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Angiotensin II has been implicated in the progression of diabetic retinopathy, which is characterized by altered microvasculature, oxidative stress, and neuronal dysfunction. The signaling induced by angiotensin II can occur not only via receptor-mediated calcium release that causes vascular constriction, but also through a pathway whereby angiotensin II activates NADPH oxidase to elicit the formation of reactive oxygen species (ROS). In the current study, we administered the angiotensin II receptor antagonist candesartan (or vehicle, in untreated animals) in a rat model of type 1 diabetes in which hyperglycemia was induced by injection of streptozotocin (STZ). Eight weeks after the STZ injection, untreated diabetic rats were found to have a significant increase in tissue levels of angiotensin converting enzyme (ACE; p < 0.05) compared to non-diabetic controls, a 33% decrease in retinal blood flow rate ( p < 0.001), and a dramatic increase in p22phox (a subunit of the NADPH oxidase). The decrease in retinal blood flow, and the increases in retinal ACE and p22phox in the diabetic rats, were all significantly attenuated ( p < 0.05) by the administration of candesartan in drinking water within one week. Neither STZ nor candesartan induced any changes in tissue levels of superoxide dismutase (SOD-1), 4-hydroxynonenal (4-HNE), or nitrotyrosine. We conclude that one additional benefit of candesartan (and other angiotensin II antagonists) may be to normalize retinal blood flow, which may have clinical benefits in diabetic retinopathy.

Electrophilic aldehyde (4-hydroxynonenal; 4-HNE), formed after lipid peroxidation, is a mediator of mitochondrial dysfunction and implicated in both the pathogenesis and the progression of cardiovascular disease. Manganese superoxide dismutase (MnSOD), a nuclear-encoded antioxidant enzyme, catalyzes the dismutation of superoxide radicals (O 2 •- ) in mitochondria. To study the role of MnSOD in the myocardium, we generated a cardiomyocyte-specific SOD2 (SOD2Δ) deficient mouse strain. Unlike global SOD2 knockout mice, SOD2Δ mice reached adolescence; however, they die at ~4 months of age due to heart failure. Ultrastructural analysis of SOD2Δ hearts revealed altered mitochondrial architecture, with prominent disruption of the cristae and vacuole formation. Noninvasive echocardiographic measurements in SOD2 Δ mice showed dilated cardiomyopathic features such as decreased ejection fraction and fractional shortening along with increased left ventricular internal diameter. An increased incidence of ventricular tachycardia was observed during electrophysiological studies of the heart in SOD2Δ mice. Oxidative phosphorylation (OXPHOS) measurement using a Seahorse XF analyzer in SOD2Δ neonatal cardiomyocytes and adult cardiac mitochondria displayed reduced O 2 consumption, particularly during basal conditions and after the addition of FCCP (H + ionophore/uncoupler), compared to that in SOD2fl hearts. Measurement of extracellular acidification (ECAR) to examine glycolysis in these cells showed a pattern precisely opposite that of the oxygen consumption rate (OCR) among SOD2Δ mice compared to their SOD2 fl littermates. Analysis of the activity of the electron transport chain complex identified a reduction in Complex I and Complex V activity in SOD2Δ compared to SOD2fl mice. We demonstrated that a deficiency of SOD2 increases reactive oxygen species (ROS), leading to subsequent overproduction of 4-HNE inside mitochondria. Mechanistically, proteins in the mitochondrial respiratory chain complex and TCA cycle (NDUFS2, SDHA, ATP5B, and DLD) were the target of 4-HNE adduction in SOD2Δ hearts. Our findings suggest that the SOD2 mediated 4-HNE signaling nexus may play an important role in cardiomyopathy., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background The contribution of glucocorticoids to sexual dimorphism in the heart is essentially unknown. Therefore, we sought to determine the sexually dimorphic actions of glucocorticoid signaling in cardiac function and gene expression. To accomplish this goal, we conducted studies on mice lacking glucocorticoid receptors (GR) in cardiomyocytes (cardioGRKO mouse model). Methods and Results Deletion of cardiomyocyte GR leads to an increase in mortality because of the development of spontaneous cardiac pathology in both male and female mice; however, females are more resistant to GR signaling inactivation in the heart. Male cardioGRKO mice had a median survival age of 6 months. In contrast, females had a median survival age of 10 months. Transthoracic echocardiography data showed phenotypic differences between male and female cardioGRKO hearts. By 3 months of age, male cardioGRKO mice exhibited left ventricular systolic dysfunction. Conversely, no significant functional deficits were observed in female cardioGRKO mice at the same time point. Functional sensitivity of male hearts to the loss of cardiomyocyte GR was reversed following gonadectomy. RNA-Seq analysis showed that deleting GR in the male hearts leads to a more profound dysregulation in the expression of genes implicated in heart rate regulation (calcium handling). In agreement with these gene expression data, cardiomyocytes isolated from male cardioGRKO hearts displayed altered intracellular calcium responses. In contrast, female GR-deficient cardiomyocytes presented a response comparable with controls. Conclusions These data suggest that GR regulates calcium responses in a sex-biased manner, leading to sexually distinct responses to stress in male and female mice hearts, which may contribute to sex differences in heart disease, including the development of ventricular arrhythmias that contribute to heart failure and sudden death.

Individuals with inflammatory bowel diseases (IBD) have an elevated risk of ocular inflammation. Both the anterior and posterior eye can be affected by IBD, although posterior eye dysfunction is more likely to go undetected. Little investigative attention has been directed toward the mechanisms of ocular dysfunction with IBD; however, given the prevalence of anemia in IBD and the effects of anemia on the retina, we examined the association between retinal function (electroretinography, ERG) and the anemia induced by experimental IBD, and we tested for a potential retinal benefit of acutely attenuating anemia (via red blood cell (RBC) infusion). Colitis was induced in mice in a model involving drinking water ingestion of dextran sodium sulfate (DSS), with untreated drinking water administered to controls. A subset of the DSS mice was infused with RBCs to attenuate the severity of the anemia induced by DSS. ERG signals (a-waves, b-waves, and oscillatory potential amplitudes and implicit times) were compared between the three groups of mice to evaluate retinal function. ERG amplitudes were significantly decreased in DSS mice compared to controls, with the amplitudes demonstrating a positive correlation with hematocrit, that is, the lowest ERG amplitudes were found with the most severe cases of anemia. An acute infusion of RBCs into DSS mice provided an improvement in the oscillatory potential implicit times, but no significant improvements in other ERG parameters. Despite the association between anemia and ERG signals in DSS-induced colitis, acute RBC infusion may only partially attenuate the associated retinal dysfunction., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Patients with inflammatory bowel disease suffer not only from gut inflammation, but also from extraintestinal manifestations of the disease, including ocular pathology. The mechanisms causing ocular inflammation in these patients are unknown. The purpose of the current study was to investigate the possible vascular changes occurring in the retina using a mouse model of acute colitis, that is, ingestion of dextran sodium sulfate (DSS). Intravital microscopy of anesthetized mice revealed that DSS caused a significant 30-40% decrease in retinal red blood cell velocities, and a 45% decrease in total retinal blood flow, but no changes in intraocular pressure. To determine whether the decreases in retinal perfusion could be inhibited by an angiotensin II receptor antagonist, losartan was administered by eye drops in a subset of the mice prior to the intravital microscopy measurements. Topical losartan was able to largely attenuate the altered hemodynamics induced by DSS. We conclude that angiotensin II might be a possible target for reducing the vascular changes occurring distantly in the eye during colitis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Ocular disease is known widely to occur in a subset of patients experiencing inflammatory bowel diseases. Although this extraintestinal manifestation has been recognized for a number of years, the pathogenetic mechanisms responsible for this distant organ inflammatory response are unknown., Methods: In the current study, we used a T-cell transfer model of chronic colitis in mice in which we quantified colonic inflammation, ocular function (electroretinography), ocular blood flow (intravital microscopy of the retina), intraocular pressure, and retinal hypoxia., Results: Ocular function in colitic mice was significantly impaired, with decreases in retinal b-wave amplitudes and oscillatory potentials. Moreover, retinal a waves and oscillatory potentials were delayed. Retinal blood flow was significantly reduced in the colitic mice, and this decrease in perfusion coupled with significant decreases in hematocrit would decrease oxygen delivery to the eye. Accordingly, mice with severe colitis showed increased levels of immunostaining for the hypoxia-dependent probe pimonidazole. Finally, intraocular pressures were found to be reduced in the colitic mice., Conclusions: Ocular disease occurs in a mouse model of chronic colitis, with retinal dysfunction seeming to be related to insufficient perfusion and oxygen delivery.

Background: Approximately one-half of patients with inflammatory bowel disease (IBD) suffer from anemia, with the most prevalent cause being iron deficiency. Accompanying the anemia are increases in erythropoietin, a plasma protein that can initiate the feedback production of new red blood cells. Anemia also occurs in animal models that are used to investigate the mechanisms of IBD; however, the extent to which iron deficiency produces the anemia in these animal models is unknown. Also unknown in the different animal models of IBD is whether the anemia upregulates the production of erythropoietin or, alternatively, whether a decrease in erythropoietin contributes to the induction of anemia., Methods: Two mouse models of colitis were used in this study: (1) acute 6-day ingestion of dextran sodium sulfate and (2) T-cell transfer into lymphopenic recipient mice. Measurements included indices of colitis severity, hematocrit, blood hemoglobin, plasma erythropoietin, serum iron concentration, plasma iron-binding capacities, transferrin saturation, and tissue iron concentrations., Results: Both models of colitis induced significant decreases in hematocrit, blood hemoglobin, and transferrin saturation, with the spleen and liver showing a decrease in iron content in the T-cell transfer model. Additionally, both models of colitis demonstrated significant increases in plasma erythropoietin and plasma iron-binding capacities., Conclusions: The measurements of iron, whether in acute (dextran sodium sulfate) or chronic (T-cell transfer) models of colitis, were generally consistent with iron-deficient anemia, with large increases in erythropoietin indicative of tissue hypoxia. These changes in animal models of colitis are similar to those found in human IBD.

Purpose: Reports of altered retinal blood flow in experimental models of type I diabetes have provided contrasting results, which leads to some confusion as to whether flow is increased or decreased. The purpose of our study was to evaluate early diabetes-induced changes in retinal blood flow in diabetic rats, using two distinctly different methods., Methods: Diabetes was induced by injection of streptozotocin (STZ), and retinal blood flow rate was measured under anesthesia by a microsphere infusion technique, or by an index of flow based on the mean circulation time between arterioles and venules. Measurements in STZ rats were compared to age-matched nondiabetic controls. In addition, the retinal distribution of fluorescently-labeled red blood cells (RBCs) was viewed by confocal microscopy in excised flat mounts., Results: Retinal blood flow rate was found to decrease by approximately 33% in the STZ rats compared to controls (P < 0.001) as assessed by the microsphere technique. However, in striking contrast, the mean circulation time through the retina was found to be almost 3× faster in the STZ rats (P < 0.01). This contradiction could be explained by flow redistribution through the superficial vessels of the diabetic retina, with this possibility supported by our observation of significantly fewer RBCs flowing through the deeper capillaries., Conclusions: We conclude that retinal blood flow rate is reduced significantly in the diabetic rat, with a substantial decrease of flow through the capillaries due to shunting of blood through the superficial layer, allowing rapid transit from arterioles to venules.

The mechanisms by which microvascular alterations contribute to the pathogenesis of the inflammatory bowel diseases (IBDs; Crohn's disease, ulcerative colitis) have not been clearly delineated. The purpose of the current study was to characterize the inflammatory events, microvascular alterations, and blood cell changes that occur in a mouse model of IBD. In this model, CD4(+) T-lymphocytes obtained from interleukin-10-deficient mice were injected intraperitoneally into lymphopenic, recombinase-activating gene-1 deficient (RAG(-/-)) mice. Two groups of control mice were also included: RAG(-/-) mice and C57BL/6 mice that were injected with phosphate-buffered saline but did not receive the T-cells. Four weeks later, the RAG(-/-) mice that had received the T-cell transfer showed significant signs of colonic inflammation, but without significant decreases in either body weight or mean arterial blood pressure. T-cell transfer increased the volume % of circulating platelets, while decreasing the number of circulating red blood cells. Additionally, the T-cell transfer tended to increase the circulating numbers of both lymphocytes and neutrophils when compared to unmanipulated RAG(-/-) mice. First-order colonic arterioles and venules tended to dilate in the colitic mice; however, the dilation was considerably more substantial with higher numbers of circulating leukocytes. The possibility that circulating inflammatory cells initiate the microvascular alterations in colitis warrants further investigation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Hypoxia has been reported to be associated with the colonic inflammation observed in a chemically induced mouse model of self-limiting colitis, suggesting that low tissue oxygen tension may play a role in the pathophysiology of inflammatory tissue injury. However, no studies have been reported evaluating whether tissue hypoxia is associated with chronic gut inflammation. Therefore, the objective of the present study was to determine whether hypoxia is produced within the colon during the development of chronic gut inflammation., Methods: Adoptive transfer of CD4(+) T cells obtained from interleukin-10-deficient (IL-10(-/-)) mice into lymphopenic recombinase-activating gene-1-deficient (RAG(-/-)) mice induces chronic colonic inflammation, with the inflammation ranging from mild to severe as determined by blinded histological analyses. Colonic blood flow, hematocrit, and vascular density were determined using standard protocols, whereas tissue hypoxia was determined using the oxygen-dependent probe pimonidazole., Results: Adoptive transfer of IL-10(-/-) CD4(+) T cells into RAG(-/-) recipients induced chronic colonic inflammation that ranged from mild to severe at 8 weeks following T-cell transfer. The colitis was characterized by bowel wall thickening, goblet cell dropout, and inflammatory infiltrate. Surprisingly, we found that animals exhibiting mild colonic inflammation had increased hypoxia and decreased systemic hematocrit, whereas mice with severe colitis exhibited levels of hypoxia and hematocrit similar to healthy controls. In addition, we observed that the extent of hypoxia correlated inversely with hematocrit and vascular density., Conclusions: Changes in hematocrit, vascular density, and inflammatory state appear to influence the extent of tissue oxygenation in the T-cell-mediated model of chronic gut inflammation., (Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.)

We have built a simple mechanical system to emulate the fast-start performance of fish. The system consists of a thin metal beam covered by a urethane rubber, the fish body and an appropriately shaped tail. The body form of the mechanical fish was modeled after a pike species and selected because it is a widely-studied fast-start specialist. The mechanical fish was held in curvature and hung in water by two restraining lines, which were simultaneously released by a pneumatic cutting mechanism. The potential energy in the beam was transferred into the fluid, thereby accelerating the fish. We measured the resulting acceleration, and calculated the efficiency of propulsion for the mechanical fish model, defined as the ratio of the final kinetic energy of the fish and the initially stored potential energy in the body beam. We also ran a series of flow visualization tests to observe the resulting flow patterns. The maximum start-up acceleration was measured to be around 40 m s(-2), with the maximum final velocity around 1.2 m s(-1). The form of the measured acceleration signal as function of time is quite similar to that of type I fast-start motions studied by Harper and Blake (1991 J. Exp. Biol. 155 175-92). The hydrodynamic efficiency of the fish was found to be around 10%. Flow visualization of the mechanical fast-start wake was also analyzed, showing that the acceleration peaks are associated with the shedding of two vortex rings in near-lateral directions.

Background: Adoptive transfer of naive T-lymphocyte subsets into lymphopenic mice initiates chronic gut inflammation that mimics several aspects of inflammatory bowel disease (IBD). Patients with IBD can have profound alterations in intestinal blood flow, but whether the same is true in the T-cell transfer model has yet to be determined., Methods: In the current study, chronic intestinal inflammation was induced in recombinase-activating gene-1-deficient (RAG(-/-)) mice by adoptive transfer of CD4(+) T-lymphocytes obtained from interleukin-10 deficient (IL-10(-/-)) mice., Results: Four weeks later, widespread colonic inflammation was observed in the reconstituted recipients, in contrast to 2 control sets of mice injected with a different subset of lymphocytes or with vehicle alone. We observed that the resulting pathology induced in the reconstituted RAG(-/-) mice was divided distinctly into 2 subsets: 1 with blood flow near normal with very high inflammation scores, and the other with severely attenuated blood flow but with much lower signs of inflammation. Colonic and ileal blood flow rates in the latter subset of CD4(+) mice averaged only approximately 30% compared to the mice with higher inflammation scores. The lower blood flow rates were associated with greatly reduced red blood cell concentrations in the tissue, suggesting a possible loss of vascular density., Conclusions: In this model of chronic intestinal inflammation, mild inflammation was associated with significant decreases in blood flow.

Background: Ingestion by mice of dextran sodium sulfate (DSS) induces colonic vasoconstriction and inflammation, with some of the effects potentially mediated by the vasoconstrictor endothelin-1 (ET-1)., Methods: In this study, mice given 5% 40 kD DSS for 5-6 days had elevated colonic immunostaining for ET-1 and platelet endothelial cell adhesion molecule-1 (PECAM-1). Increased ET-1 can induce microvascular constriction; however, the increase in PECAM-1 is consistent with angiogenesis that could decrease flow resistance., Results: Our measurements of intestinal blood flow, via infused microspheres, suggests that these 2 factors may offset each other, with only a nonsignificant tendency for a DSS-induced decrease in flow. Daily administration of the endothelin converting enzyme inhibitor SM-19712 (15 mg/kg) attenuated DSS-induced increases in colonic immunostaining of ET-1 and PECAM-1., Conclusions: SM-19712 attenuated histologic signs of tissue injury and inflammation induced by DSS, and decreased the extent of loose stools and fecal blood. However, the inhibitor did not significantly decrease DSS-induced colon shortening or tissue levels of myeloperoxidase (an indicator of neutrophil infiltration).