Upregulation Of Nerve Growth Factor Research Articles

FNDC5/irisin mediates the protective effects of Innovative theta-shaking exercise on mouse memory

As a passive motion and non-invasive treatment, theta-shaking exercise is considered an alternative to traditional active exercise for slowing down brain ageing. Here, we studied the influence of theta-shaking exercise on fibronectin type III domain containing 5/irisin (FNDC5/irisin) in the anterior nucleus of the thalamus, hippocampus, and medial prefrontal cortex (ATN–HPC–MPFC). Further, we assessed memory in senescence-accelerated prone mice (SAMP-10 mice) using a behavioural test to confirm the protective effect of theta-shaking exercise against age-related memory decline. SAMP-10 mice were subjected to theta-shaking exercise for 9–30 weeks. Mice then performed the T-maze test and passive avoidance task. Immunohistochemical analysis and ELISA were used to assess FNDC5/irisin, nerve growth factor (NGF), and neurotrophin 4/5 (NT4/5) expression in the ATN–HPC–MPFC. In the shaking group, FNDC5 was locally upregulated within the hippocampus and MPFC area rather than exhibiting even distribution throughout brain tissue. Irisin levels were generally higher in the control group. Meanwhile, hippocampal NGF levels were significantly higher in the shaking group, with no differences noted in neurotrophin levels. Theta-shaking preserved normal neurons in certain sub-regions. However, no beneficial changes in neuronal density were noted in the ATN. Theta-shaking exercise positively affects memory function in SAMP-10 mice. FNDC5 upregulation and higher levels of NGF, along with the potential involvement of irisin, may have contributed to the preservation of normal neuronal density in the hippocampus and MPFC subregions.

Micropatterned photothermal double-layer periosteum with angiogenesis-neurogenesis coupling effect for bone regeneration.

The abundant neurovascular network in the periosteal fibrous layer is essential for regulating bone homeostasis and repairing bone defects. However, the majority of the current studies only focus on the structure or function, and most of them merely involve osteogenesis and angiogenesis, lacking an in-depth study of periosteal neurogenesis. In this study, a photothermal double-layer biomimetic periosteum with neurovascular coupling was proposed. The outer layer of biomimetic periosteum is a conventional electrospinning membrane to prevent soft tissue invasion, and the inner layer is an oriented nanofiber membrane to promote cell recruitment and angiogenesis. From the perspective of functional bionics, based on the whitlockite (WH) similar to bone composition, we doped Nd (the trivalent form of neodymium element) in it as the inducing element of photothermal response to prepare photothermal whitlockite (Nd@WH). The sustained release of Mg2+ in Nd@WH can effectively promote the up-regulation of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF). The release of Ca2+ and PO4 3- ions and photothermal osteogenesis jointly promote bone regeneration. Under the combined effect of structure and function, the formation of nerves, blood vessels, and related collagens greatly simulates the microenvironment of extracellular matrix and periosteum regeneration and ultimately promotes bone regeneration. In this study, physical and chemical characterization proved that the bionic periosteum has good flexibility and operability. The in vitro cell experiment and in vivo calvarial defect model verified that PPCL/Nd@WH biomimetic periosteum had excellent bone tissue regeneration function compared with other groups. Finally, PPCL/Nd@WH provides a new idea for the design of bionic periosteum.

Sympathetic activation in heart failure.

Sympathetic activation has been long appreciated exclusively as a fundamental compensatory mechanism of the failing heart and, thus, welcome and to be supported. In the initial clinical phases of heart failure (HF), the sympathetic nervous system overdrive plays a compensatory function aimed at maintaining an adequate cardiac output despite the inotropic dysfunction affecting the myocardium. However, when the sympathetic reflex response is exaggerated it triggers a sequence of unfavourable remodelling processes causing a further contractile deterioration that unleashes major adverse cardiovascular consequences, favouring the HF progression and the occurrence of fatal events. Eventually, the sympathetic nervous system in HF was demonstrated to be a ‘lethality factor’ and thus became a prominent therapeutic target. The existence of an effective highly specialized intracardiac neuronal network immediately rules out the old concept that sympathetic activation in HF is merely the consequence of a drop in cardiac output. When a cardiac damage occurs, such as myocardial ischaemia or a primary myocardial disorder, the adaptive capability of the system may be overcame, leading to excessive sympatho-excitation coupled with attenuation till to abolishment of central parasympathetic drive. Myocardial infarction causes, within a very short time, both a functional and anatomical remodelling with a diffuse up-regulation of nerve growth factor (NGF). The subsequent nerve sprouting signal, facilitated by a rise in the levels of NGF in the left stellate ganglion and in the serum, triggers an increase in cardiac nerve density in both peri-infarct and non-infarcted areas. Finally, NFG production decreases over time, supposedly as an adaptative response to the prolonged exposure to sympathetic overactivity, leading in the end to a reduction in sympathetic nerve density. Accordingly, NGF levels were markedly reduced in patients with severe congestive heart failure. The kidney is the other key player of the sympathetic response to HF as it indeed reacts to under-perfusion and to loop diuretics to preserve filtration at the cost of many pathological consequences on its physiology. This vicious loop ultimately participates to the chronic and disruptive sympathetic overdrive.In conclusion, sympathetic activation is the natural physiological consequence to life stressors but also to any condition that may harm our body. It is the first system of reaction to any potential life-threatening event. However, in any aspect of life over reaction is never effective but, in many instances, is, actually, life threatening. One for all is the case of ischaemia-related ventricular fibrillation which is, strongly facilitated by sympathetic hyperactivity. The take home message? When, in a condition of harm, everybody is yelling failure is just around the corner.

MP49-05 INTRAVESICAL NERVE GROWTH FACTOR-TARGETING THERAPY USING LIPOSOME-CONJUGATED ANTISENSE IN RATS WITH STRESS-INDUCED BLADDER HYPERSENSITIVITY

You have accessJournal of UrologyCME1 May 2022MP49-05 INTRAVESICAL NERVE GROWTH FACTOR-TARGETING THERAPY USING LIPOSOME-CONJUGATED ANTISENSE IN RATS WITH STRESS-INDUCED BLADDER HYPERSENSITIVITY Tetsuichi Saito, Pradeep Tyagi, Taro Igarashi, Tomonori Minagawa, Teruyuki Ogawa, Osamu Ishizuka, and Naoki Yoshimura Tetsuichi SaitoTetsuichi Saito More articles by this author , Pradeep TyagiPradeep Tyagi More articles by this author , Taro IgarashiTaro Igarashi More articles by this author , Tomonori MinagawaTomonori Minagawa More articles by this author , Teruyuki OgawaTeruyuki Ogawa More articles by this author , Osamu IshizukaOsamu Ishizuka More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002624.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Psychological stress has been implicated as an etiological factor of bladder hypersensitive disorders such as bladder pain syndrome/interstitial cystitis (BPS/IC). Also, upregulation of nerve growth factor (NGF) in the bladder, which is detected in BPS/IC patients, is shown to be an important factor inducing bladder hypersensitivity in animal models. Thus. this study investigated the relationship of NGF in the bladder and bladder hypersensitivity caused by psychological stress induced by water avoidance stress (WAS) in rats, and examined whether an intravesical therapy using liposome-conjugated NGF antisense oligonucleotide (OND) can improve the WAS-induced bladder hypersensitive condition. METHODS: Female Wistar rats were used and divided into sham control, WAS and WAS with OND treatment groups. The WAS with OND rats were treated with NGF antisense OND on the day before the first WAS session. After 1-hour WAS or sham sessions once daily for 10 days, the rats were evaluated by cystometry or pain behavior measurements followed by bladder histology. In another set of animals without functional evaluation, the bladder was harvested for molecular studies. RESULTS: Rats exposed to 10-days WAS showed shortened inter-contraction intervals (ICI) and increased pain behaviors, compared to control rats, which were improved with OND treatment (Fig 1-a,b,c). Protein assay showed increased NGF levels in the bladder mucosa in WAS rats, which were decreased by OND treatment (Fig 1-d). RT-PCR showed increases of TRPV1 and TRPA1 and a decrease in Cx43 in the bladder mucosa of WAS rats compared to control rats, which were improved in WAS rats treated with OND (Fig 1-e). Bladder histology revealed urothelial exfoliation in WAS rats, which was recovered with OND treatment (Fig 1-f,g). CONCLUSIONS: Psychological stress induced by WAS elicited bladder hypersensitivity, which was improved by NGF antisense OND treatment. Thus, liposome-based local suppression of upregulated NGF in the bladder could be an effective therapeutic modality for bladder overactivity and enhanced pain sensation induced by psychological stress. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e854 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tetsuichi Saito More articles by this author Pradeep Tyagi More articles by this author Taro Igarashi More articles by this author Tomonori Minagawa More articles by this author Teruyuki Ogawa More articles by this author Osamu Ishizuka More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF DownloadLoading ...

Increased nerve fibers and decreased perivascular adipose tissue in coronary arteries of acute myocardial infarction patients indicate structural changes before AMI episode

Abstract Acute myocardial infarction (AMI) is caused by ruptured atheroslerotic plaques. The influence of nerves on both plaque formation and vascular tone might play a role in AMIs. Nerve sprouting can occur following AMI throughout the myocardium and coronary arteries. High densities of sympathetic nerves are associated with cardiac arrhythmias, occurring most often in the first month following an AMI. The imbalance of the nervous system is caused by the upregulation of nerve growth factor from diseased cardiomyocytes. In addition to cardiomyocytes, adipose tissue activates nerves as well. Neural remodelling in the ventricle occurs one week following AMI in dogs. In humans, however, it is unknown how much time passes following an AMI before neural remodelling. In addition, the role of adipose tissue in neural remodelling is largely unknown and the amount of nerve fibres in coronary arteries in AMI patients has not been examined yet. The purpose of this study is determining both the amount of neural remodelling and changes in adipose tissue at different time points following an AMI. Coronary arteries were obtained at autopsy from AMI patients (N=23) and controls who did not have any kind of heart disease (N=7). The infarct related area from the heart was also obtained from AMI patients. AMI patients were subdivided based on time passed following infarction. Nerve fibres were visualised by immunohistochemistry, using a S100 antibody. Activation of nerves was visualised by neuropeptide Y (NPY). Adipose tissue was visualised by use of a Elastica Von Gieson (EvG) staining. In the coronary arteries, S100+ nerve fibres and NPY+ nerve fibres were quantified by using a light microscope and then calculated per mm2 of the adventitia surface areas. Fat tissue was quantified by measuring the total area, intima and media of blood vessels. AMI patients had an increase of nerves in their coronary arteries compared to control patients. Comparing the different phases of AMI, an increase in AMI patients with phase 1 infarctions was found compared to AMI patients with phase 3 infarctions. No differences were found based on the specific coronary arteries. Next we looked at the ventricles, which had no increase of nerves in AMI patients. A significant decrease of PVAT was found in both the left ventricle and coronary arteries of AMI patients. As AMI patients have an increase of S100+ nerves during the early stage of infarction, these nerves are presumably upregulated before the MI episode. All coronary arteries, including those not related to the infarction, have an increase of S100+ nerves, hinting at activated arteries during MI. Decrease of PVAT is associated with vascular dysfunction and occurs during aging. Whether the increase of nerves and a decrease of PVAT cause an AMI is unknown. As there is little time following an phase 1 infarct for upregulation of nerves, however, it can be assumed nerves are atleast increased before the AMI episode. Funding Acknowledgement Type of funding sources: None.

Manual Acupuncture at ST37 Modulates TRPV1 in Rats with Acute Visceral Hyperalgesia via Phosphatidylinositol 3-Kinase/Akt Pathway.

Acupuncture can significantly ameliorate inflammatory pain in acute visceral hyperalgesia. Hyperalgesia is attenuated by inflammatory mediators that activate transient receptor potential vanilloid 1 (TRPV1), and TRPV1 is regulated by nerve growth factor (NGF)-induced phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, it is unknown whether NGF-induced PI3K/Akt pathway is associated with manual acupuncture (MA). In this study, the effect and mechanism of MA at Shangjuxu (ST37) and Quchi (LI11) were examined using an acetic acid-induced rat model with visceral hyperalgesia. We demonstrated that MA at ST37 significantly decreased abdominal withdrawal reflex (AWR) scores, proinflammatory cytokine expression (TNF-α, IL-1β, and IL-6), and TRPV1 protein and mRNA expression in rats with acute visceral hyperalgesia compared with the untreated controls, while MA at LI11 showed no effect. The effects of MA at ST37 were reversed after treatment with the PI3K agonist IGF-1 30 min before MA. In rats with visceral hyperalgesia, the upregulation of NGF, tropomyosin-receptor-kinase A (TrkA), PI3K, and phosphorylation-Akt (p-Akt) was decreased by MA at ST37, indicating that TRPV1 regulation via the NGF-induced PI3K/Akt pathway plays a vital role in the effects of MA-mediated amelioration of acute visceral hyperalgesia.

Acceleration of TAA-Induced Liver Fibrosis by Stress Exposure Is Associated with Upregulation of Nerve Growth Factor and Glycopattern Deviations.

Liver fibrosis results from many chronic injuries and may often progress to cirrhosis and hepatocellular carcinoma (HCC). In fact, up to 90% of HCC arise in a cirrhotic liver. Conversely, stress is implicated in liver damage, worsening disease outcome. Hence, stress could play a role in disrupting liver homeostasis, a concept that has not been fully explored. Here, in a murine model of TAA-induced liver fibrosis we identified nerve growth factor (NGF) to be a crucial regulator of the stress-induced fibrogenesis signaling pathway as it activates its receptor p75 neurotrophin receptor (p75NTR), increasing liver damage. Additionally, blocking the NGF decreased liver fibrosis whereas treatment with recombinant NGF accelerated the fibrotic process to a similar extent than stress challenge. We further show that the fibrogenesis induced by stress is characterized by specific changes in the hepatoglycocode (increased β1,6GlcNAc-branched complex N-glycans and decreased core 1 O-glycans expression) which are also observed in patients with advanced fibrosis compared to patients with a low level of fibrosis. Our study facilitates an understanding of stress-induced liver injury and identify NGF signaling pathway in early stages of the disease, which contributes to the established fibrogenesis.

CS nerve growth factor regulates sevoflurane anesthesiainduced nerve injury in nerve cells

Purpose: Sevoflurane is the most commonly used anesthetic agent for surgery. However, it is associated with deficiency in learning and memory abilities. The study was aimed at investigating the role of nerve growth factor (NGF) in sevoflurane anesthesia-induced nerve injury.Methods: RT-qPCR assay was applied to measure expressions of NGF, miR-98-5p and other factors related to apoptosis. CCK-8 assay was used for detecting cell viability while luciferase reporter assay was employed to measure binding condition between miR-98-5p and NGF. Expressions of proteins in PI3K/AKT/mTOR signaling pathway was measured with western blot.Results: Sevoflurane reduced cell viability of RGC-5 cells, promoted apoptosis and reduced the expression of NGF. In sevoflurane-induced RGC-5 cells, over-expression of NGF promoted cell viability with reduced apoptosis. Also, there was reduction in the protein expression of PI3K/AKT/mTOR signaling pathway by sevoflurane, while up-regulation of NGF promoted the expressions of these proteins. In the presence of PI3K inhibitor, reduction cell viability was reduced but apoptosis increased. Luciferase reporter assay detected MiR-98-5p as the target gene of NGF and its overexpression restored high cell viability in the over-expressed NGF. The rate of apoptosis and expressions of proteins was also restored with up-regulation of miR-98-5p.Conclusion: Sevoflurane caused damage to nerve cells, while over-expression of NGF reduced the injury through PI3K/AKT/mTOR signaling pathway and suppression of miR-98-5p.
 Keywords: Nerve growth factor, Sevoflurane, Nerve injury, Anesthesia, miR-98-5p

Interleukin-10 Reduces Neurogenic Inflammation and Pain Behavior in a Mouse Model of Type 2 Diabetes.

PurposeNeurogenic inflammation is a major component of chronic neuropathic pain. Previously, we established the db/db mouse as an animal model of painful diabetic neuropathy (PDN) of type 2 diabetes. In the current study, we investigate the roles of interleukin (IL)-10, an anti-inflammatory cytokine, in the development of neurogenic inflammation and pain behavior in db/db mouse.Materials and MethodsWe first studied IL-10 expression in lumbar dorsal root ganglion (LDRG) neurons of db/db mice using immunohistochemistry, immunoblots, and reverse transcription polymerase chain reaction during the period of pain behavior (from 8 to 16 wk of age). To determine if the reduced IL-10 expression mediates the mechanical allodynia in db/db mice, we administered recombinant mouse IL-10 or saline (control) intraperitoneally to control db/+ and db/db mice starting at 8 wk of age. IL-10 treatment was repeated every other day for 2 wk until the mice reached 10 wk of age.ResultsDuring the period of pain behavior, reduction of IL-10 protein and gene expression was detected in LDRG of db/db mice. Treatment with recombinant IL-10, from 8 to 10 wk of age, alleviates pain behaviors in db/db mice without affecting other diabetic parameters. In parallel, IL-10 treatment reduced the upregulation of nerve growth factor (NGF), inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and high-affinity NGF receptor (Trk A) in LDRG, as well as the numbers of iNOS-positive Langerhans cells and CD-68-positive dermal dendritic cells in the hind-foot-pad skin in db/db mice.ConclusionOur findings suggest that the reduction in neuronal IL-10 increases inflammatory phenomena, ultimately contributing to PDN. These results suggest that the dysregulation of cytokine-mediated inflammation contributes to the development of PDN in db/db mice. Targeting this pathophysiologic mechanism could be an effective approach for treating PDN in type 2 diabetes.

Role of CD14-positive cells in inflammatory cytokine and pain-related molecule expression in human degenerated intervertebral discs.

Multiple human and animal studies suggest that the upregulation of inflammatory cytokines and other pain-related molecules in degenerated or injured intervertebral discs (IVDs) may cause discogenic low back pain (LBP). We previously reported that macrophages in injured IVD in mice produced inflammatory cytokines, but not other pain-related molecules. CD14 is a monocyte marker expressed mainly by macrophages. The aim of the current study was to evaluate the role of CD14-positive cells in inflammatory cytokine and pain-related molecule expression in human degenerated IVD. IVD samples were harvested from 14 patients, including 10 with lumbar spinal stenosis, four with adult spinal deformity, and one with lumbar disc herniation during spinal interbody fusion surgery. Harvested IVD-derived mononuclear cells were obtained and CD14-positive (+) and CD14-negative (-) cells were separated using CD14 antibody and streptavidin-labeled magnetic beads. Inflammatory cytokines messenger RNA (mRNA) in the CD14(+) and CD14(-) cells, including tumor necrosis factor ɑ (TNFA), in, terleukin-1β (IL1B) and IL6, were determined using quantitative polymerase chain reaction (qPCR) and their expression levels were compared. To evaluate factors controlling the regulation of pain-related molecules mRNA expression, cultured CD14(-) and CD14(+) cells from IVDs were stimulated with recombinant human TNF-ɑ and IL-1β and levels of pain-related molecules, including calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF) were determined using qPCR. Levels of TNFA, IL1B, IL6, and NGF in CD14(+) cells were significantly increased compared with those in CD14(-) cells (TNFA, p = 0.006; IL1B, p = .017; IL6, p = .010; NGF, p = .027). Following TNFA stimulation, NGF levels were significantly increased in CD14(-) and CD14(+) cells (CD14(-), p = .003; CD14(+), p < .001) and CGRP was significantly increased in CD14(-) IVD cells (p = .040). Following IL1B stimulation, NGF levels were significantly increased in CD14(-) cells (p = .004). CD14(+) cells had higher TNFA, IL1B, IL6, and NGF expressions than CD14(-) cells in human degenerated IVDs. Additionally, TNFA stimulation promoted the upregulation of NGF and CGRP in CD14(-) cells. These findings suggested that CD14(+) cells directly and indirectly contributed to inflammatory cytokine and pain-related molecule expression in human degenerated IVD. CD14(+) cells might be important in the pathological mechanism of chronic discogenic LBP in humans.

Involvement of the G-Protein-Coupled Receptor 4 in the Increased Expression of RANK/RANKL/OPG System and Neurotrophins by Nucleus Pulposus Cells under the Degenerated Intervertebral Disc-Like Acidic Microenvironment.

Intervertebral disc (IVD) degeneration is associated with local inflammation and increased expression of neurotrophins. Acidic microenvironment is believed to cause the progression of IVD degeneration. However, there is a paucity of information regarding the relationship between acidic microenvironment and the inflammation and expression of neurotrophins in IVD. G-protein-coupled receptor 4 (GPR4) is a pH-sensing receptor, which can activate the inflammation and increase the expression levels of nerve growth factor in acidic microenvironment. In this study, culture media with pH 7.2 (representing the normal IVD-like acidic condition) and pH 6.5 (degenerated IVD-like acidic condition) were prepared. The gene and protein expression levels of GPR4 in SD rat nucleus pulposus cells were determined under the acidic conditions. And cyclic AMP (cAMP), the second messenger of GPR4, was assayed. Furthermore, the expression levels of receptor activator of nuclear factor κ B (RANK), RANKL ligand (RANKL), osteoprotegerin (OPG), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were also determined. To clarify the involvement of GPR4 in the upregulation of the expression of RANK/RANKL/OPG system and neurotrophins, gene knockdown and forced expression of GPR4 and inhibiting its downstream cAMP accumulation and Ca2+ mobilization were performed. The alternation of the expression levels of matrix metalloproteinase-3 (MMP-3), MMP-13, and aggrecanase-2 (ADAMTS-5) were evaluated by RT-PCR and western blot. The results showed that GPR4 was expressed in rat nucleus pulposus cells, and the expression was upregulated under the degenerated IVD-like acidic microenvironment. cAMP accumulation levels were increased under the degenerated IVD-like acidic culture conditions. The expression levels of RANK, RANKL, OPG, NGF, and BNDF were significantly upregulated under the degenerated IVD-like acidic microenvironment. GPR4 knockdown and reduction of cAMP by the inhibitor SQ22536 abolished the upregulation of the expression of RANK, RANKL, OPG, NGF, and BNDF under the degenerated IVD-like acidic microenvironment. On the opposite, acidosis-induced cAMP accumulation and upregulation of RANK, RANKL, OPG, NGF, and BNDF were further promoted by GPR4 overexpression. The expression levels of MMP-3, MMP-13, and ADAMTS-5 were upregulated under the degenerated IVD-like acidic condition, which can be promoted or attenuated by GPR4 overexpression or knockdown, respectively. We concluded that GPR4-mediated cAMP accumulation was involved in the increased expression of RANK/RANKL/OPG system and neurotrophins by nucleus pulposus cells under the degenerated IVD-like acidic microenvironment.

Green synthesis of silver nanoparticles by Nigella sativa extract alleviates diabetic neuropathy through anti-inflammatory and antioxidant effects.

Green synthesis of silver nanoparticles has gained great interest among scientists. In view of this data, we conducted this study to identify the ameliorative effect of green synthesis of silver nanoparticles using Nigella sativa extract in diabetic neuropathy induced experimentally. In this study, 50 adult male albino rats were used and they were randomly divided into five groups; the first group was the healthy control group, the second group were the diabetic neuropathy diabetic neuropathy induced, Groups (3-6) diabetic neuropathy induced group and treated with silver nanoparticles, Nigella sativa extract and green synthesized silver nanoparticles using Nigella sativa extract respectively. Biochemical parameters including diabetic, inflammatory and antioxidant biomarkers were evaluated. Brain histopathology was also performed. Results revealed substantial rise in glucose, AGE, aldose reductase with insulin reduction in diabetic neuropathy induced group as compared to healthy control. Also, inflammatory markers increased significantly in diabetic neuropathy induced group. A remarkable change in oxidative status was observed in the same group. Furthermore, significant decline in nitrotyrosin level was observed. Regarding gene expression, we found significant down regulation in brain TKr A accompanied by upregulation of nerve growth factor in diabetic neuropathy group comparing with healthy control. Several treatments for diabetic neuropathy remarkably ameliorate all the investigated biomarkers. Histological findings are greatly relied on for the results achieved in this study. Therefore, it can be established that green synthesis of silver nanoparticles in combination with Nigella sativa extract could be a newly neuroprotective agents against inflammation and oxidative stress characterizing diabetic neuropathy through their antidiabetic, anti-inflammatory and anti-oxidants effects.

Abstract 7: Nerve Growth Factor Derives From Pericytes And Smooth Muscle Cells After Extremity Trauma

Purpose: Neurotrophic factors like nerve growth factor (NGF) have been described in the literature as a crucial regulator in developmental biology and inflammation of musculoskeletal tissues. While extremity trauma has been shown to elicit re-engagement of developmental programs like NGF upregulation and invasion of new sensory nerve endings in a mature organism, the source of NGF has remained largely unknown. In a validated extremity trauma mouse model, we have previously demonstrated a significant attenuation of injury site re-innervation by sensory fibers following inhibiting of afferent neural signaling and NGF binding of its receptor TrkA. Given this significant impact of NGF on sensory reinnervation of injured soft tissues and downstream bone formation, we examined the source of NGF after extremity trauma using single cell transcriptomic and reporter proteomic technologies. Methods: A 30% dorsal burn and Achilles transection was performed. The tendon site tissues were harvested from baseline (t0) and day 3, 7, and 21 (n=3-4/group) after induction. Samples were prepared for library generation on a 10x Genomics Chromium Controller, sequenced on a Illumina HiSeq 4000, and analyzed with Cell Ranger Software for pre-processing and alignment to the mm10 genome. Downstream analyses including unsupervised clustering and canonical correlation analyses were performed with Seurat. Immunofluorescent (IF) labeling of cellular markers including αSMA was performed using NGF-eGFP reporter mice at baseline, and at 1,3 and 9 weeks after injury (n=2-3/group). Results: To localize cell specific Ngf expression from injured soft tissue, 9 cell clusters were defined across all timepoints: mesodermal (Prrx1) populations including Acta2+ pericyte/vascular smooth muscle (SMC) and Pdgfra+ mesenchymal cells, two Pecam1+ endothelium, and four inflammatory cell populations (mixed, B cell, T cell, and neutrophil). Ngf was found uniquely enriched in the pericyte/SMC cluster in a composite view. These pericytes/SMCs were found in increasing number and Ngf expression across timepoints, peaking at day 21. To further characterize this joint cluster (1273 cells), the pericyte/SMC cluster was isolated and blindly re-clustered to produce new sub-clusters, distinguishing Pdgfrb (platelet derived growth factor receptor beta)highPrrx1highAbcc9+ pericytes from Acta2highPln+Myh11high SMCs; an uncharacterized cluster with 24 cells was discarded from analysis. Of these two sub-clusters, vascular SMCs demonstrated the highest Ngf expression at baseline (mean normalized Ngf expression of 0.52 [in 41% of cells] vs 0.28 [in 23% of cells]. IF of NGF-eGFP reporter mice show robust NGF co-localization with αSMA+ SMCs. Conclusions: This is the first work characterizing the pericyte and vascular SMC as a major contributor to NGF signaling at an extremity injury site. The fine resolution defining the cellular source of NGF provides insight into potential mechanisms correlating nascent nerve and vascular growth given the intersection of these pathways at the level of pericytes and myofibroblastic SMCs that will inform future candidate therapeutics to improve extremity trauma healing and re-innervation.

Mechanisms underlying immobilization-induced muscle pain in rats.

We investigated the mechanisms underlying immobilization-induced muscle pain in rats. In rat skeletal muscle, pressure pain threshold (PPT) of the gastrocnemius muscle was measured, and nerve growth factor (NGF) level, peripheral nerve fiber density, macrophage number, and interleukin-1β (IL-1β) mRNA expression were examined. An NGF receptor inhibitor was injected intramuscularly to assess the relationship between PPT and NGF levels. Immobilization resulted in a decrease in PPT and increases in NGF level, C-fiber density, M1 macrophage number, and IL-1β mRNA expression. Injection of NGF receptor inhibitor reversed the decrease in PPT. NGF upregulation may be a major contributor to immobilization-induced muscle pain. The increases in C-fiber density, M1 macrophage number, and IL-1β mRNA expression may be related to immobilization-induced muscle pain.

Differential impact of psychological and psychophysical stress on low back pain in mice.

Low back pain (LBP) is a highly prevalent and disabling condition whose initiating factors are poorly understood. It is known that psychological and physical stress is associated with LBP but the causal relationship, mechanisms, and mediators have not been elucidated, and a preclinical model enabling the investigation of causality and thereby critically contributing to clinical translation does not exist. In this study, we first established and characterized a myofascial LBP model in mice based on nerve growth factor (NGF) injection into the low back muscles. Second, we investigated the effect of 2 different stress paradigms on this mouse LBP model by applying the chronic unpredictable stress and vertical chronic restraint stress (vCRS) paradigms, to mimic psychological and psychophysical stress, respectively. In these studies, we combined longitudinal behavioral tests with gene and protein expression analysis in the muscle, dorsal root ganglia, and spinal cord. Nerve growth factor-induced LBP was characterized by long-lasting local and plantar mechanical hypersensitivity, cold hyperalgesia, decreased grip strength and wheel running activity, and time-dependent changes of neuropeptide and glial markers in the spinal cord. Interestingly, the exposure to chronic unpredictable stress slightly worsened pain behavior, whereas vCRS primed and highly aggravated pain in this LBP model, by causing per se the intramuscular upregulation of endogenous NGF and increased spinal astrocyte expression. Our mouse model, particularly the combination of NGF injection and vCRS, suggests that similar mechanisms are important in nonspecific LBP and might help to investigate certain aspects of stress-induced exacerbation of pain.

Nerve growth factor induced farnesoid X receptor upregulation modulates autophagy flux and protects hepatocytes in cholestatic livers

Upregulation of nerve growth factor (NGF) in parenchymal hepatocytes has been shown to exert hepatoprotective function during cholestatic liver injury. However, the modulatory role of NGF in regulation of liver autophagy remains unclear. This study aimed to scrutinize the regulatory role of NGF in hepatic expression of farnesoid X receptor (FXR), a bile acid (BA)-activated nuclear receptor, and to determine its cytoprotective effect on BA-induced autophagy and cytotoxicity. Livers of human hepatolithiasis and bile duct ligation (BDL)-induced mouse cholestasis were used for histopathological and molecular detection. The regulatory roles of NGF in autophagy flux and FXR expression, as well as its hepatoprotection against BA cytotoxicity were examined in cultured hepatocytes. FXR downregulation in human hepatolithiasis livers showed positive correlation with hepatic NGF levels. NGF administration upregulated hepatic FXR levels, while neutralization of NGF decreased FXR expression in BDL-induced cholestatic mouse livers. In vitro studies demonstrated that NGF upregulated FXR expression, increased cellular LC3 levels, and exerted hepatoprotective effect in cultured primary rat hepatocytes. Conversely, autophagy inhibition abrogated NGF-driven cytoprotection under BA exposure, suggesting involvement of NGF-modulated auophagy flux. Although FXR agonistic GW4064 stimulation did not affect auophagic LC3 levels, FXR activity inhibition significantly potentiated BA-induced cytotoxicity and increased cellular p62/SQSTM1 and Rab7 protein in SK-Hep1 hepatocytes. Moreover, FXR gene silencing abolished the protective effect of NGF under BA exposure. These findings support that NGF modulates autophagy flux via FXR upregulation and protects hepatocytes against BA-induced cytotoxicity. NGF/FXR axis is a novel therapeutic target for treatment of cholestatic liver diseases.

Magnesium protects mouse hippocampal HT22 cells against hypoxia-induced injury by upregulation of miR-221.

Magnesium (Mg2+ ) has been shown to exert neuroprotective effects against hypoxia. However, it still remains elusive whether Mg2+ protected mouse hippocampal HT22 cells against hypoxia-evoked damages. Therefore, we aimed to investigate the function of Mg2+ and mechanisms associated with microRNA-221 (miR-221). HT22 cells were exposed to 3% O2 for 24 hours to induce hypoxic damages with 21% as a normoxic culture condition. The damages were monitored by viability, migration, and apoptosis of HT22 cells with or without Mg2+ pretreatment. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied to examine the alteration of miR-221, miR-210, and miR-17-5p. Transduction was carried out to artificially alter the expression of miR-221 and nerve growth factor (NGF), which was confirmed by qRT-PCR or Western blot assays. To blunt phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and nuclear factor κB (NF-κB), LY294002 (10 µM) and BAY 11-7082 (10 µM) were used. We observed Mg2+ protected HT22 cells against hypoxia-induced damages by upregulating miR-221. Further, miR-221 positively regulated NGF expression. Overexpression of NGF alleviated cell injury, while suppression of NGF aggravated cell injury. Moreover, miR-221 elevated NGF by inducing phosphorylation of regulators in PI3K/AKT and NF-κB transduction cascades and then alleviated cell injury. In conclusion, Mg2+ protected HT22 cells against hypoxia-induced damages by upregulation of miR-221 and NGF. These findings provided insights into the development of improved strategies for clinical application.

Disc degeneration induces a mechano-sensitization of disc afferent nerve fibers that associates with low back pain

We aimed to investigate mechano-sensitivity at the afferent nerve fibers projecting to degenerated intervertebral disc (IVD) and nociceptive behaviour in a rat model of low back pain (LBP). Animal model with LBP was established by lumbar 4/5 IVD puncture and nucleus pulposus aspiration. Invivo single nerve recordings (n=121) were introduced to measure discharge frequency at the afferent nerve fiber innervating the IVD during mechanical stimulations (von Frey filament or intradiscal pressure). Nerve growth factor (NGF) expression levels in the IVD (n=20) were assessed by Western blot. LBP-related behaviour (n=22) was assessed by measuring changes in rearing, mechanical paw-withdrawal threshold, and dynamic weight bearing in a freely walking rat. Inhibitory effect of morphine on the neuronal excitability (n=19) and painful behaviour (n=28) was also assessed. Compared to those with sham or naïve IVD, animal group with degenerated IVD displayed the sensitized neuronal responses and painful behaviour, with hyperexcitability of the afferent nerve fibers in any range of mechanical stimulations (von Frey filament stimulation; 1, 2, and 26g; intradiscal pressure, 1,500-3,000mm Hg), strong upregulation of NGF (200-250 % increase), and LBP-like behaviour such as failure of rearing, front limbs-dependent walking pattern, and hypersensitivity in hind-paws. However, the neuronal hyperexcitability and pain behaviour were attenuated after local (30 μM) or systemic (3mgkg-1) morphine administration. Our study suggests that enhanced mechano-sensitivity at the afferent nerve fiber innervating degenerated IVD is deeply correlated with LBP development, which supports the hypothesis that hyperexcited responses at the nerve fibers represent a decisive source of LBP.

A conditionally immortalized Gli1-positive kidney mesenchymal cell line models myofibroblast transition.

Glioma-associated oncogene homolog-1 (Gli1)-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis, but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KGli1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation, and display robust myofibroblast differentiation upon treatment with transforming growth factor-β (TGF-β). Coculture of KGli1 cells with endothelium stabilizes capillary formation. Single-cell RNA sequencing (scRNA-seq) analysis during differentiation identified autocrine ligand-receptor pair upregulation and a strong focal adhesion pathway signal. This led us to test the serum response factor inhibitor CCG-203971 that potently inhibited TGF-β-induced pericyte-to-myofibroblast transition. scRNA-seq also identified the unexpected upregulation of nerve growth factor (NGF), which we confirmed in two mouse kidney fibrosis models. The Ngf receptor Ntrk1 is expressed in tubular epithelium in vivo, suggesting a novel interstitial-to-tubule paracrine signaling axis. Thus, KGli1 cells accurately model myofibroblast activation in vitro, and the development of this cell line provides a new tool to study resident mesenchymal stem cell-like progenitors in health and disease.

Epalrestat, an Aldose Reductase Inhibitor, Restores Erectile Function in Streptozocin-induced Diabetic Rats

Epalrestat, an aldose reductase inhibitor (ARI), was adopted to improve the function of peripheral nerves in diabetic patients. The aim of this study was to investigate whether epalrestat could restore the erectile function of diabetic erectile dysfunction using a rat model. From June 2016, 24 rats were given streptozocin (STZ) to induce the diabetic rat model, and epalrestat was administered to ten diabetic erectile dysfunction (DED) rats. Intracavernous pressure (ICP) and mean systemic arterial pressure (MAP), levels of aldose reductase (AR), nerve growth factor (NGF), neuronal nitric oxide synthase (nNOS), α-smooth muscle antigen (α-SMA), and von Willebrand factor (vWF) in the corpus cavernosum were analyzed. We discovered that epalrestat acted on cavernous tissue and partly restored erectile function. NGF and nNOS levels in the corpora were increased after treatment with epalrestat. We also found that the content of α-SMA-positive smooth muscle cells and vWF-positive endothelial cells in the corpora cavernosum were declined. Accordingly, epalrestat might improve erectile function by increasing the upregulation of NGF and nNOS to restore the function of the dorsal nerve of the penis.