Reversal of cerebral ischaemia and hypoxia and of sickness behaviour by megadose sodium ascorbate in ovine Gram-negative sepsis.

BACKGROUND: The mechanisms by which megadose sodium ascorbate improves clinical status in experimental sepsis is unclear. We determined its effects on cerebral perfusion, oxygenation, and temperature, and plasma levels of inflammatory biomarkers, nitrates, nitrites, and ascorbate in ovine Gram-negative sepsis. METHODS: Sepsis was induced by i.v. infusion of live Escherichia coli for 31 h in unanaesthetised Merino ewes instrumented with a combination sensor in the frontal cerebral cortex to measure tissue perfusion, oxygenation, and temperature. Fluid resuscitation at 23 h was followed by i.v. megadose sodium ascorbate (0.5 g kg-1 over 30 min+0.5 g kg-1 h-1 for 6.5 h) or vehicle (n=6 per group). Norepinephrine was titrated to restore mean arterial pressure (MAP) to 70-80 mm Hg. RESULTS: At 23 h of sepsis, MAP (mean [sem]: 85 [2] to 64 [2] mm Hg) and plasma ascorbate (27 [2] to 15 [1] µM) decreased (both P<0.001). Cerebral ischaemia (901 [58] to 396 [40] units), hypoxia (34 [1] to 19 [3] mm Hg), and hyperthermia (39.5 [0.1]°C to 40.8 [0.1]°C) (all P<0.001) developed, accompanied by malaise and lethargy. Sodium ascorbate restored cerebral perfusion (703 [121] units], oxygenation (30 [2] mm Hg), temperature (39.2 [0.1]°C) (all PTreatment<0.05), and the behavioural state to normal. Sodium ascorbate slightly reduced the sepsis-induced increase in interleukin-6, returned VEGF-A to normal (both PGroupxTime<0.01), and increased plasma ascorbate (20 000 [300] µM; PGroup<0.001). The effects of sodium ascorbate were not reproduced by equimolar sodium bicarbonate. CONCLUSIONS: Megadose sodium ascorbate rapidly reversed sepsis-induced cerebral ischaemia, hypoxia, hyperthermia, and sickness behaviour. These effects were not reproduced by an equimolar sodium load.

Feasibility of endovascular stimulation of the femoral nerve using a stent-mounted electrode array.

Objective.Electrical stimulation of peripheral nerves has long been a treatment option to restore impaired neural functions that cannot be restored by conventional pharmacological therapies. Endovascular neurostimulation with stent-mounted electrode arrays is a promising and less invasive alternative to traditional implanted electrodes, which typically require invasive implantation surgery. In this study, we investigated the feasibility of endovascular stimulation of the femoral nerve using a stent-mounted electrode array and compared its performance to that of a commercially available pacing catheter.Approach.In acute animal experiments, a pacing catheter was implanted unilaterally in the femoral artery to stimulate the femoral nerve in a bipolar configuration. Electromyogram of the quadriceps and electroneurogram of a distal branch of the femoral nerve were recorded. After retrieval of the pacing catheter, a bipolar stent-mounted electrode array was implanted in the same artery and the recording sessions were repeated.Main Results.Stimulation of the femoral nerve was feasible with the stent-electrode array. Although the threshold stimulus intensities required with the stent-mounted electrode array (at 100-500µs increasing pulse width, 2.17 ± 0.87 mA-1.00 ± 0.11 mA) were more than two times higher than the pacing catheter electrodes (1.05 ± 0.48 mA-0.57 ± 0.28 mA), we demonstrated that, by reducing the stimulus pulse width to 100µs, the threshold charge per phase and charge density can be reduced to 0.22 ± 0.09µC and 24.62 ± 9.81µC cm-2, which were below the tissue-damaging limit, as defined by the Shannon criteria.Significance.The present study is the first to reportin vivofeasibility and efficiency of peripheral nerve stimulation using an endovascular stent-mounted electrode array.

Differential responses of cerebral and renal oxygenation to altered perfusion conditions during experimental cardiopulmonary bypass in sheep.

We tested whether the brain and kidney respond differently to cardiopulmonary bypass (CPB) and to changes in perfusion conditions during CPB. Therefore, in ovine CPB, we assessed regional cerebral oxygen saturation (rSO2 ) by near-infrared spectroscopy and renal cortical and medullary tissue oxygen tension (PO2 ), and, in some protocols, brain tissue PO2 , by phosphorescence lifetime oximetry. During CPB, rSO2 correlated with mixed venous SO2 (r = 0.78) and brain tissue PO2 (r = 0.49) when arterial PO2 was varied. During the first 30 min of CPB, brain tissue PO2 , rSO2 and renal cortical tissue PO2 did not fall, but renal medullary tissue PO2 did. Nevertheless, compared with stable anaesthesia, during stable CPB, rSO2 (66.8 decreasing to 61.3%) and both renal cortical (90.8 decreasing to 43.5 mm Hg) and medullary (44.3 decreasing to 19.2 mm Hg) tissue PO2 were lower. Both rSO2 and renal PO2 increased when pump flow was increased from 60 to 100 mL kg-1 min-1 at a target arterial pressure of 70 mm Hg. They also both increased when pump flow and arterial pressure were increased simultaneously. Neither was significantly altered by partially pulsatile flow. The vasopressor, metaraminol, dose-dependently decreased rSO2 , but increased renal cortical and medullary PO2 . Increasing blood haemoglobin concentration increased rSO2 , but not renal PO2 . We conclude that both the brain and kidney are susceptible to hypoxia during CPB, which can be alleviated by increasing pump flow, even without increasing arterial pressure. However, increasing blood haemoglobin concentration increases brain, but not kidney oxygenation, whereas vasopressor support with metaraminol increases kidney, but not brain oxygenation.

Effects of furosemide, acetazolamide and amiloride on renal cortical and medullary tissue oxygenation in non-anaesthetised healthy sheep.

It has been proposed that diuretics can improve renal tissue oxygenation through inhibition of tubular sodium reabsorption and reduced metabolic demand. However, the impact of clinically used diuretic drugs on the renal cortical and medullary microcirculation is unclear. Therefore, we examined the effects of three commonly used diuretics, at clinically relevant doses, on renal cortical and medullary perfusion and oxygenation in non-anaesthetised healthy sheep. Merino ewes received acetazolamide (250 mg; n = 9), furosemide (20 mg; n = 10) or amiloride (10 mg; n = 7) intravenously. Systemic and renal haemodynamics, renal cortical and medullary tissue perfusion and P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ , and renal function were then monitored for up to 8 h post-treatment. The peak diuretic response occurred 2 h (99.4 ± 14.8 mL/h) after acetazolamide, at which stage cortical and medullary tissue perfusion and P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ were not significantly different from their baseline levels. The peak diuretic response to furosemide occurred at 1 h (196.5 ± 12.3 mL/h) post-treatment but there were no significant changes in cortical and medullary tissue oxygenation during this period. However, cortical tissue P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ fell from 40.1 ± 3.8 mmHg at baseline to 17.2 ± 4.4 mmHg at 3 h and to 20.5 ± 5.3 mmHg at 6 h after furosemide administration. Amiloride did not produce a diuretic response and was not associated with significant changes in cortical or medullary tissue oxygenation. In conclusion, clinically relevant doses of diuretic agents did not improve regional renal tissue oxygenation in healthy animals during the 8 h experimentation period. On the contrary, rebound renal cortical hypoxia may develop after dissipation of furosemide-induced diuresis.

Mega-dose sodium ascorbate: a pilot, single-dose, physiological effect, double-blind, randomized, controlled trial.

BACKGROUND: Mega-dose sodium ascorbate (NaAscorbate) appears beneficial in experimental sepsis. However, its physiological effects in patients with septic shock are unknown. METHODS: We conducted a pilot, single-dose, double-blind, randomized controlled trial. We enrolled patients with septic shock within 24 h of diagnosis. We randomly assigned them to receive a single mega-dose of NaAscorbate (30 g over 1 h followed by 30 g over 5 h) or placebo (vehicle). The primary outcome was the total 24 h urine output (UO) from the beginning of the study treatment. Secondary outcomes included the time course of the progressive cumulative UO, vasopressor dose, and sequential organ failure assessment (SOFA) score. RESULTS: We enrolled 30 patients (15 patients in each arm). The mean (95% confidence interval) total 24-h UO was 2056 (1520-2593) ml with placebo and 2948 (2181-3715) ml with NaAscorbate (mean difference 891.5, 95% confidence interval [- 2.1 to 1785.2], P = 0.051). Moreover, the progressive cumulative UO was greater over time on linear mixed modelling with NaAscorbate (P < 0.001). Vasopressor dose and SOFA score changes over time showed faster reductions with NaAscorbate (P < 0.001 and P = 0.042). The sodium level, however, increased more over time with NaAscorbate (P < 0.001). There was no statistical difference in other clinical outcomes. CONCLUSION: In patients with septic shock, mega-dose NaAscorbate did not significantly increase cumulative 24-h UO. However, it induced a significantly greater increase in UO and a greater reduction in vasopressor dose and SOFA score over time. One episode of hypernatremia and one of hemolysis were observed in the NaAscorbate group. These findings support further cautious investigation of this novel intervention. Trial registration Australian New Zealand Clinical Trial Registry (ACTRN12620000651987), Date registered June/5/2020.

Selective efferent vagal stimulation in heart failure.

Patients diagnosed with heart failure have high rates of mortality and morbidity. Based on promising preclinical studies, vagal nerve stimulation has been trialled in these patients using whole nerve electrical stimulation, but the results have been mixed. This is, at least in part, due to an inability to selectively recruit the activity of specific fibres within the vagus with whole nerve electrical stimulation, as well as not knowing which the 'therapeutic' fibres are. This symposium review focuses on a population of cardiac-projecting efferent vagal fibres with cell bodies located within the dorsal motor nucleus of the vagus nerve and a new method of selectively targeting these projections as a potential treatment in heart failure. NEW FINDINGS: What is the topic of this review? Selective efferent vagal stimulation in heart failure. What advances does it highlight? Selectively targeting a population of cardiac-projecting efferent vagal fibres with cell bodies within the dorsal motor nucleus of the vagus using optogenetics slows the progression of heart failure in rats.

Renal arterial infusion of tempol prevents medullary hypoperfusion, hypoxia, and acute kidney injury in ovine Gram-negative sepsis.

AIM: Renal medullary hypoperfusion and hypoxia precede acute kidney injury (AKI) in ovine sepsis. Oxidative/nitrosative stress, inflammation, and impaired nitric oxide generation may contribute to such pathophysiology. We tested whether the antioxidant and anti-inflammatory drug, tempol, may modify these responses. METHODS: Following unilateral nephrectomy, we inserted renal arterial catheters and laser-Doppler/oxygen-sensing probes in the renal cortex and medulla. Noanesthetized sheep were administered intravenous (IV) Escherichia coli and, at sepsis onset, IV tempol (IVT; 30 mg kg-1 h-1 ), renal arterial tempol (RAT; 3 mg kg-1 h-1 ), or vehicle. RESULTS: Septic sheep receiving vehicle developed renal medullary hypoperfusion (76 ± 16% decrease in perfusion), hypoxia (70 ± 13% decrease in oxygenation), and AKI (87 ± 8% decrease in creatinine clearance) with similar changes during IVT. However, RAT preserved medullary perfusion (1072 ± 307 to 1005 ± 271 units), oxygenation (46 ± 8 to 43 ± 6 mmHg), and creatinine clearance (61 ± 10 to 66 ± 20 mL min-1 ). Plasma, renal medullary, and cortical tissue malonaldehyde and medullary 3-nitrotyrosine decreased significantly with sepsis but were unaffected by IVT or RAT. Consistent with decreased oxidative/nitrosative stress markers, cortical and medullary nuclear factor-erythroid-related factor-2 increased significantly and were unaffected by IVT or RAT. However, RAT prevented sepsis-induced overexpression of cortical tissue tumor necrosis factor alpha (TNF-α; 51 ± 16% decrease; p = 0.003) and medullary Thr-495 phosphorylation of endothelial nitric oxide synthase (eNOS; 63 ± 18% decrease; p = 0.015). CONCLUSIONS: In ovine Gram-negative sepsis, renal arterial infusion of tempol prevented renal medullary hypoperfusion and hypoxia and AKI and decreased TNF-α expression and uncoupling of eNOS. However, it did not affect markers of oxidative/nitrosative stress, which were significantly decreased by Gram-negative sepsis.

Splanchnic sympathetic nerve denervation improves bacterial clearance and clinical recovery in established ovine Gram-negative bacteremia.

BACKGROUND: The autonomic nervous system can modulate the innate immune responses to bacterial infections via the splanchnic sympathetic nerves. Here, we aimed to determine the effects of bilateral splanchnic sympathetic nerve denervation on blood pressure, plasma cytokines, blood bacterial counts and the clinical state in sheep with established bacteremia. METHODS: Conscious Merino ewes received an intravenous infusion of Escherichia coli for 30 h (1 × 109 colony forming units/mL/h) to induce bacteremia. At 24 h, sheep were randomized to have bilaterally surgically implanted snares pulled to induce splanchnic denervation (N = 10), or not pulled (sham; N = 9). RESULTS: Splanchnic denervation did not affect mean arterial pressure (84 ± 3 vs. 84 ± 4 mmHg, mean ± SEM; PGroup = 0.7) compared with sham treatment at 30-h of bacteremia. Splanchnic denervation increased the plasma levels of the pro-inflammatory cytokine interleukin-6 (9.2 ± 2.5 vs. 3.8 ± 0.3 ng/mL, PGroup = 0.031) at 25-h and reduced blood bacterial counts (2.31 ± 0.45 vs. 3.45 ± 0.11 log10 [CFU/mL + 1], PGroup = 0.027) at 26-h compared with sham treatment. Plasma interleukin-6 and blood bacterial counts returned to sham levels by 30-h. There were no differences in the number of bacteria present within the liver (PGroup = 0.3). However, there was a sustained improvement in clinical status, characterized by reduced respiratory rate (PGroup = 0.024) and increased cumulative water consumption (PGroup = 0.008) in splanchnic denervation compared with sham treatment. CONCLUSION: In experimental Gram-negative bacteremia, interrupting splanchnic sympathetic nerve activity increased plasma interleukin-6, accelerated bacterial clearance, and improved clinical state without inducing hypotension. These findings suggest that splanchnic neural manipulation is a potential target for pharmacological or non-pharmacological interventions.

Associations Between Systemic and Cerebral Inflammation in an Ovine Model of Cardiopulmonary Bypass.

BACKGROUND: Intraoperative inflammation may contribute to postoperative neurocognitive disorders after cardiac surgery requiring cardiopulmonary bypass (CPB). However, the relative contributions of general anesthesia (GA), surgical site injury, and CPB are unclear. METHODS: In adult female sheep, we investigated (1) the temporal profile of proinflammatory and anti-inflammatory cytokines and (2) the extent of microglia activation across major cerebral cortical regions during GA and surgical trauma with and without CPB (N = 5/group). Sheep were studied while conscious, during GA and surgical trauma, with and without CPB. RESULTS: Plasma tumor necrosis factor-alpha (mean [95% confidence intervals], 3.7 [2.5-4.9] vs 1.6 [0.8-2.3] ng/mL; P = .0004) and interleukin-6 levels (4.4 [3.0-5.8] vs 1.6 [0.8-2.3] ng/mL; P = .029) were significantly higher at 1.5 hours, with a further increase in interleukin-6 at 3 hours (7.0 [3.7-10.3] vs 1.8 [1.1-2.6] ng/mL; P < .0001) in animals undergoing CPB compared with those that did not. Although cerebral oxygen saturation was preserved throughout CPB, there was pronounced neuroinflammation as characterized by greater microglia circularity within the frontal cortex of sheep that underwent CPB compared with those that did not (0.34 [0.32-0.37] vs 0.30 [0.29-0.32]; P = .029). Moreover, microglia had fewer branches within the parietal (7.7 [6.5-8.9] vs 10.9 [9.4-12.5]; P = .001) and temporal (7.8 [7.2-8.3] vs 9.9 [8.2-11.7]; P = .020) cortices in sheep that underwent CPB compared with those that did not. CONCLUSIONS: CPB enhanced the release of proinflammatory cytokines beyond that initiated by GA and surgical trauma. This systemic inflammation was associated with microglial activation across 3 major cerebral cortical regions, with a phagocytic microglia phenotype within the frontal cortex, and an inflammatory microglia phenotype within the parietal and temporal cortices. These data provide direct histopathological evidence of CPB-induced neuroinflammation in a large animal model and provide further mechanistic data on how CPB-induced cerebral inflammation might drive postoperative neurocognitive disorders in humans.

The effects of recruitment of renal functional reserve on renal cortical and medullary oxygenation in non-anesthetized sheep.

AIM: Recruitment of renal functional reserve (RFR) with amino acid loading increases renal blood flow and glomerular filtration rate. However, its effects on renal cortical and medullary oxygenation have not been determined. Accordingly, we tested the effects of recruitment of RFR on renal cortical and medullary oxygenation in non-anesthetized sheep. METHODS: Under general anesthesia, we instrumented 10 sheep to enable subsequent continuous measurements of systemic and renal hemodynamics, renal oxygen delivery and consumption, and cortical and medullary tissue oxygen tension (PO2 ). We then measured the effects of recruitment of RFR with an intravenous infusion of 500 ml of a clinically used amino acid solution (10% Synthamin® 17) in the non-anesthetized state. RESULTS: Compared with baseline, Synthamin® 17 infusion significantly increased renal oxygen delivery mean ± SD maximum increase: (from 0.79 ± 0.17 to 1.06 ± 0.16 ml/kg/min, p < 0.001), renal oxygen consumption (from 0.08 ± 0.01 to 0.15 ± 0.02 ml/kg/min, p < 0.001), and glomerular filtration rate (+45.2 ± 2.7%, p < 0.001). Renal cortical tissue PO2 increased by a maximum of 26.4 ± 1.1% (p = 0.001) and medullary tissue PO2 increased by a maximum of 23.9 ± 2.8% (p = 0. 001). CONCLUSIONS: In non-anesthetized healthy sheep, recruitment of RFR improved renal cortical and medullary oxygenation. These observations might have implications for the use of recruitment of RFR for diagnostic and therapeutic purposes.

Effects of changes in inspired oxygen fraction on urinary oxygen tension measurements.

BACKGROUND: Continuous measurement of urinary PO2 (PuO2) is being applied to indirectly monitor renal medullary PO2. However, when applied to critically ill patients with shock, its measurement may be affected by changes in FiO2 and PaO2 and potential associated O2 diffusion between urine and ureteric or bladder tissue. We aimed to investigate PuO2 measurements in septic shock patients with a fiberoptic luminescence optode inserted into the urinary catheter lumen in relation to episodes of FiO2 change. We also evaluated medullary and urinary oxygen tension values in Merino ewes at two different FiO2 levels. RESULTS: In 10 human patients, there were 32 FiO2 decreases and 31 increases in FiO2. Median pre-decrease FiO2 was 0.36 [0.30, 0.39] and median post-decrease FiO2 was 0.30 [0.23, 0.30], p = 0.006. PaO2 levels decreased from 83 mmHg [77, 94] to 72 [62, 80] mmHg, p = 0.009. However, PuO2 was 23.2 mmHg [20.5, 29.0] before and 24.2 mmHg [20.6, 26.3] after the intervention (p = 0.56). The median pre-increase FiO2 was 0.30 [0.21, 0.30] and median post-increase FiO2 was 0.35 [0.30, 0.40], p = 0.008. PaO2 levels increased from 64 mmHg [58, 72 mmHg] to 71 mmHg [70, 100], p = 0.04. However, PuO2 was 25.0 mmHg [IQR: 20.7, 26.8] before and 24.3 mmHg [IQR: 20.7, 26.3] after the intervention (p = 0.65). A mixed linear regression model showed a weak correlation between the variation in PaO2 and the variation in PuO2 values. In 9 Merino ewes, when comparing oxygen tension levels between FiO2 of 0.21 and 0.40, medullary values did not differ (25.1 ± 13.4 mmHg vs. 27.9 ± 15.4 mmHg, respectively, p = 0.6766) and this was similar to urinary oxygen values (27.1 ± 6.17 mmHg vs. 29.7 ± 4.41 mmHg, respectively, p = 0.3192). CONCLUSIONS: Changes in FiO2 and PaO2 within the context of usual care did not affect PuO2. Our findings were supported by experimental data and suggest that PuO2 can be used as biomarker of medullary oxygenation irrespective of FiO2.

Vascular remodeling in sheep implanted with endovascular neural interface.

Objective.The aim of this work was to assess vascular remodeling after the placement of an endovascular neural interface (ENI) in the superior sagittal sinus (SSS) of sheep. We also assessed the efficacy of neural recording using an ENI.Approach.The study used histological analysis to assess the composition of the foreign body response. Micro-CT images were analyzed to assess the profiles of the foreign body response and create a model of a blood vessel. Computational fluid dynamic modeling was performed on a reconstructed blood vessel to evaluate the blood flow within the vessel. Recording of brain activity in sheep was used to evaluate efficacy of neural recordings.Main results.Histological analysis showed accumulated extracellular matrix material in and around the implanted ENI. The extracellular matrix contained numerous macrophages, foreign body giant cells, and new vascular channels lined by endothelium. Image analysis of CT slices demonstrated an uneven narrowing of the SSS lumen proportional to the stent material within the blood vessel. However, the foreign body response did not occlude blood flow. The ENI was able to record epileptiform spiking activity with distinct spike morphologies.Significance. This is the first study to show high-resolution tissue profiles, the histological response to an implanted ENI and blood flow dynamic modeling based on blood vessels implanted with an ENI. The results from this study can be used to guide surgical planning and future ENI designs; stent oversizing parameters to blood vessel diameter should be considered to minimize detrimental vascular remodeling.

Influence of moderate hypothermia on renal and cerebral haemodynamics and oxygenation during experimental cardiopulmonary bypass in sheep.

AIM: Cardiac surgery requiring cardiopulmonary bypass (CPB) can result in renal and cerebral injury. Intraoperative tissue hypoxia could contribute to such organ injury. Hypothermia, however, may alleviate organ hypoxia. Therefore, we tested whether moderate hypothermia (30°C) improves cerebral and renal tissue perfusion and oxygenation during ovine CPB. METHODS: Ten sheep were studied while conscious, under stable anesthesia, and during 3 h of CPB. In a randomized within-animal cross-over design, five sheep commenced CPB at a target body temperature of 30°C (moderate hypothermia). After 90 min, the body temperature was increased to 36°C (standard procedure). The remaining five sheep were randomized to the opposite order of target body temperature. RESULTS: Compared with the standard procedure, moderately hypothermic CPB reduced renal oxygen delivery (-34.8% ± 19.6%, P = 0.003) and renal oxygen consumption (-42.7% ± 35.2%, P = 0.04). Nevertheless, moderately hypothermic CPB did not significantly alter either renal cortical or medullary tissue PO2 . Moderately hypothermic CPB also did not significantly alter cerebral perfusion, cerebral tissue PO2 , or cerebral oxygen saturation compared with the standard procedure. Compared with the anesthetized state, the standard procedure reduced renal medullary PO2 (-21.0 ± 13.8 mmHg, P = 0.014) and cerebral oxygen saturation (65.0% ± 7.0% to 55.4% ± 9.6%, P = 0.022) but did not significantly alter either renal cortical or cerebral PO2 . CONCLUSION: Ovine experimental CPB leads to renal medullary tissue hypoxia. Moderately hypothermic CPB did not improve cerebral or renal tissue oxygenation. In the kidney, this is probably because renal tissue oxygen consumption is matched by reduced renal oxygen delivery.

Preclinical safety study of a fully implantable, sub-scalp ring electrode array for long-term EEG recordings.

OBJECTIVE: Long-term electroencephalogram (EEG) recordings can aid diagnosis and management of various neurological conditions such as epilepsy. In this study we characterize the safety and stability of a clinical grade ring electrode arrays by analyzing EEG recordings, fluoroscopy, and computed tomography (CT) imaging with long-term implantation and histopathological tissue response. APPROACH: Seven animals were chronically implanted with EEG recording array consisting of four electrode contacts. Recordings were made bilaterally using a bipolar longitudinal montage. The array was connected to a fully implantable micro-processor controlled electronic device with two low-noise differential amplifiers and a transmitter-receiver coil. An external wearable was used to power, communicate with the implant via an inductive coil, and store the data. The sub-scalp electrode arrays were made using medical grade silicone and platinum. The electrode arrays were tunneled in the subgaleal cleavage plane between the periosteum and the overlying dermis. These were implanted for 3-7 months before euthanasia and histopathological assessment. EEG and impedance were recorded throughout the study. MAIN RESULTS: Impedance measurements remained low throughout the study for 11 of 12 channels over the recording period ranged from 3 to 5 months. There was also a steady amplitude of slow-wave EEG and chewing artifact (noise). The post-mortem CT and histopathology showed the electrodes remained in the subgaleal plane in 6 of 7 sheep. There was minimal inflammation with a thin fibrotic capsule that ranged from 4 to 101µm. There was a variable fibrosis in the subgaleal plane extending from 210 to 3617µm (S3-S7) due to surgical cleavage. One sheep had an inflammatory reaction due to electrode extrusion. The passive electrode array extraction force was around 1N. SIGNIFICANCE: Results show sub-scalp electrode placement was safe and stable for long term implantation. This is advantageous for diagnosis and management of neurological conditions where long-term, EEG monitoring is required.

Role of perioperative hypotension in postoperative acute kidney injury: a narrative review.

Perioperative hypotension is common and associated with poor outcomes, including acute kidney injury (AKI). The mechanistic link between perioperative hypotension and AKI is at least partly a consequence of the susceptibility of the kidney, and particularly the renal medulla, to ischaemia and hypoxia. Several critical gaps in our knowledge lead to uncertainty about when and how to intervene to prevent AKI attributable to perioperative hypotension. First, although we know that the risk of AKI varies with both the severity and duration of hypotensive episodes, 'safe' levels of arterial pressure have not been identified. Second, there have been few adequately powered clinical trials of interventions to avoid perioperative hypotension. Thus, most evidence surrounding perioperative hypotension is observational rather than based on randomised clinical trials. This means that the link between perioperative hypotension and AKI may represent association (where both phenomena reflect illness severity) rather than causation. Third, there is little information regarding the relative risks and benefits of various clinically available therapies (e.g. vasoconstrictors, i.v. fluids, or both) to treat and prevent perioperative hypotension, particularly with regard to renal medullary perfusion and oxygenation. Fourth, there are currently no validated, clinically feasible methods for real-time clinical monitoring of renal perfusion or oxygenation. Thus, future developments in perioperative kidney-protective strategies must rely on the development of methods to better monitor renal perfusion and oxygenation in the perioperative period, and thereby guide timing, intensity, type, and duration of interventions.

Dynamic responses of renal oxygenation at the onset of cardiopulmonary bypass in sheep and man.

INTRODUCTION: The renal medulla is susceptible to hypoxia during cardiopulmonary bypass (CPB), which may contribute to the development of acute kidney injury. But the speed of onset of renal medullary hypoxia remains unknown. METHODS: We continuously measured renal medullary oxygen tension (MPO2) in 24 sheep, and urinary PO2 (UPO2) as an index of MPO2 in 92 patients, before and after induction of CPB. RESULTS: In laterally recumbent sheep with a right thoracotomy (n = 20), even before CPB commenced MPO2 fell from (mean ± SEM) 52 ± 4 to 41 ±5 mmHg simultaneously with reduced arterial pressure (from 108 ± 5 to 88 ± 5 mmHg). In dorsally recumbent sheep with a medial sternotomy (n = 4), MPO2 was even more severely reduced (to 12 ± 12 mmHg) before CPB. In laterally recumbent sheep in which a crystalloid prime was used (n = 7), after commencing CPB, MPO2 fell abruptly to 24 ±6 mmHg within 20-30 minutes. MPO2 during CPB was not improved by adding donor blood to the prime (n = 13). In patients undergoing cardiac surgery, UPO2 fell by 4 ± 1 mmHg and mean arterial pressure fell by 7 ± 1 mmHg during the 30 minutes before CPB. UPO2 then fell by a further 12 ± 2 mmHg during the first 30 minutes of CPB but remained relatively stable for the remaining 24 minutes of observation. CONCLUSIONS: Renal medullary hypoxia is an early event during CPB. It starts to develop even before CPB, presumably due to a pressure-dependent decrease in renal blood flow. Medullary hypoxia during CPB appears to be promoted by hypotension and is not ameliorated by increasing blood hemoglobin concentration.

Renal Denervation in Combination With Angiotensin Receptor Blockade Prolongs Blood Pressure Trough During Hemorrhage.

Majority of patients with hypertension and chronic kidney disease (CKD) undergoing renal denervation (RDN) are maintained on antihypertensive medication. However, RDN may impair compensatory responses to hypotension induced by blood loss. Therefore, continuation of antihypertensive medications in denervated patients may exacerbate hypotensive episodes. This study examined whether antihypertensive medication compromised hemodynamic responses to blood loss in normotensive (control) sheep and in sheep with hypertensive CKD at 30 months after RDN (control-RDN, CKD-RDN) or sham (control-intact, CKD-intact) procedure. CKD-RDN sheep had lower basal blood pressure (BP; ≈9 mm Hg) and higher basal renal blood flow (≈38%) than CKD-intact. Candesartan lowered BP and increased renal blood flow in all groups. 10% loss of blood volume alone caused a modest fall in BP (≈6-8 mm Hg) in all groups but did not affect the recovery of BP. 10% loss of blood volume in the presence of candesartan prolonged the time at trough BP by 9 minutes and attenuated the fall in renal blood flow in the CKD-RDN group compared with CKD-intact. Candesartan in combination with RDN prolonged trough BP and attenuated renal hemodynamic responses to blood loss. To minimize the risk of hypotension-mediated organ damage, patients with RDN maintained on antihypertensive medications may require closer monitoring when undergoing surgery or experiencing traumatic blood loss.

Renal and Cerebral Hypoxia and Inflammation During Cardiopulmonary Bypass.

Cardiac surgery-associated acute kidney injury and brain injury remain common despite ongoing efforts to improve both the equipment and procedures deployed during cardiopulmonary bypass (CPB). The pathophysiology of injury of the kidney and brain during CPB is not completely understood. Nevertheless, renal (particularly in the medulla) and cerebral hypoxia and inflammation likely play critical roles. Multiple practical factors, including depth and mode of anesthesia, hemodilution, pump flow, and arterial pressure can influence oxygenation of the brain and kidney during CPB. Critically, these factors may have differential effects on these two vital organs. Systemic inflammatory pathways are activated during CPB through activation of the complement system, coagulation pathways, leukocytes, and the release of inflammatory cytokines. Local inflammation in the brain and kidney may be aggravated by ischemia (and thus hypoxia) and reperfusion (and thus oxidative stress) and activation of resident and infiltrating inflammatory cells. Various strategies, including manipulating perfusion conditions and administration of pharmacotherapies, could potentially be deployed to avoid or attenuate hypoxia and inflammation during CPB. Regarding manipulating perfusion conditions, based on experimental and clinical data, increasing standard pump flow and arterial pressure during CPB appears to offer the best hope to avoid hypoxia and injury, at least in the kidney. Pharmacological approaches, including use of anti-inflammatory agents such as dexmedetomidine and erythropoietin, have shown promise in preclinical models but have not been adequately tested in human trials. However, evidence for beneficial effects of corticosteroids on renal and neurological outcomes is lacking. © 2021 American Physiological Society. Compr Physiol 11:1-36, 2021.

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to >10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in >1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.