While it is known that opioid receptors (ORs) are densely expressed in both the brain and periphery, it is widely accepted that hypoxic effects of opioids result solely from their direct action in the CNS. To examine the role of peripheral ORs in triggering brain hypoxia, we used oxygen sensors in freely moving rats to examine how naloxone-HCl and naloxone-methiodide, the latter which is commonly believed to be peripherally restricted, affect brain oxygen responses induced by intravenous heroin at low, human-relevant doses. Similar to naloxone-HCl, naloxone-methiodide at a relatively low dose (2 mg/kg) fully blocked heroin-induced decreases in brain oxygen levels. As measured by mass spectrometry, naloxone-methiodide was found to be ~40-fold less permeable than naloxone-HCl across the blood-brain barrier, thus acting as a selective blocker of peripheral ORs. Despite this selectivity, a low but detectable amount of naloxone was found in brain tissue after naloxone-methiodide administration, potentially influencing our results. Therefore, we examined the effects of naloxone-methiodide at a very low dose  and found that this drug still powerfully attenuates heroin-induced brain oxygen responses. These data demonstrate the role of peripheral ORs in triggering heroin-induced respiratory depression and subsequent brain hypoxia. While this side-effect of opioids is minor following their therapeutic use, it is a leading cause of coma and death when highly potent opioid drugs such as heroin or fentanyl are self-administered at high doses via routes that provide rapid drug delivery to the CNS.Respiratory depression that leads to brain hypoxia appears to be the most dangerous effect of opioid drugs9. This basic mechanism has solid experimental support: respiratory activity has been shown to rapidly decrease following intracerebral, intercisternal, and local intra-brain microinjections of various opioid agonists12. This suggests an important role for centrally located ORs in descending mechanisms regulating respiratory activity. However, ORs are also abundantly expressed in afferents of sensory nerves innervating blood vessels, skin and internal organs, including lung airways14. Therefore, in addition to central targets, systemically administered opioid drugs directly interact with peripherally located ORs and change afferent inputs to the CNS, thus inducing neural effects and leading to subsequent changes in physiological parameters.It is generally believed that respiratory depression results from the direct interaction of opioid drugs with opioid (mainly μ) receptors (ORs) expressed on brainstem neurons of the breathing center as well as other central neurons7. However, respiratory depression induced by morphine and fentanyl is also attenuated by large-dose naloxone-methiodide , a quaternary analog of naloxone which is believed to be impermeable to the BBB and thus would interact only with the peripheral pool of ORs20. Naloxone-MET is also able to affect nociceptive and gastro-intestinal responses induced by morphine16, fentanyl15, and oxycodone21.Since opioid drugs interact with both centrally and peripherally located ORs, comparing the effects of opioid antagonists which cross the blood-brain barrier (BBB) with ones that are unable to cross this barrier could be an effective tool for examining the role of peripheral receptors in triggering the neural and physiological effects of opioids. Naloxone-HCl, a highly potent non-selective opioid antagonist that easily crosses the BBB and interacts with both centrally and peripherally located ORs, fully blocks respiratory depression as well as other physiological and behavioral effects of morphine, heroin and fentanyl22 and dose-dependently decrease following administration of various opioid drugs23. As in our previous studies, oxygen recordings were conducted in the nucleus accumbens (NAc), a deep, ventrally located structure critically involved in sensorimotor integration and drug reinforcement25.The primary goal of this study was to evaluate the role of peripheral ORs in mediating brain hypoxia induced by intravenous (iv) heroin in awake, freely-moving rats. The use of oxygen sensors coupled with amperometry allowed us to directly examine drug-induced changes in brain oxygen levels—a functional and integrative output of respiratory activity. While respiratory activity is the primary means for oxygen delivery into brain tissue, oxygen dissolved in the brain’s extracellular space is a more clinically relevant parameter and its decrease below physiological range is the direct cause of death during opioid overdose. Another critical advantage of this technology is its second-scale temporal resolution that allows for the observation of rapid drug-induced oxygen changes under physiologically relevant conditions. As shown previously, brain oxygen levels rapidly increase following exposure to various sensory stimuli20. Heroin was delivered at a low, physiologically relevant dose (0.1 mg/kg) that is optimal for maintaining iv self-administration26; heroin at this dose induces moderate brain hypoxia27. In contrast to previous studies, in which large doses of opioid antagonists were often used, the dose of naloxone-HCl was relatively small (0.2 mg/kg). Naloxone-MET was delivered at 2.0 mg/kg to compensate for its 10–15-fold lower affinity for μ-opioid receptors30. To clarify the mechanisms underlying heroin-induced NAc oxygen responses and their changes following administration of opioid antagonists we conducted two additional experiments, using the same experimental protocol but monitoring different parameters. In the first experiment, we conducted oxygen recordings in the subcutaneous space, a densely vascularized location with no  metabolic activity of its own, thus a valuable proxy of blood oxygen levels, which directly reflect the efficiency of respiratory activity31. In the second experiment, we examined drug-induced changes in locomotion and temperature in the NAc, temporal muscle, and skin. This three-point temperature-recording paradigm makes it possible to evaluate the effects of drugs on brain metabolic activity and peripheral vascular tone32.This study followed a three-part progression, in which each subsequent experiment was designed to answer questions arising from the previous experiment. Initially, we examined how the effects of iv heroin would be affected by naloxone-HCl and naloxone-MET, assuming as other researchers that naloxone-MET does not enter brain tissueAlthough it is generally accepted that naloxone-MET does not permeate the BBB (see above), we found a lack of objective proof to support this claim. To make sure that our results were not influenced by possible entry of naloxone-MET into brain tissue, in the second stage of the study we used liquid chromatography-mass spectrometry (LC-MS) to examine the extent to which both naloxone analogs, when delivered systemically, permeate the brain.in vivo form of naloxone-MET, were found in brain tissue.Finally, our third stage of the study was conducted in response to our LC-MS findings. In this oxygen experiment we examined whether and how the effects of iv heroin would be affected by systemic administration of naloxone-MET at a lower dose (0.24 mg/kg), the molar equivalent of the low dose of naloxone-HCl used in our experiments. At this dose, no reliable amounts of methyl-naloxone, the For proper assessment of the effects of opioid antagonists on heroin-induced responses, it is important to examine these responses and their underlying mechanisms in intact rats preceding the use of antagonists. Therefore, at the first stage of our analysis, we examined changes in oxygen levels in the NAc and subcutaneous space, temperature parameters, and locomotion induced by iv heroin in intact, drug-free rats.23,2093 = 16.51, p < 0.0001; Fig. 17,1547 = 26.96, p < 0.0001; Fig. When analyzed with slow, 1-min time resolution, iv heroin induced a biphasic effect on NAc oxygen levels . Then oxygen levels began to increase, reaching maximum levels at ~5 min, slowly returning to baseline thereafter. In contrast, oxygen levels in the subcutaneous space decreased with shorter latencies (~30 s) but reached nadir at a later time (~180 s), slowly returning toward baseline thereafter. More precise relationships between heroin-induced changes in oxygen levels in the NAc and subcutaneous space were examined by using time-dependent correlation analysis , naloxone-MET (2.0 mg/kg), and saline on heroin-induced changes in NAc oxygen levels. To establish the link between oxygen decrease and respiratory depression, we also examined how naloxone-MET (2.0 mg/kg) affects heroin-induced oxygen responses in the subcutaneous space. Because of variability in baseline oxygen levels and long duration of heroin responses, the data are shown as relative, percent change vs. pre-injection baseline (=100%) for 60 min following heroin injections. To better represent the initial changes in heroin responses, Supplemental Fig. 7,637 = 5.26, 3.65, 6.35, respectively; p < 0.001). In each case, oxygen levels rapidly decreased within a couple of minutes following heroin injection, but then increased above baseline. No significant changes in the strength of heroin-induced NAc oxygen decrease were found when the area under the curve was analyzed . While the positive phase of the heroin response was restored when heroin was injected 150 min after naloxone , the initial negative phase was still absent. Blockade of NAc oxygen decrease induced by naloxone-HCl was significant when data were analyzed as an area under the curve . The decrease in oxygen was partially restored but oxygen levels increased following the second post-naloxone-MET injection . Significant between-group differences were also detected by analyzing the areas under the curves  that can be attributed to the stressful nature of the subcutaneous injection procedure.Similar to naloxone-HCl, administration of naloxone-MET altered the heroin-induced NAc oxygen responses, but the pattern of changes was different Fig. . When heAlthough naloxone-MET fully blocked the initial decreases in NAc oxygen levels, suggesting blockade of respiratory depression, it was unable to block the second, increasing phase of the heroin response. To confirm the blockade of respiratory depression and understand the possible mechanisms underlying brain oxygen increases, we examined how naloxone-MET affects heroin-induced oxygen changes in the subcutaneous space Fig. .3,273 = 5.24, p < 0.001) that greatly exceeds the decrease observed in the NAc. When heroin was delivered 30 min after naloxone-MET, this rapid and strong drop in subcutaneous oxygen was absent. Oxygen levels in this case slightly decreased, but the change was slow, weak, and statistically not significant. However, the heroin-induced decrease in oxygen reappeared again at a similar strength when the next heroin injections was administered,150 min after naloxone-MET . In contrast to the transient increases in NAc oxygen elicited by all sc injections, oxygen levels in the subcutaneous space showed a weak, transient decrease immediately after the injection.In this experiment we found that the initial heroin injection induced a strong (~75% of baseline) and relatively prolonged (~40 min) decrease in subcutaneous oxygen levels  and the response was almost fully restored when the next heroin injection was done 150 min after naloxone-HCl .In contrast to naloxone-HCl, naloxone-MET only slightly attenuated heroin-induced temperature responses . In the second experiment, we injected both naloxone analogs at lower equimolar doses . In addition to brain samples, we also measured concentrations of the same substances in kidney and liver.As shown in Table 7,637 = 1.91, p < 0.05), but the first decreasing phase was greatly attenuated. Oxygen decrease was almost fully restored following the second post-naloxone-MET heroin injection . Strong attenuation of the heroin-induced oxygen response was evident when analyzing the area under the curve  would be affected by sc administration of naloxone-MET at a lower dose 0.24 mg/kg), the molar equivalent to naloxone HCl at 0.2 mg/kg. As shown in Fig.  mg/kg, tIn addition to strong attenuation of heroin-induced brain oxygen decrease, naloxone-MET also increased the onset latency of the heroin response Fig. . In cont2 in peripheral blood, the use of oxygen sensors coupled with high-speed amperometry allowed us to directly monitor heroin-induced changes in brain oxygen levels and quantitatively assess brain hypoxia—the functional output of respiratory activity and a clinically relevant parameter.This study’s aim was to examine the possible role of peripheral ORs in triggering respiratory depression and subsequent brain hypoxia induced by opioid drugs. While it is usually believed that these effects result exclusively from the direct interaction of opioid drugs and their metabolites with centrally located ORs, ORs are also abundantly expressed on afferent terminals of visceral sensory nerves innervating blood vessels and internal organs. Therefore, these receptors can be affected by opioids before reaching central substrates and at higher concentrations than those occurring in brain tissue. This study was designed to explore this mechanism with respect to heroin, a prototypical opioid drug of abuse, used at low, behaviorally-relevant doses. In contrast to most studies focused on ventilation aspects of respiration or changes in O20 and this was our assumption when we initiated this study. While our first experiment revealed that naloxone-MET at a relatively low dose fully blocks heroin-induced decreases in NAc oxygen, supporting the critical role of peripheral ORs, this conclusion can be valid only if naloxone-MET cannot cross the BBB and affect central ORs. Our second experiment was designed to verify this assumption, using highly sensitive LC-MS measurements of naloxone and methyl-naloxone in brain tissue. Finally, based on the results of these measurements, we conducted the last experiment, in which we examined how the hypoxic effects of iv heroin are affected by naloxone-MET at a much lower dose (0.5 μmol/kg or 0.24 mg/kg) that did not result in any detectable levels of methyl-naloxone in the brain.As a tool for assessing the role of peripheral ORs, we employed an antagonist strategy and examined how heroin-induced NAc oxygen responses are affected by two naloxone analogs—one, which easily crosses the BBB and another, which has no or limited penetration of the BBB. Multiple studies using this strategy hold the belief that naloxone-MET cannot cross the BBBThis study produced two major findings. First, we provided the first objective analysis of BBB permeability of naloxone-HCl and naloxone-MET, which has major implications for past and future research. Second, we confirmed that interaction of opioids with peripheral ORs plays an important role in triggering brain hypoxia induced by heroin at low, human-relevant doses.Respiratory depression as a cause of NAc oxygen decrease induced by heroin. Consistent with our previous studies27, iv heroin induced a biphasic down-up change in NAc oxygen levels. In the subcutaneous space—a densely-vascularized but metabolically inactive area—heroin induced a monophasic oxygen decrease, which was much stronger and more prolonged than in the brain. The initial oxygen decrease in both locations was rapid and tightly correlated, suggesting respiratory depression and subsequent decreases in blood oxygen levels as its cause. However, heroin engages an opposing mechanism that acts to increase brain oxygen levels, causing heroin-induced hypoxia to be weaker in the brain than in the periphery and determining rebound-like brain oxygen increases. As shown previously22, brain oxygen levels can increase due to neural activation with subsequent dilation of cerebral vessels and enhanced cerebral blood flow independently of changes in its blood levels. While cerebral vasodilation can occur due to neural activation via a neuro-vascular coupling mechanism33 and due to post-hypoxia accumulation of CO2, a strong vasodilator35, it also results from redistribution of arterial blood from the periphery to the brain due to skin vasoconstriction confirmed in our thermorecording experiment. While oxygen decreases resulting from respiratory depression are dose-dependent, being responsible for robust hypoxia following high-dose heroin exposure27, the opposing vascular effect is more tonic, weaker, and is limited by the natural propensity of cerebral vessels to dilate.Naloxone-MET potently inhibits heroin-induced NAc oxygen decreases. Consistent with the high affinity of naloxone-HCl to ORs, this drug at a relatively small dose (0.2 mg/kg or 0.5 μmol/kg) fully blocked both oxygen and temperature responses induced by heroin. While heroin-induced hyperthermia reappeared again at the same strength when heroin was injected 2.5 hours after naloxone-HCl, the decreasing phase of NAc oxygen response was still absent at this time. Thus, heroin-induced respiratory depression is more sensitive to full blockade of ORs than other physiological effects, particularly drug-induced changes in brain temperature and vascular tone.Naloxone-MET at a higher dose (2.0 mg/kg or 4.3 μmol/kg) fully blocked heroin-induced NAc oxygen decreases, pointing at the role of peripheral ORs in triggering this centrally-mediated effect of heroin. However, blockade of peripheral ORs had minimal inhibitory effects on heroin-induced temperature responses (+30 min) and it did not affect heroin-induced brain oxygen increases, indicating their mediation via central ORs. Additional support for the role of peripheral ORs in triggering heroin-induced respiratory depression was obtained in experiments with subcutaneous oxygen measurements. While heroin injected prior to naloxone-MET rapidly and strongly decreased subcutaneous oxygen levels, this effect was absent when the second heroin injection was delivered 30 min after naloxone-MET. The oxygen decrease was basically restored when the next heroin injection was made 2.5 hours after naloxone-MET. However, heroin after naloxone-MET still induced a small tonic decrease in subcutaneous oxygen, resulting from constriction of skin blood vessels as shown in our thermorecording experiment.The problem: BBB permeability of naloxone-MET and its possible metabolic degradation. Although these findings were impressive, blockade of heroin-induced oxygen decreases by naloxone-MET could still be due to two different factors. First, some amount of this drug can cross the BBB, causing a blockage of central ORs. Second, naloxone-MET sample could be contaminated with naloxone or metabolized into this substance in vivo, thus affecting our results. These considerations led us to conduct LC-MS measurements to assess BBB permeability of both naloxone analogs.th of the amount of naloxone found in the brain at an equimolar, 1.704 mg/kg dose (1:40 permeability ratio), but only 1/6th of the experimentally used 0.2 mg/kg dose of naloxone-HCl. Though this is significant, the affinity of naloxone-MET to μ ORs is 10–15 times less than that for naloxone, suggesting the overall influence to be very small. While we conducted a high-quality brain perfusion, we cannot be fully confident that this procedure was able to remove all naloxone-MET contained in cerebral vessels. This inescapable factor could also affect the selectivity ratio obtained in our measurements.These measurements resulted in two major findings. First, approximately 5 ng/mL of methyl-naloxone was found in the brain after 4.3 μmol/kg of naloxone-MET was administered. This is approximately 1/40th of the naloxone found in the brain after injection of naloxone-HCl at the same dose , it is possible to speculate that some of the effects of naloxone-MET at the larger doses could be attributed to naloxone found in the brain. This could be due to two factors: original sample contamination or in-vivo metabolism of methyl-naloxone into naloxone. First, upon analyzing the standard naloxone-MET sample obtained from Sigma-Aldrich it was found that it contained ~1% of naloxone. This 1% could possibly be the reason for detecting naloxone within brain tissue. There is also a possibility that methyl-naloxone is converted into naloxone in vivo. While the brain tissue contained low but detectable levels of naloxone after the large-dose naloxone-MET injection, only traces of this substance were found in both liver and kidney. While this finding argues against systemic metabolic transformation of naloxone-MET into naloxone, we cannot fully exclude this mechanism for brain tissue.The second unexpected finding was that naloxone was found in rats injected with naloxone-MET at a higher dose. Although the amount was very small (2.3 ng/mL) and it is only ~1/94Our measurements of BBB permeability of naloxone-MET could have significant implications for past and present work with this drug. While our data confirm that naloxone-MET has limited BBB permeability, low but quantifiable amounts of naloxone were found in brain tissue. When naloxone-MET is injected at high doses, the effects seen can be the result of the contribution of centrally acting methyl-naloxone and naloxone. Thus, results from prior studies using naloxone-MET could have been affected by both naloxone analogs, though this contribution appears significant only when using high doses of naloxone-MET.Peripheral component of heroin-induced brain hypoxia. Based on results obtained in our LC-MS study, we conducted an additional experiment using naloxone-MET at a much lower dose (0.24 mg/kg), which did not result in any detectable amounts of brain naloxone or methyl-naloxone. Though our data can be theoretically affected by naloxone, the amount of naloxone in the brain will be equivalent to a 0.0014 mg/kg dose of naloxone-HCl. This amount is well below the detection limits of our measurements and naloxone at this dose is thought to induce no effects.37. Activation of these receptors by heroin and its active metabolites creates a neural signal that rapidly reaches the brain stem neurons via sensory pathways, and by modulating descending neural signals, it changes respiration. The activation of these receptors by endogenous opioid peptides is mild and possibly insignificant under physiological conditions, but their selective over-activation by highly potent opioid agonists has much stronger physiological effects.In this experiment NAc oxygen decreases were strongly attenuated by naloxone-MET. The area under the curve for the decreasing phase of oxygen response was ~7 times smaller than that for the original heroin response. Therefore, this experiment underscores the importance of peripherally located ORs in triggering brain hypoxia, a clinically important effect of opioids. These receptors are expressed on the terminals of visceral sensory nerves that innervate blood vessels, carotid bodies, and internal organs and they are also found in the neuromuscular components of the chest wall and diaphragm (pulmonary C-fiber or lung J receptors)30. Thus, the small dose of a weaker antagonist could result in peripheral ORs still being open and triggering hypoxia.When analyzed with fast time resolution, the contribution of peripheral receptors was even more obvious, demonstrating that the latency of heroin-induced NAc oxygen decrease after naloxone-MET was twice as long as that from a normal, pre-treatment heroin injection. This latency could be attributed to the time it takes for heroin to enter the CNS. The appearance of an oxygen decrease post naloxone-MET could result from the inability of naloxone-MET at this low dose to block the effective pool of peripheral ORs. Although we had to decrease the dose of naloxone-MET because of permeability, the affinity of naloxone-MET to μ-opioid receptors is 10 to 15 times less than naloxone-HCl38. While the descending efferent signals from the CNS are the ultimate cause of respiratory depression, our study demonstrates that the blockade of peripheral ORs either blocks or strongly attenuates heroin-induced afferent inputs from the periphery to the CNS, thus significantly diminishing respiratory depression and subsequent brain oxygen decreases. Since heroin in this study was used at relatively low but behaviorally relevant doses, these findings cannot be generalized to high-dose opioid use and overdose, when the direct action of these drugs and its metabolites in the CNS will have larger effects on respiratory depression and subsequent brain hypoxia. Under these conditions, the relative contribution of the peripherally triggered component becomes weaker and this mechanism is overshadowed by the more powerful direct action of opioids on central neurons.Respiratory depression is usually viewed as a consequence of the direct actions of opioid drugs on “respiratory neurons” located in the brain stem and medulla40. It is often suggested that these drugs would negate the problem of respiratory depression. Our current data demonstrate that though there would be less respiratory depression and brain hypoxia, these drugs are still able to trigger brain hypoxia, which could be significant at high doses. More experimentation will be needed to better understand the effects of peripheral opioids and the functional role of peripheral opioid receptors.Peripherally acting opioid drugs have recently been promoted for pain relief to avoid central side-effects57 adult male Long-Evans rats (Charles River Laboratories) weighing 460 ± 40 g at the time of surgery were used in this study. Rats were individually housed in a climate-controlled animal colony maintained on a 12–12 light-dark cycle with food and water available ad libitum. All procedures were approved by the NIDA-IRP Animal Care and Use Committee and complied with the Guide for the Care and Use of Laboratory Animals . Maximal care was taken to minimize the number of experimental animals and any possible discomfort or suffering at all stages of the study.In this study, we describe the results of three types of experiments . In our electrochemical experiments (n = 24 rats), we examined how naloxone-HCl, naloxone-MET, and saline affect NAc oxygen responses induced by iv heroin in awake, freely moving rats. In two additional electrochemical experiments (n = 7 rats), we examined heroin-induced changes in subcutaneous oxygen levels and the influence of naloxone-MET on such heroin-induced oxygen responses. In the thermorecording experiments (n = 12 rats) conducted with the same protocol we examined how these same treatments affect heroin-induced changes in NAc, temporal muscle, and skin temperatures. Finally, we used LCMS measurements to quantitatively examine how both naloxone analogs enter brain tissue (n = 14 rats).22. Briefly, under general anesthesia , rats were chronically implanted with a Pt-Ir oxygen sensor  into the NAc shell and the subcutaneous space. Target coordinates for the right NAc were: AP + 1.2 mm, ML ± 0.8 mm, and DV + 7.6 mm from the skull surface, according to coordinates of the rat brain atlas41. The subcutaneous sensor was implanted under the skin in the medio-frontal area of the rat’s head. The sensor was secured with dental acrylic to three stainless steel screws threaded into the skull. During the same surgery, rats were also implanted with a chronic jugular catheter, which ran subcutaneously to the head mount. Rats were allowed a minimum of 5 days of post-operative recovery and at least 3 daily habituation sessions (~6 h each) to the recording environment. Jugular catheters were flushed daily with 0.2 ml heparinized saline to maintain patency.Surgical procedures for electrochemical assessment of oxygen have been described in detail elsewhere43. Briefly, under the same anesthesia protocol, rats were implanted with a jugular catheter and three copper-constantan thermocouple electrodes in the NAc shell, temporal muscle, and subcutaneously along the nasal ridge with the tip ~15 mm anterior to bregma. Probes were secured with dental acrylic to three screws threaded into the skull. Rats were given a minimum of 5 days to recover post operation. Rats were also allowed at least 3 habituation sessions (~6 h each) to the recording environment prior to experimentation. Jugular catheters were flushed daily with 0.2 ml heparinized saline to maintain patency.Surgical procedures for thermorecording experiments have been described in detail elsewherein vivo oxygen detection we used Pinnacle oxygen sensors coupled with high-speed amperometry. These sensors were prepared from 180 μm Pt-Ir wire and had a sensing area of 0.025 mm2 at the tip. The active electrode was incorporated with an integrated Ag/AgCl reference electrode. Dissolved oxygen is reduced on the active surface of these sensors, held at a stable potential of −0.6 V vs. the reference electrode, producing an amperometric current. The current from the sensor was then transmitted via a potentiostat  to the computer and recorded at 1-s intervals, using PAL software (Pinnacle Technology).For 44. The sensors produced linear current changes with increases in oxygen concentrations within a wide range of brain oxygen concentrations (0–40 μM). Substrate sensitivity of oxygen sensors varied from 0.67 to 1.41 nA/1 μM (mean = 0.84 nA/1 μM). Oxygen sensors were also tested by the manufacturer for their selectivity toward electroactive substances such as dopamine (0.4 μM) and ascorbate (250 μM), none of which had significant effects on current measurements.Oxygen sensors were calibrated at 37 °C by the manufacturer (Pinnacle Technology) according to a standard protocol described elsewhereAt the onset of each experiment, rats were briefly anesthetized (<2 min) with isoflurane. During this time either the electrochemical or temperature sensors were connected to the recording instrument via an electrically shielded flexible cable and a multi-channel electrical swivel. A catheter extension was used to allow for stress and cue-free drug delivery from outside the cage. Testing began approximately 120 min after the temperature or electrochemical sensors were connected to the recording instruments, allowing for baseline currents to stabilize.In our electrochemical experiments, we examined how four types of treatments  affect changes in NAc oxygen induced by iv heroin . Both naloxone analogs were obtained from Sigma-Aldrich. Rats in these experiments received three heroin injections during an ~8-hour recording session. 120 min after the initial heroin injection, rats received a subcutaneous (sc) injection of one of three testing drugs . Second and third heroin injections were delivered 30 and 150 min after the injection of each testing drug. At the end of a session, rats were injected with Equithesin (0.6–0.8 ml) to safely disconnect the animals from the recording instruments. Data with naloxone analogs and saline were obtained in four different groups of rats. In two additional electrochemical experiments, we used a similar protocol to examine how heroin (0.1 mg/kg) affects oxygen levels in the subcutaneous space and how naloxone-MET (2 mg/kg) affects heroin-induced changes in subcutaneous oxygen levels.45. Control data in both experiments  were obtained in separate groups of rats, which were recorded for two daily sessions. Data with both naloxone analogs were obtained in two different groups of rats, with each rat receiving one injection of naloxone-HCl and one injection of naloxone-MET in a counter-balanced manner.A similar protocol was maintained in our thermorecording experiments, in which rats received three heroin injections—one before and two at 30 and 150 min after the injection of each testing drug. In these experiments, we also monitored locomotion activity using 4 infrared motion detectors (Med Associates) as previously describedThese experiments were conducted in drug-naive rats, which were anesthetized with isoflurane, injected with naloxone HCl, naloxone-MET, or saline, and perfused with room-temperature heparinized saline . Because of our expectation of limited permeability of naloxone-MET, in the first experiment, both naloxone analogs were used at a 4.3 μmol/kg dose  to ensure sufficient quantity for detection in brain tissue. In the second measurement experiment, we used low doses of these drugs  identical to those used in our last oxygen experiment. At the time of initial anesthesia, rats were injected with 1000 units of heparin to prevent blood coagulation that could affect brain levels of measured substances. Perfusion began at 30 min after the injection of tested drugs. Similar to oxygen and temperature experiments, both naloxone analogs were injected subcutaneously in 0.4 ml of saline. Brains were rapidly extracted, cut in half, and frozen on dry ice and later transferred into a freezer at −20 °C. We also took one lobe of liver and left kidney as control peripheral organs.Brain, liver and kidney samples were homogenized in 2 mL of water per gram of tissue. For extraction, 200 μL of the tissue homogenates were mixed with 800 µL acetonitrile. The samples were then vortexed for 30 min and centrifuged at 14000 rpm for 15 minutes before being stored at −20 °C for 1 hour. Next, the supernatant was transferred and evaporated at 50 °C under nitrogen. Finally, the samples were resuspended in 200 µL of 6% methanol in 0.1% formic acid for mass analysis. Samples were analyzed on a Dionex UltiMate 3000 HPLC coupled to an Orbitrap Velos with a HESI source . A Kinetex® 2.6 µm C18 100 Å (100 × 2.1 mm) column from Phenomenex  was used for liquid chromatography separation. Liquid chromatography conditions were as follow: column temperature, 30 °C; mobile phase A (aqueous) 0.1% formic acid and mobile phase B (organic) was methanol; flow-rate, 0.3 mL/mi; gradient, from 6% to 100% mobile phase B in 8 min; sample injection volume, 5 μL. ESI–MS parameters were: source voltage = 4 kV; positive ion mode; mass resolution = 60,000. A standard curve was generated for naloxone-HCl and naloxone-MET from 1 to 5000 ng/mL. The limit of quantification was 5 ng/mL and the limit of detection was 1 ng/mL for both naloxone-HCl and naloxone-MET. The retention time for naloxone was 3.55 and for naloxone-MET 3.35 minutes. Standard samples of naloxone-MET with high concentrations showed a mass peak corresponding to naloxone (~1%), suggesting the presence of a very small amount of naloxone in the sample.When electrochemical and temperature recordings were completed, rats were deeply anesthetized with isoflurane, decapitated, the brains were extracted, and stored in 10% formalin solution. Later, the brains were sectioned and analyzed for verification of the locations of cerebral implants and tissue damage.Electrochemical data was analyzed with slow (1-min) and rapid (10-s) time resolution. Because each individual sensor differed in substrate sensitivity, currents were converted into concentrations according to sensitivity calibrations provided by the manufacturer. Data were then converted into % changes in concentration of oxygen. Values from one minute prior to injection were averaged and used to set the 100% baseline. One-way repeated measure ANOVAs (followed by Fisher LSD post-hoc tests) were used to evaluate statistical significance of drug/antagonist-induced oxygen changes. Decreases in oxygen were also represented as the area under the curve (AUC) calculated for the time interval of oxygen decrease during the first (control) heroin injection in each treatment group. To determine this parameter, we subtracted 100 from each 10-s oxygen percent value, summated all the points within a time interval of mean decrease in pre-treatment control and then averaged these values to determine the means and standard errors for the initial, pre-treatment and two post-treatment heroin injections. These mean values were statistically compared using Student’s t-test. Similar procedures were used to analyze temperature data. In addition to drug-induced temperature changes in the NAc, temporal muscle and skin, we also determined NAc-muscle and skin-muscle differentials, which reflect changes in intra-brain heat production due to metabolic activation/inhibition and in peripheral heat loss due to changes in skin vascular tone (vasoconstriction/vasodilation). Changes in temperature parameters are shown as changes relative to pre-injection baseline (set to 0 °C).Supplementary Information.


Met tyrosine kinase inhibitors (Met-TKIs) subjected to ongoing clinical trials are a promising option for Met-amplified gastric cancer (GC), but how to optimize their antitumor activity especially with combination schemes remains unclear. Since autophagy is known to be initiated by Met-TKIs, we investigated its underlying mechanisms and therapeutic potentials of Met-TKIs combined with autophagy inhibitors against Met-amplified GC. As expected, four Met-TKIs induced autophagy in Met-amplified GC cells marked by p62 degradation, LC3-II accumulation and increased LC3-positive puncta. Autophagy flux activation by Met-TKIs was further validated with combined lysosomal inhibitors, bafilomycin A1 (Baf A1) and hydroxychloroquine (HCQ). Molecular investigations reveal that autophagy induction along with mTOR and ULK1 de-phosphorylation upon Met-TKI treatment could be relieved by hepatocyte growth factor (HGF) and mTOR agonist MHY1485 (MHY), suggesting that autophagy was initiated by Met-TKIs via Met/mTOR/ULK1 cascade. Intriguingly, Met-TKIs further suppressed cell survival and tumor growth in the presence of autophagy blockade in Met-amplified GC preclinical models. Thus, these findings indicate Met/mTOR/ULK1 cascade responsible for Met-TKI-mediated autophagy and Met-TKIs combined with autophagy inhibitors as a promising choice to treat Met-amplified GC. Receptor tyrosine kinase Met  receptor) is a promising target for Met-addicted GC. The HGF/Met pathway broadly participates in GC survival, invasion and metastasis2, and aberrant activation of HGF/Met pathway represented by Met overexpression and gene amplification frequently occurs in GC3. Met overexpression and amplification were found in 39% and 7% of advanced GC in our previous study, respectively4. Growing evidence suggests Met gene amplification rather than protein overexpression as a true oncogenic driver and a predictive marker for Met-TKIs in GC7. Several Met tyrosine kinase inhibitors (Met-TKIs) including crizotinib (Criz) and volitinib (Voli) against Met-amplified GC are being investigated for this reason.Despite recent improvements in anticancer therapeutics, clinically available drugs for gastric cancer (GC) are limited, and hence GC remains a leading cause of mortality in China8. Therefore, understanding signaling pathway changes resulting from Met-TKI treatment are very critical to develop novel combination strategies for improving Met-TKI efficacy, especially in the Met-amplified subpopulation. Based on accumulating data, autophagy is frequently induced by drug exposure and acts as an attractive molecular target to potentiate efficacy of anticancer treatment19. Autophagy, a cellular adaptive response to stresses including anticancer agents, is an evolutionally conserved proteolytic process involving lysosomal degradation and recycling damaged cellular components and energy to maintain homeostasis20. Of note, protective autophagy rising in most contexts poses an opportunity for autophagy inhibitor-based combination therapies. Autophagy blockade has been applied concurrently with either chemotherapies or targeted therapies to optimize their efficacy in various cancers in preclinical studies22. Regarding GC, a previous study roughly revealed that targeting autophagy initiated by Met-TKIs improved Met-TKI efficacy in vitro23; however, Met-TKI-associated autophagy flux alterations, mechanisms underlying autophagy induced by Met-TKIs and therapeutic potentials of dual targeting Met/autophagy in Met-amplified GC, especially in vivo, remain far from clear. Hence, this study aims at these issues to deepen our understanding of potentials of optimizing Met-TKI efficiency with targeting autophagy in Met-addicted GC.Targeted drugs usually elicit better antitumor activity when combined with chemotherapy or other inhibitors due to known or unknown mechanisms25 were treated with various doses and duration of Met-TKIs. Met-TKIs actioned on Met-amplified GC cells, indicated by remarkable de-phosphorylation of Met  and hydroxychloroquine (HCQ); Fig. Met-amplified GC cellsMet Fig. . Of noteMet Fig. . LC3-posMet Fig. . Thus, M26. Met, mTOR, and ULK1 were de-phosphorylated when Met-TKIs induced autophagy, suggesting that mTOR inactivation and ULK1 activation were responsible for autophagy initiation by Met-TKIs  and its autophagy downstream effector ULK1KIs Fig. . As a liKIs Fig. . Of inteKIs Fig.  but alsoKIs Fig. . MeanwhiKIs Fig. . Taken t20. Given Met-TKI-mediated autophagy presented in our Met-amplified GC cells, dual inhibition of Met and autophagy was proposed. Compared to controls, Met-TKIs or the autophagy inhibitor HCQ alone inhibited cell viability to some extent, while the combination groups had a better response in Met-amplified GC cells  interference was employed to further confirm augmented effects of autophagy blockade on Met-TKI cellular lethality. As anticipated, both Atg5 and BECN1 knockdown could sensitize the Met-amplified MKN45 cell to Met-TKIs . However, efficacy of Met-TKIs alone is limited in Met-addicted GC. Different approaches represented by combination designs, therefore, are being studied to improve overall response.Continuous efforts to optimize Met-targeted therapeutic strategies led to the identification of Met amplification as a possible driver event of GC30. Protective autophagy frequently occurs after drug stimulation, indicative of potential benefits with combined autophagy inhibitors. Based on published results31, our data further display autophagy induction after exposure to Met-TKIs in Met-amplified GC preclinical models  pathway’s role during the process as reported in lung cancer17 merit future investigations.Molecular mechanisms underlying Met-TKI-induced autophagy in GC are unclear. De-phosphorylation of mTOR and ULK1  involves in autophagylls Fig. . Met/mTOlls Fig. . These p19, in accordance with potentiated anticancer activity of Met-TKIs plus autophagy inhibition against Met-amplified GC preclinical models  but early-phase apoptotic cells were almost unaffected . Similar to the low Met amplification rate, none of our established 50 GC PDXs featured Met amplification43. Efficacy of Met-TKIs plus autophagy inhibitors can be further verified when Met-amplified PDXs are available. On the other hand, our previous studies revealed a pMet-positive rate of 6% in patients with advanced GC, a strong association between pMet expression and Met amplification, and Voli-elicited strong antitumor activity in a GC PDX model with pMet expression43. Indeed, GC cells (MKN45 and SNU5) were pMet positive and Met-TKI-based treatment obviously inhibited Met phosphorylation in this work  models were applied. Met amplification occurred in about 7% of patients with advanced GC in our retrospective studyrk Figs. . Thus, prk Figs. , indicatMet-TKIs induced protective autophagy via Met/mTOR/ULK1 cascade and Met-TKIs combined with autophagy inhibitors augmented antitumor activity in Met-amplified GC preclinical models, shedding light upon this combination strategy in the treatment of Met-amplified GC.6. Primary antibodies against Met, pMet (Y1234/1235), p62, LC3, mTOR, pmTOR (S2448), pULK1 (S757), Atg5, Beclin-1, NBR1, and secondary horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies were purchased from Cell Signal Technology . Anti-β-actin primary antibody was purchased from Sigma-Aldrich .PHA-665752 (PHA), SU11274 (SU), Criz, Baf A1, HCQ, and MHY were purchased from Selleck Chemicals . Voli was kindly provided by AstraZeneca . HGF was purchased from Life Technologies . Reagents were formulated and stored following the manufacturer’s protocols for in vitro and in vivo experiments. Voli was formulated in a 0.5% (v/v) carboxymethylcellulose sodium solution for mice2.GC cell lines, MKN45 and NCI-N87, were provided by Professor Youyong Lv , while SNU5 was obtained from Hutchison Medi Pharma . MKN45 and NCI-N87 cells were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum (Gibco BRL) and 1% penicillin and streptomycin , while SNU5 cells were cultured in Iscove's modified Dulbecco's medium (Gibco BRL) replenished with 20% fetal bovine serum and 1% penicillin and streptomycin. Cells were incubated in a humidified incubator (37 °C) with 5% COCells were lysed using a CytoBuster protein extraction reagent  in the presence of protease and phosphatase inhibitor cocktail tablets . Protein concentration was measured by a BCA Protein Assay Kit . Soluble lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Merck Millipore). After blocking with 5% bull serum albumin  or fat-free milk, membranes were probed with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. Signals were visualized using Amersham Imager 600  after incubation with Clarity Western ECL substrate . Protein expressions were quantified using ImageJ Version 1.48 software and normalized to β-actin level followed by calculations of relative ratios to controls.Cells  were planted into a 35 mm glass bottom dish  and incubated overnight in complete medium. After drug treatment, cells were fixed with 4% paraformaldehyde  for 10 min, permeabilized with 0.5% Triton X-100 (Amresco) for 20 min, and blocked with normal goat serum  for 30 min at room temperature. Cells were probed with primary antibody against LC3B  at 4 °C overnight and Alexa Fluor 488-conjugated goat anti-rabbit IgG  in the dark for 1 h at room temperature, whereas nuclei were counterstained with 4′,6-diamidino-2-phenylindole  in the dark for 5 min at 4 °C. All reagents were diluted in phosphate-buffered saline (PBS) and all steps were followed by PBS-washing for three times. Images were captured with the ZEN version 2012 software  using a laser scanning confocal microscope LSM 780 (Zeiss). Dots of specific green signals within cells were considered to be LC3-positive puncta.Cells were seeded at a density of 4000 cells/well in 96-well plates and incubated overnight in complete medium. After drug treatment, cell viability was measured using a CCK-8 kit  according to the manufacturer’s protocol. Absorbance was measured at 450 nm using a spectrophotometer.Atg5 and BECN1 siRNA kits were purchased from RiboBio . Cells were seeded in 6-well plates and transfected at about 80% confluence with Atg5/BECN1 siRNAs and their corresponding negative control siRNA by Lipofectamine 3000  according to the manufacturer’s protocol. Cells transfected for 72 h were harvested for immunoblotting analysis.After drug treatment, cells were double-stained with Annexin V-phycoerythrin (PE) and 7-amino-actinomycin (7-AAD) (BD Biosciences) at room temperature in the dark as described in the vendor’s protocol, followed by flow cytometry analysis within 1 h (BD Biosciences). We analyzed proportions of apoptotic cells using FlowJo Version 7.6.1 software .7 cells/ml. Then, 100 μl cell suspension was inoculated subcutaneously in the right flank of 6-week-old female Balb/c nude mice . When tumor volume reached approximately 100–250 mm3, mice bearing GC cells were randomly assigned to treatment groups and given daily PBS, Criz , and Voli  alone or in combination with HCQ ) for 21 days (n = 5). Tumor size and body weight were measured every 3 days and tumor volume (V) was calculated by formula: V = L × W2/2 . After the final drug administration, mice were killed and tumors were stripped for western blots. All animal experiments were approved by Peking University Cancer Hospital’s Institutional Animal Care and Use Committee and complied with the internationally recognized Animal Research: Reporting of in vivo Experiments guideline.MKN45 cells detached with trypsin/EDTA (Gibco BRL) and SNU5 cells were collected and re-suspended with PBS to a final concentration of 1 × 10t-test, one-way or two-way analysis of variance (ANOVA) with post hoc Bonferroni correction; otherwise, data were expressed as median with interquartile range and compared using nonparametric Mann–Whitney U-test. P < 0.05 was considered statistically significant.All data were representative of three independent experiments and analyzed by GraphPad Prism 5 software. Continuous variables in line with normal distribution were expressed as means ± SD and compared using two-tailed Student’s Figure S1FigureS2supplemental material Figure legend


Background: Worldwide, a large proportion of neonates are prescribed antibiotics without having infections leading to increased antimicrobial resistance, disturbance of the evolving microbiota, and increasing the risk of various chronical diseases. Comparing practice between different hospitals/settings is important in order to optimize antibiotic stewardship.Aim: To investigate and compare the potential for improved antibiotic stewardship in neonates in two Norwegian hospitals with different academic culture, with emphasis on antibiotic exposure in unconfirmed infections, treatment length/doses, CRP values and the use of broad-spectrum antibiotics (BSA). All types of infections were investigated, but the main focus was on early-onset sepsis (EOS).Methods: We conducted a prospective observational cohort study of antibiotic use in a Norwegian university hospital (UH) and a district hospital (DH), 2017. Unconfirmed infections were defined as culture negative infections that neither fulfilled the criteria for clinical infection .Results: Ninety-five neonates at the DH and 89 neonates at the UH treated with systemic antibiotics were included in the study. In total, 685 prescriptions (daily doses) of antibiotics were given at the DH and 903 at the UH. Among term and premature infants (≥ 28 weeks), 82%  of the treatments for suspected EOS were for unconfirmed infections, and average treatment length in unconfirmed infections was 3.1 days . Median dose for aminoglycoside was higher for term infants at the UH  compared to the DH . At the UH, all prescriptions with aminoglycosides were gentamicin, while tobramycin accounted for 93% of all prescriptions with aminoglycosides at the DH.Conclusion: There is a potential for reduction in both antibiotic exposure and treatment length in these two neonatal units, and a systematic risk/observational algorithm of sepsis should be considered in both hospitals. We revealed no major differences between the UH and DH, but doses and choice of aminoglycosides varied significantly. Unnecessary use of antibiotics leads to increased rates of antimicrobial resistance (AMR) and is one of the main challenges in global health , 2. AMR Neonates and small children are particularly vulnerable to antibiotic exposure as the diversity of the gut microbiota increases and evolves during the first years of life . In addiWorldwide, neonates with suspected sepsis are exposed to antibiotics, although only a small proportion have a confirmed infection –13. The Fifteen of the 68 hospitals registered in the database of The Norwegian Institute of Public Health hold a neonatal unit; seven of these units are situated in university hospitals while the rest are situated in smaller district hospitals . The university hospitals hold many academic positions and are expected to be in the frontline of developing clinical practice. We therefor speculate whether there are any clearly differences in antibiotic use between centrally located university hospitals and more rural located district hospitals.The aim of this study was to explore antibiotic use among neonates with and without confirmed infection, with emphasis on choice and dosing of antibiotics, treatment duration, CRP values and the use of BSA. Furthermore, we assessed whether pattern of antibiotic use in neonates differs between university and district hospitals.We designed a prospective observational cohort study, collecting data from 2017 to describe and compare antibiotic use in neonates in a Norwegian university hospital (UH)  and a district hospital (DH) .The study population consisted of all neonates admitted to the neonatal units at the UH and the DH in the study periods. The DH has a neonatal intensive care unit (NICU) consisting of 13 beds and provides regional neonatal service for neonates from gestational age (GA) 26 weeks  and offers all kinds of intensive care apart from neonatal surgery and ECMO. The UH has a NICU consisting of 27 beds and provides regional service for neonates with all GA ages and all intensive care needs apart from ECMO and thoracic/heart surgery. Both hospitals mainly treat neonates admitted from the maternity ward, but at the DH critically ill infants (<3 month) can in certain circumstances be referred to the neonatal unit from home.There are no official national guidelines for antibiotic treatments in neonates in Norway, and most hospitals have local guidelines. In 2017, both study hospitals recommended the use of an aminoglycoside in combination with ampicillin for the treatment of early-onset sepsis. For term infants, the UH recommended aminoglycoside to be dosed 6 mg/kg as one daily administration, while the DH recommended 5 mg/kg. Both hospitals recommended ampicillin to be dosed 50 mg/kg two times a day. None of the hospitals used specific algorithms/observations routines for deciding whether to start antibiotic therapy once neonatal sepsis was suspected. The communication between the laboratory and the neonatal departments is well-established in both hospitals, and both results from CRP analyses and blood cultures are easy and rapidly available for the treating clinicians. In both hospitals, positive blood cultures are alerted directly from the microbiologist in terms of a personal call to the on-duty physician.At the DH, data were collected from 1st of January−31st of December, 2017. The collection was performed by trained nurses working at the unit and double-checked by the project manager. At the UH, data were collected during 15 weeks in 2017; from 27th of March−20th of May and from 01st of November−31th of December. Data were collected by two MD‘s working at the unit and the quality control was performed by the project manager. Educational classes for data collectors were held before the start of the registration.In both hospitals, patients receiving antibiotics were identified at 08.00 a.m. by the collectors every morning by evaluating all inpatients. In these neonatal wards, outpatient treatment of infections is very uncommon, thus we only included inpatients.For registrations we modified and extended an international standardized point prevalence protocol developed by the European Center for Disease Prevention and Control (ECDC) . The datData collection included the total number of patients in the wards, the total number of patients receiving antibiotics, gender, GA at birth, birthweight, delivery mode, age and weight at the start of antibiotic treatment, type and dose  of antibiotics, route of administration, treatment duration (in days), whether it was for treatment of infection or prophylaxis, indication for treatment/prophylaxis, respiratory support (any kind), maximum CRP value and results from blood cultures. For patients receiving antibiotics at the start or end of the registration period we obtained information from previous/remaining days of antibiotic treatment.The total numbers of live births in the uptake area for both hospitals were collected from the maternity ward and also controlled with the Norwegian birth registry.Term-infants were defined as GA ≥ 37 weeks, premature infants as GA 28–36 weeks and extremely premature infants as GA 23–27 weeks. All prescribed antibiotics were included in our analyses and described in relation to prescriptions, administrations, courses, and admitted patients. One prescription was defined as a daily dose with one antibiotic, an administration was defined as one single dose with one antibiotic, and a course was defined as antibiotic therapy/prophylaxis with one or more antibiotics for a certain indication in a certain continuous time range. Each patient registered at the wards during the daily registration was regarded as one bed day. Doses in mg/kg were based on birthweight until a higher body weight was recorded, and we only compared doses in term infants. Treatment duration was defined as number of days with antibiotic exposure. The total number of live births was used as a denominator for expressing antibiotic exposure within the first 3 days of life. Antibiotics were defined as antibacterials for systemic use (J01). Broad-spectrum antibiotics were defined as second- and third-generation cephalosporins, carbapenems, piperacillin/tazobactam and quinolones, according to the National Strategy against AMR .Surgical prophylaxis was defined as antibiotics given immediately before, during or shortly after surgery to prevent infection. Medical prophylaxis was defined as antibiotics prescribed to prevent infection in patients at risk, but without infectious symptoms and without obtainment of blood culture. Cases where symptoms could be explained by infections, but also by other conditions  were not regarded as prophylaxis. Early-onset sepsis (EOS) was defined as suspected sepsis within the first 3 days of life and late-onset sepsis (LOS) when sepsis was suspected after 3 days of life. Other indications were only used if organ specific symptoms were present (such as skin infections) without suspected sepsis. In theory, all infant with clinical symptoms and exposure for blood culture were classified as sepsis treatments.Treatments for suspected sepsis were divided in three categories: Culture positive sepsis , culture negative sepsis and no sepsis. The first two categories were regarded as confirmed infections. According to recommendations from the Norwegian Neonatal Network, the diagnosis of a culture negative neonatal sepsis , should only be used if certain criteria are fulfilled; clinical symptoms, CRP >30 mg/L, at least 5 days of antibiotic therapy (or death before 5 days) and whenever other medical conditions are ruled out . Thus, wt-test and Moods median test, respectively. 95% confidence intervals of means were calculated assuming normal distribution, whereas confidence intervals of medians were calculated using the binomial distribution. Correlation was estimated using Pearson correlation coefficient. P-values <0.05 were considered significant. Because the DH discontinued their service for extremely premature infants with GA <26 weeks during the study period, antibiotic use in extremely premature infants (GA <28 week) was described without comparisons.Statistical analyses were performed using SPSS Statistics 23 . Comparisons of proportions were done using standard chi-square tests. Means and medians were compared using independent samples The study was approved by the Regional Committee for Medical and Health Research Ethics (2017/30/REK Midt) and by the Local Data Protection Officials at the two hospitals.In total, 184 patients received 207 courses and 1,588 prescriptions of antibiotics. See p = 0.023), see For term and premature infants, aminoglycosides, and ampicillin accounted for the majority of antibiotic prescriptions, namely 84% at the UH and 85% at the DH See . Use of For extremely premature infants, aminoglycosides and ampicillin accounted for 57% of the prescriptions , and BSA use accounted for 15%. For all neonates, 119 (96%) of in total 124 prescriptions with BSA were second or third generation cephalosporins and 5 (4%) were carbapenems. At the UH, all prescriptions with aminoglycosides were gentamicin, while tobramycin accounted for 93% of all prescriptions with aminoglycosides at the DH.Hundred and twenty-four (83%) out of 150 courses of antibiotics for term and premature infants were started during the first 3 days of life in both hospitals; day one , day two , day three . p = 0.452). The remaining courses were given for LOS (7%), organ-specific infections without suspected sepsis (7%) and prophylaxis (5%). See p < 0.001). Among extremely premature infants, EOS accounted for 28 (48%) out of 58 antibiotic courses, late-onset sepsis (LOS) for 24 (41%) of the courses and prophylaxis for 6 (10%) of the courses. Mean treatment duration was 4.25 days, 95% CI 3.49–5.01 (EOS + LOS). For term and premature infants, treatment of suspected EOS accounted for 121 (81%) out of 150 antibiotic courses without significant difference between the hospitals  was 99% (171/173). Among term and premature infants, four (two EOS and two LOS) out of 128  treatments for suspected sepsis revealed a positive blood culture, corresponding to 0.8/1,000 live born infants. For EOS, the numbers needed to treat for one positive blood culture was 60. Among extremely premature infants, 14 (2 EOS and 12 LOS) out of 52  treatments for sepsis included a positive blood culture (12 at the UH and 2 at the DH), corresponding to 14 out of 38 (37%) of extremely premature infants in the units. coagulase negative Staphylococcus , two treatments involved an aminoglycoside combined with ampicillin in one case and cloxacillin in the other, and one case involved a combination of vancomycin, ceftazidime, and metronidazole. Mean treatment duration for infections with Staphlococcus aureus was 8.3 days. Two of the treatments consisted of cloxacillin monotherapy for more than half of the course and the last case involved an aminoglycoside combined with ampicillin and cloxacillin. For treatment of Escherichia coli, one patient was treated with an aminoglycoside combined with ampicillin for 8 days. The other patients, that was treated with an aminoglycoside and ampicillin the first to days and with cefotaxime monotherapy the third day, died during the treatment period. Among the other patients with culture positive sepsis, no fatalities or relapse of infections were registered during the study period.Overall, the bacteria growing in the cultures were lococcus , Streptoalactiae , Staphyls aureus , and Eschia coli . Mean trAmong term infants treated with antibiotics during the first week of life, significantly higher doses of aminoglycosides were used at the UH compared to the DH . MoreoveThis study reveals that only 1/5 of treatments for suspected EOS in term and premature infants were confirmed infections. Average treatment length for unconfirmed infections was just above 3 days. No significant differences were observed between the hospitals for characteristics of EOS, but doses and choice of aminoglycosides varied between the hospitals.A strength of this study is the prospective design, that only a few collectors performed the registrations and the small share of missing data. Other studies have excluded coagulase-negative Staphylococci from epidemiological overviews of positive blood cultures because of the probability of contamination , 19. SinAn important limitation is the low power of the study to detect clinically relevant differences between the hospitals. We aimed to include extremely premature infants also from the DH, but this could not be done due to unexpected hospital centralization for infants with GA <26 weeks during the study period. Another limitation is the lack of data on maternal risk factors for EOS. We did nor register whether patients were admitted from home, from other hospitals or from the maternity ward; as described in methods we could speculate that some patients at the DH were admitted from home reflected by the significantly higher number of term and premature infants >10 days when initiating their antibiotic course. The data collection was performed by clinicians and nurses working at the respective wards, and most clinicians were aware of the study. This may have affected the data in the manner of more prudent antibiotic use than usual. However, as this possible bias was the same in both hospitals it would not affect the comparison between the hospitals. Our sampling did neither report the name of the clinicians prescribing antibiotics. It is reasonable to assume that prescription habits are difficult to change in a manner that would have a significantly impact on our results, but a minor bias can not be excluded. Finally, different registration periods at the two hospitals may have introduced a bias in relation to the seasonality of certain pathogens. One study concluded that there was no seasonal variation in the prevalence of gram-negative microbes causing LOS . AnotherFor term and premature infants, use of BSA was low in both hospitals, but the number of prescriptions was higher at the DH. This can partly be explained by four children at the DH receiving BSA after 10 days of age compared to zero at the UH. Also, three of the seven BSA treatments at the DH was for other indications than sepsis and three of the infants were critically ill in term of receiving respirator treatment. One could speculate whether doctors at the UH have a higher threshold for prescribing BSA than in the DH, but taking the low numbers of BSA treatments into account, this difference is probably not clinically relevant. However, this finding should be controlled in future studies.The difference in the doses of aminoglycosides in term infants is explained by different local guideline recommendations at the hospitals. We did not register any switch of aminoglycoside doses in term infants during the same course, and one study showed that aminoglycosides safely can be dosed 6 mg/kg every 24 h in term born infants . The higPseudomonas aeruginosa (The choice of aminoglycoside differed in the UH (gentamicin) and the DH (tobramycin) because of different local guidelines. One study found lower creatinine levels in neonates treated with tobramycin compared to gentamicin, but concluded that the clinical significance of the findings were minimal . Tobramyruginosa , but thiruginosa , 27. As The rate of antibiotic exposure during the first 3 days of life in term infants is in line with national data from 2009 to 2011 –30. The We found that 77% of antibiotic courses to term infants for suspected EOS were given to infants without confirmed infections, compared to 54% in the previous national survey . In the Mean treatment duration for unconfirmed EOS (term infants) was shorter than in the national survey from 2009 to 2011, 3 (mean) vs. 4 days (median), respectively. Extremely premature infants in our study were averagely treated 4 days (EOS+LOS). The probability of positive blood cultures beyond 24–48 h is small , and stuWe found that maximum CRP values and 95% CI increased with number of treatment days, indicating that CRP values are regarded as an important factor when deciding treatment length. A recently published study showed that CRP values >30 was uncommon in healthy term infants, supporting the decision of using 30 as cut of level for infection . Other bListeria monocytogenes could justify the use of ampicillin, but according to data from The Norwegian Institute of Public Health, only four cases of listeriosis have been reported among Norwegian children (< 1 year) from 2011 to 2018  to rule out prophylaxis. Even though we speculate that blood cultures in some cases are taken as part of an implemented routine, our results show a low use of antibiotic prophylaxis in both hospitals compared to international data .The number of extremely premature infants with a positive blood culture , is in line with international reports , 46. It Our results can be used in future antibiotic stewardship programs, including research projects, in Norwegian neonatal departments, for instance by introducing interventions/algorithms to reduce antibiotic exposure and treatment duration. A unified national guideline including clear antibiotic recommendations and dose regimes is desirable. For future surveillances, we have suggested a definition of prophylaxis.Based on our study there are no indications of major differences in the pattern of antibiotic use between university and district hospitals in Norway, but term infants at the UH were treated with higher doses of aminoglycosides and fewer daily administrations of ampicillin. Furthermore, gentamicin was the aminoglycoside of choice at the UH, while tobramycin was mainly used at the DH. Even though neonates in Norway receive less antibiotic than in other countries, this study revealed that there is a potential for reduction in both antibiotic exposure and treatment duration for neonates. A systematic risk/observational stratification of sepsis should be considered in both hospitals.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by Regional Committee for Medical and Health Research Ethics (2017/30/REK Midt). Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.CT, DB, HB, and BE were involved in the development of the protocol. CT developed the registration form and was responsible for the data collection at Ålesund hospital. AB was responsible for the data collection at Oslo University Hospital, Ullevål. CT and TM did the analyses. CT wrote the first draft. All the authors contributed to the interpretation of the data and revisions of the manuscript and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The paper deals with a concept of a practical computation method to simulate the temperature distribution in an extradosed bridge deck. The main goal of the study is to develop a feasible model of hardening of concrete consistent with in-situ measurement capabilities. The presented investigations include laboratory tests of high performance concrete, measurements of temperature evolution in the bridge deck and above all, numerical simulations of temperature field in a concrete box bridge girder. A thermal conductivity equation in the author’s program, using finite difference method has been solved. New approach for identification of the model parameters and boundary conditions (heat transfer coefficients) has been proposed. The numerical results are verified by means of a wide set of experimental tests carried out on three stages of the extradosed bridge studies. A high agreement between the concrete temperature distribution in the time and space domain was obtained. The temperature history of concrete hardening, supplemented with maturity method equations, made it possible to estimate an early-age compressive strength of the cast-in-place concrete. The proposed solution could be applied in a Structural Health Monitoring system for concrete objects. Attention to the quality, durability and rapid construction process is one of the major challenges for the concrete industry. Prediction of the temperature field in concrete objects allows us to use the proper care of young concrete as well as to estimate development of compressive strength. In the case of massive structures, it is particularly important to not exceed the temperature difference between the interior and the concrete surface. Too high temperature gradient generates an increase in thermal-shrinkage stresses, what could be the reason of exceeding the tensile strength and as a consequence cracking occurrence . KnowledIn recent years, there has been growing interest in rationalization of the building schedule. Supporting the process of designing, curing and monitoring concrete features with the use of computer technologies, provide a lot of benefits, especially for contractors ,4. ElSafThe key aspect in the prediction of temperature distribution of cast-in-place (CIP) concrete is the choice of the numerical model, which should be consistent with the measurement capabilities. The multi-field mathematical models of young concrete ,7 are weThe well-defined model parameters and initial-boundary conditions corresponding to the real environmental conditions at the construction site are the basis of reliable predictions of the in-place concrete temperature distribution. In this paper, two thermo-chemical models of hardening of concrete ,9 were uThe plurality and unrecognizability of phenomena occurring during concrete curing, leads to extensive experimental research and the development of a model, in which concrete is defined as a material with non-stationary properties. Mathematical models, available in the literature, takes into account four aspects of concrete hardening. It can be said that in the early age concrete there is a strong coupling of thermal, chemical, moisture and mechanical phenomena. In this study complex models presented i.e., in Azenha’s ,12 and DThe process of cement hydration is related to the change of the phase composition of the medium. Phase transitions are accompanied by thermal phenomena associated with the heat release, as a result of hydration reactions of cement components. As a reason of heat conduction, the temperature field appears in every area of the body. In the macroscopic approach, concrete can be treated as an isotropic and homogeneous material with constant conductivity The model proposed by Cervera et al.  consistsbility η . Hence, by Mills :(4)ξmax=r Waller :(5)ξmax=The normalized chemical affinity is directly measurable during an adiabatic test in which heat is not exchanged with the environment, thus the Equation (1) is simplified to:At the end of an adiabatic test By substituting the Equation (2) and (7) into (6) we get:Formula No. 8 allows us to calculate the normalized affinity by measuring the temperature rate The described model  is suitaThe model presented in the work of Martinelli et al. ,15 is al3 volume. Based on the results of adiabatic experiments on concrete samples, the evolution of the hydration heat can be approximated using equation . T. T32]. T2Oeq < 0.6%) and very high corrosion resistance, especially sulphate (SR3: C3A < 3%) and chloride [The used cement characterizes by a normal (N) initial strength gain, low content of alkali . The results of the presented studies were In order to determine the activation energy divided by the gas constant TM C1074 ) were cagth data a using nR=4620 K b.The cement content per cubic meter of concrete The final hydration degree Similar values of proposal  were ado3A) was restricted to lower than 3%, which affected the relatively low heat of cement hydration.The hydration heat of CEM I 52,5 N SR3/NA marked in the semi-adiabatic calorimetry test given by the cement producer after 48 h was about 302 kJ/kg. In Prediction formulas also exist for specific heat of concrete, for instance, in the work of Lura and Breugel  the valuThe heat conduction coefficient of concrete, expressed in W/(m·K), was estimated based on the thermal conductivities of the mix components using the following equation :(25)λ=WcAccording to Neville , for conThe model parameters  measured in the middle of the 93 cm thick bottom slab  for the considered specimens allowed us to plot the development of hydration degree ation 8, a. Foundetal data b, three The next step included verification of the determined constants in reliance on temperature simulations of the concrete specimens according to the Cervera’s approach and also involved a possible modification of the three parameters. For this purpose two simulations were carried out: “v1” for designated constants: ent data .The predicted adiabatic temperature evolution was consistent with Martinelli’s proposal . The resCase 1: Variable coefficient Case 2: Variable coefficient Case 3: Variable coefficient Additionally, in order to recognize the influence of The one-dimensional model (1D) was used to predict the temperature development of regular concrete slabs. In case of any irregularities such as the proximity of the protruding reinforcement or the influence of the temperature of earlier concreted parts of element, previously determined model parameters are no longer valid. Therefore, the calculations were performed for elements with clearly defined boundary conditions. This applies to the B-B section of the bottom plate of stage I, the web and the top plate (stage II) and the web monitored during III stage of research on the bridge.2. As shown in −5 was assumed, and for elements thinner than 60 cm  −4.Numerical simulations were carried out relying on experimentally determined constants for each research stage. The selected model parameters and heat transfer coefficients were adopted from the own propositions dedicated to the analyzed high-performance concrete and element thickness up to 100 cm. In this kind of structure, semi-adiabatic conditions of concrete hardening might be assumed. The prediction of concrete temperature for compressive strength assessment is the most useful for the medium-weight structure, which constitute the building contractor’s interest. The proposed relationships are not intended for massive or thin-walled concrete elements. On the basis of laboratory and field tests, the coefficient perature a, and th element b was expLiterature propositions for heat transfer coefficients for forced convection or for insulation layer are often the result of specific experimental investigations and are only relevant in this particular case. In this study, the convective heat transfer coefficient on the concrete surface protected by the formwork layer one Tenv a. In FigIn the future, to determine reliable nomograms such as , a serieThe slab was concreted in June, when the average ambient temperature of 10 days was 22.1 °C. It was assumed that throughout the analyzed period, on the bottom surface of the slab the formwork (plywood and girders) was mounted. The top surface of the plate was exposed to the weather conditions, but between 23 and 94 h it was covered with a styrofoam layer, 5 cm thick. The average wind speed in this period was approximately 16 km/h = 4.44 m/s , thus acUsing the finite difference method, the space domain was subdivided in was 1 cm . Moreoveconstant a and a dtant  and the top slab (56 cm), i.e., medium-weight elements, monitored during the second stage of bridge investigations . The recIn both monitored elements, boundary conditions had a strong influence on the temperature distribution. The concrete temperature of the web, 40 cm thick, surrounded on both sides with formwork, was very similar at all measurement points a. The toDuring concreting of overhanging section of the bridge span, the average ambient temperature of 10 days was 20.1 °C. The average wind speed in this period reached 8 km/h = 2.22 m/s . CalculaThe third stage of research on the bridge object was carried out in March, when the average ambient temperature was the lowest of the considered cases and it was around 4 °C. The average wind speed was equal in this period 13 km/h = 3.61 m/s . The iniAs before, it was assumed, that throughout the analyzed time, the web was insulated by the formwork layer. Thermophysical parameters were adopted according to The proposed one-dimensional model was able to predict well both the heating and cooling phase, which is important in relation to the occurrence of undesirable tensile stresses. However, the one-dimensional model was not recommended to describe the temperature evolution of concrete plates with undefined strictly boundary conditions, as shown in the example discussed below. It concerns the bottom slab of stage I, for which the A-A measurement cross-section was located just next to the protruding web reinforcement and could not be insulated well with the styrofoam layer. The second example with irregular boundary conditions was the top and bottom plate monitored in stage No. III, where some disorder was the temperature coming from the hardening web. For these elements, the selected model parameters were modified. The obtained solutions were satisfactory, but for special cases, the calculations should be made with a two-dimensional model.For the bottom slab, 80 cm thick (stage I) simulations in two variants were carried out. The first one for thermophysical coefficients specified for the 93 cm slab  with mod and αfc . The res and αfc a it coulsolution b.For the A-A cross-section (80 cm thick), after 24 h, the temperature difference between points p1 and p2 was 19.3 °C a. Such iIn stage III, the box section of the bridge was concreted in one step. Thus, for the upper surface of the bottom slab and the bottom surface of the top slab, the influence of the temperature from the embedded web was significant. Therefore, a replacement heat transfer coefficient was adopted, indicating it with a symbol αsconc . The oth results  were satThe thermal changes in the top plate differed the most . The maxThe one-dimensional approach has several limitations. Early-age concrete is difficult for modeling because it is a complex material, which is additionally subjected to transformations as a result of cement hydration. The contractors use different types of formwork, insulation and the weather is changing all the time during construction process. Due to this fact, the 1–D way is not universal. Nevertheless, this fundamental method gives a possibility to control the early age behavior of concrete with quite good agreement. In the case of 1–D problem we could solve the partial differential equation using the own code of the finite difference method. There are no hardware restrictions and calculation time is short. A numerical, one-dimensional model is always a good complement to the measuring system. Many researchers ,9,12 useAs mentioned at the beginning of the paper, the in-place concrete temperature measurements were performed with a focus on estimation the real concrete compressive strength. The contractor planned to prestress every typical segment after reaching 60% of characteristic strength for class C 60/75, so it was exactly 45 MPa for cubic strength. Due to that fact, the maturity method was implemented ,41. The Based on the numerically obtained maps of the temperature field and the maturity–strength relationship expressed in the equivalent age For on-line monitoring of concrete structures, the numerical model of the hardening concrete must be relatively simple, so that it can be an integral part of the SHM system.To register temperature of cast-in-place concrete it was suggested to use 1-wire digital sensors and install them on one measuring strip.The performed tests of cubic specimens curing under isothermal and semi-adiabatic conditions demonstrated the potential of experimental identification of the adiabatic hydration curve and thus model parameters: The generated temperature of young concrete depends on the element dimensions and the weather conditions. Therefore, two novel nomograms for determination The boundary conditions were responsible for the heat exchange with the environment and had a huge impact on the rate gain of concrete temperature, the temperature drop and on the extreme temperature value. Therefore, the suggestions of connective heat transfer coefficient for the concrete surface with a formwork layer To reliably forecast thermal changes in concrete in a multi-segment structure, it was necessary to carry out preliminary tests on the starting section. The temperature should be measured inside the concrete element and on the formwork surface. Additionally, the ambient temperature as well as the wind speed needs to be registered.The numerical evolutions of temperature of high-performance concrete were positively verified in experimental measurements. The daily fluctuations of ambient temperature improved the accuracy of estimated temperature, however the assumption of constant air temperature provided very good results.The considered bridge deck proved that the FDM method was appropriate for structural elements with clearly defined boundary conditions as well as quite regular and simple geometry . In the case of more complex geometry, the calculations of the temperature evolution should be performed using the finite element method. The maturity method gave possibilities to estimate compressive strength distribution with an indication of time to achieve a required strength. The reduced air temperature resulted in a lower initial temperature of the mixture and a lower self-heating temperature, which delayed the development of the concrete strength, which was clearly visible for section No. 3.18.The knowledge of the temperature and strength development of concrete before the starting construction process allowed us to select temperature measurement points, to modify the designed concrete mixture and to take proper care of early-age concrete.This paper provided the guidelines to predict the temperature field in a concrete structure based on limited input data. The proposed solution, taking into account the thermal and chemical aspect of the hardening concrete, allowed us to simulate successfully the temperature changes in cast-in-place concrete of box bridge girder. The main results of the experimental and numerical studies could be summarized as follows: