BACKGROUND AND PURPOSE:Intraplaque neovessels (INVs) are considered important contributors to carotid plaque vulnerability. The purpose of this study was to examine whether differences in INV distribution affect plaque vulnerability. METHODS:The study cohort comprised 110 patients with significant stenosis of the carotid artery who had undergone carotid endarterectomy. The distribution of INVs within carotid plaques was assessed by immunohistochemical studies using anti-CD-34 antibody as a marker for endothelial cells. First, we divided the patients into M group and S group depending on the numbers of INVs in middle and shoulder region. Next, we categorized carotid plaques into four categories according to the distributions of INVs: Shoulder, Middle, Mixed, and Scarce. We then compared total area of intraplaque hemorrhage, cholesterol, and calcification, width of thinnest fibrous cap, and number of INVs between the four categories of plaque. RESULTS:The area of intraplaque hemorrhage was significantly larger in the M group than in the S group (P = 0.011). Meanwhile, symptomatic carotid stenosis was significantly more frequently associated with the Middle and Mixed than the Shoulder and Scarce categories (P < 0.01). The area of intraplaque hemorrhage was significantly different between the four groups (P = 0.022). Rupture of the fibrous cap was more frequently detected in the Middle and Mixed than the other categories (P = 0.002). CONCLUSIONS:INVs in the middle region of carotid plaques are strongly associated with symptomatic carotid stenosis, intraplaque hemorrhage, and rupture of the fibrous cap. Our findings indicate that the distribution of INVs may affect plaque vulnerability.

Recent genetic studies have shown that genetic loci with significant effects in whole-genome quantitative trait loci (QTL) analyses were lost or weakened in congenic strains. Characterisation of the genetic basis of this attenuated QTL effect is important to our understanding of the genetic mechanisms of complex traits. We previously found that a consomic strain, B6-Chr6C MSM , which carries chromosome 6 of a wild-derived strain MSM/Ms on the genetic background of C57BL/6J, exhibited lower home-cage activity than C57BL/6J. In the present study, we conducted a composite interval QTL analysis using the F2 mice derived from a cross between C57BL/6J and B6-Chr6C MSM . We found one QTL peak that spans 17.6 Mbp of chromosome 6. A subconsomic strain that covers the entire QTL region also showed lower home-cage activity at the same level as the consomic strain. We developed 15 congenic strains, each of which carries a shorter MSM/Ms-derived chromosomal segment from the subconsomic strain. Given that the results of home-cage activity tests on the congenic strains cannot be explained by a simple single-gene model, we applied regression analysis to segregate the multiple genetic loci. The results revealed three loci (loci 1–3) that have the effect of reducing home-cage activity and one locus (locus 4) that increases activity. We also found that the combination of loci 3 and 4 cancels out the effects of the congenic strains, which indicates the existence of a genetic mechanism related to the loss of QTLs.

Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4), the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.

Age-related disappearance of the LH surge is one of major biomarkers of reproductive aging in female rats. Kisspeptin neurons in the hypothalamic anteroventral periventricular nucleus (AVPV) are proposed as the critical regulator of the preovulatory LH surge in response to estrogenic positive feedback. Here we investigated the possible involvement of the AVPV kisspeptin neurons in the disappearance of the LH surge in middle-age rats. Middle-age rats exhibiting persistent estrus (M-PE) did not show an LH surge although neither Kiss1 mRNA nor peptide in the AVPV was differentially expressed when compared to young rats exhibiting normal estrous cycles (YN). M-PE released LH in response to exogenous kisspeptin in a similar dose-dependent manner as YN, suggesting that their GnRH neurons still maintained responsiveness to kisspeptin. To investigate the estrogenic positive feedback effect on kisspeptin neurons in the AVPV, rats were ovariectomized and supplemented with estradiol (OVX + E2). We performed in situ hybridization and immunohistochemistry for Kiss1 mRNA and cFos, respectively, and found that M-PE exhibited a significantly lower percentage of Kiss1 mRNA positive neurons with cFos immunoreactivity, although the total number of kisspeptin neurons was not different from that in cyclic rats. Furthermore, OVX + E2 M-PE did not show the surge-like LH release under high estradiol administration while YN did. Thus our current study suggests that the reduced responsiveness of the AVPV kisspeptin neurons to estrogenic positive feedback presumably results in the decrease in kisspeptin secretion from neurons and eventually causes the age-related disappearance of the LH surge in middle age female rats.

Based on crosses among inbred strains derived principally from M. m. domesticus, sucrose octaacetate (SOA) aversion in laboratory mice has been thought for many years to be controlled by a single genetic locus (Soa) located on distal chromosome (Chr) 6. To expand knowledge of the genetic basis underlying SOA aversion, we have studied the M. m. molossinus derived strain (MSM) and MSM consomic strains on a M. m. domesticus (C57BL/6J: B6) host background. Using two-bottle preference procedures, MSM mice avoided 0.1 mM and 1 mM SOA while B6 mice were indifferent to 0.1 mM and exhibited slight aversion to 1 mM SOA. Preference tests of 16 available consomic strains implicated Chr 2, 4 and 15 in SOA aversion in addition to the prominent effect of the known Soa locus on Chr 6 (implicated by study of two congenic strains). The originally defined Soa locus is presumably associated with one or more members of the cluster of Tas2r genes on distal Chr 6 that code for bitter taste receptors. Our results point to the possible role of established Tas2r genes on Chr 2 and 15, as well as to genes not coding for bitter receptors (Chr 4), in SOA aversion. SOA aversion emerges from this consomic screen as being definitively under polygenic control. The genetic diversity captured by the MSM model system is shown to be a valuable tool to complement the limited genetic variation in the commonly used stocks derived from M m. domesticus.

The activity of mice in their home cage is influenced greatly by the cycle of light and dark. In addition, home-cage activity shows remarkable time-dependent changes that result in a prominent temporal pattern. The wild-derived mouse strain MSM/Ms (MSM) exhibits higher total activity in the home cage than does C57BL/6 (B6), a commonly used laboratory strain. In addition, there is a clear strain difference in the temporal pattern of home-cage activity. This study aimed to clarify the genetic basis of strain differences in the temporal pattern of home-cage activity between MSM and B6. Through the comparison of temporal patterns of home-cage activity between B6 and MSM, the pattern can be classified into five temporal components: (1) resting phase, (2) anticipation phase, (3) 1st phase, (4) 2nd phase, and (5) 3rd phase. To identify quantitative trait loci (QTLs) involved in these temporal components, we used consomic strains established from crosses between B6 and MSM. Five consomic strains, for Chrs 2T (telomere), 3, 4, 13, and 14, showed significantly higher total activity than B6. In contrast, the consomic strains of Chrs 6C (centromere), 7T, 9, 11, and 15 were less active than B6. This indicates that multigenic factors regulate the total activity. Further analysis showed an impact of QTLs on the temporal components of home-cage activity. The present data showed that each temporal component was regulated by different combinations of multigenic factors, with some overlap. These temporal component-related QTLs are important to understand fully the genetic mechanisms that underlie home-cage activity.

By creating the Visible Burrow System (VBS) Bob Blanchard found a way to study the interaction of genetics, physiology, environment, and adaptive significance in a model with broad validity. The VBS changed the way we think about anxiety and affective disorders by allowing the mechanisms which control them to be observed in a dynamic setting. Critically, Blanchard used the VBS and other models to show how behavioral systems like defense are dependent upon context and behavioral elements unique to the individual. Inspired by the VBS, we developed a Stress Alternatives Model (SAM) to further explore the multifaceted dynamics of the stress response with a dichotomous choice condition. Like the VBS, the SAM is a naturalistic model built upon risk-assessment and defensive behavior, but with a choice of response: escape or submission to a large conspecific aggressor. The anxiety of novelty during the first escape must be weighed against fear of the aggressor, and a decision must be made. Both outcomes are adaptively significant, evidenced by a 50/50 split in outcome across several study systems. By manipulating the variables of the SAM, we show that a gradient of anxiety exists that spans the contextual settings of escaping an open field, escaping from aggression, and submitting to aggression. These findings correspond with increasing levels of corticosterone and increasing levels of NPS and BDNF in the central amygdala as the context changes. Whereas some anxiolytics were able to reduce the latency to escape for some animals, only with the potent anxiolytic drug antalarmin (CRF1R-blocker) and the anxiogenic drug yohimbine (α2 antagonist) were we able to reverse the outcome for a substantial proportion of individuals. Our findings promote a novel method for modeling anxiety, offering a distinction between low-and-high levels, and accounting for individual variability. The translational value of the VBS is immeasurable, and it guided us and many other researchers to seek potential clinical solutions through a deeper understanding of regional neurochemistry and gene expression in concert with an ecological behavioral model.

The goal of this study was to determine the adiposity of a range of rat strains, including a panel of consomics, to estimate heritability. To that end, we assessed the body fat distribution and organ weights of groups of adult male rats from 3 outbred strains, 11 inbred strains and 22 consomic strains. We measured the weights of the gonadal, retroperitoneal, mesenteric, femoral, subscapular and pericardial white fat depots, the subscapular brown fat depot, the kidneys, liver, heart, spleen, and brain. Strains were compared for the measured weight of each of these adipose depots and organs, and also for these weights adjusted statistically for body size. All individual adipose depot and organ weights were highly heritable, in most cases h2 > 0.50. The fourteen inbred and outbred rat strains were not very different in body length but there was a three-fold difference in body weight, and up to a twenty-fold difference in the weight of some adipose depots. Comparison of the FHH-Chr nBN consomic strains with the FHH host strain revealed 98 quantitative trait loci (QTLs) for body composition and organ weight, with the introgressed chromosome reducing weight or adiposity in most cases. These results can be used to guide the choice of appropriate rat strains for future studies of the genetic architecture of obesity and body size.

Endogenous renal dopamine is a major physiological regulator of renal ion transport; however its intracellular signaling pathways are not thoroughly understood. The present study examined the role of 20-hydroxyeicosatetraenoic acid (20-HETE), the major cytochrome P450 (CYP4A) metabolite of arachidonic acid formed in the renal cortex, on the natriuretic response to dopamine in Sprague Dawley rats. Infusion of dopamine (1.5 μg/kg/min, i.v.) increased urine flow (1.9 fold over basal), sodium excretion (UNaV, 2.7 fold), fractional sodium excretion (FENa, 3.3 fold) and proximal and distal delivery of sodium by 1.5‐ and 2-fold respectively. Administration of two inhibitors of the synthesis of 20-HETE, 1-aminobenzotriazole (ABT) and N-hydroxy-N′-(‐4-butyl-2-methylphenyl)formamidine (HET0016) reduced the response to dopamine by 65%. Induction of the renal expression of CYP4A enzymes with clofibrate did not alter the response to dopamine. The natriuretic response to dopamine was lower in Dahl salt-sensitive rats in comparison to an SS.BN5 consomic strain in which transfer of chromosome 5 from Brown Norway to Dahl salt-sensitive rats upregulates the renal expression of CYP4A protein and the production of 20-HETE. Treatment with HET0016 blocked the renal effects of dopamine in SS.BN5 rats. We also examined the influence of 20-HETE in the natriuretic response to acute volume expansion that is in part mediated via the release of endogenous dopamine. The increase in urine flow, UNaV, FENa and distal FENa following volume expansion was markedly reduced in rats treated with ABT. These results suggest that 20-HETE plays at least a permissive role in the natriuretic response to dopamine.

Consomic strains have recently attracted attention as an advantageous method to screen for genes related to developmental, physiological, and behavioral phenotypes. Recently, a new set of consomic strains was established from the Japanese wild‐derived mouse strain MSM/Ms and C57BL/6JJcl. By analyzing the entire consomic panel, we were able to identify a number of chromosomes associated with anxiety‐like behaviors in the open‐field (OF) test, a light–dark box and an elevated plus maze. Detailed observation of the OF behavior allowed us to identify chromosomes associated with those ethological traits, such as stretch attend, rearing, and jumping. Repeated OF test trials have different meanings for animals, and we found that some chromosomes responded to only the first or second trial, while others were consistent across both trials. By examining both male and female mice, sex‐dependent effects were found in several measurements. Principal component analysis of anxiety‐like behaviors extracted five factors: ‘general locomotor activity’, ‘thigmotaxis’, ‘risk assessment’, ‘open‐arm exploration’ and ‘autonomic emotionality’. We mapped chromosomes associated with these five factors of emotionality.

In higher animal cells, the principal limitation of gene-targeting technology is the extremely low efficiency of targeted integration, which occurs three to four orders of magnitude less frequently than random integration. Assuming that random integration mechanistically involves non-homologous end-joining (NHEJ), inactivation of this pathway should reduce random integration and may enhance gene targeting. To test this possibility, we examined the frequencies of random and targeted integration in NHEJ-deficient chicken DT40 and human Nalm-6 cell lines. As expected, loss of NHEJ resulted in drastically reduced random integration in DT40 cells. Unexpectedly, however, this was not the case for Nalm-6 cells, indicating that NHEJ is not the sole mechanism of random integration. Nevertheless, we present evidence that NHEJ inactivation can lead to enhanced gene targeting through a reduction of random integration and/or an increase in targeted integration by homologous recombination. Most intriguingly, our results show that, in the absence of functional NHEJ, random integration of targeting vectors occurs more frequently than non-targeting vectors (harboring no or little homology to the host genome), implying that suppression of NHEJ-independent random integration events is needed to greatly enhance gene targeting in animal cells.