We present a new family of fluorescent probes with varying emission colors for selectively imaging hydrogen peroxide (H 2 O 2 ) generated at physiological cell signaling levels. This structurally homologous series of fluorescein-and rhodol-based reporters relies on a chemospecific boronate-tophenol switch to respond to H 2 O 2 over a panel of biologically relevant reactive oxygen species (ROS) with tunable excitation and emission maxima and sensitivity to endogenously produced H 2 O 2 signals, as shown by studies in RAW 264.7 macrophages during the phagocytic respiratory burst and A431 cells in response to EGF stimulation. We further demonstrate the utility of these reagents in multicolor imaging experiments by using one of the new H 2 O 2 -specific probes, Peroxy Orange 1 (PO1), in conjunction with the green-fluorescent highly reactive oxygen species (hROS) probe, APF. This dual-probe approach allows for selective discrimination between changes in H 2 O 2 and hypochlorous acid (HOCl) levels in live RAW 264.7 macrophages. Moreover, when macrophages labeled with both PO1 and APF were stimulated to induce an immune response, we discovered three distinct types of phagosomes: those that generated mainly hROS, those that produced mainly H 2 O 2 , and those that possessed both types of ROS. The ability to monitor multiple ROS fluxes simultaneously using a palette of different colored fluorescent probes opens new opporunities to disentangle the complex contributions of oxidation biology to living systems by molecular imaging.

Background Targeted temperature management (TTM) is endorsed by various guidelines to improve neurologic outcomes following cardiac arrest. Shivering, a consequence of hypothermia, can counteract the benefits of TTM. Despite its frequent occurrence, consensus guidelines provide minimal guidance on the management of shivering. The purpose of this study was to evaluate the impact of a pharmacologic antishivering protocol in patients undergoing TTM following cardiac arrest on the incidence of shivering. Methods A retrospective observational cohort study at a large academic medical center of adult patients who underwent TTM targeting 33 °C following out-of-hospital (OHCA) or in-hospital cardiac arrest (IHCA) was conducted between January 2013 and January 2019. Patients were included in the preprotocol group if they received TTM prior to the initiation of a pharmacologic antishivering protocol in 2015. The primary outcome was incidence of shivering between pre- and postprotocol patients. Secondary outcomes included time from arrest (IHCA) or admission to the hospital (OHCA) to goal body temperature, total time spent at goal body temperature, and percentage of patients alive at discharge. All pharmacologic agents listed as part of the antishivering protocol were recorded. Results Fifty-one patients were included in the preprotocol group, and 80 patients were included in the postprotocol group. There were no significant differences in baseline characteristics between the groups, including percentage of patients experiencing OHCA (75% vs. 63%, p = 0.15) and time from arrest to return of spontaneous circulation (17.5 vs. 17.9 min, p = 0.96). Incidence of patients with shivering was significantly reduced in the postprotocol group (57% vs. 39%, p = 0.03). Time from arrest (IHCA) or admission to the hospital (OHCA) to goal body temperature was similar in both groups (5.1 vs. 5.3 h, p = 0.57), in addition to total time spent at goal body temperature (17.7 vs. 18 h, p = 0.93). The percentage of patients alive at discharge was significantly improved in the postprotocol group (35% vs. 55%, p = 0.02). Patients in the postprotocol group received significantly more buspirone (4% vs. 73%, p < 0.01), meperidine (8% vs. 34%, p < 0.01), and acetaminophen (12% vs. 65%, p < 0.01) as part of the pharmacologic antishivering protocol. Use of neuromuscular blockade significantly decreased post protocol (19% vs. 6%, p = 0.02). Conclusions In patients undergoing TTM following cardiac arrest, the implementation of a pharmacologic antishivering protocol reduced the incidence of shivering and the use neuromuscular blocking agents. Prospective data are needed to validate the results and further evaluate the safety and efficacy of an antishivering protocol on clinical outcomes.

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