Effects of Cultured Adrenal Chromaffin Cell Implants on Hindlimb Reflexes of the 6-OHDA Lesioned Rat

The effects of implantation of cultured adrenal medullary cells on the recovery of neurotransmitter specific reflex activity were studied in the rat spinal cord using electrophysiological testing methods. Cell suspensions of cultured neonatal adrenal medullary chromaffin (AM) cells (which produce catecholamines), or Schwann (Sc) cells (controls) were implanted into the lumbar region of the spinal cord 2 weeks after catecholamine (CA) denervation by intracisternal injection of 6-hydroxydopamine (6-OHDA). All cells were taken from 7 day neonates and cultured for 10 days in the presence of nerve growth factor (NGF). Three months after implantation, the extent of implant-associated recovery of reflex activity was determined by measuring electromyogram (EMG) activity and force associated with the long latency component of the hindlimb withdrawal reflex (which is CA modulated). After the electrophysiological testing, rats were anesthetized, and the spinal cords were rapidly removed and frozen. Spinal cords were sectioned longitudinally, and implanted cells were visualized using glyoxylic acid techniques. Labelled sections were examined to determine cell survival. Results indicate that 1) chromaffin cells survive for 3 months in the segments of the cord into which they have been implanted and 2) rats implanted with AM cells have significantly more forceful withdrawal reflexes than those that received Sc cells or received no implant after lesioning

Use of different RT-QuIC substrates for detecting CWD prions in the brain of Norwegian cervids

Chronic wasting disease (CWD) is a highly contagious prion disease affecting captive and free-ranging cervid populations. CWD has been detected in United States, Canada, South Korea and, most recently, in Europe (Norway, Finland and Sweden). Animals with CWD release infectious prions in the environment through saliva, urine and feces sustaining disease spreading between cervids but also potentially to other non-cervids ruminants (e.g. sheep, goats and cattle). In the light of these considerations and due to CWD unknown zoonotic potential, it is of utmost importance to follow specific surveillance programs useful to minimize disease spreading and transmission. The European community has already in place specific surveillance measures, but the traditional diagnostic tests performed on nervous or lymphoid tissues lack sensitivity. We have optimized a Real-Time Quaking-Induced Conversion (RT-QuIC) assay for detecting CWD prions with high sensitivity and specificity to try to overcome this problem. In this work, we show that bank vole prion protein (PrP) is an excellent substrate for RT-QuIC reactions, enabling the detection of trace-amounts of CWD prions, regardless of prion strain and cervid species. Beside supporting the traditional diagnostic tests, this technology could be exploited for detecting prions in peripheral tissues from live animals, possibly even at preclinical stages of the disease

Detection of Protease-Resistant Cervid Prion Protein in Water From a CWD-Endemic Area

Chronic wasting disease (CWD) is the only known transmissible spongiform encephalopathy affecting free-ranging wildlife. Although the exact mode of natural transmission remains unknown, substantial evidence suggests that prions can persist in the environment, implicating components thereof as potential prion reservoirs and transmission vehicles. CWD-positive animals may contribute to environmental prion load via decomposing carcasses and biological materials including saliva, blood, urine and feces. Sensitivity limitations of conventional assays hamper evaluation of environmental prion loads in soil and water. Here we show the ability of serial protein misfolding cyclic amplification (sPMCA) to amplify a 1.3 x 10-7 dilution of CWD-infected brain homogenate spiked into water samples, equivalent to approximately 5 x 107 protease resistant cervid prion protein (PrPCWD) monomers. We also detected PrPCWD in one of two environmental water samples from a CWD endemic area collected at a time of increased water runoff from melting winter snow pack, as well as in water samples obtained concurrently from the flocculation stage of water processing by the municipal water treatment facility. Bioassays indicated that the PrPCWD detected was below infectious levels. These data demonstrate detection of very low levels of PrPCWD in the environment by sPMCA and suggest persistence and accumulation of prions in the environment that may promote CWD transmission

A model of type 2 diabetes in the guinea pig using sequential diet-induced glucose intolerance and streptozotocin treatment

Type 2 diabetes is a leading cause of morbidity and mortality among noncommunicable diseases, and additional animal models that more closely replicate the pathogenesis of human type 2 diabetes are needed. The goal of this study was to develop a model of type 2 diabetes in guinea pigs, in which diet-induced glucose intolerance precedes β-cell cytotoxicity, two processes that are crucial to the development of human type 2 diabetes. Guinea pigs developed impaired glucose tolerance after 8 weeks of feeding on a high-fat, high-carbohydrate diet, as determined by oral glucose challenge. Diet-induced glucose intolerance was accompanied by β-cell hyperplasia, compensatory hyperinsulinemia, and dyslipidemia with hepatocellular steatosis. Streptozotocin (STZ) treatment alone was ineffective at inducing diabetic hyperglycemia in guinea pigs, which failed to develop sustained glucose intolerance or fasting hyperglycemia and returned to euglycemia within 21 days after treatment. However, when high-fat, high-carbohydrate diet-fed guinea pigs were treated with STZ, glucose intolerance and fasting hyperglycemia persisted beyond 21 days post-STZ treatment. Guinea pigs with diet-induced glucose intolerance subsequently treated with STZ demonstrated an insulin-secretory capacity consistent with insulin-independent diabetes. This insulin-independent state was confirmed by response to oral antihyperglycemic drugs, metformin and glipizide, which resolved glucose intolerance and extended survival compared with guinea pigs with uncontrolled diabetes. In this study, we have developed a model of sequential glucose intolerance and β-cell loss, through high-fat, high-carbohydrate diet and extensive optimization of STZ treatment in the guinea pig, which closely resembles human type 2 diabetes. This model will prove useful in the study of insulin-independent diabetes pathogenesis with or without comorbidities, where the guinea pig serves as a relevant model species

Amyloid-β and Proinflammatory Cytokines Utilize a Prion Protein-Dependent Pathway to Activate NADPH Oxidase and Induce Cofilin-Actin Rods in Hippocampal Neurons

<div><p>Neurites of neurons under acute or chronic stress form bundles of filaments (rods) containing 1∶1 cofilin∶actin, which impair transport and synaptic function. Rods contain disulfide cross-linked cofilin and are induced by treatments resulting in oxidative stress. Rods form rapidly (5–30 min) in >80% of cultured hippocampal or cortical neurons treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% of maximum response in ∼6 h) occurs in a subpopulation (∼20%) of hippocampal neurons upon exposure to soluble human amyloid-β dimer/trimer (Aβd/t) at subnanomolar concentrations. Here we show that proinflammatory cytokines (TNFα, IL-1β, IL-6) also induce rods at the same rate and within the same neuronal population as Aβd/t. Neurons from prion (PrP^C)-null mice form rods in response to glutamate or antimycin A, but not in response to proinflammatory cytokines or Aβd/t. Two pathways inducing rod formation were confirmed by demonstrating that NADPH-oxidase (NOX) activity is required for prion-dependent rod formation, but not for rods induced by glutamate or energy depletion. Surprisingly, overexpression of PrP^C is by itself sufficient to induce rods in over 40% of hippocampal neurons through the NOX-dependent pathway. Persistence of PrP^C-dependent rods requires the continuous activity of NOX. Removing inducers or inhibiting NOX activity in cells containing PrP^C-dependent rods causes rod disappearance with a half-life of about 36 min. Cofilin-actin rods provide a mechanism for synapse loss bridging the amyloid and cytokine hypotheses for Alzheimer disease, and may explain how functionally diverse Aβ-binding membrane proteins induce synaptic dysfunction.</p></div

Proinflammatory cytokine dose-response curves and time course of rod formation in dissociated hippocampal neurons.

<p>(A) Percent of neurons with rods at 20 hr after treatment with TNFα, IL-1β and IL-6 show a similar dose-response. The maximum response level of approximately 20% of the neurons was reached at ∼50 ng/ml for each cytokine. Higher doses of TNFα kill the neurons within 12–24 hr, so 50 ng/ml was selected for further studies on treatments of up to 24 h. (B) The time course of rod formation in dissociated hippocampal neurons treated with 50 ng/ml TNFα is remarkably similar to that for Aβd/t which is used at ∼250 pM concentration <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095995#pone.0095995-Davis1" target="_blank">[7]</a>. Six and 16 h time points were performed with IL-1β and IL6 and they are not significantly different from TNFα and Aβ responses at the same times. Significance values with respect to untreated or zero time controls: *(p<0.01), **(p<0.05), ## (p<0.005). Error bars in this and all subsequent figures are standard deviations.</p

Both Aβd/t and TNFα utilize an NADPH oxidase-dependent pathway for rod formation, whereas glutamate does not.

<p>Hippocampal neurons were either untreated or infected with adenovirus for expressing DNp22^PHOX for 48 h prior to day 5 in culture. Some cultures were pre-treated 1 h with NOX inhibitorsTG6-227 (1 µM), ML171 (500 nM) or apocynin (1 µM). In the continued presence of the NOX inhibitors (or in neurons expressing DNp22 for 48 h), neurons were treated for 20 h with Aβd/t (∼250 pM) or TNFα (50 ng/ml), or for 30 min with glutamate (150 µM) before fixing, immunostaining for cofilin and quantifying the percent of neurons with rods. Rod response to Aβd/t and TNFα, but not glutamate, was significantly (# p<0.001) reduced by each of the NOX inhibitors.</p

The cellular prion protein, PrP^C, is required for rod formation from Aβd/t and proinflammatory cytokines, but not from rods induced by glutamate or mitochondrial inhibitors.

<p>(A) Percent of neurons with rods 20 h after treatment with Aβd/t or proinflammatory cytokines measured in dissociated neurons from FVB wild type mice or from the PrP^C-null mouse made in the FVB background. All of the decreases in the response of PrP^C-null neurons are significant with respect to their wild type controls (* p<0.01; ** p<0.05). (B) Rod formation is significant (# p<0.001) with respect to untreated neurons but does not differ significantly (NS) between hippocampal neurons from wild type (WT) and PrP^C-null mice in response to excitotoxic levels of glutamate (150 µM) or ATP-depletion (10 mM NaN₃, 2 mM 2-deoxyglucose) demonstrating that neither of these rod-inducing stresses utilize a PrP^C-dependent pathway.</p