Inhibition of Plexin C1 Protects Against Hepatic Ischemia-Reperfusion Injury.

OBJECTIVES: Hepatic ischemia-reperfusion injury is a disease pattern that is associated with an acute inflammatory reaction. It is well known that neutrophils play an essential role in the early phase of hepatic ischemia-reperfusion injury and determine the extent of tissue damage. Hepatic ischemia-reperfusion injury can result in organ failure, which is linked to high mortality. Recent data indicate that the neuronal guidance receptor Plexin C1 is involved in the control of the acute inflammatory response and, as such, modulates the transmigration of neutrophils. Hence, we investigated the functional role of Plexin C1 in a mouse model of early hepatic ischemia-reperfusion injury. DESIGN: Animal study. SETTING: University experimental laboratory. SUBJECTS: Wild-type, PLXNC1 and chimeric mice. INTERVENTIONS: Hepatic ischemia-reperfusion injury or sham operation. MEASUREMENTS AND MAIN RESULTS: We found that the functional inhibition of Plexin C1 in wild-type mice treated with an anti-Plexin C1 antibody and a Semaphorin 7A peptide reduced hepatic ischemia-reperfusion injury, as measured by the levels of lactate dehydrogenase, aspartate, and alanine aminotransferase. This reduction in ischemia-reperfusion injury was accompanied by reduced numbers of neutrophils in ischemic hepatic tissue and reduced serum levels of inflammatory cytokines. Experiments using Plexin C1 receptor-deficient (PLXNC1) mice also demonstrated decreased hepatic ischemia-reperfusion injury. Studies of chimeric mice revealed that the hematopoietic Plexin C1 knockout is crucial for reducing the extent of hepatic ischemia-reperfusion injury. CONCLUSIONS: These results describe a role for Plexin C1 during ischemia-reperfusion injury, highlight the role of hematopoietic Plexin C1 in the development of hepatic ischemia-reperfusion injury, and suggest that Plexin C1 is a potential drug target.

Mechanisms by which a CACNA1H mutation in epilepsy patients increases seizure susceptibility.

T-type calcium channels play essential roles in regulating neuronal excitability and network oscillations in the brain. Mutations in the gene encoding Cav3.2 T-type Ca(2+) channels, CACNA1H, have been found in association with various forms of idiopathic generalized epilepsy. We and others have found that these mutations may influence neuronal excitability either by altering the biophysical properties of the channels or by increasing their surface expression. The goals of the present study were to investigate the excitability of neurons expressing Cav3.2 with the epilepsy mutation, C456S, and to elucidate the mechanisms by which it influences neuronal properties. We found that expression of the recombinant C456S channels substantially increased the excitability of cultured neurons by increasing the spontaneous firing rate and reducing the threshold for rebound burst firing. Additionally, we found that molecular determinants in the I-II loop (the region in which most childhood absence epilepsy-associated mutations are found) substantially increase the surface expression of T-channels but do not alter the relative distribution of channels into dendrites of cultured hippocampal neurons. Finally, we discovered that expression of C456S channels promoted dendritic growth and arborization. These effects were reversed to normal by either the absence epilepsy drug ethosuximide or a novel T-channel blocker, TTA-P2. As Ca(2+)-regulated transcription factors also increase dendritic development, we tested a transactivator trap assay and found that the C456S variant can induce changes in gene transcription. Taken together, our findings suggest that gain-of-function mutations in Cav3.2 T-type Ca(2+) channels increase seizure susceptibility by directly altering neuronal electrical properties and indirectly by changing gene expression.

A potassium leak channel silences hyperactive neurons and ameliorates status epilepticus.

OBJECTIVE: To develop a constitutively active K(+) leak channel using TREK-1 (TWIK-related potassium channel 1; TREK-M) that is resistant to compensatory down-regulation by second messenger cascades, and to validate the ability of TREK-M to silence hyperactive neurons using cultured hippocampal neurons. To test if adenoassociated viral (AAV) delivery of TREK-M could reduce the duration of status epilepticus and reduce neuronal death induced by lithium-pilocarpine administration. METHODS: Molecular cloning techniques were used to engineer novel vectors to deliver TREK-M via plasmids, lentivirus, and AAV using a cytomegalovirus (CMV)-enhanced GABRA4 promoter. Electrophysiology was used to characterize the activity and regulation of TREK-M in human embryonic kidney (HEK-293) cells, and the ability to reduce spontaneous activity in cultured hippocampal neurons. Adult male rats were injected bilaterally with self-complementary AAV particles composed of serotype 5 capsid into the hippocampus and entorhinal cortex. Lithium-pilocarpine was used to induce status epilepticus. Seizures were monitored using continuous video-electroencephalography (EEG) monitoring. Neuronal death was measured using Fluoro-Jade C staining of paraformaldehyde-fixed brain slices. RESULTS: TREK-M inhibited neuronal firing by hyperpolarizing the resting membrane potential and decreasing input resistance. AAV delivery of TREK-M decreased the duration of status epilepticus by 50%. Concomitantly it reduced neuronal death in areas targeted by the AAV injection. SIGNIFICANCE: These findings demonstrate that TREK-M can silence hyperexcitable neurons in the brain of epileptic rats and treat acute seizures. This study paves the way for an alternative gene therapy treatment of status epilepticus, and provides the rationale for studies of AAV-TREK-M's effect on spontaneous seizures in chronic models of temporal lobe epilepsy.

Recovery of residual curarization after red blood cell transfusion.

BACKGROUND: The muscle-relaxing effects of succinylcholine are terminated via hydrolysis by plasma cholinesterase. There are multiple genetic variants of this enzyme and clinical circumstances that might influence the activity of plasma cholinesterase and eventually lead to prolonged neuromuscular blockade following succinylcholine application. CASE REPORT: Here, we report a parturient woman with atonic bleeding who suffered significant blood loss (hemoglobin 6.0 g•dL-¹). For surgical curettage, general anesthesia was performed by using short-acting succinylcholine. By the end of the 105-minute procedure, the patient's trachea was extubated. After extubation she showed signs of the prolonged neuromuscular blocking action of succinylcholine. At this time, the patient received an AB0-compatible red blood cell transfusion and recovered instantly from neuromuscular blockade. The plasma cholinesterase (3.200 U•L-¹) was below the normal range (4.900-12.000 U•L-¹). Patient's blood DNA analysis revealed heterozygously the genetic K variant of plasma cholinesterase. After red blood cell transfusion, serum potassium was elevated (5.7 mmol•L-¹; 4.4 mmol•L-¹ prior to transfusion). CONCLUSIONS: Pregnancy, blood loss and genetic variation contributed to impairment of plasma cholinesterase. Due to high-speed red blood cell transfusion, hemolytic release of erythrocyte cholinesterase might have terminated the neuromuscular blocking succinylcholine effect.

TTA-P2 is a potent and selective blocker of T-type calcium channels in rat sensory neurons and a novel antinociceptive agent.

Several agents that are preferential T-type calcium (T-channel) blockers have shown promise as being effective in alleviating acute and chronic pain, suggesting an urgent need to identify even more selective and potent T-channel antagonists. We used small, acutely dissociated dorsal root ganglion (DRG) cells of adult rats to study the in vitro effects of 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2), a derivative of 4-aminomethyl-4-fluoropiperdine, on T currents, as well as other currents known to modulate pain transmission. We found that TTA-P2 potently and reversibly blocked DRG T currents with an IC(50) of 100 nM and stabilized channel in the inactive state, whereas high-voltage-activated calcium and sodium currents were 100- to 1000-fold less sensitive to channel blocking effects. In in vivo studies, we found that intraperitoneal injections of 5 or 7.5 mg/kg TTA-P2 reduced pain responses in mice in phases 1 and 2 of the formalin test. Furthermore, TTA-P2, at 10 mg/kg i.p., selectively and completely reversed thermal hyperalgesia in diabetic rats treated with streptozocin but had no effect on the nociceptive response of healthy animals. The antihyperalgesic effects of TTA-P2 in diabetic rats were completely abolished by administration of oligonucleotide antisense for Ca(V)3.2 isoform of T channels. Thus, TTA-P2 is not only the most potent and selective blocker of T channels in sensory neurons yet described, but it also demonstrates the potential for the pharmacological effectiveness of this approach in addressing altered nociceptive responses in animal models of both inflammatory and neuropathic pain.

[Resuscitation algorithms for cardiac arrest and arrhythmias]. / Reanimation: Was ist neu? Notfallbehandlung von Herzstillstand und Arrhythmien.

Recently, guidelines for cardiopulmonary resuscitation have been updated by the European Resuscitation Council. Here we focus on cardiac arrest and arrhythmia algorithms for primary healthcare providers incorporating new recommendations.

[Postexposure prophylaxis after sexual assault]. / Infektionsprophylaxe nach sexueller Gewalt.

Relevant exposure to important infectious pathogens can occur during sexual assault. If there is a latent period between exposure and illness due to an infection with pathogens, a postexposure prophylaxis can effectively inhibit the infection. In the present review article possible postexposure prophylaxis treatment for tetanus, hepatitis B, HIV and hepatitis A are discussed with a focus on the time window within which a specific regimen should be started and in which temporal order. These recommendations are based on the epidemiologic conditions in Germany. Moreover, the two most frequent sexually transmitted bacterial infections, namely Neisseria gonorrhoea and Chlamydia trachomatis are presented, as victims of sexual assault in particular often do not return for control investigations in an outpatient setting.

[Acute aortic dissection: be aware of signs and symptoms]. / Ist es eine Aortendissektion.

Acute aortic dissection is a rare medical emergency accompanied by high mortality. For ensuring fast diagnosis and delivery of appropriate medical treatment the knowledge of signs and symptoms is crucial. Here we summarize clinical presentation according to existing studies, focus on diagnosis and introduce to contemporary treatment of this disease.

[Unexpected increase in extravascular lung water after acute lung injury]. / Unerwarteter Anstieg des extravasalen Lungenwassers nach akutem Lungenversagen.

HISTORY AND CLINICAL FINDING: We report on a 27-year-old female patient presenting with pneumonia and developing acute respiratory distress syndrome. Using transpulmonary thermodilution, an elevated extravascular lung water was detected (17 ml/kg). Patient required lung-protective mechanical ventilation and received antibiotic therapy. Negative fluid balance was targeted. Under this treatment, respiratory function improved, inflammation parameters declined, and extravascular lung water was recurrent (10 ml/kg). Subsequently, extravascular lung water increased to 29 ml/kg. DIAGNOSIS: In a chest x-ray, the central venous catheter tip was dislocated into the right internal jugular vein. 7 days before, the catheter was shown to be in projection of the Vena cava superior in a previous chest x-ray. THERAPY AND COURSE: After insertion of a new central venous catheter, extravascular lung water was quantified at 10 ml/kg again. After 10 days of mechanical ventilation the patient was successfully extubated. CONCLUSION: As mechanism for this catheter dislocation, we propose a Valsalva maneuver or spontaneous movements of the upper body of the patient. Sudden increase in extravascular lung water may reflect central venous catheter tip dislocation and chest x-ray should be considered to verify catheter tip position.

Scientific habilitations as an indicator of research productivity in Germany - a comparative analysis of several clinical specialties / Habilitationen als Kriterium für akademische Produktivität medizinischer Fakultäten: eine vergleichende Analyse mehrerer Fachdisziplinen.

BACKGROUND: As a part of the German academic system the scientific habilitation is a significant individual qualification in research and teaching. In a pilot project, we recently reported the number of habilitations in anesthesiology, visceral surgery, gynecology and internal medicine. Here, we analyze habilitations from 13 additional clinical specialties. METHODS: Habilitations in clinical specialties published in the German Medical Journal (Dtsch Arztebl) between 2010 and 2017 were quantitatively analyzed. For data validation, the dean offices of all German medical schools were requested to forward all accomplished habilitations. The percentage of women and the numbers with respect to the specialty were analyzed for each university hospital. Data are presented as medians (interquartile ranges). RESULTS: In this study, 2,264 accomplished habilitations were analyzed. Annually, 45 (36-56) habilitations were reported in orthopedic/trauma surgery, 40 (36-48) in radiology and 37 (29-46) in neurology, while the medians in the other analyzed specialties were lower. The highest percentage of women earning a habilitation degree was reported for pediatrics (37 %), dermatology (33 %), and ophthalmology (32 %). CONCLUSIONS: Here, the output of completed habilitations from 13 medical specialties was analyzed for each German medical school. As a habilitation stands for a sustainable scientific contribution, this study may help to identify further career development needs for junior and for female scientists.

Xenon reduces N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic transmission in the amygdala.

BACKGROUND: The neuronal and molecular targets of the inhalational general anesthetic xenon are a matter of debate. The current knowledge is largely based on studies using neurons in culture or heterologous expression systems. In the current study, the authors evaluated for the first time the effect of xenon on synaptic transmission in the basolateral amygdala in an in vitro brain slice preparation of the mouse. METHODS: A patch clamp technique was used to evaluate the effects of xenon on N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs), as well as on gamma-aminobutyric acid type A receptor-mediated inhibitory postsynaptic currents. The currents were either evoked upon electrical stimulation (NMDA-eEPSCs, AMPA-eEPSCs) or upon focal, laser-guided photolysis of caged l-glutamate (p-NMDA-Cs, p-AMPA-Cs). In addition, the authors investigated the effects of xenon on miniature EPSCs. RESULTS: Xenon reversibly reduced basal synaptic transmission but had no effect on gamma-aminobutyric acid type A receptor-mediated inhibitory synaptic transmission. Xenon concentration-dependently diminished NMDA-eEPSCs and p-NMDA-Cs to the same amount. Likewise, xenon-induced reduction of AMPA-eEPSCs and p-AMPA-Cs did not differ. Xenon did not affect the frequency of miniature EPSCs but reduced their amplitude. CONCLUSIONS: In the current study, xenon considerably depressed NMDA and AMPA receptor-mediated synaptic transmission in the basolateral amygdala without affecting inhibitory synaptic transmission. The results provide evidence that the effects of xenon on NMDA- and AMPA-EPSCs are primarily mediated via postsynaptic mechanisms.

Botulinum toxin B increases intrinsic muscle activity in organotypic spinal cord-skeletal muscle co-cultures.

In organotypic spinal cord-skeletal muscle co-cultures, motoneurons are driven by locomotor commands and induce contractions in surrounding muscle fibres. Using these co-cultures, it has been shown that effects of organophosphorus compounds on neuromuscular synapses can be determined in vitro. In the present study we aimed to extend this in vitro tool for pharmacologic testing of botulinum toxin B. This neurotoxin is widely used for the treatment of dystonia. Besides its effects on the neuromuscular junction, botulinum toxins may also act at centrally located synapses. Incubation with botulinum toxin B (Neurobloc(®)) induced a significant increase in muscular activity after 24, 48 and 72h. Application of the NMDA- and AMPA-receptor antagonists AP5 (20µM) and CNQX (15µM) induced a similar augmentation of muscle activity after 48 and 72h, respectively. Administration of the glycine- and GABA(A)-receptor antagonists strychnine (1µM) and bicuculline (100µM) did not alter intrinsic muscle activity. In contrast, application of a non-depolarizing muscle relaxant rocuronium bromide reduced the muscle activity in a dose-dependent manner. Our findings suggest that glutamatergic synapses in the spinal cord are more sensitive to botulinum toxin B than synaptic contacts between spinal motoneurons and muscle fibres.

Self-regeneration of neuromuscular function following soman and VX poisoning in spinal cord-skeletal muscle cocultures.

Aside from nerve agents, various highly toxic pesticides belong to the group of organophosphorus (OP) compounds, thereby causing a large number of intoxications every year. Unfortunately, there are still shortcomings in the current treatment for OP poisoning and research on novel therapeutic options is restricted in several aspects. In this study we investigated the suitability of organotypic cocultures for pharmacological in vitro studies involving OP compounds. These slice cultures are derived from murine spinal cord and muscle tissue forming functional neuromuscular synapses, which trigger spontaneous contractions of muscle fibers. Using video microscopy to quantify muscle activity, we assessed the viability of cocultures after exposure to soman and VX, and the associated loss and recovery of neuromuscular function. Antidotal treatment was not provided. The application of nerve agents led to an almost complete loss of muscle activity. However, cell cultures regained equivalent muscular function to the control situation three and seven days after intoxication. In summary, the tested in vitro system could be a promising tool for the investigation of long term effects and therapeutic options for OP poisoning.

Spinal cord--skeletal muscle cocultures detect muscle-relaxant action of botulinum neurotoxin A.

The mouse LD50 assay is routinely used for potency testing of botulinum toxins. Unfortunately, this test is associated with severe pain and distress in animals and requires large quantities of mice. Here we used cocultures of spinal cord and muscle tissue as an alternative for probing botulinum toxins. Cocultures were prepared from mouse embryonic tissue (C57/BL6J) and cultured for 24-27 days. In these cultures spontaneous muscle activity was quantified in sham- and botulinum toxin-treated cultures for up to 3 days by video microscopy. At a concentration of 58 fmol/L or higher, incobotulinumtoxin A significantly reduced the frequency of muscle contractions within 24 hours after incubation. Hence, nerve-muscle-cultures are similar sensitive as the mouse LD50 assay. The limit of detection, as observed in our study, is close to the most sensitive cell-based bioassays, capable to detect concentrations of botulinum neurotoxin A between 30 and 50 fmol/L. However, spontaneous muscle activity of individual cultures displayed considerable fluctuations when evaluated on a day-to-day basis. Generally, the authors would like to emphasize, that in its present form, this in vitro assay might be too laborious for botulinum toxin potency testing. Thus, methodical improvements to decrease data variability are the next milestone to be passed towards developing this model into an assay that can be utilized for reducing animal experimentation.

Opposing actions of sevoflurane on GABAergic and glycinergic synaptic inhibition in the spinal ventral horn.

BACKGROUND: The ventral horn is a major substrate in mediating the immobilizing properties of the volatile anesthetic sevoflurane in the spinal cord. In this neuronal network, action potential firing is controlled by GABA(A) and glycine receptors. Both types of ion channels are sensitive to volatile anesthetics, but their role in mediating anesthetic-induced inhibition of spinal locomotor networks is not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: To compare the effects of sevoflurane on GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) whole-cell voltage-clamp recordings from ventral horn interneurons were carried out in organotypic spinal cultures. At concentrations close to MAC (minimum alveolar concentration), decay times of both types of IPSCs were significantly prolonged. However, at 1.5 MAC equivalents, GABAergic IPSCs were decreased in amplitude and reduced in frequency. These effects counteracted the prolongation of the decay time, thereby decreasing the time-averaged GABAergic inhibition. In contrast, amplitudes and frequency of glycinergic IPSCs were not significantly altered by sevoflurane. Furthermore, selective GABA(A) and glycine receptor antagonists were tested for their potency to reverse sevoflurane-induced inhibition of spontaneous action potential firing in the ventral horn. These experiments confirmed a weak impact of GABA(A) receptors and a prominent role of glycine receptors at a high sevoflurane concentration. CONCLUSIONS: At high concentrations, sevoflurane mediates neuronal inhibition in the spinal ventral horn primarily via glycine receptors, and less via GABA(A) receptors. Our results support the hypothesis that the impact of GABA(A) receptors in mediating the immobilizing properties of volatile anesthetics is less essential in comparison to glycine receptors.