1.

HYPDXIC INDUCTION OF THE ANThAPOPTOTIC PROTEIN, BCL-2, IN TUMOUR CELLS IS MEDIATED BY VASCULAR ENDOTHELIAL. GROWTH FACTOR

MP BAiu, Thi HARMEY, DA Foiiy, DJ Bouct-uEi-HAEs Dimwr OF SURGERY, ROYAL COLLEGE. OF SURGEONS IN IRELAND EDUCATION AND RESEARCH CENTRE, BEAUMONT HOSPITAL, DUBLIN, IRELAND

Angiogenesis is essential for tumour growth and metastasis. Vascular endothelial growth factor (VEGF) is one of the most potent pro-angiogenic cytokines identified to date and has been implicated in a number of tumour types. Tumour cells under hypoxic conditions produce VEGF to stimulate angiogenesis. The ability of tumour cells to adapt to periods of hypoxia is important for their survival. The authors have previously shown that VEGF inhibits tumour cell apoptosis by up-regulating the anti-apoptotic protein, Bcl-2. In this study, the authors hypothesise that hypoxia increases tumour cell expression of Bcl-2 through a VEGF-mediated mechanism. Murine (4T1) and human (MDA-MB-231) mammary adenocarcinoma cells were grown under normoxic and hypoxic conditions. Hypoxia was achieved by culturing tumour cells in an anaerobic chamber and flushing with a purified gas consisting of 5% CO 21 95% N 2 . The resulting dissolved oxygen concentration was 2%. The effect of hypoxia on VEGF protein expression was examined by western blot analysis and enzyme-linked immunosorbent assay (ELISA). &1-2 expression was examined in response to hypoxia by western blot. The effect of anti-VEGF polyclonal antibody (1mg/mi) on hypoxia-induced Bcl-2 expression was also examined. Hypoxia significantly increased the expression of VEGF protein by both tumour cell lines (4T1; normoxia 0.146pg/mg protein, hypoxia 0.363pg1mg protein, . MDA; normoxia 6.73pg1mg protein, hypoxia 8.00pglmg protein, p.cO.OS). Expression of the anti-apoptotic protein, Bcl-2, was upregulated in response to a hypoxic growth environment. Neutralising antibody to VEGF blocked this hypoxia-induced expression of Bcl-2. Densitometry analysis was performed in each experiment to account for equal loading. For VEGF and Bcl-2 protein expression, densitometric values for cells grown under normoxia, normoxia+anti-VEGF, hypoxia and hypoxia+anti-VEGF were as follows: 4T1 VEGF; 100%, 92%, 138% 5 117%. 4T1 Bcl-2; 100%, 63% 1,125% 1 100%. MDA VEGF; 100%, 89%, 180%,124%. MDA Bcl-2; 100%, 75%,152% and 110%. The results show - that hypoxia, via the induction of VEGF, confers a survival advantage to tumour cells by increasing Bcl-2. Thus, in addition to its role as an endothelial cell mitogen, VEGF may act as a tumour cell survival factor in areas of tumour hypoxia.

2.

A • POTENTIAL ROLE FOR STATINS IN PREVENTING MICROVASCULAR INJURY?

M JOYCE, CJ Ktux, G CiiEN, G MCGitaL, DC WiNma, E KAY', DJ Boanait-Hxas DEPARTMENT OF SURGERY AND PATHOLOGY', ROYAL COLLEGE OF SURGEONS IN IRELAND, BREtmorr HosPrrAL, DUBLIN, Iita.&r

The UK small aneurysm trial has recently reported that pulmonary dysfunction is still a major cause of morbidity and mortality in patients undergoing elective abdominal aortic aneurysm repair. It is an injury characterised by microvascular leakage and neutrophil infiltration within lung tissue. In vitro experiments have shown that pravastatin has a positive effect on the vascular endothelium causing an upregulation of constitutive endothelial nitric oxide synthase (ecNOS). The authors hypothesise that pravastatM attenuates lower torso ischaemiareperfusion induced endothelial dysfunction in the lung and this. is associated with an upregulation of ecNOS.

Male Sprague-Dawley tats were randomised into three groups (n=7 per group). All animals were anaesthetised during experimentation using inhalational halothane. All groups including controls underwent laparotomy and exposure of the aorta. Ischaemia was induced by cross-clamping the infrarenal aorta for 30 minutes followed by reperfusion for two hours. The pre-treated group were administered pravastatin, 4mg/kg per day, over the preceding five days.. The parameters used to assess lung injury included: wet to dry lung weight ratio (W:D), myeloperoxidase activity (MPO), protein concentration (BALprot) and neutrophil count (BAL PMN) of bronchoaveolar lavage fluid. Western blotting was used to determine the expression of ecNOS within lung tissue.

I Pravastatin

Results

Control

I-R group

MPO activity (units/g) BALprot (mg/ml) BAL PMN (IHPF) Wet:dry ratio Densitometric values for ecNOS relative to control

4.97 (0.49) 159.32 (36.27) 4.94 (1.27) 4.96 (0.23)

7.67 (0.51)*1 5.7 (0.28) 544.87 (355)* 362.29 (41.14) 26.12 (3.2)*9.8 (1.42) 7.63 (0.31)*5.72 (0.46)

100%

78%

170%

Data is expressed as meanaSEM. Analysis with ANOVA, post-hoc scheffe test applied to positive results. *p<005 vs control and pravastatin group. These data show for the first time that pravastatin attenuates ischemia-reperfusion induced lung injury. This was associated with a dramatic upregulation of ecNOS in the treated group. The authors believe that pravastatin may have clinical applications in patients undergoing elective vascular procedures.

3. UPREGULA11ON OFCONSflIUTIVE ENDO1HEUAL NITRIC OXIDE SYNTHASE, BY A HMG-COA REDUCTASE INHIBITOR, ATTENUATES ISCHAEMIA-REPERFUSION INDUCED RENAL DYSFUNCTION M JOYCE, CJ KELLY, G C,ia, DC WnsrrEx, G McGREAL, C CONDRON, A LEAH, DJ BOUcFIIER-HAYES. DEPARTMENT OF SURGERY, Rovu. COLLEGE OF SURGEONS IN IRELAND, Bawrior HosPITAL, DUBLIN, IRELAND Renal dysfunction due to ischaemia-reperfusion may occur following suprarenal aortic cross-clamping or kidney transplantation. Inhibition of constitutive endothelial nitric oxide synthase (ecNOS) has been implicated in renal rejection. Independent of their lipid lowering effects, statins provide protection against hypoxia, in vitro by upregulating constitutive endothelial nitric oxide synthase (ecNOS) production. The authors hypothesised that pravastatin would attenuate ischaemia-reperfusion renal injury. Male Sprague-Dawley rats (n=21) were randomised into three equal groups. Animals were anaesthesised for the duration of the experiment with inhalational halothane. The ischaemia-reperfusion group had a unilateral nephrectomy with contralateral clamping of the renal vascular pedicle for 45 minutes followed by reperfusion for two hours. The treatment group received pravastatin for five days (4mg/kg per day) and were also subjected to an ischaemia-reperfusion injury. The control group had a unilateral nephrectomy only. The parameters used to assess renal dysfunction included: urine production, glomerular filtration rate (GFR) and total urinary protein leakage (TUPL). Western blotting was used to determine the expression of ecNOS within renal tissue. Results Control Urine production (ml/hour) GFR (ml/hour) TUPL (g/l) Densitometric values for #eCNOS relative to control

0.61 (0.13) 14.14 (0.9) 1.67 (0.12) 100 (8)%

I-R group

Pravastatin

(ØØ5)*

0.62 (0.2) # 0.11 1.31 (05)*8.6 (0.97)# 7.21 (1.3)*3.76 (0.7)# 74 (10) % 170 (13)%*

Data is expressed as mean (SEM). Analysis with ANOVA, post-hoc scheffe test. control; #p<0.05 vs IR group and pravastatin group. 174

*<005

vs

Irish Journal of Medical Science • Volume 170 • Number 4 • Supplement 3

Ischaemia-reperfusion causes a renal injury manifested by oliguria, decreased GFR and increased protein leakage. Pravastatin attenuates IRinduced renal injury by mechanism of action involving upregulation of ecNOS. Thus, pravastatin may play a role in modulating renal impairment following aortic or transplantation surgery.

5.

PROTEINS ABERRANT IN ALZHEIMER'S DISEASE MODULATE THEIR EXPRESSION DURING MEMORY CONSOLIDATION IN THE HIP POCAMPAL DENTATE GYRUS OF ADULT WISTAR RATS

L CoNsoY, CM REGAN

4. UPPER AIRWAY PRESSURE-FLOW RELA11ONSHIPS AND ELECTROMYOGRAM ACTIVITY DURING CENTRAL APNOEA IN AWAKE GOATS KD O'HaoiAi 1 , JK HERivtAN 2 , GE BISGARD 2 DEPARTMENTS OF HUMAN ANATOMY AND PHYSIOLOGY', UNIVERSITY COLLEGE DUBLIN, IRELAND, COMPARATIVE BIOSCIENCES 2 , UNIVERSITY OF WISCONSIN-MADISON, USA

The authors recently demonstrated that central apnoeas induced by systemic administration of clonidine in awake goats are associated with tonic activation of laryngeal and pharyngeal adductor muscles resulting in active airway closure.' The authors undertook the present study to establish whether active closure of the upper airway is a common feature of central apnoea that is independent of the initiating cause of the apnoea. Eleven adult female goats (47-8 1kg) were studied. At* least two weeks prior to study, electromyogram (EMG) wire electrodes were inserted into the middle pharyngeal constrictor (MPC) muscle under general anaesthesia (1-5% halothane, 40% nitrous oxide, balance oxygen). The goats were trained to stand in a stanchion whilst wearing a tight-fitting facemask. During several experimental recording sessions in each animal the authors recorded inspired and expired airflows, subglottic tracheal pressure, mask (mouth) pressure and MPC EMG activity. Pressure-flow relationships and EMG activity were analysed during the prolonged expiratory duration of spontaneous or induced central apnoeas and were compared to data obtained from the five consecutive control breaths immediately preceding the central apnoea. . The authors recorded a total of 382 spontaneous central apnoeas; 169 were preceded by an augmented breath or sigh. In separate trials on different days, central apnoea was induced by systemic administration of dopamine (10-50mg/kg, 96 trials) or by raising the inspired fraction of 0 2 from 0.10 to 1.00 during poikilocapnic conditions (17 trials). Apnoeas of variable duration were observed in all animals. Continuous tonic activation of the MPC at a level significantly greater than that observed in control breaths (p
165-76.

DEPARTMENT OF PHARMACOLOGY, CONWAY INSTITUTE, NATIONAL UNWERSITY OF IRELAND, DUBLIN, IRELAND

Low-density lipoprotein receptor related protein (LRP) is a receptor for apolipoprotein E (Apo E), a carrier molecule for low- and high-density lipoproteins, of which certain alleles predict risk of Alzheimer's disease (AD). The LRP-Apo E complex is known to facilitate endocytosis, a mechanism by which membrane proteins are recycled and/or degraded.' amyloid precursor protein (APP), a synaptic protein from which amyloid fragments have been associated with neurodegenerative events in AD, may be degraded by endocytosis. The authors investigated if APP expression was related to LRP-Apo E complex formation during the periods of increased endocytosis and synapse formation that occur transiently in the hippocampal dentate gyrus at two to four hours and six hours respectively, following avoidance conditioning in the adult Wistar rat. 2 Animals were trained in a step-through, light-dark passive avoidance paradigm and sacrificed at increasing post-training times.2 The hippocampus was quickly excised and the dentate gyrus dissected and homogenised in 0.32M sucrose. The post-training expression of dentate APP, LRP or Apo E was determined by immunoblotting procedures using specific monoclonal antibodies and quantitative densitometric analysis. 2 APP protein expression was found to be 'significantly reduced in the two to four hours post-training period as compared to that observed in naive animals (two hours 73.84±7.14% n=4; four hours 92.04±0.3% n3; p
6. DOES AN EXERCISE-INDUCED CHANGE IN SERUM [IGFBP-.I]:[IGFBP3] ALTER IGF/IGFBP-I BINDING IN MAN? R KEOGH, PM JAKEMAN HUMAN SCIENCE RESEARCH CENTRE,

UNivaitsrn' OF UMERICK, Isnun

Insulin-like growth factor (IGF) bioactivity in serum is principally

Irish Journal of Medical Science • Volume 170 • Number 4 • Supplement 3

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Biomedical Section Meetings 5th January 2001

GPHb/IUa immunoprecipitated, it was found to be cross-linked to NGR. This confirmed that NGR does bind to GPIIb/IIla. However platelets from patients with Glanzmann's thrombasthenia (GPIlbfllIa deficient platelets), were found to adhere as effectively as normal platelets to both fibrinogen and S. sanguis. These results suggest that resting platelets adhere to fibrinogen in an NGR-dependent manner, which would appear to be distinct from GPIIbfIlla. The vitronectin receptor (avb3) antibody, LM609 partially inhibits platelet adhesion to S. sanguis 13379. Although specific avb3 antagonists failed to inhibit the adhesion. To identify this receptor, S. sanguis was incubated with platelets, the platelets were lysed and the bound proteins eluted, of which six bands were identified. Future experiments will involve a trypsin digestion of these proteins and then identified via matrix-assisted laser desorption/ionisation (MALDI) in combination with time-of-flight (TOF) mass analyser. From this it is hoped that each of the bands will be sequenced and the NGR receptor identified from one or more of these proteins.

regulated by two of the six known binding proteins (IGFBPs). Ninety per cent of IGF are sequestered by a stable concentration (1 OOnM) of growth hormone-dependent IGFBP-3, the remainder associated with a fluctuating concentration (2-22nM) of the insulin-dependent IGFBP-I. Distribution of IGF between the binding proteins can be determined empirically or theoretically from the concentration and affinity of the binding proteins for IGF. Association constants for IGF range from 5.6x10 9 to 4.4x10 10 M and 3.6x10 9 to 2.3x10 10 M for IGFBP-3 and IGFBP-I respectively.' Prolonged exercise significantly increases (2-20 fold) the [IGFBP-I1 and hence the ratio of [IGFBPI]:[lGFBP3]. 2 This study examined whether the increase in [IGFBP-J] relative to [IGFBP-3] resulted in a redistribution of IGF between binding proteins as predicted by the theoretical model. With ethical approval, blood samples were collected from 14 healthy (age 24±5 years, meantSD) volunteers pre- and post-prolonged exercise at 60% maximal heart rate. IGF-I & II, IGFBP-I and IGFBP-3 were measured by ELISA (DSL, London). Immunoprecipitation of IGFBP-I allowed for the determination of IGF bound to IGFBP-I. The %IGF bound to IGFBP-I experimentally derived and calculated are presented in Table 1. The serum [IGFBP-I]:[IGFBP-3] ranged from 0.005 to 0.383. The %IGF-I bound to IGFBP-I was highly variable within this range -and did not conform to the theoretical values (r 2 =0.049). However, in selected samples containing a high [IGFBP-I]:[IGFBP-3], there is evidence of a substantial redistribution of IGF. Data are presented in Table 1.

8. COMPARISON OF WOOL ZINC CONTENT IN TEXEL EWES FOLLOWING SUPPLEMENTATION WITH ZINC SULPHATE AND BIOPLEX ZINC JP RYAN, P KEARNS, T QUINN DEPARTMENT OF VETERINARY PHYSIOLOGY AND BIOCHEMISTRY, VETERINARY COLLEGE, UNIVERSITY COLLEGE DUBLIN, NUI, DUBLIN, IRELAND

Table 1. Theoietied and empirical values for %IGFbound to IGFBP-I (range) n= 27. % bound to BP-! empirical % bound to BP-I theoretical References:

I

IGF-I

IGF-II

0.0-14.1 2.03-50.40

0.0-5.7 0.04-7.12

Forty adult Texel ewes were kept outdoors and fed SOOg grass nuts per day plus silage ad libitum for the 100 day duration of the trial (February to May, 1999). The sheep were divided into four groups of 10 at random and each group was supplemented with additional zinc sulphate or Bioplex zinc as indicated in the table. Bioplex zinc is a chelate of amino acids and short chain peptides from hydrolysed soya protein, containing 15% zinc by weight, as supplied by Alitech (Ireland) Ltd. Samples of wool were taken from each sheep at the start and at the end of the trial and the total zinc in each sample was determined using a Perkin Elmer graphite furnace atomic absorption spectrophotometer (HGA 800/AAnalyst 100). The results expressed as mean zinc levels±SE for each group of 10 observations are summarised in the following Table

1. Westwood M, Gibson JM, White A. Purification and characterisation of the insulin-like growth factor-binding protein-1 phosphoform found in normal plasma. Endocrinol 1997; 138: 1130-36. 2. Keogh R, Jakeman PM. Exercise-induced changes in insulin-like growth factor (IGF) bioavailability in man: evidence of reduced affinity of IGFBP-I for IGFs. J Physiol 1999; 523: 231.

7. PLATELET ADHESION TO FIBRINOGEN - A ROLE FOR A NOVEL NGR-DEPENDENT RECEPTOR

Integrins are the major family of cell adhesion molecules. To date, two ligand recognition sequences with the amino acid sequence, Arg-GlyAsp (RGD) and Asn-Gly-Arg (NGR, a novel vitronectin receptor recognition motif) have been identified. Previous work has shown that platelets bind to Streptococcus sanguis in an RGD/NGR dependent manner. Selective GPIIb/IIIa antagonists failed to inhibit the adhesion of platelets to S. sanguis, suggesting the existence of an NGR-dependent receptor on platelets. The authors further investigated the function of this receptor. Fibrinogen (50mg/mI) was coated on a 96 well ELISA plate at 37°C for 2 hours. The number of adherent platelets was determined by assaying the intracellular acid phosphatase content. S. sanguis strain 133-79 was coated on a petri dish for 2 hours, platelets added, lysed and the supernatant eluted with 1M NaCl and separated by SDS PAGE. Gels were then stained with either Silver or Coomassie Blue stain. NGR, RGD and orbofibn (GPIIbI[IIa antagonist) inhibited the adhesion to fibrinogen. This suggested that GPIIbflIla is the NGR receptor on fibrinogen. When platelets were cross-linked with NGR and

176

IBiP1ex zinc

Zinc sulphate

Supplement

C McMANUs, S KEiuuGAr, P HARRIOT, D FirzGEaAw, D Cox DEPARTMENr OF Cuicu. PHARMAcoLoGY, Royi. COLLEGE OF SURGEONS IN IRELAND, Dusui, Iitaur.o

75mg 150mg Zn/sheep/day Initial wool Zn (mgfkg) 173±34 148±25 425 ±49* 411 ±40** Final wool Zn (mg/kg) Wool Zn increase (mg/kg) 252±63 263±47 Plasma Zn increase (mg/I) 1 0.12tO.12c 0.23±0.13a

75mg 150mg 161t33 192±38 4737* 343 ±28* 247±56 162±40 P.S0±0.lOd 0.52z0.08b,e

All treatments gave significantly higher wool zinc contents than the corresponding initial samples, as indicated by Student's paired twotail t-test. * p<0.005 p<0.0005. Comparisons between groups were also determined using Fischer's protected least significance difference (PLSD) ;

test: ab p<0.1; cd p<0.05; cc p<0.01.

The authors previously showed that Bioplex zinc is more bioavailable than zinc sulphate as determined by overall increases in plasma zinc levels.' In this study, however, no significant differences were observed between any of the wool groups indicating no differences in bioavailability of wool zinc in the supplement range studied, despite increases in plasma zinc, suggesting that zinc may be incorporated equally from both sources into wool. Reference:

1. Ryan JP, Kearns P, Quinn T. Comparison of plasma zinc levels in sheep following supplementation with zinc sulphate and Bioplex zinc. Biochem Soc Trans 2000; 28:

p

A26

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MEASUREMENT OF SEVOFLURANE IN ARTIFICIAL CEREBROSPINAL FLUID BY GAS CHROMATO GRAPHIC HEADSPACE ANALYSIS

9.

DA CRONIN, J McHiE 2 , D MoRIARTY 2 , C HERRON3 , JJ O'CoNoR3 DEPARTMENTS OF FOOD, SCIENCE, UNIVERSITY COLLEGE DUBLIN, ANAESTHETICS', MATER MIsEMcoiwiE HOSPITAL, HUMAN ANATOMY AND PHYSIOLOGY', UNIVERSITY COLLEGE, DUBLIN, IRELAND

Sevoflurane (sevorane) is now one of the most commonly used general anaesthetic agents in Ireland and is known to affect synaptic transmission at clinically relevant concentrations. However, its precise site and mechanism of action at central synapses has yet to be elucidated. The authors have used rat brain slices in submerged artificial cerebrospinal fluid (aCSF)1 that can be bubbled with sevoflurane via a vaporiser. However, the exact concentration of sevoflurane in aCSF and thus acting at central synapses cannot be relied on from vaporiser settings. The authors have used gas chromatographic (GC) headspace methodology for the determination of the concentration of sevoflurane in aCSF perfusate samples. Sevoflurane from 0.5 to 2ml aliquots of perfusate was allowed to vaporise within a sealed flask held at 25°C. After equilibration, imI vapour samples were injected with a Hamilton series 1000 gas tight syringe onto a GC column for measurement of the sevoflurane content of the headspace. A 2m-glass column packed with S% OV-17 on silanised Chromosorb W, connected to a flame ionisation detector and operated at a temperature of 30°C, was used for the analysis. Peak areas were recorded using a Spectra Physics ChromJet computing integrator. A calibration graph for a series of standards was linear over the concentration range 0-120mg/l sevoflurane (r2=0.998, n=3). Vapoiiser settings of 0.5, 1.0, 2.0, 3.0 and 4.0% perfused onto the brain slices gave rise to a linear relationship between the gas phase concentration of the analyte and the amount dissolved in the aCSF solution (r 2 =0.997; n=3). Inhibition of excitatory post-synaptic potential amplitude was 5.1±0.9, 19.84:1.2 and 61.4±4.7% control at concentrations of 0.11±0.01, 0.45±0.05 and 0.74a0.09mM sevoflurane (n=6, mean*SEM). In conclusion, this method for determination of sevoflurane concentration in aCSF perfusate is rapid and simple to perform. It eliminates the need for solvent extraction of the compound and thereby the requirement for the use of a selective (electron capture) GC detector to measure it without interference from solvent or solvent impurity peaks. Reference: 1. Frizelle HP, Moriarty DC, O'Connor JJ. The combined efects of halothane and lamotrigine on excitatory postsynaptic potentials and use-dependent block in the rat dentate gyrus in vitro. Anesth Anaig 1999; 89: 496-501.

10.

BRACHIAL PLEXUS BLOCK: A NEW APPROACH

AF Btcim1, T FARRELL 1 , C Cooy 2 DEPARTMENT OF ANATOMY 1 , Royi. COLLEGE OF SURGEONS IN IRELAND AND DPamiarr OF ANAEsThEsiA2, ADELAIDE AND MEATH HOSPITAL, DUBLiN, IRELAND

complications, would involve less manipulation of the arm and could be used in the sitting or lying position. The viability of this new approach was demonstrated by the injection of SO axillae in 25 cadavers with small amounts of methylene blue (0.5ml or lml). Well-defined anatomical landmarks were used to direct the needle to the target site. With the arm adducted and the elbow flexed to 90 degrees the needle was inserted at a point 2cm lateral to the midpoint of the lateral border of the scapula, where the lateral border of the scapula is understood as stretching from the posterior tubercle of the acromion process to the inferior angle. The needle was directed towards a point 2cm below the tip of the coracoid process. The appropriate depth of insertion was dependent upon the physique of the cadaver and was typically about 7cm. The area of distribution of the injected material was established by dissection of the axilla from the anterior approach. In 80% of the specimens all cords of the plexus were stained with dye, and in all cases some staining of the plexus was achieved. In those cases where full staining was not achieved the shoulder region was either particularly bulky or only 0.5ml of dye was used. These results suggest that there is potential for this new approach to be developed in the clinical context, where it will add to the armamentarium of the anaesthetist. This technique of regional anaesthesia may be used in contexts where previously a general anaesthetic had been indicated.

11. THE AX1LLARY SHEATH AND ITS CONTENTS AF Brn, T FARRELL DEPARTMENT OF ANATOMY, RoY.i. COLLEGE OF SURGEONS IN IRELAND, DUBLIN, IRELAND

The axillary sheath remains a controversial topic in anatomy, with little agreement found among the standard texts on either its extent or its contents. In particular, various authors hold contradictory opinions regarding the axillary vein, which some' describe as lying within the sheath while othersz claim that the sheath surrounds only the artery and the brachial plexus. This controversy may partly be due to the difficulty of distinguishing fascial layers in embalmed tissue. To provide a definitive resolution to this controversy 20 sheaths in 10 cadavers were meticulously dissected, specifically looking for the fascial layers that constitute the sheath. In two of the cadavers the dissections were carried out prior to embalming. The dissections especially concentrated on the area posterior to pectoralis minor where the cords of the brachial plexus are closely related to the axillary artery. As the brachial plexus is often used for regional anaesthesia, this area behind pectoralis minor is of particular interest since local anaesthetic injected here may be expected to reach all parts of the brachial plexus. In all cases, the sheath was found to invest the axillary vein as well as the artery and the nervous plexus. Furthermore, all of the contents of the sheath were found to be enveloped in separate compartments. References: 1. Moore KL. Clinically Oriented Anatomy 1992: 528. 2. McMinn RMH. Last's Anatomy 1994: 423.

Regional anaesthesia adequate for surgery of the upper limb may frequently be achieved by brachial plexus blockade. Three standard approaches to the plexus, the interscalene, the infraclavicular and the axillary, are described. However, these approaches all have disadvantages with considerable risk of pneumothorax, phrenic nerve or stellate ganglion block, and difficulty in positioning the arm of the injured patient. A new approach from the posterior aspect of the axilla would avoid or lessen the risk of these

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177

12. EFFECTS OF DIETARY CREATINE SUPPLEMENTATION ON ISOLATED RAT SKELETAL MUSCLE CONTRACTILE PROPERTIES M GAGNON', M MAGUIRE2 , M MACDERM0U 1 , A BRADFORD 1 Dumtrrr OF PHYSIOLOGY', ROYAL COLLEGE. OF SURGEONS IN IRELAND, DUBLIN, SLEEP DISORDERS PROGRAM 2 , ENDOCRINE DivisIoN, RFB 485, BRIGHAM AND WoitEri's HOSPITAL, HARVARD MEDICAL SCHOOL, BOSTON, MA, USA

In humans, dietary creatine supplementation increases creatine and phosphocreatine concentrations in skeletal musclel 2 and improves isokinetic torque production and fatigue resistance. 1 However, some studies have reported no effect of creatine supplementation on exercise performance 2 Rats were fed a standard diet (n=10), 250-270mg/kg per day of creatine for 10 days (n=8) or -guanidinopropionate, which depletes muscle creatine, as 0.8% of diet for seven days (n=6). The potential benefits of creatine on limb, respiratory and upper airway muscles were examined by measuring contractile properties in isolated extensor digitorum longus, diaphragm and sternohyoid muscle respectively in Krebs solution at 30°C. Creatine treatment had no effect on specific twitch and tetanic tension, contraction time, half-relaxation time, twitch/tetanic tension ratio, the tension-frequency relationship or fatigue in all three muscles. 3-Guanidinopropionate had no effect on the twitch and tetanic tension, contraction time, half-relaxation time, twitch/tetanus tension ratio or tension-frequency relationship but significantly increased (ANOVA, p
13. CALCIUM INVOLVEMENT IN NEUROKININ A AND CARBACHOL-INDUCED CONTRACTIONS OF GUINEA PIG COLONIC SMOOTH MUSCLE IN VITRO A O'RIoRDAN, T QUINN, A BIRD DEPARTMENT OF VETERINARY PHYSIOLOGY AND BIOCHEMISTRY, VETERINARY COLLEGE, UNWER5rrY COLLEGE DusuN, NATIONAL UNIVERSITY OF IRELAND

In the guinea pig colon, the major excitatory neurotransmitters are acetylcholine and the tachykinins. Of the tachykinins, neurokinin A (NKA) and substance P have been shown to be potent contractile agonists of guinea pig colonic smooth muscle.1 In this study, the authors compared the contribution of voltage-operated calcium channels which can be blocked by nifedipine (an L-type voltage gated calcium channel antagonist), receptor-operated calcium channels, which can be blocked by SK&F 96365 (a putative receptor and voltage-operated calcium channel antagonist) and intracellular calcium to contractions induced by NKA and carbachol in isolated strips of guinea pig colonic circular smooth muscle in vitro.

178

Colonic circular smooth muscle strips were suspended under ig of tension in a physiological solution at 37*C gassed with 95% 0 2/5% CO 2 . Mechanical activity was recorded isometrically. Statistical analysis was carried out using the Student's unpaired two tailed t-test or analysis of variance, when applicable. NKA (1010-3x10 7 M) produced concentration-dependent contractions of colonic smooth muscle which were abolished by nifedipine (10 M) and in a calcium-free solution containing 1mM EDTA (p<0.0005). SK&F 96365 (lOS t4) attenuated the contractile response to NKA (p
14. KCL AND N-METHYL D-ASPARTATE INDUCED ACI1VA110N OF MEDIAL PREFRONTAL - VENTRAL TEGMENTAL GLUTAMATE TRANSMISSION DIFFERENTIALLY AFFECTS VENTRAL TEGMENTAL fly-AMINO BUTRYIC ACID TRANSMISSION IN CONSCIOUS RAT BRAIN M HARm, WT O'CooR Di'utmm-rr OF HUMAN ANATOMY AND PHYSIOLOGY, CONWAY INSTITUTE OF BIOMOLECULAR AND BIOMEDICAL RESEARCH, UNivEitsrry COLLEGE DUBLIN, IRELAND

Carlsson et a!' proposed that mesolimbic dopamine is controlled by cortiofugal glutamate neurons, either directly or via ventral tegmental area (VTA) y-amino butyric acid (GABA) interneurons acting as accelerators and brakes respectively. Normally there is a balance between the accelerator and the brake, however enhanced mesolimbic dopamine activity (i.e. hyperdopaminergia) may be induced by failure of the brake i.e. the VTA GABA interneurons - a condition that may underlie schizophreniform symptomatology. In the present study the authors looked for evidence to support this hypothesis in intact conscious brain. Thus, they employed dual probe microdialysis whereby one microdialysis probe (4mm membrane length, OSmm outer diameter; Carnegie Medicine AB, Stockholm) was inserted into the medial prefrontal cortex (mPFC) and another into the ipsilateral VTA (1mm membrane) under isoflurane/air (5%-1.9%, 0.41/mm.) anaesthesia to compare the effects of intra-mPFC perfusion with the depolarising agent KCI and the glutamate receptor agonist N-methyl Daspartate (NMDA) on mPFC-VTA glutamate transmission, by monitoring glutamate and GABA release in the VTA. These studies were performed 24 to 48 hours after probe implantation in intact conscious brain. Data are expressed as meanaSEM (n=4-8) and statistical analysis was performed using a two tailed paired Student's t-test. The experiments were approved by the Department of Health (B100/3013). Basal dialysate VTA glutamate and GABA levels were 0.232±0.037pM and 1 1.74a1.3nM respectively. Intra-mPFC KCI (100mM, 20 minutes) induced a rapid and reversible increase in VTA glutamate (+71.48%±14.29%, p<0.001 vs control) but not VTA GABA release (-5.26%±2.65%, p=0.681). In contrast, mPFC NMDA (10, 100 and 300mM, 20 minutes) was associated with a rapid and reversible concentration-dependent increase in VTA glutamate (+8.11%:t4.48%,

Irish Journal of Medical Science 9 Volume 170 • Number 4 • Supplement 3

p=0.202; +109.56%±17.94% and +72.57%±18.99% both p<0.01) and GABA release (+4.84%±4.47%, p=0.13 +29.99%±11.95% and +37.35%±9.22% both p
15. AMPHETAMINE INHIBITS LOCAL y-AMINO BUTRYIC ACID AND INCREASES LOCAL AND INTRA-MEDIAL PREFRONTAL CORTICAL - VENTRAL TEGMENTAL GLUTAMATE TRANSMISSION IN INTACT CONSCIOUS RAT BRAIN PJ MAGEE, B NicNioc,nL, M HARm, WT O'Cor-moR DEPARTMENT OF HUMAN ANATOMY AND PHYSIOLOGY, CONWAY INSTITUTE OF BIOMOLECULAR AND BIOMEDICAL RESEARCH, UNIVERSITY COLLEGE DUBLIN, IRELAND

Recent evidence suggests that disorders of the mesocortical and mesolimbic nerve circuits are implicated in schizophrenia. Mesocortical and mesolimbic dopamine pathways project from the A N dopamine cell bodies in the ventral tegmental area (VTA) to directly and indirectly (via the nucleus accumbens-ventral pallidum and inediodorsal thalamus) influence glutamate and 1-amino butyric acid (GABA) transmission in the medial prefrontal cortex (mPfc). These nerve circuits are completed by a glutamate projection from the mPfc to the VTA. These nerve pathways are extremely sensitive to antipsychotic agents. In this study, the authors employed microdialysis whereby one microdialysis probe was inserted into the mPfc and another into the ipsilateral VTA in intact conscious rat brain in order to investigate the effects of intra-mPfc perfusion with the indirect dopamine agonist, amphetamine (1 and 1000pM, 60 minutes) on local and VTA dialysate glutamate and GABA release. Data are expressed as meanaSEM (n=47) and statistical analysis was performed using a one tailed paired Student's t-test. The experiments were approved by the Department of Health (B100/3069). Basal dialysate glutamate and GABA levels in the mPfc were 0.42±0.05pM (n=4) and 48.57±4.96nM (n=S) respectively and in the VTA were 0.27:0.06pM (n6) and 17.32±4.49nM (n=S). Intra-mPfc perfusion with 1000pM amphetamine induced a rapid (20 minutes) increase in local glutamate release (+53.01±17.87%, p=0.03 vs pretreatment levels) which was maximal 80 minutes after amphetamine perfusion commenced. This increase in local glutamate release was paralleled by a rapid reversible decrease in local GABA release (-55.41:4.2%, p<0.001 vs pre-treatment levels). Intra-mPfc amphetamine was also associated with a delayed increase in VTA glutamate (+54.30a19.55%, p=0.03 vs pre-treatment levels) but not GABA release. Amphetamine (ljiM) perfusion did not influence glutamate or GABA release in either the mPfc or the VTA. Thus the authors show that amphetamine, which induces symptoms of schizophrenia in otherwise normal individuals, suppresses local mPfc GABA mediated inhibition and activates local and mPfc-VTA glutamate transmission. This data suggests that the

'core defect' in schizophrenia may be a loss of mPfc GABA interneurons 1 leading to excessive mPfc-VTA glutamate transmission and to mesolimbic hyperdopaminergia. These findings aid the understanding of schizophrenia at the network level and of how amphetamine may exert its psychoactive effects. Reference: 1. Benes FM, McSparren J, Bird ED, Sangiovanni JP, Vincent SL. Deficits in small interneurons in prefrontal and cingulate cortices of schizophrenic and schizoaffective patients. Arch Gen Psychiatry

1991; 48: 996-1001.

16.

EVIDENCE FOR A DIFFERENTIAL NEURONAL POOL FOR STRIATAL ASPARTATE AND GLUTAMATE IN INTACT BRAIN

B Nic NiocAiLL, WT O'CoroR DEPARTMENT OF HUMAN ANATOMY AND PHYSIOLOGY, CONWAY INSTITUTE OF BIMOLECULAR AND Bioizoiciu. RESEARCH, UNIVERSITY COLLEGE, DUBLIN, IRELAND The neostriatum represents the major input region of the basal ganglia where afferent information including a significant nigrostriatal dopamine input is processed by local interneurons. Striatal glutamate is derived from afferent cortico- and thalamostriatal terminals. Until recently, striatal aspartate was believed to be mainly co-stored with glutamate. In this study the authors investigated the role of striatal dopamine D 2 and N-methyl D-aspartate (NMDA) receptor regulation of striatal aspartate and glutamate release in male Sprague-Dawley rats awake freely moving rats and halothane anaesthetised mice wild type control and R6/1 transgenic mice as a model. The findings provide strong evidence for a differential neuronal pool for aspartate and glutamate in the striatum. The experiments were approved by the Department of Health, Ireland and the local ethics committee. Data are expressed as meanaSEM (n=3-7, unless otherwise indicated) and statistical analysis was performed using a two tailed paired Student's t-test. Basal dialysate aspartate and glutamate levels were 0.45a0.048jsM (n=122) and 7.728a0.77jaM (n=143) respectively. Intrastriatal pergolide (50 and SOOnM, 60 minutes) increased local aspartate (+75±25% p=O.OS, and +106t5O% time, p<0.05, 180 minutes) but not glutamate levels. In a separate study in halothane-Anaesthetised transgenic mouse model of Huntington's disease, basal siriatal dialysate glutamate levels were reduced by 42% in transgenic mice while aspartate levels did not differ from those observed in control mice. Furthermore, intra-striatal NMDA (lOO)tM, 15 minutes) was associated with greater aspartate (at 15 minutes) release in the transgenic mice compared to controls (+120±27% vs +35±15%, p<0.05 respectively) while glutamate release rapidly increased to the same extent in both groups (transgenic; +74±21% vs control; +75±46%). Taken together, the authors provide strong evidence for a differential striatal dopamine and NMDA receptor regulation of aspartate and glutamate which strengthens the evidence for a neuronal pool for aspartate (i.e. local interneurons) separate to that for glutamate (i.e. cortico- and thalamostriatal afferent terminals). This finding which may be important in developing new drugs for the treatment of basal ganglia disorders.

17.

PPARy AGONISTS I 5DEOXY-PROSTAGLANDIN J 2 AND CIGUTAZONE INDUCE CARDIAC CELL DEATH

M CAHiLL, DJ FrrzGERALD, MR KENEALY CENTRE FOR CARDIOVASCULAR SCIENCE, ROYAL COLLEGE OF SURGEONS IN IRELAND, DUBLIN, IRELAND

Cardiomyocyte programmed cell death occurs as a component of ischaemic heart disease, transplant rejection and doxorubicin toxicity.

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Biomedical Section Meetings

In each case, there is induction of cyclooxygenase and infiltrating inflammatory cells may further contribute to local prostaglandin formation. A novel series of prostaglandin receptors, the peroxisome proliferator-activated receptors (PPAR) that regulate lipid metabolism and cell growth are expressed in cardiac cells. The authors have previously shown that one of the prostaglandins, prostacyclin is cytoprotective. In this study the authors explored the effects of 15deoxy-delta-12,14-PGJ 2 (15d-PGJ 2 that is generated from PGD2 by dehydration and isomerisation. In contrast to prostacyclin, which is a PPARyand d agonist, 15d-PGJ 2 binds and activates PPAR)'. 15d-PGJ 2 induced dose-dependent morphological evidence of apoptosis of rat neonatal cardiomyocytes over 24 hour, with effects seen at a concentration as low as 2.5pM. A similar effect was seen with the synthetic PPARg agonist ciglitazone with apoptosis evident at a concentration of 6pM and both compounds induced DNA degradation. Both 15d-PGJ 2 and ciglitazone activated caspase-3 activity in a dose-dependezit manner and this was blocked by the caspase-3 inhibitor AC-Asp-Met-Gln-Asp-CHO. 15d-PGJ 2 also induced cleavage of procaspase-3 with the appearance of the active fragment at S hour and loss of the parent protein over 24 hour. PGE and PGF 2 did not provoke apoptosis or caspase-3 activation. Electrospray-tandem mass spectrometry showed that -the cells were capable of generating 15d-PGJ 2 on addition of arachidonic acid and this was blocked by a cyclooxygenase inhibitor. However, the main product generated from the cells was prostacyclin. In conclusion prostaglandins exert differential effects on cardiomyocytes. 15d-PGJ 2 induces cell death, as does ciglitazone, possibly as both are PPARy agonists. )

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