TH Expression Research Articles

Targeting alpha-synuclein with a microRNA-embedded silencing vector in the rat substantia nigra: Positive and negative effects

Background: Alpha-synuclein (SNCA) downregulation shows therapeutic potential for synucleinopathies, including Parkinson’s disease (PD). Previously we showed that human (h)SNCA gene silencing using a short hairpin (sh)RNA in rat substantia nigra (SN) protects against a hSNCA-induced forelimb deficit, but not dopamine (DA) neuron loss. Furthermore, the shRNA increases cell death in vitro, but the same target sequence embedded in a microRNA30 transcript (mir30-hSNCA) does not. Objective: Examine hSNCA gene silencing using mir30-hSNCA in vivo. Methods: Rats were stereotaxically injected into one SN with adeno-associated virus serotype 2/8 (AAV)-hSNCA, AAV-hSNCA plus AAV-mir30-SNCA or AAV-hSNCA plus a control non-silencing mir30-embedded siRNA and DA neuron markers and associated behavior were examined. Results: AAV2/8-mediated SN hSNCA expression induces a forelimb deficit and tyrosine hydroxylase-immunoreactive (TH-IR) neuron loss. hSNCA gene silencing using mir30-hSNCA protects against this forelimb deficit at 2m and ameliorates TH-IR neuron loss. Striatal (ST) TH-IR fiber density and DA markers, assessed by western blot, are unaffected by AAV-hSNCA alone. Co-expression of either silencing vector reduces ST TH-IR fibers, panTH in SN and Ser40 phosphorylated TH in SN and ST, but does not affect vesicular monoamine transporter-2. However, hSNCA gene silencing promotes partial TH-IR fiber recovery by 2m. Co-expression of either silencing vector also induces SN inflammation, although some recovery was observed by 2m in hSNCA-silenced SN. Conclusion: hSNCA gene silencing with AAV-mir30-hSNCA has positive effects on forelimb behavior and SN DA neurons, which are compromised by inflammation and reduced TH expression, suggesting that AAV2/8-mir30-hSNCA-mediated gene silencing, although promising in vitro, is not a candidate for therapeutic translation for PD.

Neuroprotective Effects of Tetramethylpyrazine against Dopaminergic Neuron Injury in a Rat Model of Parkinson's Disease Induced by MPTP

Parkinson's disease (PD) is the second most prevalent progressive neurodegenerative disease. Although several hypotheses have been proposed to explain the pathogenesis of PD, apoptotic cell death and oxidative stress are the most prevalent mechanisms. Tetramethylpyrazine (TMP) is a biological component that has been extracted from Ligusticum wallichii Franchat (ChuanXiong), which exhibits anti-apoptotic and antioxidant roles. In the current study, we aimed to investigate the possible protective effect of TMP against dopaminergic neuron injury in a rat model of Parkinson's disease induced by MPTP and to elucidate probable molecular mechanisms. The results showed that TMP could notably prevent MPTP-induced dopaminergic neurons damage, reflected by improvement of motor deficits, enhancement of TH expression and the content of dopamine and its metabolite, DOPAC. We observed MPTP-induced activation of mitochondrial apoptotic death pathway, evidenced by up-regulation of Bax, down-regulation of Bcl-2, release of cytochrome c and cleavage of caspase 3, which was significantly inhibited by TMP. Moreover, TMP could prevent MPTP-increased TBARS level and MPTP-decreased GSH level, indicating the antioxidant role of TMP in PD model. And the antioxidant role of TMP attributes to the prevention of MPTP-induced reduction of Nrf2 and GCLc expression. In conclusion, in MPTP-induced PD model, TMP prevents the down-regulation of Nrf2 and GCLc, maintaining redox balance and inhibiting apoptosis, leading to the attenuation of dopaminergic neuron damage. The effectiveness of TMP in treating PD potentially leads to interesting therapeutic perspectives.

Anti-inflammatory effects of cell-based therapy with tyrosine hydroxylase-positive catecholaminergic cells in experimental arthritis

ObjectivesStudies in rheumatoid arthritis (RA), osteoarthritis (OA) and mice with arthritis demonstrated tyrosine hydroxylase-positive (TH+) cells in arthritic synovium and parallel loss of sympathetic nerve fibres. The exact function of...

Morphological and Behavioral Impact of AAV2/5-Mediated Overexpression of Human Wildtype Alpha-Synuclein in the Rat Nigrostriatal System

The discovery of the involvement of alpha-synuclein (α-syn) in Parkinson’s disease (PD) pathogenesis has resulted in the development and use of viral vector-mediated α-syn overexpression rodent models. The goal of these series of experiments was to characterize the neurodegeneration and functional deficits resulting from injection of recombinant adeno-associated virus (rAAV) serotype 2/5-expressing human wildtype α-syn in the rat substantia nigra (SN). Rats were unilaterally injected into two sites in the SN with either rAAV2/5-expressing green fluorescent protein (GFP, 1.2 x 1013) or varying titers (2.2 x 1012, 1.0 x 1013, 5.9 x 1013, or 1.0 x 1014) of rAAV2/5-α-syn. Cohorts of rats were euthanized 4, 8, or 12 weeks following vector injection. The severity of tyrosine hydroxylase immunoreactive (THir) neuron death in the SN pars compacta (SNpc) was dependent on vector titer. An identical magnitude of nigrostriatal degeneration (60-70% SNpc THir neuron degeneration and 40-50% loss of striatal TH expression) was observed four weeks following 1.0 x 1014 titer rAAV2/5-α-syn injection and 8 weeks following 1.0 x 1013 titer rAAV2/5-α-syn injection. THir neuron degeneration was relatively uniform throughout the rostral-caudal axis of the SNpc. Despite equivalent nigrostriatal degeneration between the 1.0 x 1013 and 1.0 x 1014 rAAV2/5-α-syn groups, functional impairment in the cylinder test and the adjusting steps task was only observed in rats with the longer 8 week duration of α-syn expression. Motor impairment in the cylinder task was highly correlated to striatal TH loss. Further, 8 weeks following 5.9 x 1013 rAAV2/5-α-syn injection deficits in ultrasonic vocalizations were observed. In conclusion, our rAAV2/5-α-syn overexpression model demonstrates robust nigrostriatal α-syn overexpression, induces significant nigrostriatal degeneration that is both vector and duration dependent and under specific parameters can result in motor impairment that directly relates to the level of striatal TH denervation.

Gonadal hormone dependent developmental plasticity of catecholamine:β2-adrenoceptor signaling complex in male rat thymus: Putative implications for thymopoiesis

The study was undertaken considering that: i) androgens affect β2-adrenoceptor (AR)-mediated catecholamine (CA) action in many tissues; and ii) peripubertal changes in both circulating androgen and thymic CA levels are implicated in rat thymic involution. Its aims were to: i) explore putative effects of the late prepubertal orchidectomy on thymic CA:β2-AR complex in young adult rats, and ii) delineate the direct effects of testicular hormone withdrawal on the CA:β2-AR complex from those elicited secondarily through altered influence of this complex components on each other's availability. Upon showing that prepubertal orchidectomy augmented the efficacy of thymopoiesis through increasing the thymocyte surface density of Thy-1, whose expression is negatively regulated by β2-AR-mediated signaling, we examined the effects of orchidectomy and 14-day-long propranolol (PROP) treatment in orchidectomized (ORX) and sham-ORX rats on thymic norepinephrine (NE) concentration and metabolism and β2-AR expression. Orchidectomy, despite an increase in the average NE amount per thymocyte and total thymocyte NE content, diminished thymic NE concentration. This decrease reflected the diminished density of CA-synthesizing nerve fibers, CD68+ macrophages, cortical (aminopeptidase A+), and medullary (UEA-1+) thymic epithelial cells (TECs) and their CA content (probably due to lessened TH expression accompanied by increased MAO-A expression). Moreover, orchidectomy decreased the surface β2-AR expression on thymocytes, CD68+ macrophages and OX-62+ dendritic cells, but increased its expression on the TECs. In sham-ORX rats, PROP reduced thymic NE concentration by diminishing TH expression in the thymic cells. Additionally, PROP in thymocytes and thymic stromal cells diminished and enhanced the β2-AR mRNA expression, respectively. However, in ORX rats PROP did not significantly affect CA(NE):β2-AR complex components. This indicated that prepubertal orchidectomy affects ability of young adult rats to respond to β-AR blockade by altering thymic NE and β2-AR availability. Collectively, the results showed that testicular hormones contribute to alterations in thymus/thymopoiesis during the critical peripubertal period by shaping modulatory sympathetic influence and CA autocrine/paracrine action within the organ.

Geraniol attenuates α-synuclein expression and neuromuscular impairment through increase dopamine content in MPTP intoxicated mice by dose dependent manner

Parkinson’s disease (PD) is characterized by progressive loss of dopamine (DA) neurons in the nigrostriatal system and by the presence of Lewy bodies (LB), proteinaceous inclusions mainly composed of filamentous α-synuclein (α-Syn) aggregates. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was adopted to generate PD models in C57BL/6 mice. In the present study, we investigated the effect of geraniol (GE) against α-Syn aggregation on MPTP induced mouse model of PD in dose dependant manner. When pretreatment of GE improved neuromuscular impairment, TH expressions and decreases α-Syn expressions in MPTP intoxicated PD mice by dose dependent manner. In addition, we confirmed that sub-chronic administration of MPTP in mice leads to permanent neuromuscular deficits and depletion of dopamine and its metabolites. Our results suggest that GE is beneficial for the treatment of PD associated with neuromuscular disability and LB aggregation.

Changes of Th17 cells, Treg cells and Th17/Treg cell-associated cytokines in patients with tubercu-lous pleurisy and significance of the changes

Objective To investigate the significance of Th 17 cells, Treg cells and Th17/Treg cell-associated cytokines in the development of tuberculous pleurisy through detecting the expressions of Th 17 cells and Treg cells （CD4＋CD25＋Foxp3＋） in CD4＋T cells, analyzing concentrations of IL-17, IL-23, IL-6＆nbsp;and TGF-βin serum of patients with tuberculous pleurisy and healthy controls and measuring levels of IL -17, IL-23, IL-6 and TGF-βin hydrothorax of patients with tuberculous pleurisy .Methods Flow cytometry was used to detect expressions of Th 17 and Treg cells in peripheral blood of patients with tuberculous pleurisy and healthy controls.ELISA method was performed for quantitative detection of concentrations of IL -17, IL-23, IL-6 and TGF-βin serum and hydrothorax .Differences and correlations between the above measured data were analyzed by the statistical software SPSS 17.0.Results Compared with the healthy control group , the expressions of Th17 cells in peripheral blood of the patients were significantly increased （1.02%±0.20%vs.0.89%±0.13%, P=0.002〈0.05）, while the expressions of Treg cells were significantly decreased （4.64%±0.77%vs.5.10%±0.90%, P=0.000〈0.05）.Correspondingly, the ratio of Th17/Treg cells （0.25±0.07） in the patients were significantly higher than that in the healthy controls （0.17±0.05, P=0.000〈0.05）.Concentrations of IL-17, IL-23 and IL-6 in peripheral blood and in hydrothorax of the pa-tients were （17.49±3.94） ng/L, （90.42±23.06） ng/L, （4.54±1.02） ng/L and （26.13±5.98） ng/L, （122.26±31.71） ng/L, （5.31±0.74） ng/L respectively, which were remarkably higher than the levels of IL-17 （14.45±3.81） ng/L, IL-23 （77.55±20.26） ng/L and IL-6 （4.26±0.91） ng/L in control group. In tuberculous pleurisy group , concentrations of IL-17, IL-23 and IL-6 in hydrothorax were significantly higher than those in peripheral blood with P values of 0.000, 0.000 and 0.003.There was no difference be-tween IL-6 levels in peripheral blood from patients and IL-6 levels in peripheral blood from the healthy con-trols, P=0.274.In comparison with the control group , TGF-βlevels in peripheral blood and in hydrothorax of the patients were significantly decreased （3.95 ng/L±0.79 ng/L, 3.12 ng/L±0.77 ng/L vs.3.32 ng/L ±0.80 ng/L） .In tuberculous pleurisy patients , the expression of Th17 cells in peripheral blood was nega-tively correlated with Treg cells in peripheral blood （r=-0.684, P=0.000〈0.05）, but was positively relat-ed to the levels of IL-17, IL-23 and IL-6 （r=0.479, 0.441, 0.326, P=0.013, 0.015, 0.017）.TGF-βlevel had significantly positive correlations with Treg cells in the peripheral blood of the patients （r=0.297, P=0.024）, but no significant correlation with Th17 cells was found （r=0.091, P=0.659）.Conclusion Th17/Treg cell-associated cytokines might regulate the expressions of Th 17 and Treg cells and inflammatory reaction.Changes of Th17 cells, Treg cells and related cytokines might be important immunopathological mechanisms for tuberculous pleurisy . Key words: Th17 cell; Treg cell; Tuberculous pleurisy; Flow cytometry; Cytokine

Effect of ginsenoside Rg3 on tyrosine hydroxylase and related mechanisms in the forced swimming-induced fatigue rats

Ethnopharmacological relevanceGinsenoside Rg3 has shown multiple pharmacological activities and been considered as one of the most promising approaches for fatigue treatment. However, little is known about the cellular and molecular mechanisms of Rg3 on anti-fatigue and the effect of Rg3 on dopaminergic system has not been reported yet. The major aim of this study is to investigate the effect of Rg3 on TH expression and the related biochemical parameters, such as PKAα, ERK1/2, Akt and α-synuclein in brain of fatigue rats. Materials and methodsWeight-loaded forced swimming was performed to establish an animal model of fatigue. Rg3 (10mg/kg, 50mg/kg and 100mg/kg) was intragastrically administrated before swimming. The effect of Rg3 on the expression and phosphorylation of TH and TH-related proteins in fatigue rats or in SH-SY5Y cells was assessed with western blotting. HPLC was used to examine the level of DA and DOPAC in the fatigue rats tissues. ResultsTH and phosphorylated TH were decreased in different brain regions of which ventral midbrain were less affected in weight-loaded forced swimming rats. Pretreatment with Rg3 significantly suppressed fatigue-induced decrease expression of TH and TH phosphorylation. Also treatment with Rg3 reversed the decrease expression of PKAα as well as the phosphorylation of ERK1/2 and Akt which were induced by weight-loaded forced swimming. Moreover, weight-loaded swimming could induce the increase expression of α-synuclein in hippocampus and midbrain, while suppressed α-synuclein expression in striatum and prefrontal cortex. Furthermore, Rg3 could induce the increase of TH expression and phosphorylation which was accompanied with elevated expression and phosphorylation of related kinase proteins in vitro, while the inhibitors of kinase proteins could suppress these effects of Rg3. In addition, HPLC results showed that Rg3 could reverse the weight-loaded swimming-induced increase of DOPAC/DA ratio. ConclusionOur data suggest that fatigue can induce the decrease of DA which might partially result from the change of TH expression and phosphorylation, and Rg3 can reverse these fatigue-induced changes. The underling mechanisms may include the activity changes of PKAα, ERK1/2, Akt and α-synuclein.

Prognostic impact and detection of minimal residual disease (MRD) in neuroblastoma patients.

10055 Background: Bone marrow (BM) involvement and MRD detection in neuroblastoma (NB) seems to be useful tool for patients’ prognosis, stratification and risk-adapted treatment. Real-time PCR (RQ-PCR) of tumor-specific gene transcripts and flow cytometry (FC) are commonly applied for this purpose. Aim. RQ-PCR MRD marker definition, its qualitative concordance with FC and prognostic impact in NB patients. Methods: We analyzed 331 BM samples from 57 NB patients and 26 ‘normal’ BM samples from 20 patients without malignancies for PHOX2B, TH, ELAVL4 and GD2 genes expression. BM samples were defined as positive either in case of positivity for PHOX2B or in case of NB cells presence in BM. 326 BM samples from 52 NB patients were analyzed by RQ-PCR and FC together. Results: PHOX2B and TH were not detecting in normal BM samples. Out of 224 negative BM samples TH was identified in 5 only, while ELAVL4 and GD2 expression was detected in the majority of normal and negative BM samples: 20 and 15 from 26 normal; 209 and 197 out of 224 negative samples correspondingly. TH, ELAVL4 and GD2 expression in positive BM samples was significantly higher comparing to negative and normal BM samples.Threshold levels of each gene expression were established by ROC-analysis and subsequently applied for overall correct prediction (OCP) calculation. OCP for PHOX2B and TH achieved 0.994 and 0.952, while OCP for ELAVL4 and GD2 were significantly lower: 0.828 and 0.767 respectively. Analytical sensitivity of RQ-PCR for PHOX2B achieved 1E-06, while sensitivity of FC ranged from 1E-03 to 1E-05. In 193(59.2%) out of 326 evaluated samples BM was negative by both methods. 38(11.7%) samples were negative by FC but positive by RQ-PCR for PHOX2B expression. 31(9.5%) samples were positive by FC but negative by RQ-PCR. 64(19.6%) samples were positive by both techniques. Thus qualitative concordance between RQ-PCR and FC achieved 78.8%. Conclusions: Patients with detectable PHOX2B expression have lower event-free survival comparing to patients with PHOX2B negative BM: 0.30±0.10 and 0.77±0.27 respectively, p=0.002 and overall survival is 0.51±0.11 and 0.79±0.07 correspondingly, p=0.083. Median of follow up in our NB patients series is 42 months, ranged from 1 to 156.

Comparison of Taqman low density array (TLDA) five-gene assay for tumor cells in bone marrow and blood with histologic bone marrow examination and imaging for disease assessment and outcome in patients with recurrent/refractory neuroblastoma (NBL): A new approaches to neuroblastoma therapy (NANT) study.

10039 Background: Accurate quantification of tumor burden in NBL patients is needed to define homogenous populations for therapy and establish response criteria that predict outcome. The 5-gene TLDA assay was developed for quantification of NBL cells in bone marrow (BM) and blood (BLD). Methods: Expression of CHGA, DCX, DDC, PHOX2B, and TH (NBL genes) was quantified with TLDA and reported as the geometric mean cycle threshold for the 5 genes (DGS=detection gene score; inversely related to tumor content, 40=negative). Sixty-three patients with recurrent/refractory NBL had TLDA performed on 107 BM and 99 BLD samples (66 paired) at 140 time points. Based on central review of reports, tumor longest diameter (LD) on CT/MRI (n=118), and number of 123I-MIBG avid sites (n=120) were recorded. Percentage of tumor cells in BM was from institutional reports of bilateral BM aspirates/biopsies (n=109). Overall response was assessed per NANT Response Criteria. Spearman rank correlation was performed. Results:TLDA detected tumor cells in 62/99(63%) BLD (average DGS=37.45) and 91/107(85%) BM samples (average DGS=33.42). 39/91(42%) with positive BM TLDA were negative by morphology. BLD and BM TLDA were correlated r = 0.6540, p< 0.0001 with stronger correlation with lower BM DGS scores. The BM and BLD DGS correlated with % BM involvement (BM r= -0.63, p<0.0001; BLD r= -0.35, p=0.0023) and number of MIBG sites (BM r=-0.34, p=0.001, BLD r=-0.51 p<0.0001) but not LD. Number of MIBG sites was also correlated with % BM involvement (r= 0.45, p<0.001) and LD (r= 0.28, p=0.0039). Analysis of 43 BM pairs demonstrated decreasing DGS correlated with overall progressive disease (r= 0.39, p=0.01). Conclusions: This TLDA assay detects NBL cells in both BM and BLD at high rates, and frequently detects tumor cells when BM morphology is negative. Quantification of tumor with DGS correlates with % BM involvement and number MIBG sites. TLDA may provide an additional parameter to delineate response in NBL. Clinical trial information: NCT01587300.

Parkinson's disease cybrids, differentiated or undifferentiated, maintain morphological and biochemical phenotypes different from those of control cybrids

SH-SY5Y, control, and Parkinson's disease (PD) cybrids prepared from an Indian population were differentiated using retinoic acid (RA) for understanding their dopaminergic characteristics and neuritogenesis. Undifferentiated control and PD cybrids exhibited higher levels of TH mRNA, but lower c-RET expression, short neurites, low neuritic density, and low proportion of cells with neurites compared with the undifferentiated parent cell line, SH-SY5Y. The expression levels of DAT and Ptx3 were similar to SH-SY5Y. PD cybrids showed poor viability and lower differentiating potency than SH-SY5Y or control cybrids. RA treatment for 6 days elevated c-RET expression and corrected the neuritic morphology of the control, but not of PD cybrids. Cell viability was found to be reduced in differentiated control and PD cybrids. TH expression level was significantly elevated in SH-SY5Y following RA treatment, but not in both the cybrids. In differentiated control and PD cybrids, the TH immunofluorescence intensity was significantly lower compared with SH-SY5Y cells. MitoTracker Green fluorescence intensity of the mitochondria was higher in differentiated PD cybrids. Dopamine released into the medium was unaffected in the differentiated SH-SY5Y or in the control cybrids but was significantly elevated in PD cybrids. These results suggest that PD cybrids, differentiated or undifferentiated, maintained morphological and biochemical phenotypes significantly different from those of the control cybrids, or the differentiated SH-SY5Y cells, and therefore could be an ideal cellular model of the disease for pharmacological screening of drugs and for investigation of the pathophysiology of PD.

Moderate and severe perinatal asphyxia induces differential effects on cocaine sensitization in adult rats

Perinatal asphyxia (PA) increases the likelihood of suffering from dopamine-related disorders, such as ADHD and schizophrenia. Since dopaminergic transmission plays a major role in cocaine sensitization, the purpose of this study was to determine whether PA could be associated with altered behavioral sensitization to cocaine. To this end, adult rats born vaginally (CTL), by caesarean section (C+), or by C+ with 15 min (PA15, moderate PA) or 19 min (PA19, severe PA) of global anoxia were repeatedly administered with cocaine (i.p., 15 mg/kg) and then challenged with cocaine (i.p., 15 mg/kg) after a 5-day withdrawal period. In addition, c-Fos, FosB/ΔFosB, DAT, and TH expression were assessed in dorsal (CPu) and ventral (NAcc) striatum. Results indicated that PA15 rats exhibited an increased locomotor sensitization to cocaine, while PA19 rats displayed an abnormal acquisition of locomotor sensitization and did not express a sensitized response to cocaine. c-Fos expression in NAcc, but not in CPu, was associated with these alterations in cocaine sensitization. FosB/ΔFosB expression was increased in all groups and regions after repeated cocaine administration, although it reached lower expression levels in PA19 rats. In CTL, C+, and PA15, but not in PA19 rats, the expression of TH in NAcc was reduced in groups repeatedly treated with cocaine, independently of the challenge test. Furthermore, this reduction was more pronounced in PA15 rats. DAT expression remained unaltered in all groups and regions studied. These results suggest that moderate PA may increase the vulnerability to drug abuse and in particular to cocaine addiction.

108 INSULIN-LIKE GROWTH FACTOR-1 AS AN IMPORTANT ENDOGENOUS GROWTH FACTOR FOR THE RECOVERY FROM STRESS URINARY INCONTINENCE IN RATS WITH SIMULATED CHILDBIRTH INJURY

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research (II)1 Apr 2013108 INSULIN-LIKE GROWTH FACTOR-1 AS AN IMPORTANT ENDOGENOUS GROWTH FACTOR FOR THE RECOVERY FROM STRESS URINARY INCONTINENCE IN RATS WITH SIMULATED CHILDBIRTH INJURY Yasuhiro Sumino, Satoru Yoshikawa, Hiromitsu Mimata, and Naoki Yoshimura Yasuhiro SuminoYasuhiro Sumino Oita, Japan More articles by this author , Satoru YoshikawaSatoru Yoshikawa Pittsburgh, PA More articles by this author , Hiromitsu MimataHiromitsu Mimata Oita, Japan More articles by this author , and Naoki YoshimuraNaoki Yoshimura Pittsburgh, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1487AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Insulin-like growth factor-1 (IGF-1) plays an important role in cell proliferation, survival and regeneration in various tissues.We previously identified the significant upregulation of exogenous IGF-1 and its receptors is associated with the recovery from stress urinary incontinence (SUI) induced by stimulated childbirth trauma in rats (2012AUA). In the present study, we examined the functional role of endogenous IGF-1 for the recovery from SUI induced by simulated childbirth trauma using an IGF-1 receptor inhibitor. METHODS Simulated birth trauma was induced by vaginal distension (VD) in female SD rats. An IGF-1 receptor antagonist, JB-1 (10 and 100μg/kg/day) or vehicle (saline) was continuously delivered from 1 day before VD using subcutaneous osmotic pumps. At 7, 14 and 21 days after VD, the effect of JB-1 treatment was examined by functional analyses (leak point pressure; LPP, urethral baseline pressure; UBP and urethral responses during passive increment in intravesicular pressure) and biomolecular analyses in urethral tissues (phosphorylation of Akt by Western blotting, apoptotic changes by TUNEL staining and peripheral adrenergic and cholinergic nerve density using immunohistochemistry of tyrosine hydroxylase; TH and vesicular acetylcholine transporter; VAChT, respectively). RESULTS In functional analyses, vehicle-treated rats had significantly reduced LPP, UBP and urethral responses after 7 days, with a recovery to the normal level 14 and 21 days after VD. However, in the JB-1 treated group (100μg/kg/day), LPP, UBP and urethral responses were still significantly reduced 21 days after VD. In biomolecular analyses, TUNEL positive cells were significantly increased in urethral tissues of JB-1 treated rats 7days after VD compared to vehicle-treated rats (P < 0.05). JB-1 treatment also prolonged the decrease of both TH and VAChT expression in urethral tissues (14 and 21 days after VD) although the reduction of positive stained area in vehicle-treated rats returned to normal levels at 14 days after VD (P < 0.05). Phosphorylation of urethral Akt in JB-1 treated rats tended to decrease 7 and 14 days after VD compared to vehicle-treated rats (not significant). CONCLUSIONS Suppression of endogenous IGF-1 activity delayed the recovery from SUI induced by simulated childbirth trauma in rats. Thus, IGF-1 is likely to be an important endogenous mediator for the functional recovery from childbirth-related SUI, suggesting that IGF-1 treatment could be effective for the treatment of SUI in women. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e43-e44 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yasuhiro Sumino Oita, Japan More articles by this author Satoru Yoshikawa Pittsburgh, PA More articles by this author Hiromitsu Mimata Oita, Japan More articles by this author Naoki Yoshimura Pittsburgh, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Morphine administration modulates expression of Argonaute 2 and dopamine‐related transcription factors involved in midbrain dopaminergic neurons function

Alterations in transcription factors that regulate the development and maintenance of dopamine (DA) neurons (such as Nurr1 and Pitx3) play an important role in the pathogenesis of addiction diseases. We have examined the effects of acute and chronic morphine and morphine withdrawal on TH expression and activity as well as expression of Nurr1, Pitx3 and Ago2 in the ventral tegmental area (VTA) and nucleus accumbens (NAc) of the rat. Rats were injected acutely with morphine and decapitated 1 or 2 h later. Another set of rats were made dependent on morphine by implantation of two morphine pellets. Precipitated withdrawal was induced by injection of naloxone. Ago2, Pitx3, Nurr1, total TH (tTH), TH phosphorylated at Ser31 and at Ser40, and 3,4-Dihydroxyphenylacetic acid, and DA determination in the VTA and/or NAc were measured using immunoblotting, HPLC and immunofluorescence. Acute morphine produced a marked increase in TH activity and DA turnover in the NAc, concomitantly with increased Nurr1 and Pitx3 expression in the VTA. In contrast, precipitated morphine withdrawal decreased TH activation, TH expression and did not increase DA turnover in the NAc. These effects paralleled decreases in Ago2 expression, which was accompanied by increased Nurr1 and Pitx3, TH activity and normalized TH protein levels in the VTA. The combined decrease in Ago2 and increases in Nurr1 and Pitx3 might represent some of the mechanisms that served to protect against accumbal TH regulation observed in morphine withdrawn rats, which may be critical for DA bioavailability to influence behaviour.

Pleiotrophin is involved in the amniotic epithelial cell-induced differentiation of human umbilical cord blood-derived mesenchymal stem cells into dopaminergic neuron-like cells

We have reported that human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) are capable of differentiating into dopaminergic (DA) neuron-like cells upon being induced by amniotic epithelial cells (AECs). However, what factor(s) is involved in the differentiation process has not been explored out thoroughly. Because pleiotrophin (PTN) is known to exert important trophic effects on DA neurons, in the present study, we investigated whether PTN is released by AECs and whether it is involved in the differentiation of hUCB-MSCs into DA neuron-like cells. The expression and secretion of PTN by AECs were detected by immunofluorescence, RT-PCR and ELISA. The hUCB-MSCs were isolated and treated with AEC-conditioned medium (ACM) or recombinant human PTN. Compared to the controls, a higher proportion of treated cells differentiated into DA neuron-like cells, indicated by the increased expression of TH and DAT and the increased dopamine content. These results indicate that PTN released by AECs acts as a synergetic factor with other neurotrophic factors and is involved in the differentiation of hUCB-MSCs into DA neuron-like cells. We suggest that ACM, which contains PTN and other neurotrophic factors, could potentially be used as an agent to promote the differentiation of DA neuron-like cells from hUCB-MSCs for cell therapy of Parkinson's disease without creating legal or ethical issues.

Dopaminergic Dysregulation in Prefrontal Cortex of Rhesus Monkeys Following Cocaine Self-Administration

Chronic cocaine administration regulates the expression of several proteins related to dopaminergic signaling and synaptic function in the mesocorticolimbic pathway, including the prefrontal cortex. Functional abnormalities in the prefrontal cortex are hypothesized to be due in part to the expression of proteins involved in dopamine signaling and plasticity. Adult male rhesus monkeys self-administered cocaine (i.v.) under limited (n = 4) and extended access conditions (n = 6). The abundance of surrogate markers of dopamine signaling and plasticity in the dorsolateral prefrontal cortex (DLPFC), orbitofrontal cortex (OFC), and anterior cingulate cortex (ACC) were examined: glycosylated and non-glycosylated forms of the dopamine transporter (efficiency of dopamine transport), tyrosine hydroxylase (TH; marker of dopamine synthesis) and phosphorylated TH at Serine 30 and 40 (markers of enzyme activity), extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK 2), and phosphorylated ERK1 and ERK2 (phosphorylates TH Serine 31; markers of synaptic plasticity), and markers of synaptic integrity, spinophilin and post-synaptic density protein 95 (roles in dopamine signaling and response to cocaine). Extended cocaine access increased non-glycosylated and glycosylated DAT in DLPFC and OFC. While no differences in TH expression were observed between groups for any of the regions, extended access induced significant elevations in pTHSer31 in all regions. In addition, a slight but significant reduction in phosphorylated pTHSer40 was found in the DLPFC. Phosphorylated ERK2 was increased in all regions; however, pERK1 was decreased in ACC and OFC but increased in DLPFC. PSD-95 was increased in the OFC but not in DLPFC or ACC. Furthermore, extended cocaine self-administration elicited significant increases in spinophilin protein expression in all regions. Results from the study provide insight into the biochemical alterations occurring in primate prefrontal cortex.

Exposure to the polybrominated diphenyl ether mixture DE-71 damages the nigrostriatal dopamine system: Role of dopamine handling in neurotoxicity

In the last several decades polybrominated diphenyl ethers (PBDEs) have replaced the previously banned polychlorinated biphenyls (PCBs) in multiple flame retardant utilities. As epidemiological and laboratory studies have suggested PCBs as a risk factor for Parkinson's disease (PD), the similarities between PBDEs and PCBs suggest that PBDEs have the potential to be neurotoxic to the dopamine system. The purpose of this study was to evaluate the neurotoxic effects of the PBDE mixture, DE-71, on the nigrostriatal dopamine system and address the role of altered dopamine handling in mediating this neurotoxicity. Using an in vitro model system we found DE-71 effectively caused cell death in a dopaminergic cell line as well as reducing the number of TH+ neurons isolated from VMAT2 WT and LO animals. Assessment of DE-71 neurotoxicity in vivo demonstrated significant deposition of PBDE congeners in the brains of mice, leading to reductions in striatal dopamine and dopamine handling, as well as reductions in the striatal dopamine transporter (DAT) and VMAT2. Additionally, DE-71 elicited a significant locomotor deficit in the VMAT2 WT and LO mice. However, no change was seen in TH expression in dopamine terminal or in the number of dopamine neurons in the substantia nigra pars compacta (SNpc). To date, these are the first data to demonstrate that exposure to PBDEs disrupts the nigrostriatal dopamine system. Given their similarities to PCBs, additional laboratory and epidemiological research should be considered to assess PBDEs as a potential risk factor for PD and other neurological disorders.

Phox2b Influences the Development of a Caudal Dopaminergic Subset

The developing mesodiencephalic dopaminergic (mdDA) neuronal field can be subdivided into several molecularly distinct domains that arise due to spatiotemporally distinct origins of the neurons and distinct transcriptional pathways controlling these neuronal subsets. Two large anatomically and functionally different subdomains are formed that eventually give rise to the SNc and VTA, but more subsets exist which require detailed characterization in order to better understand the development of the functionally different mdDA subsets, and subset-specific vulnerability. In this study, we aimed to characterize the role of transcription factor Phox2b in the development of mdDA neurons. We provide evidence that Phox2b is co-expressed with TH in a dorsal-caudal subset of neurons in the mdDA neuronal field during embryonic development. Moreover, Phox2b transcripts were identified in FAC-sorted Pitx3 positive neurons. Subsequent analysis of Phox2b mutant embryos revealed that in the absence of Phox2b, a decrease of TH expression occurred specifically in the midbrain neuronal subset that normally co-expresses Phox2b with TH. Our data suggest that Phox2b is, next to the known role in the development of the oculomotor complex, involved in the development of a specific caudal mdDA neuronal subset.

The Influence of NaIO3-Induced Retinal Degeneration on Intra-retinal Layer and the Changes of Expression Profile/Morphology of DA-ACs and mRGCS

Sodium iodate (NaIO(3))-induced retina injury is one of models that is commonly used to study various retinal diseases caused by retinal pigment epithelium (RPE) injury such as AMD. Previous researches have revealed that RPE and photoreceptors are main impaired objects in this model. By comparison, intra-retinal layer has not been studied in detail after NaIO(3) administration. In this study, we present evidences that intra-retinal neurons can be directly injured by NaIO(3) at early stage and that the morphology had taken obvious changes, the decreased areas of dendritic fields of dopaminergic amacrine cells (DA-ACs), horizontal cells, and melanopsin-expressing retinal ganglion cells (mRGCs). Moreover, we found that miRNA 133b that was considered specifically to express in midbrain dopaminergic neurons was markedly upregulated in retinal DA-ACs after NaIO(3) administration. The overexpression of mir-133b negatively regulated the expression of pitx3, an important transcription factor, and led to a series of deficits of DA-ACs such as TH and D2 receptor expression and DA producing, which may play a causative role in pathological events of horizontal cells and mRGCs. After mir-133b was interfered with mir-133b/RNAi, not only those deficits were rescued, but also the amplitude of b-wave and summed OPs of ERG were improved significantly. In conclusion, our data demonstrate, for the first time, that intra-retinal neurons can be directly injured by NaIO(3) at early stage, and that mir-133b level effectively controls synaptic contacts or neural interactions among DA-ACs, horizontal cells, and mRGCs. Delivering mir-133b/RNAi intravitreally can rescue NaIO(3)-induced failure and improve visual function by restoring synaptic contacts.

Neuroprotection by Silencing iNOS Expression in a 6-OHDA Model of Parkinson’s Disease

In this study, we investigated the protective role of silenced iNOS expression in neuron death in the nigrostriatal pathway in a 6-OHDA animal model of Parkinson's disease (PD). The animal model was established by intrastriatal infusion of a single dose of 6-OHDA. Silencing of iNOS expression was established by intrastriatal infusion of adenovirus-carried iNOS-targeted small interfering RNA (siRNA). Apomorphine-induced rotation behavior was measured. Expression of iNOS, OX-42, and TH; levels of DA, DOPAC, and HVA in the striatum; and the levels of p53 phosphorylation, Bax, and cleaved caspase-3 (CC3) in the substantia nigra were measured. We demonstrated that co-infusion of 6-OHDA with adenovirus expressing siRNA of iNOS blocked the activation of microglia, iNOS transcription, and p53-Bax-CC3 apoptotic cascade as well as significantly blocking 6-OHDA-induced decreases in DA, DOPAC, HVA, and TH levels and effectively decreased rotation number. Our study highlighted the role of iNOS in the neurodegeneration of nigrostriatal dopaminergic neurons in the 6-OHDA animal model of PD and suggested that the microglial activation-iNOS-p53-Bax-CC3 apoptotic pathway plays a key role in the neurodegeneration in the 6-OHDA model.