Left ventricle function and post-transcriptional events with exercise training in pigs.

BACKGROUND: Standardized exercise protocols have been shown to improve overall cardiovascular fitness, but direct effects on left ventricular (LV) function, particularly diastolic function and relation to post-transcriptional molecular pathways (microRNAs (miRs)) are poorly understood. This project tested the central hypothesis that adaptive LV remodeling resulting from a large animal exercise training protocol, would be directly associated with specific miRs responsible for regulating pathways relevant to LV myocardial stiffness and geometry. METHODS AND RESULTS: Pigs (n = 9; 25 Kg) underwent a 4 week exercise training protocol (10 degrees elevation, 2.5 mph, 10 min, 5 days/week) whereby LV chamber stiffness (KC) and regional myocardial stiffness (rKm) were measured by Doppler/speckle tracking echocardiography. Age and weight matched non-exercise pigs (n = 6) served as controls. LV KC fell by approximately 50% and rKm by 30% following exercise (both p < 0.05). Using an 84 miR array, 34 (40%) miRs changed with exercise, whereby 8 of the changed miRs (miR-19a, miR-22, miR-30e, miR-99a, miR-142, miR-144, miR-199a, and miR-497) were correlated to the change in KC (r ≥ 0.5 p < 0.05) and mapped to matrix and calcium handling processes. Additionally, miR-22 and miR-30e decreased with exercise and mapped to a localized inflammatory process, the inflammasome (NLRP-3, whereby a 2-fold decrease in NLRP-3 mRNA occurred with exercise (p < 0.05). CONCLUSION: Chronic exercise reduced LV chamber and myocardial stiffness and was correlated to miRs that map to myocardial relaxation processes as well as local inflammatory pathways. These unique findings set the stage for utilization of myocardial miR profiling to identify underlying mechanisms by which exercise causes changes in LV myocardial structure and function.

Changes in Myocardial Microstructure and Mechanics With Progressive Left Ventricular Pressure Overload.

This study assessed the regional changes in myocardial geometry, microstructure, mechanical behavior, and properties that occur in response to progressive left ventricular pressure overload (LVPO) in a large animal model. Using an index of local biomechanical function at early onset of LVPO allowed for prediction of the magnitude of left ventricular chamber stiffness (Kc) and left atrial area at LVPO late timepoints. Our study found that LV myocardial collagen content alone was insufficient to identify mechanisms for LV myocardial stiffness with progression to heart failure with preserved ejection fraction (HFpEF). Serial assessment of regional biomechanical function might hold value in monitoring the natural history and progression of HFpEF, which would allow evaluation of novel therapeutic approaches.

Application of Hybrid Matrix Metalloproteinase-Targeted and Dynamic 201Tl Single-Photon Emission Computed Tomography/Computed Tomography Imaging for Evaluation of Early Post-Myocardial Infarction Remodeling.

BACKGROUND: The induction of matrix metalloproteinases (MMPs) and reduction in tissue inhibitors of MMPs (TIMPs) plays a role in ischemia/reperfusion (I/R) injury post-myocardial infarction (MI) and subsequent left ventricular remodeling. We developed a hybrid dual isotope single-photon emission computed tomography/computed tomography approach for noninvasive evaluation of regional myocardial MMP activation with 99mTc-RP805 and dynamic 201Tl for determination of myocardial blood flow, to quantify the effects of intracoronary delivery of recombinant TIMP-3 (rTIMP-3) on I/R injury. METHODS: Studies were performed in control pigs (n=5) and pigs following 90-minute balloon occlusion-induced ischemia/reperfusion (I/R) of left anterior descending artery (n=9). Before reperfusion, pigs with I/R were randomly assigned to intracoronary infusion of rTIMP-3 (1.0 mg/kg; n=5) or saline (n=4). Three days post-I/R, dual isotope imaging was performed with 99mTc-RP805 and 201Tl along with contrast cineCT to assess left ventricular function. RESULTS: The ischemic to nonischemic ratio of 99mTc-RP805 was significantly increased following I/R in saline group (4.03±1.40), and this ratio was significantly reduced with rTIMP-3 treatment (2.22±0.57; P=0.03). This reduction in MMP activity in the MI-rTIMP-3 treatment group was associated with an improvement in relative MI region myocardial blood flow compared with the MI-saline group and improved myocardial strain in the MI region. CONCLUSIONS: We have established a novel hybrid single-photon emission computed tomography/computed tomography imaging approach for the quantitative assessment of regional MMP activation, myocardial blood flow, and cardiac function post-I/R that can be used to evaluate therapeutic interventions such as intracoronary delivery of rTIMP-3 for reduction of I/R injury in the early phases of post-MI remodeling.

Delivery of a matrix metalloproteinase-responsive hydrogel releasing TIMP-3 after myocardial infarction: effects on left ventricular remodeling.

Although improvements in timing and approach for early reperfusion with acute coronary syndromes have occurred, myocardial injury culminating in a myocardial infarction (MI) remains a common event. Although a multifactorial process, an imbalance between the induction of proteolytic pathways, such as matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of metalloproteinase (TIMPs), has been shown to contribute to this process. In the present study, a full-length TIMP-3 recombinant protein (rTIMP-3) was encapsulated in a specifically formulated hyaluronic acid (HA)-based hydrogel that contained MMP-cleavable peptide cross-links, which influenced the rate of rTIMP-3 release from the HA gel. The effects of localized delivery of this MMP-sensitive HA gel (HAMMPS) alone and containing rTIMP-3 (HAMMPS/rTIMP-3) were examined in terms of the natural history of post-MI remodeling. Pigs were randomized to one of the following three different groups: MI and saline injection (MI/saline group, 100-µl injection at nine injection sites, n = 7), MI and HAMMPS injection (MI/HAMMPS group; 100-µl injection at nine injection sites, n = 7), and MI and HAMMPS/rTIMP-3 injection (MI/HAMMPS/rTIMP-3 group; 20-µg/100-µl injection at nine injection sites, n = 7). Left ventricular (LV) echocardiography was serially performed up to 28 days post-MI. LV dilation, as measured by end-diastolic volume, and the degree of MI wall thinning were reduced by ~50% in the HAMMPS/rTIMP-3 group ( P < 0.05). Furthermore, indexes of heart failure progression post-MI, such as LV filling pressures and left atrial size, were also attenuated to the greatest degree in the HAMMPS/rTIMP-3 group. At 28 days post-MI, HAMMPS/rTIMP-3 caused a relative reduction in the transcriptional profile for myofibroblasts as well as profibrotic pathways, which was confirmed by subsequent histochemistry. In conclusion, these findings suggest that localized delivery of a MMP-sensitive biomaterial that releases a recombinant TIMP holds promise as a means to interrupt adverse post-MI remodeling. NEW & NOTEWORTHY The present study targeted a myocardial matrix proteolytic system, matrix metalloproteinases (MMPs), through the use of a recombinant tissue inhibitor of MMPs incorporated into a MMP-sensitive hydrogel, which was regionally injected using a large animal model of myocardial infarction. Left ventricular geometry and function and indexes of myocardial remodeling were improved with this approach and support the advancement of localized therapeutic strategies that specifically target the myocardial matrix.

Regional and temporal changes in left ventricular strain and stiffness in a porcine model of myocardial infarction.

The aim of the present study was to serially track how myocardial infarction (MI) impacts regional myocardial strain and mechanical properties of the left ventricle (LV) in a large animal model. Post-MI remodeling has distinct regional effects throughout the LV myocardium. Regional quantification of LV biomechanical behavior could help explain changes in global function and thus advance clinical assessment of post-MI remodeling. The present study is based on a porcine MI model to characterize LV biomechanics over 28 days post-MI via speckle-tracking echocardiography (STE). Regional myocardial strain and strain rate were recorded in the circumferential, radial, and longitudinal directions at baseline and at 3, 14, and 28 days post-MI. Regional myocardial wall stress was calculated using standard echocardiographic metrics of geometry and Doppler-derived hemodynamic measurements. Regional diastolic myocardial stiffness was calculated from the resultant stress-strain relations. Peak strain and phasic strain rates were nonuniformly reduced throughout the myocardium post-MI, whereas time to peak strain was increased to a similar degree in the MI region and border zone by 28 days post-MI. Elevations in diastolic myocardial stiffness in the MI region plateaued at 14 days post-MI, after which a significant reduction in MI regional stiffness in the longitudinal direction occurred between 14 and 28 days post-MI. Post-MI biomechanical changes in the LV myocardium were initially limited to the MI region but nonuniformly extended into the neighboring border zone and remote myocardium over 28 days post-MI. STE enabled quantification of regional and temporal differences in myocardial strain and diastolic stiffness, underscoring the potential of this technique for clinical assessment of post-MI remodeling. NEW & NOTEWORTHY For the first time, speckle-tracking echocardiography was used to serially track regional biomechanical behavior and mechanical properties postmyocardial infarction (post-MI). We found that changes initially confined to the MI region extended throughout the myocardium in a nonuniform fashion over 28 days post-MI. Speckle-tracking echocardiography-based evaluation of regional changes in left ventricular biomechanics could advance both clinical assessment of left ventricular remodeling and therapeutic strategies that target aberrant biomechanical behavior post-MI.

Intracoronary delivery of recombinant TIMP-3 after myocardial infarction: effects on myocardial remodeling and function.

Ischemia-reperfusion (IR) and myocardial infarction (MI) cause adverse left ventricular (LV) remodeling and heart failure and are facilitated by an imbalance in matrix metalloproteinase (MMP) activation and the endogenous tissue inhibitors of metalloproteinase (TIMPs). We have identified that myocardial injections of recombinant TIMP-3 (rTIMP-3; human full length) can interrupt post-MI remodeling. However, whether and to what degree intracoronary delivery of rTIMP-3 post-IR is feasible and effective remained to be established. Pigs (25 kg) underwent coronary catheterization and balloon occlusion of the left anterior descending coronary artery (LAD) for 90 min whereby at the final 4 min, rTIMP-3 (30 mg, n = 9) or saline was infused in the distal LAD. LV echocardiography was performed at 3-28 days post-IR, and LV ejection fraction (EF) and LV end-diastolic volume were measured. LV EF fell and LV end-diastolic volume increased from baseline (pre-IR) values (66 ± 1% and 40 ± 1 ml, respectively, means ± standard deviation) in both groups; however, the extent of LV dilation was reduced in the rTIMP-3 group by 40% at 28 days post-IR (P < 0.05) and the fall in LV EF was attenuated. Despite equivalent plasma troponin levels (14 ± 3 ng/ml), computed MI size at 28 days was reduced by over 45% in the rTIMP-3 group (P < 0.05), indicating that rTIMP-3 treatment abrogated MI expansion post-IR. Plasma NH2-terminal pro-brain natriuretic peptide levels, an index of heart failure progression, were reduced by 25% in the rTIMP-3 group compared with MI saline values (P < 0.05). Although the imbalance between MMPs and TIMPs has been recognized as a contributory factor for post-MI remodeling, therapeutic strategies targeting this imbalance have not been forthcoming. This study is the first to demonstrate that a relevant delivery approach (intracoronary) using rTIMP can alter the course of post-MI remodeling.NEW & NOTEWORTHY Myocardial ischemia and reperfusion injury remain significant causes of morbidity and mortality whereby alterations in the balance between matrix metalloproteinase and tissue inhibitor of metalloproteinase have been identified as contributory biological mechanisms. This novel translational study advances the concept of targeted delivery of recombinant proteins to modify adverse myocardial remodeling in ischemia-reperfusion injury.

Pseudouridine synthase 1 deficient mice, a model for Mitochondrial Myopathy with Sideroblastic Anemia, exhibit muscle morphology and physiology alterations.

Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1(-/-) animals. Pus1(-/-) mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1(-/-) mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1(-/-) mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1(-/-) mice.

Hematologic and serum biochemical values of 4 species of Peromyscus mice and their hybrids.

Deer mice (Peromyscus maniculatus) and congeneric species are used in a wide variety of research applications, particularly studies of developmental, physiologic, and behavioral characteristics associated with habitat adaptation and speciation. Because peromyscine mice readily adapt to colony conditions, animals with traits of interest in the field are moved easily into the laboratory where they can be studied under controlled conditions. The purpose of this study was to determine the serum chemistry and hematologic parameters of 4 frequently used species from the Peromyscus Genetic Stock Center species (P. californicus, P. leucopus, P. maniculatus, and P. polionotus) and to determine quantitative differences in these parameters among species and between sexes. Triglyceride values were substantially higher in female compared with male mice in all 4 species. Similar cross-species differences in MCH were present. Overall there was considerable interspecific variation for most blood parameters, with little evidence for covariation of any 2 or more parameters. Because crosses of P. maniculatus and P. polionotus produce fertile offspring, segregation analyses can be applied to determine the genetic basis of any traits that differ between them, such as their 3.8- and 2.1-fold interspecific differences in cholesterol and triglyceride levels, respectively. The current data provide a set of baseline values useful for subsequent comparative studies of species experiencing different circumstances, whether due to natural variation or anthropogenic environmental degradation. To enable such comparisons, the raw data are downloadable from a site maintained by the Stock Center (http://ww2.biol.sc.edu/∼peromyscus).

Caring for Peromyscus spp. in research environments.

Peromyscus spp. are the most abundant native North American mammals. They have gained popularity as research animals in the last 20 years, and this trend is expected to continue as new research tools, such as whole genome sequences, baseline physiological data and others, become available. Concurrently, advances have been made in the recommendations for the care of laboratory animals. The authors provide insight into how the Peromyscus Genetic Stock Center successfully breeds and maintains several stocks of deer mice and related species. This information is beneficial to researchers that plan to include Peromyscus spp. in their research programs.

SDF-1-CXCR4 differentially regulates autoimmune diabetogenic T cell adhesion through ROBO1-SLIT2 interactions in mice.

AIMS/HYPOTHESIS: We had previously reported that stromal cell-derived factor 1 (SDF-1) mediates chemorepulsion of diabetogenic T cell adhesion to islet microvascular endothelium through unknown mechanisms in NOD mice. Here we report that SDF-1-mediated chemorepulsion occurs through slit homologue (SLIT)2-roundabout, axon guidance receptor, homologue 1 (Drosophila) (ROBO1) interactions. METHODS: C-X-C receptor (CXCR)4 and ROBO1 protein expression was measured in mouse and human T cells. Parallel plate flow chamber adhesion and detachment studies were performed to examine the molecular importance of ROBO1 and SLIT2 for SDF-1-mediated T cell chemorepulsion. Diabetogenic splenocyte transfer was performed in NOD/LtSz Rag1(-/-) mice to examine the effect of the SDF-1 mimetic CTCE-0214 on adoptive transfer of diabetes. RESULTS: CXCR4 and ROBO1 protein expression was elevated in diabetic NOD/ShiLtJ T cells over time and coincided with the onset of hyperglycaemia. CXCR4 and ROBO1 expression was also increased in human type 1 diabetic T cells, with ROBO1 expression maximal at less than 1 year post diagnosis. Cell detachment studies revealed that immunoneutralisation of ROBO1 prevented SDF-1-mediated chemorepulsion of NOD T cell firm adhesion to TNFα-stimulated islet endothelial cells. SDF-1 increased NOD T cell adhesion to recombinant adhesion molecules, a phenomenon that was reversed by recombinant SLIT2. Finally, we found that an SDF-1 peptide mimetic prevented NOD T cell adhesion in vitro and significantly delayed adoptive transfer of autoimmune diabetes in vivo. CONCLUSIONS/INTERPRETATION: These data reveal a novel molecular pathway, which regulates diabetogenic T cell recruitment and may be useful in modulating autoimmune diabetes.

Genetic deficiency of Itgb2 or ItgaL prevents autoimmune diabetes through distinctly different mechanisms in NOD/LtJ mice.

OBJECTIVE: Insulitis is an important pathological feature of autoimmune diabetes; however, mechanisms governing the recruitment of diabetogenic T-cells into pancreatic islets are poorly understood. Here, we determined the importance of leukocyte integrins beta(2)(Itgb2) and alphaL (ItgaL) in developing insulitis and frank diabetes. RESEARCH DESIGN AND METHODS: Gene-targeted mutations of either Itgb2 or ItgaL were established on the NOD/LtJ mouse strain. Experiments were performed to measure insulitis and diabetes development. Studies were also performed measuring mutant T-cell adhesion to islet microvascular endothelial cells under hydrodynamic flow conditions. T-cell adhesion molecule profiles and adoptive transfer studies were also performed. RESULTS: Genetic deficiency of either Itgb2 or ItgaL completely prevented the development of hyperglycemia and frank diabetes in NOD mice. Loss of Itgb2 or ItgaL prevented insulitis with Itgb2 deficiency conferring complete protection. In vitro hydrodynamic flow adhesion studies also showed that loss of Itgb2 completely abrogated T-cell adhesion. However, ItgaL deficiency did not alter NOD T-cell adhesion to or transmigration across islet endothelial cells. Adoptive transfer of ItgaL-deficient splenocytes into NOD/Rag-1 mice did not result in development of diabetes, suggesting a role for ItgaL in NOD/LtJ T-cell activation. CONCLUSIONS: Together, these data demonstrate that genetic deficiency of Itgb2 or ItgaL confers protection against autoimmune diabetes through distinctly different mechanisms.

Stromal cell-derived factor-1/CXCL12 stimulates chemorepulsion of NOD/LtJ T-cell adhesion to islet microvascular endothelium.

OBJECTIVE: Diabetogenic T-cell recruitment into pancreatic islets facilitates beta-cell destruction during autoimmune diabetes, yet specific mechanisms governing this process are poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1) controls T-cell recruitment, and genetic polymorphisms of SDF-1 are associated with early development of type 1 diabetes. RESEARCH DESIGN AND METHODS: Here, we examined the role of SDF-1 regulation of diabetogenic T-cell adhesion to islet microvascular endothelium. Islet microvascular endothelial cell monolayers were activated with tumor necrosis factor-alpha (TNF-alpha), subsequently coated with varying concentrations of SDF-1 (1-100 ng/ml), and assayed for T-cell/endothelial cell interactions under physiological flow conditions. RESULTS: TNF-alpha significantly increased NOD/LtJ T-cell adhesion, which was completely blocked by SDF-1 in a dose-dependent manner, revealing a novel chemorepulsive effect. Conversely, SDF-1 enhanced C57BL/6J T-cell adhesion to TNF-alpha-activated islet endothelium, demonstrating that SDF-1 augments normal T-cell adhesion. SDF-1 chemorepulsion of NOD/LtJ T-cell adhesion was completely reversed by blocking G(i)alpha-protein-coupled receptor activity with pertussis toxin. CXCR4 protein expression was significantly decreased in NOD/LtJ T-cells, and inhibition of CXCR4 activity significantly reversed SDF-1 chemorepulsive effects. Interestingly, SDF-1 treatment significantly abolished T-cell resistance to shear-mediated detachment without altering adhesion molecule expression, thus demonstrating decreased integrin affinity and avidity. CONCLUSIONS: In this study, we have identified a previously unknown novel function of SDF-1 in negatively regulating NOD/LtJ diabetogenic T-cell adhesion, which may be important in regulating diabetogenic T-cell recruitment into islets.

Sildenafil promotes ischemia-induced angiogenesis through a PKG-dependent pathway.

BACKGROUND: Peripheral artery disease (PAD) is a prevalent cardiovascular disorder that results in tissue ischemia which can progress to critical limb ischemia. Restoration of tissue perfusion in the setting of chronic ischemia through stimulation of arteriogenesis and angiogenesis remains a key therapeutic target for PAD. However, experimental therapeutics, including growth factor and gene therapy, have had little clinical success indicating the need for a better understanding of molecular pathways required for therapeutic angiogenesis. METHODS AND RESULTS: Here we report that phosphodiesterase-5 inhibition by sildenafil significantly increases vascular perfusion, tissue blood flow, and vascular density during chronic ischemia of the mouse hind limb. Importantly, sildenafil therapy did not alter any of these parameters in nonischemic limbs. Sildenafil increased tissue cGMP levels independently of increases in nitric oxide production, and sildenafil therapy stimulated angiogenesis in ischemic limbs of eNOS-/- and iNOS-/- mice. Lastly, sildenafil-mediated angiogenic activity was blocked by inhibition of protein kinase G using the PKG antagonist DT-3. CONCLUSIONS: These data demonstrate that sildenafil therapy results in increased angiogenic activity through a PKG-dependent pathway that is independent of nitric oxide production or NOS activity and identify the angiogenic therapeutic potential of sildenafil for critical limb ischemia.

Regulation of endothelial glutathione by ICAM-1 governs VEGF-A-mediated eNOS activity and angiogenesis.

Previous studies suggest that inflammatory cell adhesion molecules may modulate endothelial cell migration and angiogenesis through unknown mechanisms. Using a combination of in vitro and in vivo approaches, herein we reveal a novel redox-sensitive mechanism by which ICAM-1 modulates endothelial GSH that controls VEGF-A-induced eNOS activity, endothelial chemotaxis, and angiogenesis. In vivo disk angiogenesis assays showed attenuated VEGF-A-mediated angiogenesis in ICAM-1(-/-) mice. Moreover, VEGF-A-dependent chemotaxis, eNOS phosphorylation, and nitric oxide production were impaired in ICAM-1(-/-) mouse aortic endothelial cells (MAEC) compared to WT MAEC. Decreasing intracellular GSH in ICAM-1(-/-) MAEC to levels observed in WT MAEC with 150 microM buthionine sulfoximine restored VEGF-A responses. Conversely, GSH supplementation of WT MAEC with 5 mM glutathione ethyl ester mimicked defects observed in ICAM-1(-/-) cells. Deficient angiogenic responses in ICAM-1(-/-) cells were associated with increased expression of the lipid phosphatase PTEN, consistent with antagonism of signaling pathways leading to eNOS activation. PTEN expression was also sensitive to GSH status, decreasing or increasing in proportion to intracellular GSH concentrations. These data suggest a novel role for ICAM-1 in modulating VEGF-A-induced angiogenesis and eNOS activity through regulation of PTEN expression via modulation of intracellular GSH status.

Loss of cortical function in mice after decapitation, cervical dislocation, potassium chloride injection, and CO2 inhalation.

Electroencephalograms (EEG) and visual evoked potentials (VEP) in mice were recorded to evaluate loss of cortical function during the first 30 s after euthanasia by various methods. Tracheal cannulae (for positive-pressure ventilation, PPV) and cortical surface electrodes were placed in mice anesthetized with inhaled halothane. Succinylcholine was used to block spontaneous breathing in the mice, which then underwent continuous EEG recording. Photic stimuli (1 Hz) were presented to produce VEPs superimposed on the EEG. Anesthesia was discontinued immediately before euthanasia. Compared with that obtained before euthanasia, EEG activity during the 30-s study period immediately after euthanasia was significantly decreased after cervical dislocation (at 5 to 10 s), 100% PPV-CO2 (at 10 to 15 s), decapitation (at 15 to 20 s), and cardiac arrest due to KCl injection (at 20 to 25 s) but not after administration of 70% PPV-CO2. Similarly, these euthanasia methods also reduced VEP amplitude, although 100% PPV-CO2 treatment affected VEP amplitude more than it did EEG activity. Thus, 100% PPV-CO2 treatment significantly decreased VEP beginning 5 to 10 s after administration, with near abolition of VEP by 30 s. VEP amplitude was significantly reduced at 5 to 10 s after cervical dislocation and at 10 to 15 s after decapitation but not after either KCl or 70% PPV-CO2 administration. The data demonstrate that 100% PPV-CO2, decapitation, and cervical dislocation lead to rapid disruption of cortical function as measured by 2 different methods. In comparison, 70% PPV-CO2 and cardiac arrest due to intracardiac KCl injection had less rapid effects on cortical function.

Regulation of dextran sodium sulfate induced colitis by leukocyte beta 2 integrins.

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders whose etiology remains unknown. Reports have shown that infiltration of leukocytes into intestinal tissue is a pathognomonic hallmark for this disease. Leukocyte beta(2) integrins are heterodimeric adhesion membrane proteins that are exclusively expressed on leukocytes and participate in immune cell adhesion and activation. In this study, we examined the pathophysiological role of the beta(2) integrins CD18, CD11a, and CD11b in the pathogenesis of dextran sodium sulfte (DSS)-induced experimental colitis. Disease activity was measured by daily assessment of clinical parameters including stool consistency, weight loss, occult blood, and gross rectal bleeding. Histopathological changes including severity of inflammation, surface epithelial/crypt damage, and depth of injury were also determined. The CD18 null and CD11a null mice had significantly lower disease activity and cumulative histopathological scores compared to wild-type mice. Interestingly, CD11b null mice did not show protection against DSS colitis and displayed increased disease activity compared to wild-type mice. Examination of specific leukocyte populations in the distal colon from various mice revealed significant attenuation of neutrophil and macrophage infiltrates in CD18, CD11a, and CD11b null mice. Surprisingly, the CD11b null mice showed a significant increase in plasma cell infiltration in response to DSS suggesting that this molecule may influence plasma cell function during colitis. This study demonstrates that genetic loss of CD18 or CD11a is protective during experimental colitis and that CD11b may serve a regulatory role during development of disease.

Eradication of Syphacia muris from food-restricted rats without environmental decontamination.

Rodent pinworms rarely cause clinical disease, but infestation can affect experimental results. Our facility maintained a colony of Wistar rats for behavioral pharmacology studies that had been infested with Syphacia muris for > 15 years. The laboratory in which the animals were housed encompassed several rooms and contained a variety of complex behavioral equipment, including > 60 operant chambers. Several prior attempts to eliminate the pinworms were unsuccessful because of inadequate duration of treatment and incomplete environmental decontamination. Many of the rats in this colony were food-restricted as part of behavioral studies. Pinworms were eliminated from these animals by treating them with 450 ppm fenbendazole-containing feed for 3 consecutive weeks followed by 6 weeks of alternating every other week with standard rodent diet. Rats not on food restriction protocols were treated on the same schedule with 150 ppm fenbendazole-containing feed. Environmental decontamination of eggs from the behavioral equipment was not attempted. One year after treatment, the colony has remained free of S. muris. We adapted previously published protocols to our situation, including the problem of food-restricted rats and unfeasible environmental decontamination, to eradicate S. muris from our colony.

CD18 deficiency protects against multiple low-dose streptozotocin-induced diabetes.

Leukocyte recruitment into pancreatic islets is believed to play an important pathophysiological role in autoimmune diabetes. Previous reports have suggested that several different adhesion molecules may be involved in leukocyte recruitment during autoimmune diabetes, including members of the leukocyte beta(2) integrins. Here we report that a gene-targeted deficiency of the beta(2) integrin, CD18, protects against multiple low-dose streptozotocin-induced autoimmune diabetes. CD18 null mice displayed lower blood glucose values throughout the study, with only 10% of these mice eventually developing diabetes compared to 95% in the control group. Importantly, the development of insulitis was markedly absent in the CD18 null mice, suggesting that members of this integrin subfamily predominately regulate leukocyte infiltration into pancreatic islets. This study demonstrates that the beta(2) integrins play a key pathophysiological role in the development of multiple low-dose streptozotocin-induced autoimmune diabetes.

Cystatin m: a novel candidate tumor suppressor gene for breast cancer.

The contribution of pericellular proteolysis to tumor progression is well documented. To better understand protease biology and facilitate clinical translation, specific proteolytic systems need to be better defined. In particular, the precise role of endogenous protease inhibitors still needs to be deciphered. We reported previously that cystatin M, a potent endogenous inhibitor of lysosomal cysteine proteases, significantly suppressed in vitro cell proliferation, migration, and Matrigel invasion. Here, we show that scid mice orthotopically implanted with breast cancer cells expressing cystatin M show significantly delayed primary tumor growth and lower metastatic burden in the lungs and liver when compared with mice implanted with mock controls. The incidence of metastasis, however, appeared to be unaltered between the cystatin M group and the control group. Experimental metastasis assays suggest that cystatin M suppressed tumor cell proliferation at the secondary site. By using laser capture microdissection and quantitative reverse transcription-polymerase chain reaction, we found consistent expression of cystatin M in normal human breast epithelial cells, whereas expression was decreased by 86% in invasive ductal carcinoma (IDC) cells of stage I to IV patients. Complete loss of expression of cystatin M was observed in two of three IDCs from stage IV patients. Immunohistochemical studies confirmed that expression of cystatin M in IDCs was partially or completely lost. We propose cystatin M as a novel candidate tumor suppressor gene for breast cancer.