The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the "cuff model" of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.     

6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27

The control of glucose metabolism and the cell cycle must be coordinated in order to guarantee sufficient ATP and anabolic substrates at distinct phases of the cell cycle. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are well established regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent allosteric activator of 6-phosphofructo-1-kinase (Pfk-1). PFKFB3 is overexpressed in human cancers, regulated by HIF-1α, Akt and PTEN, and required for the survival and growth of multiple cancer types. Although most functional studies of the role of PFKFB3 in cancer progression have invoked its well-recognized function in the regulation of glycolysis, recent observations have established that PFKFB3 also traffics to the nucleus and that its product, F2,6BP, activates cyclin-dependent kinases (Cdks). In particular, F2,6BP stimulates the Cdk-mediated phosphorylation of the Cip/Kip protein p27 (threonine 187), which in turn results in p27''s ubiquitination and proteasomal degradation. As p27 is a potent suppressor of the G1/S transition and activator of apoptosis, we hypothesized that the known requirement of PFKFB3 for cell cycle progression and prevention of apoptosis may be partly due to the ability of F2,6BP to activate Cdks. In this study, we demonstrate that siRNA silencing of endogenous PFKFB3 inhibits Cdk1 activity, which in turn stabilizes p27 protein levels causing cell cycle arrest at G1/S and increased apoptosis in HeLa cells. Importantly, we demonstrate that the increase in apoptosis and suppression of the G1/S transition caused by siRNA silencing of PFKFB3 expression is reversed by co-siRNA silencing of p27. Taken together with prior publications, these observations support a model whereby PFKFB3 and F2,6BP function not only as regulators of Pfk-1 but also of Cdk1 activity, and therefore serve to couple glucose metabolism with cell proliferation and survival in transformed cells.The homodimeric bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB) phosphorylate fructose 6-phosphate (F6P) to fructose-2,6-bisphosphate (F2,6BP), which in turn activates 6-phosphofructo-1-kinase and glycolytic flux to lactate.¹ Of the four genes encoding distinct PFKFB isozymes (PFKFB1-4), PFKFB3 is distinguished by the presence of multiple copies of the AUUUA instability motif in its 3''untranslated region,² a very high kinase:phosphatase activity ratio (740 : 1),³ increased protein expression in rapidly proliferating transformed cells,² solid tumors and leukemias^{2, 4, 5} and regulation by several proteins essential for tumor progression (e.g. HIF-1α,⁶ Akt⁷ and PTEN^{8, 9}). Not surprisingly, heterozygous genomic deletion of the pfkfb3 gene has been found to reduce both the glucose metabolism and growth of Ras-transformed tumors in syngeneic mice.¹⁰In recent studies, we unexpectedly observed that PFKFB3 trafficked to the nucleus of multiple cell lines via a highly conserved nuclear localization motif in the C-terminal domain.¹¹ Although the precise role of nuclear PFKFB3 is unknown, ectopic expression of wild-type PFKFB3 in the nucleus was found to stimulate cellular proliferation without affecting glycolysis, suggesting a novel role for nuclear F2,6BP in regulating the cell cycle.¹¹ Moreover, the addition of F2,6BP to total cell lysates was found to increase the cyclin-dependent kinase (Cdk)-dependent phosphorylation of its substrate p27 at threonine 187 (T187), a posttranslational modification that targets p27 for degradation (i.e. high Cdk activity suppresses p27 levels).¹¹ Given that p27 can potently block the G1/S transition and stimulate apoptosis, these data indicated that PFKFB3-mediated production of F2,6BP in the nucleus may directly stimulate Cdks to phosphorylate T187-p27, targeting p27 for degradation by the proteasome and allowing cells to both proliferate and evade apoptosis. Furthermore, these data signified that PFKFB3 may not only be essential for the regulation of glycolysis in the cytoplasm but also for the control of the cell cycle in the nucleus.Based on these prior studies, we postulated that selective inhibition of PFKFB3 would suppress Cdk1 activity, which in turn would reduce the phosphorylation of T187-p27, resulting in increased p27 expression, reduced G1/S transition and increased apoptosis. We provide evidence to support this chain of biochemical and cellular events after PFKFB3 inhibition as well as direct verification that p27 itself is required for the simultaneous suppression of G1/S transition and induction of apoptosis caused by PFKFB3 inhibition. Given that PFKFB3 inhibitors are entering phase I trials for the treatment of advanced cancers,¹² we believe that this new mechanism of action may facilitate the development of rational phase I/II trials that combine other apoptosis-activating agents that disrupt p27 function (e.g. Cdk1 inhibitors) as well as potential biomarkers such as p27 that may demonstrate the on-target effects of PFKFB3 inhibitors in biopsies and resected tumors. From a broader perspective, these data provide further support for the concept that PFKFB3 may be an essential coupler of glucose metabolism and cell cycle progression.     

Is decreased bone mineral density associated with development of scoliosis? A bipedal osteopenic rat model

Background

The etiology of AIS remains unclear, thus various hypotheses concerning its pathomechanism have been proposed. To date, biomechanical modeling has not been used to thoroughly study the influence of the abnormal growth profile (i.e., the growth rate of the vertebral body during the growth period) on the pathomechanism of curve progression in AIS. This study investigated the hypothesis that AIS progression is associated with the abnormal growth profiles of the anterior column of the spine.

Methods

A finite element model of the spinal column including growth dynamics was utilized. The initial geometric models were constructed from the bi-planar radiographs of a normal subject. Based on this model, five other geometric models were generated to emulate different coronal and sagittal curves. The detailed modeling integrated vertebral body growth plates and growth modulation spinal biomechanics. Ten years of spinal growth was simulated using AIS and normal growth profiles. Sequential measures of spinal alignments were compared.

Results

(1) Given the initial lateral deformity, the AIS growth profile induced a significant Cobb angle increase, which was roughly between three to five times larger compared to measures utilizing a normal growth profile. (2) Lateral deformities were absent in the models containing no initial coronal curvature. (3) The presence of a smaller kyphosis did not produce an increase lateral deformity on its own. (4) Significant reduction of the kyphosis was found in simulation results of AIS but not when using the growth profile of normal subjects.

Conclusion

Results from this analysis suggest that accelerated growth profiles may encourage supplementary scoliotic progression and, thus, may pose as a progressive risk factor.     

Guanylin and functional coupling proteins in the hepatobiliary system of rat and guinea pig

Guanylin, a bioactive intestinal peptide, is involved in the cystic fibrosis transmembrane conductance (CFTR)-regulated electrolyte/water secretion in various epithelia. In the present work we report on the expression and cellular localization of guanylin and its affiliated signaling and effector proteins, including guanylate cyclase C (Gucy2c), Proteinkinase GII (Pkrg2), CFTR and the solute carrier family 4, anion exchanger, member 2 (Slc4a2) in the hepatobiliary system of rat and guinea pig. Localization studies in the liver and the gallbladder revealed that guanylin is located in the secretory epithelial cells of bile ducts of the liver and of the gallbladder, while Gucy2c, Pkrg2, CFTR, and Slc4a2 are confined exclusively to the apical membrane of the same epithelial cells. Based on these findings, we assume that guanylin is synthesized as an intrinsic peptide in epithelial cells of the hepatobiliary system and released luminally into the hepatic and cystic bile to regulate electrolyte secretion by a paracrine/luminocrine signaling pathway.     

Sequence-based genotyping for marker discovery and co-dominant scoring in germplasm and populations

Conventional marker-based genotyping platforms are widely available, but not without their limitations. In this context, we developed Sequence-Based Genotyping (SBG), a technology for simultaneous marker discovery and co-dominant scoring, using next-generation sequencing. SBG offers users several advantages including a generic sample preparation method, a highly robust genome complexity reduction strategy to facilitate de novo marker discovery across entire genomes, and a uniform bioinformatics workflow strategy to achieve genotyping goals tailored to individual species, regardless of the availability of a reference sequence. The most distinguishing features of this technology are the ability to genotype any population structure, regardless whether parental data is included, and the ability to co-dominantly score SNP markers segregating in populations. To demonstrate the capabilities of SBG, we performed marker discovery and genotyping in Arabidopsis thaliana and lettuce, two plant species of diverse genetic complexity and backgrounds. Initially we obtained 1,409 SNPs for arabidopsis, and 5,583 SNPs for lettuce. Further filtering of the SNP dataset produced over 1,000 high quality SNP markers for each species. We obtained a genotyping rate of 201.2 genotypes/SNP and 58.3 genotypes/SNP for arabidopsis (n?=?222 samples) and lettuce (n?=?87 samples), respectively. Linkage mapping using these SNPs resulted in stable map configurations. We have therefore shown that the SBG approach presented provides users with the utmost flexibility in garnering high quality markers that can be directly used for genotyping and downstream applications. Until advances and costs will allow for routine whole-genome sequencing of populations, we expect that sequence-based genotyping technologies such as SBG will be essential for genotyping of model and non-model genomes alike.     

Kainic acid-induced changes in the opioid/nociceptin system and the stress/toxicity pathways in the rat hippocampus

Excitotoxicity is a contributing factor to the pathogenesis of acute or chronic neurodegenerative disease states. Kainic acid (KA) is an excitotoxic substance and the administration of it to rodents induces seizure activity (status epilepticus, SE) and leads to neurodegeneration. In this study the effect of KA-induced excitotoxicity on the G-protein activations and the gene expression levels of the opioid/nociceptin system receptors as MOPr, KOPr, DOPr, ORL-1, and PNOC (N/OFQ) were investigated, and the regulator effect of naloxone (Nal) on the gene expressions of the opioid system receptors against KA-induced seizures in the rat hippocampus was tested. In addition, the expression levels of stress-toxicity genes were assessed in the hippocampus following KA-induced excitotoxicity in order to determine the potential genetic targets which can be helpful for neuroprotective interventions. Our results indicate that the KA-induced excitotoxicity increased the mRNA levels of MOPr, DOPr, KOPr, PNOC, and ORL-1. However, G-protein activations of MOPr, DOPr, and KOPr remained relatively unchanged while both the potency and efficacy of N/OFQ were significantly increased. The PCR array data showed that KA-induced excitotoxicity altered the expression levels of genes in the cellular stress or toxicity pathways. Our data suggests that the induction of the opioid/nociceptin system may be involved in the cellular stress response following a neurodegenerative insult and that the genes modulated by the KA-treatment in the stress-toxicity pathways may be evaluated as targets of potential neuroprotective interventions.     

Next-generation sequencing of experimental mouse strains

Since the turn of the century the complete genome sequence of just one mouse strain, C57BL/6J, has been available. Knowing the sequence of this strain has enabled large-scale forward genetic screens to be performed, the creation of an almost complete set of embryonic stem (ES) cell lines with targeted alleles for protein-coding genes, and the generation of a rich catalog of mouse genomic variation. However, many experiments that use other common laboratory mouse strains have been hindered by a lack of whole-genome sequence data for these strains. The last 5?years has witnessed a revolution in DNA sequencing technologies. Recently, these technologies have been used to expand the repertoire of fully sequenced mouse genomes. In this article we review the main findings of these studies and discuss how the sequence of mouse genomes is helping pave the way from sequence to phenotype. Finally, we discuss the prospects for using de novo assembly techniques to obtain high-quality assembled genome sequences of these laboratory mouse strains, and what advances in sequencing technologies may be required to achieve this goal.     

Removal of chromate anions from aqueous stream by a cationic surfactant-modified yeast

The removal of chromate anions (CrO(4)(2-)) from aqueous solution by a cationic surfactant-modified yeast was studied in a batch system. Cetyl trimethyl ammonium bromide (CTAB) was used for biomass modification; it substantially improved the biosorption efficiency. The influences of solution pH, CrO(4)(2-) anion concentrations and biomass concentration on the biosorption efficiency were investigated. The biosorption of chromate anions by modified yeast was strongly affected by pH. The optimum pH for biosorption of CrO(4)(2-) by modified yeast was 4.5-5.5. Zeta potential values of modified and unmodified yeast were determined at various pH values. Concentrations ranging from 5.2 to 208 mg/l Cr(VI) were tested and the biosorptive removal efficiency of the metal ions from aqueous solution was more than 99.5%. Freundlich and Langmuir isotherms were used to evaluate the data and the regression constants were determined.