WHAT IS BASELINE FOR IL-1-BETA, IL-6 AND TNF-ALPHA IN A CRUSH-INJURY MOUSE MODEL?

2.50
Hdl Handle:
http://hdl.handle.net/10755/211579
Type:
Research Study
Title:
WHAT IS BASELINE FOR IL-1-BETA, IL-6 AND TNF-ALPHA IN A CRUSH-INJURY MOUSE MODEL?
Abstract:
Wounded soldiers suffer from hypobaric hypoxia (low blood oxygenation) after crush injury when transported by air for medical treatment at elevations of 8000 feet and above, from the battle field to a remote treatment facility. Since oxygen is an essential component of wound healing, acute hypobaric hypoxia exacerbates combat injuries and prolongs recovery.  Providing external oxygen during the flight is not sufficient to augment hypoxia and our hypothesis is to reverse the effects of hypobaric hypoxia to the wounds by giving a single dose of estrogen during the flight. In order for us to study this phenomenon, we developed a mouse model to study the effects of estrogen in skeletal mouse model after crush injury and hypoxia. Every research project that focuses on inflammatory processes in skeletal muscle injury has to establish baseline values to which the injury model and the treatment course is compared to. Here we  report on the process, decisions and outcomes of our time 0 gene expression experiments studying three cytokines interleukin 1 beta (IL-1β), Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) in normal, hypobaric and crush injured mice. Following tissue harvest, tissue was shipped from the University of Nevada to the University of Washington on dry ice. Reverse transcription was performed on previously extracted RNA with Applied Biosystems High Capacity cDNA Reverse Transcription (RT) Kit. Reverse transcription PCR experiments compared gene expression of 3 pro-inflammatory cytokine primers (TNFa, IL-1 β, IL-6) to 18S endogenous control with gastrocnemius cDNA. Means and standard error (SE) were recorded. The relative level of gene expression for the experimental sample was computed using a normobaric sample pool. The pools consisted of (n=5) normobaric mice, separated into right and left gastrocnemius  muscle. Very little variation was observed for IL-1β, IL-6 and TNFα regardless of right or left muscle, injury or control, male and female sex. Best optimal pressure for crush injury was 45 PSI and comparisons ranged from 40-55. Switching to an alternate reverse transcription kit improved detection of cytokines and significantly lowered the standard error between runs. Establishing baseline values in a mouse model is critical to normalize later values accordingly to these standard samples. Pooled samples are an excellent way to limit variability and establish reliable control and injury detection values in cytokine research.
Keywords:
Oxygen; Wound healing; Hypobaric hypoxia treatment
Repository Posting Date:
20-Feb-2012
Date of Publication:
20-Feb-2012
Other Identifiers:
5511
Sponsors:
Western Institute of Nursing

Full metadata record

DC FieldValue Language
dc.typeResearch Studyen_GB
dc.titleWHAT IS BASELINE FOR IL-1-BETA, IL-6 AND TNF-ALPHA IN A CRUSH-INJURY MOUSE MODEL?en_GB
dc.identifier.urihttp://hdl.handle.net/10755/211579-
dc.description.abstractWounded soldiers suffer from hypobaric hypoxia (low blood oxygenation) after crush injury when transported by air for medical treatment at elevations of 8000 feet and above, from the battle field to a remote treatment facility. Since oxygen is an essential component of wound healing, acute hypobaric hypoxia exacerbates combat injuries and prolongs recovery.  Providing external oxygen during the flight is not sufficient to augment hypoxia and our hypothesis is to reverse the effects of hypobaric hypoxia to the wounds by giving a single dose of estrogen during the flight. In order for us to study this phenomenon, we developed a mouse model to study the effects of estrogen in skeletal mouse model after crush injury and hypoxia. Every research project that focuses on inflammatory processes in skeletal muscle injury has to establish baseline values to which the injury model and the treatment course is compared to. Here we  report on the process, decisions and outcomes of our time 0 gene expression experiments studying three cytokines interleukin 1 beta (IL-1β), Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) in normal, hypobaric and crush injured mice. Following tissue harvest, tissue was shipped from the University of Nevada to the University of Washington on dry ice. Reverse transcription was performed on previously extracted RNA with Applied Biosystems High Capacity cDNA Reverse Transcription (RT) Kit. Reverse transcription PCR experiments compared gene expression of 3 pro-inflammatory cytokine primers (TNFa, IL-1 β, IL-6) to 18S endogenous control with gastrocnemius cDNA. Means and standard error (SE) were recorded. The relative level of gene expression for the experimental sample was computed using a normobaric sample pool. The pools consisted of (n=5) normobaric mice, separated into right and left gastrocnemius  muscle. Very little variation was observed for IL-1β, IL-6 and TNFα regardless of right or left muscle, injury or control, male and female sex. Best optimal pressure for crush injury was 45 PSI and comparisons ranged from 40-55. Switching to an alternate reverse transcription kit improved detection of cytokines and significantly lowered the standard error between runs. Establishing baseline values in a mouse model is critical to normalize later values accordingly to these standard samples. Pooled samples are an excellent way to limit variability and establish reliable control and injury detection values in cytokine research.en_GB
dc.subjectOxygenen_GB
dc.subjectWound healingen_GB
dc.subjectHypobaric hypoxia treatmenten_GB
dc.date.available2012-02-20T12:03:24Z-
dc.date.issued2012-02-20T12:03:24Z-
dc.date.accessioned2012-02-20T12:03:24Z-
dc.description.sponsorshipWestern Institute of Nursingen_GB
All Items in this repository are protected by copyright, with all rights reserved, unless otherwise indicated.