Propranolol prevents cerebral blood flow changes and pain-related behaviors in migraine model mice

Propranolol, a β-adrenergic receptor blocker, is one of the most commonly used prophylactic drugs for migraines. Cortical spreading depression (CSD) is the propagation wave of neuronal excitation along with cerebral blood flow (CBF) changes over the cerebral cortex and has been implicated in the pathological process of migraine auras and its pain response. However, the effect of propranolol on CSD-related CBF changes and behavioral responses remains poorly understood. In this study, we measured CSD-related CBF responses using a micro-device with a green light emitting diode (LED) and micro-complementary-metal-oxide-semiconductor (CMOS) image sensor and evaluated pain-related reduced locomotor activity in mice. An injection of KCl into the visual cortex led to CSD-related CBF changes; however, propranolol prevented the increase in CBF as well as delayed the propagation velocity in KCl-induced CSD. Furthermore, an injection of KCl reduced locomotor activity and induced freezing behavior in awake and freely moving mice, which were prevented by propranolol treatment. These results suggest that the modulation of CSD-related CBF responses by the blockade of β-adrenergic receptor contributes to its prophylactic effects on migraines.     

Participation of macrophages in atherosclerotic lesion morphology in LDLr-/- mice

Lystbeige (beige) mice crossed with LDL receptor-deficient (LDLr-/-) mice had a distinct atherosclerotic lesion morphology that was not observed in LDLr-/- mice. This morphology is often associated with a stable plaque phenotype. We hypothesized that macrophage expression of the beige mutation accounted for this distinct morphology. Cultured bone marrow-derived macrophages from LDLr-/- and beige,LDLr-/- mice were compared for their ability to accumulate cholesterol, efflux cholesterol, migrate in response to chemotactic stimuli through Matrigel-coated membranes, and express matrix metalloproteinase 9 (MMP9). No differences in cholesterol metabolism were identified. Beige,LDLr-/- macrophage invasion in vitro appeared to be less than LDLr-/- macrophage invasion but did not achieve significance. Nevertheless, tumor necrosis factor-alpha-induced MMP9 expression, secretion, and enzymatic activity of beige,LDLr-/- macrophages were all significantly decreased compared with those of LDLr-/- macrophages (P < 0.05). For in vivo analyses of macrophage function, bone marrow transplantation (BMT) studies were performed. LDLr-/- mice and beige,LDLr-/- mice were irradiated and reconstituted with wild-type or beige bone marrow from mice expressing green fluorescent protein (GFP). Identification of GFP cells provided for direct identification of donor-derived cells within lesions. Only expression of the beige mutation in the BMT recipients altered the macrophage location and collagen content of the lesions. These results suggested that impaired macrophage function by itself did not account for the stable lesion morphology of beige,LDLr-/- double-mutant mice.     

Course of Brugia malayi infection in C57BL/6J NOS2 +/+ and -/- mice.

Previous results from our laboratory using pharmacological approaches suggested a role for nitric oxide (NO) in the host defense against the human filarial parasite, Brugia malayi. We sought to determine whether a complementary genetic approach, using mice homozygous for a targeted mutation in the gene encoding inducible nitric oxide-synthase (NOS2), would confirm our observation. We hypothesized that such mice would exhibit some deficit in their ability to clear B. malayi. Our data show that the course of infection in NOS2-/- mice is the same as in wild-type mice. Thus, peritoneal cellular responses to infection are similar in NOS2-/- and wild-type mice, with the exception that T cells form a higher percentage of total peritoneal cells in the former. We find virtually no serum IgE in NOS2-/- mice, suggesting a less robust Th2 response. In contrast, NOS2-/- mice demonstrate an early rise in IgG2a titers compared to B6 +/+ mice. Our data suggest that NO is not an obligate requirement for the elimination of B. malayi from the peritoneal cavities of mice.     

Leukocyte-derived hepatic lipase increases HDL and decreases en face aortic atherosclerosis in LDLr-/- mice expressing CETP

In addition to hepatic expression, cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) are expressed by human macrophages. The combined actions of these proteins have profound effects on HDL structure and function. It is not known how these HDL changes influence atherosclerosis. To elucidate the role of leukocyte-derived HL on atherosclerosis in a background of CETP expression, we studied low density lipoprotein receptor-deficient mice expressing human CETP (CETPtgLDLr(-/-)) with a leukocyte-derived HL deficiency (HL(-/-) BM). HL(-/-) bone marrow (BM), CETPtgLDLr(-/-) mice were generated via bone marrow transplantation. Wild-type bone marrow was transplanted into CETPtgLDLr(-/-) mice to generate HL(+/+) BM, CETPtgLDLr(-/-) controls. The chimeras were fed a high-fat, high-cholesterol diet for 14 weeks to promote atherosclerosis. In female HL(-/-) BM, CETPtgLDLr(-/-) mice plasma HDL-cholesterol concentration during high-fat feeding was decreased 27% when compared with HL(+/+) BM, CETPtgLDLr(-/-) mice (P < 0.05), and this was associated with a 96% increase in en face aortic atherosclerosis (P < 0.05). In male CETPtgLDLr(-/-) mice, leukocyte-derived HL deficiency was associated with a 16% decrease in plasma HDL-cholesterol concentration and a 25% increase in aortic atherosclerosis. Thus, leukocyte-derived HL in CETPtgLDLr(-/-) mice has an atheroprotective role that may involve increased HDL levels.     

Numerical simulation of local blood flow in the carotid and cerebral arteries under altered gravity

A computational fluid dynamics (CFD) approach was presented to model the blood flows in the carotid bifurcation and the brain arteries under altered gravity. Physical models required for CFD simulation were introduced including a model for arterial wall motion due to fluid-wall interactions, a shear thinning fluid model of blood, a vascular bed model for outflow boundary conditions, and a model for autoregulation mechanism. The three-dimensional unsteady incompressible Navier-Stokes equations coupled with these models were solved iteratively using the pseudocompressibility method and dual time stepping. Gravity source terms were added to the Navier-Stokes equations to take the effect of gravity into account. For the treatment of complex geometry, a chimera overset grid technique was adopted to obtain connectivity between arterial branches. For code validation, computed results were compared with experimental data for both steady-state and time-dependent flows. This computational approach was then applied to blood flows through a realistic carotid bifurcation and two Circle of Willis models, one using an idealized geometry and the other using an anatomical data set. A three-dimensional Circle of Willis configuration was reconstructed from subject-specific magnetic resonance images using an image segmentation method. Through the numerical simulation of blood flow in two model problems, namely, the carotid bifurcation and the brain arteries, it was observed that the altered gravity has considerable effects on arterial contraction/dilatation and consequent changes in flow conditions.     

Monounsaturated fatty acyl-coenzyme A is predictive of atherosclerosis in human apoB-100 transgenic, LDLr-/- mice

ACAT2, the enzyme responsible for the formation of cholesteryl esters incorporated into apolipoprotein B-containing lipoproteins by the small intestine and liver, forms predominantly cholesteryl oleate from acyl-CoA and free cholesterol. The accumulation of cholesteryl oleate in plasma lipoproteins has been found to be predictive of atherosclerosis. Accordingly, a method was developed in which fatty acyl-CoA subspecies could be extracted from mouse liver and quantified. Analyses were performed on liver tissue from mice fed one of four diets enriched with one particular type of dietary fatty acid: saturated, monounsaturated, n-3 polyunsaturated, or n-6 polyunsaturated. We found that the hepatic fatty acyl-CoA pools reflected the fatty acid composition of the diet fed. The highest percentage of fatty acyl-CoAs across all diet groups was in monoacyl-CoAs, and values were 36% and 46% for the n-3 and n-6 polyunsaturated diet groups and 55% and 62% in the saturated and monounsaturated diet groups, respectively. The percentage of hepatic acyl-CoA as oleoyl-CoA was also highly correlated to liver cholesteryl ester, plasma cholesterol, LDL molecular weight, and atherosclerosis extent. These data suggest that replacing monounsaturated with polyunsaturated fat can benefit coronary heart disease by reducing the availability of oleoyl-CoA in the substrate pool of hepatic ACAT2, thereby reducing cholesteryl oleate secretion and accumulation in plasma lipoproteins.     

Antisense oligonucleotide inhibition of cholesteryl ester transfer protein enhances RCT in hyperlipidemic,CETP transgenic,LDLr-/- mice

Due to their ability to promote positive effects across all of the lipoprotein classes, cholesteryl ester transfer protein (CETP) inhibitors are currently being developed as therapeutic agents for cardiovascular disease. In these studies, we compared an antisense oligonucleotide (ASO) inhibitor of CETP to the CETP small molecule inhibitor anacetrapib. In hyperlipidemic CETP transgenic (tg) mice, both drugs provided comparable reductions in total plasma cholesterol, decreases in CETP activity, and increases in HDL cholesterol. However, only mice treated with the antisense inhibitor showed an enhanced effect on macrophage reverse cholesterol transport, presumably due to differences in HDL apolipoprotein composition and decreases in plasma triglyceride. Additionally, the ASO-mediated reductions in CETP mRNA were associated with less accumulation of aortic cholesterol. These preliminary findings suggest that CETP ASOs may represent an alternative means to inhibit that target and to support their continued development as a treatment for cardiovascular disease in man.     

SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I-/- mice.

Serum amyloid A (SAA) is an acute phase protein of unknown function that is involved in systemic amyloidosis and may also be involved in atherogenesis. The precise role of SAA in these processes has not been established. SAA circulates in plasma bound to high density lipoprotein-3 (HDL3). The pathway for the production of SAA-containing HDL is not known. To test whether apolipoprotein (apo)A-I-HDL is required in the production of SAA-HDL, we analyzed the lipopolysaccharide (LPS)-induced changes in apoA-I+/+ and apoA-I-/- mice. In apoA-I+/+ mice, after injection of LPS, remodeling of HDL occurred: total cholesterol increased and apoA-I decreased slightly and shifted to lighter density. Dense (density of HDL3) but large (size of HDL2 ) SAA-containing particles were formed. Upon fast phase liquid chromatography fractionation of plasma, >90% of SAA eluted with HDL that was enriched in cholesterol and phospholipid and shifted "leftward" to larger particles. Non-denaturing immunoprecipitation with anti-mouse apoA-I precipitated all of the apoA-I but not all of the SAA, confirming the presence of SAA-HDL devoid of apoA-I. In the apoA-I-/- mice, which normally have very low plasma lipid levels, LPS injection resulted in significantly increased total and HDL cholesterol. Greater than 90% of the SAA was lipid associated and was found on dense but large, spherical HDL particles essentially devoid of other apolipoproteins.We conclude that serum amyloid A (SAA) is able to sequester lipid, forming dense but large HDL particles with or without apoA-I or other apolipoproteins. The capacity to isolate lipoprotein particles containing SAA as the predominant or only apolipoprotein provides an important system to further explore the biological function of SAA.     

Total and regional cerebral blood flow during moderate and severe exercise in miniature swine.

To determine the influence of exercise on cerebral blood flow, we ran 14 swine at 3-6 mph and at 0-10% grades on a treadmill for 30 min at moderate and severe levels of exercise. Measuring heart rate, cardiac output, and aortic pressure via implanted probes, we injected 15-mum radiolabeled microspheres via the left atrium before and during exercise. We measured their distribution by gamma spectrometry, determining total cerebral blood flow, regional blood flow, and ratio of flow to gray and white matter. Heart rate, cardiac output, and aortic pressure rose progressively with increasing exercise. Total cerebral flow resembled that reported in humans, i.e., it did not change significantly with exercise. Regional flow distribution also failed to change significantly with exercise. The ratio of gray to white matter flow did not change except to the cerebellum where it rose significantly from resting values at both moderate and severe exercise. Gray matter received more flow than white matter during all three conditions of observation. Cerebral blood flow was remarkably constant during even severe exercise.     

Paucity of V-D-D-J rearrangements and VH replacement events in lupus prone and nonautoimmune TdT-/- and TdT+/+ mice

CDR3 regions containing two D segments, or containing the footprints of V(H) replacement events, have been reported in both mice and humans. However, the 12-23 bp rule for V(D)J recombination predicts that D-D rearrangements, which would occur between 2 recombination signal sequences (RSSs) with 12-bp spacers, should be extremely disfavored, and the cryptic RSS used for V(H) replacement is very inefficient. We have previously shown that newborn mice, which lack TdT due to the late onset of its expression, do not contain any CDR3 with D-D rearrangements. In the present study, we test our hypothesis that most D-D rearrangements are due to fortuitous matching of the second apparent D segment by TdT-introduced N nucleotides. We analyzed 518 sequences from adult MRL/lpr- and C57BL/6 TdT-deficient B cell precursors and found only two examples of CDR3 with D-D rearrangements and one example of a potential V(H) replacement event. We examined rearrangements from pre-B cells, marginal zone B cells, and follicular B cells from mice congenic for the Lbw5 (Sle3/5) lupus susceptibility loci and from other strains of mice and found very few examples of CDR3 with D-D rearrangements. We assayed B progenitor cells, and cells enriched for receptor editing, for DNA breaks at the "cryptic heptamer" but such breaks were rare. We conclude that many examples of apparent D-D rearrangements in the mouse are likely due to N additions that fortuitously match short stretches of D genes and that D-D rearrangements and V(H) replacement are rare occurrences in the mouse.     

Phenotypic characterization of transgenic mice harboring Nf1+/- or Nf1-/- osteoclasts in otherwise Nf1+/+ background

Skeletal abnormalities in neurofibromatosis type 1 syndrome (NF1) are observed in ～50% of patients. Here, we describe the phenotype of Nf1_Ocl mouse model with Nf1‐deficient osteoclasts. Nf1_Ocl mice with Nf1^+/? or Nf1^?/? osteoclasts in otherwise Nf1^+/+ background were successfully generated by mating parental Nf1flox/flox and TRAP‐Cre mice. Contrary to our original hypothesis, osteoporotic or fragile bone phenotype was not observed. The µCT analysis revealed that tibial bone marrow cavity, trabecular tissue volume, and the perimeter of cortical bone were smaller in Nf1 mice compared to Nf1 control mice. Nf1 mice also a displayed narrowed growth plate in the proximal tibia. In vitro analysis showed increased bone resorption capacity and cytoskeletal changes including irregular cell shape and abnormal actin ring formation in Nf1^?/? osteoclasts. Surprisingly, the size of spleen in Nf1 mice was two times larger than in controls and histomorphometric analysis showed splenic megakaryocytosis. In summary, Nf1Ocl mouse model presented with a mild but specific bone phenotype. This study shows that NF1‐deficiency in osteoclasts may have a role in the development of NF1‐related skeletal abnormalities, but Nf1‐deficiency in osteoclasts in Nf1^+/+ background is not sufficient to induce skeletal abnormalities analogous to those observed in patients with NF1. J. Cell. Biochem. 113: 2136–2146, 2012. © 2012 Wiley Periodicals, Inc.     

Implication of adenosine A2A receptors in hypotension-induced vasodilation and cerebral blood flow autoregulation in rat pial arteries

This study aimed to evaluate the role for adenosine A2A receptors in the autoregulatory vasodilation to hypotension in relation with cerebral blood flow (CBF) autoregulation in rat pial arteries. Changes in pial artery diameters were observed directly through a closed cranial window. Vasodilation induced by adenosine was markedly suppressed by ZM 241385 (1 micromol/l, A2A antagonist) and alloxazine (1 micromol/l, A2B antagonist), but not by 8-cyclopentyltheophylline (CPT, 1 micromol/l, A1 antagonist). CGS-21680-induced vasodilation was more strongly inhibited by ZM 241385 (25.3-fold; P<0.05) than by alloxazine. In contrast, 5'-N-ethylcarboxamido-adenosine (NECA)-induced vasodilation was more prominently suppressed by alloxazine (12.0-fold; P<0.001) than by ZM 241385. The autoregulatory vasodilation in response to acute hypotension of the pial arteries was significantly suppressed by ZM 241385, but not by CPT and alloxazine. Consistent with this finding, the lower limit of CBF autoregulation significantly shifted to a higher blood pressure by 1 micromol/l of ZM 241385 (53.0+/-3.9 mm Hg to 69.2+/-2.9 mm Hg, P<0.01) and 10 micromol/l of glibenclamide (54.7+/-6.5 mm Hg to 77.9+/-4.2 mm Hg, P<0.001), but not by CPT and alloxazine. Thus, it is suggested that adenosine-induced vasodilation of the rat pial artery is mediated via activation of adenosine A2A and A2B receptors, but not by A1 subtype, and activation of adenosine A2A receptor preferentially contributes to the autoregulatory vasodilation via activation of ATP-sensitive K+ channels in response to hypotension and maintenance of CBF autoregulation.     

DHA improves cognition and prevents dysfunction of entorhinal cortex neurons in 3xTg-AD mice

Defects in neuronal activity of the entorhinal cortex (EC) are suspected to underlie the symptoms of Alzheimer's disease (AD). Whereas neuroprotective effects of docosahexaenoic acid (DHA) have been described, the effects of DHA on the physiology of EC neurons remain unexplored in animal models of AD. Here, we show that DHA consumption improved object recognition (↑12%), preventing deficits observed in old 3xTg-AD mice (↓12%). Moreover, 3xTg-AD mice displayed seizure-like akinetic episodes, not detected in NonTg littermates and partly prevented by DHA (↓50%). Patch-clamp recording revealed that 3xTg-AD EC neurons displayed (i) loss of cell capacitance (CC), suggesting reduced membrane surface area; (ii) increase of firing rate versus injected current (F-I) curve associated with modified action potentials, and (iii) overactivation of glutamatergic synapses, without changes in synaptophysin levels. DHA consumption increased CC (↑12%) and decreased F-I slopes (↓21%), thereby preventing the opposite alterations observed in 3xTg-AD mice. Our results indicate that cognitive performance and basic physiology of EC neurons depend on DHA intake in a mouse model of AD.     

Cardiovascular effects of centrally administered endothelin-1 and its relationship to changes in cerebral blood flow

Intracerebroventricular (i.c.v.) injection of endothelin-1 (ET-1; 100 ng, i.c.v.) produced an initial pressor (24%) (peak at 3 min following ET-1 administration) and a delayed depressor (−40%) (30 and 60 min following ET-1 administration) effects in urethane anesthetized rats. The pressor effect of ET-1 was due to an increase (21%) in cardiac output, while the depressor effect of ET-1 was associated with a marked decrease (−46%) in cardiac output. Stroke volume significantly decreased at 30 and 60 min after the administration of ET-1. No change in total peripheral vascular resistance and heart rate was observed following central administration of ET-1. The effects of ET-1 on blood pressure, cardiac output and stroke volume were not observed in BQ123 (10 μg, i.c.v.) treated rats. Blood flow to the cerebral hemispheres, cerebellum, midbrain and brain stem was not affected at 3 min, but a significant decrease in blood flow to all the regions of the brain was observed at 30 and 60 min following central administration of ET-1. BQ123 pretreatment completely blocked the central ET-1 induced decrease in blood flow to the brain regions. It is concluded that the pressor effect of centrally administered ET-1 is not accompanied by a severe decrease in brain blood flow, however, a subsequent decrease in blood pressure is associated with a decrease in blood flow to the brain. The cardiovascular effects of ET-1 including decrease in brain blood flow are mediated through central ET_A receptors.     

大鼠脑缺血/再灌注过程中血流量和脑组织含水量的变化趋势*

目的:研究大鼠脑缺血/再灌注过程中血流量及与脑组织水含量变化的趋势。方法:选取5只成年SD雄性大鼠(n=5),参照改良Zea-Longa线栓法制备大鼠大脑中动脉缺血/再灌注模型,2 h后拔出线栓。利用PeriCam PSI血流灌注成像系统实时监测大鼠在缺血前及缺血5 min、30 min、1 h、2 h、再灌注5 min、30 min、1 h、2 h、4 h、6 h及24 h的血流灌注量,记录在ROI(感兴趣区)测量的数值。再选取15只成年SD雄性大鼠,分为Control组、缺血2 h、再灌注30 min、4 h及24 h组(n=3)。正常组不做任何处理,实验组按上述线栓法制备MCAO模型。取新鲜脑组织用干湿重法测定其左、右半球的水含量。结果:栓塞时缺血侧血流量逐渐下降,缺血2 h下降最低(P＜0.05);再灌注早期血流量恢复较大(P＜0.05),30 min时显著下降(P＜0.05),4 h明显上升(P＜0.05),24 h再次上升(P＜0.05)但低于缺血前血流量(P＞0.05)。脑组织水含量测量,缺血2 h组和再灌注30 min组与正常组无明显差异(P＞0.05);再灌4 h组和再灌24 h组明显增高(P＜0.05),且再灌24 h组明显高于再灌4 h组(P＜0.05)。结论:大鼠脑缺血/再灌注过程中血流量和脑组织中水含量的变化存在一定的规律,且脑组织中水含量与再灌注过程中血流量的变化有一定关系。     

Comparison of antigen-specific T cell responses in autoimmune MRL/Mp-lpr/lpr and MRL/Mp-+/+ mice

The MRL-1 mouse develops severe autoimmune disease characterized by high titers of autoantibodies at an early age (3 to 5 mo). The congeneic MRL-n mouse, which differs only in the lymphoproliferative (lpr) gene, exhibits no such pathologic or serologic abnormalities at the same age. We examined antigen-specific T cell responses in the MRL-1 mouse and compared them to age- and sex-matched MRL-n controls. We found broad defects in these responses in the MRL-1 mouse; an inability to generate primary allospecific and hapten-specific cytolytic T lymphocytes (CTL), secondary hapten- and virus-specific CTL, as well as a deficient proliferative response to hapten and natural antigens and a weak delayed-type hypersensitivity response were demonstrated. Our data furthermore suggest a lack of interleukin 2 (IL 2) acceptor sites in the proliferating T cell, while suggesting no such lack on CTL precursors. In fact, the deficient CTL responses in MRL-1 mice can be restored to levels seen in MRL-n by the in vitro addition of IL 2. The implications of these findings and the possible explanations for the relative deficit in helper function in the MRL-1 mouse are discussed.