Release Of Nerve Growth Factor Research Articles

Electrospun PCL/Alginate/Nanoclay Nerve Conduit with Olfactory Ectomesenchymal Stem Cells for Nerve Regeneration.

Biocompatible and biodegradable nerve growth conduits (NGCs) provide a promising alternative to conventional nerve grafting for peripheral nerve regeneration. Incorporating nanoclay (NC) has been shown to increase the hydrophilicity and flexibility of polymeric scaffolds. In the present study, poly caprolactone-alginate (PCL-ALG) conduits with varying percentages of NC (0.1%, 0.2%, and 0.5%) were fabricated using the electrospinning technique. The conduit containing 0.5% NC showed a greater increase in elongation (33%) and porosity, reaching 95% with the lowest contact angle (10°). For in vitro, human olfactory ectomesenchymal stem cells (OE-MSCs) were used as a favorable choice for neuronal differentiation owing to the origin from the neural crest. The viability and proliferation of OE-MSCs were maintained after 5 days on scaffolds with 0.5% NC, as confirmed by the MTT assay, cell adhesion analysis, and live/dead staining. Furthermore, the impact of 0.5% PCL-ALG-NC on the paracrine activity of OE-MSCs was studied for a period of 7 days. Our results indicated that human OE-MSCs, when cocultured with PC12 cells on NGC, have the capability to release nerve growth factor levels of up to 1392.83 pg/mL. In summary, the electrospun PCL-ALG conduit containing an optimal NC dosage (0.5%) and seeded with human OE-MSCs shows promising outcomes as NGC scaffold for peripheral nerve regeneration.

(132) MICONAZOLE VAGINAL CREAM AND ACQUIRED NEUROPROLIFERATIVE VESTIBULODYNIA

Abstract Introduction The vestibular epithelium is non-keratinized and is thus susceptible to injury from an allergic reaction. Topical pharmaceutical agents applied to the genitals for various medical indications, such as miconazole vaginal cream to treat vaginal yeast infections, may absorb through the non-keratinized vestibular epithelium. Active and inactive ingredients in miconazole vaginal cream include miconazole nitrate, benzoic acid, cetyl alcohol, isopropyl myristate, polysorbate 60, potassium hydroxide, propylene glycol, purified water and stearyl alcohol. Patients without vestibular pain previously have reported applying miconazole vaginal cream to their genitals and immediately experienced excruciating burning that resulted in persistent unrelenting severe pain throughout the vestibule, symptoms consistent with acquired neuroproliferative vestibulodynia (NPV). Objective To review reports of clinic patients who used miconazole vaginal cream prior to a diagnosis of acquired NPV to provide awareness of the management of this condition. Methods We performed a chart review of individuals with vulvas presenting to a single sexual medicine clinic between June 2019 and June 2023 to identify those who met the following inclusion criteria. They were clinically suspected to have acquired NPV after an allergic reaction following use of miconazole vaginal cream. They failed conservative treatments for provoked vestibular pain and underwent vestibulectomy. Their excised vestibular specimens revealed an excess of CD117 immunopositive stained cells (>8 per high-power field), consistent with mast cells, and PGP9.5 immunopositive stained area, consistent with nerves, histopathologically confirming the diagnosis of NPV. In addition, demographic data, prior diagnoses and treatments, results from validated questionnaires, pertinent physical exam findings and treatment outcomes were collected. Results A total of 7 individuals met inclusion criteria. Mean age at the time of exposure to miconazole vaginal cream was 23 (range 18-29) years. Mean age at presentation to our sexual medicine facility was 28 (range 22-33) years, thus a delay in diagnosis of miconazole vaginal cream-induced acquired NPV of 5 (range 1-12) years. 71% were previously diagnosed and unsuccessfully managed as hormonally-mediated vestibulodynia and 29% as interstitial cystitis. The mean pain domain score and overall score for the Female Sexual Function Index was 1.2/6 (range 0-3.2/6) and 11.7/36 (range 4.2-18.1/36), respectively. A total score of <26 is indicative of sexual dysfunction. The mean Female Sexual Distress Scale-Revised score was 32.2/52 (range 21-39/52), consistent with significant distress. Conclusions Our cohort of patients with miconazole vaginal cream-induced acquired NPV experienced several years of misdiagnoses, unsuccessful treatments, significant pain, and distress. It is hypothesized that miconazole vaginal cream induces NPV in genetically susceptible individuals in part because the vestibular epithelium is non-keratinized and therefore more easily absorbs the ingredients in the miconazole vaginal cream, which may result in an allergic response in the subepithelial region of the vestibule. This allergic reaction results in accumulation of mast cells in the subepithelial stroma that release nerve growth factor leading to neuroproliferation with subsequent allodynia and hyperalgesia. In individuals who develop yeast infections, use of systemic strategies and rather than topical medication is encouraged to avoid potential allergic reactions and resultant acquired NPV. Disclosure No.

Effects of TNF-α on Behaviour and Inflammation in Rats with Rotator Cuff Injury through NGF.

Rotator cuff injury is a common injury that includes inflammation, partial tearing, or complete tearing of the rotator cuff tendon. In cases of rotator cuff tears (RCTs), Tumor Necrosis Factor-alpha (TNF-α) can trigger the release of nerve growth factor (NGF). TNF-α is an important inflammatory mediator that affects rotator cuff activity and increased NGF expression is observed in RCTs. Therefore, this study aimed to investigate whether inhibition of TNF-α could reduce behavioural responses and inflammation levels in rats through NGF. A rat RCT model was established, and the CatWalk gait analysis system was used for behavioural assessment. Immunohistochemistry was used to detect NGF protein levels in tendon tissue. Hematoxylin eosin (HE) staining was used to observe histopathological changes. The expressions of Interleukin-1beta (IL-1β) and Cyclooxygenase-2 (COX2) were detected by western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR). The expression of apoptosis protein Bcl-2-associated X (Bax), B-cell lymphoma 2 (Bcl-2), and Cysteine-aspartic acid protease-3 (Caspase-3) were detected using WB. Oxidative stress markers, namely Reactive Oxygen Species (ROS), Malondialdehyde (MDA), and Superoxide Dismutase (SOD) were quantified in tissues using an ELISA kit. In the RCT model, elevated NGF protein expression, noticeable atrophy in the supraspinatus muscle tissue, and substantial fat infiltration were observed. The levels of IL-1β, COX2, apoptosis, and oxidative stress were all increased. TNF-α inhibition resulted in decreased NFG expression, decreased tissue fibrosis, and improved tendon atrophy. Moreover, when TNF-α was inhibited, the expressions of IL-1β and Cox2 were reduced and both apoptosis and oxidative stress were decreased. The results showed that inhibiting TNF-α had the potential to reduce inflammation levels and behavioural responses in rats. TNF-α can affect behaviour and inflammation in rats with RCTs through NGF, and TNF-α inhibition can improve rotator cuff injury.

Ultrasound-Responsive Aligned Piezoelectric Nanofibers Derived Hydrogel Conduits for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) are considered as promising treatment strategy and frontier trend for peripheral nerve regeneration, while their therapeutic outcomes are limited by the lack of controllable drug delivery and available physicochemical cues. Herein, novel aligned piezoelectric nanofibers derived hydrogel NGCs with ultrasound (US)-triggered electrical stimulation (ES) and controllable drug release for repairing peripheral nerve injury are proposed. The inner layer of the NGCs is the barium titanate piezoelectric nanoparticles (BTNPs)-doped polyvinylidene fluoride-trifluoroethylene [BTNPs/P(VDF-TrFE)] electrospinning nanofibers with improved piezoelectricity and aligned orientation. The outer side of the NGCs is the thermoresponsive poly(N-isopropylacrylamide) hybrid hydrogel with bioactive drug encapsulation. Such NGCs can not only induce neuronal-oriented extension and promote neurite outgrowth with US-triggered wireless ES, but also realize the controllable nerve growth factor release with the hydrogel shrinkage under US-triggered heating. Thus, the NGC can positively accelerate the functional recovery and nerve axonal regeneration of rat models with long sciatic nerve defects. It is believed that the proposed US-responsive aligned piezoelectric nanofibers derived hydrogel NGCs will find important applications in clinic neural tissue engineering.

Locally produced nerve growth factor is a novel regulator of (juxta)glomerular and collecting duct functions in the kidney

The dense network of sympathetic efferent and sensory afferent nerves innervating the kidney is well recognized, however the elements and the functional importance of neurotrophin signaling in renal tissues are much less known. Peripherally innervated tissues are known to produce and release nerve growth factor (NGF) which belongs to the neurotrophin family of neuronal signaling proteins and is essential for the growth and survival of peripheral sensory and sympathetic nerves. This study aimed to characterize the expression and role of NGF in the kidney. We hypothesized that NGF is produced by multiple renal tubular and vascular cell types and helps to maintain hemodynamic and tubular transport functions. Local NGF expression was studied using histological sections of kidneys harvested from NGF-GFP reporter mice. To address the functional importance of NGF signaling, a new mouse model was generated by crossing Ren1d-Cre and NGF-floxed mice and Cre/lox-mediated knockout of NGF from cells of the renin lineage co-labeled with the multicolor reporter Confetti (Ren1d-Confetti-NGF KO). WT and NGF KO mice were placed in metabolic cages for urine collections and analysis, and systolic blood pressure (SBP) was measured with tail-cuff pletysmography. Intercalated cells of the collecting duct (CD) showed by far the highest NGF expression among all renal cell types based on the reporter signal in NGF-GFP mice, in addition to select cells in the renal interstitium including juxtaglomerular (JG) renin cells. Since both CD cells and JG cells are part of the renin lineage, the applied genetic strategy using Ren1d-Confetti-NGF KO mice was ideal, and showed diminished NGF expression compared to WT. Urine excretion rate and albuminuria were 3-fold increased in Ren1d-Confetti-NGF KO vs. WT mice, while there was no difference in SBP between WT and KO groups. Immunohistochemistry showed aberrant CD structure with multiple small, undifferentiated cells with apparently lost polarity and reduced AQP2 labeling in CD principal cells in Ren1d-Confetti-NGF KO mice compared to WT. In addition, the characteristic strong apical membrane AQP2 (in principal cells) and H+-ATPase (in intercalated cells) labeling in WT mice was diminished in Ren1d-Confetti-NGF KO mice. JG renin cell number per nephron was significantly reduced in Ren1d-Confetti-NGF KO (2.5±1.0) vs. WT mice (6.2±1.5, n=4 mice each). In summary, our results suggest high level of NGF expression in the adult kidney that plays important physiological functional roles in maintaining collecting duct water and electrolyte reabsorption and acid secretion, and intrarenal renin production. Deficient intrarenal NGF production may cause kidney disease, suggesting the importance of altered renal neurotrophin signaling in pathology development. DK064234, DK123564, DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Decellularized brain extracellular matrix based NGF-releasing cryogel for brain tissue engineering in traumatic brain injury

Traumatic brain injuries(TBI) pose significant challenges to human health, specifically neurological disorders and related motor activities. After TBI, the injured neuronal tissue is known for hardly regenerated and recovered to their normal neuron physiology and tissue compositions. For this reason, tissue engineering strategies that promote neuronal regeneration have gained increasing attention. This study explored the development of a novel neural tissue regeneration cryogel by combining brain-derived decellularized extracellular matrix (ECM) with heparin sulfate crosslinking that can perform nerve growth factor (NGF) release ability. Morphological and mechanical characterizations of the cryogels were performed to assess their suitability as a neural regeneration platform. After that, the heparin concnentration dependent effects of varying NGF concentrations on cryogel were investigated for their controlled release and impact on neuronal cell differentiation. The results revealed a direct correlation between the concentration of released NGF and the heparin sulfate ratio in cryogel, indicating that the cryogel can be tailored to carry higher loads of NGF with heparin concentration in cryogel that induced higher neuronal cell differentiation ratio. Furthermore, the study evaluated the NGF loaded cryogels on neuronal cell proliferation and brain tissue regeneration in vivo. The in vivo results suggested that the NGF loaded brain ECM derived cryogel significantly affects the regeneration of brain tissue. Overall, this research contributes to the development of advanced neural tissue engineering strategies and provides valuable insights into the design of regenerative cryogels that can be customized for specific therapeutic applications.

Hybrid Electrically Conductive Hydrogels with Local Nerve Growth Factor Release Facilitate Peripheral Nerve Regeneration.

It is challenging to treat peripheral nerve injury (PNI) clinically. As the gold standard for peripheral nerve repair, autologous nerve grafting remains a critical limitation, including tissue availability, donor-site morbidity, immune rejection, etc. Recently, conductive hydrogels (CHs) have shown potential applications in neural bioengineering due to their good conductivity, biocompatibility, and low immunogenicity. Herein, a hybrid electrically conductive hydrogel composed of acrylic acid derivatives, gelatin, and heparin with sustained nerve growth factor (NGF) release property was developed. The rat sciatic nerve injury (SNI) model (10 mm long segment defect) was used to investigate the efficacy of these hydrogel conduits in facilitating peripheral nerve repair. The results showed that the hydrogel conduits had excellent conductivity, mechanical properties, and biocompatibility. In addition, NGF immobilized in the hydrogel conduits had good sustained release characteristics. Finally, functional recovery and electrophysiological evaluations, together with histological analysis, indicated that the hydrogel conduits immobilizing NGF had superior effects on motor recovery, axon growth, and remyelination, thereby significantly accelerating the repairing of the sciatic nerve. This study demonstrated that hybrid electrically conductive hydrogels with local NGF release could be effectively used for PNI repair.

Neurotrophic and Neuroprotective Effects of Hericium erinaceus

Hericium erinaceus is a valuable mushroom known for its strong bioactive properties. It shows promising potential as an excellent neuroprotective agent, capable of stimulating nerve growth factor release, regulating inflammatory processes, reducing oxidative stress, and safeguarding nerve cells from apoptosis. The active compounds in the mushroom, such as erinacines and hericenones, have been the subject of research, providing evidence of their neuroprotective effects. Further research and standardization processes for dietary supplements focused on H. erinaceus are essential to ensuring effectiveness and safety in protecting the nervous system. Advancements in isolation and characterization techniques, along with improved access to pure analytical standards, will play a critical role in achieving standardized, high-quality dietary supplements based on H. erinaceus. The aim of this study is to analyze the protective and nourishing effects of H. erinaceus on the nervous system and present the most up-to-date research findings related to this topic.

Synthesis and secretion of Nerve Growth Factor is regulated by Nitric Oxide in bladder cells in vitro under a hyperglycemic environment

Urine samples of female patients with overactive bladder (OAB) are characterized by low levels of nerve growth factor (NGF) and elevated concentrations of nitric oxide (NO) compared to healthy controls. We therefore examined how NO might regulate NGF synthesis using rat bladder smooth muscle (SMCs) and urothelial (UROs) cells in culture. In UROs, incubation in hyperglycemic conditions to mimic insulin insensitivity present in the OAB cohort increased secretion of NO and concomitantly decreased NGF, except when the NO synthase inhibitor, l-NAME (1 mM) was present. Sodium nitroprusside (SNP) (300 μM, 24 h), a NO generator, decreased NGF levels and decreased cyclic GMP (cGMP) content, a process validated by the cGMP synthase inhibitor ODQ (100 μM). Alternatively, SNP increased mRNA of both NGF and matrix metalloproteinase-9 (MMP-9). MMP-9 knockout of UROs by Crispr-Cas9 potently decreased the effect of SNP on NGF, implying a dependent role of NO on MMP-9. On the other hand, matrix metalloproteinase-7 (MMP-7) activity was increased by SNP, which taken together with increase in NGF mRNA, suggests a compensatory mechanism. In SMCs, hyperglycemic conditions had the same effect on extracellular content of NO and NGF than in UROs. SNP also decreased NGF secretion but increased cGMP content. Stable permeable analogs of cGMP 8-(4-Chlorophenylthio)-cGMP (1 mM) and N2,2′-O-Dibutyryl-cGMP (3 mM) inhibited NGF release. NGF and MMP-9 mRNA expression was unchanged by SNP. Deletion of MMP-9 in SMCs by Crispr-Cas9 did not alter the effect of SNP. Finally, SNP decreased MMP-7 activity, diminishing the conversion of proNGF to NGF. These results demonstrate that enhanced NO secretion triggered by high glucose decreases NGF secretion through pathways unique for each cell type that involve cGMP and proteases MMP-7 and MMP-9. These results might help to explain our observations from the urine from patients with OAB associated with metabolic syndrome.

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.

Alginate/gum arabic-based biomimetic hydrogel enriched with immobilized nerve growth factor and carnosine improves diabetic wound regeneration

Diabetic foot ulcers (DFUs) often remain untreated because they are difficult to heal, caused by reduced skin sensitivity and impaired blood vessel formation. In this study, we propose a novel approach to manage DFUs using a multifunctional hydrogel made from a combination of alginate and gum arabic. To enhance the healing properties of the hydrogel, we immobilized nerve growth factor (NGF), within specially designed mesoporous silica nanoparticles (MSN). The MSNs were then incorporated into the hydrogel along with carnosine (Car), which further improves the hydrogel's therapeutic properties. The hydrogel containing the immobilized NGF (SiNGF) could control the sustain release of NGF for >21 days, indicating that the target hydrogel (AG-Car/SiNGF) can serve as a suitable reservoir managing diabetic wound regeneration. In addition, Car was able to effectively reduce inflammation and significantly increase angiogenesis compared to the control group. Based on the histological results obtained from diabetic rats, the target hydrogel (AG-Car/SiNGF) reduced inflammation and improved re-epithelialization, angiogenesis, and collagen deposition. Specific staining also confirmed that AG-Car/SiNGF exhibited improved tissue neovascularization, transforming growth factor-beta (TGFβ) expression, and nerve neurofilament. Overall, our research suggests that this newly developed composite system holds promise as a potential treatment for non-healing diabetic wounds.

The microglial challenge to encapsulated cell mediated drug delivery in brain tissue

AbstractBackgroundDementia disorders affect millions of people worldwide, among which Alzheimer’s disease (AD) accounts for 60% cases. Treatment of these disorders are constrained due to inefficient brain availability of peripherally administered drugs. Conversely, direct intracerebroventricular injections are counterintuitive due to unspecific side‐effects. Recently, Aducanumab has shown potential to clear amyloid plaques from AD brain, but its efficacy to revive clinical symptoms are under debate. In this regard, direct delivery of regenerative biological drugs utilizing encapsulated cell biodelivery (ECB) has gained much attention. Conversely, how the pathological microenvironment of the AD brain affects these encapsulated drug‐delivering cells are scarcely understood. The ECB approach nullifies immune reaction from host tissue by protecting drug‐delivering cells from direct physical contact with the host tissue. Thus, in this study we probe the effect of factors released from activated human microglial cells (HMC3 cell line) on ECB devices harboring nerve growth factor (NGF) releasing cells (NGC0211; modified human ARPE‐19 cells).MethodECB devices harboring NGC0211 cells were manufactured and maintained in DMEM/F12 culture medium containing 10% serum until used. HMC3 cells were activated using AD related molecules including 1µM amyloid beta (Aβ40 and Aβ42) peptides and 10ng/mL inflammatory cytokines (interferon‐γ, IFN‐γ; interleukin‐1β, IL‐1β; or both) for 24hrs, and a cell free microglia conditioned medium (MCM) was obtained. ECB devices were then exposed to various MCMs, and NGF release was followed for several weeks assessed by ELISA (DY256, R&amp;D systems). Similarly, non‐encapsulated NGC0211 cells grown in multi‐well slides were exposed to MCMs and cellular proliferation (Ki67 expression) along with senescence parameters (SA‐β‐galactosidase) were evaluated.ResultWe found that factors released from activated microglia hampered NGF release from ECB devices. Simultaneously, MCMs reduced Ki67 expression and elevated SA‐β‐galactosidase activity in NGC0211 cells.ConclusionActivated microglia, a common feature of all neurodegenerative conditions, impair NGF release, cellular proliferation and induce senescence in NGC0211 cells. Similar impact of microglia on other types of drug‐delivering cells (stem cells or genetically modified) needs to be studied. Our study highlights one of the probable underlying causes for impaired long‐term cell mediated drug delivery in neurodegenerative diseases and helps guide improvements in the technologies.

(022) Durability of Vestibulectomy Surgery for Neuroproliferative Vestibulodynia

Abstract Introduction In 2006, Goldstein et al reported on 134 patients who underwent complete vestibulectomy surgery with vaginal advancement flap reconstruction for neuroproliferative vestibulodynia (NPV). Ninety-seven of 104 participants (93%) noted being satisfied/very satisfied with surgical outcome. The mean duration of follow up was 26 months (12 – 72 months). NPV is a genital pain condition hypothesized to be caused by an accumulation of excess mast cells in the vestibular epithelium and sub-epithelial stroma that release nerve growth factor. This promotes sub-epithelial stromal nerve ending proliferation (Figure 1). The excess nerve endings result in bothersome, persistent provoked vestibular allodynia and hyperalgesia. Durability of post-operative outcomes past 72 months has not been previously characterized. It is yet unknown if the immunohistochemical pathologic findings of excess mast cells and nerve endings ever reaccumulate in the vestibule, causing recurrent allodynia, hyperalgesia and entrance dyspareunia. Objective We assessed the long-term surgical outcome durability (&amp;gt;6 years) of resolution of pain from total vestibulectomy with vaginal advancement flap reconstruction. Methods We routinely perform post-operative follow-up of surgical patients who undergo complete vestibulectomy surgery with vaginal advancement flap reconstruction for NPV. We identified patients who claimed successful resolution of their entrance dyspareunia at least 72 months after complete vestibulectomy surgery. We contacted them to determine whether or not they continued to have resolution of their entrance dyspareunia. Results 57 patients have had complete vestibulectomy surgery with vaginal advancement flap reconstruction from 2007 – 2016 (&amp;gt; 6 years ago). 41 patients responded to long term follow-up contact. 34 patients (78%) were satisfied/very satisfied with surgical outcome in the first 72 months post-operatively. All 34 (100%) continued to note excellent durability of their surgical result, still claiming being satisfied/very satisfied with surgical outcome, a mean of 9.3 years post-op (range 5-14 years). Conclusions Based on this small series, it appears that if a patient is a treatment responder to complete vestibulectomy surgery, there is strong durability of the surgical result. These data suggest that in treatment responders, there is no evidence of re-accumulation of excess mast cells and nerve endings in the introitus. These data support that the pathology of NPV lies in the endodermal vestibule and that complete excision is an important long term treatment strategy in many NPV patients. Disclosure No

(136) Long-term Durability of Response to Complete Vestibulectomy Surgery for Neuroproliferative Vestibulodynia

Abstract Introduction In 2006, Goldstein et al published on the surgical outcomes of the largest-to-date patient series undergoing complete vestibulectomy with vaginal advancement flap reconstruction for neuroproliferative vestibulodynia (NPV). One hundred and thirty-four patients underwent surgery over a 5 year period, and 93% of the 104 patients who agreed to participate were satisfied/very satisfied with their surgical outcome. The mean time to follow up was 26 months, range 12 – 72 months. NPV is hypothesized to be, in part, a result of accumulation of excess mast cells in the epithelial basement membrane and sub-epithelial stroma that may release nerve growth factor promoting sub-epithelial nerve ending proliferation (Figure 1). The excess nerve endings are hypothesized to result in bothersome, persistent, and unrelenting provoked vestibular allodynia and hyperalgesia that are characteristic of NPV. Durability of post-operative outcomes past 72 months has yet to be addressed. It is still unknown if the pathologic findings of excess mast cells and nerve endings ever regenerate in the vestibule and cause recurrent allodynia, hyperalgesia and entrance dyspareunia. Objective We wished to assess, for the first time, the long-term surgical outcome durability data (&amp;gt;5 years) of those NPV patients who claimed successful resolution of their entrance dyspareunia following complete vestibulectomy surgery in the first 5 years post-operatively. Methods We routinely perform post-operative follow-up of surgical patients who undergo complete vestibulectomy surgery with vaginal advancement flap reconstruction for NPV. We identified those who claimed successful resolution of their entrance dyspareunia following complete vestibulectomy surgery in the first year post-operatively and thereafter for the next 4 years, and asked them if they continued to have resolution of their entrance dyspareunia. Results Fifty-seven patients had complete vestibulectomy with vaginal advancement flap reconstruction between 2007 and 2017 (&amp;gt; 5 years ago). Forty-one patients responded to the routine contact for long term follow-up. Thirty-four patients (78%) had resolution of pain in the first year post-operatively. Upon repeat interview, all 34 (100%) continued to note excellent durability of their surgical result, a mean of 9.3 years after their vestibulectomy (range 5-14 years). Conclusions Based on this small series, it appears that if a patient is a treatment responder following complete vestibulectomy, there is strong durability of the surgical result with no evidence of recurrent allodynia or hypoalgesia. These data suggest that in treatment responders, there is no evidence of re-accumulation of excess mast cells and nerve endings in the introitus. These data support that the pathology of NPV lies in the endodermal vestibule and that complete excision may be an important long-term treatment strategy in many NPV patients. Disclosure No

3, 3’-diindolylmethane enhances macrophage efferocytosis and subsequently relieves visceral pain via the AhR/Nrf2/Arg-1-mediated arginine metabolism pathway

Background3, 3’-diindolylmethane (DIM), a classical aryl hydrocarbon receptor (AhR) agonist, has been shown to relieve neuropathic pain, but few studies have reported the efficacy of DIM in visceral pain under colitis condition. PurposeThis study aimed to investigate the effect and mechanism of DIM on visceral pain under colitis condition. MethodsCytotoxicity was performed using the MTT assay. RT-qPCR and ELISA assays were applied to determine the expression and release of algogenic substance P (SP), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Flow cytometry was used to examine the apoptosis and efferocytosis. The expression of Arg-1-arginine metabolism-related enzymes was detected using western blotting assays. ChIP assays were used to examine the binding of Nrf2 to Arg-1. Mouse models of dextran sulfate sodium (DSS) were established to illustrate the effect of DIM and validate the mechanism in vivo. ResultsDIM did not directly affect expressions and release of algogenic SP, NGF and BDNF in enteric glial cells (EGCs). However, when co-cultured with DIM-pre-treated RAW264.7 cells, the release of SP and NGF was decreased in lipopolysaccharides-stimulated EGCs. Furthermore, DIM increased the number of PKH67+ F4/80+ cells in the co-culture system of EGCs and RAW264.7 cells in vitro and alleviated visceral pain under colitis condition by regulating levels of SP and NGF as well as values of electromyogram (EMG), abdominal withdrawal reflex (AWR) and tail-flick latency (TFL) in vivo, which was significantly inhibited by efferocytosis inhibitor. Subsequently, DIM was found to down-regulate levels of intracellular arginine, up-regulate levels of ornithine, putrescine and Arg-1 but not extracellular arginine or other metabolic enzymes, and polyamine scavengers reversed the effect of DIM on efferocytosis and release of SP and NGF. Moving forward, Nrf2 transcription and the binding of Nrf2 to Arg-1-0.7 kb was enhanced by DIM, AhR antagonist CH223191 abolished the promotion of DIM on Arg-1 and efferocytosis. Finally, nor-NOHA validated the importance of Arg-1-dependent arginine metabolism in DIM-alleviated visceral pain. ConclusionDIM enhances macrophage efferocytosis in an arginine metabolism-dependent manner via “AhR-Nrf2/Arg-1” signals and inhibits the release of SP and NGF to relieve visceral pain under colitis condition. These findings provide a potential therapeutic strategy for the treatment of visceral pain in patients with colitis.

Enzyme-Mediated Nerve Growth Factor Release from Nanofibers Using Gelatin Microspheres.

Spinal cord injury is a complex environment, with many conflicting growth factors present at different times throughout the injury timeline. Delivery of multiple growth factors has received mixed results, highlighting a need to consider the timing of delivery for possibly antagonistic growth factors. Cell-mediated degradation of delivery vehicles for delayed release of growth factors offers an attractive way to exploit the highly active immune response in the spinal cord injury environment. In this study, growth factor-loaded gelatin microspheres (GMS) combined with methacrylated hyaluronic acid (MeHA) were electrospun to create GMS fibers (GMSF) for delayed release of growth factors (GFs). GMS were successfully combined with MeHA while electrospinning, with an average fiber diameter of 365 ± 10 nm and 44% ± 8% fiber alignment. GMSF with nerve growth factor (NGF) was tested on dissociated chick dorsal root ganglia cells. We further tested the effect of M1 macrophage-conditioned media (M1CM) to simulate macrophage invasion after spinal cord injury for cell-mediated degradation. We hypothesized that neurons grown on GMSF with loaded NGF would exhibit longer neurites in M1CM, showing a release of functional NGF, as compared with controls. GMSF in M1CM was significantly different from MeHA in serum-free media (SFM) and M0-conditioned media (M0CM), as well as GMSF in M0CM (p < 0.05). Moreover, GMSF + NGF in all media conditions were significantly different from MeHA in SFM and M0CM (p < 0.05). The goal of this study was to develop a biomaterial system where drug delivery is triggered by immune response, allowing for more control and longer exposure to encapsulated drugs. The spinal cord injury microenvironment is known to have a robust immune response, making this immune-medicated drug release system particularly significant for directed repair.

Influence of chrysin on D-galactose induced-aging in mice: Up regulation of AMP kinase/liver kinase B1/peroxisome proliferator-activated receptor-γ coactivator 1-α signaling pathway.

Adenosine monophosphate kinase/liver kinase B1/peroxisome proliferator-activated receptor-γ coactivator 1-α (AMPK/LKB1/PGC1α) pathway has a vital role in regulating age-related diseases. It controls neurogenesis, cell proliferation, axon outgrowth, and cellular energy homeostasis. AMPK pathway also regulates mitochondrial synthesis. The current study evaluated the effect of chrysin on D-galactose (D-gal) induced-aging, neuron degeneration, mitochondrial dysfunction, oxidative stress, and neuroinflammation in mice. The mice were allocated randomly into four groups (10 each group): Group 1: normal control group, Group 2: D-gal group, Groups 3 and 4: chrysin (125 and 250 mg/kg, respectively). Groups 2-4 were injected with D-gal (200 mg/kg/day; s.c) for 8weeks to induce aging. Groups 3 and 4 were orally gavaged every day concurrent with D-gal. At the end of experiment, behavioral, brain biochemical and histopathological changes were monitored. Chrysin administration elevated discrimination ratio in object recognition, Y Maze percentage alternation, locomotor activity and brain contents of AMPK, LKB1, PGC1α, NAD (P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), nerve growth factor (NGF) (neurotrophin-3; NT-3), and seretonin as well as reduced brain contents of tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), advanced glycation end products (AGEs) and glial fibrillary acidic protein (GFAP) compared to D-gal-treated mice. Chrysin also alleviated cerebral cortex and white matter neurons degeneration. Chrysin protects against neurodegeneration, improves mitochondrial autophagy and biogenesis as well as activates antioxidant genes expression. In addition, chrysin ameliorates neuroinflammation and stimulates the release of NGF and serotonin neurotransmitter. So, chrysin has a neuroprotective effect in D-gal induced-aging in mice.

Zn2+-responsive palladium nanoclusters synergistically manage Alzheimer’s disease through neuroprotection and inhibition of oxidative stress

The current focus on inhibiting β-Amyloid (Aβ) aggregation has shown great limitations for the management of Alzheimer's disease (AD). Multi-target strategies that synergistically exert neuroprotective effects and alleviate oxidative stress are effective strategies to modulate the AD microenvironment. Herein, a novel Zn2+-responsive nano-drug delivery platform (NWP) is developed using palladium nanoclusters (Pd NCs) as carriers, loaded with nerve growth factor (NGF), capped by supramolecular nanovalves containing carboxyl pillars[5]arenes (WP5) for the treatment of AD. Pd NCs have both OH elimination and catalase activities to alleviate oxidative stress. The larger specific surface area could significantly improve the loading capacity of NGF. The supramolecular nanovalve constructed by WP5 achieves precise targeting and controlled release of NGF with Zn2+ responsiveness, thereby improving the utilization of loading drugs. In vitro and in vivo experiments demonstrates that NWP effectively scavenges reactive oxygen species (ROS), protects neuronal cells from oxidative damage and modulates microglial polarization (the pro-inflammatory M1-phenotype microglial transition to the anti-inflammatory M2-phenotype), thus reducing the release of pro-inflammatory factors and alleviating neuroinflammation. Furthermore, intracerebral injection of NWP significantly ameliorates the learning and memory deficits of AD model mice. Overall, this Zn2+-responsive multi-target therapy provides a new option for AD.

Ultrasound‐Responsive Composited Conductive Silk Conduits for Peripheral Nerve Regeneration

Peripheral nerve injuries are challenging to repair clinically due to their limited regenerative capacity. Herein, a novel poly(3,4‐ethylenedioxythiophene):poly(4‐styrene sulfonate) (PEDOT:PSS) composited conductive silk conduit with ultrasound (US)‐triggered active release for peripheral nerve regeneration is presented. The conduit is composed of silk fibroin inverse opal tubular scaffolds and the secondary hydrogel filler that contains thermosensitive material (poly(N‐isopropylacrylamide), PNIPAM), PEDOT:PSS, and nerve growth factor (NGF). Benefiting from the integration of these functional materials and active factors, the silk fibroin conduits show excellent biocompatibility, flexibility, conductivity, and bioactivity. In particular, owing to the thermal effect of US and the temperature responsiveness of PNIPAM, the secondary filling hydrogel undergoes volume shrinkage upon US triggering, allowing the responsive NGF release. Besides, the electrical conductivity of PEDOT:PSS has been affirmed to promote neuron and axon outgrowth. Through in vivo experiments, the synergistic effect of PEDOT:PSS and US‐triggered delivery of NGF on accelerating injured nerve repair are demonstrated. These results reveal the practical value of the proposed intelligent conductive silk conduits with US responsiveness for nerve regeneration in preclinical studies.

Triterpenoids from the leaves of Abies koreana and their biological activities

Seven undescribed triterpenoids, abikoranes A–G, along with three known triterpenoids were isolated from the leaves of Abies koreana E. H. Wilson. The structures of compounds were elucidated by 1D and 2D NMR, HRMS, ECD, specific rotation, and DP4+ analysis. Abikorane A represents the second example of nor-3,4-seco-17,14-friedo-lanostane triterpenoid. Among the isolates, some compounds showed strong cytotoxic activities against some of four tested cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-116) with the values of IC50 0.89–9.62 μM, inhibited lipopolysaccharide-stimulated nitric oxide production with IC50 values of 11.57–15.16 μM, and exhibited significant nerve growth factor release effect (192.54 ± 12.33%) from C6 glioma cells.