Methods: Excitotoxic retinal injury was induced with intravitreal injection of NMDA in Sprague-Dawley rats. All treatments were given as pre-, co-, and post-treatment with NMDA. Seven days post-injection, the retinas were processed for measurement of the expression of NOS isoforms using immunostaining and enzyme-linked immunosorbent assay (ELISA), retinal 3-NT content using ELISA, retinal histopathological changes using hematoxylin and eosin (H&E) staining, and retinal cell apoptosis using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining.
Results: As observed on immunohistochemistry, the treatment with NMDA caused a 4.53-fold increase in retinal nNOS expression compared to the PBS-treated rats (p<0.001). Among the MgAT-treated groups, only the pretreatment group showed significantly lower nNOS expression than the NMDA-treated group with a 2.00-fold reduction (p<0.001). Among the TAU-treated groups, the pre- and cotreatment groups showed 1.84- and 1.71-fold reduction in nNOS expression compared to the NMDA-treated group (p<0.001), respectively, but remained higher compared to the PBS-treated group (p<0.01). Similarly, iNOS expression in the NMDA-treated group was significantly greater than that for the PBS-treated group (2.68-fold; p<0.001). All MgAT treatment groups showed significantly lower iNOS expression than the NMDA-treated groups (3.58-, 1.51-, and 1.65-folds, respectively). However, in the MgAT co- and post-treatment groups, iNOS expression was significantly greater than in the PBS-treated group (1.77- and 1.62-folds, respectively). Pretreatment with MgAT caused 1.77-fold lower iNOS expression compared to pretreatment with TAU (p<0.05). In contrast, eNOS expression was 1.63-fold higher in the PBS-treated group than in the NMDA-treated group (p<0.001). Among all treatment groups, only pretreatment with MgAT caused restoration of retinal eNOS expression with a 1.39-fold difference from the NMDA-treated group (p<0.05). eNOS expression in the MgAT pretreatment group was also 1.34-fold higher than in the TAU pretreatment group (p<0.05). The retinal NOS expression as measured with ELISA was in accordance with that estimated with immunohistochemistry. Accordingly, among the MgAT treatment groups, only the pretreated group showed 1.47-fold lower retinal 3-NT than the NMDA-treated group, and the difference was significant (p<0.001). The H&E-stained retinal sections in all treatment groups showed statistically significantly greater numbers of retinal cell nuclei than the NMDA-treated group in the inner retina. However, the ganglion cell layer thickness in the TAU pretreatment group remained 1.23-fold lower than that in the MgAT pretreatment group (p<0.05). In line with this observation, the number of apoptotic cells as observed after TUNEL staining was 1.69-fold higher after pretreatment with TAU compared to pretreatment with MgAT (p<0.01).
Conclusions: MgAT and TAU, particularly with pretreatment, reduce retinal cell apoptosis by reducing retinal nitrosative stress. Pretreatment with MgAT caused greater improvement in NMDA-induced changes in iNOS and eNOS expression and retinal 3-NT levels than pretreatment with TAU. The greater reduction in retinal nitrosative stress after pretreatment with MgAT was associated with lower retinal cell apoptosis and greater preservation of the ganglion cell layer thickness compared to pretreatment with TAU.
METHODS: Sprague Dawley rats were intravitreally injected with ET1. MgAT and TAU were administered as pre-, co-, or posttreatment. Subsequently, the expression of NOS isoforms was detected in retina by immunohistochemistry, retinal nitrotyrosine level was estimated using ELISA, and retinal cell apoptosis was detected by TUNEL staining.
RESULTS: Intravitreal ET1 caused a significant increase in the expressions of nNOS and iNOS while eNOS expression was significantly reduced compared to vehicle treated group. Administration of both MgAT and TAU restored the altered levels of NOS isoform expression, reduced retinal nitrosative stress and retinal cell apoptosis. The effect of MgAT, however, was greater than that of TAU alone.
CONCLUSIONS: MgAT and TAU prevent ET1-induced retinal cell apoptosis by reducing retinal nitrosative stress in Sprague Dawley rats. Addition of TAU to Mg seems to enhance the efficacy of TAU compared to when given alone. Moreover, the pretreatment with MgAT/TAU showed higher efficacy compared to co- or posttreatment.
METHODS: The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing.
RESULTS: The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS -786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele).
CONCLUSIONS: Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C variants.
METHODS: A cross-section study using retrospective data over a 2-year period (1999-2000) involved 101 archival, formalin-fixed, paraffin-embedded tissue samples of colorectal cancers that were surgically resected in a tertiary referral.
RESULTS: COX-2 production was detected in adjacent normal tissue in 34 sample (33.7%) and in tumour tissue in 60 samples (59.4%). More tumours expressed iNOS (82/101, 81.2%) than COX-2. No iNOS expression was detected in adjacent normal tissue. Intense beta-catenin immunoreactivity at the cell-to-cell border. Poorly differentiated tumours had significantly lower total beta-catenin (p = 0.009) and COX-2 scores (p = 0.031). No significant relationships were established between pathological stage and beta-catenin, COX-2 and iNOS scores.
CONCLUSIONS: the accumulation of beta-catenin does not seem to be sufficient to activate pathways that lead to increased COX-2 and iNOS expression. A high proportion of colorectal cancers were found to express COX-2 and a significant number produced iNOS, suggesting that their inhibitors may be potentially useful as chemotherapeutic agents in the management of colorectal cancer.
METHODS: BV2 microglial cells c for 24 h, pre-treated with EPA for 24 h prior to LPS induction for another 24 h. Surface expression of CD11b and CD40 on BV2 cells was analyzed by flow cytometry. ELISA was employed to measure the production of pro-inflammatory mediators i.e. nitric oxide (NO) and tumor necrosis factor (TNF)-α. Western blotting technique was used to determine the expression of inducible nitric oxide synthase (iNOS), myeloid differentiation protein 88 (MYD88), nuclear factor kappa B (NF-κB), caspase-1, and mitogen activated protein kinase (MAPK).
RESULTS: Qualitative and quantitative analyses of the EPA using a validated ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method indicated the presence of phyllanthin, hypophyllanthin, niranthin, ellagic acid, corilagin, gallic acid, phyltetralin, isolintetralin and geraniin. EPA suppressed the production of NO and TNFα in LPS-activated BV2 microglial cells. Moreover, EPA attenuated the expression of MyD88, NF-κB and MAPK (p-P38, p-JNK and p-ERK1/2). It also inhibited the expression of CD11b and CD40. EPA protected against LPS-induced microglial activation via MyD88 and NF-κB signaling in BV2 microglial cells.
CONCLUSIONS: EPA demonstrated neuroprotective effects against LPS-induced microglial cells activation through the inhibition of TNFα secretion, iNOS protein expression and subsequent NO production, inhibition of NF-κB and MAPKs mediated by adapter protein MyD88 and inhibition of microglial activation markers CD11b and CD40.