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.

Immobilization of actively thromboresistant assemblies on sterile blood-contacting surfaces.

Rapid one-step modification of thrombomodulin with alkylamine derivatives such as azide, biotin, and PEG is achieved using an evolved sortase (eSrtA) mutant. The feasibility of a point-of-care scheme is demonstrated herein to site-specifically immobilize azido-thrombomodulin on sterilized commercial ePTFE vascular grafts, which exhibit superior thromboresistance compared with commercial heparin-coated grafts in a primate model of acute graft thrombosis.

Clinical utility of pharmacogenetic testing in compounding pharmacy.

Pharmacists must consider all factors when dosing medication for a patient. Until recently, however, one key piece to this puzzle was missing--genetics. This invisible piece of the puzzle can now be utilized with pharmacogenetic testing. By using pharmacogenetic testing, the compounding pharmacist will be able to better predict disease risk as well as the pharmacodynamic and pharmacokinetic actions of the prescriptions their patients are taking. Pharmacogenetics is poised to become the standard of care that not only physicians are embracing, but pharmacists can utilize to better personalize their patient's needs.

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.

Inhibition of Factor XII-Mediated Activation of Factor XI Provides Protection Against Experimental Acute Ischemic Stroke in Mice.

Blood coagulation factor XI (FXI) is an established risk factor for acute ischemic stroke (AIS) and thrombosis, but is also needed for normal hemostasis. Contact factor XII (FXII), an upstream activator of FXI, also contributes to experimental stroke, but is not required for hemostasis. We investigated whether selectively inhibiting FXII-mediated FXI activation, while leaving other FXI and FXII functions intact, could improve the outcome of experimental AIS in mice. Twenty-four hours before induction of AIS by placement of a filament into the internal carotid artery for 60 min, mice were anticoagulated with an antibody directed against the apple 2 domain of FXI. This antibody selectively reduces the prothrombotic activation of FXI by FXIIa but does not affect activated FXI or hemostatic activation of FXI by thrombin, thus leaving hemostasis intact in mice and primates. In this model of AIS/reperfusion injury, mice that received the antibody before AIS displayed less ischemic damage, manifested as reduced cerebral infarction and fibrin deposition (thrombosis), increased cortical reperfusion, and improved neurological behavior. Further, the antibody-anticoagulated mice had no detectable hemostasis impairment. Consistent with the neuroprotective phenotype of FXII-deficient mice, our data suggest that a single molecular event, FXII-mediated FXI activation, contributes to the development of experimental AIS.

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.

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.

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.

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.

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 R (max) 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.

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.

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