Treatment with a cannabinoid receptor 2 agonist decreases severity of established cystitis.

PURPOSE: We investigated whether treatment with the selective cannabinoid receptor 2 agonist GP1a would ameliorate the severity of experimental cystitis. We determined the association of referred hyperalgesia and increased urinary frequency after establishing cystitis in mice by intravesical instillation of acrolein. MATERIALS AND METHODS: Cystitis was induced by intravesical instillation of acrolein in female C57BL/6NH mice. Mice were treated with GP1a (10 mg/kg intraperitoneally) or vehicle 3.5, 22 and 30 hours after instillation of acrolein. Mice were tested for mechanical sensitivity of hind paws. Short-term voluntary voiding was assessed by quantifying urine spots of freely moving mice. Bladders were collected, weighed and processed for immunohistochemical, histological and immunoblotting analysis. RESULTS: At 48 hours after acrolein instillation the bladder of all mice showed histological evidence of inflammation. The severity of edema and increase in bladder weight were inhibited in cannabinoid receptor 2 agonist treated animals (p <0.05). Neither cystitis nor treatment with GP1a or AM630 (selective cannabinoid receptor 2 antagonist) plus GP1a appeared to alter cannabinoid receptor 2-like immunoreactivity abundance in urothelium. Mechanical sensitivity was significantly increased after acrolein and the increase was attenuated in cannabinoid receptor 2 agonist treated mice (p <0.05). The number of small diameter urine spots was significantly increased after acrolein and treatment with GP1a attenuated this increase (p <0.05). GP1a effects were prevented by AM630. CONCLUSIONS: Treatment with a selective cannabinoid receptor 2 agonist decreased severity of established acrolein induced cystitis and inhibited bladder inflammation associated increased referred mechanical sensitivity and increased bladder urinary frequency. Our data indicate that cannabinoid receptor 2 is a potential therapeutic target for treatment of painful inflammatory bladder diseases.

Activation of cannabinoid receptor 2 inhibits experimental cystitis.

Cannabinoids have been shown to exert analgesic and anti-inflammatory effects, and the effects of cannabinoids are mediated primarily by cannabinoid receptors 1 and 2 (CB1and CB2). Both CB1 and CB2 are present in bladders of various species, including human, monkey, and rodents, and it appears that CB2 is highly expressed in urothelial cells. We investigated whether treatment with the CB2 agonist GP1a alters severity of experimental cystitis induced by acrolein and referred mechanical hyperalgesia associated with cystitis. We also investigated whether the mitogen-activated protein kinases (MAPK), ERK1/2, p38, and JNK are involved in the functions of CB2. We found that treatment with the selective CB2 agonist GP1a (1-10 mg/kg, ip) inhibited the severity of bladder inflammation 3 h after intravesical instillation of acrolein in a dose-dependent manner, and inhibition reached significance at a dose of 10 mg/kg (P < 0.05). Treatment with GP1a (10 mg/kg) inhibited referred mechanical hyperalgesia associated with cystitis (P < 0.05). The inhibitory effects of the CB2 agonist were prevented by the selective CB2 antagonist AM630 (10 mg/kg, sc). We further demonstrated the inhibitory effects of CB2 appear to be at least partly mediated by reducing bladder inflammation-induced activation of ERK1/2 MAPK pathway. The results of the current study indicate that CB2 is a potential therapeutic target for treatment of bladder inflammation and pain in patients.

Inhibition of fatty acid amide hydrolase suppresses referred hyperalgesia induced by bladder inflammation.

OBJECTIVE: • To determine (i) the presence of fatty acid amide hydrolase (FAAH) in the urinary bladder; (ii) whether or not endogenous fatty acid ethanolamides are synthesized by the bladder; (iii) the effects of FAAH inhibition on referred hyperalgesia associated with acute bladder inflammation in rats. MATERIALS AND METHODS: • Immunohistochemistry and immunoblotting were performed to detect FAAH in the bladder. Acrolein (1 mM, 400 µL) was instilled into bladders of female Wistar rats to induce cystitis. Referred mechanical hyperalgesia was assessed by application of Von Frey monofilaments to the hind paws. • Animals were killed 4, 24, 48 and 72 h after acrolein instillation, and the fatty acid ethanolamide content of bladders was measured using isotope-dilution liquid chromatography/mass spectrometry. • Other rats were treated with the FAAH inhibitor URB597 (0.3 mg/kg, i.p.) after the induction of cystitis, and the mechanical sensitivity of the hind paws was determined. RESULTS: • Immunohistochemistry and immunoblotting showed the presence of FAAH in the bladder, with greatest abundance in the urothelium. • Acrolein-induced cystitis increased fatty acid ethanolamide content (including anandamide) in the bladder in a time-dependent manner. Inhibition of FAAH diminished referred hyperalgesia associated with acute bladder inflammation. CONCLUSIONS: • The results obtained in the present study indicate that (i) FAAH is present in the urinary bladder; (ii) fatty acid ethanolamides are increased during bladder inflammation; (iii) inhibition of FAAH could be an effective therapeutic approach for the treatment of bladder pain. • These results raise the possibility that inhibitors of enzymes responsible for metabolism of fatty acid ethanolamides could inhibit pain associated with bladder inflammation.

Tumor necrosis factor-α induces expression and release of interleukin-6 by human urothelial cells.

OBJECTIVE: We examined the effects of tumor necrosis factor-α (TNF-α) on expression and release of interleukin-6 (IL-6) by human urothelial cells (HUCs) and investigated whether the effects of TNF-α are mediated by mitogen-activated protein kinase (MAPK) pathways. MATERIALS AND METHODS: HUCs were treated with TNF-α at 1-10 ng/ml for 2-24 h. Expression of IL-6 and TNF-α receptor 1 (TNFR1) mRNAs were examined by real-time PCR. The release of IL-6 into culture medium was determined by ELISA. The presence of TNFR1 protein and TNF-α-induced activation of MAPK pathways was examined by immunoblotting analysis. The effects of selective blockers of MAPK pathways on TNF-α-induced IL-6 expression and release were determined. RESULTS: TNF-α increased IL-6 mRNA expression and stimulated release of IL-6 in a concentration- and time-dependent manner. The effects of TNF-α were mediated by TNFR1. TNF-α induced phosphorylation of ERK1/2 and JNK, and TNF-α-induced IL-6 expression and release were inhibited by selective ERK1/2 and JNK blockers. CONCLUSIONS: These results demonstrate that TNF-α increases expression and release of IL-6 by HUCs and that the effects of TNF-α are mediated by TNFR1. Also, the ERK1/2 and JNK pathways are involved in TNF-α-induced expression and release of IL-6 in HUCs and may represent therapeutic targets in inflammatory urinary tract diseases.

Models of inflammation of the lower urinary tract.

Inflammation of the lower urinary tract occurs frequently in people. The causes remain obscure, with the exception of urinary tract infection. Animal models have proven useful for investigating and assessing mechanisms underlying symptoms associated with lower urinary tract inflammation and options for suppressing these symptoms. This review will discuss various animal models of lower urinary tract inflammation, including feline spontaneous (interstitial) cystitis, neurogenic cystitis, autoimmune cystitis, cystitis induced by intravesical instillation of chemicals or bacterial products (particularly lipopolysaccharide or LPS), and prostatic inflammation initiated by transurethral instillation of bacteria. Animal models will continue to be of significant value in identifying mechanisms resulting in bladder inflammation, but the relevance of some of these models to the causes underlying clinical disease is unclear. This is primarily because of the lack of understanding of causes of these disorders in people. Comparative and translational studies are required if the full potential of findings obtained with animal models to improve prevention and treatment of lower urinary tract inflammation in people is to be realized.

[Detection of pear injury based on visible-near infrared spectroscopy and multispectral image].

A new approach to detect the injury degree and time of pear based on visible-near infrared spectroscopy and multispectral image has been proposed. Firstly, visible-near infrared spectroscopy combined with partial least squares (PLS) and least squares-support vector machine (LS-SVM) was used for pear injury degree and time prediction. The result indicated that these two methods both have good performances in predicting pear injury degree in the late period. The LS-SVM method is more accurate in predicting the injury time of light pear injury, but its overall result of injury time prediction is not as good as that for the PLS method. Then, the multispectral image was used to predict the time of pear injury. The result shows that for different degrees of pear injury, the prediction models based on LS-SVM have a better performance with correlation coefficients around 5.85. The result of this study can be used to detect the injury degree and time of pear rapidly and non-destructively, and provide a new approach to pear classification.

LncRNA-SNHG7 Enhances Chemotherapy Resistance and Cell Viability of Breast Cancer Cells by Regulating miR-186.

BACKGROUND: Clinical tolerance to trastuzumab greatly affects the therapeutic effect in breast cancer (BC). Long-chain non-coding RNA (lncRNA) plays an important role in the development of trastuzumab resistance, in which SNHG7 can promote the epithelial mesenchymal transformation (EMT) of breast cancer cells into, while EMT is related to trastuzumab resistance of breast cancer cells. OBJECTIVE: To investigate whether lncRNA-SNHG7 can enhance chemotherapy resistance and cell viability of BC cells by regulating miR-186. METHODS: SK-BR-3 and SNHG7 of HER2+BC cells were induced to enhance the resistance of BC cells to trastuzumab by regulating miR-186, and to regulate the expression levels of SNHG7 and miR-186. The sensitivity of drug-resistant cells to trastuzumab and the changes of cell proliferation, migration, apoptosis, and EMT were measured and verified by tumorigenesis in vivo. The effects of miR-186 on SNHG7 were investigated through rescue experiments; the regulatory relationship between the expression of SNHG7 and miR-186 was verified by the double luciferase reporter (DLR) and the mechanism of SNHG7 was explored. RESULTS: Down-regulation of SNHG7 or up-regulation of miR-186 could increase the sensitivity of BC cells to trastuzumab, inhibit the proliferation, migration and EMT, and promote apoptosis. Compared with the down-regulation of SNHG7 or miR-186 alone, simultaneous down-regulation of SNHG7 and miR-186 on drug-resistant cells brought notably lower sensitivity to trastuzumab and apoptosis rate, and higher proliferation and apoptosis ability. The DLR showed that miR-186 could specifically inhibit the expression of SNHG7. The results of tumorigenesis in vivo revealed that down-regulation of SNHG7 or up-regulation of miR-186 could improve the therapeutic effect of trastuzumab and reduce the tumor volume, and miR-186 could also antagonize the effect of SNHG7. CONCLUSION: Down-regulation of SNHG7-targeted miR-186 can reverse trastuzumab resistance of BC cells, inhibit the proliferation, migration, and EMT levels of BC cells, and promote apoptosis.

HIF-2α regulates CD44 to promote cancer stem cell activation in triple-negative breast cancer via PI3K/AKT/mTOR signaling.

BACKGROUND: Breast cancer is a common malignant tumor that seriously threatens women's health. Breast cancer stem cell (CSC)-like cell population may be the main factor for breast cancer metastasis. Therefore, targeted therapy for CSCs has great potential significance. Hypoxia-inducible factor is a transcription factor widely expressed in tumors. Studies have shown that down-regulation of the hypoxia signaling pathway inhibits tumor stem cell self-renewal and increases the sensitivity of stem cells to radiotherapy and chemotherapy mediated by hypoxia-inducible factor-2α (HIF-2α). However, the specific mechanism remains unclear and further research is necessary. AIM: To investigate the effect of HIF-2α down-regulation on stem cell markers, microsphere formation, and apoptosis in breast cancer cell line MDA-MB-231 under hypoxia and its possible mechanism. METHODS: Immunohistochemistry was used to detect the expression of HIF-2α and CD44 in triple-negative breast cancer (TNBC) and non-TNBC tissues. Double-labeling immunofluorescence was applied to detect the co-expression of HIF-2α and CD44 in MDA-MB-231 cells and MCF-7 cells. HIF-2α was silenced by RNA interference, and the expression of CD44 and transfection efficiency were detected by real-time fluorescent quantitative PCR. Further, flow cytometry, TdT-mediated X-dUTP nick end labeling, and mammosphere formation assays were used to evaluate the effect of HIF-2α on CSCs and apoptosis. The possible mechanisms were analyzed by Western blot. RESULTS: The results of immunohistochemistry showed that HIF-2α was highly expressed in both TNBC and non-TNBC, while the expression of CD44 in different molecular types of breast cancer cells was different. In in vitro experiments, it was found that HIF-2α and CD44 were expressed almost in the same cell. Compared with hypoxia + negative-sequence control, HIF-2α small interfering ribonucleic acid transfection can lower the expression of HIF-2α and CD44 mRNA(P < 0.05), increase the percentage of apoptotic cells (P < 0.05), and resulted in a reduction of CD44+/CD24- population (P < 0.05) and mammosphere formation (P < 0.05) in hypoxic MDA-MB-231 cells. Western blot analysis revealed that phosphorylated protein-serine-threonine kinase (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR) levels in MDA-MB-231 decreased significantly after HIF-2α silencing (P < 0.05). CONCLUSION: Down-regulation of HIF-2α expression can inhibit the stemness of human breast cancer MDA-MB-231 cells and promote apoptosis, and its mechanism may be related to the CD44/phosphoinosmde-3-kinase/AKT/mTOR signaling pathway.

Role of mast cells and protease-activated receptor-2 in cyclooxygenase-2 expression in urothelial cells.

Mast cells have been shown to play a role in development and persistence of various inflammatory bladder disorders. Mast cell-derived tryptase specifically activates protease-activated receptor-2 (PAR-2), and PAR-2 is known to be involved in inflammation. We investigated whether mast cells participate in increase of cyclooxygenase-2 (COX-2) protein abundance in urothelium/suburothelium of bladders of mice subsequent to cyclophosphamide (CYP)-induced bladder inflammation. We also used primary cultures of human urothelial cells to investigate cellular mechanisms underlying activation of PAR-2 resulting in increased COX-2 expression. We found that treatment of mice with CYP (150 mg/kg ip) increased COX-2 protein abundance in bladder urothelium/suburothelium 3, 6, and 24 h after CYP (P < 0.01), and increased COX-2 protein abundance was prevented by treatment of mice with the mast cell stabilizer sodium cromolyn (10 mg/kg ip) for 4 consecutive days before CYP treatment. Incubation of freshly isolated mouse urothelium/suburothelium with a selective PAR-2 agonist, 2-furoyl-LIGRLO-amide (3 microM), also increased COX-2 protein abundance (P < 0.05). We further demonstrated that 2-furoyl-LIGRLO-amide (3 microM) increased COX-2 mRNA expression and protein abundance in primary cultures of human urothelial cells (P < 0.01), and the effects of PAR-2 activation were mediated primarily by the ERK1/2 MAP kinase pathway. These data indicate that there are functional interactions among mast cells, PAR-2 activation, and increased expression of COX-2 in bladder inflammation.

Lidocaine prevents referred hyperalgesia associated with cystitis.

AIMS: Lidocaine produces analgesia by inhibiting excitation of nerve endings or blocking impulse conduction in peripheral nerves. This study was performed to determine whether intrathecal or intravesical administration of lidocaine prior, or subsequent, to induction of chemical cystitis in rats would block referred mechanical hyperalgesia. METHODS: Intrathecal or intravesical lidocaine was administered 15 (intrathecal) or 30 (intravesical) min before intravesical instillation of saline or 1 mM acrolein (400 microl) or 4 hr after saline or acrolein instillation in female Wistar rats. Mechanical sensitivity of hind paws was determined at 24 hr prior to any treatment (baseline) and, 4, 24, and 48 hr after intravesical instillation of acrolein or saline. Also, nerve growth factor (NGF) content was measured in bladder and dorsal root ganglia (DRG). RESULTS: Pre-treatment with intrathecal or intravesical lidocaine attenuated acrolein-induced referred mechanical hyperalgesia of the hind paws. Lidocaine administered after acrolein instillation did not alter referred hyperalgesia. Lidocaine treatment prior to or after induction of cystitis reduced NGF content in the bladder. CONCLUSIONS: These results indicate that pre-treatment with lidocaine attenuates referred hyperalgesia associated with cystitis. Lidocaine treatment 4 hr after induction of cystitis failed to prevent referred hyperalgesia despite a similar decrease in bladder NGF. Neurourol. Urodynam. (c) 2009 Wiley-Liss, Inc.

Analysis of factors potentially predicting prognosis of colorectal cancer.

BACKGROUND: Accurate assessment of the prognosis after colorectal cancer surgery is of great significance in patients with colorectal cancer. However, there is no systematic analysis of factors affecting the prognosis of colorectal cancer currently. AIM: To systematically analyze the influence of clinical data and serological and histological indicators on the prognosis of patients with colorectal cancer, and to explore the indicators that can accurately assess the prognosis of patients with colorectal cancer. METHODS: A total of 374 patients with colorectal cancer were enrolled. The clinical data, tumor-node-metastasis (TNM) stage, and Dukes stage were recorded. All patients received examinations including carcinoembryonic antigen (CEA), carbohydrate antigen 199, C-reactive protein, albumin, D-dimer, and fibrinogen as well as routine blood tests one week before surgery. The tumor location, size, depth of invasion, lymph node metastasis, and distant metastasis were recorded during surgery. The pathological tissue typing and expression of proliferating cell nuclear antigen (PCNA) and p53 were observed. All patients were followed for 3 years, and patients with endpoint events were defined as a poor prognosis group, and the remaining patients were defined as a good prognosis group. The differences in clinical data, serology, and histology were analyzed between the two groups. Multivariate COX regression was used to analyze the independent influencing factors for the prognosis of colorectal cancer. The receiver operating characteristic curve was used to evaluate the predictive value of each of the independent influencing factors and their combination for the prognosis of colorectal cancer. RESULTS: The follow-up outcomes showed that 81 patients were in the good prognosis group and 274 patients in the poor prognosis group. The TNM stage, PCNA, Glasgow prognostic score (GPS), neutrophil-lymphocyte ratio (NLR), C-reactive protein/albumin ratio (CAR), D-dimer, and CEA were independent influencing factors for the prognosis of colorectal cancer (P = 0.000). NLR had the highest predictive power for colorectal cancer prognosis [area under the receiver operating characteristic curve (AUC) = 0.925], followed by D-dimer (AUC = 0.879) and GPS (AUC = 0.872). The accuracy of the combination of all indicators in predicting the prognosis of colorectal cancer was the highest (AUC = 0.973), which was significantly higher than that of any of the indicators alone (P < 0.05). The sensitivity and specificity of the combination were 92.59% and 90.51%, respectively. CONCLUSION: The independent influence factors for the prognosis of colorectal cancer include TNM stage, PCNA, GPS, NLR, CAR, D-dimer, and CEA. The combined assessment of the independent factors is the most accurate predictor of the prognosis after colorectal cancer surgery.

Analysis of B-ultrasound and contrast-enhanced ultrasound characteristics of different hepatic neuroendocrine neoplasm.

BACKGROUND: Hepatic neuroendocrine neoplasm (hNEN) is a highly heterogeneous tumor. The exact identification of the source and malignant degree of hNEN is important. However, there is a lack of information regarding diagnosis of hNEN with imaging. In addition, no studies have compared the imaging between hNEN and hepatocellular carcinoma (HCC) and among different sources and malignant degrees of hNEN. AIM: To compare the ultrasound characteristics between hNEN and HCC and among different sources and malignant degrees of hNEN. METHODS: A total of 55 patients with hNEN were recruited and defined as the hNEN group. Among them, 35 cases of hNET were defined as the hNET group. Twenty cases of hepatic neuroendocrine carcinoma (hNEC) were defined as the hNEC group. Among the 55 lesions, 29 were transferred from the pancreas, 20 were from the gastrointestinal tract, and six were from other sites. In total, 55 patients with HCC were recruited and defined as the HCC group. The characteristic differences of B-mode ultrasound and contrast-enhanced ultrasound (CEUS) between hNEN and HCC and among different sources and malignant degrees of hNEN were compared. RESULTS: In the hNEN group, the proportions of multiple liver lesions, unclear borders, and high echo lesions were higher than those in the HCC group. The proportions of non-uniform echo and peripheral acoustic halo were lower than those in the HCC group (P < 0.05). The washout to iso-enhancement time and washout to hypo-enhancement time were lower than those in the HCC group (P < 0.05). The characteristics of B-ultrasound and CEUS among different sources of hNEN were similar, and the differences were not statistically significant (P > 0.05). B-mode ultrasound characteristics of hNET and hNEC were similar. The proportions of low enhancement at portal venous phase, non-uniform enhancement forms, and combined tumor vasculature in the hNEC group were larger than those in the hNEN group (P < 0.05). CONCLUSION: Compared with HCC, hNEN showed multiple intrahepatic lesions, uniform high echo, uniform high enhancement at arterial phase, and rapid washout. Low enhancement at portal venous phase, overall non-uniform enhancement form, and the proportion of combined tumor vasculature in hNEC were larger than those in hNET.

Roles of estrogen receptor alpha and beta in modulating urothelial cell proliferation.

We reported previously that both subtypes of estrogen receptors, ERalpha and ERbeta, are expressed by human urothelial cells and mediate estrogen-induced cell proliferation in these cells. The aim of this study was to determine the extent to which each ER subtype contributes to urothelial cell proliferation and their possible involvement in the regulation of the cell cycle. We compared the expression of ERalpha and ERbeta mRNAs and protein quantitatively in primarily cultured human bladder urothelial cells obtained from six individuals with three immortalized urothelial (E6, E7, and UROtsa) and two bladder cancer cell lines (HTB-9 and T24). We found that all these cells express similar levels of ERbeta, but immortalized and cancer cells express much higher amounts of ERalpha than primary cells. Higher levels of ERalpha mRNA were also observed in the biopsies of bladder transitional cell carcinoma compared with sample from the same bladder unaffected by tumor. Using the ERalpha-selective agonist PPT, the ERbeta-selective agonist DPN, and specific small interfering RNA against ERalpha or ERbeta, we found that ERbeta predominantly mediates estrogen-induced G1/S transition and cell proliferation in the primary urothelial cells. By contrast, ERalpha predominantly mediates estrogen-induced G1/S transition and cell proliferation in bladder cancer cell lines. Furthermore, we found that 17beta-estradiol (E(2)) rapidly induces phosphorylation of extracellular signal-regulated kinases, but U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, does not affect E(2)-induced urothelial cell proliferation. E(2) up-regulated cyclin D1 and cyclin E expression in both the primary and bladder cancer cells, and the cancer cells have higher cyclin D1 and cyclin E expression during G0/G1 phases. Our data suggest that estrogen exerts its effects through different ER subtypes in urothelial cells. Increased expression of ERalpha may contribute to early induction of cyclin D1 and cyclin E during the cell cycle in bladder cancer cells.

The G protein-coupled receptor GPR30 inhibits human urothelial cell proliferation.

We have previously shown that estrogen stimulates cell proliferation in both normal and transformed urothelial cells mainly through activation of the two primary estrogen receptors (ERs), ERalpha and ERbeta. A growing body of evidence suggests that estrogen also initiates nongenomic effects that cannot be explained by activation of primary ERs. In the present study, we observed that urothelial cells express high amounts of GPR30, a G protein-coupled receptor recently identified as a candidate for membrane-associated estrogen binding. Membrane- impermeable bovine serum albumin-conjugated 17beta-estradiol and the specific GPR30 agonist G-1 both inhibited urothelial cell proliferation in a concentration-dependent manner. Transient overexpression of GPR30 inhibited 17beta-estradiol (E2)-induced cell proliferation. Decreased GPR30 expression caused by specific small interfering RNA increased E2-induced cell proliferation. These results indicate that membrane-associated inhibitory effects of E2 on cell proliferation correlate with abundance of GPR30. Although E2 induced a significant increase in caspase-3/7 activity, G-1 did not, suggesting that the GPR30-mediated inhibitory effect on cell proliferation was not caused by apoptosis. Furthermore, we found that G-1 failed to induce c-fos, c-jun, and cyclin D1 expression, and GPR30 overexpression abolished E2-induced c-fos, c-jun, and cyclin D1 expression. However, inactivation of GPR30 by small interfering RNA increased c-fos, c-jun, and cyclin D1 expression. These results suggest that GPR30-mediated inhibition of urothelial cell proliferation is the result of decreased cyclin D1 by down-regulation of activation protein-1 signaling.

Bacterial cystitis is accompanied by increased peripheral thermal sensitivity in mice.

PURPOSE: Visceral inflammation and pain associated with chemical cystitis produce increased sensitivity to noxious stimuli in the sacral dermatomes. We determined whether a similar sensitization occurs in response to bacterial cystitis. MATERIALS AND METHODS: Bacterial cystitis was induced by intravesical instillation of Escherichia coli 1677 in female C57BL/6N and C3H/OuJ mice (Jackson Laboratories, Bar Harbor, Maine). C3H/HeJ mice (Jackson Laboratories) served as a control because C3H/HeJ mice lack functional toll-like receptor 4, which is an essential component of cellular recognition of bacterial lipopolysaccharide. Hind paw sensitivity to thermal stimulus was quantitatively determined 1, 2, 7 and 14 days after infection. RESULTS: Intravesical instillation of E. coli produced infection in all strains of mice. Infection persisted in all C3H/OuJ and C3H/HeJ mice but it spontaneously cleared in some C57BL/6N mice. Increased sensitivity to thermal stimuli was observed in C57BL/6N and C3H/OuJ mice starting 1 to 2 days after E. coli instillation and it was still present 14 days after instillation. Increased sensitivity to thermal stimuli did not occur in C3H/HeJ mice. CONCLUSIONS: E. coli induced cystitis produced increased sensitivity to peripheral thermal stimuli in mice with competent toll-like receptor 4.

Cannabinoid receptor 2 is increased in acutely and chronically inflamed bladder of rats.

Cannabinoid receptors 1 and 2 (CB1 and CB2) are G-protein coupled receptors that are expressed throughout the body. Cannabinoid receptors are expressed in the urinary bladder and may affect bladder function. The purpose of this study was twofold: to confirm the presence of cannabinoid receptors in the bladder, the L6/S1 spinal cord, and dorsal root ganglia (DRG), and to determine the effects of acute and chronic bladder inflammation on expression of cannabinoid receptors. Acute or chronic bladder inflammation was induced in rats by intravesical administration of acrolein. Abundance of CB1 and CB2 protein and their respective mRNA was determined using immunoblotting and quantitative real-time PCR, respectively. We confirmed the presence of CB1 and CB2 receptor protein and mRNA in bladder, L6-S spinal cord, and DRG. Acute bladder inflammation induced increased expression of CB2, but not CB1, protein in the bladder detrusor. Chronic bladder inflammation increased expression of bladder CB2 protein and mRNA but not CB1 protein or mRNA. Expression of CB1 or CB2 in spinal cord or DRG was unaffected by acute or chronic bladder inflammation. CB1 and CB2 receptors are present in the bladder and its associated innervation, and CB2 receptors are up-regulated in bladder after acute or chronic inflammation. CB2 receptors may be a viable target for pharmacological treatment of bladder inflammation and associated pain.

[Study on influence of gamma-ray treatment on spectral characteristic of rapeseed].

After being treated by gamma-ray, the spectral characteristic of rapeseed would be changed. Based on the principle, a rapid and nondestructive method by using visible and near infrared spectroscopy was proposed to discriminate rapeseeds (Brassica nupus) treated by different dosages of gamma-ray. Partial least square (PLS) method and BP neural network (BPNN) were applied to establish the discrimination model, and the influences of different pretreatment methods of original spectra data, data transformation methods of PIS principal components and the.selection of node number of hidden layers of BP neural network model on prediction precision were compared and discussed. In the experiment, 184 samples were treated by gamma-ray with 5 different dosages (50, 100, 150, 200 Gy, and the samples without gamma-ray treatment). Then spectra tests were performed on the 184 samples using a spectrophotometer (325-1 075 nm). One hundred thiry five samples were selected randomly for model calibration and the left 49 samples were used for prediction. As a result, the optimal model was established and the parameters of the model were shown as follows. The original spectra data were pretreated by smoothing media filter, multiplicative scatter correction and Savitzky-Golay derivatives, then 6 PLS principal components were selected by using partial least square method. After being transformed by using natural logarithm transformation method, the 6 PLS principal components were used as the input layer factors to establish the BP neural network model and the node number of hidden layers was selected as 4 or 9. The prediction precision of the optimal model to distinguish the untreated samples from gamma-ray treated samples was 100%. The precision of predicting the dosages of gamma-ray treatment of all samples achieved 85.71%. It can be concluded that the proposed method for estimating the influence of different gamma-ray dosages on the spectral characteristic of treated rapeseeds was feasible.

Potential of Endocannabinoids to Control Bladder Pain.

Bladder-related pain is one of the most common forms of visceral pain, and visceral pain is among the most common complaints for which patients seek physician consultation. Despite extensive studies of visceral innervation and treatment of visceral pain, opioids remain a mainstay for management of bladder pain. Side effects associated with opioid therapy can profoundly diminish quality of life, and improved options for treatment of bladder pain remain a high priority. Endocannabinoids, primarily anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are endogenously-produced fatty acid ethanolamides with that induce analgesia. Animal experiments have demonstrated that inhibition of enzymes that degrade AEA or 2-AG have the potential to prevent development of visceral and somatic pain. Although experimental results in animal models have been promising, clinical application of this approach has proven difficult. In addition to fatty acid amide hydrolase (FAAH; degrades AEA) and monacylglycerol lipase (MAGL; degrades 2-AG), cyclooxygenase (COX) acts to metabolize endocannabinoids. Another potential limitation of this strategy is that AEA activates pro-nociceptive transient receptor potential vanilloid 1 (TRPV1) channels. Dual inhibitors of FAAH and TRPV1 or FAAH and COX have been synthesized and are currently undergoing preclinical testing for efficacy in providing analgesia. Local inhibition of FAAH or MAGL within the bladder may be viable options to reduce pain associated with cystitis with fewer systemic side effects, but this has not been explored. Further investigation is required before manipulation of the endocannabinoid system can be proven as an efficacious alternative for management of bladder pain.

Nerve growth factor mediates peripheral mechanical hypersensitivity that accompanies experimental cystitis in mice.

Increased sensitivity to somatic stimuli has been noted in the presence of visceral inflammation. Cystitis was induced by intraperitoneal injection of cyclophosphamide (CYP) in female mice. Sensitivity of hind paws to mechanical stimuli was determined prior to and 4, 9 and 24 h after CYP, and sensitivity of the tail to thermal stimuli was determined prior to, 4 and 24 h after CYP treatment. To investigate the role of nerve growth factor (NGF) in these processes, other groups of mice received NGF antiserum, normal serum, or K252a intravenously 30 min after CYP administration. CYP induced bladder inflammation that was not ablated by treatment with NGF antiserum or K252a. Sensitivity to mechanical stimuli was increased 4 and 9 h after CYP administration. This was reversed by NGF antiserum or K252a but not by normal serum. After 24 h, no differences were observed in withdrawal threshold among groups. None of the treatments had any effect on sensitivity to thermal stimuli. To further investigate the role of NGF in this process, NGF was instilled into the bladders of mice in the presence or absence of intravenous NGF antiserum. Four hours after intravesical instillation of NGF, the threshold of the hind paws to mechanical stimulation was significantly decreased, and this effect was reversed by prior treatment with NGF antiserum. This model of visceral pain causes increased sensitivity to peripheral application of mechanical stimuli. This effect is at least partially mediated by NGF, and the bladder may be the source of NGF in this process.

Chronic intermittent hypoxia alters ventilatory and metabolic responses to acute hypoxia in rats.

We determined the effects of chronic exposure to intermittent hypoxia (CIH) on chemoreflex control of ventilation in conscious animals. Adult male Sprague-Dawley rats were exposed to CIH [nadir oxygen saturation (SpO2), 75%; 15 events/h; 10 h/day] or normoxia (NORM) for 21 days. We assessed the following responses to acute, graded hypoxia before and after exposures: ventilation (V̇e, via barometric plethysmography), V̇o2 and V̇co2 (analysis of expired air), heart rate (HR), and SpO2 (pulse oximetry via neck collar). We quantified hypoxia-induced chemoreceptor sensitivity by calculating the stimulus-response relationship between SpO2 and the ventilatory equivalent for V̇co2 (linear regression). An additional aim was to determine whether CIH causes proliferation of carotid body glomus cells (using bromodeoxyuridine). CIH exposure increased the slope of the V̇e/V̇co2/SpO2 relationship and caused hyperventilation in normoxia. Bromodeoxyuridine staining was comparable in CIH and NORM. Thus our CIH paradigm augmented hypoxic chemosensitivity without causing glomus cell proliferation.