Association between cytokines, nitric oxide, hemodynamic and microcirculation in a porcine model of sepsis.

Systemic inflammation and hemodynamic or microvascular alterations are a hallmark of sepsis and play a role in organs hypoperfusion and dysfunction. Pimobendan, an inodilator agent, could be an interesting option for inotropic support and microcirculation preservation during shock. The objectives of this study were to evaluate effect of pimobendan on cytokine and nitric oxide (NO) release and investigate whether changes of macro and microcirculation parameters are associated with the release of cytokines and NO in pigs sepsis model. After circulatory failure, induced by intravenous inoculation of live Pseudomonas aeruginosa, eight animals were treated with pimobendan and eight with placebo. Pimobendan did not affect cytokines secretion (TNF-α, IL-6 and IL-10), but decreased time-dependently NO release. Data of macro and microcirculation parameters, NO and TNF- α recorded at the time of circulatory failure (Thypotension) and the time maximum of production cytokines was used for analyses. A positive correlation was observed between TNF-α and cardiac index (r = 0.55, p = 0.03) and a negative with systemic vascular resistance (r = -0.52, p = 0.04). Positive correlations were seen both between IL-10, 30 min after resuscitation (T30min), and systolic arterial pressure (r = 0.57, p = 0.03) and cardiac index (r = 0.67, p = 0.01), and also between IL-6, taken 2 h after resuscitation and systolic arterial pressure (r = 0.53, p = 0.04). Negative correlations were found between IL-10 and lactate, measured resuscitation time (r = -0.58, p = 0.03). Regarding microcirculation parameters, we observed a positive correlation between IL-6 and IL-10 with the microvascular flow index (r = 0.52, p = 0.05; r = 0.84, p = 0.0003) and a negative correlation with the heterogeneity index with TNF-α and IL-10 (r = -0.51, p = 0.05; r = -0.74, p = 0.003) respectively. NO derivatives showed a positive correlation with temperature gradient (r = 0.54, p = 0.04). Pimobendan did not show anti-inflammatory effects in cytokines release. Our results also, suggest changes of macro- and microcirculation are associated mainly with low levels of IL-10 in sepsis.

How I do it: simplified craniotomy for acute subdural hematoma in the elderly.

CONTEXT: Acute subdural hematoma (ASH) is responsible for significant morbidity and mortality in the elderly. As military neurosurgeons, we perform a simplified technique using a linear skin incision and a small craniotomy bone flap in order to ease perioperative tolerance. METHODS: The patient lies supine, a pad under the shoulder ipsilateral to the ASH, the head completely rotated on the other side and placed on a circular pad, without head clamp. The linear frontotemporal skin incision should be twice the size of the bone flap's diameter, allowing to access the whole subdural space. Care is taken to obtain complete decompression of the temporal fossa in order to alleviate uncal herniation. A subdural drain can be placed, and the subdural space is filled with warm saline solution in order to create a closed drainage system. CONCLUSION: The patient is allowed to sit at postoperative day 1 and to walk at postoperative day 2. Simplified craniotomy for ASH allows to reduce operative time and provides faster functional recovery.

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) might increase the risk of anastomotic leakage compared to HIPEC: an experimental study.

BACKGROUND: Pressurized intraperitoneal aerosol chemotherapy (PIPAC) and hyperthermic intraperitoneal chemotherapy (HIPEC) are technics proposed to treat patients with peritoneal carcinomatosis, in different settings. There is some concern about an over-risk of anastomotic leakage (AL) with PIPAC jeopardizing a combination with cytoreductive surgery. This study used a healthy swine model to compare the postoperative AL rate between PIPAC and HIPEC with digestive resection and to analyze macrocirculation and microcirculation parameters. METHODS: Segmental colonic resection with a handsewn anastomosis was performed on 16 healthy pigs; 8 pigs had a PIPAC procedure with 7.5 mg/m2 cisplatin (PIPAC group), and 8 pigs had a closed HIPEC procedure with 70 mg/m2 cisplatin and 42 °C as the target intraperitoneal temperature (HIPEC group). Pigs were kept alive for 8 days, then sacrificed and autopsied to look for AL, which was defined as local abscess or digestive fluid leakage when pressure was applied to the anastomosis. Food intake, weight, and core temperature were monitored postoperatively. Macrocirculation (heart rate, systolic blood pressure) and microcirculation parameters (percentage of perfused vessels, perfused vessels density, DeBacker score) were evaluated intraoperatively at five timepoints. Results were compared between pigs with AL and those without. RESULTS: The HIPEC group had no AL, but 3 of 8 pigs (37.5%) had AL in the PIPAC group (p = 0.20). Heart rate and core temperature showed perioperative increases in the HIPEC group. Intraoperatively, heart rate was higher in the HIPEC group at the two last timepoints (123 vs. 93 bpm, p = 0.031, and 110 vs. 85 bpm, p = 0.010, at timepoints 3 and 4, respectively). Other macrocirculatory and microcirculatory parameters showed no significant differences. CONCLUSION: In this healthy swine model, PIPAC might have increased AL incidence compared to HIPEC. This potential over-risk did not seem to be related to changes in the microcirculation. PIPAC should probably not be used with digestive resection and should be avoided in cases of perioperative serosal injury.

PIPAC versus HIPEC: cisplatin spatial distribution and diffusion in a swine model.

Purpose: Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a novel approach for delivering intraperitoneal chemotherapy and offers perspective in the treatment of peritoneal carcinomatosis. Concept is based on a 12 mmHg capnoperitoneum loaded with drug changed in microdoplets. It was postulated to guarantee a more homogeneous drug distribution and tissular uptake than hyperthermic intraperitoneal chemotherapy (HIPEC). The aim of this study was to compare cisplatin peritoneal distribution and pharmacokinetic between HIPEC and PIPAC procedures in a healthy swine model.Methods: Two groups of eight pigs underwent either HIPEC with cisplatin (70 mg/m2) at 43 °C for 60 min, or PIPAC with cisplatin (7.5 mg/m2) for 30 min. Postoperatively, peritoneal areas were biopsied allowing peritoneal cavity cartography. Tissular and plasmatic cisplatin concentrations were analyzed.Results: Cisplatin distribution was heterogeneous in both the groups with higher concentrations obtained closed to the delivery sites. Median total platinum peritoneal concentration by pig was higher in the HIPEC group than in the PIPAC group (18.0 µg/g versus 4.3 µg/g, p < .001) but the yield was 2.2 times better with PIPAC. Platinum concentrations were higher in the HIPEC group in all stations. At each time-point, cisplatin plasmatic concentrations were higher in the HIPEC group (p < .001) but beneath the toxicity threshold.Conclusions: With doses used in clinical practice, HIPEC guaranteed a higher cisplatin peritoneal uptake than PIPAC in this swine model. Spatial drug distribution was heterogeneous with both technics, with hotspots closed to the drug delivery sites. Nevertheless, considering the dose ratio, IP drug uptake yield was better with PIPAC.

Gut and sublingual microvascular effect of esmolol during septic shock in a porcine model.

INTRODUCTION: Esmolol may efficiently reduce heart rate (HR) and decrease mortality during septic shock. An improvement of microcirculation dissociated from its macrocirculatory effect may a role. The present study investigated the effect of esmolol on gut and sublingual microcirculation in a resuscitated piglet model of septic shock. METHODS: Fourteen piglets, anesthetized and mechanically ventilated, received a suspension of live Pseudomonas aeruginosa. They were randomly assigned to two groups: the esmolol (E) group received an infusion of esmolol, started at 7.5 µg⋅kg(-1)⋅min(-1), and progressively increased to achieve a HR below 90 beats⋅min(-1). The control (C) group received an infusion of Ringer's lactate solution. HR, mean arterial pressure (MAP), cardiac index (CI), stroke index (SI), systemic vascular resistance (SVR), arterio-venous blood gas and lactate were recorded. Oxygen consumption (VO2), delivery (DO2) and peripheral extraction (O2ER) were computed. Following an ileostomy, a laser Doppler probe was applied on ileal mucosa to monitor gut microcirculatory laser Doppler flow (GMLDF). Videomicroscopy was also used on ileal mucosa and sublingual areas to evaluate mean flow index (MFI), heterogeneity, ratio of perfused villi and proportion of perfused vessels. Resuscitation maneuvers were performed following a defined algorithm. RESULTS: Bacterial infusion induced a significant alteration of the gut microcirculation with an increase in HR. Esmolol produced a significant time/group effect with a decrease in HR (P <0.004) and an increase in SVR (P <0.004). Time/group effect was not significant for CI and MAP, but there was a clear trend toward a decrease in CI and MAP in the E group. Time/group effect was not significant for SI, O2ER, DO2, VO2, GMLDF and lactate. A significant time/group effect of ileal microcirculation was found with a lower ileal villi perfusion (P <0.025) in the C group, and a trend toward a better MFI in the E group. No difference between both groups was found regarding microcirculatory parameters in the sublingual area. CONCLUSIONS: Esmolol provided a maintenance of microcirculation during sepsis despite its negative effects on macrocirculation. Some parameters even showed a trend toward an improvement of the microcirculation in the gut area in the esmolol group.

Microbial risk assessment of Nocardia cyriacigeorgica in polluted environments, case of urban rainfall water.

Urban Infiltration Basins (UIBs) are used to manage urban runoff transfers and feed aquifers. These UIBs can accumulate urban pollutants and favor the growth of potentially pathogenic biological agents as Nocardia. OBJECTIVES: To assess the spatio-temporal dynamics of pathogenic Nocardia in UIBs and to stablish phylogenetic relationships between clinical and UIB N. cyriacigeorgica strains. To assess pathogenicity associated with environmental N. cyriacigeorgica using an animal model, and to identify genetic elements that may be associated to its virulence. METHODS: A well-characterized UIB in terms of chemical pollutants from Lyon area was used in this study during a whole year. Cultural and Next-Generation-Sequencing methods were used for Nocardia detection and typing. Clinical and environmental isolates phylogenetic relationships and virulences were compared with Multilocus-Sequence-Analysis study together with a murine model. RESULTS: In autumn, N. cyriacigeorgica and N. nova were the pathogenic most prevalent species in the UIB. The complex N. abscessus/asiatica was also detected together with some other non-pathogenic species. The presence of pathogenic Nocardia was positively correlated to metallic trace elements. Up to 1.0 × 103 CFU/g sediment of N. cyriacigeorgica and 6 OTUs splited in two different phylogroups were retrieved and were close to clinical strains. The EML446 tested UIB isolate showed significant infectivity in mice with pulmonary damages similar to clinical clone (GUH-2). CONCLUSION: Hsp65 marker-based metabarcoding approach allowed detecting N. cyriacigeogica as the most abundant Nocardia pathogenic species in a UIB. Metal trace elements-polluted environments can be reservoirs of pathogenic Nocardia which may have a similar virulence to clinical strains.

Feasibility study and direct extraction of endogenous free metallic cations combining hemodialysis and chelating polymer.

In this article, we report the conception and the use of dialysis-based medical device for the extraction of metals. The medical device is obtained by addition in the dialysate of a functionalized chitosan that can chelate endogenous metals like iron or copper. This water-soluble functionalized chitosan is obtained after controlled reacetylation and grafting of DOTAGA. Due to the high mass of chitosan, the polymer cannot cross through the membrane and the metals are trapped in the dialysate during hemodialysis. Copper extraction has been evaluated in vitro using an hemodialysis protocol. Feasibility study has been performed on healthy sheep showing no acute toxicity througout the entire dialysis procedure and first insights of metallic extraction even on healthy animals.

Performance of the Parasympathetic Tone Activity (PTA) index to assess the intraoperative nociception using different premedication drugs in anaesthetised dogs.

The dynamic variations of the Parasympathetic Tone Activity (PTA) index were evaluated to assess nociception in dogs undergoing anaesthetic protocols with different premedication drugs. Sixty-six dogs, divided into three groups of 22 dogs each, were given different premedication drugs "morphine" (0.2 mg/kg), "morphine + medetomidine (5 µg/kg)", "morphine + acepromazine (0.03 mg/kg)", then similarly induced and maintained under general anaesthesia. The PTA, HR (heart rate) and MAP (mean arterial pressure) were assessed at S (steady-state), Cut (cutaneous incision), PTAE (PTA Event, assessed 1 min before a significant PTA decrease), HDR (Haemodynamic reaction, assessed 5 min before an increase of ≥ 20% in HR and/or MAP). For each group, the dynamic variation of PTA (ΔPTA) was calculated and a Receiver Operating Characteristics (ROC) curve was performed to detect if any of the premedication drugs may alter the performance of PTA index to predict intraoperative haemodynamic reactions. In all groups, a haemodynamic reaction was detected after Cut, PTAE and HDR and was preceded with a significant decrease of PTA, except for "morphine + medetomidine" group which showed a significant drop of PTA only at PTAE and HDR. The ΔPTA showed a fair performance in all groups (a mean [95% CI] AUC of 0.73 [0.62-0.82], 0.70 [0.59-0.79] and 0.71 [0.59-0.80] respectively in morphine, morphine + ACP and morphine + medetomidine). Although ΔPTA was rather altered by the premedication drug, it was able to predict, to a certain extent, haemodynamic reactions in all groups.

Comprehensive assessment of coronary pulse wave velocity in anesthetized pigs.

BACKGROUND: Coronary stiffness represents a new paradigm for interventional cardiology and can be assessed by coronary pulse wave velocity (CoPWV). Assessing CoPWV is complex because of the coexistence of backward and forward waves. OBJECTIVES: Evaluate the feasibility, repeatability, and capacity of methods assessing CoPWV to detect predictable velocity changes. METHODS: CoPWV was measured from distal and proximal pressure guidewires in the left anterior descending artery of 10 pigs under general anesthesia. Four methods were studied: the tangent intersection method applied to the forward (FW) and backward (BK) waves, as well as the dicrotic notch (DIC) and template matching (TM) methods. All were evaluated at baseline, during various arterial pressure and heart rate conditions, during simulated flow limitation (balloon inflation), and after increasing coronary stiffness (stent insertion). RESULTS: All the methods were significantly different between them (p ≤ .05) showing a systematic trend toward higher CoPWV when compared to the FW method (.05 < p<.10). Results were found to be significantly correlated only between the BK and FW methods and between the DIC and TM methods (p ≤ .05). CoPWV increased with arterial pressure increase, this increase being significant for the DIC and TM methods and partly for the FW method (p ≤ .05). Conversely, heart rate had no systematic impact on CoPWV. The lowest variability was found for the DIC and TM methods (p ≤ .05). Only the BK and TM methods remained applicable during flow limitation; stent increased CoPWV when measured by the BK method only (p ≤ .05). CONCLUSION: Although CoPWV can be measured by various methods, the BK and TM methods seem the most appropriate for clinical studies.

ß-Glucan as Trained Immunity-Based Adjuvants for Rabies Vaccines in Dogs.

The mechanisms of trained immunity have been extensively described in vitro and the beneficial effects are starting to be deciphered in in vivo settings. Prototypical compounds inducing trained immunity, such as ß-glucans, act through epigenetic reprogramming and metabolic changes of innate immune cells. The recent advances in this field have opened new areas for the development of Trained immunity-based adjuvants (TIbAs). In this study, we assessed in dogs the potential immune training effects of ß-glucans as well as their capacity to enhance the adaptive immune response of an inactivated rabies vaccine (Rabisin®). Injection of ß-glucan from Euglena gracilis was performed 1 month before vaccination with Rabisin® supplemented or not with the same ß-glucan used as adjuvant. Trained innate immunity parameters were assessed during the first month of the trial. The second phase of the study was focused on the ability of ß-glucan to enhance adaptive immune responses measured by multiple immunological parameters. B and T-cell specific responses were monitored to evaluate the immunogenicity of the rabies vaccine adjuvanted with ß-glucan or not. Our preliminary results support that adjuvantation of Rabisin® vaccine with ß-glucan elicit a higher B-lymphocyte immune response, the prevailing factor of protection against rabies. ß-glucan also tend to stimulate the T cell response as shown by the cytokine secretion profile of PBMCs re-stimulated ex vivo. Our data are providing new insights on the impact of trained immunity on the adaptive immune response to vaccines in dogs. The administration of ß-glucan, 1 month before or simultaneously to Rabisin® vaccination give promising results for the generation of new TIbA candidates and their potential to provide increased immunogenicity of specific vaccines.

ß-Glucan-Induced Trained Immunity in Dogs.

Several observations in the world of comparative immunology in plants, insects, fish and eventually mammals lead to the discovery of trained immunity in the early 2010's. The first demonstrations provided evidence that innate immune cells were capable of developing memory after a first encounter with some pathogens. Trained immunity in mammals was initially described in monocytes with the Bacille Calmette-Guerin vaccine (BCG) or prototypical agonists like ß-glucans. This phenomenon relies on epigenetic and metabolic modifications leading to an enhanced secretion of inflammatory cytokines when the host encounters homologous or heterologous pathogens. The objective of our research was to investigate the trained immunity, well-described in mouse and human, in other species of veterinary importance. For this purpose, we adapted an in vitro model of trained innate immunity in dogs. Blood enriched monocytes were stimulated with ß-glucans and we confirmed that it induced an increased production of pro-inflammatory and anti-microbial compounds in response to bacterial stimuli. These results constitute the first demonstration of trained immunity in dogs and confirm its signatures in other mammalian species, with an implication of cellular mechanisms similar to those described in mice and humans regarding cellular epigenetics and metabolic regulations.

The effects of 10% hypertonic saline, 0.9% saline and hydroxy ethyl starch infusions on hydro-electrolyte status and adrenal function in healthy conscious dogs.

The purpose of this study was to investigate the influence of different saline and colloid solutions on adrenal steroid secretion in dogs. Six healthy male Beagles underwent three infusion cycles: 10 min infusion of 30 ml/kg of NaCl 0.9%, 5 ml/kg of hydroxy ethyl starch, or 5 ml/kg of NaCl 10%. Plasma osmolality, hematocrit, total solids, cortisol and aldosterone levels were measured at 0, 5, 15, 30, 60, 120, 180 and 240 min after beginning infusion. Plasma ACTH levels were measured at 0, 15 and 240 min. An identical timing of sampling was applied during a control session omitting the fluid infusion. Osmolality, sodium, chloride and cortisol levels were found to be significantly higher with hypertonic saline solute compared to control. All fluid infusions lead to lowered plasma potassium, hematocrit, total solids and aldosterone values. ACTH concentrations did not show significant changes with any of the infusion cycles. The increase in cortisol levels suggests that hypertonic saline infusion could be interesting in critical care resuscitation, particularly in patients who are suffering from relative adrenal insufficiency.

Influence of aging on adrenal responsiveness in a population of eleven healthy beagles.

The present study aimed at investigating the effects of aging on the adrenal cortex response of cortisol and aldosterone in dogs. A population of healthy adult Beagles was evaluated twice at a five-year interval. At each evaluation, plasma basal cortisol and aldosterone, cortisol and aldosterone following ACTH-stimulation, sodium, and potassium concentrations and arterial blood pressure were measured. We observed significantly (p<0.05) greater sodium, urea and creatinine concentrations with aging. Nevertheless urea and creatinine remained within our laboratory reference ranges. This study showed a highly significant age-related elevation of basal cortisol (p<0.01). Inversely, both aldosterone following ACTH-stimulation levels and difference between aldosterone following ACTH-stimulation and basal aldosterone values plummeted significantly (p<0.01) with aging. In conclusion, the evaluation of the adrenal cortex function in dogs should take in consideration the age of the individuals.

Mice Survival and Plasmatic Cytokine Secretion in a "Two Hit" Model of Sepsis Depend on Intratracheal Pseudomonas Aeruginosa Bacterial Load.

Sepsis is characterized by pro- and anti-inflammatory responses following infection. While inflammation is responsible for widespread organ damage, anti-inflammatory mediators lead to immunoparalysis increasing susceptibility to secondary infections (nosocomial pneumonia). We aimed to investigate the impact of bacterial load on survival and cytokine release in a two-hit murine (C57BL/6J) model of CLP followed by P. aeruginosa pneumonia. Plasmatic TNFα, IL-6, IL-10, sTNFr I and II were quantified until 13 days. At D5, splenocytes were processed for immunological assays or mice were intratracheally instilled with Pseudomonas aeruginosa (5.106, 2.107 and 108 CFU) to evaluate survival and cytokines production. TNFα, sTNFrs, IL-6 and IL-10 increased 2h post CLP. TNFα and sTNFrs declined respectively one and two days later. In CLP mice, IL-6 and IL-10 remained high for the whole experiment, as compared to Sham. At D5, for CLP mice, whereas total T cells population (CD3+) decreased, Treg fraction (CD4+/CD25+) increased. In parallel, T cells proliferation and LPS-stimulated splenocytes ability to release TNFα decreased. At D13, survival was 100% after 5.106 CFU, 50% for CLP mice after 2.107 CFU and 0% for CLP and Sham after 108 CFU. After instillation, IL-10 and IL-6 increased and appeared to be dose and time dependent. Pseudomonas was detected in all CLP and Sham's lungs; in spleen and liver only in CLP at 2.107 CFU, and in CLP and Sham at 108 CFU. We demonstrated that post-CLP immunosuppression followed by Pseudomonas aeruginosa lung instillation increases mortality reactivates cytokines secretion and is associated with systemic dissemination in septic mice depending on bacterial load.

In vivo evaluation of immediately loaded stainless steel and titanium orthodontic screws in a growing bone.

The present work intends to evaluate the use of immediate loaded orthodontic screws in a growing model, and to study the specific bone response. Thirty-two screws (half of stainless steel and half of titanium) were inserted in the alveolar bone of 8 growing pigs. The devices were immediately loaded with a 100 g orthodontic force. Two loading periods were assessed: 4 and 12 weeks. Both systems of screws were clinically assessed. Histological observations and histomorphometric analysis evaluated the percent of "bone-to-implant contact" and static and dynamic bone parameters in the vicinity of the devices (test zone) and in a bone area located 1.5 cm posterior to the devices (control zone). Both systems exhibit similar responses for the survival rate; 87.5% and 81.3% for stainless steel and titanium respectively (p = 0.64; 4-week period), and 62.5% and 50.0% for stainless steel and titanium respectively (p = 0.09; 12-week period). No significant differences between the devices were found regarding the percent of "bone-to-implant contact" (p = 0.1) or the static and dynamic bone parameters. However, the 5% threshold of "bone-to-implant contact" was obtained after 4 weeks with the stainless steel devices, leading to increased survival rate values. Bone in the vicinity of the miniscrew implants showed evidence of a significant increase in bone trabecular thickness when compared to bone in the control zone (p = 0.05). In our study, it is likely that increased trabecular thickness is a way for low density bone to respond to the stress induced by loading.

Renal effect of meloxicam versus ketoprofen in anaesthetized pseudo-normovolaemic piglets.

Due to renal COX-2 constitutive expression, meloxicam is presumably deleterious for kidney function in critical situations. The present study investigates the influence of intravenous meloxicam on renal parameters and compares it with a nonselective COX inhibitor, ketoprofen. Piglets (n = 6 in each group) were treated with ketoprofen (2 mg.kg(-1)), meloxicam (0.2 mg.kg(-1)), or saline at the beginning of anaesthesia. Under intravenous anaesthesia, pigs were instrumented for cardiovascular, respiratory, and renal function evaluation, including urinary flow (UF), glomerular filtration rate (GFR), and renal blood flow (RBF). After baseline data collection (U0), data collection consisted of six 20-minute periods (U1 to U6). In all groups, the time course of cardiovascular and respiratory parameters remained within normal ranges. A small decrease in cardiac output and an increase in mean systemic arterial blood pressure (p = 0.002) occurred in all groups. In the placebo group, a similar decrease was observed for RBF and cardiac output, with troughs of -10.1% +/- 6.8%, and -12.9% +/- 3.2%, respectively. GFR and UF, however, remained stable over time in this group. Ketoprofen significantly decreased UF (-29.3% +/- 5.5% max at U3), with similar decreases in GFR and RBF. Meloxicam induced a transient (at U2) and small decrease in UF with no difference, at any time point, with the placebo group. The renal effects of meloxicam appear minimal and transient in anaesthetized piglets. This study demonstrates the safety of meloxicam for preemptive surgical analgesia under conditions of normovolaemia. Fluid therapy appears recommended to prevent any renal dysfunction.

Effects of intravenous perfusion of glucagon on renal blood flow in conscious sheep.

The effect of glucagon on renal haemodynamics in sheep is controversial. In this study we have examined the effects of perfused glucagon on renal blood flow (RBF) in six conscious sheep bilaterally implanted with transit-time ultrasonic flow metering probes around the renal arteries. Glucagon was perfused intravenously over 90 min at doses of 3.12, 6.25, 12.5, 25, 50 and 100 ng kg(-1) min(-1). Mean RBF was calculated over 10 min periods. Blood samples were taken to monitor the time course of the changes in glycaemia and glucagonaemia. The perfusions of glucagon induced rapid and progressive dose-dependent increases in RBF (9-19.2 %, P < 0.05) and glycaemia (29-155 %, P < 0.05) for doses of 25-100 ng kg(-1) min(-1). High positive correlations were found between the increases in RBF and glucagonaemia (R2 = 0.95) and between the increases in RBF and glycaemia (R2 = 0.96). At the lowest doses of glucagon (3.12-12.5 ng kg(-1) min(-1)), the increase in RBF was highly significant; however, the rise in glucose level was not. At the highest doses (25-100 ng kg(-1) min(-1)) the time course of the changes in RBF was parallel to that of glucagonaemia throughout the perfusion time. However, between minutes 45 and 90 of the glucagon perfusion, the increase in RBF was the inverse of the change in glycaemia, which decreased. One hour after the end of the 50 and 100 ng kg(-1) min(-1) perfusions, the glucose levels were still significantly higher than the baseline, while the RBF values were not. These results are consistent with the idea that the enhanced RBF cannot be attributed to a rise in blood glucose level. They also show that the haemodynamic response to glucagon perfusion was more sensitive than the metabolic response. It is concluded that the intravenous perfusion of physiological doses of glucagon induced a highly sensitive dose-dependent increase in RBF in sheep.

Optimal anticoagulation strategy in haemodialysis with heparin-coated polyacrylonitrile membrane.

BACKGROUND: Binding of polycationic unfractionated heparin onto the modified AN69 polyacrylonitrile membrane, whose surface electronegativity has been neutralized by layering polyethyleneimine (AN69ST), produces stable coating. We investigated whether the heparin-coated membrane was suitable for regular haemodialysis with low heparin doses. METHODS: Sheep were instrumented for extracorporeal circulation perfusing a dialyser equipped with either the AN69ST or the original AN69 membrane. Dialysis sessions were performed after priming the dialyser with heparinized saline. The session was conducted without systemic administration of heparin. In chronic haemodialysis patients, the AN69ST membrane was tested for safety, clotting and thrombin generation according to protocols of 4-h haemodialysis sessions with tapered heparin doses. The goal was to define optimal heparin requirements with the heparin-coated membrane in the setting of continuous or intermittent administration of heparin. Both unfractionated and low molecular weight heparin (LMWH) (enoxaparin) were tested. RESULTS: In sheep, systemic heparin-free haemodialysis was conducted for 6 h without clotting using the heparin-coated dialyser. In the same conditions, massive clotting was observed within 90 min of dialysis with the native AN69 membrane. In man, through kinetic measurements of activated partial thromboplastin time (APTT), heparin anti-Xa concentration and thrombin-anti-thrombin complexes levels (TAT), significant dialyser clotting was avoided when APTT and anti-Xa concentration at 180 min of dialysis, were maintained at >40 s and >0.2 IU/ml, respectively. With the AN69ST heparin-coated membrane, thrombin generation was reduced then suppressed, as compared with the original AN69, primed in the same conditions. Safety of haemodialysis conducted with the AN69ST heparin-coated membrane and low doses of unfractionated heparin (50% reduction of the reference dose) was validated by a survey of 2590 sessions in 32 patients. Doses of LMWH were also safely reduced by 50%. In addition, haemodialysis without systemic administration of heparin was possible with minor risk of clotting. CONCLUSION: During the rinsing phase, the ionic interactions between the new AN69ST polyacrylonitrile membrane and unfractionated heparin induce stable heparin coating. This allows a significant reduction of systemic anticoagulant requirements without increasing the risk of clotting, both in the experimental setting and in the chronic haemodialysis patients. Further studies are required to assess this advantage in patients with acute renal failure and at risk of bleeding and to reduce the metabolic consequences of long-term treatment with heparin.