Cecal Resection with Bipolar Sealing Devices in a Rat Model.

Background: Bipolar sealing devices are routinely used to seal blood vessels. The aim of the study is to evaluate the feasibility and safety of colonic sealing with the use of the bipolar energy devices in rats as model for experimental appendectomy. Methods: Seventy-five male Wistar rats underwent a cecal resection with four different bipolar sealing devices or a linear stapler. The harvesting procedure was performed immediately or at postoperative day (POD) 7. The sealing front bursting pressure (BP) was measured in both groups. At POD7, the resection line was clinically examined and the hydroxyproline (HDP) levels were determined. Hematoxylin and Eosin (H&E) staining was used for histopathological evaluation of the sealing front as well. Results: There was no mortality and no insufficiency. The BPs between the bipolar sealing devices showed no statistical differences. The early phase of the seal (POD 0) provides a low BP with an 30.8% increase until POD 7. The BPs in the stapler group showed significant better values. The hydroxyproline levels did not differ statistically between the groups. Histopathologically, there were more signs of ischemic necrosis in the stapler group than in the sealing devices groups. Conclusion: The resection and sealing of the cecum as an experimental appendectomy model with the use of bipolar energy devices proved feasible and safe in rats. The different energy devices in this study produce comparable results. To justify clinical practice in humans, several studies on the underlying mechanisms of early stage wound healing are needed.

Transcutaneous sacral nerve stimulation for intraoperative verification of internal anal sphincter innervation.

BACKGROUND: The current standard for pelvic intraoperative neuromonitoring (pIONM) is based on intermittent direct nerve stimulation. This study investigated the potential use of transcutaneous sacral nerve stimulation for non-invasive verification of pelvic autonomic nerves. METHODS: A consecutive series of six pigs underwent low anterior rectal resection. For transcutaneous sacral nerve stimulation, an array of ten electrodes (cathodes) was placed over the sacral foramina (S2 to S4). Anodes were applied on the back, right and left thigh, lower abdomen, and intra-anally. Stimulation using the novel method and current standard were performed at different phases of the experiments under electromyography of the autonomic innervated internal anal sphincter (IAS). KEY RESULTS: Transcutaneous stimulation induced increase of IAS activity could be observed in each animal under specific cathode-anode configurations. Out of 300 tested configurations, 18 exhibited a change in the IAS activity correlated with intentional autonomic nerve damage. The damage resulted in a significant decrease of the relative area under the curve of the IAS frequency spectrum (P<.001). Comparison of the IAS spectra under transcutaneous and direct stimulation revealed no significant difference (after rectal resection: median 5.99 µV•Hz vs 7.78 µV•Hz, P=.12; after intentional nerve damage: median -0.27 µV•Hz vs 3.35 µV•Hz, P=.29). CONCLUSIONS AND INFERENCES: Non-invasive selective transcutaneous sacral nerve stimulation could be used for verification of IAS innervation.

Peripheral interactions between cannabinoid and opioid systems contribute to the antinociceptive effect of crotalphine.

BACKGROUND AND PURPOSE: Crotalphine is an antinociceptive peptide that, despite its opioid-like activity, does not induce some of the characteristic side effects of opioids, and its amino acid sequence has no homology to any known opioid peptide. Here, we evaluated the involvement of the peripheral cannabinoid system in the crotalphine effect and its interaction with the opioid system. EXPERIMENTAL APPROACH: Hyperalgesia was evaluated using the rat paw pressure test. Involvement of the cannabinoid system was determined using a selective cannabinoid receptor antagonist. Cannabinoid and opioid receptor activation were evaluated in paw slices by immunofluorescence assays using conformation state-sensitive antibodies. The release of endogenous opioid peptides from skin tissue was measured using a commercial enzyme immunoassay (EIA). KEY RESULTS: Both p.o. (0.008-1.0 µg·kg(-1) ) and intraplantar (0.0006 µg per paw) administration of crotalphine induced antinociception in PGE2 -induced hyperalgesia. Antinociception by p.o. crotalphine (1 µg·kg(-1) ) was blocked by AM630 (50 µg per paw), a CB2 receptor antagonist, and by antiserum anti-dynorphin A (1 µg per paw). Immunoassay studies confirmed that crotalphine increased the activation of both κ-opioid (51.7%) and CB2 (28.5%) receptors in paw tissue. The local release of dynorphin A from paw skin was confirmed by in vitro EIA and blocked by AM630. CONCLUSIONS AND IMPLICATIONS: Crotalphine-induced antinociception involves peripheral CB2 cannabinoid receptors and local release of dynorphin A, which is dependent on CB2 receptor activation. These results enhance our understanding of the mechanisms involved in the peripheral effect of crotalphine, as well as the interaction between the opioid and cannabinoid systems.

Comparative analysis of in situ versus ex situ perfusion on micro circulation in liver procurement--an experimental trial in a porcine model.

INTRODUCTION: The Achilles heel of liver transplantation remains the biliary system. The crucial step for liver preservation is effective rinsing and perfusion of the peribiliary plexus (PBP). Due to the physiology of the vascular tree, it seems almost impossible to achieve the necessary physiologic ranges of pressure and flow by the in situ perfusion technique. We investigated the role of additional ex situ perfusion via the hepatic artery in this animal model. MATERIALS AND METHODS: Fifteen German Landrace pigs underwent standardized multiorgan procurement. In situ perfusion and additional ex situ perfusion were performed consecutively. Meanwhile the external pressure applied to the perfusion system was increased stepwise. To visualize the effects on the liver parenchyma and PBP, we administered colored microparticles (MPs; 10 µm). Frozen sections of the explanted liver were studied histologically by quantitative evaluation of the MPs. RESULTS: Ex situ perfusion was able to build up significantly higher values of pressure (P < .001) and flow (P < .001) than in situ perfusion. Those of ex situ perfusion reached physiological levels under application of an external pressure of 200 mm Hg. Considering the liver parenchyma, significantly higher amounts of MPs originating from ex situ perfusion were evident (P < .001) and PBP (P < .001). CONCLUSION: MPs provide an appropriate tool to determine organ perfusion quantitatively in experimental models. Considering flow, pressure, and microcirculation, we consider that additional ex situ perfusion of the liver is more effective than in situ perfusion.

Neuroprotective effect of ceftriaxone on the penumbra in a rat venous ischemia model.

OBJECTIVE: Glutamate transporter-1 (GLT-1) maintains low concentrations of extracellular glutamate by removing glutamate from the extracellular space. It is controversial, however, whether upregulation of GLT-1 is neuroprotective under all ischemic/hypoxic conditions. Recently, a neuroprotective effect of preconditioning with a ß-lactam antibiotic ceftriaxone (CTX) that increases expression of GLT-1 has been reported in animal models of focal ischemia. On the other hand, it is said that CTX does not play a neuroprotective role in an in vitro study. Thus, we examined the effect of CTX on ischemic injury in a rat model of two-vein occlusion (2VO). This model mimics venous ischemia during, e.g. tumor surgery, a clinical situation that is best suitable for pretreatment with CTX. METHODS: CTX (100mg/kg, 200mg/kg per day) or vehicle (0.9% NaCl) was intraperitoneally injected into Wistar rats for 5days before venous ischemia (n=57). Then, animals were prepared for occlusion of two adjacent cortical veins (2VO) by photothrombosis with rose bengal that was followed by KCl-induced cortical spreading depression (CSD). Infarct volume was evaluated with hematoxylin and eosin (H&E) staining 2days after venous occlusion. [(3)H]MK-801, [(3)H]AMPA and [(3)H]Muscimol ligand binding were examined autoradiographically in additional two groups without 2VO (n=5/group). Animals were injected either with NaCl (vehicle) or CTX 200mg/kg for 5days in order to evaluate whether NMDA, AMPA and GABAA ligand binding densities were affected. RESULTS: CTX pretreatment reduced infarct volume compared to vehicle pretreatment (p<0.05). The effect of CTX pretreatment was attenuated by administration of the GLT-1 inhibitor, dihydrokainate (DHK) 30min before 2VO. CTX had no effect on the number of spontaneous spreading depressions after 2VO. Analysis of quantitative receptor autoradiography showed no statistically significant difference between rats after administration with CTX compared to control rats. CONCLUSIONS: Pretreatment with CTX has neuroprotective potential without effect on NMDA, AMPA and GABAA receptor density and spontaneous spreading depression. This effect can be abolished by GLT-1 inhibition, indicating that upregulation of GLT-1 is an important mechanism for neuroprotective action in penumbra-like conditions, e.g. if neurosurgeons plan to occlude cerebral veins during tumor surgery.

Erythropoietin neuroprotection is enhanced by direct cortical application following subdural blood evacuation in a rat model of acute subdural hematoma.

Recombinant human erythropoietin (EPO) has been successfully tested as neuroprotectant in brain injury models. The first large clinical trial with stroke patients, however, revealed negative results. Reasons are manifold and may include side-effects such as thrombotic complications or interactions with other medication, EPO concentration, penetration of the blood-brain-barrier and/or route of application. The latter is restricted to systemic application. Here we hypothesize that EPO is neuroprotective in a rat model of acute subdural hemorrhage (ASDH) and that direct cortical application is a feasible route of application in this injury type. The subdural hematoma was surgically evacuated and EPO was applied directly onto the surface of the brain. We injected NaCl, 200, 2000 or 20,000IU EPO per rat i.v. at 15min post-ASDH (400µl autologous venous blood) or NaCl, 0.02, 0.2 or 2IU per rat onto the cortical surface after removal of the subdurally infused blood t at 70min post-ASDH. Arterial blood pressure (MAP), blood chemistry, intracranial pressure (ICP), cerebral blood flow (CBF) and brain tissue oxygen (ptiO2) were assessed during the first hour and lesion volume at 2days after ASDH. EPO 20,000IU/rat (i.v.) elevated ICP significantly. EPO at 200 and 2000IU reduced lesion volume from 38.2±0.6mm(3) (NaCl-treated group) to 28.5±0.9 and 22.2±1.3mm(3) (all p<0.05 vs. NaCl). Cortical application of 0.02IU EPO after ASDH evacuation reduced injury from 36.0±5.2 to 11.2±2.1mm(3) (p=0.007), whereas 0.2IU had no effect (38.0±9.0mm(3)). The highest dose of both application routes (i.v. 20,000IU; cortical 2IU) enlarged the ASDH-induced damage significantly to 46.5±1.7 and 67.9±10.4mm(3) (all p<0.05 vs. NaCl). In order to test whether Tween-20, a solvent of EPO formulation 'NeoRecomon®' was responsible for adverse effects two groups were treated with NaCl or Tween-20 after the evacuation of ASDH, but no difference in lesion volume was detected. In conclusion, EPO is neuroprotective in a model of ASDH in rats and was most efficacious at a very low dose in combination with subdural blood removal. High systemic and topically applied concentrations caused adverse effects on lesion size which were partially due to increased ICP. Thus, patients with traumatic ASDH could be treated with cortically applied EPO but with caution concerning concentration.

Effects of a small acute subdural hematoma following traumatic brain injury on neuromonitoring, brain swelling and histology in pigs.

An acute subdural hematoma (ASDH) induces pathomechanisms which worsen outcome after traumatic brain injury, even after a small hemorrhage. Synergistic effects of a small ASDH on brain damage are poorly understood, and were studied here using neuromonitoring for 10 h in an injury model of controlled cortical impact (CCI) and ASDH. Pigs (n = 32) were assigned to 4 groups: sham, CCI (2.5 m/s), ASDH (2 ml) and CCI + ASDH. Intracranial pressure was significantly increased above sham levels by all injuries with no difference between groups. CCI and ASDH reduced ptiO(2) by a maximum of 36 ± 9 and 26 ± 11%, respectively. The combination caused a 31 ± 11% drop. ASDH alone and in combination with CCI caused a significant elevation in extracellular glutamate, which remained increased longer for CCI + ASDH. The same two groups had significantly higher peak lactate levels compared to sham. Somatosensory evoked potential (SSEP) amplitude was persistently reduced by combined injury. These effects translated into significantly elevated brain water content and histological damage in all injury groups. Thus, combined injury had stronger effects on glutamate and SSEP when compared to CCI and ASDH, but no clear-cut synergistic effects of 2 ml ASDH on trauma were observed. We speculate that this was partially due to the CCI injury severity.

The effect of a gap-junction blocker, carbenoxolone, on ischemic brain injury and cortical spreading depression.

Cortical spreading depression (CSD) has been shown to cause secondary cell loss in experimental models of brain injury and in patients, and blocking of CSD is a potential neuroprotective strategy. Here we tested the hypothesis that gap junctions affect CSD under physiological conditions as well as infarct development in a rat two-vein occlusion model suited to study pathophysiology of the penumbra (n = 71). We applied the gap junction blocker carbenoxolone (CBX) or saline intra-ventricularly. Interestingly, CBX temporarily increased systemic blood pressure and cortical blood flow (41% and 53%, 15 min after 250 µg CBX). We induced CSD with cortical microinjection of potassium chloride (KCl), counted how many spontaneous CSDs after CSD induction were elicited and measured the propagation velocity. After 250 µg CBX administration, significant 37.5 ± 6.5 additional CSDs were seen. CSD velocity increased significantly after 50 µg and 250 µg CBX. Occlusion of two adjacent cortical veins using Rose Bengal dye and fiberoptic illumination followed by 250 µg CBX or saline showed a significant more than doubling of infarct volumes 7 days after CBX. The current experiments provide evidence that CBX can accelerate the initiation and propagation of CSD suggesting opening of gap junctions is not required for CSD propagation. Blocking gap junctions worsens outcome from focal cerebral ischemia. Hence, measures intended to improve spatial buffering via astroglial gap junctions could have therapeutic potential in disease processes involving CSD.

Selective pelvic autonomic nerve stimulation with simultaneous intraoperative monitoring of internal anal sphincter and bladder innervation.

BACKGROUND: Pelvic autonomic nerve preservation avoids postoperative functional disturbances. The aim of this feasibility study was to develop a neuromonitoring system with simultaneous intraoperative verification of internal anal sphincter (IAS) activity and intravesical pressure. METHODS: 14 pigs underwent low anterior rectal resection. During intermittent bipolar electric stimulation of the inferior hypogastric plexus (IHP) and the pelvic splanchnic nerves (PSN), electromyographic signals of the IAS and manometry of the urinary bladder were observed simultaneously. RESULTS: Stimulation of IHP and PSN as well as simultaneous intraoperative monitoring could be realized with an adapted neuromonitoring device. Neurostimulation resulted in either bladder or IAS activation or concerted activation of both. Intravesical pressure increase as well as amplitude increase of the IAS neuromonitoring signal did not differ significantly between stimulation of IHP and PSN [6.0 cm H(2)O (interquartile range [IQR] 3.5-9.0) vs. 6.0 cm H(2)O (IQR 3.0-10.0) and 12.1 µV (IQR 3.0-36.7) vs. 40.1 µV (IQR 9.0-64.3)] (p > 0.05). CONCLUSIONS: Pelvic autonomic nerve stimulation with simultaneous intraoperative monitoring of IAS and bladder innervation is feasible. The method may enable neuromonitoring with increasing selectivity for pelvic autonomic nerve preservation.

Online signal processing of internal anal sphincter activity during pelvic autonomic nerve stimulation: a new method to improve the reliability of intra-operative neuromonitoring signals.

AIM: Intra-operative neuromonitoring is increasingly applied in several surgical disciplines and has been introduced to facilitate pelvic autonomic nerve preservation. Nevertheless, it has been considered a questionable tool for the minimization of risk, as the results are variable and might be misleading. The aim of the present experimental study was to develop an intra-operative neuromonitoring system with improved reliability for monitoring pelvic autonomic nerve function. METHOD: Fifteen pigs underwent low anterior rectal resection with pelvic autonomic nerve preservation. Intra-operative neuromonitoring was performed under autonomic nerve stimulation with observation of electromyographic signals of the internal anal sphincter and bladder manometry. As the internal anal sphincter frequency spectrum during stimulation was found to be mainly in the range of 5-20 Hz, intra-operative neuromonitoring signals were postoperatively processed by implementation of matching band pass filters. RESULTS: In 10 preliminary experiments, signal processing was performed offline in the postoperative analysis. Of 163 stimulations intra-operatively assessed by the surgeon as positive responses, 135 (83%) were confirmed after signal processing. In the following five consecutive experiments intra-operative online signal processing was realized and demonstrated reliable intra-operative neuromonitoring signals of internal anal sphincter activity with significant increase during pelvic autonomic nerve stimulation [0.5 µV (interquartile range = 0.3-0.7) vs 4.8 µV (interquartile range = 2.5-7.5); P < 0.001]. CONCLUSION: Online signal processing of internal anal sphincter activity aids reliable identification of pelvic autonomic nerves with potential for improvement of intra-operative neuromonitoring in pelvic surgery.

Dynamic in vivo imaging of microvasculature and perfusion by miniaturized confocal laser microscopy.

INTRODUCTION: Microvasculature and associated pathologies mandate dynamic imaging. We evaluated a novel miniaturized confocal laser scanning probe for in vivo visualization of blood vessels, blood flow, cell tracking and perfusion in both healthy rodents and disease models. METHODS: The hand-held confocal microscopy system allowed a 500- to 2,400-fold magnification at a dynamically variable imaging depth. Different intravital stains were used alone or in combination for tissue, nuclear, plasma and vascular endothelial cell staining and for blood flow visualization, and targeted staining for individual cell populations. RESULTS: Precision optical sectioning yielded high-resolution images in vivo. Leucocyte-endothelium interactions in brain microvasculature were followed in serial sections. A microthrombosis was identified after sequential injection of FITC-labelled erythrocytes, FITC-dextran and acriflavine. Glomerular alterations were visualized in the MRL/lprmouse model of lupus nephritis. DISCUSSION: Intravital confocal microscopy with a miniaturized hand-held probe combines high-resolution subsurface imaging in real time for dynamic visualization of vessels, cells, blood flow and associated pathologies, permitting a truly comprehensive vascular imaging in vivo at the cellular level.

A new model of skull base reconstruction following expanded endonasal or transoral approaches--long-term results in primates.

OBJECTIVE: The direct endonasal or transoral transclival approaches to the skull base permit effective minimally invasive surgery along the clivus region. Developing consistently effective techniques to prevent cerebrospinal fluid (CSF) leaks and their consequences (infections and healing processes with long and complicated recoveries) remains a major challenge. In this study, we tested over a long period a method of bone reconstruction newly developed by us, which makes use of a specially designed elastic silicone plug that can be employed for bone replacement after minimally invasive skull base surgery without risk of postoperative CSF leaks. After acute testing of plug efficiency in a pig model, which showed a 100% closure of the bone defect without CSF leak, we now tested the long-term accuracy of the plugs. METHODS: In 3 primates, we used an endoscope-controlled transoral transclival approach and after opening the dura we simulated a CSF leakage. We inserted the plug into the bone defect and closed the mucosa of the oral cavity with stitches. The follow-up included blood, weight, and wound control 1, 4 and 8 weeks postoperatively. Social behavior, such as reintegration and postoperative eating abnormalities, was also studied. The aims of this study were: (1) testing the biocompatibility of the material; (2) development of infection against the foreign body; (3) effects of the plug on the surrounding bone, and (4) development of CSF leakages during the postoperative phase. RESULTS: Clinically no infection was seen. Wound healing, immediate and long-term postoperative social behavior of the animals, feeding and body weight were normal. No CSF leakages developed. The histological examination of the clivus bone showed no abnormalities. The implant was covered by fibrous layer; there was no bone atrophy but osteoid formation. CONCLUSION: This novel medical device allows easy, fast and uncomplicated, leak-proof closure of bone defects after minimally invasive craniotomies as seen in transsphenoidal or transoral skull base approaches.

Flow and pressure during liver preservation under ex situ and in situ perfusion with University of Wisconsin solution and histidine-tryptophan-ketoglutarate solution.

Effective preservation of liver grafts is the first essential step for successful liver transplantation. Insufficient perfusion leads to ischemic-type biliary lesions after transplantation. Perfusion of the graft can be performed either in situ or ex situ, with gravity flow or pressure-controlled. Mainly University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions are used widespread in clinical liver transplantation. Due to a persistent lack of data, we performed this systematic investigation of in situ and ex situ perfusion of liver grafts with HTK (low-viscous) and UW (high-viscous) solutions at different pressure steps on the perfusion solution (gravity flow, 50, 100, 150, and 200 mm Hg). End points were perfusion flow and pressure in the hepatic artery. A pig model was used with n = 8 pigs randomized to each (HTK and UW) group. In situ perfusion was ineffective for both solutions at any pressure on the perfusate bag. Ex situ perfusion showed significantly improved flow and pressure in the hepatic artery and, therefore, was highly effective. No major differences between HTK and UW solutions could be detected. Therefore, an additional ex situ perfusion of the hepatic artery should be mandatory in every liver procurement.

Dynamics of reductive TCE dechlorination in two distinct H(2) supply scenarios and at various temperatures.

Anaerobic microbial dechlorination of trichloroethene (TCE) by a mixed, Dehalococcoides containing culture was investigated at different temperatures (4-60 degrees C) using propionate and lactate as a slow- and fast-releasing hydrogen (H(2)) source, respectively. Distinct temperature-dependent dynamics of substrate fermentation and H(2) levels could explain observed patterns of dechlorination. While varying the temperature caused changes in rate, the overall pattern of dechlorination was characteristic of the supplied electron donor. Feeding cultures with a rapidly fermentable substrate such as lactate generally resulted in high H(2) concentrations and fast and complete dechlorination accompanied by rapid methanogenesis. In contrast, low H(2) release rates resulting from fermentation of propionate were associated with 2 to 3-fold longer time frames necessary for complete dechlorination at intermediate temperatures (15-30 degrees C). A lag-phase prior to dechlorination of cis-dichloroethene (cDCE), together with a characteristic build-up of H(2) and methane, was consistently observed at slow H(2) supply. At temperatures of 10 degrees C and lower, the system remained in this lag phase and no dechlorination past cDCE was observed within the experimental time frame. However, when lactate was the substrate, complete dechlorination of TCE occurred within 74 days at 10 degrees C, accompanied by methane production. The choice of fermentable substrate decisively influenced the rate and degree of dechlorination at an electron donor/TCE ratio as high as 666:1. Temperature-dependent H(2) levels resulting from fermentation of different substrates could be satisfactorily explained through thermodynamic calculations of the Gibbs free energy yield assuming a constant metabolic energy threshold of -20 kJ/(mol reaction).

Filtration through nylon membranes negatively affects analysis of arsenic and phosphate by the molybdenum blue method.

Filtering synthetic arsenic- or phosphate-containing solutions (1.5-47.6mumol/L) with nylon syringe filters significantly reduced absorbances (by 6-74%) when analyzed with the colorimetric molybdenum blue method. Filtering the same solutions with cellulose acetate syringe filters yielded no significant differences as compared to unfiltered controls. The detrimental effect of nylon membranes was also observed when pure Milli-Q water was filtered and subsequently spiked with arsenic(III) or phosphate suggesting that some compound(s) eluting from the filter membranes interfere with the color formation in the assay. Consequently, we caution against using nylon filters when filtering water samples for the determination of arsenic or phosphate with the molybdenum blue method.

Caspase-dependent cell death involved in brain damage after acute subdural hematoma in rats.

Traumatic brain injury is associated with acute subdural hematoma (ASDH) that worsens outcome. Although early removal of blood can reduce mortality, patients still die or remain disabled after surgery and additional treatments are needed. The blood mass and extravasated blood induce pathomechanisms such as high intracranial pressure (ICP), ischemia, apoptosis and inflammation which lead to acute as well as delayed cell death. Only little is known about the basis of delayed cell death in this type of injury. Thus, the purpose of the study was to investigate to which extent caspase-dependent intracellular processes are involved in the lesion development after ASDH in rats. A volume of 300microL blood was infused into the subdural space under monitoring of ICP and tissue oxygen concentration. To asses delayed cell death mechanisms, DNA fragmentation was measured 1, 2, 4 and 7 days after ASDH by TUNEL staining, and the effect of the pan-caspase inhibitor zVADfmk on lesion volume was assessed 7 days post-ASDH. A peak of TUNEL-positive cells was found in the injured cortex at day 2 after blood infusion (53.4+/-11.6 cells/mm(2)). zVADfmk (160ng), applied by intracerebroventricular injection before ASDH, reduced lesion volume significantly by more than 50% (vehicle: 23.79+/-7.62mm(3); zVADfmk: 9.06+/-4.08). The data show for the first time that apoptotic processes are evident following ASDH and that caspase-dependent mechanisms play a crucial role in the lesion development caused by the blood effect on brain tissue.

Substrate phosphorylation affects degradation and interaction to endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme.

Recent findings from our laboratory suggest that intracellular peptides containing putative post-translational modification sites (i.e., phosphorylation) could regulate specific protein interactions. Here, we extend our previous observations showing that peptide phosphorylation changes the kinetic parameters of structurally related endopeptidase EP24.15 (EC 3.4.24.15), neurolysin (EC 3.4.24.16), and angiotensin-converting enzyme (EC 3.4.15.1). Phosphorylation of peptides that are degraded by these enzymes leads to reduced degradation, whereas phosphorylation of peptides that interacted as competitive inhibitors of these enzymes alters only the K(i)'s. These data suggest that substrate phosphorylation could be one of the mechanisms whereby some intracellular peptides would escape degradation and could be regulating protein interactions within cells.

Substrate phosphorylation affects degradation and interaction to endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme

Recent findings from our laboratory suggest that intracellular peptides containing putative post-translational modification sites (i.e., phosphorylation) could regulate specific protein interactions. Here, we extend our previous observations showing that peptide phosphorylation changes the kinetic parameters of structurally related endopeptidase EP24.15 (EC 3.4.24.15), neurolysin (EC 3.4.24.16), and angiotensin-converting enzyme (EC 3.4.15.1). Phosphorylation of peptides that are degraded by these enzymes leads to reduced degradation, whereas phosphorylation of peptides that interacted as competitive inhibitors of these enzymes alters only the Ki's. These data suggest that substrate phosphorylation could be one of the mechanisms whereby some intracellular peptides would escape degradation and could be regulating protein interactions within cells.

Brain oxygen monitoring: in-vitro accuracy, long-term drift and response-time of Licox- and Neurotrend sensors.

BACKGROUND: Oxygen tension sensors have been used to monitor tissue oxygenation in human brain for several years. The working principals of the most frequently used sensors, the Licox (LX) and Neurotrend (NT), are different, and they have never been validated independently for correct measurement in vitro. Therefore, we tried to clarify if the two currently available sensors provide sufficient accuracy and stability. METHOD: 12 LX oxygen tension sensors and NT sensors were placed into a liquid-filled tonometer chamber. The solution was kept at 37 +/- 0.2 degrees C and equilibrated with five calibration gases containing different O(2)- and CO(2)-concentrations. After equilibration, readings were taken for each gas concentration (accuracy test). Afterwards, the sensors were left in 3% O(2) and 9% CO(2) and readings were taken after 24, 48, 72, 96 and 120 hours (drift test). Thereafter, a 90% response time test was performed transferring sensors from 1% to 5% oxygen concentration and back, using pre-equilibrated tonometers. FINDINGS: All Licox oxygen probes [12] were used for this study. Two of 14 Neurotrend sensors did not calibrate, revealing a failure rate of 14% for NT. Oxygen tension during the accuracy test was measured as follows: 1% O(2) (7.1 mmHg): LX 6.5 +/- 0.4, NT 5.3 +/- 2.3 mmHg, 2% O(2) (14.2 mmHg): LX 12.9 +/- 0.6, NT 12.1 +/- 2.2 mmHg, 3% O(2) (21.4 mmHg): LX 19.8 +/- 0.7, NT 19.4 +/- 2.4 mmHg, 5% O(2) (35.8 mmHg): LX 33.4 +/- 1.0 mmHg, NT 33.5 +/- 2.9 mmHg, 8% O(2) (57.0 mmHg): 53.8 +/- 1.5, NT 53.6 +/- 3.3 mmHg. After 120 hours in 3% O(2) (21 mmHg), LX measured 19.8 +/- 1.9 mmHg, NT 17.9 +/- 4.7 mmHg. 90% response time from 1% to 5%/5% to 1% oxygen concentration was 129 +/- 27/174 +/- 26 sec for LX, 55 +/- 19/98 +/- 39 sec for NT. CONCLUSIONS: Both systems are measuring oxygen tension sufficiently, but more accurately with LX probes. NT sensors read significantly lower pO(2) in 1% O(2) and show an increasing deviation with higher oxygen concentrations which was due to two of twelve probes. A slight drift towards lower oxygen tension readings for both sensors but more pronounced for the NT does not impair long-term use. NT measures pCO(2) and pH very accurately.