BDNF and cAMP are neuroprotective in a porcine model of traumatic optic neuropathy.

Traumatic optic neuropathy (TON) is a devastating condition that can occur after blunt or penetrating trauma to the head, leading to visual impairment or blindness. Despite these debilitating effects, no clinically available therapeutic targets neuroprotection or promotes axon regeneration in this or any optic neuropathy. Limited data in large-animal models are a major obstacle to advancing treatments toward clinical therapeutics. To address this issue, we refined a surgical model of TON in Yucatan minipigs. First, we validated the model by demonstrating visual impairment by flash visual-evoked potential and retinal ganglion cell degeneration and death. Next, we developed and optimized a delivery method and nontoxic dosing of intravitreal brain-derived neurotrophic factor (BDNF) and cAMP. Finally, we showed that intravitreal injection of BDNF and cAMP rescued visual function and protected against retinal ganglion cell death and optic nerve axon degeneration. Together these data in a preclinical large-animal model advance our understanding of and ability to model TON and further identify and develop candidate clinical therapeutics.

FoxO3 normalizes Smad3-induced arterial smooth muscle cell growth.

Transition of arterial smooth muscle (ASM) from a quiescent, contractile state to a growth-promoting state is a hallmark of cardiovascular disease (CVD), a leading cause of death and disability in the United States and worldwide. While many individual signals have been identified as important mechanisms in this phenotypic conversion, the combined impact of the transcription factors Smad3 and FoxO3 in ASM growth is not known. The purpose of this study was to determine that a coordinated, phosphorylation-specific relationship exists between Smad3 and FoxO3 in the control of ASM cell growth. Using a rat in vivo arterial injury model and rat primary ASM cell lysates and fractions, validated low and high serum in vitro models of respective quiescent and growth states, and adenoviral (Ad-) gene delivery for overexpression (OE) of individual and combined Smad3 and/or FoxO3, we hypothesized that FoxO3 can moderate Smad3-induced ASM cell growth. Key findings revealed unique cellular distribution of Smad3 and FoxO3 under growth conditions, with induction of both nuclear and cytosolic Smad3 yet primarily cytosolic FoxO3; Ad-Smad3 OE leading to cytosolic and nuclear expression of phosphorylated and total Smad3, with almost complete reversal of each with Ad-FoxO3 co-infection in quiescent and growth conditions; Ad-FoxO3 OE leading to enhanced cytosolic expression of phosphorylated and total FoxO3, both reduced with Ad-Smad3 co-infection in quiescent and growth conditions; Ad-FoxO3 inducing expression and activity of the ubiquitin ligase MuRF-1, which was reversed with concomitant Ad-Smad3 OE; and combined Smad3/FoxO3 OE reversing both the pro-growth impact of singular Smad3 and the cytostatic impact of singular FoxO3. A primary takeaway from these observations is the capacity of FoxO3 to reverse growth-promoting effects of Smad3 in ASM cells. Additional findings lend support for reciprocal antagonism of Smad3 on FoxO3-induced cytostasis, and these effects are dependent upon discrete phosphorylation states and cellular localization and involve MuRF-1 in the control of ASM cell growth. Lastly, results showing capacity of FoxO3 to normalize Smad3-induced ASM cell growth largely support our hypothesis, and overall findings provide evidence for utility of Smad3 and/or FoxO3 as potential therapeutic targets against abnormal ASM growth in the context of CVD.

Human platelet lysate delivered via an ocular wound chamber for the treatment of corneal epithelial injuries.

Current strategies to address corneal surface defects are insufficient to successfully resolve damage caused by injury and/or disease. To address this issue, we have developed an ocular wound chamber (OWC) that creates a fluid-filled environment by encompassing damaged ocular and periocular tissues allowing for the continuous delivery of therapeutics. This study tested human platelet lysate (hPL) as a treatment for corneal epithelial defects when used with the OWC. Corneal epithelial injuries were created in anesthetized guinea pigs by debridement of the central cornea. An OWC was placed over the injured eye and animals randomly grouped followed by injection of either 20% hPL, 100% hPL, or vehicle (balanced salt solution, BSS) into the chamber. Eyes were assessed at 0, 24, 48, and 72 h using intraocular pressure (IOP), optical coherence tomography (OCT), and fluorescein imaging. Whole globes were histologically processed, and hematoxylin and eosin (H&E) stained. No differences in IOP were recorded as a result of corneal wounding, chamber placement, and/or therapeutic application. OCT images demonstrated increased corneal swelling at 48 h and 72 h in the vehicle group compared to 20% hPL. Fluorescein staining showed increased corneal re-epithelialization in the 20% and 100% hPL groups at 48 h compared to vehicle only. H&E staining revealed increased stromal cellular infiltrate in the BSS group. This study demonstrates the delivery of hPL via the OWC improves corneal re-epithelialization and supports the expanded usage of the chamber in combination with hPL to manage a variety of corneal surface injuries, diseases and/or periocular conditions.

Traumatic Optic Nerve Injury Elevates Plasma Biomarkers of Traumatic Brain Injury in a Porcine Model.

A diagnosis of traumatic brain injury (TBI) is typically based on patient medical history, a clinical examination, and imaging tests. Elevated plasma levels of glial fibrillary acidic protein (GFAP), ubiquitin c-terminal hydrolase L1 (UCH-L1), and neurofilament light chain (NFL) have been observed in numerous studies of TBI patients. It is reasonable to view traumatic optic neuropathy (TON) as a focal form of TBI. The purpose of this study was to assess if circulating GFAP, UCH-L1, and NFL are also elevated in a porcine model of TON. Serum levels of GFAP, UCH-L1, and NFL were measured immediately before optic nerve crush and 1 h post-injury in 10 Yucatan minipigs. Severity of optic nerve crush was confirmed by visual inspection of the optic nerve at time of injury, loss of visual function as measured by flash visual evoked potential (fVEP) at 7 and 14 days, and histological analysis of axonal transport of cholera toxin-ß (CT-ß) within the optic nerve. Post-crush concentrations of GFAP, UCH-L1, and NFL were all significantly elevated compared with pre-crush concentrations (p < 0.01, p = 0.01, and p < 0.01, respectively). The largest increase was observed for GFAP with the post-injury median concentration increasing nearly sevenfold. The use of these TBI biomarkers for diagnosing and managing TON may be helpful for non-ophthalmologists in particular in diagnosing this condition. In addition, the potential utility of these biomarkers for diagnosing other optic nerve and/or retinal pathologies should be evaluated.

Healthcare costs and outcomes for patients undergoing tracheostomy in an Australian tertiary level referral hospital.

OBJECTIVE: Patients undergoing tracheostomy represent a unique cohort, as often they have prolonged hospital stay, require multi-disciplinary, resource-intensive care, and may have poor outcomes. Currently, there is a lack of data around overall healthcare cost for these patients and their outcomes in terms of morbidity and mortality. The objective of the study was to estimate healthcare costs and outcomes associated in tracheostomy patients at a tertiary level hospital in South Australia. DESIGN: Retrospective review of prospectively collected data in patients who underwent tracheostomy between July 2009 and May 2015. METHODS: Overall healthcare-associated costs, length of mechanical ventilation, length of intensive care unit stay, and mortality rates were assessed. RESULTS: A total of 454 patients with tracheostomies were examined. Majority of the tracheostomies (n = 386 (85%)) were performed in intensive care unit patients, predominantly using bedside percutaneous approach (85%). The median length of hospital stay was 44 (29-63) days and the in-hospital mortality rate was 20%. Overall total cost of managing a patient with tracheostomy was median $192,184 (inter-quartile range $122560-$295553); mean 225,200 (range $5942-$1046675) Australian dollars. There were no statistically significant differences in any of the measured outcomes, including costs, between patients who underwent percutaneous versus surgical tracheostomy and patients who underwent early versus late tracheostomy in their intensive care unit stay. Factors that predicted (adjusted R 2 = 0.53) the cost per patient were intensive care unit length of stay and hospital length of stay. CONCLUSION: Hospitalised patients undergoing tracheostomy experience high morbidity and mortality and typically experience highly resource-intensive and costly healthcare.

Use of an ocular wound chamber for the prevention of exposure keratopathy in a guinea pig model.

Current therapies available to treat and heal ocular surface injuries and periocular burns are frequently inadequate, costly, and labor intensive. To address these limitations, we have employed a flexible, semitransparent ocular wound chamber (OWC) to provide protection as well as a watertight seal to allow for the constant delivery of therapeutics to the ocular surface and surrounding periocular tissue. This study demonstrates the safety and utilization of the OWC on uninjured eyes and in our exposure keratopathy model. For initial safety studies (N = 3 per group), the eyelids remained intact and the eye uninjured. A blepharotomy (N = 6 per group) was performed to remove the upper and lower eyelids surrounding the left (OS) eye to create our exposure keratopathy model. Right (OD) eyes served as uninjured controls in all studies. Following OWC placement, 0.5 mL HPMC gel or balanced saline solution (BSS) was injected into the chamber. Animals were monitored daily and fully assessed via white light, fluorescein, and OCT imaging at least through 72 hours post OWC placement. In studies that included a blepharotomy, skin samples were analyzed by multiplex cytokine analysis. Results of safety experiments revealed no significant differences between treatment groups in corneal thickness, fluorescein staining, OCT imaging, or histological eye or skin sections when compared to control eyes. In our exposure keratopathy model, OWC treated eyes showed significantly less fluorescein uptake and also were found to have significantly lower levels of cytokines IL-13 and IL-5 in skin samples. These results demonstrate for the first time that treatment using the OWC device is not only safe, but significantly protects against blepharotomy-induced exposure keratopathy. As a whole, this study advances our overall efforts to develop a feasible solution to treat ocular surface injuries, infections, and periocular burns.

The utilization of an ocular wound chamber on corneal epithelial wounds.

PURPOSE: Currently available ocular moisture chambers are not adequate to manage the treatment of periocular burns, corneal injuries, and infection. The purpose of these studies was to demonstrate that a flexible, semi-transparent ocular wound chamber device adapted from technology currently used on dermal wounds is safe for use on corneal epithelial injuries. MATERIALS AND METHODS: A depilatory cream (Nair™, 30 seconds) was utilized to remove the excess hair surrounding the left eyes of anesthetized Institute Armand Frappier (IAF) hairless, female guinea pigs (Crl:HA-Hrhr). A 4 mm corneal epithelium defect was created using a corneal rust ring remover (Algerbrush®II). Epithelial defects were either left untreated or the eyes were fitted with an ocular wound chamber and 0.5 mL of hydroxypropyl methylcellulose (HPMC) gel (GenTeal®) or HPMC liquid (GenTeal®) was injected into each chamber (N=5 per group). At 0, 24, 48, and 72 hours fluorescein and optical coherence tomography imaging was collected and the intraocular pressure (IOP) was measured. H&E staining was performed on corneal and eyelid skin samples and evaluated by a veterinary pathologist. RESULTS: Corneal epithelial wounds demonstrated 100% closure rates when left untreated or treated with an ocular wound chamber containing HPMC gel at 72 hours while wounds treated with an ocular wound chamber containing HPMC liquid were 98% healed. No significant differences were found in corneal thickness and wound healing, IOP, or eyelid skin pathology in any treatment group when compared to controls. CONCLUSIONS: This study indicates that adapted wound chamber technology can be safely used on sterile, corneal epithelial wounds without adverse effects on periocular or ocular tissue when filled with a liquid or gel.

Making the cut: Innovative methods for optimizing perfusion-based migration assays.

Application of fluid shear stress to adherent cells dramatically influences their cytoskeletal makeup and differentially regulates their migratory phenotype. Because cytoskeletal rearrangements are necessary for cell motility and migration, preserving these adaptations under in vitro conditions and in the presence of fluid flow are physiologically essential. With this in mind, parallel plate flow chambers and microchannels are often used to conduct in vitro perfusion experiments. However, both of these systems currently lack capacity to accurately study cell migration in the same location where cells were perfused. The most common perfusion/migration assays involve cell perfusion followed by trypsinization which can compromise adaptive cytoskeletal geometry and lead to misleading phenotypic conclusions. The purpose of this study was to quantitatively highlight some limitations commonly found with currently used cell migration approaches and to introduce two new advances which use additive manufacturing (3D printing) or laser capture microdissection (LCM) technology. The residue-free 3D printed insert allows accurate cell seeding within defined areas, increases cell yield for downstream analyses, and more closely resembles the reported levels of fluid shear stress calculated with computational fluid dynamics as compared to other residue-free cell seeding techniques. The LCM approach uses an ultraviolet laser for "touchless technology" to rapidly and accurately introduce a custom-sized wound area in otherwise inaccessible perfusion microchannels. The wound area introduced by LCM elicits comparable migration characteristics compared to traditional pipette tip-induced injuries. When used in perfusion experiments, both of these newly characterized tools were effective in yielding similar results yet without the limitations of the traditional modalities. These innovative methods provide valuable tools for exploring mechanisms of clinically important aspects of cell migration fundamental to the pathogenesis of many flow-mediated disorders and are applicable to other perfusion-based models where migration is of central importance. © 2016 International Society for Advancement of Cytometry.

Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls. Intriguingly, FL-VASP/239D abrogated the stimulatory effects of FL-VASP/WT and FL-VASP/239A cells on PKG activity. In turn, pharmacologic blockade of PKG in the presence of BAY60 reversed the inhibitory effect of BAY60 on naïve ASM cell migration. Taken together, we demonstrate for the first time that BAY60 inhibits ASM cell migration through cGMP/PKG/VASP signaling yet through mechanisms independent of pVASP·S239 and that FL-VASP overexpression regulates PKG activity in rat ASM cells. These findings implicate BAY60 as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as CAD and also establish a unique mechanism through which VASP controls PKG activity.

Addition of indapamide to frusemide increases natriuresis and creatinine clearance, but not diuresis, in fluid overloaded ICU patients.

BACKGROUND: Fluid and sodium overload are a common problem in critically ill patients. Frusemide may result in diuresis in excess of natriuresis. The addition of indapamide may achieve a greater natriuresis, and also circumvent some of the problems associated with frusemide. The objective of this study was to examine the effect of adding indapamide to frusemide on diuresis, natriuresis, creatinine clearance and serum electrolytes. METHODS: Fluid overloaded ICU patients were randomised to either intravenous frusemide (Group F) or intravenous frusemide and enteral indapamide (Group F + I). Comprehensive exclusion criteria were applied to address confounders. 24 hour urine was analysed for electrolytes and creatinine. Serum electrolytes were measured before and 24 hours after administration of diuretics. RESULTS: Forty patients (20 in each group) were included in the study. The groups were similar in their baseline characteristics. Over the 24 h study period, patients in Group F + I, had a larger natriuresis (P = 0.01), chloride loss (P = 0.01) and kaliuresis (P = 0.047). Patients in Group F + I also had a greater 24 hour urinary creatinine clearance (P = 0.01). The 24 hour urine volume and fluid balance was similar between the groups. Patients in Group F had an increase in serum sodium (P = 0.04), while patients in Group F + I had a decrease in both serum chloride (P = 0.01) and peripheral oedema (P < 0.001) during the study duration. CONCLUSION: In fluid overloaded ICU patients, addition of indapamide to frusemide led to a greater natriuresis and creatinine clearance. Such a strategy might be utilised in optimising sodium balance in ICU patients.

AMP-activated protein kinase inhibits transforming growth factor-ß-mediated vascular smooth muscle cell growth: implications for a Smad-3-dependent mechanism.

Dysfunctional vascular growth is a major contributor to cardiovascular disease, the leading cause of morbidity and mortality worldwide. Growth factor-induced activation of vascular smooth muscle cells (VSMCs) results in a phenotypic switch from a quiescent, contractile state to a proliferative state foundational to vessel pathology. Transforming growth factor-ß (TGF-ß) is a multifunctional signaling protein capable of growth stimulation via Smad signaling. Although Smad signaling is well characterized in many tissues, its role in VSM growth disorders remains controversial. Recent data from our lab and others implicate the metabolic regulator AMP-activated protein kinase (AMPK) in VSM growth inhibition. We hypothesized that AMPK inhibits VSMC proliferation by reducing TGF-ß-mediated growth in a Smad-dependent fashion. Treatment of rat VSMCs with the AMPK agonist AICAR significantly decreased TGF-ß-mediated activation of synthetic Smad2 and Smad3 and increased inhibitory Smad7. Flow cytometry and automated cell counting revealed that AICAR reversed TGF-ß-mediated cell cycle progression at 24 h and elevated cell numbers at 48 h. TGF-ß/Smad signaling increased the G0/G1 inducers cyclin D1/cyclin-dependent kinase (CDK) 4 and cyclin E/CDK2; however, AICAR reversed these events while increasing cytostatic p21. The specific role of Smad3 in AMPK-mediated reversal of TGF-ß-induced growth was then explored using adenovirus-mediated Smad3 overexpression (Ad-Smad3). Ad-Smad3 cells increased cell cycle progression and cell numbers compared with Ad-GFP control cells, and these were restored to basal levels with concomitant AICAR treatment. These findings support a novel AMPK target in TGF-ß/Smad3 for VSMC growth control and support continued investigation of AMPK as a possible therapeutic target for reducing vascular growth disorders.

Experimental Rat and Mouse Carotid Artery Surgery: Injury & Remodeling Studies.

In cardiovascular research, translation of benchtop findings to the whole body environment is often critical in order to gain a more thorough and comprehensive clinical evaluation of the data with direct extrapolation to the human condition. In particular, developmental and/or pathophysiologic vascular growth studies often employ in vitro approaches such as cultured cells or tissue explant models in order to analyze specific cellular, molecular, genetic and/or biochemical signaling factors under pristine controlled conditions. However, validation of in vitro data in a whole body setting complete with neural, endocrine and other systemic contributions provides essential proof-of-concept from a clinical perspective. Several well-characterized experimental in vivo models exist that provide excellent proof-of-concept tools with which to examine vascular growth and remodeling in the whole body. This article will examine the rat carotid artery balloon injury model, the mouse carotid artery wire denudation injury model, and rat and mouse carotid artery ligation models with particular emphasis on minimally invasive surgical access to the site of intervention. Discussion will include key scientific and technical details as well as caveats, limitations, and considerations for practical use for each of these valuable experimental models.

Inadvertent sodium loading in critically ill patients.

BACKGROUND: Recommended daily intake of sodium is 1- 2mmol/kg. Sodium administration is rarely separated from fluid administration in critically ill patients. OBJECTIVE: To estimate the amount of sodium administered to patients who were invasively ventilated, and to investigate whether sodium administration affected oxygenation, length of stay in ICU and serum sodium level. DESIGN, PARTICIPANTS AND SETTING: Retrospective audit of adult patients who received invasive mechanical ventilation for ≥ 5 days in a tertiary-level intensive care unit. MAIN OUTCOME MEASURES: Total sodium administered from resuscitation and maintenance fluids, infusions, flushes, medicines, transfusions, enteral feeds and total parenteral nutrition; oxygenation, length of ICU stay and serum sodium level. RESULTS: 13 men and 7 women were included. Their median age was 71.9 years (range, 19.8-89.2 years). Median duration of mechanical ventilation was 9 days (range, 6-20 days) and median ICU stay 11.6 days (range, 6-21 days). Median APACHE II score was 29 (range, 18-41). Daily sodium administration was 225.5mmol (151-355mmol). The median daily net fluid balance was 351mL (range, - 759 to +1125mL) and median daily fluid intake was 2352mL (range, 1437- 3798mL). Daily sodium administered correlated with net fluid balance (P<0.001; r=0.35). Of total sodium administered, infusions contributed 22.2% (1.2%-39.9%); drugs 21.6% (0.0-35.5%); flushes 17.4% (9.3%-24.5%); enteral feeds 17% (0.0-39.5%); resuscitation 16.0% (2.5%-36.9%); maintenance fluids 5.8% (0.0-24.0%); transfusions 3.9% (0.0-9.5%) and parenteral nutrition 0.1% (0.0-2.6%). CONCLUSION: Sodium administration to this cohort of critically ill patients requiring prolonged mechanical ventilation was high. Further studies should examine ways of limiting the amount of sodium administered to such patients and to examine if this influences patient outcomes.

The role of estrogen in the developmental appearance of sensory-motor behaviors in the zebrafish (Danio rerio): the characterization of the "listless" model.

The brain is a steroidogenic organ and is thus dependent on estrogen for many aspects of its development and maintenance in both males and females. The purpose of this study was to develop a model to investigate the effect of estrogen on zebrafish sensory-motor (S-M) maturation through mechanisms found in the central nervous (CNS) and peripheral nervous (PNS) systems. In these experiments the aromatase inhibitor (AI), 4-hydroxy androstenedione (4-OH-A), which blocks estrogen synthesis, was used to diminish estrogen's effects on zebrafish CNS and PNS development. During these various treatments the zebrafish were analyzed for neurological deficits, including tactile response, swimming movements, vestibular behavior, pectoral fin and eye movements. Over a three to five day time period (48-168 h post fertilization), in response to AI treatment, none of these S-M behaviors were developmentally expressed creating a "listless" or non-responding condition. Furthermore, when the AI was removed from the treatment medium the S-M behaviors were fully expressed over a two to three day time period. Most importantly, when estrogen was added at the same time as the AI in a co-treatment paradigm, normal developmental appearance of S-M behaviors was rescued in all neurological parameters measured. Furthermore, the addition of estrogen alone after AI washout accelerated the recovery of the tactile response during the first 24 h of treatment. Treatment of developing zebrafish with the selective estrogen receptor blocker ICI 182,780 mimicked the deficit in S-M behaviors caused by AI treatment. This deficit was overcome by low concentrations of estrogen in a co-treatment paradigm with high ICI levels indicating the possibility of a non-genomic mechanism for estrogen's actions on the developmental expression of these S-M behaviors. Eventually, AI exposed fish died of cardiac arrest 4 to 5 days after the start of treatment; however, AI/estrogen co-treatment allowed for 90-100% survival and the maintenance of normal heart rates during this time period. In conclusion, these studies have demonstrated that the presence of estrogen in the early developing zebrafish embryo is necessary for the proper developmental expression of critical nervous system S-M behaviors necessary for survival, as well as the health of the cardiovascular system. These studies also establish a unique "listless" model for further analysis of estrogen's role in the development of brain, brainstem, and spinal cord circuitry related to the maturation of these behavioral and cardiovascular phenomena.

Conventional coagulation and thromboelastograph parameters and longevity of continuous renal replacement circuits.

OBJECTIVE: To determine the relationship between conventional and thromboelastograph (TEG) coagulation parameters and continuous renal replacement therapy (CRRT) circuit longevity. DESIGN: Conventional coagulation and TEG parameters were measured at the commencement of and during CRRT. Time to circuit cessation was measured and only circuits reaching a predetermined rise from baseline in the pressure gradient across the haemofilter were diagnosed as failing due to clotting. All other circuits were excluded from analysis. SETTING: A general critical care unit of a metropolitan tertiary hospital. PATIENTS AND PARTICIPANTS: Fourteen consecutive patients requiring CRRT were studied. The CRRT technique used was continuous veno-venous haemodialysis. INTERVENTIONS: Thromboelastograph measurements were made prior to the commencement of CRRT and daily thereafter for each circuit. The international normalised ratio (INR), activated partial thromboplastin time (APTT) and platelet numbers were measured at commencement and 8 hourly thereafter. Heparin was used for anticoagulation unless considered contraindicated. MEASUREMENTS AND RESULTS: Forty-seven circuits with a mean (SD) circuit life of 33.0 (30.2) h were entered. Twenty-five circuits fulfilled circuit clotting criteria; the mean circuit life was 30.8 (22.1) h. Heparin anticoagulation was found to prolong circuit life significantly despite adequate mean circuit life, 33.2 (35.7) h, in heparin-free circuits. The starting APTT and the TEG variables reaction time (R) and coagulation time (RK) were significantly correlated. The starting APTT, starting RK and mean time taken for the amplitude to increase from 2 to 20 mm (K) were predictive of circuit life. None of these variables predicted which patients would need heparin. CONCLUSION: While TEG variables more closely predicted circuit longevity than conventional coagulation variables, the clinical benefit of TEG monitoring of anticoagulation for CRRT would appear to be minimal.