Quantifying AAV (hM3Dq) transfection in neocortical cells as a guide to DREADDs control of trauma-induced epileptogenesis

Le néocortex déclenche des activités paroxystiques suite aux lésions cérébrales traumatiques. Avec des blessures cérébrales pénétrantes, la déafférentation s'accompagne d'une longue période silencieuse du cortex atteint, et d'une augmentation des périodes d'hyperpolarisation du néocortex entourant la lésion. L'activité synchrone du réseau neuronal après une période de latence conduirait à l'épileptogenèse. Des tentatives de modifier la nature des crises ont été effectuées à l'aide de modèles animaux. L'une des plus prometteuse étant la chimiogenèse par l'injection des vecteurs viraux afin d'anéantir la réponse de certains récepteurs couplés aux protéines G aux ligands habituels, alors qu'ils réagissent aux médicaments désirés, comme N-oxyde de clozapine. Ces récepteurs appelés « DREADDs » exclusivement activés par ces médicaments ont été étudiés pour réduire le nombre et la sévérité des crises. Selon les résultats non-publiés de notre laboratoire, l'utilisation d'un DREADD excitateur près de «undercut» serait antiépileptogène. Nous pensons qu'une excitation ciblée pourrait optimiser l'effet antiépileptogène. L'excitation est directement liée aux neurones transduits. Nous postulons qu'une transduction optimale des neurones pourrait être atteinte par un dosage optimisé du virus injecté, tant au titre viral qu'au volume injecté. Pour prouver notre hypothèse nous avons utilisé trois différentes titrations de AAV2/8 et injecté différents volumes de ces titrations aux souris. Le volume de transfection corticale et le nombre des neurones transduits ont été quantifiés. Avec la titration E11gc/ml aucune transfection n'a été observée. Avec la titration E12gc/ml une corrélation quasi-linéaire a été observée entre le volume viral injecté, et le volume cortical transfecté, ainsi que le nombre de neurones transduits. Avec la titration E13gc/ml, une meilleure corrélation a été observée à la transduction neuronale qu'à la transfection corticale, par rapport au volume viral injecté. En conclusion, la titration E12gc/ml paraît être un meilleur choix pour nos futures études, la fiabilité de la titration E13gc/ml n'ayant pas été démontrée.The neocortex is the origin of paroxysmal activities that occur after traumatic brain injuries. In penetrating brain injuries, deafferentation causes long silent periods in affected cortex and increased hyperpolarization period in neocortical tissue around the injury. The synchronous neural network activity after a latent period may lead to epileptogenesis. Some attempts to alter seizures were done using animal models. One of the most promising involves chemogenetic tools via AAV viral vector injection to make some G protein-coupled receptors unresponsive to their natural ligands, and activated by the desired drug, such as clozapine-N-oxide. These designer receptors exclusively activated by designer drugs (DREADDs) have been studied in reducing the numbers and severity of seizures. According to unpublished works in our lab, using excitatory DREADDs in the vicinity of undercut was antiepileptogenic. We believe there could be an optimal level of excitation for yielding an optimal antiepileptogenic response. This excitation is in direct relation with neurons transfected. We hypothesize that the optimal neuronal transduction might be achieved with optimal dosage of virus delivered, in terms of viral titration and the volume of virus injected. To test this, we used three different titrations of AAV2/8 and we injected different volumes of these titrations in adult mice. Cortical transfection volume and number of neuronal transductions were estimated. With E11gc/ml titration, no transfection was visible. With E12gc/ml titration, an almost linear correlation was observed between the volume of virus injected and the number of neurons transduced and the cortical volume of transfection. With E13gc/ml titration the correlation between the injected AAV volume and the number of neuronal transductions was still good but there was a poor correlation between AAV volume and transfection volume. We concluded that E12gc/ml titration was a more reliable option for our further studies. The reliability of E13gc/ml titration needs to be proven

Internal Consistency and Convergent Validity of the Inventory of Hyperacusis Symptoms.

OBJECTIVES: The aim was to assess the internal consistency and convergent and discriminant validity of a new questionnaire for hyperacusis, the Inventory of Hyperacusis Symptoms (IHS; Greenberg & Carlos 2018), using a clinical population. DESIGN: This was a retrospective study. Data were gathered from the records of 100 consecutive patients who sought help for tinnitus and/or hyperacusis from an audiology clinic in the United Kingdom. The average age of the patients was 55 years (SD = 13 years). Audiological measures were the pure-tone average threshold (PTA) and uncomfortable loudness levels (ULL). Questionnaires administered were: IHS, Tinnitus Handicap Inventory (THI), Hyperacusis Questionnaire (HQ), Insomnia Severity Index, Generalized Anxiety Disorder, and Patient Health Questionnaire-9. RESULTS: Cronbach's alpha for the 25-item IHS questionnaire was 0.96. Neither the total IHS score nor scores for any of its five subscales were correlated with the PTA of the better or worse ear. This supports the discriminant validity of the IHS, as hyperacusis is thought to be independent of the PTA. There were moderately strong correlations between IHS total scores and scores for the HQ, Tinnitus Handicap Inventory, Generalized Anxiety Disorder, and Patient Health Questionnaire-9, with r = 0.58, 0.58, 0.61, 0.54, respectively. Thus, although IHS scores may reflect hyperacusis itself, they may also reflect the coexistence of tinnitus, anxiety, and depression. The total score on the IHS was significantly different between patients with and without hyperacusis (as diagnosed based on ULLs or HQ scores). Using the HQ score as a reference, the area under the receiver operating characteristic for the IHS was 0.80 (95% confidence interval = 0.71 to 0.89) and the cutoff point of the IHS with highest overall accuracy was 56/100. The corresponding sensitivity and specificity were 74% and 82%. CONCLUSIONS: The IHS has good internal consistency and reasonably high convergent validity, as indicated by the relationship of IHS scores to HQ scores and ULLs, but IHS scores may also partly reflect the co-occurrence of tinnitus, anxiety, and depression. We propose an IHS cutoff score of 56 instead of 69 for diagnosing hyperacusis

Reproduction biology of Astacus leptodactylus eichwaldi in the Iranian coastal water of the Caspian Sea (Bandar Anzali)

Sampling of Astacus leptodactylusc were conducted in order to determination of biometrical and biological parameters suchas length, weight, sex ratio, fecundity and natural reproduction time. Two transect were selected at 49 34 and 49 36 geographical position on east Caspian Sea near to Anzali city. Metallic folding trap with Silurus glanis as attractive diet were used to catch Astacus leptodactylusc at each line the traps were set on depth of 35, 45, 55 and 65 (5 trap at each depth). Random sampling from each depth on tow lin for one year were conducted and the biometry performed on catched Astacus leptodactylusc where their sext uality and their ration were determined for eacd month , season and whole year. Absolute fecundity determined by cooking Astacus leptodactylusc , taking out the ovary weighing and counting them .Working fecundity assesed by separating eggs from their swiming leges while enomerate all egg . Complete randomized test of ANOVA for analysing the data were employed. The results showed average length and welght were calculated 122/07±1/74mm and 57/96±1/81gr respectively. Average absolute fecundity was 310/22 ±10/72 eggs , average working fecundity was 251/84±8/84 eggs, Average ovary weight was determined 4.31±0.619 gr and average number of eggs in one gr was 74/52±1/53 eggs. The sextual ratio in all year long was 1:1.32. The reproduction season is about seven month from Feburary to July and the moulting of males occurs two times each year. One of time is at May and the other is in September. The female molt thtina as the male start for second time

HT2008-56155 EFFECTS OF SYNGAS ASH PARTICLE SIZE ON DEPOSITION AND EROSION OF A FILM COOLED LEADING EDGE

Abstract The paper investigates the deposition and erosion caused by Syngas ash particles in a film cooled leading edge region of a representative turbine vane. The carrier phase is predicted using Large Eddy Simulation for three blowing ratios of 0.4, 0.8 and 1.2. Three ash particle sizes of 1, 5, and 10 microns are investigated using Lagrangian dynamics. The 1 micron particles with momentum Stokes number St = 0.03 (based on approach velocity and cylinder diameter), follow the flow streamlines around the leading edge and few particles reach the blade surface. The 10 micron particles, on the other hand with a high momentum Stokes number, St = 3, directly impinge on the surface, with blowing ratio having a minimal effect. The 5 micron particles with St = 0.8, show the largest receptivity to coolant flow and blowing ratio. On a number basis, 85-90% of the 10 micron particles, 40-50% of 5 micron particles and less than 1% of 1 micron particles deposit on the surface. Overall there is a slight decrease in percentage of particles deposited with increase in blowing ratio. On the other hand, the potential for erosive wear is highest in the coolant hole and is mostly attributed to 5 micron particles. It is only at B.R.=1.2 that 10 micron particles contribute to erosive wear in the coolant hole