Inflammatory cytokine receptor blockade in a rodent model of mild traumatic brain injury.

In rodent models of traumatic brain injury (TBI), both Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNFα) levels increase early after injury to return later to basal levels. We have developed and characterized a rat mild fluid percussion model of TBI (mLFP injury) that results in righting reflex response times (RRRTs) that are less than those characteristic of moderate to severe LFP injury and yet increase IL-1α/ß and TNFα levels. Here we report that blockade of IL-1α/ß and TNFα binding to IL-1R and TNFR1, respectively, reduced neuropathology in parietal cortex, hippocampus, and thalamus and improved outcome. IL-1ß binding to the type I IL-1 receptor (IL-1R1) can be blocked by a recombinant form of the endogenous IL-1R antagonist IL-1Ra (Kineret). TNFα binding to the TNF receptor (TNFR) can be blocked by the recombinant fusion protein etanercept, made up of a TNFR2 peptide fused to an Fc portion of human IgG1. There was no benefit from the combined blockades compared with individual blockades or after repeated treatments for 11 days after injury compared with one treatment at 1 hr after injury, when measured at 6 hr or 18 days, based on changes in neuropathology. There was also no further enhancement of blockade benefits after 18 days. Given that both Kineret and etanercept given singly or in combination showed similar beneficial effects and that TNFα also has a gliotransmitter role regulating AMPA receptor traffic, thus confounding effects of a TNFα blockade, we chose to focus on a single treatment with Kineret.

A rodent model of mild traumatic brain blast injury.

One of the criteria defining mild traumatic brain injury (mTBI) in humans is a loss of consciousness lasting for less than 30 min. mTBI can result in long-term impairment of cognition and behavior. In rats, the length of time it takes a rat to right itself after injury is considered to be an analog for human return to consciousness. This study characterized a rat mild brain blast injury (mBBI) model defined by a righting response reflex time (RRRT) of more than 4 min but less than 10 min. Assessments of motor coordination relying on beam-balance and foot-fault assays and reference memory showed significant impairment in animals exposed to mBBI. This study's hypothesis is that there are inflammatory outcomes to mTBI over time that cause its deleterious effects. For example, mBBI significantly increased brain levels of interleukin (IL)-1ß and tumor necrosis factor-α (TNFα) protein. There were significant inflammatory responses in the cortex, hippocampus, thalamus, and amygdala 6 hr after mBBI, as evidenced by increased levels of the inflammatory markers associated with activation of microglia and macrophages, ionized calcium binding adaptor 1 (IBA1), impairment of the blood-brain barrier, and significant neuronal losses. There were significant increases in phosphorylated Tau (p-Tau) levels, a putative precursor to the development of neuroencephalopathy, as early as 6 hr after mBBI in the cortex and the hippocampus but not in the thalamus or the amygdala. There was an apparent correlation between RRRTs and p-Tau protein levels but not IBA1. These results suggest potential therapies for mild blast injuries via blockade of the IL-1ß and TNFα receptors.

Substrate and flow characteristics associated with White Sturgeon recruitment in the Columbia River Basin.

A study was conducted to identify habitat characteristics associated with age 0+ White Sturgeon (Acipenser transmontanus Richardson, 1863) recruitment in three reaches of the Columbia River Basin: Skamania reach (consistent recruitment), John Day reach (intermittent/inconsistent recruitment), and Kootenai reach (no recruitment). Our modeling approach involved numerous steps. First, we collected information about substrate, embeddedness, and hydrodynamics in each reach. Second, we developed a set of spatially explicit predictor variables. Third, we built two habitat (probability) models with Skamania reach training data where White Sturgeon recruitment was consistent. Fourth, we created spawning maps of each reach by populating the habitat models with in-reach physical metrics (substrate, embeddedness, and hydrodynamics). Fifth, we examined model accuracy by overlaying spawning locations in Skamania and Kootenai reaches with habitat predictions obtained from probability models. Sixth, we simulated how predicted habitat changed in each reach after manipulating physical conditions to more closely match Skamania reach. Model verification confirmed White Sturgeon generally spawned in locations with higher model probabilities in Skamania and Kootenai reaches, indicating the utility of extrapolating the models. Model simulations revealed significant gains in White Sturgeon habitat in all reaches when spring flow increased, gravel/cobble composition increased, or embeddedness decreased. The habitat models appear well suited to assist managers when identifying reach-specific factors limiting White Sturgeon recruitment in the Columbia River Basin or throughout its range.

Variability in a commercially available enzyme-linked immunosorbent assay system. I. Assay variability.

Three experiments were conducted to characterize the variation in enzyme-linked immunosorbent assay (ELISA) kits for infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV). Expt. 1 was carried out to determine the variation in assay results when the same pools of low-, medium-, and high-titered serum were assayed. Significant variation occurred among separate lots and among test plates within the same lots for the IBV and IBDV assays. In most cases, variability between days and among technicians was not significant. Coefficients of variation were larger than is acceptable for immune-type assays. In the IBDV assay with high-titered serum, most of the wells in the plates reached maximum absorbance and were not capable of detecting titers above 1:8000-1:9000. Expt. 2 was conducted to determine the effects of varying the length of the ortho-phenylene-diamine (OPD) incubation time upon assay results. Either 7-, 12-, or 15-minute OPD incubation times were used. Incubation time significantly affected mean titer at all combinations of assay types and times, except determinations on the low-titered IBV samples. Expt. 3 was conducted to determine the effects of three different dilution methods on observed IBDV titer. The use of non-standard dilutions had significant effects on observed titer. In the medium- and high-titered samples, the use of two different dilution methods at 1:5000 rather than 1:500 resulted in titers that were three to four times those observed at the 1:500 dilution.

Variability in a commercially available enzyme-linked immunosorbent assay system. II. Laboratory variability.

An experiment was conducted to determine the amount of variability that occurred in enzyme-linked immunosorbent assays when samples from common serum pools were assayed in five different labs on three consecutive days. Low- (approximately 1:2000), medium- (approximately 1:4000), and high-titered (approximately 1:8000) serum pools were distributed to five poultry industry laboratories that cooperated in the study. Results varied significantly among different laboratories and among different days in the same lab. Variation among days within the same laboratory and among laboratories were large. The greatest variability occurred among labs. Correlations between mean daily titer and laboratory ambient temperature were small and not significant. The amount of variability within and among different laboratories that were observed indicate that single determinations on individual serum samples are not likely to give a reliable estimate of antibody titer. The large variability within labs further indicates the need for standard reference pools of positive serum to be included in assays in order to substantiate assay results.

Characterization of lower temperature storage limitations of fresh-extended porcine semen.

Irreversible damage caused by cold shock has been assumed to occur when boar semen is exposed to temperatures below 15 degrees C. Identification of the lower critical temperature at which extended boar semen undergoes cold shock, however, has yet to be defined. The aims of this study were to 1) identify the cold-shock critical temperature and time on extended boar semen as assessed by sperm motility and morphology, and 2) determine the effects on fertility of using extended porcine semen exposed to this critical temperature and time. For Objective 1, ejaculates from 18 boars were collected, analyzed and extended in Androhep to 50 x 10(6) sperm/mL. Doses (4 x 10(9) sperm) from each ejaculate were exposed to 5 storage temperatures (8, 10, 12, 14 and 17 degrees C). Sperm motility and morphology (including acrosomes) were assessed following collection and at 12-h intervals for 48-h. Decreases in sperm motility occurred within the first 12-h at all temperatures. Sample motility dropped below 70% within 12-h in the 8 degrees C group and by 48-h in the 10 degrees C group. Sample motility was > 75% in the 12, 14 and 17 degrees C (control) groups throughout the trial. The percentage of morphologically abnormal sperm cells, including acrosomes, did not change within or between treatment groups over the 48-h storage period. In Objective 2, boar ejaculates (n = 9) were handled as in the first objective and were equally divided into treated (12 degrees C for < or = 60-h) and control (17 degrees C for < or = 60-h) groups. Using a timed, double insemination technique, 135 sows were bred by AI using either 12 degrees C (n = 74) or 17 degrees C (n = 61) extended, stored semen. No differences were observed in the farrowing rate (93 vs 95%), total offspring born (11.58 vs 11.61) or number live born (10.68 vs 10.63) between 12 and 17 degrees C groups, respectively. The results demonstrate that acceptable fertility can be obtained with Androhep extended boar semen exposed to temperatures as low as 12 degrees C for up to 60-h, and that cold shock appears to occur in vitro when extended boar semen is exposed to storage temperatures below 12 degrees C.