Investigation of West Nile Virus Infection Rates in Cx. tarsalis at Medicine Lake Wildlife Refuge and Ninepipe Wildlife Refuge 2011

This study focused on the infection rate of West Nile virus in two Culex tarsalis mosquito populations in Montana. Medicine Lake Wildlife Refuge (MLWR) and Ninepipe Wildlife Refuge (NWR) Cx. tarsalis populations were chosen due to the variance in past viral incidence. Viral incidence at MLWR was thought to be higher due to higher infection rate in the Cx. tarsalis population possibly from favorable environmental factors. This study suggests, however, that high viral incidence may be a result of high Cx. tarsalis population as a proportion of the total mosquito population. Samples were homogenized and purified for RNA using a series of centrifugations through a specialized filter. A RT-PCR and Taqman assay then determined viral presence. No positive samples were collected from either collection site; a pooled infection rate program utilizing a 95% confidence interval determined there was no statistical difference in infection rates. Future studies can build upon this research by increasing sample number, recording differing temperatures and precipitation levels at both sites, and incorporating a temporal study in addition to the spatial analysis

PHENOTYPIC CHARACTERIZATION AND TREATMENT OF A NOVEL MOUSE MODEL FOR LIMB-GIRDLE MUSCULAR DYSTROPHY 2i

Muscular dystrophies are a subset of inherited neuromuscular diseases that are characterized by weakened muscle structure and muscle wasting. Treatment options for these diseases are limited to treating the symptoms and not the disease. However, recombinant adeno-associated virus (rAAV) vector gene therapies have become a promising area of study for potential cures. The benefits of rAAV therapy for muscular dystrophies are as follows: 1) a high tropism for skeletal and cardiac muscle among vector serotypes, 2) they allow for production of native protein or functional truncated to restore muscle function, 3) the use of muscle-specific promoters maintains vector specificity, and 4) there is no incorporation of the transgene into genomic DNA. Unfortunately, most gene therapy clinical trials do not complete all trial phases, wasting substantial time and money. This is due to limited animal models for the extensive subtypes of muscular dystrophy and variations in phenotypic presentation in animal and human pathology. Additionally, animal models are not sufficiently challenged or tested with assays that are translatable and non-invasive for human patients. The goal of this work was to isolate whole animal testing metrics for a novel limb-girdle muscular dystrophy 2i (LGMD2i) mouse model by recording gait analysis and exercise capacity. We found that the mice had myopathic gaits, dilated cardiomyopathy, impaired respiration, and sustained muscle damage with forced exercise. We then tested this rigorous exercise protocol on aged mice injected with an AAV serotype 6 gene therapy. Results indicate that the disease phenotype is ameliorated by the drug, preventing further muscle damage and decreased respiratory capacity caused by the exercise schedule. Overall, the studies have shown that systemic whole animal testing of dystrophic models provide useful assays for disease characterization. These assays can be used to challenge drug therapies, as demonstrated in the LGMD2i mice with the rAAV6 vector, to determine the efficacy of future clinical trials

Trendelenburg-Like Gait, Instability and Altered Step Patterns in a Mouse Model for Limb Girdle Muscular Dystrophy 2i

Limb-girdle muscular dystrophy type 2i (LGMD2i) affects thousands of lives with shortened life expectancy mainly due to cardiac and respiratory problems and difficulty with ambulation significantly compromising quality of life. Limited studies have noted impaired gait in patients and animal models of different muscular dystrophies, but not in animal models of LGMD2i. Our goal, therefore, was to quantify gait metrics in the fukutin-related protein P448L mutant (P448L) mouse, a recently developed model for LGMD2i. The Noldus CatWalk XT motion capture system was used to identify multiple gait impairments. An average galloping body speed of 35 cm/s for both P448L and C57BL/6 wild-type mice was maintained to ensure differences in gait were due only to strain physiology. Compared to wild-type mice, P448L mice reach maximum contact 10% faster and have 40% more paw surface area during stance. Additionally, force intensity at the time of maximum paw contact is roughly 2-fold higher in P448L mice. Paw swing time is reduced in P448L mice without changes in stride length as a faster swing speed compensates. Gait instability in P448L mice is indicated by 50% higher instances of 3 and 4 paw stance support and conversely, 2-fold fewer instances of single paw stance support and no instance of zero paw support. This leads to lower variation of normal step patterns used and a higher use of uncommon step patterns. Similar anomalies have also been noted in muscular dystrophy patients due to weakness in the hip abductor muscles, producing a Trendelenburg gait characterized by "waddling" and more pronounced shifts to the stance leg. Thus, gait of P448L mice replicates anomalies commonly seen in LGMD2i patients, which is not only potentially valuable for assessing drug efficacy in restoring movement biomechanics, but also for better understanding them

Maternal high-fat diet during lactation impairs thermogenic function of brown adipose tissue in offspring mice

Maternal obesity and high-fat diet (HFD) predisposes offspring to obesity and metabolic diseases. Due to uncoupling, brown adipose tissue (BAT) dissipates energy via heat generation, mitigating obesity and diabetes. The lactation stage is a manageable period for improving the health of offspring of obese mothers, but the impact of maternal HFD during lactation on offspring BAT function is unknown. To determine, female mice were fed either a control or HFD during lactation. At weaning, HFD offspring gained more body weight and had greater body fat mass compared to the control, and these differences maintained into adulthood, which correlated with glucose intolerance and insulin resistance in HFD offspring. Adaptive thermogenesis of BAT was impaired in HFD offspring at weaning. In adulthood, HFD offspring BAT had lower Ucp1 expression and thermogenic activity. Mechanistically, maternal HFD feeding during lactation elevated peripheral serotonin, which decreased the sensitivity of BAT to sympathetic β3-adrenergic signaling. Importantly, early postnatal metformin administration decreased serotonin concentration and ameliorated the impairment of offspring BAT due to maternal HFD. Our data suggest that attenuation of BAT thermogenic function may be a key mechanism linking maternal HFD during lactation to persisted metabolic disorder in the offspring

Gait summary.

<p>(A) Visual representation of the step cycle over time in wild-type (WT) and P448L mice. Each bar signifies a single step cycle lasting the indicated time. Stand phase represents a greater proportion of the cycle, maximum contact is reached earlier and swing time is less in P448L mice while stride length is unaffected due to compensatory faster swing speeds. (B) Visual representation of differences in initial dual stance (IDS), single stance (SS), and terminal dual stance (TDS) between WT and P448L mice (gray boxes = paw in contact with surface; lines = periods of IDS, SS or TDS). The longer IDS and TDS and shorter SS in P448L mice reflect longer periods of contralateral paws touching the walkway during the gait cycle and, conversely, less time with only one contralateral paw touching. This was also reflected in greater instances of 3 and 4 paw support in P448L mice. (C) Hildebrand plot of the common gait patterns: AA (RF-RH-LF-LH), AB (LF-RH-RF-LH), CA (RF-LF-RH-LH) and CB (LF-RF-LH-RH). Percentages indicate relative use of each step pattern in each mouse group. The most common step pattern in both was the alternating AB pattern, although P448L mice used it significantly less and compensated with an increase use of the radial AA pattern.</p

Stance and Contralateral Paws.

<p>(A,B) Fore paw and hindpaw measurement of ground contact duration for a single paw while the contralateral paw is airborne. (C,D) The time contralateral paws simultaneously contact the ground at the beginning of a step and (E,F) at the end of a step. (G,H) The distance between contralateral paws while both are in stance phase. (FP = fore paw, HP = hindpaw, n = 12; *p<0.05; **p≤0.01, ***p≤0.001).</p

Paw Contact/Print Area.

<p>Fore paw and hindpaw measurements of print length (A,B), width (C,D) and area (E,F) recorded from the entire stance phase. (FP = fore paw, HP = hindpaw, n = 12; ****p≤0.0001).</p

Paw Support.

<p>(A) Percentage of run duration that no paws, a single paw, three paws or four paws were simultaneously touching the surface. (B) Percentage of run duration that two diagonal paws (RF-LH or LF-RH), contralateral paws (girdle, RF-LF or RH-LH) or lateral paws (RF-RH or LF-LH) were simultaneously touching. Students t-test was performed between WT and P448L in each group and combined into a single panel. (n = 12; *p<0.05; **p≤0.01, ***p≤0.001, ****p≤<u>0.0001</u> & denotes 0 recorded instances).</p

Gait metric overview.

<p>(A) Single paw representation of gait cycle between point A and B along a walkway. Separate gait metrics are defined by solid black lines within the gait cycle. (B) Screen capture images of the Noldus Catwalk System software showing print measurements and recorded intensities for the left hind paw of a P448L mouse. Light displacement leaves a green paw print on the walkway as shown and is used to derive print surface area metrics and intensity. The latter is an indication of weight supported by the paw. Intensity is measured in real time, graphed and used to calculate the highest intensity pixel in the print, Max Intensity (black line), and the Mean Intensity of all pixels (green area under curve). (C) Summary of support metrics. Solid prints and arrows represent a support pair, the opposite pairs are represented as fragmented prints and arrows. (D) Common step patterns include the alternate patterns AA and AB and the cruciate patterns CA and CB. Numbers and arrows indicate step order. The right front paw was arbitrarily chosen as the initial step.</p