Compartmented Cultures Research Articles

Differential susceptibility of human motor neurons to infection with Usutu and West Nile virus

West Nile virus (WNV) and Usutu virus (USUV) are closely related flaviviruses with differing capacities to cause neurological disease in humans. WNV is thought to use a transneural route of neuroinvasion along motor neurons and causes severe motor deficits. The potential for use of transneural routes of neuroinvasion by USUV has not been investigated experimentally, and evidence from the few clinical case reports of USUV-associated neuroinvasive disease is lacking. We hypothesised that, compared with WNV, USUV is less able to infect motor neurons, and therefore determined the susceptibility of human induced pluripotent stem cell (iPSC)-derived spinal cord motor neurons to infection. Both viruses could grow to high titres in iPSC-derived neural cultures. However, USUV could not productively infect motor neurons due to restriction by the antiviral response, which was not induced upon WNV infection. Inhibition of the antiviral response allowed for widespread infection and transportation of USUV along motor neurons within a compartmented culture system. These results show a stark difference in the ability of these two viruses to evade initiation of intrinsic antiviral immunity. Our data suggests that USUV cannot infect motor neurons in healthy individuals but in case of immunodeficiency may pose a risk for motor-related neurological disease and transneural invasion.

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Neuronal expression of herpes simplex virus-1 VP16 protein induces pseudorabies virus escape from silencing and reactivation.

Alpha herpesvirus (α-HV) particles enter their hosts from mucosal surfaces and efficiently maintain fast transport in peripheral nervous system (PNS) axons to establish infections in the peripheral ganglia. The path from axons to distant neuronal nuclei is challenging to dissect due to the difficulty of monitoring early events in a dispersed neuron culture model. We have established well-controlled, reproducible, and reactivateable latent infections in compartmented rodent neurons by infecting physically isolated axons with a small number of viral particles. This system not only recapitulates the physiological infection route but also facilitates independent treatment of isolated cell bodies or axons. Consequently, this system enables study not only of the stimuli that promote reactivation but also the factors that regulate the initial switch from productive to latent infection. Adeno-associated virus (AAV)-mediated expression of herpes simplex-1 (HSV-1) VP16 alone in neuronal cell bodies enabled the escape from silencing of incoming pseudorabies virus (PRV) genomes. Furthermore, the expression of HSV VP16 alone reactivated a latent PRV infection in this system. Surprisingly, the expression of PRV VP16 protein supported neither PRV escape from silencing nor reactivation. We compared transcription transactivation activity of both VP16 proteins in primary neurons by RNA sequencing and found that these homolog viral proteins produce different gene expression profiles. AAV-transduced HSV VP16 specifically induced the expression of proto-oncogenes including c-Jun and Pim2. In addition, HSV VP16 induces phosphorylation of c-Jun in neurons, and when this activity is inhibited, escape of PRV silencing is dramatically reduced.IMPORTANCEDuring latency, alpha herpesvirus genomes are silenced yet retain the capacity to reactivate. Currently, host and viral protein interactions that determine the establishment of latency, induce escape from genome silencing or reactivation are not completely understood. By using a compartmented neuronal culture model of latency, we investigated the effect of the viral transcriptional activator, VP16 on pseudorabies virus (PRV) escape from genome silencing. This model recapitulates the physiological infection route and enables the study of the stimuli that regulate the initial switch from a latent to productive infection. We investigated the neuronal transcriptional activation profiles of two homolog VP16 proteins (encoded by HSV-1 or PRV) and found distinct gene activation signatures leading to diverse infection outcomes. This study contributes to understanding of how alpha herpesvirus proteins modulate neuronal gene expression leading to the initiation of a productive or a latent infection.

Foliar-applied potassium improved mycorrhizal Goji (Lycium barbarum L.) growth of the potassium free - compartment in a compartmented culture system

Arbuscular mycorrhizal (AM) fungi promote plant mineral nutrient acquisition, and AM fungal hyphae can transport water and nutrients. Potassium has significant effects on plant photosynthesis, stomatal conductance, and other physiological processes. However, it is little known whether foliar-applied potassium in one plant could affect the nearby plant that connected by the AM fungal hyphae network. We analyzed the effects of foliar-applied potassium (2% K2SO4) in the potassium additional compartment (potassium was foliar-sprayed on plant in this compartment) on growth, photosynthesis, chlorophyll fluorescence parameters, potassium content, and the relative expression of potassium channel/transporter genes (LbHAK, LbKAT3, LbAKT2, LbKT1, and LbSKOR) of mycorrhizal Lycium barbarum in the potassium-free compartment (potassium was not foliar-sprayed on plant in this compartment) via AM fungal hyphae network of a three compartments culture system (the system contains three compartments, namely two root compartments on the left and right and a hyphal compartment in the middle). Compared with inoculation of Rhizophagus irregularis, foliar-applied potassium improved growth, photosynthesis, and potassium content of mycorrhizal L. barbarum in the potassium-free compartment, but had no effect on mycorrhizal L. barbarum chlorophyll fluorescence parameters. Additionally, foliar-applied potassium increased the relative expressions of LbHAK, LbKAT3 LbKT1 and LbSKOR in roots of mycorrhizal plants in the potassium-free compartment, while the relative expression of LbAKT2 in shoots of mycorrhizal plant was increased in the potassium-free compartment. This study suggests that AM fungal hyphae network transports potassium to the potassium-free compartment, then potassium regulates the expression of potassium channel/transporter genes to promote potassium uptake and transport, thus improves photosynthesis, and ultimately, promotes the growth of mycorrhizal L. barbarum in the potassium-free compartment.

Mesenchymal stem cells and local tacrolimus delivery synergistically enhance neurite extension.

The aim of this study was to investigate the combined effect of mesenchymal stem cells (MSC) and local delivery of tacrolimus (FK506) on nerve regeneration when applied to nerve autografts and decellularized allografts. A three-dimensional in vitro compartmented cell culture system consisting of a neonatal dorsal root ganglion adjacent to a nerve graft was used to evaluate the regenerating neurites into the peripheral nerve scaffold. Nerve autografts and allografts were treated with (i) undifferentiated MSCs, (ii) FK506 (100 ng/mL) or (iii) both (N = 9/group). After 48 hours, neurite extension was measured to quantify nerve regeneration and stem cell viability was evaluated. Stem cell viability was confirmed in all MSC-treated grafts. Neurite extension was superior in autografts treated with FK506, and MSCs and FK506 combined (p < 0.001 and p = 0.0001, respectively), and autografts treated with MSCs (p = 0.12) were comparable to untreated autografts. In allografts, FK506 treatment and combined treatment were superior to controls (p < 0.001 and p = 0.0001, respectively), and treatment with MSCs (p = 0.09) was comparable to controls. All autograft groups were superior compared to their respective allograft treatment group (p < 0.05) in neurite extension. Alone, either MSC or FK506 treatment improved neurite outgrowth, and combined they further enhanced neurite extension in both autografts and allografts.

QS5: The Effect of Stem Cells and Local Tacrolimus on Neurite Extension

Purpose:Application of mesenchymal stem cells (MSCs) or tacrolimus (FK506), an FDA approved immunosuppressant, to nerve grafts has been a topic of interest to enhance peripheral nerve regeneration. The aim of this study was to investigate the combined effect of MSCs and local delivery of FK506 on nerve regeneration when applied to nerve autografts and decellularized nerve allografts.Methods:A three-dimensional (3D) in vitro compartmented cell culture system, validated by Tajdaran et al (2019), consisted of rat neonatal dorsal root ganglion (DRG) adjacent to rat nerve autograft or decellularized allograft. This model was used to evaluate regenerating neurites from the DRG into the peripheral nerve scaffold. Nerve autografts and decellularized allografts were augmented with (i) dynamic undifferentiated MSC seeding, (ii) local application of FK506 (100 ng/mL) or (iii) both (N=9/group). Local application was ensured by isolating the central system (i.e. DRG side) from the peripheral system (i.e. nerve graft side), where treatment was applied. After 48-hours of incubation, DRG-nerve graft constructs were collected, fixed, sectioned and stained against neurofilament-160 to measure neurite extension. CD90 staining was used to confirm stem cell characterization.Results:All grafts treated with MSCs confirmed CD90 expression. Compared to untreated autografts, neurite extension in autografts treated with FK506 and autografts treated with MSCs and FK506 combined were found superior (P<0.001 and P=0.0001, respectively), and comparable to autografts treated with MSCs (P=0.12). Compared to untreated allografts, allografts treated with FK506, and allografts treated with MSCs and FK506 were found superior (P<0.001 and P=0.0001, respectively), and allografts treated with MSCs were found comparable (P=0.09). All autograft groups were found superior compared to their respective allograft treatment group (P<0.05). Solely allografts receiving combined treatment were found superior to untreated autografts (P<0.05).Conclusion:MSCs or FK506 treatment improved neurite outgrowth and when combined, this resulted in significant synergistic neurite extension in both autografts and allografts in comparable patterns. Schematic overview of 3D compartmented cell culture system for isolated evaluation of treatment with MSCs and local FK506 in vitro. A 3.5 mm autograft or allograft with or without undifferentiated MSC seeding is attached to a DRG. DRG-nerve graft constructs are placed through a silicone isolator in the middle of a 24-wells plate to isolate the DRG from the nerve graft. FK506 containing media was added to the nerve graft side.

Local FK506 dose-dependent study using a novel three-dimensional organotypic assay.

Local administration of FK506, an FDA approved immunosuppressant with neuroregenerative properties, is a promising technique to achieve improved peripheral nerve regeneration while preventing the side effects associated with the systemic administration of this drug. Although considerable research has been devoted to the development of clinically suitable systems for local delivery of FK506 to the site of nerve injury and repair, the optimal dose of FK506 for enhancement of axon regeneration in the peripheral nerve has not yet been established. To this end, we devised a three-dimensional (3D) organotypic assay capable of mimicking the peripheral nerve. This assay consisted of a neonatal rat dorsal root ganglion (DRG) extending its neurites into the native peripheral nerve scaffold provided by an acellular nerve allograft (ANA). A novel 3D compartmented cell culture system was adapted from the 3D organotypic assay to achieve local delivery of FK506 just to the growing neurites in vitro and establish the required local dose of FK506 for peripheral nerve regeneration. A bimodal dose response was observed by culturing the entire DRG-ANA construct with media containing different concentrations of FK506. Low drug concentration of 1 pg/ml and high drug concentration of 100 ng/ml lead to the longest neurite extension in vitro. Furthermore, regardless of the FK506 concentration, concentrating the drug to the growing neurites resulted in significant increase in both neurite extension and neurite density, an effect that was not observed with the FK506 delivery to both neurites and neural cell bodies within DRG. The findings in this study provide valuable insight into the optimal local dose of FK506 for peripheral nerve regeneration. Furthermore, for the first time, this study suggests the potential interaction of FK506 with axons at the level of the growth cone.

The HDAC6 Inhibitor Trichostatin A Acetylates Microtubules and Protects Axons From Excitotoxin-Induced Degeneration in a Compartmented Culture Model.

Axon degeneration has been implicated as a pathological process in several neurodegenerative diseases and acquired forms of neural injury. We have previously shown that stabilizing microtubules can protect axons against excitotoxin-induced fragmentation, however, the alterations of microtubules following excitotoxicity that results in axon degeneration are currently unknown. Hence, this study investigated whether excitotoxicity affects the post-translational modifications of microtubules and microtubule-associated proteins, and whether reversing these changes has the potential to rescue axons from degeneration. To investigate microtubule alterations, primary mouse cortical neurons at 10 days in vitro were treated with 10 or 25 μM kainic acid to induce excitotoxicity and axon degeneration. Post-translational modifications of microtubules and associated proteins were examined at 6 h following kainic acid exposure, relative to axon degeneration. While there were no changes to tyrosinated tubulin or MAP1B, acetylated tubulin was significantly (p < 0.05) decreased by 40% at 6 h post-treatment. To determine whether increasing microtubule acetylation prior to kainic acid exposure could prevent axon fragmentation, we investigated the effect of reducing microtubule deacetylation with the HDAC6 inhibitor, trichostatin A. We found that trichostatin A prevented kainic acid-induced microtubule deacetylation and significantly (p < 0.05) protected axons from fragmentation. These data suggest that microtubule acetylation is a potential target for axonal protection where excitotoxicity may play a role in neuronal degeneration.

Local NGF and GDNF levels modulate morphology and function of porcine DRG neurites, In Vitro.

Nerve terminals of primary sensory neurons are influenced by their environment through target derived trophic factors, like nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF). In mice, subpopulations of DRG neurons express receptors either for NGF or GDNF and therefore differentially respond to these neurotrophic factors. We probed neurite endings from porcine DRG neurons cultured in either NGF or GDNF and examined their shape, elongation and stimulus-evoked CGRP release. A compartmentalized culture system was employed allowing spatial separation of outgrown neurites from their somata and use of different growth factors in the compartments. We show that neurites of GDNF cultured somata extend into lateral compartments without added growth factor, unlike neurites of NGF cultured ones. Neurites of NGF cultured somata extend not only into NGF- but also into GDNF-containing compartments. GDNF at the site of terminals of NGF responsive somata led to a strong neurite arborization and formation of large growth cones, compared to neurites in medium with NGF. Functionally, we could detect evoked CGRP release from as few as 7 outgrown neurites per compartment and calculated release per mm neurite length. CGRP release was detected both in neurites from NGF and GDNF cultured somata, suggesting that also the latter ones are peptidergic in pig. When neurites of NGF cultured somata were grown in GDNF, capsaicin evoked a lower CGRP release than high potassium, compared to those grown in NGF. Our experiments demonstrate that the compartmented culture chamber can be a suitable model to assess neurite properties from trophic factor specific primary sensory neurons. With this model, insights into mechanisms of gain or loss of function of specific nociceptive neurites may be achieved.

Compartmented neuronal cultures reveal two distinct mechanisms for alpha herpesvirus escape from genome silencing.

Alpha herpesvirus genomes encode the capacity to establish quiescent infections (i.e. latency) in the peripheral nervous system for the life of their hosts. Multiple times during latency, viral genomes can reactivate to start a productive infection, enabling spread of progeny virions to other hosts. Replication of alpha herpesviruses is well studied in cultured cells and many aspects of productive replication have been identified. However, many questions remain concerning how a productive or a quiescent infection is established. While infections in vivo often result in latency, infections of dissociated neuronal cultures in vitro result in a productive infection unless lytic viral replication is suppressed by DNA polymerase inhibitors or interferon. Using primary peripheral nervous system neurons cultured in modified Campenot tri-chambers, we previously reported that reactivateable, quiescent infections by pseudorabies virus (PRV) can be established in the absence of any inhibitor. Such infections were established in cell bodies only when physically isolated axons were infected at a very low multiplicity of infection (MOI). In this report, we developed a complementation assay in compartmented neuronal cultures to investigate host and viral factors in cell bodies that prevent establishment of quiescent infection and promote productive replication of axonally delivered genomes (i.e. escape from silencing). Stimulating protein kinase A (PKA) signaling pathways in isolated cell bodies, or superinfecting cell bodies with either UV-inactivated PRV or viral light particles (LP) promoted escape from genome silencing and prevented establishment of quiescent infection but with different molecular mechanisms. Activation of PKA in cell bodies triggers a slow escape from silencing in a cJun N-terminal kinase (JNK) dependent manner. However, escape from silencing is induced rapidly by infection with UVPRV or LP in a PKA- and JNK-independent manner. We suggest that viral tegument proteins delivered to cell bodies engage multiple signaling pathways that block silencing of viral genomes delivered by low MOI axonal infection.

3D compartmented model to study the neurite-related toxicity of Aβ aggregates included in collagen gels of adaptable porosity

Insoluble deposits of β-amyloid (Aβ) are associated to neurodegenerative pathologies, in particular Alzheimer’s Disease (AD). The toxicity of synthetic amyloid-like peptides has been largely demonstrated and shown to depend upon their aggregation state. However, standard 2D cell culture conditions are not well suited to study the role of the close vicinity of Aβ aggregates and growing neurites in the degenerative process. Here, we have designed a compartmented set-up where model neural cells are differentiated on the surface of Aβ-containing collagen matrices. The average pore size can be modulated, from below 0.2μm to more than 0.5μm by simple treatment with collagenase, to respectively hamper or permit neurite outgrowth towards the depth of the matrix. Dense Aβ aggregates (Congo red and ThT-positive) were obtained inside the collagen matrix with a homogeneous distribution and dimensions similar to those observed in post-mortem brain slices from Alzheimer’s patients. The aggregates are not toxic to cells when the pore size is small, in spite of relatively high concentrations of 0.05–0.62mg of peptide per gram of collagen (equivalent to 11.3–113μM). In contrast, on Aβ-containing matrices with large pores, massive neural death is observed when the cells are seeded in the same conditions. It is the first time to our knowledge that Aβ aggregates with a typical morphology of dense plaques are obtained within a porous biomimetic matrix, and are shown to be toxic only when accessible to differentiating cells. Statement of SignificanceInsoluble deposits of β-amyloid (Aβ) are associated to neurodegenerative pathologies, in particular Alzheimer’s Disease (AD). In this study, we have formed Aβ aggregates directly inside a biomimetic collagen matrix loaded with growth factors to induce the differentiation of PC12 or SH-SY6Y cells. For the first time, we show that when the contact between cells and Aβ aggregates is allowed by opening up the matrix porosity, the close vicinity with aggregates induces neurite dystrophy. The compartmented 3D culture model developed and used in this study is a valuable tool to study the cytotoxicity of preformed dense Aβ aggregates and proves that contact between the aggregates and neurons is required to induce neurodegenerative processes.

Two Modes of the Axonal Interferon Response Limit Alphaherpesvirus Neuroinvasion.

ABSTRACTInfection by alphaherpesviruses, including herpes simplex virus (HSV) and pseudorabies virus (PRV), typically begins at epithelial surfaces and continues into the peripheral nervous system (PNS). Inflammatory responses are induced at the infected peripheral site prior to invasion of the PNS. When the peripheral tissue is first infected, only the innervating axons are exposed to this inflammatory milieu, which includes the interferons (IFNs). The fundamental question is how do PNS cell bodies respond to these distant, potentially damaging events experienced by axons. Using compartmented cultures that physically separate neuron axons from cell bodies, we found that pretreating isolated axons with beta interferon (IFN-β) or gamma interferon (IFN-γ) significantly diminished the number of herpes simplex virus 1 (HSV-1) and PRV particles moving in axons toward the cell bodies in a receptor-dependent manner. Exposing axons to IFN-β induced STAT1 phosphorylation (p-STAT1) only in axons, while exposure of axons to IFN-γ induced p-STAT1 accumulation in distant cell body nuclei. Blocking transcription in cell bodies eliminated antiviral effects induced by IFN-γ, but not those induced by IFN-β. Proteomic analysis of IFN-β- or IFN-γ-treated axons identified several differentially regulated proteins. Therefore, unlike treatment with IFN-γ, IFN-β induces a noncanonical, local antiviral response in axons. The activation of a local IFN response in axons represents a new paradigm for cytokine control of neuroinvasion.

Manipulation of the N-terminal sequence of the Borna disease virus X protein improves its mitochondrial targeting and neuroprotective potential.

To favor their replication, viruses express proteins that target diverse mammalian cellular pathways. Due to the limited size of many viral genomes, such proteins are endowed with multiple functions, which require targeting to different subcellular compartments. One salient example is the X protein of Borna disease virus, which is expressed both at the mitochondria and in the nucleus. Moreover, we recently demonstrated that mitochondrial X protein is neuroprotective. In this study, we sought to examine the mechanisms whereby the X protein transits between subcellular compartments and to define its localization signals, to enhance its mitochondrial accumulation and thus, potentially, its neuroprotective activity. We transfected plasmids expressing fusion proteins bearing different domains of X fused to enhanced green fluorescent protein (eGFP) and compared their subcellular localization to that of eGFP. We observed that the 5-16 domain of X was responsible for both nuclear export and mitochondrial targeting and identified critical residues for mitochondrial localization. We next took advantage of these findings and constructed mutant X proteins that were targeted only to the mitochondria. Such mutants exhibited enhanced neuroprotective properties in compartmented cultures of neurons grown in microfluidic chambers, thereby confirming the parallel between mitochondrial accumulation of the X protein and its neuroprotective potential.-Ferré C. A., Davezac, N., Thouard, A., Peyrin, J. M., Belenguer, P., Miquel, M.-C., Gonzalez-Dunia, D., Szelechowski, M. Manipulation of the N-terminal sequence of the Borna disease virus X protein improves its mitochondrial targeting and neuroprotective potential.

Effects of root iron plaque on selenite and selenate dynamics in rhizosphere and uptake by rice (Oryza sativa)

Iron plaque commonly forms on rice root surfaces under flooded conditions. However, little is known about the role of iron plaque in Se movement from rhizosphere to plant. A compartmented soil-quartz sand culture system was applied to investigate the effects of iron plaque on Se uptake by rice and document Se speciation dynamics in soil solution under flooded conditions. Rice seedlings were planted in a nylon bag containing sand and surrounded by soil amended with Se combined at rates of 0 or 0.5 mg of Se kg−1 of soil in the form of selenate or selenite. Plants were harvested at seedling stage and at maturity. Fe and Se concentrations in soil solution, in DCB extracts and Se concentrations in rice roots and shoots were determined. A visible reddish coating on root surfaces could be observed when sampled at the seedling stage and at maturity. Iron plaque on root surfaces greatly influenced Se uptake and had a higher affinity for selenite. The distribution of Se in different components of rice seedlings followed iron plaque > shoot > root, while it followed shoot > root > iron plaque at maturity. The addition of Se initially increased the soluble Se concentrations, and selenate was the main species in soil solution. Iron plaque greatly influenced Se uptake and might act as a pool to Se uptake during rice growth. Selenate or selenite addition to paddy soil showed the same effect in increasing Se concentrations in rice grain.

Specific Retrograde Transduction of Spinal Motor Neurons Using Lentiviral Vectors Targeted to Presynaptic NMJ Receptors

To understand how receptors are involved in neuronal trafficking and to be able to utilize them for specific targeting via the peripheral route would be of great benefit. Here, we describe the generation of novel lentiviral vectors with tropism to motor neurons that were made by coexpressing onto the lentiviral surface a fusogenic glycoprotein (mutated sindbis G) and an antibody against a cell-surface receptor (Thy1.1, p75(NTR), or coxsackievirus and adenovirus receptor) on the presynaptic terminal of the neuromuscular junction. These vectors exhibit binding specificity and efficient transduction of receptor positive cell lines and primary motor neurons in vitro. Targeting of each of these receptors conferred to these vectors the capability of being transported retrogradely from the axonal tip, leading to transduction of motor neurons in vitro in compartmented microfluidic cultures. In vivo delivery of coxsackievirus and adenovirus receptor-targeted vectors in leg muscles of mice resulted in predicted patterns of motor neuron labeling in lumbar spinal cord. This opens up the clinical potential of these vectors for minimally invasive administration of central nervous system-targeted therapeutics in motor neuron diseases.

Diagnostic accuracy of a point-of-care urine bacteriologic culture test in dogs

To determine diagnostic accuracy of a compartmented bacteriologic culture and antimicrobial susceptibility testing plate (CCSP) for detection of bacterial urinary tract infection (UTI) in dogs and antimicrobial susceptibility testing of bacterial isolates. Evaluation study. 62 frozen, previously characterized bacterial isolates from canine urine cultures and 147 canine urine samples. The study was conducted in 2 phases: preliminary assay validation (phase 1) and diagnostic validation (phase 2). For phase 1, the frozen bacterial isolates were revitalized and tested with the CCSP and with standard aerobic microbiological culture (SAMC). For phase 2, the urine samples were tested with the CCSP and SAMC in parallel. For phase 1, after 24 hours of culture, 46 of 62 (74%) bacterial isolates had growth on the CCSP and all (100%) had growth in SAMC. For bacterial isolates with growth, the CCSP allowed correct identification of 45 of 46 (98%) isolates. Isolates yielding no growth on the CCSP were gram-positive cocci (Staphylococcus spp [n = 7] and Enterococcus spp [9]). In phase 2, the overall diagnostic accuracy of the CCSP, compared with SAMC, was 94% (sensitivity, 81%; specificity, 99%). The positive predictive value was 98% and negative predictive value was 92%. Susceptibility results for enrofloxacin and trimethoprim-sulfamethoxazole as determined with the CCSP had greatest concordance with those determined by SAMC (71% and 96%, respectively), compared with other antimicrobial susceptibilities. Use of the CCSP led to accurate exclusion of UTI in dogs without a UTI but was less reliable for diagnosis of UTI, particularly infections caused by gram-positive cocci. Standard aerobic microbiological culture remains the gold standard for detection of UTI in dogs.

Excitotoxin-induced caspase-3 activation and microtubule disintegration in axons is inhibited by taxol

BackgroundAxon degeneration, a key pathological event in many neurodegenerative diseases and injury, can be induced by somatodendritic excitotoxin exposure. It is currently unclear, however, whether excitotoxin-induced axon degeneration is mechanistically similar to Wallerian degeneration, which occurs following axon transection, but does not involve axonal caspase activation.ResultsWe have used mouse primary cortical neurons at 9 days in vitro, in a compartmented culture model that allows separation of the axon from the soma, to examine the pathological cascade of excitotoxin-induced axon degeneration. Excitotoxicity induced by chronic exposure to kainic acid, resulted in axonal fragmentation, which was associated with activation of caspase-3 in the axonal compartment. To examine the role of microtubules in these events, the microtubule-stabilizing agent, taxol, was added to either the axonal or somatodendritic compartment. Our results demonstrated that microtubule stabilization of axons resulted in a significant reduction in the number of fragmented axons following excitotoxin exposure. Interestingly, taxol exposure to either the somatodendritic or axonal compartment resulted in reduced caspase-3 activation in axons, suggesting that caspase activation is a downstream event of microtubule destabilization and involves signalling from the cell soma.ConclusionThese data suggest that excitotoxin-induced axon degeneration shows some mechanistic differences to Wallerian degeneration, and that microtubule stabilization may assist in protecting nerve cells from excitotoxic effects.

Nerve growth factor acts through the TrkA receptor to protect sensory neurons from the damaging effects of the HIV-1 viral protein, Vpr

Distal sensory polyneuropathy (DSP) with associated neuropathic pain is the most common neurological disorder affecting patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Viral protein R (Vpr) is a neurotoxic protein encoded by HIV-1 and secreted by infected macrophages. Vpr reduces neuronal viability, increases cytosolic calcium and membrane excitability of cultured dorsal root ganglion (DRG) sensory neurons, and is associated with mechanical allodynia in vivo. A clinical trial with HIV/AIDS patients demonstrated that nerve growth factor (NGF) reduced the severity of DSP-associated neuropathic pain, a problem linked to damage to small diameter, potentially NGF-responsive fibers. Herein, the actions of NGF were investigated in our Vpr model of DSP and we demonstrated that NGF significantly protected sensory neurons from the effects of Vpr. Footpads of immunodeficient Vpr transgenic (vpr/RAG1−/−) mice displayed allodynia (p<0.05), diminished epidermalinnervation (p<0.01) and reduced NGF mRNA expression (p<0.001) compared to immunodeficient (wildtype/RAG1−/−) littermate control mice. Compartmented cultures confirmed recombinant Vpr exposure to the DRG neuronal perikarya decreased distal neurite extension (p<0.01), whereas NGF exposure at these distal axons protected the DRG neurons from the Vpr-induced effect on their cell bodies. NGF prevented Vpr-induced attenuation of the phosphorylated glycogen synthase-3 axon extension pathway and tropomyosin-related kinase A (TrkA) receptor expression in DRG neurons (p<0.05) and it directly counteracted the cytosolic calcium burst caused by Vpr exposure to DRG neurons (p<0.01). TrkA receptor agonist indicated that NGFacted through the TrkA receptor to block the Vpr-mediated decrease in axon outgrowth in neonatal and adult rat and fetal human DRG neurons (p<0.05). Similarly, inhibiting the lower affinity NGF receptor, p75, blocked Vpr’s effect on DRG neurons. Overall, NGF/TrkA signaling or p75 receptor inhibition protects somatic sensory neurons exposed to Vpr, thus laying the groundwork for potential therapeutic options for HIV/AIDS patients suffering from DSP.

Reversing Roles for Innervation

Tumor necrosis factor (TNF) receptor in target tissues may guide the innervation of TNF-positive sympathetic axons in postnatal development.