role in growth, differentiation and controlling cancer cell development.

Recent progress in the Nerve Growth Factor (NGF) research has shown that this factor acts not only outside its classical domain of the peripheral and central nervous system, but also on non-neuronal and cancer cells. This latter observation has led to divergent hypothesis about the role of NGF, its specific distribution pattern within the tissues and its implication in induction as well as progression of carcinogenesis. Moreover, other recent studies have shown that NGF has direct clinical relevance in certain human brain neuron degeneration and a number of human ocular disorders. These studies, by suggesting that NGF is involved in a plethora of physiological function in health and disease, warrant further investigation regarding the true role of NGF in carcinogenesis. Based on our long-lasting experience in the physiopathology of NGF, we aimed to review previous and recent in vivo and in vitro NGF studies on tumor cell induction, progression and arrest. Overall, these studies indicate that the only presence of NGF is unable to generate cell carcinogenesis, both in normal neuronal and non-neuronal cells/tissues. However, it cannot be excluded the possibility that the co-expression of NGF and pro-carcinogenic molecules might open to different consequence. Whether NGF plays a direct or an indirect role in cell proliferation during carcinogenesis remains to demonstrate.

Nerve growth factor and autophagy: effect of nasal anti-NGF-antibodies administration on Ambra1 and Beclin-1 expression in rat brain.

Nerve growth factor (NGF) exerts protective actions in the healthy and diseased nervous system. Intranasal administration is a suitable and safe strategy to deliver NGF to CNS neurons. We investigated whether nasal anti-NGF-antibody (ANA) administration affects neuronal autophagy, in view of its putative regulatory role in this process. We focused on olfactory bulbs (OB), neocortex (Cx), hippocampus (HF) and septal complex (SC), known to be NGF-responsive and autophagically active. Our combined molecular/morphological results demonstrate that intranasally administered ANA reaches brain NGF-target neurons and lowers the levels of endogenous NGF and its receptors. Treatment also affects - in a brain region-dependent manner - the expression of the autophagic proteins Beclin-1 and Ambra1, as well as that of proteins belonging to the Bcl2 family, namely Bax and Bcl-2, reflecting apoptotic dysregulation. This study provides a nongenetically modified, NGF-defective animal model, representing a suitable tool to investigate novel properties of the neurotrophin, especially in relation to autophagy.

NGF and VEGF effects on retinal ganglion cell fate: new evidence from an animal model of diabetes.

PURPOSE: To investigate if the survival effects of nerve growth factor (NGF) eyedrops on retinal ganglion cell (RGCs) are related to vascular endothelial growth factor (VEGF) in a rat model of diabetic retinopathy. METHODS: Diabetes was induced in adult rats by streptozotocin injection and changes in the NGF/TrkA and VEGF retina levels were related to the progression of RGC loss. Diabetic rats were subjected to administration of NGF eyedrops or intraocular injection of anti-NGF antibody. All morphologic, immunohistochemical, and biochemical analyses were performed on whole retinas dissected after 7 or 11 weeks after diabetes induction. RESULTS: Diabetes was successfully induced in rats as shown by glycemic levels >250 mg/dL. The NGF levels increased in diabetic retinas at 7 weeks and decreased at 11 weeks, while VEGF levels increased at all time points. The RGC loss in diabetic retinopathy worsened with anti-NGF administration, which did not alter retina VEGF levels significantly. Administration of NGF eyedrops restored TrkA levels in the retina, and protected RGCs from degeneration without influencing VEGF levels. CONCLUSIONS: The early increase of NGF in diabetic retina might be an endogenous response for protecting RGCs from degeneration. This protective mechanism is impaired at 11 weeks following diabetes induction, and results in a marked RGC degeneration that is improved by exogenous NGF administration and worsened by anti-NGF. The observed NGF-induced neuroprotection on damaged RGCs was not associated with changes in VEGF retina levels, which were constantly high in diabetic rats and were not altered by anti-NGF administration.

Homage to Rita Levi-Montalcini, the queen of modern neuroscience.

The first cell growth factor, nerve growth factor (NGF), was discovered by Rita Levi-Montalcini (RLM) in the early 1950s. Originally identified as neurite outgrowth-stimulating factor, later studies revealed that non-neuronal cells, including immune cells, endothelial cells, cardiomyocytes, pancreatic beta cells, prostate epithelial and adipose tissue cells, were also targets for and/or sources of NGF. Nerve growth factor is well recognised as mediating multiple biological phenomena, ranging from the neurotrophic through immunotrophic and epitheliotrophic to metabotrophic effects. Consequently, NGF and other members of the neurotrophin family are implicated in the pathogenesis of a large spectrum of neuronal and non-neuronal diseases, ranging from Alzheimer's and other neurodegenerative diseases to atherosclerosis and cardiometabolic disorders. Recent studies have demonstrated the therapeutic potentials of NGF in these conditions, including ocular and cutaneous diseases. NGF TrkA receptor antagonists emerged as novel drugs for pain, prostate and breast cancer, melanoma and urinary bladder syndromes. Here, we briefly describe the 'unpredictable' ideogenesis of the discovery of NGF, a eureka in the neuroscience.

Brain cholinergic markers and Tau phosphorylation are altered in experimental type 1 diabetes: normalization by electroacupuncture.

Diabetes often correlates with tau phosphorylation and the development of Alzheimer's disease. Both are associated with brain cholinergic dysfunction that could benefit from nerve growth factor (NGF)-based therapies. Electroacupuncture (EA) improves brain NGF availability and action. Here we assessed the variations of NGF and tau phosphorylation in the cortex and hippocampus, as well as the expression of choline acetyltransferase in the basal forebrain following diabetes induction and EA in adult rats. We found that EA counteracts diabetes-associated tau hyperphosphorylation and decreases in NGF and choline acetyltransferase, suggesting a possible beneficial effect of EA on brain cholinergic system in diabetes.

The multiple life of nerve growth factor: tribute to rita levi-montalcini (1909-2012).

At the end of the 19(th) century, it was envisaged by Santiago Ramon y Cajal, but not, proven, that life at the neuronal level requires trophic support. The proof was obtained in the early 1950's by work initiated by Rita Levi-Montalcini (RLM) discovering the nerve growth factor (NGF). Today, NGF and its relatives, collectively designated neurotrophins, are well recognized as mediators of multiple biological phenomena in health and disease, ranging from the neurotrophic through immunotrophic and epitheliotrophic to metabotrophic effects. Consequently, NGF and other neurotrophins are implicated in the pathogenesis of a large spectrum of neuronal and non-neuronal diseases, from Alzheimer's and other neurodegenerative diseases to atherosclerosis and other cardiometabolic diseases. Recent studies demonstrated the therapeutic potentials of NGF in these diseases, including ocular and cutaneous diseases. Furthermore, NGF TrkA receptor antagonists emerged as novel drugs for pain, prostate and breast cancer, melanoma, and urinary bladder syndromes. Altogether, NGF's multiple potential in health and disease is briefly described here.

Capsaicin-induced corneal sensory denervation and healing impairment are reversed by NGF treatment.

PURPOSE: We aimed to evaluate the nerve growth factor (NGF) pathway and its influence on corneal healing mechanisms in normal conditions and in an animal model of corneal denervation induced by capsaicin. METHODS: Peripheral sensory damage was induced in rat pups by subcutaneous injection of capsaicin and the effects evaluated by hot-plate test, corneal nerve count, and tear secretion. Corneal damage was induced in capsaicin-treated and -untreated rats by epithelial scraping. Healing rate; NGF pathway (NGF, tyrosine kinase A [TrkA], p75); and the stem cell marker p63 were evaluated by RT-PCR, ELISA, Western blot, and immunohistochemistry. The effects of exogenous NGF administration as eye drop formulation were also tested. RESULTS: Capsaicin treatment induced a significant reduction of peripheral sensitivity, corneal innervation, tear secretion, and corneal healing rate. The ocular effects of capsaicin treatment were associated with an NGF pathway alteration. NGF eye drop treatment aided corneal healing mechanisms through a significant increase in the NGF receptors TrkA and p75, and in the stem cell marker p63. CONCLUSIONS: In this study, we show that an alteration in the NGF pathway is responsible for a delay in corneal healing in an animal model of sensory denervation. Moreover, we show that NGF eye drop administration modulates corneal innervation, epithelial cell healing, and corneal stem cells. These findings may trigger further research on the role of the NGF pathway in limbal stem cell deficiency.

Nerve growth factor: from the early discoveries to the potential clinical use.

The physiological role of the neurotrophin nerve growth factor (NGF) has been characterized, since its discovery in the 1950s, first in the sensory and autonomic nervous system, then in central nervous, endocrine and immune systems. NGF plays its trophic role both during development and in adulthood, ensuring the maintenance of phenotypic and functional characteristic of several populations of neurons as well as immune cells. From a translational standpoint, the action of NGF on cholinergic neurons of the basal forebrain and on sensory neurons in dorsal root ganglia first gained researcher's attention, in view of possible clinical use in Alzheimer's disease patients and in peripheral neuropathies respectively. The translational and clinical research on NGF have, since then, enlarged the spectrum of diseases that could benefit from NGF treatment, at the same time highlighting possible limitations in the use of the neurotrophin as a drug. In this review we give a comprehensive account for almost all of the clinical trials attempted until now by using NGF. A perspective on future development for translational research on NGF is also discussed, in view of recent proposals for innovative delivery strategies and/or for additional pathologies to be treated, such as ocular and skin diseases, gliomas, traumatic brain injuries, vascular and immune diseases.

Bevacizumab eye drop treatment stimulates tear secretion in rats through changes in VEGF and NGF lacrimal gland levels.

VEGF and NGF are known to modulate corneal healing, neovascularisation and tear secretion. While a VEGF-NGF cross talk has been recently shown to modulate corneal healing in rats, it is not known whether it also plays a role in the regulation of lacrimal function. In this study we aim to investigate the effects of anti-VEGF eye drop treatment on lacrimal gland function and on the local expression of VEGF and NGF in rats. Tear function was measured in 3 months old rats by modified Schirmer test at baseline and after 3 weeks of topical anti-VEGF eye drop treatment. Whole lacrimal glands from rats were removed after treatment and analysed by ELISA for VEGF and NGF levels. To investigate if the effects of anti-VEGF were mediated by changes in the NGF-pathway, we repeated the experiments in RCS rats, a strain with NGF-pathway impairment associated with decreased tear flow. After topical treatment with anti-VEGF eye drops, an increase in tear secretion was observed in both wild-type and RCS rats. A significant decrease of VEGF levels was also observed in lacrimal glands of both RCS and SD rats, accompanied by a significant increase in NGF levels. Inhibition of VEGF at the ocular surface in rats results in changes of tear function and lacrimal gland levels of VEGF and NGF. Further studies on the VEGF/NGF cross-talk at the ocular surface may expand our knowledge on the pathogenesis of several diseases characterized by tear dysfunction.

Effect of eye NGF administration on two animal models of retinal ganglion cells degeneration.

The aim of this study was to investigate the effect of nerve growth factor (NGF) administration on retinal ganglion cells (RGCs) in experimentally induced glaucoma (GL) and diabetic retinopathy (DR). GL was induced in adult rats by injection of hypertonic saline into the episcleral vein of the eye and diabetes (DT) was induced by administration of streptozoticin. Control and experimental rats were treated daily with either ocular application of NGF or vehicle solution. We found that both animal models present a progressive degeneration of RGCs and changing NGF and VEGF levels in the retina and optic nerve. We then proved that NGF eye drop administration exerts a protective effect on these models of retinal degeneration. In brief, our findings indicate that NGF can play a protective role against RGC degeneration occurring in GL and DR and suggest that ocular NGF administration might be an effective pharmacological approach.

Neuroprotective role of nerve growth factor in hypoxicischemic injury. From brain to skin.

Hypoxic-ischemic injuries (HII) of the brain, optic pathways, and skin are frequently associated with poor neurological and clinical outcome. Unfortunately, no new therapeutic approaches have been proposed for these conditions. Recently, experimental and clinical studies showed that nerve growth factor (NGF) can improve neurological deficits, visual loss and skin damage after HII. Based on these studies, we report the effects of NGF administration in different lesions of the brain, optic pathways and skin. 2.5S NGF purified and lyophilized from male mouse submaxillary glands was utilized for the treatment. NGF administration was started in absence of recovery after conventional and standardized treatment. One mg NGF was administered via the external catheter into the brain, by drop administration in the eye, and by subcutaneous administration in the skin. We treated 4 patients: 2 children with hypoxic-ischemic brain damage, an adult patient with an optic glioma-induced visual loss and a child with a severe crush syndrome of the lower left limb. After NGF treatment, we observed an amelioration of both neurological and electrophysiological function of the brain, a subjective and objective improvement of visual function, and a gradual improvement of ischemic skin lesion. No side effects were related to NGF treatment in all patients studied. Our observation shows that NGF administration may be an effective and safe adjunct therapy in patients with severe HII. The beneficial and prolonged effect on nerve function suggests a neuroprotective mechanism exerted by NGF on the residual viable neurological pathways of these patients.

Ocular application of nerve growth factor protects degenerating retinal ganglion cells in a rat model of glaucoma.

PURPOSE: Elevated intraocular pressure is a crucial pathologic event for the development of glaucoma (GL). We have reported that nerve growth factor (NGF) reaches retinal cells and the optic nerve (ON) when applied to the eye. Whether ocular application of NGF prevents or reduces damage to retinal ganglion cell (RGC) is not known. METHODS: GL was induced in adult rats by the injection of hypertonic saline into the episcleral vein of the right eye and the left eye used as control. Rats were then treated daily with ocular application of NGF or vehicle solution for 7 weeks. Retinal and ON tissues were then used for structural, immunohistochemical, and biochemical studies. RESULTS: The injection of hypertonic saline into the episcleral vein led to progressive degeneration of RGCs, with the loss of nearly 40% of these cells after 7 weeks of treatment. This cellular loss is associated with the downregulation of NGF and NGF-receptor expression in the retina and ON of the glaucomatous eye and ocular treatment with NGF significantly reduced the deficit induced by GL. CONCLUSIONS: These findings indicate that NGF can exert protective action on RGC degeneration occurring in glaucomatous retina. We suggest that ocular NGF treatment might be a suitable pharmacologic approach to investigate protective mechanisms of degenerating RGCs.

NGF and NGF-receptor expression of cultured immortalized human corneal endothelial cells.

PURPOSE: Several growth factors, including nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), play an important role in the homeostasis of the ocular surface. The involvement of both these growth factors in the pathophysiology of intraocular tissues has been extensively investigated. Despite the expression of NGF receptors by corneal endothelium, to date the role of NGF on the endothelial cell remains to be determined. Using a clonal cell line of human corneal endothelial cells, the aim of this study was to investigate the expression of the NGF-receptor and the potential partnership of NGF and VEGF in maintaining cell viability in vitro. METHODS: A human endothelial cell line (B4G12), was cultured under serum-free conditions as previously described with and without addition of different concentrations of NGF, anti-NGF-antibody (ANA), or VEGF for 4 days and these cells were used for immuno-istochemical, biochemical, and molecular analyses. RESULTS: NGF induces overexpression of NGF-receptors and synthesis and release of VEGF by endothelial cells and these cells are able to produce and secrete NGF. CONCLUSIONS: These observations indicate that human corneal endothelial cells are receptive to the action of NGF and that these cells may regulate NGF activity through autocrine/paracrine mechanisms.

Nerve growth factor in the developing and adult lacrimal glands of rat with and without inherited retinitis pigmentosa.

PURPOSE: In the present study, we investigated lacrimal function and presence of the neurotrophin nerve growth factor (NGF) and its receptors in the lacrimal gland (LG) of normal rats and rats with inherited retinitis pigmentosa (IRP). MATERIALS AND METHODS: After anesthesia, modified Schirmer tests were performed on IRP rats and Sprague Dawley (SD) rats to measure tear function. LGs of developing and adult IRP and SD rats were removed and used for histological, immunohistochemical, and biochemical analyses. RESULTS: The results showed that basal tear secretion is reduced in IRP rats as compared with SD rats. NGF and NGF receptors are expressed in the LG of both rat strains. In SD rats, these NGF markers are low during early life and more elevated in adult life. Conversely in rats with IRP, NGF and its receptors decreased in adult life. CONCLUSIONS: The role of NGF in maintaining ocular surface integrity is well known. The observations of this study further support the hypothesis that neurotrophins play a role in modulating tear secretion and probably in preventing the deleterious effects of dry eye. This hypothesis is presented and discussed.