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

La douleur neuropathique est définie comme une douleur causée par une lésion du système nerveux somato-sensoriel. Elle se caractérise par des douleurs exagérées, spontanées, ou déclenchées par des stimuli normalement non douloureux (allodynie) ou douloureux (hyperalgésie). Bien qu'elle concerne 7% de la population, ses mécanismes biologiques ne sont pas encore élucidés. L'étude des variations d'expressions géniques dans les tissus-clés des voies sensorielles (notamment le ganglion spinal et la corne dorsale de la moelle épinière) à différents moments après une lésion nerveuse périphérique permettrait de mettre en évidence de nouvelles cibles thérapeutiques. Elles se détectent de manière sensible par reverse transcription quantitative real-time polymerase chain reaction (RT- qPCR). Pour garantir des résultats fiables, des guidelines ont récemment recommandé la validation des gènes de référence utilisés pour la normalisation des données ("Minimum information for publication of quantitative real-time PCR experiments", Bustin et al 2009). Après recherche dans la littérature des gènes de référence fréquemment utilisés dans notre modèle de douleur neuropathique périphérique SNI (spared nerve injury) et dans le tissu nerveux en général, nous avons établi une liste de potentiels bons candidats: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) et L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) et hydroxymethyl-bilane synthase (HMBS). Nous avons évalué la stabilité d'expression de ces gènes dans le ganglion spinal et dans la corne dorsale à différents moments après la lésion nerveuse (SNI) en calculant des coefficients de variation et utilisant l'algorithme geNorm qui compare les niveaux d'expression entre les différents candidats et détermine la paire de gènes restante la plus stable. Il a aussi été possible de classer les gènes selon leur stabilité et d'identifier le nombre de gènes nécessaires pour une normalisation la plus précise. Les gènes les plus cités comme référence dans le modèle SNI ont été GAPDH, HMBS, Actb, HPRT1 et 18S. Seuls HPRT1 and 18S ont été précédemment validés dans des arrays de RT-qPCR. Dans notre étude, tous les gènes testés dans le ganglion spinal et dans la corne dorsale satisfont au critère de stabilité exprimé par une M-value inférieure à 1. Par contre avec un coefficient de variation (CV) supérieur à 50% dans le ganglion spinal, 18S ne peut être retenu. La paire de gènes la plus stable dans le ganglion spinal est HPRT1 et Actb et dans la corne dorsale il s'agit de RPL29 et RPL13a. L'utilisation de 2 gènes de référence stables suffit pour une normalisation fiable. Nous avons donc classé et validé Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 et 18S comme gènes de référence utilisables dans la corne dorsale pour le modèle SNI chez le rat. Dans le ganglion spinal 18S n'a pas rempli nos critères. Nous avons aussi déterminé que la combinaison de deux gènes de référence stables suffit pour une normalisation précise. Les variations d'expression génique de potentiels gènes d'intérêts dans des conditions expérimentales identiques (SNI, tissu et timepoints post SNI) vont pouvoir se mesurer sur la base d'une normalisation fiable. Non seulement il sera possible d'identifier des régulations potentiellement importantes dans la genèse de la douleur neuropathique mais aussi d'observer les différents phénotypes évoluant au cours du temps après lésion nerveuse.

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.

A musculoskeletal shoulder model based on pseudo-inverse and null-space optimization.

The goal of the present work was assess the feasibility of using a pseudo-inverse and null-space optimization approach in the modeling of the shoulder biomechanics. The method was applied to a simplified musculoskeletal shoulder model. The mechanical system consisted in the arm, and the external forces were the arm weight, 6 scapulo-humeral muscles and the reaction at the glenohumeral joint, which was considered as a spherical joint. The muscle wrapping was considered around the humeral head assumed spherical. The dynamical equations were solved in a Lagrangian approach. The mathematical redundancy of the mechanical system was solved in two steps: a pseudo-inverse optimization to minimize the square of the muscle stress and a null-space optimization to restrict the muscle force to physiological limits. Several movements were simulated. The mathematical and numerical aspects of the constrained redundancy problem were efficiently solved by the proposed method. The prediction of muscle moment arms was consistent with cadaveric measurements and the joint reaction force was consistent with in vivo measurements. This preliminary work demonstrated that the developed algorithm has a great potential for more complex musculoskeletal modeling of the shoulder joint. In particular it could be further applied to a non-spherical joint model, allowing for the natural translation of the humeral head in the glenoid fossa.

Differentially expressed gene profile in the 6-hydroxy-dopamine-induced cell culture model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons of the substantia nigra pars compacta with unknown aetiology. 6-Hydroxydopamine (6-OHDA) treatment of neuronal cells is an established in vivo model for mimicking the effect of oxidative stress found in PD brains. We examined the effects of 6-OHDA treatment on human neuroblastoma cells (SH-SY5Y) and primary mesencephalic cultures. Using a reverse arbitrarily primed polymerase chain reaction (RAP-PCR) approach we generated reproducible genetic fingerprints of differential expression levels in cell cultures treated with 6-OHDA. Of the resulting sequences, 23 showed considerable homology to known human coding sequences. The results of the RAP-PCR were validated by reverse transcription PCR, real-time PCR and, for selected genes, by Western blot analysis and immunofluorescence. In four cases, [tomoregulin-1 (TMEFF-1), collapsin response mediator protein 1 (CRMP-1), neurexin-1, and phosphoribosylaminoimidazole synthetase (GART)], a down-regulation of mRNA and protein levels was detected. Further studies will be necessary on the physiological role of the identified proteins and their impact on pathways leading to neurodegeneration in PD.

Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Experimental evaluation of an electromechanical artificial urinary sphincter in an animal model.

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The AMS 800 urinary control system is the gold standard for the treatment of urinary incontinence due to sphincter insufficiency. Despite excellent functional outcome and latest technological improvements, the revision rate remains significant. To overcome the shortcomings of the current device, we developed a modern electromechanical artificial urinary sphincter. The results demonstrated that this new sphincter is effective and well tolerated up to 3 months. This preliminary study represents a first step in the clinical application of novel technologies and an alternative compression mechanism to the urethra. OBJECTIVES: To evaluate the effectiveness in continence achievement of a new electromechanical artificial urinary sphincter (emAUS) in an animal model. To assess urethral response and animal general response to short-term and mid-term activation of the emAUS. MATERIALS AND METHODS: The principle of the emAUS is electromechanical induction of alternating compression of successive segments of the urethra by a series of cuffs activated by artificial muscles. Between February 2009 and May 2010 the emAUS was implanted in 17 sheep divided into three groups. The first phase aimed to measure bladder leak point pressure during the activation of the device. The second and third phases aimed to assess tissue response to the presence of the device after 2-9 weeks and after 3 months respectively. Histopathological and immunohistochemistry evaluation of the urethra was performed. RESULTS: Bladder leak point pressure was measured at levels between 1091 ± 30.6 cmH2 O and 1244.1 ± 99 cmH2 O (mean ± standard deviation) depending on the number of cuffs used. At gross examination, the explanted urethra showed no sign of infection, atrophy or stricture. On microscopic examination no significant difference in structure was found between urethral structure surrounded by a cuff and control urethra. In the peripheral tissues, the implanted material elicited a chronic foreign body reaction. Apart from one case, specimens did not show significant presence of lymphocytes, polymorphonuclear leucocytes, necrosis or cell degeneration. Immunohistochemistry confirmed the absence of macrophages in the samples. CONCLUSIONS: This animal study shows that the emAUS can provide continence. This new electronic controlled sequential alternating compression mechanism can avoid damage to urethral vascularity, at least up to 3 months after implantation. After this positive proof of concept, long-term studies are needed before clinical application could be considered.

Vasopressin-stimulated CFTR Cl- currents are increased in the renal collecting duct cells of a mouse model of Liddle's syndrome.

Liddle's syndrome is a genetic form of hypertension linked to Na(+) retention caused by activating mutations in the COOH terminus of the beta or gamma subunit of the epithelial sodium channel (ENaC). In this study, we used the short-circuit current (I(sc)) method to investigate the effects of deamino-8-d-arginine vasopressin (dDAVP) on Na(+) and Cl(-) fluxes in primary cultures of cortical collecting ducts (CCDs) microdissected from the kidneys of mice with Liddle's syndrome carrying a stop codon mutation, corresponding to the beta-ENaC R(566) stop mutation (L) found in the original pedigree. Compared to wild-type (+/+) CCD cells, untreated L/+ and L/L CCD cells exhibited 2.7- and 4.2-fold increases, respectively, in amiloride-sensitive (Ams) I(sc), reflecting ENaC-dependent Na(+) absorption. Short-term incubation with dDAVP caused a rapid and significant increase (approximately 2-fold) in Ams I(sc) in +/+, but not in L/+ or L/L CCD cells. In sharp contrast, dDAVP induced a greater increase in 5-nitro-2-(3-phenylpropamino)benzoate (NPPB)-inhibited apical Cl(-) currents in amiloride-treated L/L and L/+ cells than in their +/+ counterparts. I(sc) recordings performed under apical ion substituted conditions revealed that the dDAVP-stimulated apical secretion of Cl(-), which was absent in cultured CCDs lacking CFTR, was 1.8-fold greater in L/+ and 3.7-fold greater in L/L CCD cells than in their +/+ CCD counterparts. After the basal membrane had been permeabilized with nystatin and a basal-to-apical Cl(-) gradient had been imposed, dDAVP also stimulated larger Cl(-) currents across L/L and L/+ CCD layers than +/+ CCD layers. These findings demonstrate that vasopressin stimulates greater apical CFTR Cl(-) conductance in the renal CCD cells of mice with Liddle's syndrome than in wild-type mice. This effect could contribute to the enhanced NaCl reabsorption observed in the distal nephron of patients with Liddle's syndrome.

Devolatilization-induced pressure build-up: Implications for reaction front movement and breccia pipe formation

Generation of fluids during metamorphism can significantly influence the fluid overpressure, and thus the fluid flow in metamorphic terrains. There is currently a large focus on developing numerical reactive transport models, and with it follows the need for analytical solutions to ensure correct numerical implementation. In this study, we derive both analytical and numerical solutions to reaction-induced fluid overpressure, coupled to temperature and fluid flow out of the reacting front. All equations are derived from basic principles of conservation of mass, energy and momentum. We focus on contact metamorphism, where devolatilization reactions are particularly important owing to high thermal fluxes allowing large volumes of fluids to be rapidly generated. The analytical solutions reveal three key factors involved in the pressure build-up: (i) The efficiency of the devolatilizing reaction front (pressure build-up) relative to fluid flow (pressure relaxation), (ii) the reaction temperature relative to the available heat in the system and (iii) the feedback of overpressure on the reaction temperature as a function of the Clapeyron slope. Finally, we apply the model to two geological case scenarios. In the first case, we investigate the influence of fluid overpressure on the movement of the reaction front and show that it can slow down significantly and may even be terminated owing to increased effective reaction temperature. In the second case, the model is applied to constrain the conditions for fracturing and inferred breccia pipe formation in organic-rich shales owing to methane generation in the contact aureole.

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.

Scalable inference of heterogeneous reaction kinetics from pooled single-cell recordings.

Mathematical methods combined with measurements of single-cell dynamics provide a means to reconstruct intracellular processes that are only partly or indirectly accessible experimentally. To obtain reliable reconstructions, the pooling of measurements from several cells of a clonal population is mandatory. However, cell-to-cell variability originating from diverse sources poses computational challenges for such process reconstruction. We introduce a scalable Bayesian inference framework that properly accounts for population heterogeneity. The method allows inference of inaccessible molecular states and kinetic parameters; computation of Bayes factors for model selection; and dissection of intrinsic, extrinsic and technical noise. We show how additional single-cell readouts such as morphological features can be included in the analysis. We use the method to reconstruct the expression dynamics of a gene under an inducible promoter in yeast from time-lapse microscopy data.

Toll-like receptor 2 (TLR2) polymorphisms are associated with reversal reaction in leprosy.

BACKGROUND: Leprosy is characterized by a spectrum of clinical manifestations that depend on the type of immune response against the pathogen. Patients may undergo immunological changes known as "reactional states" (reversal reaction and erythema nodosum leprosum) that result in major clinical deterioration. The goal of the present study was to assess the effect of Toll-like receptor 2 (TLR2) polymorphisms on susceptibility to and clinical presentation of leprosy. METHODS: Three polymorphisms in TLR2 (597C-->T, 1350T-->C, and a microsatellite marker) were analyzed in 431 Ethiopian patients with leprosy and 187 control subjects. The polymorphism-associated risk of developing leprosy, lepromatous (vs. tuberculoid) leprosy, and leprosy reactions was assessed by multivariate logistic regression models. RESULTS: The microsatellite and the 597C-->T polymorphisms both influenced susceptibility to reversal reaction. Although the 597T allele had a protective effect (odds ratio [OR], 0.34 [95% confidence interval {CI}, 0.17-0.68]; P= .002 under the dominant model), homozygosity for the 280-bp allelic length of the microsatellite strongly increased the risk of reversal reaction (OR, 5.83 [95% CI, 1.98-17.15]; P= .001 under the recessive model). These associations were consistent among 3 different ethnic groups. CONCLUSIONS: These data suggest a significant role for TLR-2 in the occurrence of leprosy reversal reaction and provide new insights into the immunogenetics of the disease.

Changes in spring-mass model characteristics during repeated running sprints.

This study investigated fatigue-induced changes in spring-mass model characteristics during repeated running sprints. Sixteen active subjects performed 12 × 40 m sprints interspersed with 30 s of passive recovery. Vertical and anterior-posterior ground reaction forces were measured at 5-10 m and 30-35 m and used to determine spring-mass model characteristics. Contact (P < 0.001), flight (P < 0.05) and swing times (P < 0.001) together with braking, push-off and total stride durations (P < 0.001) lengthened across repetitions. Stride frequency (P < 0.001) and push-off forces (P < 0.05) decreased with fatigue, whereas stride length (P = 0.06), braking (P = 0.08) and peak vertical forces (P = 0.17) changes approached significance. Center of mass vertical displacement (P < 0.001) but not leg compression (P > 0.05) increased with time. As a result, vertical stiffness decreased (P < 0.001) from the first to the last repetition, whereas leg stiffness changes across sprint trials were not significant (P > 0.05). Changes in vertical stiffness were correlated (r > 0.7; P < 0.001) with changes in stride frequency. When compared to 5-10 m, most of ground reaction force-related parameters were higher (P < 0.05) at 30-35 m, whereas contact time, stride frequency, vertical and leg stiffness were lower (P < 0.05). Vertical stiffness deteriorates when 40 m run-based sprints are repeated, which alters impact parameters. Maintaining faster stride frequencies through retaining higher vertical stiffness is a prerequisite to improve performance during repeated sprinting.

Skin fibroblast model to study an impaired glutathione synthesis: consequences of a genetic polymorphism on the proteome.

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.

Aqueous dynamic and histological findings after deep sclerectomy with collagen implant in an animal model.

AIM: The use of an animal model to study the aqueous dynamic and the histological findings after deep sclerectomy with (DSCI) and without collagen implant. METHODS: Deep sclerectomy was performed on rabbits' eyes. Eyes were randomly assigned to receive collagen implants. Measurements of intraocular pressure (IOP) and aqueous outflow facility using the constant pressure method through cannulation of the anterior chamber were performed. The system was filled with BSS and cationised ferritin. Histological assessment of the operative site was performed. Sections were stained with haematoxylin and eosin and with Prussian blue. Aqueous drainage vessels were identified by the reaction between ferritin and Prussian blue. All eyes were coded so that the investigator was blind to the type of surgery until the evaluation was completed. RESULTS: A significant decrease in IOP (p<0.05) was observed during the first 6 weeks after DSCI (mean IOP was 13.07 (2.95) mm Hg preoperatively and 9.08 (2.25) mm Hg at 6 weeks); DS without collagen implant revealed a significant decrease in IOP at weeks 4 and 8 after surgery (mean IOP 12.57 (3.52) mm Hg preoperatively, 9.45 (3.38) mm Hg at 4 weeks, and 9.22 (3.39) mm Hg at 8 weeks). Outflow facility was significantly increased throughout the 9 months of follow up in both DSCI and DS groups (p<0.05). The preoperative outflow facility (OF) was 0.15 (0.02) micro l/min/mm Hg. At 9 months, OF was 0.52 (0.28) microl/min/mm Hg and 0.46 (0.07) micro l/min/mm Hg for DSCI and DS respectively. Light microscopy studies showed the appearance of new aqueous drainage vessels in the sclera adjacent to the dissection site in DSCI and DS and the apparition of spindle cells lining the collagen implant in DSCI after 2 months. CONCLUSION: A significant IOP decrease was observed during the first weeks after DSCI and DS. DS with or without collagen implant provided a significant increase in outflow facility throughout the 9 months of follow up. This might be partly explained by new drainage vessels in the sclera surrounding the operated site. Microscopic studies revealed the appearance of spindle cells lining the collagen implant in DSCI after 2 months.

A fully adaptive numerical approximation for a two-dimensional epidemic model with nonlinear cross-diffusion

An epidemic model is formulated by a reactionâeuro"diffusion system where the spatial pattern formation is driven by cross-diffusion. The reaction terms describe the local dynamics of susceptible and infected species, whereas the diffusion terms account for the spatial distribution dynamics. For both self-diffusion and cross-diffusion, nonlinear constitutive assumptions are suggested. To simulate the pattern formation two finite volume formulations are proposed, which employ a conservative and a non-conservative discretization, respectively. An efficient simulation is obtained by a fully adaptive multiresolution strategy. Numerical examples illustrate the impact of the cross-diffusion on the pattern formation.