Securin and separase modulate membrane traffic by affecting endosomal acidification.

Securin and separase play a key role in sister chromatid separation during anaphase. However, a growing body of evidence suggests that in addition to regulating chromosome segregation, securin and separase display functions implicated in membrane traffic in Caenorhabditis elegans and Drosophila. Here we show that in mammalian cells both securin and separase associate with membranes and that depletion of either protein causes robust swelling of the trans-Golgi network (TGN) along with the appearance of large endocytic vesicles in the perinuclear region. These changes are accompanied by diminished constitutive protein secretion as well as impaired receptor recycling and degradation. Unexpectedly, cells depleted of securin or separase display defective acidification of early endosomes and increased membrane recruitment of vacuolar (V-) ATPase complexes, mimicking the effect of the specific V-ATPase inhibitor Bafilomycin A1. Taken together, our findings identify a new functional role of securin and separase in the modulation of membrane traffic and protein secretion that implicates regulation of V-ATPase assembly and function.

Effects of propranolol on resting metabolic rate after severe head injury.

Postabsorptive resting metabolic rate (RMR), measured by indirect calorimetry, and the effect of iv propranolol administration were studied in 12 nonseptic patients with severe head injury by means of indirect calorimetry. Before propranolol RMR was moderately increased (126 +/- 10.4% of predicted values) whereas urinary excretion of catecholamines was markedly elevated (p less than .01 vs. normal values). RMR was significantly correlated with both resting heart rate (HR) (r = .72, p less than .01) and 24-h urinary N excretion (r = .85, p less than .001). The administration of iv propranolol (0.1 mg/kg) produced a rapid decrease in HR (-10 +/- 4%, p less than .001) and in RMR (-6.1 +/- 2.3%, p less than .001). Further administration of propranolol produced no additional reduction in either HR or RMR. We conclude that severely head-injured patients are moderately hypermetabolic in resting and postabsorptive conditions, and that acute iv propranolol administration induces a reduction of about one quarter of the resting hypermetabolism.

Plasmacytoid dendritic cells sense skin injury and promote wound healing through type I interferons.

Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-α/β)-producing cells that express intracellular toll-like receptor (TLR) 7 and TLR9 and recognize viral nucleic acids in the context of infections. We show that pDCs also have the ability to sense host-derived nucleic acids released in common skin wounds. pDCs were found to rapidly infiltrate both murine and human skin wounds and to transiently produce type I IFNs via TLR7- and TLR9-dependent recognition of nucleic acids. This process was critical for the induction of early inflammatory responses and reepithelization of injured skin. Cathelicidin peptides, which facilitate immune recognition of released nucleic acids by promoting their access to intracellular TLR compartments, were rapidly induced in skin wounds and were sufficient but not necessary to stimulate pDC activation and type I IFN production. These data uncover a new role of pDCs in sensing tissue damage and promoting wound repair at skin surfaces.

Differential proteoglycan expression in two spinal cord regions after dorsal root injury.

Dorsal root injury leads to reactive gliosis in the spinal cord dorsal root entry zone and dorsal column, two regions that undergo Wallerian degeneration, but have distinct growth-inhibitory properties. This disparity could in part be due to differences in the number of degenerating sensory fibers, differences in glial cell activation, and/or to differential expression of growth-inhibitory molecules such as chondroitin sulfate proteoglycans. Laser capture microdissection of these two spinal cord white matter regions, followed by quantitative analysis of mRNA expression by real-time PCR, revealed that glial marker transcripts were differentially expressed post-injury and that the chondroitin sulfate proteoglycans Brevican and Versican V1 and V2 were preferentially up-regulated in the dorsal root entry zone, but not the dorsal column. These results indicate that reactive gliosis differs between these two regions and that Brevican and Versican are potential key molecules participating in the highly inhibitory properties of the dorsal root entry zone.

The expressions of GABA and glutannate transporters are altered in the spared nerve injury model of neuropathic pain in the rat

Neuropathic pain is a common form of chronic pain, and is unsuccessfully alleviated by usual medications. Mounting evidence strongly point at non-neuronal glial cells in the spinal cord as key actors behind the persistence of pain. In particular, a change in the astrocytic capacity to regulate extracellular concentrations of neurotransmitters might account for the strengthened spinal nociceptive neurotransmission. Therefore, we investigated whether spinal expressions of GABA (GAT) and glutamate (EAAT) transporters were affected in the spared nerve injury (SNI) rat model of neuropathic pain. SNI was induced in male Sprague-Dawley rats by a unilateral section of tibial and common peroneal branches of the sciatic nerve, leaving the sural branch untouched. Western-blot analysis was performed to study the expression of GAT-1 and GAT-3 as well as EAAT-1 and EAAT-2, the main astrocytic GABA and glutamate transporters respectively. Seven days post-surgery, a significant increase in GAT-1, GAT-3 and EAAT-1 expressions is detected in both ipsilateral and contralateral sides of lumbar spinal cord in comparison to sham animals. No change in EAAT-2 signal could be detected. Furthermore, the astrocytic reaction parallels the glutamate and GABA transporters changes as we found an increased GFAP expression compared to the sham condition, in both spinal sides. Together, our results indicate that modifications in GABA and glutamate transport may occur along with SNI-associated painful neuropathy and identify spinal neurotransmitter reuptake machinery as a putative pharmacological target in neuropathic pain.

Mutism and Amnesia following High-Voltage Electrical Injury: Psychogenic Symptomatology Triggered by Organic Dysfunction?

Background: Mutism and dense retrograde amnesia are found both in organic and dissociative contexts. Moreover, dissociative symptoms may be modulated by right prefrontal activity. A single case, M.R., developed left hemiparesis, mutism and retrograde amnesia after a high-voltage electric shock without evidence of lasting brain lesions. M.R. suddenly recovered from his mutism following a mild brain trauma 2 years later. Methods: M.R.'s neuropsychological pattern and anatomoclinical correlations were studied through (i) language and memory assessment to characterize his deficits, (ii) functional neuroimaging during a standard language paradigm, and (iii) assessment of frontal and left insular connectivity through diffusion tractography imaging and transcranial magnetic stimulation. A control evaluation was repeated after recovery. Findings: M.R. recovered from the left hemiparesis within 90 days of the accident, which indicated a transient right brain impairment. One year later, neurobehavioral, language and memory evaluations strongly suggested a dissociative component in the mutism and retrograde amnesia. Investigations (including MRI, fMRI, diffusion tensor imaging, EEG and r-TMS) were normal. Twenty-seven months after the electrical injury, M.R. had a very mild head injury which was followed by a rapid recovery of speech. However, the retrograde amnesia persisted. Discussion: This case indicates an interaction of both organic and dissociative mechanisms in order to explain the patient's symptoms. The study also illustrates dissociation in the time course of the two different dissociative symptoms in the same patient.

Shoulder strength imbalances as injury risk in handball.

This study was conducted to analyze whether internal (IR) and external (ER) rotator shoulder muscles weakness and/or imbalance collected through a preseason assessment could be predictors of subsequent shoulder injury during a season in handball players. In preseason, 16 female elite handball players (HPG) and 14 healthy female nonathletes (CG) underwent isokinetic IR and ER strength test with use of a Con-Trex® dynamometer in a seated position with 45° shoulder abduction in scapular plane, at 60, 120 and 240°/s in concentric and at 60°/s in eccentric, for both sides. An imbalanced muscular strength profile was determined using -statistically selected cut-offs from CG values. For HPG, all newly incurred shoulder injuries were reported during the season. There were significant differences between HPG and CG only for dominant eccentric IR strength, ER/IR ratio at 240°/s and for IRecc/ERcon ratio. In HPG, IR and ER strength was higher, and ER/IR ratios lower for dominant than for nondominant side. The relative risk was 2.57 (95%CI: 1.60-3.54; P<0.05) if handball players had an imbalanced muscular strength profile. In youth female handball players IR and ER muscle strength increases on the dominant side without ER/IR imbalances; and higher injury risk was associated with imbalanced muscular strength profile.

Transcranial Doppler after traumatic brain injury: is there a role?

PURPOSE OF REVIEW: To present the practical aspects of transcranial Doppler (TCD) and provide evidence supporting its use for the management of traumatic brain injury (TBI) patients. RECENT FINDINGS: TCD measures systolic, mean, and diastolic cerebral blood flow (CBF) velocities and calculates the pulsatility index from basal intracranial arteries. These variables reflect the brain circulation, provided there is control of potential confounding factors. TCD can be useful in patients with severe TBI to detect low CBF, for example, during intracranial hypertension, and to assess cerebral autoregulation. In the emergency room, TCD might complement brain computed tomography (CT) scan and clinical examination to screen patients at risk for further neurological deterioration after mild-to-moderate TBI. SUMMARY: The diagnostic value of TCD should be incorporated into other findings from multimodal brain monitoring and CT scan to optimize the bedside management of patients with TBI and help guide the choice of appropriate therapies.

Cerebral extracellular lactate increase is predominantly non ischemic in patients with severe traumatic brain injury

Le cerveau est l'organe avec les besoins en énergie les plus élevés du corps humain, et le glucose est un substrat énergétique cérébral essentiel. Ces dernières décennies, la compréhension de la neuroénergétique a beaucoup évolué et un rôle du lactate comme substrat énergétique important a été mis en évidence, notamment suite à l'introduction du modèle de l'ANLS (astrocyte-neuron lactate shuttle). Selon celui-ci, les astrocytes convertissent le glucose en lactate par réaction de glycolyse, puis il est transporté jusqu'aux neurones qui l'utilisent comme source d'énergie à travers le cycle de Krebs. Chez l'homme, divers travaux récents ont montré que le lactate peut servir de « carburant » cérébral chez le sujet sain, après effort intense ou chez le patient diabétique. La régulation métabolique et le rôle du lactate après lésion cérébrale aiguë sont encore peu connus. Présentation de l'article Le but de ce travail a été d'étudier le métabolisme cérébral du lactate chez les patients atteints de traumatisme crânien (TCC) sévère. Nous avons émis l'hypothèse que l'augmentation du lactate cérébral chez ces patients n'était pas associée de manière prédominante à une hypoxie ou une ischémie mais plutôt à une glycolyse aérobie, et également à une perfusion cérébrale normale. L'étude a porté sur une cohorte prospective de 24 patients avec TCC sévère admis au service de médecine intensive du CHUV (centre hospitalier universitaire vaudois), monitorés par un système combinant microdialyse cérébrale (outil permettant de mesurer divers métabolites cérébraux, tels que le lactate, le pyruvate et le glucose), mesure de la pression cérébrale en oxygène et de la pression intracrânienne. Cet outil nous a permis de déterminer si l'élévation du lactate était principalement associée à une glycolyse active ou plutôt à une hypoxie. L'utilisation du CTde perfusion a permis d'évaluer la relation entre les deux patterns d'élévation du lactate (glycolytique ou hypoxique) et la perfusion cérébrale globale. Nos résultats ont montré que l'augmentation du lactate cérébral chez les patients avec TCC sévère était associée de manière prédominante à une glycolyse aérobie plutôt qu'à une hypoxie/ischémie. D'autre part, nous avons pu confirmer que les épisodes de lactate glycolytique étaient toujours associés à une perfusion cérébrale normale ou augmentée, alors que les épisodes de lactate hypoxique étaient associés à une hypoperfusion cérébrale. Conclusions et perspectives Nos résultats, qui ont permis de mieux comprendre le métabolisme cérébral du lactate chez les patients avec TCC sévère, soutiennent le concept que le lactate est produit dans des conditions aérobes et pourrait donc être utilisé comme source d'énergie par le cerveau lésé pour subvenir à des besoins augmentas. Etant donné que la dysfonction énergétique est une des probables causes de perte neuronale après traumatisme crânien, ces résultats ouvrent des perspectives thérapeutiques nouvelles après agression cérébrale chez l'homme, visant à tester un potentiel effet neuroprotecteur via l'administration de lactate exogène.

Epuration extrarénale continue pour l'insuffisance rénale aiguë aux soins intensifs [Continuous renal replacement therapy for acute kidney injury].

Acute kidney injury is common in critical illness and associated with important morbidity and mortality. Continuous renal replacement therapy (CRRT) enables physicians to safely and efficiently control associated metabolic and fluid balance disorders. The insertion of a large central venous catheter is required, which can be associated with mechanical and infectious complications. CRRT requires anticoagulation, which currently relies on heparin in most cases although citrate could become a standard in a near future. The choice of the substitution fluid depends on the clinical situation. A dose of 25 ml/kg/h is currently recommended.

Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.

BACKGROUND: The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. RESULTS: We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process. CONCLUSIONS: In the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.

Biomechanical characterization and in vitro mechanical injury of elderly human femoral head cartilage: comparison to adult bovine humeral head cartilage.

OBJECTIVES: In vitro mechanical injury of articular cartilage is useful to identify events associated with development of post-traumatic osteoarthritis (OA). To date, many in vitro injury models have used animal cartilage despite the greater clinical relevance of human cartilage. We aimed to characterize a new in vitro injury model using elderly human femoral head cartilage and compare its behavior to that of an existing model with adult bovine humeral head cartilage. DESIGN: Mechanical properties of human and bovine cartilage disks were characterized by elastic modulus and hydraulic permeability in radially confined axial compression, and by Young's modulus, Poisson's ratio, and direction-dependent radial strain in unconfined compression. Biochemical composition was assessed in terms of tissue water, solid, and glycosaminoglycan (GAG) contents. Responses to mechanical injury were assessed by observation of macroscopic superficial tissue cracks and histological measurements of cell viability following single injurious ramp loads at 7 or 70%/s strain rate to 3 or 14 MPa peak stress. RESULTS: Confined compression moduli and Young's moduli were greater in elderly human femoral cartilage vs adult bovine humeral cartilage whereas hydraulic permeability was less. Radial deformations of axially compressed explant disks were more anisotropic (direction-dependent) for the human cartilage. In both cartilage sources, tissue cracking and associated cell death during injurious loading was common for 14 MPa peak stress at both strain rates. CONCLUSION: Despite differences in mechanical properties, acute damage induced by injurious loading was similar in both elderly human femoral cartilage and adult bovine humeral cartilage, supporting the clinical relevance of animal-based cartilage injury models. However, inherent structural differences such as cell density may influence subsequent cell-mediated responses to injurious loading and affect the development of OA.

Beyond intracranial pressure: optimization of cerebral blood flow, oxygen, and substrate delivery after traumatic brain injury.

Monitoring and management of intracranial pressure (ICP) and cerebral perfusion pressure (CPP) is a standard of care after traumatic brain injury (TBI). However, the pathophysiology of so-called secondary brain injury, i.e., the cascade of potentially deleterious events that occur in the early phase following initial cerebral insult-after TBI, is complex, involving a subtle interplay between cerebral blood flow (CBF), oxygen delivery and utilization, and supply of main cerebral energy substrates (glucose) to the injured brain. Regulation of this interplay depends on the type of injury and may vary individually and over time. In this setting, patient management can be a challenging task, where standard ICP/CPP monitoring may become insufficient to prevent secondary brain injury. Growing clinical evidence demonstrates that so-called multimodal brain monitoring, including brain tissue oxygen (PbtO2), cerebral microdialysis and transcranial Doppler among others, might help to optimize CBF and the delivery of oxygen/energy substrate at the bedside, thereby improving the management of secondary brain injury. Looking beyond ICP and CPP, and applying a multimodal therapeutic approach for the optimization of CBF, oxygen delivery, and brain energy supply may eventually improve overall care of patients with head injury. This review summarizes some of the important pathophysiological determinants of secondary cerebral damage after TBI and discusses novel approaches to optimize CBF and provide adequate oxygen and energy supply to the injured brain using multimodal brain monitoring.