Genetic and Histological Alterations Reveal Key Role of Prostaglandin Synthase and Cyclooxygenase 1 and 2 in Traumatic Brain Injury–Induced Neuroinflammation in the Cerebral Cortex of Rats Exposed to Moderate Fluid Percussion Injury

After the initial insult in traumatic brain injury (TBI), secondary neurodegeneration occurs that is intimately associated with neuroinflammation. Prostaglandin (PG) synthases and cyclooxygenase (COX) 1 and 2 may contribute to inflammation in the brain. Temporal and spatial expression features of PG and COX1 and 2 following trauma may guide the development of antineuroinflammation strategies. Here, we examined PG synthase signaling and COX1 and 2 gene expression levels and COX-1- and 2-positive cell types and their temporal localization in TBI-induced brain in an effort to reveal their participation in the disease’s evolving neuroinflammation. Using brain samples from the cerebral cortex of rats subjected to TBI model of lateral moderate fluid percussion injury (FPI), we sought to characterize the temporal (subacute TBI) and spatial (lateral cortical lesion) brain alterations accompanying the disease progression. Temporal gene expression changes of PG synthase signaling were compared between sham-operated and TBI-treated rats using microarray pathway analysis. Moreover, we examined COX1 and 2 expression patterns and their intracellular distribution in sham-operated and TBI-treated rats by immunohistochemistry. After FPI, COX1 and 2 gene expression levels, and PGE2 synthase increased while PGD2 synthase decreased, suggesting that PGE2 and PGD2 afforded contraindicative effects of inflammation and anti-inflammation, respectively. Immunohistochemical analyses showed that both COX1 and COX2 increased in a time-dependent manner in the brain, specifically in degenerating neurons of the cortex. Interestingly, the expression of COX cell type was cell-specific, in that COX1 was particularly increased in degenerating neurons while COX2 was expressed in macrophages. In view of the dynamic temporal and spatial expression of PG, COX1 and 2 gene expression and localization in the injured brain regulating PG synthase and COX1 and 2 activity will require a careful disease-specific tailoring of treatments to abrogate the neuroinflammation-plagued secondary cell death due to TBI

Genetic and Histological Alterations Reveal Key Role of Prostaglandin Synthase and Cyclooxygenase 1 and 2 in Traumatic Brain Injury–Induced Neuroinflammation in the Cerebral Cortex of Rats Exposed to Moderate Fluid Percussion Injury

After the initial insult in traumatic brain injury (TBI), secondary neurodegeneration occurs that is intimately associated with neuroinflammation. Prostaglandin (PG) synthases and cyclooxygenase (COX) 1 and 2 may contribute to inflammation in the brain. Temporal and spatial expression features of PG and COX1 and 2 following trauma may guide the development of antineuroinflammation strategies. Here, we examined PG synthase signaling and COX1 and 2 gene expression levels and COX-1- and 2-positive cell types and their temporal localization in TBI-induced brain in an effort to reveal their participation in the disease’s evolving neuroinflammation. Using brain samples from the cerebral cortex of rats subjected to TBI model of lateral moderate fluid percussion injury (FPI), we sought to characterize the temporal (subacute TBI) and spatial (lateral cortical lesion) brain alterations accompanying the disease progression. Temporal gene expression changes of PG synthase signaling were compared between sham-operated and TBI-treated rats using microarray pathway analysis. Moreover, we examined COX1 and 2 expression patterns and their intracellular distribution in sham-operated and TBI-treated rats by immunohistochemistry. After FPI, COX1 and 2 gene expression levels, and PGE2 synthase increased while PGD2 synthase decreased, suggesting that PGE2 and PGD2 afforded contraindicative effects of inflammation and anti-inflammation, respectively. Immunohistochemical analyses showed that both COX1 and COX2 increased in a time-dependent manner in the brain, specifically in degenerating neurons of the cortex. Interestingly, the expression of COX cell type was cell-specific, in that COX1 was particularly increased in degenerating neurons while COX2 was expressed in macrophages. In view of the dynamic temporal and spatial expression of PG, COX1 and 2 gene expression and localization in the injured brain regulating PG synthase and COX1 and 2 activity will require a careful disease-specific tailoring of treatments to abrogate the neuroinflammation-plagued secondary cell death due to TBI

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes.

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted "bidirectional analysis," which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples

A Unique Primer with an Inosine Chain at the 5'-Terminus Improves the Reliability of SNP Analysis Using the PCR-Amplified Product Length Polymorphism Method.

Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3'-terminus of each primer. To use this method at least two allele-specific primers and one "counter-primer", which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3'-terminus, and another primer should have a few non-complementary flaps at the 5'-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5'-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs

Robustness evaluation of a PCR-APLP method using UV-irradiated template DNA.

<p>A and B show the results of robustness analysis using the sense and antisense primer sets, respectively. Lanes 1–5 correspond to female DNA damaged by 0, 0.5, 1.0, 5.0, and 10 J UV irradiation; lanes 6–10 to male DNA damaged by 0, 0.5, 1.0, 5.0, and 10 J UV irradiation; and lane 11 to the negative control. Lanes 1 and 6 are female and male positive controls, respectively. M indicates the 10-bp ladder markers. The results were reproduced in three independent assays.</p

Tm values of competing primers.

<p>Prb denotes probe. ‘α’ indicates that the 3′-terminus of the probe is complementary to the target primer, and ‘β’ denotes that the 3′-terminus of the probe is non-complementary to the target primer. 1^st indicates that the probe was used to measure the Tm of the target primer at the first PCR cycle, and 2^nd denotes that the probe was used to measure the Tm of the target primer in later PCR cycles. Mean values ± SD are shown for triplicate assays.</p><p>Tm values of competing primers.</p

DNA sexing of Jomon samples by our method.

<p>A and B show the results of sex determination using the sense and antisense primer sets, respectively. M indicates the 10-bp ladder marker. The results were reproduced in three independent assays. Typical results of bidirectional analysis are shown because amounts of template DNA were not constant among ancient samples.</p

Comparison between PCR-APLP SNP analyses using the inosine flap primer and primer with a flap of ordinary non-complementary bases.

<p>Yellow, blue, red, and white open boxes indicate human mtDNA haplogroups N, R, F, and N9, respectively. LM indicates 10 bp ladder marker. Lanes 1 and 6: haplogroup D4a mtDNA; lanes 2 and 7: haplogroup B4a mtDNA; lanes 3 and 8: haplogroup F1b mtDNA; lanes 4 and 9, haplogroup N9b mtDNA; and lanes 5 and 10, haplogroup A mtDNA.</p

Sex determination by PowerPlex^® ESX17 Fast using UV-irradiated template DNA.

<p>A and C show female and male DNA with no UV irradiation, respectively. B and D show UV irradiation-exposed female (0.5 J) and male (0.2 J) DNA, respectively. The results were reproduced in three independent assays.</p