Abnormal connexin expression in human chronic wounds.

BACKGROUND: Regulated alteration of connexin expression has been shown to be integral to acute wound repair. Downregulation of the gap-junction protein connexin 43 at the wound edge has been correlated with keratinocyte and fibroblast migration, while abnormal overexpression of connexin 43 significantly perturbs healing, as shown in the streptozotocin diabetic rodent impaired healing model. OBJECTIVES: To examine the protein expression levels of connexin 43, in addition to connexins 26 and 30, in a variety of human chronic wounds. METHODS: Wound-edge punch biopsies and a matched control from the arm were taken from a cohort of patients with venous leg, diabetic foot or pressure ulcers. Wound connexin expression in each patient was compared with that in a matched, nonwounded arm punch. Tissue was sectioned, stained and imaged by confocal microscopy using identical parameters per patient to permit quantification. RESULTS: Epidermal connexin 43, connexin 26 and connexin 30, and dermal connexin 43 were discovered to be strikingly upregulated in every ulcer from all three wound types, pointing to connexin upregulation as a common feature between chronic wounds. CONCLUSIONS: This result supports efforts to target connexin 43 to promote cell migration and wound healing in chronic ulcers.

The effect of STAT3 inhibition on status epilepticus and subsequent spontaneous seizures in the pilocarpine model of acquired epilepsy.

Pilocarpine-induced status epilepticus (SE), which results in temporal lobe epilepsy (TLE) in rodents, activates the JAK/STAT pathway. In the current study, we evaluate whether brief exposure to a selective inhibitor of the JAK/STAT pathway (WP1066) early after the onset of SE affects the severity of SE or reduces later spontaneous seizure frequency via inhibition of STAT3-regulated gene transcription. Rats that received systemic WP1066 or vehicle at the onset of SE were continuously video-EEG monitored during SE and for one month to assess seizure frequency over time. Protein and/or mRNA levels for pSTAT3, and STAT3-regulated genes including: ICER, Gabra1, c-myc, mcl-1, cyclin D1, and bcl-xl were evaluated in WP1066 and vehicle-treated rats during stages of epileptogenesis to determine the acute effects of WP1066 administration on SE and chronic epilepsy. WP1066 (two 50mg/kg doses) administered within the first hour after onset of SE results in transient inhibition of pSTAT3 and long-term reduction in spontaneous seizure frequency. WP1066 alters the severity of chronic epilepsy without affecting SE or cell death. Early WP1066 administration reduces known downstream targets of STAT3 transcription 24h after SE including cyclin D1 and mcl-1 levels, known for their roles in cell-cycle progression and cell survival, respectively. These findings uncover a potential effect of the JAK/STAT pathway after brain injury that is physiologically important and may provide a new therapeutic target that can be harnessed for the prevention of epilepsy development and/or progression.

Acute administration of the small-molecule p75(NTR) ligand does not prevent hippocampal neuron loss or development of spontaneous seizures after pilocarpine-induced status epilepticus.

Neurotrophins, such as brain-derived neurotrophic factor (BDNF), are initially expressed in a precursor form (e.g., pro-BDNF) and cleaved to form mature BDNF (mBDNF). After pilocarpine-induced status epilepticus (SE), increases in neurotrophins regulate a wide variety of cell-signaling pathways, including prosurvival and cell-death machinery in a receptor-specific manner. Pro-BDNF preferentially binds to the p75 neurotrophin receptor (p75(NTR) ), whereas mBDNF is the major ligand of the tropomyosin-related kinase receptor. To elucidate a potential role for p75(NTR) in acute stages of epileptogenesis, rats were injected prior to and at onset of SE with LM11A-31, a small-molecule ligand that binds to p75(NTR) to promote survival signaling and inhibit neuronal cell death. Modulation of early p75(NTR) signaling and its effects on electrographic SE, SE-induced neurodegeneration, and subsequent spontaneous seizures were examined after LM11A-31 administration. Despite an established neuroprotective effect of LM11A-31 in several animal models of neurodegenerative disorders (e.g., Alzheimer's disease, traumatic brain injury, and spinal cord injury), high-dose LM11A-31 administration prior to and at onset of SE did not reduce the intensity of electrographic SE, prevent SE-induced neuronal cell injury, or inhibit the progression of epileptogenesis. Further studies are required to understand the role of p75(NTR) activation during epileptogenesis and in seizure-induced cell injury in the hippocampus, among other potential cellular pathologies contributing to the onset of spontaneous seizures. Additional studies utilizing more prolonged treatment with LM11A-31 are required to reach a definite conclusion on its potential neuroprotective role in epilepsy.

Pain vs comfort scores after Caesarean section: a randomized trial.

BACKGROUND: The use of negative words, such as 'sting' and 'pain', can increase patient pain and anxiety. We aimed to determine how pain scores compare with comfort scores and how the technique of pain assessment affects patient perceptions and experiences after operation. METHODS: After Caesarean section, 300 women were randomized before post-anaesthesia review. Group P women were asked to rate their pain on a 0-10-point verbal numerical rating scale (VNRS), where '0' was 'no pain' and '10' was 'worst pain imaginable'. Group C women were asked to rate comfort on a 0-10-point VNRS, where '0' was 'no comfort' and '10' was 'most comfortable'. All women were asked whether the Caesarean wound was bothersome, unpleasant, associated with tissue damage, and whether additional analgesia was desired. RESULTS: The median (inter-quartile range) VNRS pain scores was higher than inverted comfort scores at rest, 2 (1, 4) vs 2 (0.5, 3), P=0.001, and movement, 6 (4, 7) vs 4 (3, 5), P<0.001. Group P women were more likely to be bothered by their Caesarean section, had greater VNRS 'Bother' scores, 4 (2, 6) vs 1 (0, 3), P<0.001, perceived postoperative sensations as 'unpleasant' [relative risk (RR) 3.05, 95% confidence interval (CI) 2.20, 4.23], P<0.001, and related to tissue damage rather than healing and recovery (RR 2.03, 95% CI 1.30, 3.18), P=0.001. Group P women were also more likely to request additional analgesia (RR 4.33, 95% CI 1.84, 10.22), P<0.001. CONCLUSIONS: Asking about pain and pain scores after Caesarean section adversely affects patient reports of their postoperative experiences.

Ges, A human GTPase of the Rad/Gem/Kir family, promotes endothelial cell sprouting and cytoskeleton reorganization.

Rad, Gem/Kir, and mRem (RGK) represent a unique GTPase family with largely unknown functions (Reynet, C., and C.R. Kahn. 1993. Science. 262:1441-1444; Cohen, L., R. Mohr, Y. Chen, M. Huang, R. Kato, D. Dorin, F. Tamanoi, A. Goga, D. Afar, N. Rosenberg, and O. Witte. Proc. Natl. Acad. Sci. USA. 1994. 91:12448-12452; Maguire, J., T. Santoro, P. Jensen, U. Siebenlist, J. Yewdell, and K. Kelly. 1994. Science. 265:241-244; Finlin, B.S., and D.A. Andres. 1997. J. Biol. Chem. 272:21982-21988). We report that Ges (GTPase regulating endothelial cell sprouting), a human RGK protein expressed in the endothelium, functions as a potent morphogenic switch in endothelial cells (ECs). Ges function is sufficient to substitute for angiogenic growth factor/extracellular matrix (ECM) signals in promoting EC sprouting, since overexpression of Ges in ECs cultured on glass leads to the development of long cytoplasmic extensions and reorganization of the actin cytoskeleton. Ges function is also necessary for Matrigel-induced EC sprouting, since this event is blocked by its dominant negative mutant, Ges(T94N), predicted to prevent the activation of endogenous Ges through sequestration of its guanine nucleotide exchange factor. Thus, Ges appears to be a key transducer linking extracellular signals to cytoskeleton/morphology changes in ECs.

Effects of age on the pharmacokinetics of tramadol and its active metabolite, O-desmethyltramadol following intravenous administration to foals.

REASONS FOR PERFORMING STUDY: Tramadol is an analgesic agent used in man and a number of veterinary species. The pharmacokinetics and behavioural effects of tramadol and its active metabolite have been described in mature horses, but not in young foals. OBJECTIVES: To characterise the pharmacokinetics, metabolism and some induced behavioural and physiological responses following i.v. tramadol administration in the same group of foals on 4 different occasions, from a few days after birth to age 43 days. STUDY DESIGN: Experimental. METHODS: Tramadol was administered i.v. (3 mg/kg bwt) to a group of 8 foals on 4 separate occasions at ages 6­8, 13­15, 20­22 and 40­43 days. Blood samples were collected prior to administration and at multiple times until 48 h post administration. Blood samples were analysed for tramadol and metabolite concentrations and pharmacokinetics determined at each age. Behavioural and physiological effects were also assessed. RESULTS: The average volume of distribution was 5.10, 4.63, 4.02 and 3.84 l/kg bwt and clearance 3.44, 3.08, 3.14 and 2.69 l/h/kg bwt when foals were aged 6­8, 13­15, 20­22 and 40­43 days, respectively. There was not a significant difference in the elimination half-life between age groups (1.52, 1.73, 1.13 and 1.51 for ages 6­8, 13­15, 20­22 and 40­43 days, respectively). The metabolites produced were the same as in mature horses; however, glucuronidation capability, appeared to increase with increasing age. Tramadol administration was well tolerated at all ages studied with sedation noted in the 3 older age groups. CONCLUSIONS: Tramadol appears to be consistently well tolerated following i.v. administration of 3 mg/kg bwt to foals ranging in age from 1 to 6 weeks. Although analgesic concentrations in foals have yet to be established, the results of this study support further study of tramadol for clinical use in foals.

Comparison of Ins(1,4,5)P3 receptors from rat cerebellum and bovine adrenal cortex.

Ins(1,4,5)P3 receptors in adrenal cortical and cerebellar membranes can be distinguished by their affinities for Ins(1,4,5)P3 as well as the potencies with which heparin and Mg2+ inhibit binding. We have found that the differences in Ins(1,4,5)P3 affinity and heparin inhibition are maintained upon receptor solubilization and purification. In contrast to this, heparin-agarose affinity purification of solubilized cerebellar receptors reduces the potency of Mg2+ inhibition to that in adrenal cortex. These results suggest that Ins(1,4,5)P3 receptors in adrenal cortex are structurally distinct from those in cerebellum. Monoclonal antibodies raised against C- and N-terminal regions of mouse cerebellar Ins(1,4,5)P3 receptors recognize 250-300-kDa proteins in both rat cerebellum and bovine adrenal cortex.

Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae.

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.

Cyclic ADP-ribose-induced Ca2+ release from rat brain microsomes.

Cyclic ADP-ribose (cADPR), an endogenous NAD+ metabolite in many mammalian and invertebrate tissues, is a potent mediator of calcium mobilization in sea urchin eggs. Our results show that cADPR also stimulates calcium release from rat brain microsomes, marked release occurring over the concentration range 10-250 nM. This is not inhibited by concentrations of heparin which completely abolish inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. Ryanodine (100 microM) inhibits the cADPR response. Our results are consistent with cADPR being an endogenous messenger mediating Ca2+ release from ryanodine-sensitive pools in brain.

Characterisation of rat superficial superior colliculus neurones: firing properties and sensitivity to GABA.

Physiological, pharmacological and morphological properties of superficial superior colliculus neurones (n=93) were characterised using whole-cell patch-clamp recordings in rat brain slices. Six cell types (narrow- and wide-field vertical, horizontal, piriform, marginal and stellate) were identified based on Lucifer Yellow labelling but no cell type-specific spike pattern could be identified. Resting membrane potentials were homogeneous (mean: -67.1 +/- 0.7 mV, n=48), and spike frequencies ranged from 10 to 70 Hz (80 pA current injection). About 66% of the cells displayed regular and sustained spike production, throughout all neuronal categories. Rebound spikes and spontaneous activity were observed frequently in all cell types. Synaptically evoked action potentials appeared as single spikes (mean amplitude: 76.0 +/- 3.2 mV, n=34) followed by a fast after-hyperpolarising potential (mean amplitude: 25.4 +/- 1.4 mV, n=34) and variable late potentials (late after-depolarising and/or -hyperpolarising). Pharmacologically, a characterisation using GABA and its subtype-specific agonists indicated a strong inhibitory influence of this transmitter system on >90% of cells. The GABA(A) receptor agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (100 microM), caused a reversible hyperpolarisation (approximately 9 mV) and spike inhibition of all neurones studied. This was more pronounced for intrinsic than for synaptically evoked spikes. Assessment of the GABA(C) receptor agonist, cis-4-aminocrotonic acid (1 mM), also revealed a hyperpolarisation (approximately 3 mV) and an inhibitory action on firing, but this was not as potent and homogeneous, compared to the GABA(A) receptor agonist. Further, the GABA(B) receptor agonist, baclofen (50-100 microM), had more variable (hyperpolarising, depolarising or no change) effects on the membrane potential. It showed little modulation of current-induced action potentials but fully blocked synaptic spikes. Assessment of GABA receptor antagonist actions revealed the presence of weak tonic and strong phasic GABA(A) receptor-mediated inhibition in the superficial superior colliculus: application of the GABA(A) receptor antagonist, bicuculline (100 microM), led to a generally enhanced excitability and depolarisation (approximately 5 mV). Intrinsic firing was somewhat enhanced, but synaptic spiking was drastically potentiated and prolonged. In contrast, 1,2,5,6-tetrahydro-(pyridin-4-yl) methylphosphinic acid (TPMPA; 100 microM), the GABA(C) receptor antagonist, produced little effect on these physiological parameters. The GABA(B) receptor antagonist, CGP35348 (200 microM), caused a partial inhibition of late after-hyperpolarising potentials (approximately 30%). Uptake of GABA contributes little to endogenous inhibition in the superior colliculus slice preparation, as suggested by the action of GABA uptake inhibitors SKF89976 (50-100 microM) and nipecotic acid (200-500 microM), both had no obvious effect on physiological parameters. However, in the presence of these compounds, sub-maximal inhibitory actions of GABA were potentiated. In conclusion, different cell types in the superficial superior colliculus do not display distinct physiological properties and are subject to strong inhibitory modulation. We therefore suggest that signal processing in this brain region does not require cell type-specific encoding of information. In line with evidence provided by previous in vivo investigations, identification of visual stimuli and orientation responses appears to be realised via the network properties of the receptive fields that form topographic maps.

Inhibition by heparin of platelet accumulation in vivo.

In vivo platelet aggregation has been studied using a novel, minimally invasive technique. No aggregatory effects of heparin were observed on normal circulating platelets nor was there enhancement of aggregation of platelets during activation by intravenous injection of ADP, collagen, PAF acether or thrombin. On the contrary, high doses of heparin were found to inhibit platelet accumulation induced by ADP, collagen or PAF-acether. Inhibition of these responses necessitated doses of heparin in excess of those required for anti-coagulant effects. The present experiments do not establish a mechanism for such inhibition. Extension to other species, including man, is needed before attributing clinical relevance to the present observations.

Prolongation of rat tail bleeding time caused by oral doses of a thromboxane synthetase inhibitor which have little effect on platelet aggregation.

N (7-carboxyheptyl) imidazole is an inhibitor of platelet thromboxane synthetase that has no effect on the cyclooxygenase activity. An oral dose of the substance to rats (10 mg/kg) prolonged tail bleeding time from 170 +/- 13 sec to 284 +/- 22 sec. This oral dose also inhibited platelet thromboxane B2 production induced by collagen ex vivo but had little effect on the aggregation dose response curve. There was no effect on thrombin-induced aggregation. Neither the thrombocytopenia induced by the Arthus reaction nor thrombus formation on an implanted cotton thread were inhibited by oral doses of carboxyheptylimidazole up to 30 mg/kg. Similarly neither the prothrombin nor activated partial thromboplastin time were affected. It is postulated that this thromboxane synthetase inhibitor prolongs bleeding time nor by inhibiting platelet aggregation or blood coagulation but rather by preventing the vasoconstriction which would normally be caused by thromboxane A2.

Fibrin, red cell and platelet interactions in an experimental model of thrombosis.

1 Insertion of a cotton thread into an arteriovenous shunt of an anaesthetized rat causes an increase in the weight of the thread due to deposition of thrombus. 2 The thrombus formed was of the venous (red) type, being sensitive to heparin, yet possessed important characteristics of an arterial thrombus, in that it was dependent on platelets and on rate of blood flow. 3 Thromboxane synthetase inhibitors had no effect on thrombus deposition. 4 Cyclo-oxygenase inhibitors did not significantly depress thrombus formation at doses which inhibited platelet function ex vivo. 5 Compounds which can modify the release or action of adenosine 5' -diphosphate partly inhibited thrombus formation. 6 A depression in clotting factor levels induced by sodium warfarin led to a highly significant reduction in thrombus formation at doses which caused a prolongation of prothrombin clotting time.

Intracellular enzymes and protein synthesis in rabbit skin after thermal injury.

1. The concentrations of several intracellular enzymes in rabbit skin have been measured 5 min, 2, 6 and 24 h after thermal injury.2. At 5 min and 2 h after a burn (60 degrees C for 1 min) there was a significant fall in the enzyme activities whereas at 6 h their activities were higher than control.3. It appears that the increase in enzyme concentrations in the lymph during the first few hours after thermal injury is associated with a fall in the enzyme concentrations in the tissues and therefore might be the result of leakage of enzyme from storage sites in the injured cells.4. The second increase in enzyme concentrations in the lymph which has been observed 6-18 h after thermal injury occurs at a time when there is also an increase in the enzyme concentrations in the tissue.5. It seems unlikely that these increased activities are due to new synthesis since there was no apparent correlation between tissue enzyme concentrations and protein synthetic activity, and the changes still occurred after administration of cycloheximide.6. There was a change in the LDH isoenzyme pattern after injury towards a predominance of LDH-1. This change did not occur immediately after the burn, but was present at 2 and 6 h, and returned to normal 24 h later.

Sulfinpyrazone: a review of its pharmacological properties and therapeutic use.

Sulfinpyrazone1 has long been recognised as a potent uricosuric agent, but has more recently been studied extensively as a platelet inhibitor and antithrombotic agent. It is active in man following oral administration and has been reported to be effective in reducing the incidence of transient ischaemic attacks, thromboembolism associated with vascular and cardiac prostheses, recurrent venous thrombosis, arteriovenous shunt thrombosis and sudden cardiac death following myocardial infarcton. Sulfinpyrazone has not been demonstrated to be effective in preventing or reducing the risk of stroke or death in patients with cerebrovascular disease with a recent history of cerebral or retinal ischaemioc attacks. The normal total dose of sulfinpyrazone as an antithrombotic agent is 800mg daily. The drug has been used continuously for up to 4 years with no serious adverse reactions or laboratory abnormalities. There has been no apparent diminution of effect with time. Sulfinpyrazone is not a substitute for conventional anticoagulant agents (e.g. the coumarin derivatives) in the treatment of venous thrombosis, but is an important drug for the treatment of conditions associated with arterial thrombosis and possibly for the prophylaxis of recurrent venous thrombosis.

Comparison between the effects of ethanol and diazepam on spatial working memory in the rat.

The present study compared the effects of ethanol and diazepam on a task that allows for the assessment of both spatial working memory and the acquisition of spatial information within each day. During the first trial of each day, subjects were shown the spatial location of a food reward on a six-arm radial-arm maze. During nine subsequent free-choice trials, subjects were reinforced for returning to that same spatial location. The location of the food reward varied across days. Thus, choosing correctly on any given trial required subjects to remember where food had been received during the previous trials of that day. The effects of ethanol and diazepam on working memory were assessed by analyzing the overall number of errors committed during the nine free-choice trials of each day. The effects of ethanol and diazepam on within-day acquisition were assessed by comparing the number of errors committed during the first three trials of each day to the number of errors committed during the last three trials of each day. Ethanol and diazepam both produced dose-dependent increases in working memory errors, and both did so without impairing within-day acquisition. The results of the present study provide further evidence of the similarities between the effects of ethanol and benzodiazepine receptor agonists on learning and memory, and are consistent with the hypothesis that ethanol's potentiation of GABA at GABAA receptors contributes to the learning and memory impairments produced by ethanol.

Effects of ethanol on hippocampal place-cell and interneuron activity.

Mounting evidence suggests that ethanol exerts effects on learning and memory by altering cellular activity in the hippocampus and related structures. However, little is actually known regarding ethanol's effects on hippocampal function in awake, freely-behaving animals. The present study examines the effects of ethanol on hippocampal place-cell and interneuron activity in freely-behaving rats. Signals from individual hippocampal neurons were isolated while subjects traversed a symmetric Y-maze for food reward. Following 15 min of baseline recording, subjects were injected with one of four doses of ethanol (0.0, 0.5, 1.0 and 1.5 g/kg), and cellular activity was monitored for a 1-h time period. Following sufficient time for recovery (minimum of 3 h post injection), cellular activity was monitored for an additional 15-min period. Both 1.0 and 1.5 g/kg ethanol potently suppressed the firing of hippocampal place-cells without altering place-field locations. Ethanol did not significantly suppress out-of-field firing rates, leading to a decrease in spatial specificity (i.e. the ratio of in-field/out-of-field firing rates). Interneuron activity was not altered by 1.0 g/kg ethanol, but was occasionally suppressed by 1.5 g/kg ethanol. Results are interpreted in light of recent behavioral and electrophysiological studies examining the effects of ethanol on hippocampal function.

Prenatal choline exposure alters hippocampal responsiveness to cholinergic stimulation in adulthood.

Manipulation of dietary choline levels during gestation results in enduring neurobehavioral changes in offspring that last into adulthood. Alterations of hippocampal function and memory are among the most striking changes. Depending upon the measures assessed, prenatal choline supplementation tends to promote excitatory synaptic efficacy in hippocampal circuits while prenatal choline deficiency diminishes it. However, the mechanisms underlying these changes remain unclear. Transverse hippocampal slices were prepared from adult offspring of dams fed choline supplemented, choline deficient, or control diets. We assessed paired-pulse inhibition, and excitatory synaptic responsiveness before and after activation of cholinergic receptors with Carbachol. Prenatally choline deficient animals yielded significantly fewer electrophysiological viable hippocampal slices than did animals from either of the other two treatment groups. Among the slices tested, there were no differences in paired pulse inhibition between the treatment groups. However, transient cholinergic activation resulted in a prolonged enhancement of the amplitude of the population EPSP (pEPSP) response in slices from prenatally choline supplemented animals. These results suggest that GABA receptor-mediated inhibition remains intact after prenatal choline manipulations, and that enhancement of the excitatory responsiveness of hippocampal circuits in slices from prenatally choline supplemented rats may be related in part to an increase in cholinergic tone within the CA1 circuit.