Tranexamic acid toxicity in human periarticular tissues.

OBJECTIVES: Tranexamic acid (TXA) is an anti-fibrinolytic medication commonly used to reduce perioperative bleeding. Increasingly, topical administration as an intra-articular injection or perioperative wash is being administered during surgery. Adult soft tissues have a poor regenerative capacity and therefore damage to these tissues can be harmful to the patient. This study investigated the effects of TXA on human periarticular tissues and primary cell cultures using clinically relevant concentrations. METHODS: Tendon, synovium, and cartilage obtained from routine orthopaedic surgeries were used for ex vivo and in vitro studies using various concentrations of TXA. The in vitro effect of TXA on primary cultured tenocytes, fibroblast-like synoviocytes, and chondrocytes was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays, fluorescent microscopy, and multi-protein apoptotic arrays for cell death. RESULTS: There was a significant (p < 0.01) increase in cell death within all tissue explants treated with 100 mg/ml TXA. MTT assays revealed a significant (p < 0.05) decrease in cell viability in all tissues following treatment with 50 mg/ml or 100 mg/ml of TXA within four hours. There was a significant (p < 0.05) increase in cell apoptosis after one hour of exposure to TXA (100 mg/ml) in all tissues. CONCLUSION: The current study demonstrates that TXA caused significant periarticular tissue toxicity ex vivo and in vitro at commonly used clinical concentrations.Cite this article: M. McLean, K. McCall, I. D. M. Smith, M. Blyth, S. M. Kitson, L. A. N. Crowe, W. J. Leach, B. P. Rooney, S. J. Spencer, M. Mullen, J. L. Campton, I. B. McInnes, M. Akbar, N. L. Millar. Tranexamic acid toxicity in human periarticular tissues. Bone Joint Res 2019;8:11-18. DOI: 10.1302/2046-3758.81.BJR-2018-0181.R1.

A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis.

OBJECTIVES: Staphylococcus aureus (S. aureus) is the most commonly implicated organism in septic arthritis, a condition that may be highly destructive to articular cartilage. Previous studies investigating laboratory and clinical strains of S. aureus have demonstrated that potent toxins induced significant chondrocyte death, although the precise toxin or toxins that were involved was unknown. In this study, we used isogenic S. aureus mutants to assess the influence of alpha (Hla)-, beta (Hlb)-, and gamma (Hlg)-haemolysins, toxins considered important for the destruction of host tissue, on in situ bovine chondrocyte viability. METHODS: Bovine cartilage explants were cultured with isogenic S. aureus mutants and/or their culture supernatants. Chondrocyte viability was then assessed within defined regions of interest in the axial and coronal plane following live- and dead-cell imaging using the fluorescent probes 5-chloromethylfluorescein diacetate and propidium iodide, respectively, and confocal laser-scanning microscopy. RESULTS: Hla-producing mutants caused substantial chondrocyte death compared with the toxin-deficient control (Hla-Hlb-Hlg-), whilst mutants producing Hlb and Hlg in the absence of Hla induced minimal chondrocyte death. Coronal studies established that Hla-induced chondrocyte death started in the superficial zone of cartilage and spread to deeper layers, whereas Hlb and Hlg toxins were without significant effect. CONCLUSION: This study identified Hla as a highly potent S. aureus toxin that caused rapid chondrocyte death in bovine cartilage, with other toxins or metabolic products produced by the bacteria playing a minor role. The identification of Hla in mediating chondrocyte death may assist in the development of therapeutic strategies aimed at reducing the extent of cartilage damage during and after an episode of septic arthritis.Cite this article: I. D. M. Smith, K. M. Milto, C. J. Doherty, S. G. B. Amyes, A. H. R. W. Simpson, A. C. Hall. A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis. Bone Joint Res 2018;7:457-467. DOI: 10.1302/2046-3758.77.BJR-2017-0165.R1.

Rapid in situ chondrocyte death induced by Staphylococcus aureus toxins in a bovine cartilage explant model of septic arthritis.

OBJECTIVE: To assess in situ chondrocyte viability following exposure to a laboratory strain and clinical isolates of Staphylococcus aureus. METHODS: Bovine cartilage explants were cultured in the presence of S. aureus 8325-4 (laboratory strain), clinical S. aureus isolates or non-infected culture medium of pH values 7.4, 6.4 and 5.4. All clinical isolates were isolated from the joint aspirates of patients presenting with S. aureus-induced septic arthritis (SA). At designated time points, in situ chondrocyte viability was assessed within defined regions-of-interest in the axial and coronal plane following live- and dead-cell image acquisition using the fluorescent probes 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), respectively, and confocal laser-scanning microscopy (CLSM). Cartilage water content, following S. aureus 8325-4 exposure, was obtained by measuring cartilage wet and dry weights. RESULTS: S. aureus 8325-4 and clinical S. aureus isolates rapidly reduced in situ chondrocyte viability (>45% chondrocyte death at 40 h). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ chondrocytes to S. aureus 8325-4 toxins found SZ chondrocytes to be more susceptible to the toxins than DZ chondrocytes. Cartilage water content was not significantly altered compared to non-infected controls. CONCLUSIONS: Toxins released by S. aureus have a rapid and fatal action on in situ chondrocytes in this experimental model of SA. These data advocate the prompt and thorough removal of bacteria and their toxins during the treatment of SA.

Chondrocyte death after drilling and articular screw insertion in a bovine model.

OBJECTIVE: Intra-articular screws are used for internal fixation of osteochondral fragments after fracture or osteochondritis dissecans. This causes cartilage injury potentially leading to chondrocyte death. We have visualised/quantified the hole and zone of cell death (ZCD) in cartilage after drilling/insertion of various articular screws. METHOD: Using an ex vivo bovine model with transmitted light and confocal laser scanning microscopy (CLSM), the holes and ZCD following drilling/insertion of articular screws (cortical screw, headless variable pitch metallic screw, headless variable pitch bioabsorbable screw) were evaluated. In situ chondrocyte death was determined by live/dead cell viability assay. An imaging/quantification protocol was developed to compare hole diameter and ZCD from drilling/insertion of screws into cartilage. The effect of saline irrigation during drilling on the ZCD was also quantified. RESULTS: Screw insertion created holes in cartilage that were significantly (P ≤ 0.001) less than the diameters of the equipment used. With a 1.5 mm drill, a ZCD of 580.2 ± 124 µm was produced which increased to 637.0 ± 44 µm following insertion of a 2 mm cortical screw although this was not significant (P > 0.05). The ZCD from insertion of the variable pitch headless screws (diam. 3.5 mm) was lower for the metallic compared to the bioabsorbable design (800.9 ± 159 vs 1,236.4 ± 212 µm, respectively; P < 0.01). The ZCD from drilling was reduced ∼50% (P < 0.001) by saline irrigation. CONCLUSIONS: Cartilage injury during intra-articular screw fixation caused a ZCD around the hole irrespective of screw design. Saline irrigation significantly reduced the ZCD from drilling into cartilage.

Pre-operative predictors of the length of hospital stay in total knee replacement.

In Scotland, the number of primary total knee replacements performed annually has been increasing steadily. The price of the implant is fixed but the length of hospital stay is variable. We prospectively investigated all patients who underwent primary unilateral total knee replacement in the Scottish region of Fife, between December 1994 and February 2007 and assessed their recorded pre-operative details. The data were analysed using univariate and multiple linear regression statistical analysis. Data on the length of stay were available from a total of 2106 unilateral total knee replacements. The median length of hospital stay was eight days. The significant pre-operative risk factors for an increased length of stay were the year of admission, details of the consultant looking after the patient, the stair score, the walking-aid score and age. Awareness of the pre-operative factors which increase the length of hospital stay may provide the opportunity to influence them favourably and to reduce the time in hospital and the associated costs of unilateral total knee replacement.

Hair analysis: self-reported use of "speed" and "ecstasy" compared with laboratory findings.

Drug use histories were collected from 100 subjects recruited from the "dance scene" in and around Glasgow, Scotland. In addition, each subject donated a hair sample which was analyzed by gas chromatography/mass spectrometry (GC/MS) for amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MD MA) and 3,4-methylenedioxyethylamphetamine (MDEA). The hair samples were analyzed in two 6 cm segments or in full, ranging from 1.5 to 12 cm depending on the length of the hair. Approximately 10 mg of hair was ground to a fine powder before treatment with beta-glucuronidase/aryl sulfatase. A solid-phase extraction procedure was carried out followed by derivatization with pentafluoropropionic anhydride (PFPA). All extracts were analyzed by gas chromatography/mass spectrometry (GC/MS). Of the 139 segments analyzed, 77 (52.5%) were positive for at least one of the five amphetamines. The drug concentrations found in the hair were compared with the self-reported drug histories. A concordance of greater than 50% was found between the self-report data and levels detected in hair. However, no correlation was found between the reported number of "ecstasy" tablets consumed and the drug levels detected in hair. An increase in the average drug levels measured was observed from low to high use (number of "ecstasy" tablets/month). A large number of false negatives and a low number of false positives were observed.

Korsakoff's psychosis in Scotland: evidence for increased prevalence and regional variation.

Surveys of new long-stay mental hospital patients in Scotland find that 9% have a diagnosis of alcohol-related brain damage, mainly Korsakoff's psychosis (KP), whereas the rate was 5% in the old long-stay patients. The national hospital database shows a rise in rates of KP in figures for discharge diagnosis and for diagnosis of hospital residents during the past three decades. There is an argument for more specialized provision given the significance of this group of patients.

Hair testing for "ecstasy" (MDMA) in volunteer Scottish drug users.

The aim of the study was to compare self reported "ecstasy" use with the results of the analysis of hair harvested from the same users. Subjects were recruited by multisite chain-referral sampling within the 1994-95 "dance scene" in Glasgow. One hundred subjects donated hair after completing a lengthy interviewer-administered questionnaire. Overall gross concordance between self reported "ecstasy" use and discovery of MDMA (or related compounds) in analysed hair did not surpass 59%, and no relationship had a Cohen's kappa of more than 0.08. Within the positive concordant dataset (n = 52), scatter was considerable, with no correlation being significant, and none more strongly positive than -0.0518. The results presented here indicate that, as far as MDMA is concerned, if judged by self-report, hair does not reach a level of apparent accuracy that would permit its use as a general population estimator. However, hair testing is probably more reliable than self-report, and its accuracy could be verified independently if large-scale inter- and intra-laboratory comparative research is conducted.

Rotational bias in intact rats following intrastriatal injections of dopaminergic drugs.

Following unilateral 6-OHDA lesions of the striatum, systemic dopamine agonists produce rotation due to receptor supersensitivity. Rotation following intrastriatal dopamine agonists in intact rats also has been reported, although these studies are few and contradictory. Dorsal striatal injection (0.5 microl) of the direct dopamine agonist apomorphine failed to caused rotation. In addition, neither the D1 agonist SKF 81297, the cAMP analogue Sp-cAMPS, nor the D2 agonist quinpirole affected rotation. In contrast, the dopamine releaser amphetamine (1.1, 10.9, 108.7 mM) caused significant contralateral rotation. This effect was reversed by coinjection of the D1 antagonist SCH 23390 (3.1 mM) but not by the D2 antagonist eticlopride. Rotation was also reversed by TTX coinjections (100 microM) but not by the NMDA antagonist AP7 or the kainate/AMPA antagonist CNQX. Thus, direct dopamine agonists in the striatum failed to cause behavioral asymmetry, whereas amphetamine induced contralateral rotation. This effect is mediated primarily by D1 receptors and requires concurrent neuronal activation that appears to be independent of glutamate receptor stimulation. These results are consistent with studies of Fos induction in normosensitive animals following dopamine agonists and are discussed in terms of changes in basal ganglia output pathway activity.

Glutamate receptor agonist injections into the dorsal striatum cause contralateral turning in the rat: involvement of kainate and AMPA receptors.

Unilateral stimulation of glutamate receptors in the dorsal striatum of intact rats resulted in contralateral turning. Turning behavior was recorded for 20 min following unilateral intrastriatal injections (0.5 microliter) in chronically cannulated rats. Kainate injections caused a dose-dependent increase in contralateral rotation that was blocked by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the action potential blocker tetrodotoxin, and by increasing doses of the dopamine receptor antagonist cis-flupenthixol. Injections of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) also caused rotation that was blocked with co-injections of CNQX, tetrodotoxin or cis-flupenthixol. Neither CNQX nor tetrodotoxin injected alone caused turning. This effect is dopamine-dependent, and may result from a kainate or AMPA-induced increase in dopamine release. Glutamate receptor agonist injections into the striatum may cause contralateral turning by degrading information in ascending cortical projections and may further influence locomotion via basal ganglia output nuclei projections to the brainstem.

Contralateral turning caused by metabotropic glutamate receptor stimulation in the dorsal striatum is reversed by MCPG, TTX, and cis-flupenthixol.

Recent evidence suggests an involvement of metabotropic glutamate receptors in the physiology of the striatum. In this study, rotation was recorded in an automated rotometer for 20 min following dorsal striatal injections (0.5 microliter) in cannulated rats. The metabotropic agonist 1-aminocyclopentane-trans-1, 3-dicarboxylic acid (1S,3R-ACPD) caused dose-dependent contralateral rotation. Turning caused by 500 microM 1S,3R-ACPD was reversed by coinjections of the metabotropic antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mM) and by tetrodotoxin (100 microM). Injections of MCPG alone (10 microM, 100 microM, 1 mM) failed to elicit turning. Increasing doses of the dopamine antagonist cis-flupenthixol also reversed 1S,3R-ACPD-induced rotation. Thus unilateral striatal metabotropic glutamate receptor stimulation can cause receptor-specific rotation that may result from an increase in neural activity, and is dependent on intact dopamine neurotransmission.

The neurochemical pathology of thiamine deficiency: GABAA and glutamateNMDA receptor binding sites in a goat model.

Synaptic plasma membranes were prepared from four cerebrocortical areas from six male Angora goats made chronically thiamine deficient (TD) by the administration of AmproliumTM (600-900 mg/kg daily for 38-44 d). Four male controls were matched for age (27-30 mo). Four different radioligands were used to characterise GABAA and Glu-RNMDA receptor binding sites. There were marked, localised and contrasting changes in motor cortex, with an increase in GABAA and a decrease in Glu-RNMDA binding site densities. Less clearcut changes of a similar nature were seen in visual cortex. There was no variation in the parameters of GABA-activated [3H]diazepam binding between cortical areas in control goats, but there was a reduction in the maximal response to GABA in all areas in TD goats. There were regional variations in glutamate-activated [3H]MK-801 binding in control goat brain, and a non-selectively reduced maximal response in TD. Alterations in these indices of GABA- and glutamate-mediated neurotransmission may underlie the neurological signs of acute thiamine deficiency in these animals.

Caesarean section in chronic renal failure.

A 22-year-old woman presented for Caesarean section at 34 weeks' gestation. She had chronic renal failure, with a glomerular filtration rate of 14 mL min-1 and was treated with twice weekly dialysis. We describe the potential problems of the case including cardiovascular instability, fluid balance and some of the effects of uraemia. We also describe the anaesthetic management of the case.

Pharmacokinetics of flumazenil and midazolam.

We have studied simultaneously the pharmacokinetics of flumazenil and midazolam in 12 healthy Chinese children, aged 5-9 yr, undergoing circumcision. Two hours before operation each patient received midazolam 0.5 mg kg-1 orally for premedication and 0.5 mg kg-1 i.v. during induction. Six minutes after cessation of anaesthesia, a bolus of flumazenil 10 micrograms kg-1 was given i.v., followed by an infusion of flumazenil at 5 micrograms kg-1 min-1 which was maintained until the child could identify himself. Midazolam data were consistent with a three-compartment model with a mean (SD) elimination half-life of 107 (30) min, total body clearance of 15.4 (3.2) ml min-1 kg-1 and apparent volume of distribution at steady state of 1.9 (0.6) litre kg-1. Flumazenil data were best interpreted by a monoexponential function, with a mean terminal elimination half-life of 35.3 (13.8) min, a total plasma clearance of 20.6 (6.9) ml min-1 kg-1 and apparent volume of distribution at steady state of 1.0 (0.2) litre kg-1. No unchanged midazolam was detected in the 24-h urine sample, but 5.8-13.8% of the flumazenil dose was recovered unchanged. At the time of self identification, 4.5 (1.4) min after flumazenil administration, the mean plasma concentrations of midazolam and flumazenil were 163.1 (43.7) and 29.9 (16.1) ng ml-1, respectively.

Role of the subthalamic nucleus in the regulation of nigral dopamine neuron activity.

The influence of subthalamic nucleus (STN) afferents on dopaminergic (DA) neurons of the rat substantia nigra (SN) was investigated. Hemisections of the brain placed between the STN and the SN or located anterior to the STN caused an increase in the firing rate of DA cells without producing significant changes in their firing pattern. In contrast, electrolytic and ibotenic acid lesions of the STN resulted in 93% and 49% reductions, respectively, in the level of burst firing without affecting the firing rate of DA cells recorded in the lateral SN. Furthermore, procedures which interrupted the STN input to the SN produced rapid pacemaker-like firing in 18% of the lateral SN DA neurons recorded. Activation of the STN using single pulses of electrical stimulation caused: 1) a 20-50 msec inhibition of DA cell firing followed by an excitation, which in 35% of DA cells was accompanied by spikes occurring in a burst-like pattern, and 2) a short-latency inhibition lasting 5-25 msec in 75% of non-DA SN zona reticulata (ZR) neurons. On the other hand, stimulation of the STN for 1 minute at 20 Hz resulted in an initial decrease in DA cell burst firing followed by elevated firing rates and increased burst firing by 30-60 minutes after the stimulation. Pharmacological activation of the STN by infusion of bicuculline caused a rapid inhibition of DA cells followed by a two-fold increase in burst firing 6-14 minutes later, whereas SN ZR cells responded with an elevation in firing rate which dissipated in 6-14 minutes. Muscimol-induced STN inhibition produced complimentary biphasic changes in SN neuron firing: 1) an initial increase followed by a decrease in burst firing and firing rate of DA neurons and 2) a rapid inhibition followed by an excitation of ZR cells over a similar time course. Thus, the STN appears to exert a dual action on SN DA cells: 1) initial inhibition possibly mediated through STN excitation of the inhibitory SN ZR projections to DA cells, and 2) a facilitation of burst firing which may be a direct effect of excitatory STN afferents.

An evaluation of prolonged oximetric data acquisition.

Data derived from pulse oximetry has inherent limitations, one of which is artifactual desaturation caused by patient movement. Perioperative patterns of oxygen desaturation were studied for a mean duration of 67 hours in eight young patients following corrective spinal surgery. Pulse oximetry data were relayed to a computer using Satmaster, a program which permits storage, retrieval, signal evaluation and statistical analysis of oximetry data. Desaturation episodes were mild, of short duration and their infrequent occurrence was not increased during intravenous morphine infusion. Retrospective identification of contemporaneous artifactual changes in signal amplitude permitted the removal of artifactual desaturations from our statistical data analysis. This decreased the average time desaturated from 5.4% (220 minutes) to 4.2% (162 minutes) of the monitored period representing a 25% reduction in absolute incidence and a 35% reduction in episodic incidence of desaturation. Acquired data should be validated and inferences drawn from non-validated data must be assessed with caution.

The upgraded Finapres 2300e. A clinical evaluation of a continuous noninvasive blood pressure monitor.

Measurements from the upgraded Finapres 2300e continuous noninvasive blood pressure monitor, the Finapres 2300 and Colin oscillometric noninvasive blood pressure monitor were compared with invasive arterial line blood pressure readings. Fifteen young Chinese patients undergoing elective spinal surgery of more than 2 h duration had contemporaneous blood pressure measurements digitally recorded every minute. Data were analysed using methods described by Bland and Altman to assess agreement of noninvasive devices with an arterial line. Results showed that although the Finapres 2300e was significantly more accurate than the Finapres 2300 and Colin noninvasive blood pressure monitors it could not be recommended as a substitute for continuous arterial line blood pressure monitoring. Both Finapres devices demonstrated reductions in accuracy related to time (drift) and over-read diastolic and mean pressures by 5-8 mmHg throughout the range of mean arterial line pressures (bias). The Colin was consistently less accurate than the Finapress monitors and performed worst at low mean arterial line pressures.

Determination of topanol antioxidants in methacrylates using capillary gas-liquid chromatography.

This paper presents a method to identify and determine topanol A and topanol O antioxidants in methylmethacrylate and 11-bromoundecylmethacrylate. The method is both simple and rapid. Analysis is performed on a 10 m x 0.53 mm I.D. HP-1 capillary gas chromatography column with a temperature gradient and a high carrier gas flow-rate (16.5 ml/min). Quantitation is by internal standardisation. Validation of the method is described for both topanols at concentrations of approximately 50 ppm in methylmethacrylate and 250 ppm in 11-bromoundecylmethacrylate.