Nov 09, 2024
Traumatic Brain Injury plus Systemic Hemorrhage in a Juvenile Rat- Sribnick Lab
- Eric Sribnick1,
- Mark W Hall2,3
- 1Nationwide Children's Hospital;
- 2Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH, USA;
- 3Departmtent of Pediatrics, Division of Critical Care, Nationwide Children’s Hospital, Columbus, OH, USA
- PRECISE-TBI
Protocol Citation: Eric Sribnick, Mark W Hall 2024. Traumatic Brain Injury plus Systemic Hemorrhage in a Juvenile Rat- Sribnick Lab. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19xdplr2/v1
Manuscript citation:
- Sribnick EA, Weber MD, Hall MW. Innate immune suppression after traumatic brain injury and hemorrhage in a juvenile rat model of polytrauma. J Neuroimmunol. 2019 Dec 15;337:577073. doi: 10.1016/j.jneuroim.2019.577073. Epub 2019 Oct 15. PMID: 31670063.
- Sribnick EA, Warner T, Hall MW. Traumatic brain injury and hemorrhage in a juvenile rat model of polytrauma leads to immunosuppression and splenic alterations. J Neuroimmunol. 2021 Dec 15;361:577723. doi: 10.1016/j.jneuroim.2021.577723. Epub 2021 Sep 23. PMID: 34619426.
- Sribnick EA, Warner T, Hall MW. Granulocyte- Macrophage Colony-Stimulating Factor Reverses Immunosuppression Acutely Following a Traumatic Brain Injury and Hemorrhage Polytrauma in a Juvenile Male Rat Model. J Neurotrauma. 2024 Jul;41(13-14):e1708-e1718. doi: 10.1089/neu.2023.0169. Epub 2024 May 3. PMID: 38623766.
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: August 09, 2024
Last Modified: November 09, 2024
Protocol Integer ID: 105045
Keywords: Traumatic Brain Injury, TBI, systemic hemorrhage, immune suppression, immunoparalysis
Funders Acknowledgement:
National Institutes of Health, National Institute of Neurological Disorders and Stroke
Grant ID: K08 NS119878
Abstract
This is a protocol to describe the materials and methods utilized to perform preclinical traumatic brain injury (TBI) with polytrauma for the express purpose of inducing post-injury immune suppression. This protocol, as presented, has been validated in juvenile (post-natal day 28) rats of the Sprague Dawley breed. Our results have shown that this model leads to early immune suppression as noted in innate1 and adaptive2 immune function.
The posting of this protocol is part of the mission of the PREClinical Interagency reSearch resourcE-TBI (PRECISE-TBI, precise-tbi.org) to improve clinical translation of therapeutics by providing an online catalog and standardized protocols to reduce the variability of model usage between laboratories.
Attachments
Image Attribution
Materials
Equipment:
- standard stereotactic device with rat ear-bars (Kopf, Tujunga, CA)
- Impact One Instrument (RRID:SCR_025114; Leica, Buffalo Grove, IL )
- Metzenbaum scissors
Materials for anesthetizing:
- betadine swabs
- isopropyl alcohol
- 5% isoflurane and 2-3% oxygen mixture.
Animal Housing/Conditions
Animal Housing/Conditions
Juvenile Sprague Dawley rats (post-natal day 28) are used in all experiments. A 28 day old rat equates roughly to a preadolescent youth3. Animals were housed in a standard fashion (ad libitum access to food and water, temperature controlled conditions, a 12:12 hour light: dark cycle). Prior to injury, rats are housed in groups.
Setup
Setup
All surgeries are done using sterile conditions. Fur from operative sites is removed using clippers and antisepsis is provided using betadine swabs and isopropyl alcohol.
For the cranial surgery, stereotaxis is performed using a standard stereotactic device (Kopf, Tujunga, CA) with rat ear-bars, a rat anesthesia mask, and an appropriately sized bite bar.
For the systemic hemorrhage surgery, rats are positioned supine and the limbs are secured to the surgical drapes using lab labeling tape. Inhalational gas is delivered using the rat anesthesia mask (which is unmounted from the stereotaxic device for this portion of the procedure).
Anesthesia is delivered using a mixture of isoflurane and oxygen. Specifically, isoflurane mixture was 5% for induction and 2-3% for maintenance. The total operative time is approximately 30 minutes. Normothermia was maintained using a standard warming system.
Traumatic Brain Injury
Traumatic Brain Injury
A midline incision is made using a #15 scalpel blade. The scalp is retracted to expose the cranium. The bregma is identified as a reference point, and a single craniectomy is made over the bilateral frontal area.
The craniectomy is centered at the midline at a point 2 mm anterior to bregma, and the craniectomy is 4 mm in diameter.
Injury is made using an Impact One Instrument (Leica, Buffalo Grove, IL) which is placed gently on the exposed dura. For injury, the impactor tip was 3 mm in diameter. Injury metrics are: velocity of 4 m/s, a dwell time of 500 m/s, and a depth of 3 mm.
Following injury, hemostasis is obtained using sterile 2’x2’ guaze pads. Gauze pads are weighed prior to surgery and any bleeding at the craniectomy/injury site is accounted for by weighing the used gauze pad.
Incision closure is performed using 4-0 Monocryl suture (Ethicon, Raritan, NJ)
For sham injury, the incision, craniectomy, and closure are performed as described above. The injury is not performed.
Fixed-volume hemorrhage
Fixed-volume hemorrhage
Estimated blood volume for each rat was calculated based on body weight (70 mL/kg) 4. Hemorrhagic injury was performed following TBI by repositioning the animal from the prone position to the supine position. As with TBI, the procedure was performed under anesthesia, and standard sterile technique was used.
A 1-2 cm longitudinal incision is made at the inguinal area using Metzenbaum scissors. Scissors are then used to bluntly dissect through the subcutaneous tissue to expose the femoral artery and vein bundle. Access to the femoral vein is perfomed using a 25 gauge needle with removal of 25% of EBV. If the needle is bent 45 degrees, this can facilitate entry into the vein.
Hemostasis is obtained using gentle pressure on a sterile gauze pad.
Any shed blood is collected with sterile gauze pads, and shed blood volume calculated by weight (1 mL/g).
For sham injury animals, similar leg incision, identification of the femoral artery vein complex, and closure are performed; however, vein puncture and hemorrhage are not performed.
Animals generally recover consciousness within 5 minutes of removal from the inhalational gas. They are monitored closely for 15-30 minutes afterward. Animals can perform behavioral testing the following day.
Protocol references
Manuscript citation:
- Sribnick EA, Weber MD, Hall MW. Innate immune suppression after traumatic brain injury and hemorrhage in a juvenile rat model of polytrauma. J Neuroimmunol. 2019 Dec 15;337:577073. doi: 10.1016/j.jneuroim.2019.577073. Epub 2019 Oct 15. PMID: 31670063.
- Sribnick EA, Warner T, Hall MW. Traumatic brain injury and hemorrhage in a juvenile rat model of polytrauma leads to immunosuppression and splenic alterations. J Neuroimmunol. 2021 Dec 15;361:577723. doi: 10.1016/j.jneuroim.2021.577723. Epub 2021 Sep 23. PMID: 34619426.
- Sribnick EA, Warner T, Hall MW. Granulocyte- Macrophage Colony-Stimulating Factor Reverses Immunosuppression Acutely Following a Traumatic Brain Injury and Hemorrhage Polytrauma in a Juvenile Male Rat Model. J Neurotrauma. 2024 Jul;41(13-14):e1708-e1718. doi: 10.1089/neu.2023.0169. Epub 2024 May 3. PMID: 38623766.
Additional References
- Sribnick EA, Weber MD, Hall MW. Innate immune suppression after traumatic brain injury and hemorrhage in a juvenile rat model of polytrauma. J Neuroimmunol. 2019 Dec 15;337:577073. doi: 10.1016/j.jneuroim.2019.577073. Epub 2019 Oct 15. PMID: 31670063.
- Sribnick EA, Warner T, Hall MW. Traumatic brain injury and hemorrhage in a juvenile rat model of polytrauma leads to immunosuppression and splenic alterations. J Neuroimmunol. 2021 Dec 15;361:577723. doi: 10.1016/j.jneuroim.2021.577723. Epub 2021 Sep 23. PMID: 34619426.
- Sengupta P. The Laboratory Rat: Relating Its Age With Human's. Int J Prev Med. 2013 Jun;4(6):624-30. PMID: 23930179; PMCID: PMC3733029.
- Diehl KH, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, Vidal JM, van de Vorstenbosch C; European Federation of Pharmaceutical Industries Association and European Centre for the Validation of Alternative Methods. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J Appl Toxicol. 2001 Jan-Feb;21(1):15-23. doi: 10.1002/jat.727. PMID: 11180276.