Incidence of seropositive and seronegative rheumatoid arthritis in Denmark: a nationwide population-based study.

OBJECTIVE: To investigate and compare trends in incidence rates (IRs) of seropositive and seronegative rheumatoid arthritis (RA) in Denmark using various data sources for serostatus definition. METHOD: This nationwide population-based cohort study was based on data from Danish healthcare and clinical quality registries between 2000 and 2018. Information on anti-cyclic citrullinated peptide and immunoglobulin M rheumatoid factor was obtained, and definitions of seropositivity according to the number of applied data sources were prespecified. Annual age- and sex-standardized IRs were calculated as the number of incident seropositive and seronegative cases, divided by the number of person-years (PY) in the general population in that given year. RESULTS: An increasing temporal trend in IR of seropositive RA and a decreasing trend in seronegative RA were observed. The IRs were higher for seropositive RA than for seronegative RA from 2009 onwards, with a widening of the IR gap between 2009 and 2016 regardless of the definition of seropositivity. When combining laboratory- and physician-reported autoantibody information and ICD-10 codes, the IR of seropositive RA in 2018 was approximately twice that of seronegative RA, at 19.0 and 9.0 per 100 000 PY, respectively. The level of antibody testing increased significantly during the study period. CONCLUSIONS: The IR of seropositive RA increased over time, whereas the IR of seronegative RA decreased. Temporal IR changes may be caused by a real change in the RA serology subtypes, an increase in autoantibody testing and availability, changes in registration practice over time, or a combination of these factors.

Incidence and prevalence of rheumatoid arthritis in Denmark from 1998 to 2018: a nationwide register-based study.

OBJECTIVE: To investigate the incidence and prevalence of rheumatoid arthritis (RA) in the adult Danish population. METHOD: In this nationwide register-based cohort study, patients with incident RA between 1998 and the end of 2018 were identified using Danish administrative registries. The age- and sex-standardized incidence rate (IR), incidence proportion (IP), lifetime risk (LR), and point prevalence (PP) of RA were calculated. RA was defined as a first-time RA diagnosis registered in the Danish National Patient Registry combined with a redeemed prescription of a conventional synthetic disease-modifying anti-rheumatic drug in the following year. In addition, three different case definitions of RA were explored. RESULTS: The overall age- and sex-standardized IR of RA from 1998 to 2018 was 35.5 [95% confidence interval (CI) 35.1-35.9] per 100 000 person-years while the IP was 35.2 (95% CI 34.8-35.5) per 100 000 individuals. The IR was two-fold higher for women than for men. The LR of RA ranged from 2.3% to 3.4% for women and from 1.1% to 1.5% for men, depending on the RA case definition used. The overall PP of RA was 0.6% (95% CI 0.5-0.6%) in 2018: 0.8% (95% CI 0.7-0.8%) for women and 0.3% (95% CI 0.3-0.4%) for men. The prevalence increased about 1.5-fold from 2000 to 2018. CONCLUSION: The IR and PP were approximately two-fold higher for women than for men. The prevalence of RA in Denmark increased significantly from 2000 to 2018. The RA case definition had more impact on the results than the choice of denominator.

In vivo MR imaging of magnetically labeled human embryonic stem cells.

INTRODUCTION: Human embryonic stem cells (hES) have emerged as a potentially new therapeutic approach for treatment of heart and other diseases applying the concept of regenerative medicine. A method for in vivo visualization and tracking of transplanted hES would increase our understanding of in vivo hES behavior in both experimental and clinical settings. The aim of this study was to evaluate the feasibility of magnetic labeling and visualization of hES with magnetic resonance imaging (MRI). METHODS: hES were established and expanded according to standard procedures. After expansion, the cells were cultured under feeder free conditions and magnetically labeled by addition of dextran-coated Ferrum-oxide particles (Endorem) to the medium. Accumulation of small particles of iron-oxide (SPIO) in hES was assessed by Prussian blue staining and electron microscopy. For in vitro MRI, the labeled and unlabeled hES were examined in cell solution and after transplantation into explanted mouse heart ( approximately 100,000 cells) on a Bruker Avance DMX 500 vertical magnet at 11.75 T. A multi-slice, multi spin-echo T(2)-weighted images were obtained. For in vivo imaging, the experiments were performed on male Sprague-Dawley using Bruker Biospec 2.35 T magnet. The hES were directly injected ( approximately 500,000 cells) after surgical procedure (thoracotomy) into anterior left ventricular (LV) wall. Multi-slice T(2)-weighted gradient echo images were obtained using cardiac gating. RESULTS: hES appeared to be unaffected by magnetic labeling and maintained their ability to proliferate and differentiate. No additive agent for membrane permeabilisation was needed for facilitation of intracellular SPIO accumulation. Prussian blue and electron microscopy have revealed numerous iron particles in the cytoplasm of hES. On T(2)-weighted images, the labeled cells have shown well-defined hyopintense areas at the site of injection in anterior LV wall both in vitro and in vivo. CONCLUSIONS: It is feasible to magnetically label and visualize hES both in vitro and in vivo. MR visualization of magnetically labeled hES may be a valuable tool for in vivo tracking of hES.

Selective cerebral overexpression of growth hormone alters cardiac function, morphology, energy metabolism and catecholamines in transgenic mice.

BACKGROUND: Growth hormone (GH) has important regulatory effects on cardiac morphology and function both during normal development as well as in pathophysiological settings such as myocardial infarction (MI) and congestive heart failure (CHF). In order to investigate in more detail the interaction between GH and sympathetic nervous system (SNS) system we studied the effects of selective cerebral GH overexpression on myocardial content of catecholamines, myocardial and brain energy metabolism as well as on cardiac function during resting and stress conditions in a transgenic mouse model. METHODS: Transgenic mice with selective bovine GH overexpression under control of glial fibrillary acidic protein promoter in the brain (GFAP-bGH, n=15) were created and compared to genetically matched non-transgenic mates (Control, n=15). Cardiac morphology and function were evaluated in vivo using transthoracic echocardiography during resting and stress conditions induced pharmacologically by dopamine (D) and isoprotenolol (ISO). Myocardial and brain energy metabolism were evaluated non-invasively using in vivo volume-selective phosphorus magnetic resonance spectroscopy ((31)P MRS). Myocardial content of catecholamines was analyzed by means of HPLC. RESULTS: Compared to the C animals, the GFAP-bGH mice have showed several differences in the cardiac phenotype. Systolic (fractional shortening) and diastolic function (E/A wave ratio of mitral flow) was disturbed in the GFAP-bGH mice (both p<0.05). During the dopamine stress, there was chronotropic insufficiency in the GFAP-bGH group (p<0.01) while no difference was observed in response to isoprotenolol. Left ventricular dimensions were increased in GFAP-bGH mice (p<0.05). There was a tendency for higher body weight in GFAP-bGH compared to the control group (p=0.06) while no difference was observed in heart weight and brain weight when normalized for body weight. Myocardial content of noradrenaline was lower in the GFAP-bGH group (p<0.05). PCr/ATP ratio was higher (p<0.05) in the brain and lower in the heart (p<0.05) in the GFAP-bGH mice. CONCLUSIONS: Selective cerebral overexpression of GH results in alterations of cardiac function, morphology and metabolism in transgenic mice. Decreased myocardial content of catecholamines in the GFAP-bGH mice suggests central interaction between GH and sympathetic nervous system.

Singlet oxygen energy illumination during moderate cold ischemia prolongs the survival of concordant hamster xeno-heart transplants.

INTRODUCTION: Singlet oxygen energy (SOE) is a potent inhibitor of reactive oxygen species (ROS) in vitro and in vivo in certain dose ranges and can improve the levels of high-energy phosphates (HEP) in concordant hamster xeno-heart transplants. Some data indicate that a certain degree of cold ischemia (CI) might be beneficial to xenotransplants. We investigated if SOE illumination of hamster xeno-hearts during moderate cold ischemia (CI) improved graft survival. MATERIALS AND METHODS: Eighteen hearts from Golden Syrian hamsters (70 to 80 g) were subjected to 8 hours of CI in cold (+4 degrees C) saline solution (NaCL, 0.9%) before heterotopic cervical transplantation to Lewis rats (220 g). Among the treatment group (n = 8), SOE was produced by illuminating the hearts for 10 minutes every 30 minutes with photons at lambda 634 nm with the Oxylight equipment. Graft function was evaluated every 6 hours after transplantation with digital exam until cessation of heart beats. RESULTS: The graft survival of SOE-illuminated ischemic hamster xenografts was 2.34 +/- 0.56 versus 1.15 +/- 0.37 days in the control group (P < .05). All hearts displayed immediate graft function versus 70% in the controls (NS). CONCLUSIONS: SOE illumination at lambda 634 nm during moderate cold ischemia (+4 degrees C) can improve the survival of concordant hamster xeno-heart transplants. The exact mechanism(s) are currently unknown, but the effect might in part be exerted by a combination of reduced production of ROS and increased oxidative phosphorylation.

Ischemic preconditioning can overcome the effect of moderate to severe cold ischemia on concordant mouse xeno-heart transplants.

PURPOSE: Concordant mouse xeno-heart transplants are relatively sensitive to ischemia-reperfusion injury. We investigated the effect of an ischemic preconditioning (IPC) protocol on the functional and biochemical outcome of mouse xenohearts transplanted to the Lewis rat. MATERIAL AND METHODS: NMRI mice (30 to 40 g) were anesthetized, intubated, and mechanically ventilated. They were subjected either to a IPC protocol leading to an SaO(2) of 70% for 5 minutes followed by normoxia (defined as SaO(2) >90%) for 10 minutes (n = 9) or normoxia only (n = 11). The hearts were then heterotopically transplanted to Lewis rats (220 g). The frequencies of immediate onset and early dysfunction and late dysfunction were registered. The hearts surviving for 6 hours were explanted and the absolute concentrations of phosphocreatine and adenosine triphosphate (ATP) were determined in micromole per gram of heart tissue with high-pressure liquid chromatography. The phosphorylation ratio, PCr/ATP, a known correlate to biochemical and functional outcome, was calculated. RESULTS: Four of 11 (36.4%) of control hearts experienced immediate onset and early dysfunction versus 0% (0/9) in M hearts subjected to IPC (P = .01). Furthermore, the IPC protocol increased the PCr concentration, 15.08 +/- 1.00 versus 9.04 +/- 2.04 micromol/g in controls (P = .01), and the PCr/ATP ratio, 1.80 +/- 0.17 versus 1.27 +/- 0.21 (NS; P = .06). CONCLUSIONS: IPC provides a protective PCr overshoot overcoming the short-term effects of moderate to severe ischemic injury on mouse xeno-heart transplants.

Growth hormone improves bioenergetics and decreases catecholamines in postinfarct rat hearts.

The aims of this study were to examine, in vivo, the effects of GH treatment on myocardial energy metabolism, function, morphology, and neurohormonal status in rats during the early postinfarct remodeling phase. Myocardial infarction (MI) was induced in male Sprague Dawley rats. Three different groups were studied: MI rats treated with saline (n = 7), MI rats treated with GH (MI + GH; n = 11; 3 mg/kg x day), and sham-operated rats (sham; n = 8). All rats were investigated with 31P magnetic resonance spectroscopy and echocardiography at 3 days after MI and 3 weeks later. After 3 weeks treatment with GH, the phosphocreatine/ATP ratio increased significantly, compared with the control group (MI = 1.69 +/- 0.09 vs. MI + GH = 2.42 +/- 0.05, P < 0.001; sham = 2.34 +/- 0.08). Treatment with GH significantly attenuated an increase in left ventricular end systolic volume and end diastolic volume. A decrease in ejection fraction was prevented in GH-treated rats (P < 0.05 vs. MI). Myocardial and plasma noradrenaline levels were significantly lower in MI rats treated with GH. These effects were accompanied by normalization of plasma brain natriuretic peptide levels (sham = 124.1 +/- 8.4; MI = 203.9 +/- 34.7; MI + GH = 118.3 +/- 8.4 ng/ml; P < 0.05 vs. MI). In conclusion, GH improves myocardial energy reserve, preserves left ventricular function, and attenuates pathologic postinfarct remodeling in the absence of induction of left ventricular hypertrophy in postinfarct rats. The marked decrease in myocardial content of noradrenaline, after GH treatment, may protect myocardium from adverse effects of catecholamines during postinfarct remodeling.

Impairment of cardiac function and bioenergetics in adult transgenic mice overexpressing the bovine growth hormone gene.

Cardiovascular abnormalities represent the major cause of death in patients with acromegaly. We evaluated cardiac structure, function, and energy status in adult transgenic mice overexpressing bovine GH (bGH) gene. Female transgenic mice expressing bGH gene (n = 11) 8 months old and aged matched controls (n = 11) were used. They were studied with two-dimensional guided M-mode and Doppler echocardiography. The animals (n = 6) for each group were examined with 31P magnetic resonance spectroscopy to determine the cardiac energy status. Transgenic mice had a significantly higher body weight (BW), 53.2+/-2.4 vs. 34.6+/-3.7 g (P < 0.0001) and hypertrophy of left ventricle (LV) compared with normal controls: LV mass/BW 5.6+/-1.6 vs. 2.7+/-0.2 mg/g, P < 0.01. Several indexes of systolic function were depressed in transgenic animals compared with controls mice such as shortening fraction 25+/-3.0% vs. 39.9+/-3.1%; ejection fraction, 57+/-9 vs. 77+/-5; mean velocity of circumferential shortening, 4.5+/-0.8 vs. 7.0+/-1.1 circ/sec, p < 0.01. Creatine phosphate-to-ATP ratio was significantly lower in bGH overexpressing mice (1.3+/-0.08 vs. 2.1+/-0.23 in controls, P < 0.05). Ultrastructural examination of the hearts from transgenic mice revealed substantial changes of mitochondria. This study provides new insight into possible mechanisms behind the deteriorating effects of long exposure to high level of GH on heart function.

Harmful singlet oxygen can be helpful.

Highly reactive harmful singlet oxygen O2(1delta(g)) can be helpful while relaxing to its triplet ground state O2(3sigma(g)-). The energy emitted during this relaxation from the excited energy state is discernable at 634 nm. We report here on the effect of this energy as photon illumination and as energy transfer in air on the production of reactive oxygen species (ROS) by human monocytes, measured as isoluminol-enhanced chemiluminescence. We demonstrate up to 60% decrease in the secretion of ROS after 2-min illumination of the monocytes stimulated with phorbol myristate acetate (PMA). The results provide in vitro documentation of the utility of singlet oxygen energy in modifying cellular behaviour.

Tumour purine nucleotides and cell proliferation in response to exercise in rats.

Voluntary physical exercise can delay the onset of anorexia and cachexia in tumour-bearing rats. A substrate deviation in the host which has been hypothesised as tumour burden is reduced despite an increase in food intake. Therefore, we determined the levels of purine nucleotides, the energy charge and the cell division rate in tumours from exercising animals in the postexercise period. Tumour content of purine nucleotides was analysed by HPLC. Tumour cell kinetics was studied by flow cytometry after incorporation of bromodeoxyuridine (BrdU) into DNA. Exercising animals demonstrated a 34.4% reduction in tumour volume (P < 0.05) but a 1.31-fold increase in energy charge in tumour tissue (P < 0.05). Labelling index (LI), DNA synthesis time (Ts) and potential doubling time (Tpot) were not significantly altered. These results suggest that the influence on tumour growth is closely related to the exercise period.

Cytochrome c oxidase and purine nucleotides in skeletal muscle in tumour-bearing exercising rats.

We have previously shown that spontaneous physical exercise can delay the onset of experimental anorexia and cachexia and retard tumour growth and we now report the effects on the energy metabolism in skeletal muscle. Exercising tumour-bearing animals (TBE) had an increased maximal capacity for oxygen uptake expressed as Vmax of the cytochrome c oxidase compared with their tumour-bearing sedentary controls (TBS) [mean (S.E.) 289.9 (30.7) vs. 141.6 (11.0); P less than 0.05] but an unchanged Km value. The TBS animals had a depressed Vmax as compared with non-tumour-bearing sedentary controls (CS) [141.6 (11.0) vs. 210.1 (15.1); P less than 0.05]. Most of the purine nucleotides in the 'glycolytic' anterior tibial muscle were significantly altered in the TBE animals compared with the TBS animals, but in the mainly 'oxidative' soleus muscle only the level of inosine monophosphate (IMP) was changed. The results indicate that physical exercise can normalise the oxidative capacity and improve the energy state in skeletal muscle in the tumour-bearing host.

Coenzymes Q9 and Q10 in skeletal and cardiac muscle in tumour-bearing exercising rats.

Physical exercise increases metabolic rate, and induces both adaptational biogenesis of mitochondria in skeletal muscle and an increase in antioxidant capacity. The onset of experimental anorexia and cachexia can be delayed by voluntary exercise. As skeletal muscle is the main target for cancer cachexia, we determined the levels of coenzymes Q9 and Q10 in skeletal muscle from tumour-bearing exercising rats, and compared them to those of sedentary tumour-bearers and controls. Both tumour-bearing groups had increased levels of coenzymes Q9 and Q10 in the anterior tibial muscle (P < 0.05 for exercised animals). In the soleus muscle, only the tumour-bearing exercising animals demonstrated an increase in the levels of both coenzymes (P < 0.05). In cardiac muscle, the presence of tumour and exercise reduced the levels of coenzymes below that of sedentary controls. Exercise counteracted the anaemia in the tumour-bearing host (P < 0.05). In conclusion, the increase in antioxidant capacity in skeletal muscle indicates a defence mechanism in the tumour-bearing hosts which is augmented by physical exercise.

Selective beta1-blockade attenuates post-infarct remodelling without improvement in myocardial energy metabolism and function in rats with heart failure.

OBJECTIVE: To investigate in vivo effects of long-term selective beta1-blockade on cardiac energy metabolism, remodelling, function and plasma cytokines in a rat model of post-infarct congestive heart failure (CHF). METHODS: Myocardial infarction (MI) was induced in male rats by ligation of the left coronary artery. Three different groups of rats were studied, MI rats treated with metoprolol (n=17), MI rats treated with saline (n=14) and sham-operated rats (n=12). The treatment with metoprolol 1 mg/kg/h was initiated in the third week post-infarct for a period of 6 weeks. All rats were investigated non-invasively with volume-selective 31P magnetic resonance spectroscopy and echocardiography for evaluation of left ventricular (LV) energy metabolism, morphology and function. Plasma concentration of IL-1beta and IL-6 and density of beta-adrenergic receptors were analyzed. RESULTS: Metoprolol attenuated the increase in LV dimensions and volumes. Treatment with metoprolol had no effect on PCr/ATP and LV function. Plasma level of IL-1beta was higher and IL-6 was lower in the metoprolol group. Density of beta-adrenergic receptors was similar in all three groups. CONCLUSION: Selective beta1-blockade in rats with chronic CHF attenuates post-infarct structural remodelling, without concomitant improvement in myocardial energy metabolism and function. Improvements in myocardial energy metabolism and function do not precede and are not a prerequisite for an anti-remodelling effect of beta1-blockade in the setting of chronic CHF.

Effects of nandrolone propionate on experimental tumor growth and cancer cachexia.

We studied the tumor host response to excessive doses of an anabolic steroid (nandrolone propionate, 2.5 mg 20 g intraperitoneally every second day for 11 days) with respect to body composition and tumor cell kinetics in MCG 101 sarcoma-bearing mice (C57BL/6J) with progressive cachexia. Although survival and food intake were not affected, a significant weight gain was observed that was essentially attributed to water retention. Net protein content was increased only to a minor extent (15%), of which only the liver accounted for a significant part of the body compartments. Hepatic protein accumulation was obviously caused by decreased protein degradation, since hepatic RNA content was unchanged. After anabolic steroid administration, reduced histochemical staining of succinate dehydrogenase was observed in skeletal muscles rich in oxidative type 1 fibers, but it was not different from that of tumor-bearing control animals, which was also confirmed by measurements of citrate synthase and cytochrome c oxidase activities in skeletal muscle and liver tissue. The anabolic steroid had no significant effect on tumor growth in terms of weight progression, energy state, polyamine synthesis rate, cell division rate, and cell cycle cytocompartments. We conclude that anabolic steroid supplementation is not therapeutically beneficial in counteracting progressive weight loss in experimental cancer.

Studies of the internal flow process in polymers by 1H NMR microscopy at 500 MHz.

Nuclear Magnetic Resonance (NMR) microscopy was used to study the dynamics and diffusion of the internal water flow in erodible Hydroxypropylmethylcellulose (HPMC) and Polyvinylalcohol (PVA) polymers of pharmaceutical interest. Polymers in the form of tablets were hydrated at 37 degreesC continuously and monitored at regular intervals with a 500 MHz NMR microscope. The diffusion and spin-spin relaxation time (T2)-weighted images revealed that the gel layer was strongly attached to the core of the tablet in both HPMC and PVA polymers. Spatial distribution of self diffusion coefficients (SDCs) and T2 of the hydrating polymers were calculated from NMR microscopic images.

In vivo 31P nuclear magnetic resonance evidence of the salvage effect of ascorbate on the postischemic reperfused rat skeletal muscle.

The effect of 32 mM ascorbate on the time courses of phosphocreatine (PCr), inorganic phosphate (Pi), adenosine triphosphate (ATP) and intracellular pH in rat skeletal muscle during ischemia and reperfusion was investigated in vivo using 31P nuclear magnetic resonance (NMR) spectroscopy. Ascorbate was administered intravenously prior to induction of ischemia and at the time of reperfusion. The changes in PCr/(PCr+Pi), ATP and pH were similar in the non-treated and in the treated groups during ischemia. PCr/(PCr+Pi) fell to < 10% and ATP to approximately 30% of the preischemic values after 4 hours of arrested circulation, and pH decreased considerably. Postischemic reperfusion was followed continuously for 150 minutes. At the time of reflow, treatment with ascorbate had an immediate, positive effect on the recovery of high energy phosphates and pH. The level of PCr/(PCr+Pi) was 86% higher (p < 0.001) and the ATP level was 40% higher (p < 0.001) in the treated group than in the control group by the end of the reperfusion period. The results provide in vivo evidence for a salvaging effect of ascorbate on ischemia-reperfusion injury in skeletal muscle, probably owing to its antioxidant function and other ancillary effects, mainly its provision of additional buffer capacity.

Mouse extensor digitorum longus muscle preparation as a tool in nutrition research: a quantitative comparison to in vivo and cell culture experiments.

Incubated restrained and unrestrained extensor digitorum longus (EDL) muscles from adult non-growing mice were evaluated as a tool in non-steady state nutrition experiments. Energy state was determined by nucleotide determinations in muscles. Protein synthesis was estimated by the amount of L-[U-14C]phenylalanine incorporated into proteins, and protein balance was measured by tyrosine release from muscle proteins. Confluent cultured L6 rat muscle cells served as a reference system in steady state without hypoxia being sensitive to growth factors and regulatory peptides at physiologic concentrations. Irrespective of medium composition, incubated EDL muscles remained in negative protein balance, being unrelated to the resting tension of the incubated muscles. Energy-rich phosphates were not restored to normal levels during incubation, but protein synthesis was not attenuated by the decline in energy state. Fractional protein synthesis (0.05-0.15%/h) remained constant for up to 6 h of EDL incubation, and was comparable to protein synthesis in cultured confluent non-proliferating myocytes (0.20-0.30%/h) and to mixed leg muscles measured in vivo (0.10-0.20%/h). Protein synthesis in incubated EDL muscles reflected alterations in muscle peptide formation in vivo following either oral provision of food or parenteral injection of insulin. EDL muscles were sensitive to in vitro exposure to both insulin (60-125 microU/mL) and insulin-like growth factor 1 (IGF-1) (1000 ng/mL). The sensitivity to insulin seemed to be modified by the nutritional state (starved/fed) of the animals before sacrifice. Protein synthesis in EDL muscles was less responsive to serum-containing growth factors (IGF-1, epidermal growth factor [EGF], platelet-derived growth factor [PDGF]) compared to confluent L6 muscle cells, which probably reflected different receptor expression. Our results demonstrate that protein metabolism in incubated unrestrained mouse EDL muscles reflects in vivo protein metabolism.

Purine metabolism during microsurgical transfer of human skeletal muscle.

The effect of ischaemia followed by reperfusion on energy metabolism was studied in human skeletal muscle after microsurgical free transfer. Muscle biopsy specimens from 11 patients treated by free muscle transfer for facial palsy, injury to an extremity, or scalp defect were studied. The biopsy specimens were taken during ischaemia and after one hour of reperfusion, respectively. They were analysed for ATP to uric acid and creatine phosphate by high pressure liquid chromatography. Ischaemia lasting one or two hours affected the energy metabolism of the muscle cell as evidenced by a 50% reduction in creatine phosphate; a 20% reduction in ATP and in the energy charge; a 100% increase in inosine monophosphate, and a 700% increase in hypoxanthine and xanthine. Reperfusion for one hour improved these figures somewhat, and induced the production of uric acid. Skeletal muscle can therefore tolerate ischaemia for up to two hours in the clinical situation without permanent damage to the tissues.