Constant rate infusion of diazepam or propofol for the management of canine cluster seizures or status epilepticus

INTRODUCTION: Cluster seizures (CS) and status epilepticus (SE) in dogs are severe neurological emergencies that require immediate treatment. Practical guidelines call for constant rate infusion (CRI) of benzodiazepines or propofol (PPF) in patients with seizures not responding to first-line treatment, but to date only few studies have investigated the use of CRI in dogs with epilepsy. STUDY DESIGN: Retrospective clinical study. METHODS: Dogs that received CRI of diazepam (DZP) or PPF for antiepileptic treatment during hospitalization at the Veterinary Teaching Hospital of the University of Turin for CS or SE between September 2016 and December 2019 were eligible for inclusion. Favorable outcome was defined as cessation of clinically visible seizure activity within few minutes from the initiation of the CRI, no seizure recurrence within 24 h after discontinuation of CRI through to hospital discharge, and clinical recovery. Poor outcome was defined as recurrence of seizure activity despite treatment or death in hospital because of recurrent seizures, catastrophic consequences of prolonged seizures or no return to an acceptable neurological and clinical baseline, despite apparent control of seizure activity. Comparisons between the number of patients with favorable outcome and those with poor outcome in relation to type of CRI, seizure etiology, reason for presentation (CS or SE), sex, previous AED therapy and dose of PPF CRI were carried out. RESULTS: A total of 37 dogs, with 50 instances of hospitalization and CRI administered for CS or SE were included in the study. CRI of diazepam (DZP) or PPF was administered in 29/50 (58%) and in 21/50 (42%) instances of hospitalization, respectively. Idiopathic epilepsy was diagnosed in 21/37 (57%), (13/21 tier I and 8/21 tier II); structural epilepsy was diagnosed in 6/37 (16%) of which 4/6 confirmed and 2/6 suspected. A metabolic or toxic cause of seizure activity was recorded in 7/37 (19%). A total of 38/50 (76%) hospitalizations were noted for CS and 12/50 (24%) for SE. In 30/50 (60%) instances of hospitalization, the patient responded well to CRI with cessation of seizure activity, no recurrence in the 24 h after discontinuation of CRI through to hospital discharge, whereas a poor outcome was recorded for 20/50 (40%) cases (DZP CRI in 12/50 and PPF CRI in 8/50). Comparison between the number of patients with favorable outcome and those with poor outcome in relation to type of CRI, seizure etiology, reason for presentation (CS or SE), sex and previous AED therapy was carried out but no statistically significant differences were found. CONCLUSIONS: The present study is the first to document administration of CRI of DZP or PPF in a large sample of dogs with epilepsy. The medications appeared to be tolerated without major side effects and helped control seizure activity in most patients regardless of seizure etiology. Further studies are needed to evaluate the effects of CRI duration on outcome and complications

Duration of constant rate infusion with diazepam or propofol for canine cluster seizures and status epilepticus

IntroductionConstant rate infusion (CRI) of benzodiazepines or propofol (PPF) is a therapeutic option for cluster seizures (CS) and status epilepticus (SE) in canine patients non-responding to first-line benzodiazepines or non-anesthetics. However, specific indications for optimal duration of CRI are lacking. The aim of this study was to determine the effect of duration of anesthetic CRI on outcome and length of hospital stay in dogs with refractory seizure activity of different etiology.Study designOpen-label non-randomized clinical trial.Materials and methodsSeventy-three client-owned dogs were enrolled. Two groups [experimental (EXP) vs. control (CTRL)] were compared. The EXP group received diazepam (DZP) or PPF CRI for 12 h (±1 h) and the CTRL group received DZP or PPF CRI for 24 h (±1 h) in addition to a standardized emergency treatment protocol identical for both study groups. The historical control group was made up of a population of dogs already reported in a previously published paper by the same authors. Favorable outcome was defined as seizure cessation after CRI, no seizure recurrence, and clinical recovery. Poor outcome was defined as seizure recurrence, death in hospital or no return to acceptable clinical baseline. Univariate statistical analysis was performed.ResultsThe study sample was 73 dogs: 45 (62%) received DZP CRI and 28 (38%) received PPF CRI. The EXP group was 39 dogs (25 DZP CRI and 14 PPF CRI) and the CTRL group 34 dogs (20 DZP CRI and 14 PPF CRI). We found no statistically significant difference in outcomes between the groups. The median length of stay was 56 h (IQR, 40–78) for the ALL EXP group and 58.5 h (IQR, 48–74.5) for the ALL CTRL group (p = 0.8).ConclusionEven though a shorter DZP or PPF CRI duration was not associated with a worse outcome, the study failed to identify a clear superiority of shorter CRI duration on outcome or length of hospital stay in dogs with refractory seizure activity of different etiology

Quantum Dots pour le Ciblage en Cellules Vivantes et la Microscopie HiLo Bi-couleur

Quantum Dots (QDs) are semiconductor nanocrystals with unique optical properties. Their use as probes in biology requires their water solubilization through an adapted surface chemistry which will determine the QD final size, its optical properties and its interaction with the biological environment. We propose a new ligand for QD water solubilization, the dihydrolipoic acid - sulfobetaine (DHLA-SB), which combines simultaneously small size, good stability in a large pH range, in saturated salt solutions and during time and with very low non specific adsorption to cellular membranes. Afterwards we analyzed the intracellular behavior of DHLA-SB QDs during time and we compared it to two other QD classes. This study clearly demonstrated the surface chemistry influence on the intracellular fate of the nanoparticules and also revealed an increased stability of DHLA-SB QDs. We then functionalized the QDs with streptavidin (SA) or biotin to specifically label live and fixed cells. We demonstrated how DHLA-SB QDs-SA are able to efficiently stain a membrane receptor as well as an intracellular biotinylated protein into living cells, with a higher specificity than commercial QDs-SA (Invitrogen). Lastly we proposed to use DHLA-SB QDs as probes for a new structured illumination imaging technique, two-color Hilo microscopy, which allows to obtain an optical section (similar to a confocal image) of a thick biological sample with only one image acquisition.Les Quantum Dots (QDs) sont des nanocristaux de semiconducteurs qui possèdent des propriétés optiques hors du commun. Leur utilisation comme sondes en biologie nécessite leur solubilisation dans l'eau, grâce à une chimie de surface adaptée, qui influence la taille finale du QD, ses propriétés optiques et son interaction avec l'environnement biologique. Nous avons développé un nouveau ligand, l'acide dihydrolipoïque-sulfobétaïne (DHLA-SB), qui permet d'obtenir des QDs à la fois petits, stables dans une vaste gamme de pH, dans des solutions saturées en sel et dans le temps, et avec une très faible adsorption non spécifique sur les cellules. Nous avons observé et caractérisé le comportement intracellulaire des QDs DHLA-SB au cours du temps et nous l'avons comparé à celui de deux autres classes de QDs : cette étude a clairement montré l'influence de la chimie de surface sur le devenir intracellulaire des nanoparticules et a révélé une stabilité accrue des QDs DHLA-SB. Nous nous sommes également intéressés à la fonctionnalisation des QDs avec la streptavidine (SA) ou la biotine afin de marquer spécifiquement des cellules vivantes ou fixées. Les QDs-SA DHLA-SB obtenus ont permis de suivre un récepteur membranaire ou encore de marquer de façon spécifique une protéine biotinylée à l'intérieur de cellules vivantes, bien plus efficacement qu'avec les QDs-SA commerciaux (Invitrogen). Enfin, nous avons proposé d'utiliser les QDs DHLA-SB pour améliorer une technique de microscopie à illumination structurée, la microscopie HiLo bicouleur, et obtenir une coupe optique (type image confocale) d'échantillons biologiques épais en une seule image

Quantum Dots pour le ciblage en cellules vivantes et la microscopie Hilo bi-couleur

Les Quantum Dots (QDs) sont des nanocristaux de semiconducteurs qui possèdent des propriétés optiques hors du commun. Leur utilisation comme sondes en biologie nécessite leur solubilisation dans l eau, grâce à une chimie de surface adaptée, qui influence la taille finale du QD, ses propriétés optiques et son interaction avec l environnement biologique. Nous avons développé un nouveau ligand, l acide dihydrolipoïque-sulfobétaïne (DHLA-SB), qui permet d obtenir des QDs à la fois petits, stables dans une vaste gamme de pH, dans des solutions saturées en sel et dans le temps, et avec une très faible adsorption non spécifique sur les cellules. Nous avons observé et caractérisé le comportement intracellulaire des QDs DHLA-SB au cours du temps et nous l avons comparé à celui de deux autres classes de QDs : cette étude a clairement montré l influence de la chimie de surface sur le devenir intracellulaire des nanoparticules et a révélé une stabilité accrue des QDs DHLA-SB. Nous nous sommes également intéressés à la fonctionnalisation des QDS avec la streptavidine (SA) ou la biotine afin de marquer spécifiquement des cellules vivantes ou fixées. Les QDs-SA DHLA-SB obtenus ont permis de suivre un récepteur membranaire ou encore de marquer de façon spécifique une protéine biotinylée à l intérieur de cellules vivantes, bien plus efficacement qu avec les QDs-SA commerciaux (Invitrogen). Enfin, nous avons proposé d utiliser les QDs DHLA-SB pour améliorer une technique de microscopie à illumination structurée, la microscopie HiLo bicouleur, et obtenir une coupe optique (type image confocale) d échantillons biologiques épais en une seule imagePARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Time-lapse, photoactivation, and photobleaching imaging of nucleolar assembly after mitosis

International audienceNucleolus assembly starts in telophase with the benefit of building blocks passing through mitosis and lasts until cytokinesis generating the two independent interphasic cells. Several approaches make it possible to follow the dynamics of fluorescent molecules in live cells. Here, three complementary approaches are described to measure the dynamics of proteins during nucleolar assembly after mitosis: (1) rapid two-color 4-D imaging time-lapse microscopy that demonstrates the relative localization and movement of two proteins, (2) photoactivation that reveals the directionality of migration from the activated area, and (3) fluorescence recovery after photobleaching (FRAP) that measures the renewing of proteins in the bleached area. We demonstrate that the order of recruitment of the processing machineries into nucleoli results from differential sorting of intermediate structures assembled during telophase, the prenucleolar bodies

In nucleoli, the steady state of nucleolar proteins is leptomycin B-sensitive.

International audienceBACKGROUND INFORMATION: The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)-tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. RESULTS: The movement of PAGFP (photoactivatable GFP)-tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP-tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)-sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three-dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB-sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. CONCLUSIONS: We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB

Dividing cells regulate their lipid composition and localization

SummaryAlthough massive membrane rearrangements occur during cell division, little is known about specific roles that lipids might play in this process. We report that the lipidome changes with the cell cycle. LC-MS-based lipid profiling shows that 11 lipids with specific chemical structures accumulate in dividing cells. Using AFM, we demonstrate differences in the mechanical properties of live dividing cells and their isolated lipids relative to nondividing cells. In parallel, systematic RNAi knockdown of lipid biosynthetic enzymes identified enzymes required for division, which highly correlated with lipids accumulated in dividing cells. We show that cells specifically regulate the localization of lipids to midbodies, membrane-based structures where cleavage occurs. We conclude that cells actively regulate and modulate their lipid composition and localization during division, with both signaling and structural roles likely. This work has broader implications for the active and sustained participation of lipids in basic biology

The traffic of proteins between nucleolar organizer regions and prenucleolar bodies governs the assembly of the nucleolus at exit of mitosis

The building of nuclear bodies after mitosis is a coordinated event crucial for nuclear organization and function. The nucleolus is assembled during early G1 phase. Here, two periods (early G1a and early G1b) have been defined. During these periods, the nucleolar compartments (DFC, GC) corresponding to different steps of ribosome biogenesis are progressively assembled. In telophase, rDNA transcription is first activated and PNBs (reservoirs of nucleolar processing proteins) are formed. The traffic of the processing proteins between incipient nucleoli and PNBs was analyzed using photoactivation. We demonstrate that the DFC protein fibrillarin passes from one incipient nucleolus to other nucleoli but not to PNBs, and that the GC proteins, B23/NPM and Nop52, shuttle between PNBs and incipient nucleoli. This difference in traffic suggests a way of regulating assembly first of DFC and then of GC. The time of residency of GC proteins is high in incipient nucleoli compared to interphase nuclei, it decreases in LMB-treated early G1a cells impairing the assembly of GC. Because the assembly of the nucleolus and that of the Cajal body at the exit from mitosis are both sensitive to CRM1 activity, we discuss the fact that assembly of GC and/or its interaction with DFC in early G1a depends on shuttling between PNBs and NORs in a manner dependent on Cajal body assembly

Single-Shot Optical Sectioning Using Two-Color Probes in HiLo Fluorescence Microscopy

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate

Highly Enhanced Affinity of Multidentate versus Bidentate Zwitterionic Ligands for Long-Term Quantum Dot Bioimaging

High colloidal stability in aqueous conditions is a prerequisite for fluorescent nanocrystals, otherwise known as “quantum dots”, intended to be used in any long-term bioimaging experiment. This essential property implies a strong affinity between the nanoparticles themselves and the ligands they are coated with. To further improve the properties of the bidentate monozwitterionic ligand previously developed in our team, we synthesized a multidentate polyzwitterionic ligand, issued from the copolymerization of a bidentate monomer and a monozwitterionic one. The nanocrystals passivated by this polymeric ligand showed an exceptional colloidal stability, regardless of the medium conditions (pH, salinity, dilution, and biological environment), and we demonstrated the affinity of the polymer exceeded by 3 orders of magnitude that of the bidentate ligand (desorption rates assessed by a competition experiment). The synthesis of the multidentate polyzwitterionic ligand proved also to be easily tunable and allowed facile functionalization of the corresponding quantum dots, which led to successful specific biomolecules targeting