Quantification Method of P2X3 Receptors in Rat DRG Neurons: Western Blotting

Skeletal muscle contractions are known to evoke pressor and cardioaccelerator responses in part by stimulating P2X3 receptors found on the peripheral endings of afferents. In diabetic patients, this pressor response is exaggerated. What is currently not known is whether P2X3 receptors play a role in evoking this exaggerated response. PURPOSE: The purpose of this project was to quantify P2X3 receptors in the L4 and L5 dorsal root ganglia (DRG) neurons in both healthy and type 1 diabetic rats using western blot analysis. METHODS: We injected 50 mg/kg streptozotocin (STZ) or the vehicle (CTL) i.p in fasted female and male Sprague Dawley rats and then waited at least 7 days for the rats to become diabetic. We then performed a laminectomy in the anesthetized rats to expose the spinal cord and roots. Using a dissecting microscope, we removed the L4 and L5 DRG from the spinal column. The DRG are the cell bodies of the peripheral afferents found in the hindlimb musculature. The DRG were placed in HBSS (is this buffer?) and stored at -80°C until analysis. For quantification, samples were lysed and proteins were isolated using the NucleoSpin RNA/Protein Kit (Macherey-Nagel, Bethlehem, PA, USA). A Qubit 3.0 Fluorometer was used to quantify the protein concentration of each sample so that equal protein concentrations could then be loaded onto a Bolt Bis-Tris (4-12%) gel. Following electrophoresis, the proteins were transferred to a membrane before being probed with a rabbit polyclonal P2X3 antibody (Alomone Labs), followed by an anti-rabbit secondary antibody conjugated to alkaline phosphatase (Life Technologies). The membrane was then exposed using a ChemiDoc XRS and the results analyzed using BioRad’s Quantity One imaging software. RESULTS: We were able to detect P2X3 receptor proteins. When compared with a molecular weight ladder, P2X3 receptor proteins were 54kDa, which is similar to the molecular weight of P2X3 receptors quantified in other studies. CONCLUSION: This method of quantifying P2X3 receptors in DRG neurons allows for a comparison between non-diabetic and diabetic rats. Further analyses are required to determine whether the quantity of P2X3 receptors in L4 and L5 DRG neurons is different in diabetic rats compared to non-diabetic rats

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Intradrainage Variation in Population Structure, Shape Morphology, and Sexual Size Dimorphism in the Yellow-Blotched Sawback, \u3ci\u3eGraptemys flavimaculata\u3c/i\u3e

Graptemys flavimaculata (Yellow-blotched Sawback) is a small, highly aquatic turtle that is endemic to rivers and large creeks of the Pascagoula River system of southeastern Mississippi, USA. Little is known about geographic variation in population structure, shape morphology, and sexual size dimorphism (SSD) throughout the drainage. I captured and measured G. flavimaculata from three sites in 2005 and 2006. I analyzed female head width at two of these sites in 2008. Results indicate that body size and population structure vary across a geographic gradient; turtles from the Pascagoula River site were generally larger (both body mass and plastron length) relative to two upstream sites on two tributaries, the Leaf and Chickasawhay rivers. Additionally, body shape in females varied among populations, with Pascagoula River females having a more domed shape than upstream sites where turtles have a more streamlined shape. There was little difference in male shapes among sites. Female-biased SSD typified all three populations with SSD being less pronounced in the two upstream sites. Female head width was significantly different across sites (Pascagoula \u3e Leaf), while there was no difference among sites for male claw length. Presumably, synergistic factors influence population structure, shape morphology, and sexual size dimorphism in Graptemys flavimaculata including: 1) food availability; 2) presence of competitors; 3) thermal environment; 4) presence of alligators; and 5) fluvial conditions

The Impacts of Hurricane Katrina On a Population of Yellow-Blotched Sawbacks (\u3ci\u3eGraptemys flavimaculata\u3c/i\u3e) in the Lower Pascagoula River

The Yellow-blotched Sawback (Graptemys flavimaculata) is a riverine turtle that is endemic to the Pascagoula River system of southern Mississippi, USA. Population declines led to Federal listing as a threatened species in 1991, with the most robust population inhabiting the Lower Pascagoula River near Vancleave, MS (approx. 24 river km from the Pascagoula River mouth). We conducted a mark-resight survey of this population during the spring and summer of 2005-2006. On 29 August 2005, Hurricane Katrina entered the Mississippi Gulf Coast, the location of our study site. On 13 October 2005, we conducted a one-hour preliminary visual survey by boat through the study area and we identified eight individuals that we marked prior to Katrina\u27s landfall. This demonstrated that at least some of the 49 previously marked individuals remained in the study area. Later, we conducted more extensive mark-resight surveys within the same 3.5 km section of the river in October of 2005-2006. The population estimate for 2006 was significantly lower than the 2005 population estimate for the same stretch of river, suggesting that numbers substantially decreased through the year following the hurricane. Of the plausible explanations for this pattern, the available evidence most strongly supports a real decline in population, presumably due to the long-term impact of Hurricane Katrina. Possible reasons for such a long term effect include hurricane induced saltwater intrusion and low levels of dissolved oxygen with direct impacts on individuals or indirect impacts on the prey populations (e.g. gastropods and other aquatic macroinvertebrates). Follow-up surveys are planned to investigate further these influences on the long-term population trends of G. flavimaculata

Basking Ecology of the Yellow-Blotched Sawback (\u3ci\u3eGraptemys flavimaculata\u3c/i\u3e), an Imperiled Turtle Species of the Pascagoula River System, Mississippi, United States

Basking is an understudied aspect of turtle biology, especially considering how frequent and observable it is in some species. Researchers have suggested many physiological roles that basking likely fulfills in turtles. We documented seasonal basking behavior of the yellow-blotched sawback (Graptemys flavimaculata) on the Leaf River, a tributary of the Pascagoula River in southeastern Mississippi. We used binoculars and a spotting scope to determine G. flavimaculata individual- and population-level basking patterns throughout the main active months (April–October) and across the daily activity period; we also describe a new method to determine population basking percentage that may be useful for future aquatic turtle surveys. We found distinct differences in individual- and population-level basking behavior across months, sexes, and the daily activity period. We also documented differences in basking structures used between the sexes but found little correlation between population-level basking and several environmental temperature variable

Growth in Kyphotic Ringed Sawbacks, \u3ci\u3eGraptemys oculifera\u3c/i\u3e (Testudines: Emydidae)

Kyphosis has not been reported in any Graptemys species (map turtles and sawbacks). We captured four kyphotic individuals of Graptemys oculifera from the Pearl River system, Mississippi, with three individuals recaptured 5 to 12 yrs later; one juvenile female had measurable growth, and two adult females had negligible growth. All kyphotic individuals were females, with an overall occurrence rate of 0.10% (4 of 3830 individuals) at five G. oculifera sample sites

Distribution and Abundance of Two Imperiled \u3ci\u3eGraptemys\u3c/i\u3e Species of the Pascagoula River System

Species distribution and abundance is often difficult to delineate due to species factors (e. g., crypsis, low abundance) or to researcher sampling techniques. Species of the genus Graptemys are primarily riverine turtles and have historically been subject to declines because of anthropogenic changes to river systems. Therefore, to better inform conservation efforts, we thoroughly studied the distribution and abundance of two imperiled Graptemys species within the Pascagoula River System, Mississippi, USA: the Yellow-blotched Sawback (Graptemys flavimaculata) and the Pascagoula Map Turtle (Graptemys gibbonsi). Turtle populations were studied in 17 counties in southeastern Mississippi using four methods: mark-resight population surveys (three populations), bridge surveys (160 bridge crossings), basking density surveys without marked individuals (23 localities), and trapping (three populations). Graptemys flavimaculata was found to be present throughout its historical range, as well as in new drainage localities; abundance in historically surveyed areas was generally higher than previous surveys had reported. Graptemys gibbonsi was also found in many new localities and occurred in most of the drainages of the Pascagoula River system. However, abundance was much lower for G. gibbonsi than for G. flavimaculata throughout the Pascagoula River system and individuals were not found in several historical localities, suggesting localized extirpations. We recommend that G. gibbonsi should be listed as state Endangered in Mississippi and Louisiana, U. S. federally listed as Threatened, and upgraded to Endangered (EN) under IUCN listing guidelines. Future conservation measures should extend to protect additional riparian habitat throughout the Pascagoula River system and future surveys of other Graptemys species are warranted due to the imperiled status of this genus

Life History and Ecology of the Pascagoula Map Turtle (\u3ci\u3eGraptemys gibbonsi\u3c/i\u3e)

Graptemys gibbonsi Lovich and McCoy (Pascagoula Map Turtle) is one of the most poorly understood turtle species in North America. Following the description of the species in 1992, little formal research has been conducted on the species other than population survey work. From 2005 to 2008, we conducted ecological studies on G. gibbonsi throughout the Pascagoula River ssytem of southeastern Mississippi, USA. We captured turtles at four sites to determine population structure, growth, movements, and some aspects of reproductionand nesting. We studied a single population to examine basking ecology and diet. We used visual surveys at four sites to document population density and relative abundance. Body size varied significatnly across sites and population-level sexual size dimorphism also varied. Growth was relatively rapid in small adults, with growth being slower in larger adults. Mean minimum linear active areas were in the spring and fall relative to summer, while also being longer during morning and evening periods; females generally based on larger, more robust logs versus smaller brances chosen by males. Graptemys gibbonsi basked at lower percentages (12% less) across seasons and throughout the day relative to the sympatric G. flavimaculata. Population densities and relative abundances of G. gibbonsi were greater at middle and upstream localities compared to downstream sections of the Pascagoula River system. Diet of males (n = 8) consisted primarily of caddisfly larvae and insect fragments, while females (n =4) consumed mostly Asian clams (Corbicula spp.) Reproduction extended from April to July for females \u3e 15.4 cm plastron length, with nests (n=3) encountered on both sandbars and cutbanks

Population Structure, Status, and Conservation of Two Graptemys Species from the Pearl River, Mississippi

Graptemys is one of the least studied turtle genera in North America. Graptemys oculifera (Ringed Sawback) and Graptemys pearlensis (Pearl Map Turtle) are endemic to the Pearl River system of Mississippi and southeastern Louisiana. We studied both species near Columbia, Mississippi, on the Pearl River via a trapping and basking surveys over two years. Additionally, five sites including Columbia were trapped for 27 years to determine long-term trends in capture success and relative abundance (RA). At the Columbia site, body size distribution was bimodal for G. oculifera and atypically unimodal for G. pearlensis; G. pearlensis body lengths were smaller than museum specimens. Population estimates for G. oculifera at the Columbia site indicate a stable population over 25 years. Long-term RA trends indicated that G. pearlensis was less common than G. oculifera in all periods and at all sites from 1988 to 2013. Trends in long-term capture success for G. oculifera and G. pearlensis were negative at all sites, with significant declines at three sites for both G. oculifera and G. pearlensis. Declines occurred both upstream and downstream of a major reservoir. Therefore, a combination of factors (including altered hydrology, human disturbance, lack of recruitment, excessive sedimentation, impaired water quality, and/or the pet trade) appear to be contributing to declines. Additional conservation and protection is warranted for G. pearlensis, and current protections for G. oculifera should be extended. Future studies should continue at our long-term sites to determine whether population declines persist or whether populations stabilize. Copyright 2017 Society for the Study of Amphibians and Reptile