Sawan Hurst

Three dimensional optical angiography.

With existing optical imaging techniques three-dimensional (3-D) mapping of microvascular perfusion within tissue beds is severely limited by the efficient scattering and absorption of light by tissue. To overcome these limitations we have developed a method of optical angiography (OAG) that can generate 3-D angiograms within millimeter tissue depths by analyzing the endogenous optical scattering signal from an illuminated sample. The technique effectively separates the moving and static scattering elements within tissue to achieve high resolution images of blood flow, mapped into the 3-D optically sectioned tissue beds, at speeds that allow for perfusion assessment in vivo. Its development has its origin in Fourier domain optical coherence tomography. We used OAG to visualize the cerebral microcirculation, of adult living mice through the intact cranium, measurements which would be difficult, if not impossible, with other optical imaging techniques.

Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI

The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)-dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti-human FXI monoclonal antibody (aXIMab; 2 mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and beta-thromboglobulin (betaTG) formation measured immediately downstream from thrombi forming within collagen-coated vascular grafts. FXI inhibition with aXIMab limited platelet and fibrin deposition in 4-mm diameter grafts without an apparent increase in D-dimer release from thrombi, and prevented the occlusion of 2-mm diameter grafts without affecting template bleeding times. In comparison, pretreatment with aspirin (32 mg/kg) prolonged bleeding times but failed to prevent graft occlusion, supporting the concept that FXI blockade may offer therapeutic advantages over other antithrombotic agents in terms of bleeding complications. In whole blood, aXIMab prevented fibrin formation in a collagen-coated flow chamber, independent of factor XII and factor VII. These data suggest that endogenous FXI contributes to arterial thrombus propagation through a striking amplification of thrombin generation at the thrombus luminal surface.

Mapping of cerebro-vascular blood perfusion in mice with skin and skull intact by Optical Micro-AngioGraphy at 1.3µm wavelength

Optical micro-angiography (OMAG) was developed to achieve volumetric imaging of the microstructures and dynamic cerebrovascular blood perfusion in mice with capillary level resolution and high signal-to-background ratio. In this paper, we present a high-speed and high-sensitivity OMAG imaging system by using an InGaAs line scan camera and broadband light source at 1.3 mum wavelength for enhanced imaging depth in tissue. We show that high quality imaging of cerebrovascular blood perfusion down to capillary level resolution with the intact skin and cranium are obtained in vivo with OMAG, without the interference from the blood perfusion in the overlaying skin. The results demonstrate the potential of 1.3 mum OMAG for high-speed and high-sensitivity imaging of blood perfusion in human and small animal studies.

Inhibition of factor XI activation attenuates inflammation and coagulopathy while improving the survival of mouse polymicrobial sepsis

Severe bacterial sepsis often leads to a systemic procoagulant and proinflammatory condition that can manifest as disseminated intravascular coagulation, septic shock, and multiple organ failure. Because activation of the contact proteases factor XII (FXII), prekallikrein, and factor XI (FXI) can trigger coagulation and inflammatory responses, the contact factors have been considered potential targets for the treatment of sepsis. However, the pathogenic role of contact activation in severe infections has not been well defined. We therefore investigated whether an anticoagulant antibody (14E11) that selectively inhibits prothrombotic FXI activation by activated FXII (FXIIa) modifies the course of bowel perforation-induced peritoneal sepsis in mice. Early anticoagulation with 14E11 suppressed systemic thrombin- antithrombin complex formation, IL-6, and TNF-α levels, and reduced platelet consumption in the circulation and deposition in the blood vessels. Treatment with 14E11 within 12 hours after bowel perforation significantly improved survival compared with vehicle treatment, and the saturating dose did not increase tail bleeding. These data suggest that severe polymicrobial abdominal infection induces prothrombotic FXI activation, to the detriment of the host. Systemic anticoagulation by inhibiting FXI activation or FXIIa procoagulant activity during sepsis may therefore limit the development of disseminated intravascular coagulation without increasing bleeding risks.

Survival Advantage of Coagulation Factor XI-Deficient Mice during Peritoneal Sepsis

Anticoagulation is a rational approach to the treatment of sepsis-associated consumptive coagulopathy, but its application is limited because of the risk of excessive bleeding. Factor XI (FXI) contributes substantially to pathological blood coagulation (thrombosis), whereas it contributes only modestly to normal hemostasis. We found that FXI-deficient mice have reduced coagulopathy and increased survival relative to FXI-expressing wild-type mice during cecal ligation and puncture-induced acute peritonitis/sepsis. This finding suggests that FXI contributes to coagulopathy and/or inflammation during sepsis and that pharmacologic inhibition of FXI activity may alter the course and outcome of some infections.

Thrombin Mutant W215A/E217A Treatment Improves Neurological Outcome and Reduces Cerebral Infarct Size in a Mouse Model of Ischemic Stroke

Background and Purpose— Treatment of ischemic stroke by activation of endogenous plasminogen using tissue plasminogen activator is limited by bleeding side effects. In mice, treatment of experimental ischemic stroke with activated protein C improves outcomes; however, activated protein C also has bleeding side effects. In contrast, activation of endogenous protein C using thrombin mutant W215A/E217A (WE) is antithrombotic without hemostasis impairment in primates. Therefore, we investigated the outcome of WE-treated experimental ischemic stroke in mice. Methods— The middle cerebral artery was occluded with a filament for 60 minutes to induce ischemic stroke. Vehicle, recombinant WE, or tissue plasminogen activator was administered during middle cerebral artery occlusion or 2 hours after middle cerebral artery occlusion. Neurological performance was scored daily. Intracranial bleeding and cerebral infarct size, defined by 2,3,5-triphenyltetrazolium chloride exclusion, were determined on autopsy. Hemostasis was evaluated using tail bleeding tests. Results— WE improved neurological performance scores, increased laser Doppler flowmetry-monitored post-middle cerebral artery occlusion reperfusion of the parietal cortex, and reduced 2,3,5-triphenyltetrazolium chloride-defined cerebral infarct size versus vehicle controls. However, unlike tissue plasminogen activator, WE did not increase tail bleeding or intracranial hemorrhage. Conclusions— WE treatment is neuroprotective without hemostasis impairment in experimental acute ischemic stroke in mice and thus may provide an alternative to tissue plasminogen activator for stroke treatment.

Safety and Antithrombotic Efficacy of Moderate Platelet Count Reduction by Thrombopoietin Inhibition in Primates

Inhibition of platelet production safely reduces blood clot formation without increasing bleeding side effects in baboons. Another Way to Keep Blood Flowing Freely Blood clotting is like walking a tightrope. If clots form too easily, they can lodge in blood vessels, depriving tissue of oxygen and instigating a stroke or heart attack. The danger on the other side is that blood fails to clot and small traumas can then cause uncontrolled bleeding. For most people, regulation of blood coagulation stays upright on the tightrope and blood flows freely inside vessels but clots readily when needed. But patients with cardiovascular disease need to avoid clotting. To keep blood flowing, some patients take antiplatelet medications such as aspirin or anticoagulants like Coumadin, but these drugs can tip the clotting balance too far and result in bleeding. The results from Tucker et al. suggest another approach to avoid unwanted blood clots. By inhibiting platelet formation at the source—in bone marrow—they lower the number of circulating platelets in baboons to a value that reduces clotting but is still within the acceptable range. Other platelet functions, including bleeding, remain normal. The hormone thrombopoietin regulates the production of platelets in bone marrow by encouraging the differentiation of megakaryocytes, each of which fragments into large numbers of platelets. The authors injected baboons with an antibody to thrombopoietin to interfere with this process and so altered the number of platelets in the circulation to a value that was lower but still within the normal range. Subsequent testing showed that below a certain concentration, platelet reduction reduced thrombus formation, as shown by platelet deposition on collagen-coated grafts and by measuring the blockage of small-diameter grafts. Determining the time needed for a wound to stop bleeding showed that the platelet-lowering treatment did not change bleeding time. Baboons are similar to humans when it comes to bleeding and clotting, so these results point to an interesting possibility. Although we already have drugs that inhibit blood clotting to give when patients need them, each drug has its drawbacks. This new possibility—manipulating platelet concentrations directly—provides a new tool that we may be able to use to keep people stable on the tightrope between clotting and bleeding. Most heart attacks and strokes are caused by blood clots (thrombi) that block the vasculature. Because disease-causing arterial thrombosis depends on blood platelets, platelet inhibitors such as aspirin and clopidogrel effectively decrease the risk of thrombosis; however, they also impair platelet-dependent hemostasis that staunches bleeding from wounds and can therefore produce excessive bleeding. Experimental studies show that a reduction in the number of platelets also inhibits thrombosis, but these treatments also interfere with platelet function. Because normal hemostasis requires that the platelet concentration remain within a physiological range in the circulation, we evaluated whether lowering the number of circulating platelets—but only to a value still within the normal range—by inhibiting platelet formation in the bone marrow inhibits acute thrombogenesis in baboons. We reduced the platelet count with an inhibitor against the megakaryocyte-promoting hormone thrombopoietin and then showed that experimental occlusive thrombogenesis on collagen-coated vascular grafts was reduced, without impairment of primary hemostasis. These results suggest that suppressing platelet production without interfering with the hemostatic function of platelets may offer a safe alternative to current therapies for prevention of stroke and heart attack triggered by blood clotting.

Isoflurane is an effective alternative to ketamine/xylazine/acepromazine as an anesthetic agent for the mouse electroretinogram.

The electroretinogram (ERG) is an essential measure of retinal function for studying mouse models of retinal disease. Ketamine, in combination with xylazine and/or acepromazine, is the most commonly used anesthetic agent. Although it works well in most situations, some fragile mouse strains have high mortality rates with this ketamine cocktail. We compared isoflurane with the ketamine cocktail in a longitudinal study of light-adapted and dark-adapted ERGs in C57BL/6J mice. Waveforms were averaged, oscillatory potentials (OPs) were extracted by digital filtration, and key ERG parameters were analyzed. The ERG waveforms were qualitatively similar with both anesthetics, and the male and female ERG parameters did not show significant differences. For light-adapted ERGs, b-wave amplitude and implicit time, and wavelet index were decreased under isoflurane anesthesia, whereas for dark-adapted ERGs, a- and b-wave implicit times were decreased and wavelet index was increased. The dark-adapted b-wave amplitude showed a significant inverse correlation with animal weight and age. Rod phototransduction gain and the Naka–Rushton n and Rmax parameters were the same for both anesthetics, and only the Naka–Rushton log k parameter was significantly elevated for isoflurane anesthesia. We propose that isoflurane is a satisfactory alternative to the ketamine cocktail for anesthesia in the mouse ERG. Precise quantitative comparisons, however, should only employ study designs using isoflurane versus isoflurane, or ketamine versus ketamine. Moreover, in light of the effects of both isoflurane and the ketamine cocktail on blood glucose levels, it would be prudent to control the fasting state of the animals in quantitative ERG studies.

Laser speckle contrast imaging for the quantitative assessment of flow

The ultimate objective of laser speckle flowmetry is to infer flow velocity from observed speckle contrast. Since introduction of this concept over 25 years ago, a variety of researchers have demonstrated such a qualitative relationship (between speckle contrast and flow velocity), but a quantitative relationship has proven elusive. A fundamental reason for this failure to demonstrate a convincing quantitative relationship is that the underlying mathematics describing LSCA is identical to that of quasi-elastic light scatter (QLS). As a result, it is commonly (and erroneously) assumed that the requirements for the data acquisition, the model linking the scatter dynamics to the speckle fluctuation, and the data processing are the same as well. Here we discuss some of our recent advances towards achieving quantitative velocity estimates from laser speckle contrast measurements. This concept is free of any assumptions relating scatterer dynamics to light fluctuations and is compatible with accepted data acquisition methods, but uses an entirely new data processing scheme. Results are demonstrated with a murine model.

Measuring Cone Density in a Japanese Macaque ( Macaca fuscata ) Model of Age-Related Macular Degeneration with Commercially Available Adaptive Optics

The aim of this study was to assess the feasibility of using a commercially available high-resolution adaptive optics (AO) camera to image the cone mosaic in Japanese macaques (Macaca fuscata) with dominantly inherited drusen. The macaques examined develop drusen closely resembling those seen in humans with age-related macular degeneration (AMD). For each animal, we acquired and processed images from the AO camera, montaged the results into a composite image, applied custom cone-counting software to detect individual cone photoreceptors, and created a cone density map of the macular region. We conclude that flood-illuminated AO provides a promising method of visualizing the cone mosaic in nonhuman primates. Future studies will quantify the longitudinal change in the cone mosaic and its relationship to the severity of drusen in these animals.