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Triiodothyronine (T3) concentrations in plasma decrease during acute illness and it is unclear if this contributes to disease. Clinical and laboratory studies of T3 supplementation in disease have revealed little or no effect. It is uncertain if short term supplementation of T3 has any discernible effect in a healthy animals. Observational study of intravenous T3 (1 µg/kg/h) for 24 h in a healthy sheep model receiving protocol-guided intensive care supports (T3 group, n=5). A total of 45 endpoints were measured including hemodynamic, respiratory, renal, hematological, metabolic and endocrine parameters. Data were compared with previously published studies of sheep subject to the same support protocol without administered T3 (No T3 group, n=5). Plasma free T3 concentrations were elevated 8-fold by the infusion (pmol/l at 24 h; T3 group 34.9±9.9 vs. No T3 group 4.4±0.3, P<0.01, reference range 1.6 to 6.8). There was no significant physiological response to administration of T3 over the study duration. Supplementation of intravenous T3 for 24 h has no physiological effect on relevant physiological endpoints in healthy sheep. Further research is required to understand if the lack of effect of short-term T3 may be related to kinetics of T3 cellular uptake, metabolism and action, or acute counterbalancing hormone resistance. This information may be helpful in design of clinical T3 supplementation trials., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2024, Spandidos Publications.)

Intrahepatic islet transplantation for type 1 diabetes is limited by the need for multiple infusions and poor islet viability posttransplantation. The development of alternative transplantation sites is necessary to improve islet survival and facilitate monitoring and retrieval. We tested a clinically proven biodegradable temporizing matrix (BTM), a polyurethane-based scaffold, to generate a well-vascularized intracutaneous "neodermis" within the skin for islet transplantation. In murine models, BTM did not impair syngeneic islet renal-subcapsular transplant viability or function, and it facilitated diabetes cure for over 150 days. Furthermore, BTM supported functional neonatal porcine islet transplants into RAG-1-/- mice for 400 days. Hence, BTM is nontoxic for islets. Two-photon intravital imaging used to map vessel growth through time identified dense vascular networks, with significant collagen deposition and increases in vessel mass up to 30 days after BTM implantation. In a preclinical porcine skin model, BTM implants created a highly vascularized intracutaneous site by day 7 postimplantation. When syngeneic neonatal porcine islets were transplanted intracutaneously, the islets remained differentiated as insulin-producing cells, maintained normal islet architecture, secreted c-peptide, and survived for over 100 days. Here, we show that BTM facilitates formation of an islet-supportive intracutaneous neodermis in a porcine preclinical model, as an alternative islet-transplant site., Article Highlights: Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model., (© 2023 by the American Diabetes Association.)

Key Points: Maternal shift work increases the risk of pregnancy complications, although its effects on progeny health after birth are not clear. We evaluated the impact of a simulated shift work protocol for one-third, two-thirds or all of pregnancy on the metabolic health of sheep progeny. Simulated shift work had no effect on growth, body size, body composition or glucose tolerance in pre-pubertal or young adult progeny. Glucose-stimulated insulin secretion was reduced in adult female progeny and insulin sensitivity was increased in adult female singleton progeny. The results of the present study do not support the hypothesis that maternal shift work exposure impairs metabolic health of progeny in altricial species., Abstract: Disrupted maternal circadian rhythms, such as those experienced during shift work, are associated with impaired progeny metabolism in rodents. The effects of disrupted maternal circadian rhythms on progeny metabolism have not been assessed in altricial, non-litter bearing species. We therefore assessed postnatal growth from birth to adulthood, as well as body composition, glucose tolerance, insulin secretion and insulin sensitivity, in pre-pubertal and young adult progeny of sheep exposed to control conditions (CON: 10 males, 10 females) or to a simulated shift work (SSW) protocol for the first one-third (SSW0-7: 11 males, 9 females), the first two-thirds (SSW0-14: 8 males, 11 females) or all (SSW0-21: 8 males, 13 females) of pregnancy. Progeny growth did not differ between maternal treatments. In pre-pubertal progeny (12-14 weeks of age), adiposity, glucose tolerance and insulin secretion during an i.v. glucose tolerance test and insulin sensitivity did not differ between maternal treatments. Similarly, in young adult progeny (12-14 months of age), food intake, adiposity and glucose tolerance did not differ between maternal treatments. At this age, however, insulin secretion in response to a glucose bolus was 30% lower in female progeny in the combined SSW groups compared to control females (P = 0.031), and insulin sensitivity of SSW0-21 singleton females was 236% compared to that of CON singleton female progeny (P = 0.025). At least in this model, maternal SSW does not impair progeny metabolic health, with some evidence of greater insulin action in female young adult progeny., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

We created an excitotoxic striatal lesion model of Huntington disease (HD) in sheep, using the N-methyl-d-aspartate receptor agonist, quinolinic acid (QA). Sixteen sheep received a bolus infusion of QA (75 µL, 180 mM) or saline, first into the left and then (4 weeks later) into the right striatum. Magnetic resonance spectroscopy (MRS) and diffusion tensor imaging (DTI) of the striata were performed. Metabolite concentrations and fractional anisotropy (FA) were measured at baseline, acutely (1 week after each surgery) and chronically (5 weeks or greater after the surgeries). There was a significant decrease in the neuronal marker N-acetylaspartate (NAA) and in FA in acutely lesioned striata of the QA-lesioned sheep, followed by a recovery of NAA and FA in the chronically lesioned striata. NAA level changes indicate acute death and/or impairment of neurons immediately after surgery, with recovery of reversibly impaired neurons over time. The change in FA values of the QA-lesioned striata is consistent with acute structural disruption, followed by re-organization and glial cell infiltration with time. Our study demonstrates that MRS and DTI changes in QA-sheep are consistent with HD-like pathology shown in other model species and that the MR investigations can be performed in sheep using a clinically relevant human 3T MRI scanner., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

Melatonin is a pleiotrophic hormone, synthesised primarily by the pineal gland under the control of the suprachiasmatic nuclei (SCN). It not only provides a hormonal signal of darkness but also has neuroprotective properties. Huntington's disease (HD) is a progressive neurodegenerative disorder characterised by abnormal motor, cognitive and psychiatric symptoms. There is growing evidence, particularly from animal models, that circadian rhythms may also be disturbed in HD. We measured two circadian-regulated hormones, melatonin and cortisol, in plasma samples collected around-the-clock from normal and presymptomatic transgenic HD sheep (Ovis aries) at 5 and 7 years of age, to assess SCN-driven rhythms and the effect of genotype, sex and age. Melatonin-related precursors and metabolites (tryptophan, serotonin, kynurenine) were also measured by liquid chromatography (LC)-mass spectrometry (MS). At 5 years of age in both rams and ewes, plasma melatonin levels were significantly elevated in HD sheep. In ewes measured 2 years later, there was still a significant elevation of nocturnal melatonin. Furthermore, the daytime baseline levels of melatonin were significantly higher in HD sheep. Since increased melatonin could have global beneficial effects on brain function, we suggest that the increased melatonin measured in presymptomatic HD sheep is part of an autoprotective response to mutant huntingtin toxicity that may account, at least in part, for the late onset of disease that characterises HD., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Sepsis is a highly complex and often fatal syndrome which varies widely in its clinical manifestations, and therapies that target the underlying uncontrolled immune status in sepsis are needed. The failure of preclinical approaches to provide significant sepsis survival benefit in the clinic is often attributed to inappropriate animal disease models. It has been demonstrated that high mobility group box protein 1 (HMGB1) blockade can reduce inflammation, mortality and morbidity in experimental sepsis without promoting immunosuppression. Within this study, we explored the use of ovine anti-HMGB1 antibodies in a model of ovine septic shock incorporating intensive care supports (OSSICS). Results: Septic sheep exhibited elevated levels of HMGB1 within 12 h after the induction of sepsis. In this study, sepsis was induced in six anaesthetized adult Border Leicester × Merino ewes via intravenous instillation of E. coli and sheep monitored according to intensive care unit standard protocols for 26 h, with the requirement for noradrenaline as the primary endpoint. Septic sheep exhibited a hyperdynamic circulation, renal dysfunction, deranged coagulation profile and severe metabolic acidosis. Sheep were assigned a severity of illness score, which increased over time. While a therapeutic effect of intravenous anti-HMGB1 antibody could not be observed in this model due to limited animal numbers, a reduced bacterial dose induced a septic syndrome of much lower severity. With modifications including a reduced bacterial dose, a longer timeframe and broad spectrum antibiotics, the OSSICS model may become a robust tool for preclinical assessment of sepsis therapeutics., (Copyright: © Stevens et al.)

Key Points: Shift work impairs metabolic health, although its effects during pregnancy are not well understood We evaluated the effects of a simulated shift work protocol for one-third, two-thirds or all of pregnancy on maternal and pregnancy outcomes in sheep. Simulated shift work changed the timing of activity, disrupted hormonal and cellular rhythms, and impaired maternal glucose tolerance during early pregnancy. Gestation length was increased in twin pregnancies, whereas singleton lambs were lighter at a given gestational age if mothers were subjected to shift work conditions in the first one-third of pregnancy. Exposure to rotating night and day shifts, even if only in early pregnancy, may adversely affect maternal metabolic and pregnancy outcomes., Abstract: Shift workers are at increased risk of developing type 2 diabetes and obesity; however, the impact during pregnancy on maternal metabolism is unknown. Using a large animal model, we assessed the impact of simulated shift work (SSW) exposure during pregnancy on maternal circadian rhythms, glucose tolerance and pregnancy outcomes. Following mating, ewes were randomly allocated to a control photoperiod (CON 12 h light, 12 h dark) or to SSW, where the timing of light exposure and food presentation was reversed twice each week for one-third, two-thirds or all of pregnancy. Maternal behaviour followed SSW cycles with increased activity during light exposure and feeding. Melatonin rhythms resynchronized within 2 days of the photoperiod shift, whereas peripheral circadian rhythms were arrhythmic. SSW impaired glucose tolerance (+29%, P = 0.019) and increased glucose-stimulated insulin secretion (+32%, P = 0.018) in ewes with a singleton fetus in early but not late gestation. SSW exposure did not alter rates of miscarriage or stillbirth, although it extended gestation length in twin pregnancies (+2.4 days, P = 0.032). Relative to gestational age, birth weight was lower in singleton progeny of SSW than CON ewes (-476 g, P = 0.016). These results have implications for the large number of women currently engaged in shift work, and further studies are required to determine progeny health impacts., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Sepsis remains a significant health burden and a major clinical need exists for therapeutics to dampen the excessive and uncontrolled immune activation. Nuclear protein high mobility group box protein 1 (HMGB1) is released following cell death and is a late mediator in sepsis pathogenesis. While approaches targeting HMGB1 have demonstrated reduced mortality in pre-clinical models of sepsis, the impact of HMGB1 blockade on the complex septic inflammatory milieu and the development of subsequent immunosuppression remain enigmatic. Analysis of plasma samples obtained from septic shock patients established an association between increased HMGB1 and non-survival, higher APACHE II scores, and increased pro-inflammatory cytokine responses. Pre-clinically, administration of neutralising ovine anti-HMGB1 polyclonal antibodies improved survival in murine endotoxaemia and caecal ligation and puncture-induced sepsis models, and altered early cytokine profiles to one which corresponded to patterns observed in the surviving patient cohort. Additionally, anti-HMGB1 treated murine sepsis survivors were significantly more resistant to secondary bacterial infection and exhibited altered innate immune cell phenotypes and cytokine responses. These findings demonstrate that anti-HMGB1 antibodies alter inflammation in murine sepsis models and reduce sepsis mortality without potentiating immunosuppression.

The pronounced cachexia (unexplained wasting) seen in Huntington's disease (HD) patients suggests that metabolic dysregulation plays a role in HD pathogenesis, although evidence of metabolic abnormalities in HD patients is inconsistent. We performed metabolic profiling of plasma from presymptomatic HD transgenic and control sheep. Metabolites were quantified in sequential plasma samples taken over a 25 h period using a targeted LC/MS metabolomics approach. Significant changes with respect to genotype were observed in 89/130 identified metabolites, including sphingolipids, biogenic amines, amino acids and urea. Citrulline and arginine increased significantly in HD compared to control sheep. Ten other amino acids decreased in presymptomatic HD sheep, including branched chain amino acids (isoleucine, leucine and valine) that have been identified previously as potential biomarkers of HD. Significant increases in urea, arginine, citrulline, asymmetric and symmetric dimethylarginine, alongside decreases in sphingolipids, indicate that both the urea cycle and nitric oxide pathways are dysregulated at early stages in HD. Logistic prediction modelling identified a set of 8 biomarkers that can identify 80% of the presymptomatic HD sheep as transgenic, with 90% confidence. This level of sensitivity, using minimally invasive methods, offers novel opportunities for monitoring disease progression in HD patients.

Rationale: It is unclear how septic shock causes acute kidney injury (AKI) and whether this is associated with histological change., Objectives: We aimed to determine the nature and extent of changes in renal structure and function over time in an ovine model of septic shock., Methods: Fifteen sheep were instrumented with a renal artery flow probe and renal vein cannula. Ten were given intravenous Escherichia coli to induce septic shock, and five acted as controls. Animals were mechanically ventilated for 48 hours, while receiving protocol-guided parenteral fluids and a norepinephrine infusion to maintain mean arterial pressure. Renal biopsies were taken every 24 hours or whenever animals were oliguric for 2 hours. A renal pathologist, blinded to tissue source, systematically quantified histological appearance by light and electron microscopy for 31 prespecified structural changes., Measurements and Main Results: Sheep given E. coli developed septic shock, oliguria, increased serum creatinine, and reduced creatinine clearance (AKI), but there were no changes over time in renal blood flow between groups (P > 0.30) or over time within groups (P > 0.50). Renal oxygen consumption increased only in nonseptic animals (P = 0.01), but there was no between-group difference in renal lactate flux (P > 0.50). There was little structural disturbance in all biopsies and, although some cellular appearances changed over time, the only difference between septic and nonseptic animals was mesangial expansion on electron microscopy., Conclusions: In an intensive care-supported model of gram-negative septic shock, early AKI was not associated with changes in renal blood flow, oxygen delivery, or histological appearance. Other mechanisms must contribute to septic AKI.

Objectives: Triiodothyronine concentration in plasma decreases during septic shock and may contribute to multiple organ dysfunction. We sought to determine the safety and efficacy of administering triiodothyronine, with and without hydrocortisone, in a model of septic shock., Design: Randomized blinded placebo-controlled trial., Setting: Preclinical research laboratory., Subjects: Thirty-two sheep rendered septic with IV Escherichia coli and receiving protocol-guided sedation, ventilation, IV fluids, and norepinephrine infusion., Interventions: Two hours following induction of sepsis, 32 sheep received a 24-hour IV infusion of 1) placebo + placebo, 2) triiodothyronine + placebo, 3) hydrocortisone + placebo, or 4) triiodothyronine + hydrocortisone., Measurements and Main Results: Primary outcome was the total amount of norepinephrine required to maintain a target mean arterial pressure; secondary outcomes included hemodynamic and metabolic indices. Plasma triiodothyronine levels increased to supraphysiological concentrations with hormonal therapy. Following 24 hours of study drug infusion, the amount of norepinephrine required was no different between the study groups (mean ± SD μg/kg; placebo + placebo group 208 ± 392; triiodothyronine + placebo group 501 ± 370; hydrocortisone + placebo group 167 ± 286; triiodothyronine + hydrocortisone group 466 ± 495; p = 0.20). There was no significant treatment effect on any hemodynamic variable, metabolic parameter, or measure of organ function., Conclusions: A 24-hour infusion of triiodothyronine, with or without hydrocortisone, in an ovine model of septic shock did not markedly alter norepinephrine requirement or any other physiological parameter.

Insidious changes in behaviour herald the onset of progressive neurodegenerative disorders such as Huntington's disease (HD), sometimes years before overt symptoms are seen. Sleep and circadian disturbances are particularly disruptive symptoms in patients with neurological disorders, but they are difficult to measure in humans. Here we studied circadian behaviour in transgenic HD sheep expressing the full-length human huntingtin protein with an expanded CAG repeat mutation in the juvenile range. Young HD sheep with no other symptoms exhibited circadian behavioural abnormalities that worsened with age. The most obvious change was a disturbed evening behaviour reminiscent of 'sundowning' that is seen in some patients with dementia. There were no structural abnormalities seen with magnetic resonance imaging, even in 5-year-old HD sheep. Interestingly, detection of the circadian abnormalities depended upon their social grouping. Abnormalities emerged in sheep kept in an 'HD-only' flock, whereas the behaviour of HD sheep kept mixed with normal sheep was relatively normal. Sleep-wake abnormalities in HD patients are also likely to be hidden, and may precede overt symptoms by many years. Sleep disruption has deleterious effects, even in normal people. The knock-on effects of sleep-wake disturbance may exacerbate, or even cause symptoms such as irritability and depression that are common in early stage HD patients. HD sheep will be useful models for probing the mechanisms underlying circadian behavioural disorder in HD., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aims: To study immediate early gene, c-fos, expression as a marker of neural stress after whole of gestation exposure of the fetal mouse brain to mobile telephone-type radiofrequency fields., Methods: Using a purpose-designed exposure system at 900 MHz, pregnant mice were given a single, far-field, whole body exposure at a specific absorption rate of 4 W/kg for 60 min/day from day 1 to day 19 of gestation. Pregnant control mice were sham-exposed or freely mobile in a cage without further restraint. Immediately prior to parturition on gestational day 19, fetal heads were collected, fixed in 4% paraformaldehyde and paraffin embedded. Any stress response in the brain was detected by c-fos immunohistochemistry in the cerebral cortex, basal ganglia, thalamus, hippocampus, midbrain, cerebellum and medulla., Results: c-fos expression was of limited, but consistent, neuroanatomical distribution and there was no difference in immunoreactivity between exposed and control brains., Conclusion: In this animal model, no stress response was detected in the fetal brain using c-fos immunohistochemistry after whole of gestation exposure to mobile telephony.

Aims: To study the effect of mobile telephone exposure on blood-brain barrier (BBB) permeability in the immature brain., Methods: Using a purpose-designed exposure system at 900 MHz, pregnant mice were given a single, far-field, whole body exposure at a specific absorption rate of 4 W/kg for 60 min/day from day 1 to day 19 of gestation. Pregnant control mice were sham-exposed or freely mobile in a cage without further restraint and a positive control group with cadmium-induced BBB damage was also included. Immediately prior to parturition on gestational day 19, fetal heads were collected, fixed in Bouin's fixative and paraffin embedded. Disruption of BBB integrity was detected immunohistochemically using endogenous albumin as a vascular tracer in cerebral cortex, thalamus, basal ganglia, hippocampus, cerebellum, midbrain and medulla., Results: No albumin extravasation was found in exposed or control brains., Conclusion: In this animal model, whole of gestation exposure to global system for mobile communication-like radiofrequency fields did not produce any increase in vascular permeability in the fetal brain regions studied using endogenous albumin as a light microscopic immunohistochemical marker.

A total of 120 E mu-Pim1 heterozygous mice and 120 wild-type mice were exposed for 1 h/day 5 days/week at each of the four exposure levels in "Ferris-wheel" exposure systems for up to 104 weeks to GSM-modulated 898.4 MHz radiation at SARs of 0.25, 1.0, 2.0 and 4.0 W/kg. In addition, 120 heterozygous and 120 wild-type mice were sham-exposed; there was also an unrestrained negative control group. Four exposure levels were used to investigate whether a dose-response effect could be detected. Independent verification confirmed that the exposures in the current study were nonthermal. There was no significant difference in the incidence of lymphomas between exposed and sham-exposed groups at any of the exposure levels. A dose-response effect was not detected. The findings showed that long-term exposures of lymphoma-prone mice to 898.4 MHz GSM radiofrequency (RF) radiation at SARs of 0.25, 1.0, 2.0 and 4.0 W/kg had no significant effects when compared to sham-irradiated animals. A previous study (Repacholi et al., Radiat. Res. 147, 631-640, 1997) reported that long-term exposure of lymphoma-prone mice to one exposure level of 900 MHz RF radiation significantly increased the incidence of non-lymphoblastic lymphomas when compared to sham-irradiated animals.

Aims: To study the effect of long-term exposure to global system for mobile communication (GSM) radiofrequency fields on vascular permeability in murine brains., Methods: Using a purpose-designed exposure system at 900 MHz, mice were given a 60-minute far-field, whole body exposure on each of 5 days per week for 104 weeks at specific absorption rates (SAR) of 0.25, 1.0,2.0 and 4.0 W/kg. Control mice were sham-exposed or permitted free movement in a cage to evaluate any stress-related effects. Albumin immunohistochemistry was used to detect increased vascular permeability and the efficacy of the vascular tracer was confirmed with a positive control group exposed to a clostridial toxin known to increase vascular permeability in the brain., Results: In all exposed and control groups, albumin extravasation was minimal, often leptomeningeal, and was deemed insignificant as a maximum of three capillaries or venules in a given brain showed leakage from the very many blood vessels present in the three coronal brain sections., Conclusions: These results suggest that prolonged exposure to mobile telephone-type radiation produces negligible disruption to blood-brain barrier integrity at the light microscope level using endogenous albumin as a vascular tracer.

The effect of global system for mobile communication (GSM) radiofrequency fields on vascular permeability in the brain was studied using a purpose-designed exposure system at 898.4 MHz. Mice (n= 30) were given a single far field, whole body exposure for 60 minutes at a specific absorption rate of 4 W/kg. Control mice were also sham-exposed (n = 10) or permitted free movement in a cage (n = 10) to exclude any stress-related effects. Vascular permeability changes were detected using albumin immunohistochemistry and the efficacy of this vascular tracer was confirmed with a positive control group exposed to a clostridial toxin known to increase vascular permeability in the brain. No significant difference in albumin extravasation was detected between any of the groups at the light microscope level using the albumin marker.

The efficacy of continuous low-dose xylazine infusion following an initial loading dose in providing analgesia in sheep was examined using an algesimetry method based on a leg lifting response to an electrical stimulus. Sheep received a 5 mg intramuscular injection of xylazine followed by continuous infusion of intravenous xylazine (2mg/h) for 90 min. This treatment resulted in significant increases in the level of current required to elicit a leg lifting response (287% of baseline) and steady state analgesia was maintained from 10 min after the start of the infusion until the end of the experimental period. This protocol appears to be a simple and effective regimen for providing steady state analgesia in sheep.

Amyloid precursor protein (APP) immunocytochemistry was used to study axonal and neuronal changes in guinea pig brains exposed to tunicamycin. Substantial axonal injury was found in ischemic-hypoxic foci and more generally, but this injury was not readily appreciable in conventionally stained sections. Neuronal perikaryal APP expression was also widely distributed, possibly as an acute phase response to this neurotoxin.

The recent high prevalence of fatal bites by Brown snakes (Pseudonaja genus) has led to this study of venom yields from 66 brown snake milkings over 15 months. The amount of venom obtained from all species was higher than reported previously. Electrophoretic and Western blotting analyses of their venoms showed significantly lower avidity of Brown snake antivenom (BS-AV) for the prothrombin activator (PA) component (190 kD) than for other venom components, including the neurotoxins. The LD50 of P. inframacula has been determined for the first time. SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and Western blotting studies have shown that the Pseudonaja venoms contained proportionately more PA component than venoms of the Taipan (Oxyuranus scutellatus) or the Fierce snake (O. microlepidotus). Neutralization of the prothrombin activator of the Common Brown snake (P. textilis) (Pt-PA) by BS-AV was found to be time dependent and 40% remained unneutralized after 30 minutes incubation. Adult rats administered quantities of Pt-PA (IV) died with acute disseminated intravascular coagulation. Rats were made resistant to Pt-PA by preheparinization or by induction of tolerance to increasing quantities of Pt-PA. There is no evidence that Pt-PA has intrinsic toxicity apart from being a procoagulant. The improvement of BS-AV by addressing its deficiencies should be canvassed.

The effects of combining large doses of xylazine (1.1 mg per kg intravenously) with ketamine, methohexitone and thiopentone were studied in four experimental ponies. Onset of anaesthesia was more delayed after ketamine than after the barbiturates. Assessment of smoothness of induction and recovery indicated that all three combinations were effective and acceptable. Injection of xylazine was followed by atrioventricular (A-V) block which could be prevented by the prior administration of atropine. Blood pressure was well maintained with all three combinations of drugs. Arterial oxygen tension decreased as soon as the ponies became recumbent but there were no marked changes in arterial blood pH or carbon dioxide tension. Cardiac output was measured in one pony and was found to be least affected by ketamine. There was no great difference between the recumbency times after ketamine and methohexitone but thiopentone produced a significantly longer period of recumbency. In every instance the animals stood at the first attempt without struggling or excitement. The ability of the three drug combinations to produce surgically useful anaesthesia was not tested.

1. Choline production by various tissues of the sheep was measured by determining venous and arterial free choline concentrations in blood samples taken from various vessels in conscious multicannulated sheep. 2. Significant production of free choline occurred in the upper and lower body regions, and specifically in the heart, brain and hind-limb muscles of sheep, but there was no significant uptake or output of phosphatidylcholine across these tissues, as determined by arterio-venous differences. 3. In contrast, in the rat there were no significant arterio-venous differences in the concentrations of free choline or phosphatidylcholine across the hind-body. 4. Synthesis of phosphatidylcholine from endogenous phosphatidylethanolamine and S-adenosyl-L-[methyl-14C]methionine was measured in experiments in vitro using microsomal preparations from a variety of sheep and rat tissues. 5. The biosynthetic activity was highest in liver from sheep and rats, although the activity in sheep microsomal preparations was about one-quarter of that in rat microsomal preparations. 6. Microsomal preparations from sheep lung, kidney, gut epithelium, brain, heart and skeletal muscles also showed considerable biosynthetic activity, but in the rat the activity was virtually confined to the liver. 7. Overall, the results show a significant production of choline in extrahepatic tissues of the sheep, with skeletal muscle contributing some 60% of this extrahepatic activity. Thus the extrahepatic production of choline in the sheep, together with the extensive reutilization of bile choline, can explain the maintenance of the large endogenous body pool of choline in this species.

It has been proposed that acylation at the active site of plasmin is able to prevent its reaction with alpha 2-antiplasmin without affecting the fibrin affinity of the enzyme. To investigate the possibility that 99mTc-labelled acylplasmins are improved thrombus-detecting agents, six acylating agents were synthesised and their reaction with plasmin and the labelling of the products with 99mTc studies. Uptake of 99mTc-acylplasmins in an in vitro thrombus model was complicated by precipitation processes, which may in part account for the rapid blood clearance in rabbits and high liver uptake in mice injected with the compounds. Quantitative measurements using an in vivo rabbit thrombus model demonstrated that guanidinobenzoyl-plasmin exhibited nearly a threefold increase in thrombus uptake compared with non-acylated 99mTc-plasmin. The observed uptake is less than that obtained with 125I-fibrinogen at clinically useful time intervals post-injection but represents a significant advantage over the use of 99mTc-plasmin.