Mar 18, 2024
Version 2
Lipopolysaccharide intraperitoneal injection in rats and sickness behavior assessment V.2
- 1Department of Experimental Neuroscience, Santa Lucia Foundation IRCCS, Rome, Italy;
- 2Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy;
- 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, United States
Protocol Citation: mariangela.massarocenere 2024. Lipopolysaccharide intraperitoneal injection in rats and sickness behavior assessment . protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3zmylk5/v2Version created by Nicola Biagio Mercuri
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 18, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 96846
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020505
Abstract
This protocol is a quick guide to how to prepare a solution of LPS on the saline vehicle, how to inject a rat for an intraperitoneal injection, and how to monitor the animal after injection. The procedure must be done by trained persons and done according to local Animal Care legislation.
Materials
Equipment:
- Analytical balance.
- Vortex mixer.
- Stirrer
Reagents:
- Lipopolysaccharide (LPS) from Escherichia coli O111:B4
- Sterile saline (0.9% NaCl)
- Ethanol (70%)
1. Lipopolysaccharide (LPS) preparation
1. Lipopolysaccharide (LPS) preparation
Before starting, it is better to prepare LPS solution on the same day it will be injected. However, under minimal conditions (that could be acceptable), you can work with LPS from previous days stored at kept at 2-8°C; in this case, shake very well before use.
Weigh the animal
To prepare a dose of 5 milligrams (mg) per kilogram (kg) of the weight of the rat and obtain an injection volume of 0.5 milliliters (ml) per kg of weight, calculate the amount of reagents that must be prepared as follo
| A | B | C | D | |
| total mass of rats (Kg) | Minimum mass of LPS required (mg | Mass of LPS to be measured (mg) | Volume of saline solution to dissolve the LPS (ml) | |
| 5mg/Kg*A | B*1.75 | C/(mg/ml) |
Weigh the calculated LPS total amount and add the volume of the corresponding saline solution to reach a solution of concentration 1 mg LPS / mL
mix well the solution with a stirrer for 15 minutes at medium speed (store at 4°C if not immediately used and shake on the vortex shaker for 30 seconds before injection)
2. Intraperitoneal injection
2. Intraperitoneal injection
Disinfect the lid of the multiple-dose vial with gauze and 70% ethanol
Determine the amount of volume per rat to be injected from the LPS solution or sterile saline (0.9% NaCl)
With care remove the animal from its homecage, and restrict it appropriately to perform the i.p. injection. Thus, insert the needle with the bevel of the needle facing up into the lower quadrant of the abdomen at an angle of 30-40 degrees.
Push the plunger until the entire solution is administered
Note
Be careful:
- The needle must not move inside the abdomen, to avoid lacerating surrounding organs or blood vessels
- When injecting, if a green solution is aspirated, it may be a sign that the intestine has been penetrated; if a yellow liquid comes out, it may indicate that the bladder was punctured; and if blood comes out, it may indicate that a blood vessel was perforated.
Once the solution is administered, place the animal back into the homecage
carefully, remove the needle from the syringe and discard the syringe in the sharps bin and discard the syringe in the trash
The animal should be monitored immediately after the injection, about 10 minutes
• If bleeding occurs, place gauze and apply pressure. Once bleeding stops, clean the site with gauze and water.
• In case of peritonitis, laceration of internal organs and / or infection, consult a veterinarian to assess if the animal can continue in the experiment.
3. Sickness behavior
3. Sickness behavior
5m
Signs consisting of absent exploration and locomotion, curled body posture, irregular fur, piloerection, and closed eyes are evaluated over time (at least 72h)
Place animals individually in a transparent cage and score on a four-point scale: 0 = no signs,1 = one sign, 2 = two signs, and 3 = three or more signs. The two experimenter quantifying the sickness signs should be blind to experimental and control conditions
5m
4. Body weight
4. Body weight
Record animal body weight after each sickness behavior observation and scoring
Protocol references
Flores-Martinez, Y. M. et al. Acute
Neuroinflammatory Response in the Substantia Nigra Pars Compacta of Rats after
a Local Injection of Lipopolysaccharide. J Immunol Res 2018,
1838921 (2018)