Cerebrospinal Fluid Levels of Phosphorylated Neurofilament Heavy as a Diagnostic Marker of Canine Degenerative Myelopathy.

BACKGROUND: No definitive, antemortem diagnostic test for canine degenerative myelopathy (DM) is available. Phosphorylated neurofilament heavy (pNF-H) is a promising biomarker for nervous system diseases. HYPOTHESIS/OBJECTIVE: Cerebrospinal fluid (CSF) and serum pNF-H is a detectable biological marker for diagnosis of canine DM. ANIMALS: Fifty-three DM-affected, 27 neurologically normal, 7 asymptomatic at-risk, and 12 DM mimic dogs. METHODS: Archived CSF and serum pNF-H concentrations were determined by a commercially available ELISA. A receiver-operating characteristic (ROC) curve was generated with CSF values. RESULTS: Compared with old control dogs, median CSF pNF-H concentration was increased in all stages of DM; old dogs 5.1 ng/mL (interquartile range [IQR] 1.4-9.3) versus DM stage 1 23.9 ng/mL (IQR 20.8-29.6; P < .05) versus DM stage 2 36.8 ng/mL (IQR 22.9-51.2; P < .0001) versus DM stage 3 25.2 ng/mL (IQR 20.2-61.8; P < .001) versus DM stage 4 38.0 ng/mL (IQR 11.6-59.9; P < .01). Degenerative myelopathy stage 1 dogs had increased median CSF pNF-H concentrations compared with asymptomatic, at-risk dogs (3.4 ng/mL [IQR 1.5-10.9; P < .01]) and DM mimics (6.6 ng/mL [IQR 3.0-12.3; P < .01]). CSF pNF-H concentration >20.25 ng/mL was 80.4% sensitive (confidence interval [CI] 66.09-90.64%) and 93.6% specific (CI 78.58-99.21%) for DM. Area under the ROC curve was 0.9467 (CI 0.92-0.9974). No differences in serum pNF-H concentration were found between control and DM-affected dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: pNF-H concentration in CSF is a sensitive biomarker for diagnosis of DM. Although there was high specificity for DM in this cohort, further study should focus on a larger cohort of DM mimics, particularly other central and peripheral axonopathies.

Quantitative expression profiling in mouse spinal cord reveals changing relationships among channel and receptor mRNA levels across postnatal maturation.

Neural networks ultimately arrive at functional output via interaction of the excitability of individual neurons and their synaptic interactions. We investigated the relationships between voltage-gated ion channel and neurotransmitter receptor mRNA levels in mouse spinal cord at four different postnatal time points (P5, P11, P17, and adult) and three different adult cord levels (cervical, thoracic, and lumbosacral) using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Our analysis and data visualization are novel in that we chose a focal group of voltage-gated channel subunits and transmitter receptor subunits, performed absolute quantitation of mRNA copy number for each gene from a sample, and used multiple correlation analyses and correlation matrices to detect patterns in correlated mRNA levels across all genes of interest. These correlation profiles suggest that postnatal maturation of the spinal cord includes changes among channel and receptor subunits that proceed from widespread co-regulation to more refined and distinct functional relationships.

Expressing hNF-LE397K results in abnormal gaiting in a transgenic model of CMT2E.

Charcot-Marie-Tooth disease (CMT) is the most commonly inherited peripheral neuropathy. CMT disease signs include distal limb neuropathy, abnormal gaiting, exacerbation of neuropathy, sensory defects and deafness. We generated a novel line of CMT2E mice expressing an hNF-L(E397K) transgene, which displayed muscle atrophy of the lower limbs without denervation, proximal reduction in large caliber axons and decreased nerve conduction velocity. In this study, we showed that hNF-L(E397K) mice developed abnormal gait of the hind limbs. The identification of severe gaiting defects in combination with previously observed muscle atrophy, reduced axon caliber and decreased nerve conduction velocity suggests that hNF-L(E397K) mice recapitulate many of clinical signs associated with CMT2E. Therefore, hNF-L(E397K) mice provide a context for potential therapeutic intervention.

Seizure-induced alterations of plasma membrane calcium ATPase isoforms 1, 2 and 3 mRNA and protein in rat hippocampus.

Improper intracellular regulation of the ubiquitous second messenger, calcium, has been linked to several pathological conditions. The plasma membrane calcium ATPase (PMCA) is one of the primary systems for translocating calcium from the cytosol to the extracellular milieu. As an initial assessment of the possible involvement of PMCAs in kainate (KA)-induced neurodegeneration, we have determined the effect of KA-induced seizures upon PMCA mRNA and protein. In situ hybridization was performed on tissue from adult male Sprague-Dawley rats sacrificed at various time points following i.p. injection of KA. KA altered the expression within the hippocampal subfields for mRNAs of PMCA isoforms 1 and 2. PMCA 1 and 2 mRNAs exhibited hybridization below control levels 12-48 h post-injection within CA1 and CA3. Within the dentate gyrus, PMCA 2 mRNA hybridized below control levels 4 h post-injection, but recovered to control levels by 24 h post-injection. Alterations in combined PMCA protein levels occurred at all time points examined post-injection. These observations provide evidence that KA-induced seizures alter the PMCAs at the mRNA and protein levels, suggesting a possible role for this calcium efflux system in the neuronal degeneration inherent to this paradigm.

Long-term alterations in growth factor mRNA expression following seizures.

Although alterations in growth factor mRNA occur during neuronal insults, little is known about the long-term effects of neuronal insults on growth factor expression. We have examined the effects of prolonged post-ictal times on the expression of Brain-derivered nerve factor (BDNF) and Neurotrophin 3 (NT3) following Kainic acid (KA)-induced seizures. In situ hybridization was performed on male Sprague-Dawley rats sacrificed 1-2 weeks following intracranial ventricular KA injections. BDNF mRNA increased bilaterally 1 and 2 weeks after injections, whereas NT3 mRNA decreased contralaterally 1 week and bilaterally 2 weeks post-injection. These observations provide evidence that alterations in growth factor mRNA expression occur even after prolonged post-ictal recovery suggesting a possible role for growth factors in recovery and continued maintenance of surviving neurons within limbic seizure foci.

Cannibalism as the cause of an ontogenetic shift in habitat use by fry of the threespine stickleback.

In Crystal Lake, British Columbia, small fry (≤15 mm SL) of the threespine stickleback (Gasterosteus aculeatus) are concentrated in vegetation while larger fry are not. Because fry in all size classes feed primarily on zooplankton, even when in vegetation, we hypothesized that size-limited predation was responsible for the observed shift in habitat use with size. The major predators on fry in Crystal Lake are adult threespine stickleback, the water scorpion, Ranatra sp. (Hemiptera: Nepidae), backswimmers, Notonecta spp. (Hemiptera: Notonectidae), and dragonfly naiads of the genus Aeshna (Odonata: Aeschnidae). On the basis of distribution and hunting behavior we excluded the insects Ranatra sp., and Notonecta sp. as causal agents for this shift in resource by fry in water >0.25 m deep. Ranatra was found almost exclusively near the shoreline in water <0.25 m deep, and both insects hunted primarily as ambush predators within vegetation. Such predators seemed more likely to drive vulnerable fry from vegetation than to restrict them to it. In contrast, Aeshna naiads and adult stickleback frequently hunted outside of vegetation. In prey preference experiments the naiads did not show the decline in predation efficiency on fry >15 mm SL that would be expected if size-limited predation by this insect was responsible for the observed shift in resource use by fry. Adult stickleback only fed on fry <15 mm SL, and in an experimental situation, consumed fry at a rate 10 times greater than that exhibited by any of the insects. Predation experiments demonstrated that small fry (11-15 mm) spent more time in vegetation in the presence of adult conspecifics than they did in control pools, as would be expected if size-limited cannibalism caused small, vulnerable fry to be restricted to vegetation. Fry >15 mm SL were found outside of vegetation more often than in control treatments. The probable cause of this result is that adults become aggressive toward fry at this size, and often could be seen chasing large fry from vegetation during the experiments. Dragonfly naiads (Aeshna spp.) spent most of their time in vegetation in the experimental pools. Both size classes of fry spent less time in vegetation in the presence of dragonfly naiads than they did in control treatments, an apparent reflection of their similar vulnerabilities to these naiads. The presence of vegetation in pools reduced predation rates by adult stickleback on small fry. Because the experiments presented here indicate that fry are capable of rapidly assessing predation risk and of altering their behavior adaptively, we conclude that small fry occupy vegetation as a refuge from cannibalism. Once fry have reached the size-threshold at which they are no longer vulnerable to adult conspecifics they are able to forage farther from vegetation thereby reducing risk of predation by insects in vegetation and possibly acquiring more abundant food resources.