Administration Of Nerve Growth Factor Research Articles

Enhanced neuroprotective activity of ophthalmic delivered nerve growth factor conjugated with cell penetrating peptide against optic nerve injury

Nerve growth factor is a well characterized neurotrophic factor that play a critical role in the survival, growth, and differentiation of neurons both in central and peripheral nervous system. However, it is difficult for the conventional exogenous nerve growth factor administration delivery to the central nervous system due to the biological barrier in human bodies. Here, we validated a series of cell penetrating peptides and found that L-PenetraMax significantly enhanced the efficiency of recombinant human nerve growth factor entry into the rat retina. In the optic nerve crush mice model, eye drop administration of recombinant human nerve growth factor alone promoted retinal ganglion cell survival and axon regeneration at high dose, while the combination of recombinant human nerve growth factor with L-PenetraMax significantly enhanced the neuroprotective efficacy at lower dose, thus potentially enhancing the availability of recombinant human nerve growth factor eye drops in patients with optic neuropathy. This study provides the evidence that the noncovalent coadministration of recombinant human nerve growth factor with L-PenetraMax could be a potent strategy for the non-invasive and sustained ocular delivery of therapeutic proteins for improving the optic nerve injury.

Topical Nerve Growth Factor (NGF) restores electrophysiological alterations in the Ins2Akita mouse model of diabetic retinopathy

People suffering from diabetes mellitus commonly have to face diabetic retinopathy (DR), an eye disease characterized by early retinal neurodegeneration and microvascular damage, progressively leading to sight loss. The Ins2Akita (Akita) diabetic mouse presents the characteristics of DR and experimental drugs can be tested on this model to check their efficacy before going to the clinic. Topical administration of Nerve Growth Factor (NGF) has been recently demonstrated to prevent DR in the Akita mouse, reverting the thinning of retinal layers and protecting the retinal ganglion cells (RGCs) from death. In this study, we characterize the effects of topical NGF on neuroretina function, quantified with the electroretinogram (ERG). In particular, we show that NGF can ameliorate RGC conduction in the retina of Akita mice, which correlates with a recovery of retinal nerve fiber plus ganglion cell layer (RNFL-GCL) structure. Overall, our preclinical results highlight that topical administration of NGF could be a promising therapeutic approach for DR, being capable of exerting a beneficial impact on retinal functionality.

Neurofibromatosis type 1-associated optic pathway gliomas: pathogenesis and emerging treatments.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder associated with an increased risk of developing a variety of benign and malignant tumors. Fifteen to 20% of children with NF1 are diagnosed with an optic pathway glioma (NF1-OPG) before 7 years of age, and more than half of them experience visual decline. At present, no effective therapy is available for prevention, restoration, or even stabilization of vision loss in subjects affected by NF1-OPG. This paper aims to review the main emerging pharmacological approaches that have been recently assessed in preclinical and clinical settings. We performed a search of the literature using Embase, PubMed, and Scopus databases to identify articles regarding NF1-OPGs and their treatment up to July 1st, 2022. The reference lists of the analyzed articles were also considered a source of literature information. To search and analyze all relevant English articles, the following keywords were used in various combinations: neurofibromatosis type 1, optic pathway glioma, chemotherapy, precision medicine, MEK inhibitors, VEGF, nerve growth factor. Over the past decade, basic research and the development of genetically engineered mice models of NF1-associated OPG have shed light on the cellular and molecular mechanisms underlying the disease and inspired animal and human testing of several compounds. A promising line of research is focusing on the inhibition of mTOR, a protein kinase controlling proliferation, protein synthesis rate and cell motility that is highly expressed in neoplastic cells. Several mTOR blockers have been tested in clinical trials, the most recent of which employed oral everolimus with encouraging results. A different strategy aims at restoring cAMP levels in neoplastic astrocytes and non-neoplastic neurons, since reduced intracellular cAMP levels contribute to OPG growth and, more importantly, are the major determinant of NF1-OPG-associated visual decline. So far, however, this approach has only been attempted in preclinical studies. Stroma-directed molecular therapies - seeking to target Nf1 heterozygous brain microglia and retinal ganglion cells (RGCs) - are another fascinating field. Microglia-inhibiting strategies have not yet reached clinical trials, but preclinical studies conducted over the last 15 years have provided convincing clues of their potential. The importance of NF1-mutant RGCs in the formation and progression of OPGs also holds promise for clinical translation. The evidence of Vascular Endothelial Growth Factor (VEGF)- Vascular Endothelial Growth Factor (VEGFR) signaling hyperactivity in pediatric low-grade gliomas prompted the use of bevacizumab, an anti-VEGF monoclonal antibody, which was tested in children with low-grade gliomas or OPGs with good clinical results. Neuroprotective agents have also been proposed to preserve and restore RGCs and topical eye administration of nerve growth factor (NGF) has demonstrated encouraging electrophysiological and clinical results in a double-blind, placebo-controlled study. Traditional chemotherapy in patients with NF1-OPGs does not significantly ameliorate visual function, and its effectiveness in halting tumor growth cannot be considered a satisfactory result. Newer lines of research should be pursued with the goal of stabilizing or improving the vision, rather than reducing tumor volume. The growing understanding of the unique cellular and molecular characteristics of NF1-OPG, coupled with the recent publication of promising clinical studies, raise hope for a shift towards precision medicine and targeted therapies as a first-line treatment.

Combined treatment of nerve growth factor and transcranical direct current stimulations to improve outcome in children with vegetative state after out-of-hospital cardiac arrest

BackgroundOut-of-hospital cardiac arrest (OHCA) is one of the most dramatic events in pediatric age and, despite advanced neurointensive care, the survival rate remains low. Currently, no effective treatments can restore neuronal loss or produce significant improvement in these patients. Nerve Growth Factor (NGF) is a neurotrophin potentially able to counteract many of the deleterious effects triggered by OHCA. Transcranial Direct Current Stimulation (tDCS) has been reported to be neuroprotective in many neurological diseases, such as motor deficit and cognitive impairment. Children with the diagnosis of chronic vegetative state after OHCA were enrolled. These patients underwent a combined treatment of intranasal administration of human recombinant NGF (hr-NGF), at a total dose of 50 gamma/kg, and tDCS, in which current intensity was increased from zero to 2 mA from the first 5 s of stimulation and maintained constant for 20 min. The treatment schedule was performed twice, at one month distance each. Neuroradiogical evaluation with Positron Emission Tomography scan (PET), Single Photon Emission Computed Tomography (SPECT), Electroencephalography (EEG) and Power Spectral Density of the brain (PSD) was determined before the treatment and one month after the end. Neurological assessment was deepened by using modified Ashworth Scale, Gross Motor Function Measure, and Disability Rating Scale.ResultsThree children with a chronic vegetative state secondary to OHCA were treated. The combined treatment with hr-NGF and tDCS improved functional (PET and SPECT) and electrophysiological (EEG and PSD) assessment. Also clinical conditions improved, mainly for the reduction of spasticity and with the acquisition of voluntary finger movements, improved facial mimicry and reaction to painful stimuli. No side effects were reported.ConclusionsThese promising preliminary results and the ease of administration of this treatment make it worthwhile to be investigated further, mainly in the early stages from OHCA and in patients with better baseline neurological conditions, in order to explore more thoroughly the benefits of this new approach on neuronal function recovery after OHCA.

Reduced levels of NGF shift astrocytes toward a neurotoxic phenotype.

Nerve growth factor (NGF) is critical for neuronal physiology during development and adulthood. Despite the well-recognized effect of NGF on neurons, less is known about whether NGF can actually affect other cell types in the central nervous system (CNS). In this work, we show that astrocytes are susceptible to changes in ambient levels of NGF. First, we observe that interfering with NGF signaling in vivo via the constitutive expression of an antiNGF antibody induces astrocytic atrophy. A similar asthenic phenotype is encountered in an uncleavable proNGF transgenic mouse model (TgproNGF#72), effectively increasing the brain proNGF levels. To examine whether this effect on astrocytes is cell-autonomous, we cultured wild-type primary astrocytes in the presence of antiNGF antibodies, uncovering that a short incubation period is sufficient to potently and rapidly trigger calcium oscillations. Acute induction of calcium oscillations by antiNGF antibodies is followed by progressive morphological changes similar to those observed in antiNGF AD11 mice. Conversely, incubation with mature NGF has no effect on either calcium activity nor on astrocytic morphology. At longer timescales, transcriptomic analysis revealed that NGF-deprived astrocytes acquire a proinflammatory profile. In particular, antiNGF-treated astrocytes show upregulation of neurotoxic transcripts and downregulation of neuroprotective mRNAs. Consistent with that data, culturing wild-type neurons in the presence of NGF-deprived astrocytes leads to neuronal cell death. Finally, we report that in both awake and anesthetized mice, astrocytes in layer I of the motor cortex respond with an increase in calcium activity to acute NGF inhibition using either NGF-neutralizing antibodies or a TrkA-Fc NGF scavenger. Moreover, in vivo calcium imaging in the cortex of the 5xFAD neurodegeneration mouse model shows an increased level of spontaneous calcium activity in astrocytes, which is significantly reduced after acute administration of NGF. In conclusion, we unveil a novel neurotoxic mechanism driven by astrocytes, triggered by their sensing and reacting to changes in the levels of ambient NGF.

Is seminal nerve growth factor-induced luteinizing hormone release in camelids mediated at the hypothalamus?

Seminal nerve growth factor induces ovulation in camelids by influencing the secretion of gonadotrophin-releasing hormone (GnRH) into the portal vessels of the pituitary gland. We show that the nerve growth factor-induced release of GnRH is not mediated directly through interaction with hypothalamic neurons. Ovulation in camelids is triggered by seminal nerve growth factor (NGF). The mechanism of action of NGF appears to occur via the central nervous system. In this study, we tested the hypothesis that NGF acts in the hypothalamus to induce GnRH release. To determine if NGF-induced ovulation is associated with a rise in NGF concentrations in the cerebrospinal fluid (CSF), llamas were i) mated with an urethrostomized male, ii) mated with intact male, or given intrauterine iii) seminal plasma or i.v.) saline (Experiment 1). To characterize the luteinizing hormone (LH) response after central vs peripheral administration, llamas were treated with saline (negative control) or NGF either by i.v. or intracerebroventricular (ICV) administration (Experiment 2). To determine the role of kisspeptin, the effect of ICV infusion of a kisspeptin receptor antagonist on NGF-induced LH secretion and ovulation was tested in llamas (Experiment 3). In Experiment 1, a surge in circulating concentrations of LH was detected only in llamas mated with an intact male and those given intrauterine seminal plasma, but no changes in CSF concentrations of NGF were detected. In Experiment 2, peripheral administration (i.v.) of NGF induced an LH surge and ovulation, whereas no response was detected after central (ICV) administration. In Experiment 3, the kisspeptin receptor antagonist had no effect on the LH response to NGF. In conclusion, results did not support the hypothesis that NGF-induced ovulation is mediated via a trans-synaptic pathway within the hypothalamus, but rather through a releasing effect on tanycytes at the median eminence.

NGF-Induced Upregulation of CGRP in Orofacial Pain Induced by Tooth Movement Is Dependent on Atp6v0a1 and Vesicle Release.

The nerve growth factor (NGF) and calcitonin gene-related peptide (CGRP) play a crucial role in the regulation of orofacial pain. It has been demonstrated that CGRP increases orofacial pain induced by NGF. V-type proton ATPase subunit an isoform 1 (Atp6v0a1) is involved in the exocytosis pathway, especially in vesicular transport in neurons. The objective was to examine the role of Atp6v0a1 in NGF-induced upregulation of CGRP in orofacial pain induced by experimental tooth movement. Orofacial pain was elicited by ligating closed-coil springs between incisors and molars in Sprague–Dawley rats. Gene and protein expression levels were determined through real-time polymerase chain reaction, immunostaining, and fluorescence in situ hybridization. Lentivirus vectors carrying Atp6v0a1 shRNA were used to knockdown the expression of Atp6v0a1 in TG and SH-SY5Y neurons. The release of vesicles in SH-SY5Y neurons was observed by using fluorescence dye FM1-43, and the release of CGRP was detected by Enzyme-Linked Immunosorbent Assy. Orofacial pain was evaluated through the rat grimace scale. Our results revealed that intraganglionic administration of NGF and Atp6v0a1 shRNA upregulated and downregulated CGRP in trigeminal ganglia (TG) and trigeminal subnucleus caudalis (Vc), respectively, and the orofacial pain was also exacerbated and alleviated, respectively, following administration of NGF and Atp6v0a1 shRNA. Besides, intraganglionic administration of NGF simultaneously caused the downregulation of Atp6v0a1 in TG. Moreover, the release of vesicles and CGRP in SH-SY5Y neurons was interfered by NGF and Atp6v0a1 shRNA. In conclusion, in the orofacial pain induced by experimental tooth movement, NGF induced the upregulation of CGRP in TG and Vc, and this process is dependent on Atp6v0a1 and vesicle release, suggesting that they are involved in the transmission of nociceptive information in orofacial pain.

Topical nerve growth factor prevents neurodegenerative and vascular stages of diabetic retinopathy.

Specific and effective preventive treatment for diabetic retinopathy (DR) is presently unavailable, mostly because the early stages of the complication have been, until recently, poorly understood. The recent demonstration that the vascular phase of DR is preceded and possibly caused by the neurodegeneration of retinal ganglion cells suggests that DR could, at least theoretically, be prevented through an early neuroprotective approach. The aims of our study were to clarify the natural history of diabetes-driven retinal neurodegeneration and to verify the possibility to prevent DR using topical nerve growth factor (NGF). The results of the study show that retinal neurodegeneration, characterized by the loss of retinal ganglion cells represents a relatively early phenomenon of diabetes (between 5 and 16 weeks of age), which tends to be self-limiting in the long run. Neurodegeneration is followed by the development of DR-related vascular dysfunctions, as confirmed by the development of acellular capillaries and the loss of retinal pericytes. Both retinal neurodegeneration and subsequent vascular dysfunction can be successfully prevented by topical NGF administration. These findings suggest that: 1) The first stage of DR consists in a self-limiting retinal neurodegeneration 2) The demonstrated effectiveness of topical NGF in the prevention of DR could be rapidly translated into clinical practice.

Astrocyte Reactivity in Female Rats with Persistent Low Back Pain Following Spinal Mobilization

Low back pain (LBP) is a physiologically complex and highly disabling condition with poorly understood pathophysiology. Glial reactivity has been reported in patients with chronic LBP and astrocyte reactivity has been suggested as a potential contributor to LBP chronicity.1 Spinal mobilization (SM) is a non‐pharmacological approach with mild to moderate efficacy in treating LBP but physiological mechanisms responsible are unknown.PurposeTo investigate changes in astrocyte reactivity in the upper lumbar spinal cord following SM treatment in a Nerve‐Growth Factor (NGF)‐induced LBP pre‐clinical model. We hypothesize that the administration of trunk NGF will increase astrocyte reactivity, while SM treatment will decrease this reactivity in the upper lumbar spinal cord of female rats.MethodsFemale Sprague Dawley rats (212‐252g) were unilaterally injected with either NGF (50μl;2.0μM) or phosphate buffer saline ‐ PBS (50μl; control) on Days 0 and 5 in the left multifidus muscle under isoflurane anesthesia (1‐2%/2L O2). Ten days lapsed allowing for muscular LBP to develop in NGF animals. Treatment groups (NGF + SM [N=5]; PBS + SM [N=4]) received daily passive SM (10 min) via a feedback motor (1.2Hz, 0.9N) from Days 10 to 16, while the NGF only (N=5) group received no SM treatment. On Day 17, lumbar spinal cord (L1–L3) segments were harvested, fixated, and sectioned at 14μm. Sections were blocked and permeabilized (0.1% Triton X‐100 in 5% donkey serum) for 20 min at room temperature (RT), rinsed in PBS (6 x 5 min), and incubated with primary (Polyclonal Rabbit Anti‐Glial Fibrillary Acidic Protein ‐ 3h at RT) and secondary antibodies (IgG Highly Cross‐Adsorbed Donkey anti‐Rabbit, Alexa Fluor® 488 ‐ 1h at RT). Sections were then mounted on Fluorogel II containing 4′,6‐diamidino‐2‐phenylindole. Images of the superficial dorsal‐horn (laminas I‐III) and deep dorsal‐horn (lamina V) were captured at 20x magnification (Nikon Eclipse). Astrocyte mean fluorescent intensity (MFI) was quantified using FIJI/ImageJ® by blinded lab personnel. Analysis of variance was used to assess the differences in MFI between groups on the left and right sides of the spinal cord dorsal‐horn.ResultsNo significant differences between groups at the superficial horn level was found (p>0.05). Astrocyte MFI was statistically significantly higher in the NGF‐only group when compared to the PBS +SM group at the L3 spinal cord deep‐horn level (p=0.03). SM did not appear to decrease astrocyte reactivity at superficial or deep‐horn spinal cord levels.ConclusionOverall, these preliminary findings suggest that superficial‐horn astrocyte reactivity does not play a large mechanistic role in this female NGF‐induced LBP model despite astrocytic hyperreactivity being demonstrated among male rats with NGF‐induced LBP. Biological sex differences in glial signaling have been reported in other pre‐clinical pain models which may provide an explanation for the non‐significant NGF findings among females. Whether SM acts to decrease astrocyte reactivity in male NGF‐induced LBP rats remains to be investigated.

The Effect of Interaction NGF/p75NTR in Sperm Cells: A Rabbit Model.

Background: Nerve Growth Factor (NGF) plays an important role in the reproductive system through its receptor’s interaction (p75NTR). This paper aims to analyze the impact of NGF p75NTR in epididymal and ejaculated rabbit semen during in vitro sperm storage. Methods: Semen samples from 10 adult rabbit bucks were collected four times (n = 40) and analyzed. NGF was quantified in seminal plasma, and the basal expression of p75NTR in sperm was established (time 0). Moreover, we evaluated p75NTR, the apoptotic rates, and the main sperm parameters, at times 2–4 and 6 h with or without the administration of exogenous NGF. Results: Based on the level of p75NTR, we defined the threshold value (25.6%), and sperm were divided into High (H) and Normal (N). During sperm storage, p75NTR of H samples significantly modulated some relevant sperm parameters. Specifically, comparing H samples with N ones, we observed a reduction in motility and non-capacitated cell number, together with an increased percentage of dead and apoptotic cells. Notably, the N group showed a reduction in dead and apoptotic cells after NGF treatment. Conversely, the NGF administration on H sperm did not change either the percentage of dead cells or the apoptotic rate. Conclusion: The concentration of p75NTR on ejaculated sperm modulates many semen outcomes (motility, apoptosis, viability) through NGF interaction affecting the senescence of sperm.

Tear proteomics reveals the molecular basis of the efficacy of human recombinant nerve growth factor treatment for Neurotrophic Keratopathy

Neurotrophic Keratopathy (NK), classified as an orphan disease (ORPHA137596), is a rare degenerative corneal disease characterized by epithelial instability and decreased corneal sensitivity caused by the damage to the corneal nerves. The administration of human recombinant nerve growth factor (rhNGF) eye drops, as a licensed-in-Europe specific medication for treatment of moderate and severe NK, has added promising perspectives to the management of this disorder by providing a valid alternative to the neurotization surgery. However, few studies have been conducted to the molecular mechanism underlying the response to the treatment. Here, we carried out tears proteomics to highlight the protein expression during pharmacological treatment of NK (Data are available via ProteomeXchange with identifier PXD025408).Our data emphasized a proteome modulation during rhNGF treatment related to an increase in DNA synthesis, an activation of both BDNF signal and IL6 receptor. Furthermore, the amount of neuronal Extracellular Vesicles EVs (CD171+) correlated with the EVs carrying IL6R (CD126+) together associated to the inflammatory EVs (CD45+) in tears. Such scenario determined drug response, confirmed by an in vivo confocal microscopy analysis, showing an increase in length, density and number of nerve fiber branches during treatment. In summary, rhNGF treatment seems to determine an inflammatory micro-environment, mediated by functionalized EVs, defining the drug response by stimulating protein synthesis and fiber regeneration.

Cellular level of coenzyme Q increases with neuronal differentiation, playing an important role in neural elongations.

Deficiency of coenzyme Q has been reported in various neuro­logical diseases, and the behavior of this lipid in neurons has attracted attention. However, the behavior of this lipid in normal neurons remains unclear. In this study, we analyzed the concen­tration of coenzyme Q before and after neuronal differentiation. Nerve growth factor treatment of PC12 cells caused neurite outgrowth and neuronal differentiation, and the amount of intra­cellular coenzyme Q increased dramatically during this process. In addition, when the serum was removed from the culture medium of N1E-115 cells and the neurite outgrowth was confirmed, the intracellular coenzyme Q level also increased. To elucidate the role of the increased coenzyme Q, we administered nerve growth factor to PC12 cells with coenzyme Q synthesis inhibitors and found that coenzyme Q levels decreased, neurite outgrowth was impaired, and differentiation markers were reduced. These results indicate that coenzyme Q levels increase during neuronal differentiation and that this increase is important for neurite outgrowth.

Ultrasound delivery of a TrkA agonist confers neuroprotection to Alzheimer-associated pathologies.

Early degeneration of basal forebrain cholinergic neurons contributes substantially to cognitive decline in Alzheimer’s disease. Evidence from preclinical models of neuronal injury and aging support a pivotal role for nerve growth factor (NGF) in neuroprotection, resilience, and cognitive function. However, whether NGF can provide therapeutic benefit in the presence of Alzheimer’s disease-related pathologies still unresolved. Perturbations in the NGF signalling system in Alzheimer’s disease may render neurons unable to benefit from NGF administration. Additionally, challenges related to brain delivery remain for clinical translation of NGF-based therapies in Alzheimer’s disease. To be safe and efficient, NGF-related agents should stimulate the NGF receptor, tropomyosin receptor kinase A (TrkA), avoid activation through the p75 neurotrophin receptor (p75NTR), and be delivered non-invasively to targeted brain areas using real-time monitoring. We addressed these limitations using MRI-guided focused ultrasound (MRIgFUS) to increase blood–brain barrier permeability locally and transiently, allowing an intravenously administered TrkA agonist that does not activate p75NTR, termed D3, to enter targeted brain areas. Here, we report the therapeutic potential of selective TrkA activation in a transgenic mouse model that recapitulates numerous Alzheimer’s disease-associated pathologies. Repeated MRIgFUS-mediated delivery of D3 (D3/FUS) improved cognitive function in the TgCRND8 model of Alzheimer’s disease. Mechanistically, D3/FUS treatment effectively attenuated cholinergic degeneration and promoted functional recovery. D3/FUS treatment also resulted in widespread reduction of brain amyloid pathology and dystrophic neurites surrounding amyloid plaques. Furthermore, D3/FUS markedly enhanced hippocampal neurogenesis in TgCRND8 mice, implicating TrkA agonism as a novel therapeutic target to promote neurogenesis in the context of Alzheimer’s disease-related pathology. Thus, this study provides evidence that selective TrkA agonism confers neuroprotection to effectively counteract Alzheimer’s disease-related vulnerability. Recent clinical trials demonstrate that non-invasive blood–brain barrier modulation using MRIgFUS is safe, feasible and reversible in Alzheimer’s disease patients. TrkA receptor agonists coupled with MRIgFUS delivery constitute a promising disease-modifying strategy to foster brain health and counteract cognitive decline in Alzheimer’s disease.

Microinjection of pruritogens in NGF-sensitized human skin

Single intradermal injections of nerve growth factor (NGF) evoke prolonged but temporally distinct sensitization patterns to somatosensory stimuli. Focal administration of the non-histaminergic pruritogen cowhage but not histamine resulted in elevated itch at day 21 after NGF administration. Here, we injected bovine adrenal medulla peptide 8–22 (BAM8–22), β-alanine (β-ALA) and endothelin-1 (ET-1) into NGF-treated skin of 11 healthy volunteers and investigated the corresponding itch/pain and flare reactions. β-ALA was the weakest pruritogen, while BAM8–22 and ET-1 were equally potent as histamine. NGF did not sensitize itch or flare reactions induced by any compound, but injection and evoked pain were increased at day 21 and 49. The involvement of histamine H1 receptors in itch was explored in eight subjects after oral cetirizine. ET-1-induced itch and flare were significantly reduced. BAM8–22 and β-ALA itch were not affected, but flare responses after BAM8–22 reduced by 50%. The results indicate that a single NGF injection does not sensitize for experimentally induced itch but increases pain upon pruritogen injection. In healthy humans, pruritic and algetic processing appear differentially regulated by NGF. However, in patients suffering chronic itch, prolonged elevation of NGF-levels under inflammatory conditions may contribute to elevated itch.

Nerve Growth Factor-Based Therapy in Alzheimer's Disease and Age-Related Macular Degeneration.

Alzheimer’s disease (AD) is an age-associated neurodegenerative disease which is the most common cause of dementia among the elderly. Imbalance in nerve growth factor (NGF) signaling, metabolism, and/or defect in NGF transport to the basal forebrain cholinergic neurons occurs in patients affected with AD. According to the cholinergic hypothesis, an early and progressive synaptic and neuronal loss in a vulnerable population of basal forebrain involved in memory and learning processes leads to degeneration of cortical and hippocampal projections followed by cognitive impairment with accumulation of misfolded/aggregated Aβ and tau protein. The neuroprotective and regenerative effects of NGF on cholinergic neurons have been largely demonstrated, both in animal models of AD and in living patients. However, the development of this neurotrophin as a disease-modifying therapy in humans is challenged by both delivery limitations (inability to cross the blood–brain barrier (BBB), poor pharmacokinetic profile) and unwanted side effects (pain and weight loss). Age-related macular degeneration (AMD) is a retinal disease which represents the major cause of blindness in developed countries and shares several clinical and pathological features with AD, including alterations in NGF transduction pathways. Interestingly, nerve fiber layer thinning, degeneration of retinal ganglion cells and changes of vascular parameters, aggregation of Aβ and tau protein, and apoptosis also occur in the retina of both AD and AMD. A protective effect of ocular administration of NGF on both photoreceptor and retinal ganglion cell degeneration has been recently described. Besides, the current knowledge about the detection of essential trace metals associated with AD and AMD and their changes depending on the severity of diseases, either systemic or locally detected, further pave the way for a promising diagnostic approach. This review is aimed at describing the employment of NGF as a common therapeutic approach to AMD and AD and the diagnostic power of detection of essential trace metals associated with both diseases. The multiple approaches employed to allow a sustained release/targeting of NGF to the brain and its neurosensorial ocular extensions will be also discussed, highlighting innovative technologies and future translational prospects.

Nerve growth factor and glutamate increase the density and expression of substance P-containing nerve fibers in healthy human masseter muscles

Nocifensive behavior induced by injection of glutamate or nerve growth factor (NGF) into rats masseter muscle is mediated, in part, through the activation of peripheral NMDA receptors. However, information is lacking about the mechanism that contributes to pain and sensitization induced by these substances in humans. Immunohistochemical analysis of microbiopsies obtained from human masseter muscle was used to investigate if injection of glutamate into the NGF-sensitized masseter muscle alters the density or expression of the NMDA receptor subtype 2B (NR2B) or NGF by putative sensory afferent (that express SP) fibers. The relationship between expression and pain characteristics was also examined. NGF and glutamate administration increased the density and expression of NR2B and NGF by muscle putative sensory afferent fibers (P < 0.050). This increase in expression was greater in women than in men (P < 0.050). Expression of NR2B receptors by putative sensory afferent fibers was positively correlated with pain characteristics. Results suggest that increased expression of peripheral NMDA receptors partly contributes to the increased pain and sensitivity induced by intramuscular injection of NGF and glutamate in healthy humans; a model of myofascial temporomandibular disorder (TMD) pain. Whether a similar increase in peripheral NMDA expression occurs in patients with painful TMDs warrants further investigation.

Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3

Orthodontic tooth movement elicits alveolar bone remodeling and orofacial pain that is manifested by tooth mechanical hyperalgesia. Nerve growth factor (NGF) is upregulated in periodontium and may modulate tooth mechanical hyperalgesia. The objectives were to examine the role of NGF in tooth mechanical hyperalgesia and to elucidate the underlying mechanisms. Tooth mechanical hyperalgesia was induced by ligating closed coil springs between incisors and molars in Sprague–Dawley rats. Retrograde labeling was performed by periodontal administration of fluor-conjugated NGF and the detection of fluorescence in trigeminal ganglia (TG). Lentivirus vectors carrying NGF shRNA were employed to knockdown the expression of NGF in TG. The administration of agonists, antagonists, and virus vectors into TG and periodontium was conducted. Tooth mechanical hyperalgesia was examined through the threshold of biting withdrawal. Our results revealed that tooth movement elicited tooth mechanical hyperalgesia that could be alleviated by NGF neutralizing antibody and that NGF was upregulated in periodontium (mainly in periodontal fibroblasts) and TG. Retrograde labeling revealed that periodontal NGF was retrogradely transported to TG after day 1. Acid-sensing ion channel 3 (ASIC3) and NGF were co-expressed in trigeminal neurons and the percentage of co-expression was significantly higher following tooth movement. The administration of NGF and NGF neutralizing antibody into TG could upregulate and downregulate the expression of ASIC3 in TG, respectively. NGF aggravated tooth mechanical hyperalgesia that could be alleviated by ASIC3 antagonist (APETx2). Moreover, NGF neutralizing antibody mitigated tooth mechanical hyperalgesia that could be recapitulated by ASIC3 agonist (GMQ). NGF-based gene therapy abolished tooth mechanical hyperalgesia and downregulated ASIC3 expression. Taken together, in response to force stimuli, periodontal fibroblasts upregulated the expressions of NGF that was retrogradely transported to TG, where NGF elicited tooth mechanical hyperalgesia through upregulating ASIC3. NGF-based gene therapy is a viable method in alleviating tooth-movement-induced mechanical hyperalgesia.

Nerve Growth Factor Upregulates IL‐4 And IL‐10 in an Animal Model of Persistent Low Back Pain

Low back pain (LBP) is considered the global leading cause of years lived with disability affecting more than half a billion people. Despite the large direct and indirect impact of LBP on society, the underlying neurobiological mechanisms behind LBP are still poorly understood. Nerve Growth Factor (NGF) has been proposed to contribute to the pathophysiology of persistent pain through several pathways, including the indirect regulation of inflammatory cytokines. We hypothesize that the administration of NGF will increase pro- and decrease anti-inflammatory cytokine levels in an animal model of persistent LBP. Purpose To investigate the pro- and anti-inflammatory cytokine changes induced by nerve growth factor (NGF) intramuscular injections into the lumbar paraspinal muscles in a pre-clinical model of persistent LBP. Methods Sprague Dawley rats (n=12; 180-238g; 7 Males, 5 Females) were unilaterally injected with either NGF (50μl; 0.8μM) or phosphate buffer saline - PBS (50μl; control) on days 0 and 5 in the left multifidus muscle under isoflurane anesthesia (1-2%/2L O2). On day 12, animals (n=6/group) were anesthetized and blood collected by cardiac puncture. After coagulation (30 min), blood samples were centrifuged at 3000 rpm for 10 min, blood serum collected and frozen at −80°C until cytokine assays were performed. V-PLEX Proinflammatory Panel 2 Rat Kit (Meso Scale Discovery®) multiplex analysis was conducted to characterize the concentration of cytokines (IFN-γ, IL-10, IL-13, IL-4, IL-5, KC/GRO, TNFα, IL-1β, and IL-6) in the collected blood. Wilcoxon Rank Sum test was used to assess the differences in cytokine concentration between the NGF and PBS groups. Only values above the minimum test sensitivity for each cytokine were considered for the statistical analysis. Results IL-4 and IL-10 concentration levels were statistically significantly higher in NGF-injected animals when compared to the control group (p=0.0305; Table 1). No other significant differences between groups were found (p > 0.05; Table 1). Conclusion Our preliminary findings suggest an upregulation of anti-inflammatory cytokines identified at later stages of persistent LBP as an effect of NGF intramuscular injections. Future studies should consider investigating the early effects of NGF on cytokine regulation in persistent models of LBP to better understand the neurobiology underlying this condition.

The TrkA agonist gambogic amide augments skeletal adaptation to mechanical loading.

The periosteal and endosteal surfaces of mature bone are densely innervated by sensory nerves expressing TrkA, the high-affinity receptor for nerve growth factor (NGF). In previous work, we demonstrated that administration of exogenous NGF significantly increased load-induced bone formation through the activation of Wnt signaling. However, the translational potential of NGF is limited by the induction of substantial mechanical and thermal hyperalgesia in mice and humans. Here, we tested the effect of gambogic amide (GA), a recently identified robust small molecule agonist for TrkA, on hyperalgesia and load-induced bone formation. Behavioral analysis was used to assess pain up to one week after axial forelimb compression. Contrary to our expectations, GA treatment was not associated with diminished use of the loaded forelimb or sensitivity to thermal stimulus. Furthermore, dynamic histomorphometry revealed a significant increase in relative periosteal bone formation rate as compared to vehicle treatment. Additionally, we found that GA treatment was associated with an increase in the number of osteoblasts per bone surface in loaded limbs as well as a significant increase in the fold change of Ngf, Wnt7b, and Axin2 mRNA expression as compared to vehicle (control). To test the effect of GA on osteoblasts directly, we cultured MC3T3-E1 cells for up to 21days in osteogenic differentiation media containing NGF, GA, or vehicle (control). Media containing GA induced the significant upregulation of the osteoblastic differentiation markers Runx2, Bglap2, and Sp7 in a dose-dependent manner, whereas treatment with NGF was not associated with any significant increases in these markers. Furthermore, consistent with our in vivo findings, we observed that administration of 50nM of GA upregulated expression of Ngf at both Day 3 and Day 7. However, cells treated with the highest dose of GA (500nM) had significantly increased apoptosis and impaired cell proliferation. In conclusion, our study indicates GA may be useful for augmenting skeletal adaptation to mechanical forces without inducing hyperalgesia.

New Insights Into the Role of β-NGF/TrKA System in the Endometrium of Alpacas During Early Pregnancy.

One striking reproductive feature in South American camelids is that more than 90% of gestations are established in the left uterine horn (LUH). This phenomenon could be related to a differential vascular irrigation of the LUH. An increase of vascularization in llama endometrium was observed after systemic administration of Beta Nerve Growth Factor (β-NGF), a neurotrophin present in the uterus and placenta of various mammals that is involved in pregnancy development. We hypothesized that the β-NGF signaling pathway is related to embryo implantation in the LUH in camelids. The aim of this study was to characterize the spatial expression of β-NGF and its high-affinity receptor, TrKA, between LUH and right uterine horn (RUH) of non-pregnant (NP) and early pregnant alpacas (15 and 30 days of gestation, 15 and 30P, respectively). In addition, β-NGF, TrKA, and Vascular Endothelium Growth Factor A (VEGFA) temporal gene expression patterns and counting of blood vessels were evaluated among groups. The β-NGF and TrKA were localized in the luminal, glandular, and vascular epithelium of the alpaca uterus and in the embryonic membranes of the 30-days-old conceptus. β-NGF and TrKA immunosignal were stronger in 15P females than that of NP and 30P. In addition, TrKA signal was higher in the LUH luminal epithelium of NP and 15P alpacas than that of NP-RUH and 15P-RUH. β-NGF mRNA relative abundance was higher in the 30P-RUH than that of NP-RUH; whereas TrKA mRNA abundance only differed between 15P-RUH and NP-LUH. VEGFA mRNA relative abundance was higher in NP females compared to the LUH of 15P and 30P alpacas, and lower to their right counterparts. The number of vessels per field was higher in 15P than that of 30P. A positive correlation was observed between the number of vessels per field and β-NGF immunosignal in 15P-LUH. In contrast, the area occupied by vessels was higher in 30P alpacas than of NP and 15P females. The changes of β-NGF/TrKA expression pattern in the peri-implantation endometria between LUH and RUH and their localization in the extraembryonic membranes support the implication of the neurotrophin during implantation and pregnancy development in South American Camelids.