Work Description

Date: March 11, 2025

Dataset Title: Effect of thermoneutral housing on a murine sepsis model

Dataset Creators: J.A. Nemzek, C. Fry, G. Chan

Dataset Contact: Jean Nemzek [email protected]

Funding: Unit for Laboratory Animal Medicine Cohen comparative Medicine Research Award, University of Michigan, Ann arbor

Key Points:
- We compare the effects of housing temperature (30°C or 22°C) for one week before and immediately after induction of sepsis in mice.
- Acclimation and post-operative housing after cecal ligation and puncture at thermoneutral temperatures (30°C) resulted in higher mortality compared to standard temperatures of 22°C.
- Acclimation at thermoneutral temperatures decreased blood neutrophil counts and peritoneal macrophage counts and decreased production of interferon by splenic T cells compared to standard temperatures.

Research Overview:
Current regulatory guidelines require that housing of laboratory mice be conducted at temperatures below their thermoneutral zone. Consequently, laboratory mice have chronic cold stress creating in physiologic changes that lead to immunosuppression. This may affect responses to stimuli such as bacterial sepsis and produce animal models that do not accurately translate to the human condition. We examined the effects of ambient temperature on a common model of sepsis, cecal ligation and puncture (CLP). Effects on survival, cell counts, cytokine levels and splenocyte production of interferon were measured. Overall, acclimation under thermoneutral conditions significantly impacted the immune responses and outcomes in the CLP model.

Methodology:
Cecal Ligation and Puncture (CLP)
After 7 days of housing in temperature chambers, mice (n = 10/group) underwent a laparotomy for cecal ligation and puncture to induce sepsis and then were returned to their assigned housing temperature. Mice received pre-emptive buprenorphine-HCl (0.05mg/kg, subcutaneous) diluted in 1.0 ml saline and subsequent doses in 0.1ml saline every 12 h for 48 hours post-operatively.

Temperature measurement
Transponders (IPTT-300; BioMedic Data systems, Inc, Seaford, DE) were inserted into the peritoneal cavity to allow 72 hours of temperature readings. Temperature readings were obtained with a corresponding handheld reader.

Survival analysis: Mice were observed for up to 7 days after CLP, with loss of righting reflex used as a humane endpoint. The occurrence of death within a 24 hour period was recorded for each mouse as a "1". If the event of death did not occur within 7 days, a value of "0" (Censored) was assigned for each remaining mouse. Blank rows in the file indicate no events occurred for that 24 hour time period.

Euthanasia and sample harvest
Under anesthesia, blood was collected from the retro-orbital sinus (500 μL into 50 μL of 169 mmol EDTA), followed by cervical dislocation. Peritoneal lavage fluid (PLF) was collected by injecting 10 ml Hanks Balanced Salt Solution (Invitrogen, Grand Island, NY) containing 1:100 heparin sodium (5000 USP U/mL; Abraxis, Schaumberg, IL) into the abdomen and retrieving fluid.

Sample analysis
Cell counts: A 20-μL aliquot of blood was used for automated CBC analysis (Hemavet, Drew Scientific, Miami, FL). The remaining blood and peritoneal lavage fluid were centrifuged (2000 x g, 5 min, and 600 x g, 5 min, respectively) and supernatants stored at −80 °C for later cytokine analysis. The cell pellets from PLF samples were counted using a hemocytometer (Hausser Scientific, Horsham, PA). Slides were loaded with 1 x 105 cells by centrifugation (109 x g, 5 min), and stained with Diff-Quick (Baxter, Detroit, MI). Differential cell counts (300 cells) were performed under light microscopy.

Bacterial Cultures
Colony forming units (CFUs) in PLF were determined by serial dilution in PBS (0, 1:10, 1:100, and 1:1000) and direct plating on 5% sheep blood agar (Remel, Lenexa, KS). Plates were incubated overnight (37°C) and colonies counted on plates yielding 30 to 300 colonies.

Splenocyte culture and stimulation
Spleens were macerated, combined with 10 mL 1X PBS (ThermoFisher, Waltham, MA), filtered, and centrifuged. Cells were re-suspended in 1X PBS, diluted 1:2 with Trypan blue (ThermoFisher), and counted with a hemacytometer (Hausser Scientific, Horsham, PA). Cells were plated at 5x10^6 cells/ml in mouse lymphocyte medium, with or without 5ug/ml of ConA (MilliporeSigma). Cells were harvested from individual wells at 24 hours of culture and supernatants were stored at -80°C.

Cytokine measurements
Plasma (1:10 dilution), peritoneal lavage fluid (1:2 dilution), and the supernatant from splenocyte cultures (1:10 dilution) were assayed using sandwich ELISA kits for CXCL1/KC, IL-6, CXCL2/MIP-2, and/or IFN-γ by DuoSet ELISA kits (R&D systems, Minneapolis, MN) according to manufacturer’s instructions. Absorbance was read at 450nm on a plate reader (Biotek, Winooski, VT). The data were analyzed using KC4 software (BioTek, Winooski, VT). The lower limit of detection was determined to be 15.8 pg/ml for IL-6 or CXCL1/KC and 31.0 pg/ml for CXCL2/MIP-2 or INF-γ. Samples falling below the limit of detection were assigned a value of 1/2 the lower limit of detection.

Date Coverage: 2021-2022

Instrument and/or Software specifications: Transponders (IPTT-300; BioMedic Data systems, Inc, Seaford, DE); automated CBC analysis (Hemavet, Drew Scientific, Miami, FL); hemocytometer (Hausser Scientific, Horsham, PA); plate reader (Biotek, Winooski, VT); KC4 software (BioTek, Winooski, VT)

Files contained here:
-time point study.csv: blood and PLF cell counts; plasma and PLF cytokines
-survival study.csv: survival at hours post CLP
-splenocyte culture.csv: spleen cell counts and in vitro splenocyte production of interferon gamma
-PLF CFU.csv: bacterial colony forming units from peritoneal lavage fluid
-body temperature.csv: temperature from intraperitoneal thermistor
-Data_Dictionary.docx: comprehensive data dictionary

Related publication(s):
Chan G, et al. Impact of thermoneutral acclimation on a murine model of polymicrobial peritonitis. Under review.

Use and Access:
This data set is made available under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).

To Cite Data:
Chan G, Fry C, Nemzek J. Impact of thermoneutral acclimation on a murine model of polymicrobial peritonitis [Data set]. University of Michigan - Deep Blue. https://doi.org/10.7302/55pr-9d24