Nerve Growth Factor Protein Expression Research Articles

Immune System Induction of Nerve Growth Factor in an Animal Model of Multiple Sclerosis: Implications in Re-Myelination and Myelin Repair.

Nerve growth factor (NGF) expression is augmented during neuroinflammation. However, its function in the dorsal root ganglia (DRG) and spinal cord (SC) during experimental autoimmune encephalomyelitis (EAE), the inflammatory model of Multiple Sclerosis, is indistinct. Thus, the role of antigenically induced NGF in Lewis rats under a state of EAE was considered. NGF mRNA and protein expression were highly increased in DRG and SC tissues in animals with EAE. Between 18 and 24 days post induction (dpi), NGF mRNA and protein were elevated in the DRG, correlating with neurological recovery. In the SC, an increase in NGF protein at 12 dpi was, in contrast, preceded by neurological recovery. NGF mRNA expression became elevated in the SC at 15 dpi at the onset of neurological improvement and amelioration of EAE. This study revealed that antigenic induction of the 25 kDa pro-NGF isoform is associated with the disease course of EAE. Our findings suggest the induction of NGF represents an adaptive response against immune-mediated neuroinflammation in the DRG and SC that likely contributes to the EAE attenuation.

Expression of NGF, BDNF and their high-affinity receptors in ovine mammary glands during development and lactation.

The distribution of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their high-affinity tyrosine kinase receptors TrkA and TrkB was investigated by immunohistochemical method in the mammary gland of ewes from prepubertal stage to involution. NGF and BDNF protein expressions were strong during development of glands at prepubertal stage and during pregnancy and decreased during lactation and involution. The expressions localized in both stromal and parenchymal cells of developing gland were mainly arranged in the apical side of secretory cells during lactation. These observations were also confirmed at transcriptional level by RT-PCR analyses. The highest expression of all genes significantly occurred at prepubertal stage. NGF was then down-regulated from pregnancy to involution, and no statistical differences were observed among these stages. The receptor TrkA was also under-expressed from pregnancy to involution, and its expression significantly differed between pregnancy and 30 days of lactation and also between 30 and 60 days of lactation. BDNF was significantly down-regulated at 60 days of lactation in comparison with prepubertal stage and again between pregnancy and 30 days of lactation. The relative abundance of its receptor, TrkB, showed also a significant down-regulation at 60 days of lactation in comparison with pregnancy and involution. Among the myriad of other molecular signals involved in the mammary gland cycle, the local production of neuropeptides and their receptors could be of interest in understanding their potential role in mammary biology.

Stimulation of endothelin B receptors by IRL-1620 decreases the progression of Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by severe cognitive impairment that ultimately leads to death. Endothelin (ET) and its receptors have been considered as therapeutic targets for AD. Recent studies in our lab have shown that stimulation of ETB receptors provide significant neuroprotection following Aβ1–40 administration. It is possible that IRL-1620 may be neuroprotective due to angiogenesis. However, the effect of IRL-1620 on neurovascular remodeling following Aβ1–40 administration has not been established. The purpose of this study was to determine the effect of stimulation of ETB receptors by IRL-1620 on vascular and neuronal growth factors after Aβ1–40 administration. Rats were treated with Aβ1–40 (day 1, 7 and 14) in the lateral cerebral ventricles using stereotaxically implanted cannula and received three intravenous injections of IRL-1620 (an ETB agonist), and/or BQ788 (an ETB antagonist) at 2-h interval on day 8; experiments were performed on day 15. Rats were sacrificed for estimation of brain ETB receptors, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) expression using immunofluorescence and Western blot. In the Morris swim task, amyloid-β (Aβ)-treated rats showed a significant (p<0.0001) impairment in spatial memory. Rats treated with IRL-1620 significantly (p<0.001) reduced the cognitive impairment induced by Aβ. BQ788 treatment completely blocked IRL-1620-induced improvement in cognitive impairment. IRL-1620 treatment enhanced the number of blood vessels labeled with VEGF compared to vehicle treatment. Additionally, cells showed increased (p<0.001) positive staining for NGF in IRL-1620-treated animals. ETB, VEGF and NGF protein expression significantly (p<0.001) increased in the brain of IRL-1620-treated rats as compared to vehicle. Pretreatment with BQ788 blocked the effects of IRL-1620, thus confirming the role of ETB receptors in the neurovascular remodeling actions of IRL-1620. Results of the present study demonstrate that IRL-1620 improves both acquisition (learning) and retention (memory) on the water maze task and enhances angiogenic and neurogenic remodeling. These findings indicate that the ETB receptor may be a novel therapeutic target for AD and other neurovascular degenerative disorders.

Effect of Bushenwenyanghuayu decoction on nerve growth factor and bradykinin/bradykinin B1 receptor in a endometriosis dysmenorrhea mouse model

ObjectiveTo observe the effects of Bushenwenyanghuayu decoction (BD), a Traditional Chinese Medicine (TCM), on the serum concentration of nerve growth factor (NGF) and bradykinin (BK), and protein and mRNA levels of NGF and bradykinin B1 receptor (BKB1R) in a mouse model of endometriosis dysmenorrhea. MethodsSeventy-five experimental female BALB/c mice were randomly divided into five groups, 15 mice each: sham, model, BD high dose (61.67 g/kg), BD low dose (15.42 g/kg), and gestrinone (0.4 mg/kg) groups. All the mice except for those in the sham group underwent auto-transplantation surgery and were gavaged estradiol valerate (0.5 mg/kg, daily for 12 days) after surgery. On the 12th day, 1 h after administration, writhing response was induced by intraperitoneal injection of oxytocin at 2 U/mouse. The writhing frequency and latency were recorded and the volume of the ectopic foci was measured. The concentration of serum NGF and BK was detected by enzyme-linked immunosorbent assay, the protein expression of NGF and BKB1R was tested by immunohistochemistry and western blotting, and NGF and BKB1R mRNAs were detected by real-time PCR. ResultsCompared with the model group, the volume of the ectopic foci in the treatment groups was significantly lower (P < 0.01), the writhing frequency was decreased (P < 0.05), and the writhing latency was prolonged (P < 0.01). Compared with the sham group, serum NGF and BK levels in the model group were significantly increased (P < 0.01). There were positive correlations for writhing frequency among the NGF and BK groups (P < 0.01). The serum NGF and BK levels were significantly lower in the treatment groups than the model group (P < 0.05). The protein expression of NGF, BKB1R was significantly decreased in the treatment groups compared with the model group (P < 0.01). NGF and BKB1R mRNA expression was significantly decreased in the treatment groups compared with the model group (P < 0.01). ConclusionNGF and BK/BKB1R may play an important role in the development of endometriosis-associated dysmenorrhea, and BD was found to inhibit the development of endometriosis and relieve dysmenorrhea by influencing NGF and BK/BKB1R mRNA and protein levels.

Effects of pinacidil on changes to the microenvironment around the incision site, of a skin/muscle incision and retraction, in a rat model of postoperative pain.

The aim of the present study was to evaluate the influence of the microenvironment around an incision site, on peripheral and central sensitization. The effects of pinacidil activation of ATP-sensitive potassium (KATP) channels prior to skin/muscle incision and retraction (SMIR) surgery were assessed. A total of 24 male Sprague Dawley rats were randomly assigned to four groups: Control, sham (incision operation), SMIR (incision plus retraction 1 h after the skin/muscle incision) and pinacidil (SMIR plus pinacidil). The rats in the pinacidil group were intraperitoneally injected with pinacidil prior to the SMIR procedure. The mechanical withdrawal threshold (MWT) was determined at each time point. The microvessel density (MVD) value was determined by immunohistochemistry, and western blotting was performed to analyze the relative protein expression levels of nerve growth factor (NGF), glucose transporter protein-1 (GLUT1) and C-jun N-terminal kinases. There was a significant reduction in the levels of MVD, GLUT1 and MWT following SMIR surgery as compared with the incision alone, and a significant increase in the NGF protein expression levels. In the SMIR group, the MVD value was significantly increased seven days after surgery, as compared with three days after surgery. Additionally, intraperitoneal administration of pinacidil prior to the SMIR surgery inhibited the SMIR-induced reduction in MWT and MVD and attenuated the SMIR-induced GLUT1 reduction. The results of the present study suggest that the microenvironment around an incision site may affect the development of peripheral and central sensitization. In addition, pinacidil had an inhibitory effect on the formation of the inflammatory microenvironment around the incision site through activation of KATP channels, thereby inhibiting peripheral and central sensitization.

Effects of denervation on angiogenesis and skeletal muscle fiber remodeling of ischemic limbs

To establish a murine model of hindlimb ischemia and denervation and evaluate the effects of neural factors on angiogenesis and skeletal muscle fiber remodeling. A total of 54 mice were randomly allocated into three groups of control (n = 6), hindlimb ischemia (n = 24), hindlimb ischemia and denervation (n = 24). The model of hindlimb ischemia was established by ligating femoral artery. And the model of hindlimb ischemia and denervation was established by transecting sciatic nerve on the basis of hindlimb ischemia modeling. Gastrocnemius of ischemic hindlimbs were harvested under the condition of peritoneal cavity anesthesia at Days 7, 14, 21 and 28 postoperation. And then the mice were sacrificed. The gastrocnemius of three groups were tested by hematoxylin-eosin staining, CD31 and PCNA immunohistochemical staining and myosin ATPase staining. The protein expressions of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) were detected by enzyme-linked immunosorbent assay. At Day 7 postoperation, hindlimbs of two intervention groups showed ischemic manifestations as compared with controls. And semiquantitative assessment of ischemic damage showed the proportion level I-IV of hindlimb ischemia and denervation group was significantly higher than that of hindlimb ischemia group (33.4% vs. 16.6%, χ(2) = 7.94, P = 0.047). At Day 28 postoperation, the values of endothelial cell proliferation index [(2.4 ± 0.4)% vs. (5.7 ± 1.0)%, P = 0.000], capillary density [(0.44 ± 0.05) vs. (0.61 ± 0.07), P = 0.000], NGF protein expression [(6.71 ± 0.94) vs. (8.36 ± 0.77) pg/ml, P = 0.008] and VEGF protein expression [(11.32 ± 1.56) vs. (15.13 ± 2.93) pg/ml, P = 0.018] of hindlimb ischemia and denervation group significantly decreased as compared with hindlimb ischemia group. At Day 28 postoperation, the proportion of type II muscle fiber in gastrocnemius was significantly higher than that of hindlimb ischemia group [(84.9 ± 3.8)% vs. (78.8 ± 1.8)%, P = 0.008]. Neural factors play important roles in promoting angiogenesis and maintaining muscle fiber types in ischemic limbs. But its molecular regulatory mechanism requires further studies.

Effect of acanthopanax senticosus saponins on expression of brain-derived neurotrophic factor and nerve growth factor in rat spinal cord after acute spinal cord injury

Objective To study the effect of acanthopanax senticosus saponins(ASS)on expression of brain-derived neurotrophic factor(BDNF)and nerve growth factor(NGF)in rat spinal cord after spinal cord injury,so as to explore the protective effect of ASS against spinal cord injury.Methods A total of 45 male rats were evenly randomized into three groups:sham operation group,model group and ASS intervention group.The experimental animals were anesthetized with 10%chloral hydrate and then acute spinal cord injury model was made using modified Allen's method.After modeling the sham group received normal feeding,the model group was intraperitoneally given normal saline for 7consecutive days,and the ASS intervention group was given appropriate dose of ASS for 7consecutive days.Then ELISA was used to detect the serum malondialdehyde(MDA)and superoxide dismutase(SOD);the pathological changes of spinal cord tissue were observed,and the expression of BDNF and NGF was examined by Western blotting analysis.Results Compared with the sham group,the other two groups had significantly increased serum MDA(P0.05)and significantly decreased SOD level(P0.05),with notable spinal cord injury;besides,the two groups also had significantly increased expression of BDNF and NGF protein in the spinal cord tissue(P0.05).Compared with the model group,ASS group had significantly improved spinal cord injury,with significantly decreased serum MDA content(P0.05)and increased SOD content and BDNF,NGF expression in the spinal cord tissue(P0.05).Conclusion ASS can promote BDNF and NGF protein expression in neurons of spinal cord,improving repair of damaged neurons and protecting spinal cord against acute injury in rats.

Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells.

Preliminary studies have suggested that a characteristic element of the matrix attachment region (MAR) in human interferon-β mediates the adhesion of vectors to Chinese hamster ovary (CHO) cells. In this study, we investigated if vector adhesion increased nerve growth factor (NGF) expression in CHO cells. The MAR characteristic element sequence of human interferon-β was inserted into the multiple-cloning site of the pEGFP-C1 vector. The target NGF gene was inserted upstream of the MAR characteristic element sequence to construct the MAR/NGF expression vector. The recombinant plasmid was transfected into CHO cells and stable monoclonal cells were selected using G418. NGF mRNA and protein expression was detected by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Plasmid reduction experiments were used to determine the state of transfected plasmid in mammalian cells. The insertion of MAR into the vector increased NGF expression levels in CHO cells (1.93- fold) compared to the control. The recombinant plasmid expressing the MAR sequence was digested into a linear space vector. The inserted MAR and NGF sequences were consistent with those inserted into the plasmid before recombination. Therefore, we concluded that the MAR characteristic element mediates vector adhesion to CHO cells and enhances the stability and efficiency of the target gene expression.

Left renal nerves stimulation facilitates ischemia-induced ventricular arrhythmia by increasing nerve activity of left stellate ganglion.

Renal sympathetic nerve (RSN) activity plays a key role in systemic sympathetic hyperactivity. Previous studies have shown that cardiac sympathetic hyperactivity, especially the left stellate ganglion (LSG), contributes to the pathogenesis of ventricular arrhythmias (VAs) after acute myocardial infarction (AMI). Twenty-eight dogs received 3 hours of continuous left-sided electrical stimulation of RSN (LRS; Group-1, n = 9), sham RSN stimulation (Group-2, n = 9), or LSG ablation plus 3 hours of LRS (Group-3, n = 10) were included. AMI was induced by ligating the proximal left anterior descending coronary artery. LRS was performed using electrical stimulation on the adventitia of left renal artery at the voltage increasing the systolic blood pressure (BP) by 10%. BP, heart rate variability (HRV), serum norepinephrine (NE) level, and LSG function were measured at baseline and the end of each hour of LRS. C-fos and nerve growth factor (NGF) protein expressed in the LSG were examined in Group-1 and Group-2. Compared with baseline, 3 hours of LRS induced a significant increase in BP, sympathetic indices of HRV, serum NE level, and LSG function. The incidence of VAs in Group-1 was significantly higher than other groups. The expression of c-fos and NGF protein in the LSG was significantly higher in Group-1 than Group-2. Three hours of LRS induces both systemic and cardiac sympathetic hyperactivity and increases the incidence of ischemia-induced VAs.

Social stress induces changes in urinary bladder function, bladder NGF content, and generalized bladder inflammation in mice.

Social stress may play a role in urinary bladder dysfunction in humans, but the underlying mechanisms are unknown. In the present study, we explored changes in bladder function caused by social stress using mouse models of stress and increasing stress. In the stress paradigm, individual submissive FVB mice were exposed to C57BL/6 aggressor mice directly/indirectly for 1 h/day for 2 or 4 wk. Increased stress was induced by continuous, direct/indirect exposure of FVB mice to aggressor mice for 2 wk. Stressed FVB mice exhibited nonvoiding bladder contractions and a decrease in both micturition interval (increased voiding frequency) and bladder capacity compared with control animals. ELISAs demonstrated a significant increase in histamine protein expression with no change in nerve growth factor protein expression in the urinary bladder compared with controls. Unlike stressed mice, mice exposed to an increased stress paradigm exhibited increased bladder capacities and intermicturition intervals (decreased voiding frequency). Both histamine and nerve growth factor protein expression were significantly increased with increased stress compared with control bladders. The change in bladder function from increased voiding frequency to decreased voiding frequency with increased stress intensity suggests that changes in social stress-induced urinary bladder dysfunction are context and duration dependent. In addition, changes in the bladder inflammatory milieu with social stress may be important contributors to changes in urinary bladder function.

Biological characteristics of rat dorsal root ganglion cell and human vascular endothelial cell in mono- and co-culture.

This study aimed to evaluate the biological activity of rat dorsal root ganglion cell (DRGC) and human vascular endothelial cell (HMVEC) in mono- and co-culture. Expression levels of vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) mRNA were measured by quantitative real-time RT-PCR (qRT-PCR). Western blot analysis was used to identify VEGF and NGF protein expressions. Cell injury was assessed by measuring cell viability with methylthiazol tetrazolium (MTT) assay. The results showed that VEGF and NGF mRNA levels in the HMVEC+DRGC group were significantly higher than those in the DRGC and HMVEC groups (all p < 0.05). There were also greater increases in both VEGF and NGF protein expressions in the HMVEC+DRGC group than those in the DRGC and HMVEC groups (all p < 0.05). The results of MTT analysis revealed significant differences in cell viability among the HMVEC+DRGC group and the DRGC and HMVEC groups (all p < 0.05). In summary, our findings provide evidence that DRGC and HMVEC in co-culture may exhibit greater biological activity than DRGC in mono-culture.

Aerobic exercise inhibits sympathetic nerve sprouting and restores β-adrenergic receptor balance in rats with myocardial infarction.

BackgroundCardiac sympathetic nerve sprouting and the dysregulation of β-adrenergic receptor (β-AR) play a critical role in the deterioration of cardiac function after myocardial infarction (MI). Growing evidence indicates that exercise provides protection against MI. The aims of this study were to investigate whether aerobic exercise following MI could inhibit sympathetic nerve sprouting and restore the balance of β3-AR/β1-AR.MethodsMale Sprague-Dawley rats were divided into three groups: sham-operated control group (SC), MI group (MI), and MI with aerobic exercise group (ME). The rats in ME group were assigned to 8 weeks of exercise protocol (16 m/min, 50 min/d, 5 d/wk). The expression of nerve growth factor (NGF), the sympathetic nerve marker-tyrosine hydroxylase (TH), the nerve sprouting marker-growth associated protein 43 (GAP43), and β1- and β2-AR expression in the peri-infarct area of the left ventricle (LV) were measured by Western blot and immunohistochemistry, while β3-AR expression was determined by Western blot and immunofluorescence. Endothelial nitric oxide synthase (NOS2), phospho-NOS2 (p-NOS2), and neuronal nitric oxide synthase (NOS1) were measured by Western blot.ResultsMI increased LV end-diastolic pressure (LVEDP), and decreased LV systolic pressure (LVSP). Compared with the MI group, aerobic exercise significantly decreased LVEDP and increased LVSP. The protein expression of TH, GAP43 and NGF was significantly increased after MI, which was normalized by exercise. Compared with the SC group, the ratios of β2-AR/β1-AR and β3-AR/β1-AR were elevated in the MI group, and the protein expression of β3-AR and NOS1 increased after MI. Compared with the MI group, the ratios of β2-AR/β1-AR and β3-AR/β1-AR were normalized in the ME group, while the protein expression of β3-AR and NOS1 significantly increased, and NOS2 was activated by exercise.ConclusionsAerobic exercise inhibits cardiac sympathetic nerve sprouting, restores β3-AR/β1-AR balance and increases β3-AR expression through the activation of NOS2 and NOS1 after myocardial infarction.

PD9-01 THERAPEUTIC EFFECT OF ANTISENSE OLOGONUCLEOTIDE (OND) TARGETING NERVE GROWTH FACTOR (NGF) ON BLADDER HYPESENSITIVITY IN RAT WITH EXPERIMENTAL COLITIS

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Interstitial Cystitis1 Apr 2014PD9-01 THERAPEUTIC EFFECT OF ANTISENSE OLOGONUCLEOTIDE (OND) TARGETING NERVE GROWTH FACTOR (NGF) ON BLADDER HYPESENSITIVITY IN RAT WITH EXPERIMENTAL COLITIS Naoki Kawamorita, Satoru Yoshikawa, Yoichi Arai, Pradeep Tyagi, Michael Chancellor, and Naoki Yoshimura Naoki KawamoritaNaoki Kawamorita More articles by this author , Satoru YoshikawaSatoru Yoshikawa More articles by this author , Yoichi AraiYoichi Arai More articles by this author , Pradeep TyagiPradeep Tyagi More articles by this author , Michael ChancellorMichael Chancellor More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.783AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Pelvic organ “cross sensitization” seems to contribute to overlapping symptoms in patients with chronic pelvic pain syndrome (CPPS) including bladder pain syndrome/interstitial cystitis (BPS/IC). However, it is not known whether NGF overexpression in the bladder is involved in this cross sensitization mechanism. Therefore, we investigated whether local instillation of liposome-OND conjugates (L-OND) targeting NGF overexpression in the bladder can suppress bladder hypersensitivity in a rat model of experimental colitis. METHODS Five groups of female SD rats were used; (a) control (no treatment), (b) colitis-OND (intracolonic 2,4,6,trinitrobenzen sulfonic acid [TNBS] enema & intravesical L-OND), (c) colitis-saline (TNBS & intravesical saline), (d) sham-OND (L-OND was given without colitis) and (e) sham-saline (saline was given without colitis). A day before evoking TNBS colitis, either L-OND or saline was instilled to the bladder. (1) Behavior testing: Resiniferatoxin (0.3µM, 0.3 ml) was administered into the bladder for 1 min to evaluate nociceptive behaviors such as licking (lower abdominal licking) and freezing (motionless head-turning) for 15 min. (2) Cystometry: Saline followed by 0.1% acetic acid (AA) were continuously infused to evaluate changes in intracontraction intervals (ICIs) in conscious rats. (3) Molecular analysis: Harvested bladders were divided to mucosal and detrusor layers to measure the mRNA and protein expression of NGF by qPCR and ELISA, respectively. RESULTS (1) In the colitis-saline group, the score of freezing behavior, which represents bladder pain sensation, was significantly higher than that of other groups including the colitis-OND group. (2) The reduction rate of ICI after AA instillation in the colitis-saline group was approximately 40% in contrast to 10% in the colitis-OND group. (3) The mRNA and protein expression of NGF in the mucosa was significantly higher in the colitis-saline group compared to the colitis-OND group. CONCLUSIONS These results indicate that intravesical treatment of NGF antisense with liposome, which suppressed mucosal NGF overexpression, reduced colitis-induced bladder hypersensitivity evidenced by increased bladder pain behavior (freezing) and AA-induced bladder overactivity. Thus, the intravesical NGF antisense treatment could be effective for hypersensitive bladder symptoms in CPPS including BPS/IC, in which the cross-sensitization mechanism contributes to overlapping symptoms from different pelvic organs. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e211 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Naoki Kawamorita More articles by this author Satoru Yoshikawa More articles by this author Yoichi Arai More articles by this author Pradeep Tyagi More articles by this author Michael Chancellor More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Peripheral contribution of NGF and ASIC1a to colonic hypersensitivity in a rat model of irritable bowel syndrome

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder associated with idiopathic colonic hypersensitivity (CHS). However, recent studies suggest that low-grade inflammation could underlie CHS in IBS. The pro-inflammatory mediator nerve growth factor (NGF) plays a key role in the sensitization of peripheral pain pathways and several studies have reported its contribution to visceral pain development. NGF modulates the expression of Acid-Sensing Ion Channels (ASICs), which are proton sensors involved in sensory neurons sensitization. This study examined the peripheral contribution of NGF and ASICs to IBS-like CHS induced by butyrate enemas in the rat colon. Colorectal distension and immunohistochemical staining of sensory neurons were used to evaluate NGF and ASICs contribution to the development of butyrate-induced CHS. Systemic injection of anti-NGF antibodies or the ASICs inhibitor amiloride prevented the development of butyrate-induced CHS. A significant increase in NGF and ASIC1a protein expression levels was observed in sensory neurons of rats displaying butyrate-induced CHS. This increase was specific of small- and medium-diameter L1+S1 sensory neurons, where ASIC1a was co-expressed with NGF or trkA in CGRP-immunoreactive somas. ASIC1a was also overexpressed in retrogradely labeled colon sensory neurons. Interestingly, anti-NGF antibody administration prevented ASIC1a overexpression in sensory neurons of butyrate-treated rats. Our data suggest that peripheral NGF and ASIC1a concomitantly contribute to the development of butyrate-induced CHS NGF-ASIC1a interplay may have a pivotal role in the sensitization of colonic sensory neurons and as such, could be considered as a potential new therapeutic target for IBS treatment.

Repeated variate stress in male rats induces increased voiding frequency, somatic sensitivity, and urinary bladder nerve growth factor expression

Stress exacerbates symptoms of functional lower urinary tract disorders including interstitial cystitis (IC)/bladder pain syndrome (BPS) and overactive bladder (OAB) in humans, but mechanisms contributing to symptom worsening are unknown. These studies address stress-induced changes in the structure and function of the micturition reflex using an animal model of stress in male rats. Rats were exposed to 7 days of repeated variate stress (RVS). Target organ (urinary bladder, thymus, adrenal gland) tissues were collected and weighed following RVS. Evans blue (EB) concentration and histamine, myeloperoxidase (MPO), nerve growth factor (NGF), brain-derived neurotropic factor (BDNF), and CXCL12 protein content (ELISA) were measured in the urinary bladder, and somatic sensitivity of the hindpaw and pelvic regions was determined following RVS. Bladder function was evaluated using continuous, open outlet intravesical infusion of saline in conscious rats. Increases in body weight gain were significantly (P ≤ 0.01) attenuated by day 5 of RVS, and adrenal weight was significantly (P ≤ 0.05) increased. Histamine, MPO, NGF, and CXCL12 protein expression was significantly (P ≤ 0.01) increased in the urinary bladder after RVS. Somatic sensitivity of the hindpaw and pelvic regions was significantly (P ≤ 0.01) increased at all monofilament forces tested (0.1-4 g) after RVS. Intercontraction interval, infused volume, and void volume were significantly (P ≤ 0.01) decreased after RVS. These studies demonstrate increased voiding frequency, histamine, MPO, NGF, and CXCL12 bladder content and somatic sensitivity after RVS suggesting an inflammatory component to stress-induced changes in bladder function and somatic sensitivity.

Growth and expression of rat bone marrow mesenchymal stem cells modified by nerve growth factor in diabetic rat bladders

The aim of the present study was to determine whether rat bone marrow mesenchymal stem cells (MSCs) transfected with the nerve growth factor (NGF) gene and then transplanted into diabetic rat bladder tissues survive and continue to express NGF. A recombinant lentiviral vector carrying the NGF gene was constructed and transfected into rat bone marrow MSCs. BrdU‑labeled immunohistochemistry was used to observe NGF expression in the transfected MSCs. BrdU‑labeled and NGF‑transfected MSCs were transplanted into diabetic rat bladder tissues. BrdU‑labeled immunohistochemistry was used to observe the growth of NGF‑transfected MSCs in the tissue samples. NGF mRNA and protein expression levels in MSCs were analyzed using reverse transcription polymerase chain reaction (RT-PCR) and ELISA, respectively. The recombinant NGF gene lentiviral vector and NGF gene-modified rat bone marrow MSCs were successfully constructed. NGF gene-modified rat MSCs survived in the diabetic rat bladders 4 weeks following injection and NGF gene expression was increased. In the present study, NGF gene-modified MSCs were shown to be capable of survival in diabetic rat bladder tissues and stably expressed NGF.

Epalrestat Improves Diabetic Wound Healing via Increased Expression of Nerve Growth Factor

Aldose reductase inhibitors (ARIs) are a useful therapy for diabetic neuropathy. Nerve damage is associated with delayed wound healing of skin ulcers in diabetic patients. Therefore, we hypothesized that ARI supplementation would improve diabetic wound healing. Control and streptozotocin-induced diabetic mice were fed either control diet or diet containing the ARI Epalrestat (40 mg/kg). After 12 weeks, we created skin wounds on the backs of the mice. Wound healing was determined by measuring the reduction in wound area. The wound gap of the diabetic group was significantly larger 9 days after creating the wounds when compared to the other groups (p<0.01). Interestingly, wound healing in the diabetic mice fed Epalrestat was comparable to the non-diabetic mice. To clarify the mechanism(s) behind this improved wound healing, mRNA expression of growth factors reported to be involved in wound healing were examined. Among the growth factors investigated, only the expression of nerve growth factor (NGF) was -significantly decreased (54.0%) in the healing lesions of diabetic mice. Similarly, NGF protein expression was decreased in diabetic mice and recovered in Epalrestat treated diabetic mice. Inhibition of NGF via 2 separate inhibitors (K252a and BSO) reduced the ability of Epalrestat to improve wound healing in diabetic mice. These findings suggest that Epalrestat is a potential therapy for improving diabetic wound healing and the mechanism involves upregulation of NGF.

Expression of Nerve Growth Factor and its Receptors in the Uterus of Gilts with Endometritis Induced by Infection with Escherichia coli

Nerve growth factor (NGF) is a neurotrophin implicated in the pathophysiology of allergy, diseases of the immune system and inflammation. Expression of NGF and its receptors (TrkA and p75) has not been examined in inflamed uterine tissue. The aim of the present study was to evaluate the patterns of NGF, TrkA and p75 expression in normal and inflamed porcine uteri using real-time reverse transcriptase polymerase chain reaction (RT-PCR), western blotting and immunohistochemistry. On day 3 of the oestrous cycle, 50 ml of saline or 50 ml of Escherichia coli suspension containing 10(9) colony forming units/ml, was injected into both uterine horns of control (n = 10) and experimental (n = 10) gilts, respectively. Infected animals developed moderate or severe acute endometritis after 8 days and moderate acute or subacute endometritis after 16 days. On day 8 of the study, the expression of NGF and TrkA mRNA and protein was higher in the endometrium of gilts with endometritis than in normal tissue. NGF protein was also more prominently expressed in the endometrium of those animals with endometritis 16 days post infection. There was no difference in endometrial p75 mRNA and protein expression on either day. The myometrium of animals in both groups showed no difference in expression of any of the three molecules. Immunohistochemically, on day 8 there was greater expression of NGF and TrkA by the luminal epithelium and some glandular and endothelial cells as well as the myometrium of the uteri of gilts with endometritis compared with normal tissues. At day 16, there was greater expression of NGF by the luminal epithelium and of TrkA by some endothelial cells and the myometrium, compared with control tissue. There is therefore, up-regulation of NGF and TrkA mRNA and protein expression in the porcine uterus when affected by E. coli-induced inflammation. NGF and TrkA may have roles in this inflammatory process and/or in the innervation of the uterus.

Influence of estrogen replacement and aging on the expression of nerve growth factor in the urethra of female rats

To evaluate the expression of nerve growth factor (NGF) in the urethra of adult female rats in different hormonal status using immunohistochemical assay. Forty-eight rats (Rattus norvegicus albinus, Rodentia, Mammalia) from the CEDEME-UNIFESP laboratory animal facility were used in the study. Rats were divided into four groups: group A, 12 non-neutered rats; group B, 12 oophorectomized rats; group C, 12 castrated rats treated with 17β-estradiol for 30 days; and group D, 12 aging rats. Animals were killed by lethal injection and their urethra was removed. NGF expression was evaluated by means of immunohistochemistry using mouse monoclonal primary IgG antibody anti-NGF diluted 1:600, and read under 400× magnification. Digital analysis of the images was done by Imagelab software. The intensity of the dark brown color was used as a measure of NGF cytoplasmatic expression, and was used to quantify the percentage of epithelial and muscular layer cells showing this neurotrophin. After oophorectomy, rats showed a significant increase in NGF expression in the periurethral muscular layer. Compared with oophorectomized rats, NGF expression increased in the epithelial layer and diminished in the periurethral smooth muscle following estrogen administration. In 18-month-old rats, NGF expression was diminished in both epithelial and muscular layers. Hormonal status led to significant differences in NGF protein expression in urethral epithelium and periurethral smooth muscle.

Effect of ulinastatin on growth inhibition, apoptosis of breast carcinoma cells is related to a decrease in signal conduction of JNk-2 and NF-κB

ObjectiveThis study aims to investigate the in vitro effects of Ulinastatin (UTI) and Taxotere (TXT) on cell proliferation; cell apoptosis; xenografted tumor growth; and expression of insulin-like growth factor receptor 1 (IGF-1R), platelet-derived growth factor A (PDGFA), nerve growth factor (NGF), c-Jun N-terminal kinase 2 (JNk-2), and NF-κB in a human primary breast cancer cells and breast cancer cell line MDA-MB-231.MethodsThe cell lines cultured were divided into four groups: 1) control group, 2) UTI group, 3) TXT group, and 4) UTI+TXT group. The method of MTT essay, flow cytometry, and RT-PCR were used to detect cell proliferation, cell apoptosis, and expression of IGF-1R, PDGFA, NGF, NF-κB, JNk-2, respectively. The growth of xenografted tumor in nude mice was used to calculate the anti-tumor rate. Immunohistochemistry staining (SP) was used to detect the expression of IGF-1R, PDGFA, NGF, ki-67, caspase-3, JNk-2, and NF-κB.ResultsProliferation of human breast cancer cells and MDA-MB-231 cell lines, and growth rate of xenografted tumor decreased in order of UTI+TXT > TXT > UTI > control, apoptosis increased in the order control < UTI < TXT < UTI+TXT. The gene expression and protein expression of IGF-1R, PDGFA, NGF, NF-κB and JNk-2 in breast cancer cells was inhibited by UTI and TXT.ConclusionsUTI 1) inhibits the proliferation of human breast cancer cells and the growth of xenografted tumors, 2) induces cancer cell apoptosis, and 3) enhances the anti-tumor effect of TXT. This mechanism might be related to decreasing signal transduction of JNk-2 and NF-κB, and then expression of IGF-1R, PDGFA, NGF.