Nov 14, 2025
TBI ADRD Closed Head Injury (CHI) Standard Operating Procedure
- Gustavo A. Gomez1,
- Terese Garcia1,
- Amandine Julliene1,
- JiaMin Yan1,
- Paul R. Territo2,
- Andre Obenaus1
- 1Division of Biomedical Sciences, School of Medicine, University of California, Riverside;
- 2Stark Neuroscience Research Institute, Division of Clinical Pharmacology, School of Medicine, Indiana University
- TBI ADRD UCR/IU
Protocol Citation: Gustavo A. Gomez, Terese Garcia, Amandine Julliene, JiaMin Yan, Paul R. Territo, Andre Obenaus 2025. TBI ADRD Closed Head Injury (CHI) Standard Operating Procedure. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnrqw6l5d/v1
Manuscript citation:
The juvenile CHI version of this SOP has been published in the Journal of Visualized Experiments (Dubois et al., 2025).
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: November 13, 2025
Last Modified: November 14, 2025
Protocol Integer ID: 232375
Keywords: Closed Head Injury, Traumatic Brain Injury, Mouse, Adult, Juvenile, tbi adrd closed head injury, closed head injury, head injury, chi, standard operating procedure, standard operating procedure this standard operating procedure, aged mice
Funders Acknowledgements:
NIH NINDS
Grant ID: 1RF1NS138032-01
NIH NINDS
Grant ID: 5R01NS135556-03
NIH NINDS
Grant ID: 7R01NS119605-04
Disclaimer
The authors have nothing to disclose.
Abstract
This standard operating procedure (SOP) document describes the process for performing a closed head injury (CHI) in mice. Drs. Jerome Badaut and Andre Obenaus developed it to investigate CHIs in a juvenile equivalent age group. Here, it is adapted for studies in juvenile, adult, and aged mice.
Materials
• Weighing Scale
• Impactor controller unit (NeuroPactor™ from Neuroscience Tools, Cat#2500)
• Anesthesia induction unit (Isoflurane vaporizer)
• Charcoal filter (VETEQUIP #931401)
• Air pump (RWD Life Science #R510-30)
• Isoflurane anesthetic inhalant USP: (Covetrus SKU: 029405)
• Anesthesia mask/nose cone for use with adults only (Avantor #89012-602)
• Heating block to keep mice warm during anesthesia induction and recovery
• Timer
• Video camera to record details of the CHI
• Stereotaxic frame with stereotaxic arm equipped to mount actuator (Stoelting, cat# 900-C)
• Stereotactic embroidery frame (3D-Printed: See Appendix #2)
• Embroidery hoop (Nurge Ex-small #1: Nurge 5Pcs Plastic X-Small Square Embroidery Hoops, Cross Stich Hoop, Punch Needle Hoop Multicolor) – Available at Amazon
• 3D-printed pins to stabilize the stereotactic hoop on the frame (See Appendix #3)
• Aluminum foil (ULINE, cat# 11205)
• Insulin syringe (BD, cat# 329461)
• Super glue (Loctite, cat# 45198)
• Rulers and a marker to mark the X-axis, Y-axis, and the target site on the aluminum foil
• Scissors to cut the aluminum foil
Troubleshooting
Safety warnings
Attention items are written in Bold Italics.
*Important notes:* 1) It is crucial to avoid over tightening the impactor tip, as it can result in damage to actuator integrity. 2) IACUC recommends that piston tip is disinfected with 70% ethanol before a CHI for each mouse.
Ethics statement
This protocol follows institutional regulations consistent with the National Institutes of Health Guide for the Care
and Use of Laboratory Animals and the ARRIVE guidelines for reporting animal research. Experimental procedures
involving animals comply with protocol No. 30608, approved by the Institutional Animal Care and Use Committee
(IACUC) at the University of California, Riverside, School of Medicine, and protocol No. 25046 approved by IACUC
at Indiana University.
Before start
Both chambers are placed on a heating block warmed to 37°C.
*Important note:* Half of the recovery chamber rests directly on the heating block and the other half is not in contact with the heating block; this reduces the possibility that an animal will overheat during recovery.
A timer is used to monitor the exposure time to the isoflurane anesthetic gas, and to record the time it takes the animals to recover after the CHI. Documentation of experimental parameters and metrics recorded throughout the procedure are noted on the CHI Experiment Data Sheet (see Appendix #4, 5).
SCOPE
The scope of this SOP includes:
- List of materials, required equipment, and their setup
- Describe the setup and preparation of equipment at different steps of the procedure
- List parameters specific for different age groups
- Walk investigator through the procedure (with tips where appropriate)
- Attention items are written in Bold & Italics
RESPONSIBILITIES
- SOP Author: responsible for production of the SOP and its described procedures
- SOP Owner: responsible for reviewing and approving changes to the SOP
- Staff: responsible for adhering to SOP guidelines
- NO CHANGES to this SOP are authorized without the explicit approval of the SOP Owner (See Appendix 1)
Introduction
The CHI model described in this SOP was initially created to investigate the effect of a mild traumatic brain injury on the gliovascular unit and white matter tracts at a human pediatric equivalent age in mice (Rodriguez-Grande et al., 2018). This model has resulted in the identification of distinct spatiotemporal glial responses (Clément et al., 2020), with transient cerebrovascular perturbations resulting in localized hypoxia and edema (Ichkova et al., 2020). Importantly, this model has shown that animals injured at the juvenile stage develop neurological and behavioral deficits later in life (Leyba et al., 2023; Obenaus et al., 2023). Here, we adapted the model to investigate age-dependent effects of a CHI at three epochs of a lifespan in mouse: postnatal day 17 (P17), for which the model was developed; 8-month-old, reflective of middle age in mice; and 12-month-old, reflective of a mature adult age in humans (Flurkey et al., 2007). Our objective is to investigate whether mild/moderate CHIs at different ages increase the propensity of mice to develop Alzheimer’s Disease and Related Dementias.
The juvenile CHI version of this SOP has been published in the Journal of Visualized Experiments (Dubois et al., 2025), and the experimenter is urged to review it for an overview of the procedure. Note key aspects differ from the published version and this SOP.
Procedure Flow Chart
CHI Equipment setup
There are three primary components to CHI induction: 1) A NeuroPactor Controller (Neuroscience Tools) which regulates the velocity and dwell-time, and an actuator housing on which the impactor tip is attached. The impactor tip is the end on the piston which impacts the head of the anesthetized mouse. The actuator is mounted on a manual stereotaxic unit. 2) A stereotactic apparatus has a manipulator arm to which the impactor is attached. The stereotactic apparatus should have two rails attached to which the 3D printed embroidery frame can be attached (See Figure 3 below). 3) Induction and recovery chambers. The induction chamber is connected to an isoflurane vaporizer via standard plastic tubing. The recovery chamber with ambient air ports is used for assessing righting time and time to explore. Both chambers are placed on a heating block warmed to 37°C.
*Important note: Half of the recovery chamber rests directly on the heating block and the other half is not in contact with the heating block; this reduces the possibility that an animal will overheat during recovery. A timer is used to monitor the exposure time to the isoflurane anesthetic gas, and to record the time it takes the animals to recover after the CHI. Documentation of experimental parameters and metrics recorded throughout the procedure are noted on the CHI Experiment Data Sheet (see Appendix #4, 5).
Figure 1. CHI Equipment setup
Impactor Assembly
Figure 2. Impactor Assembly
(A) A screwable metal tip (50.8 mm long) is secured onto the bottom of the actuator. The metal impactor tip is then covered with the rubber from a syringe plunger that is glued to the smooth end of the piston. This tip is replaced every 6 months unless there is cracking of the rubber tip before 6 months. (B, C) The plunger of a 0.5 mL insulin syringe is shown here. (D) The plunger of a 0.5 mL insulin syringe is shown here. (D) The rubber end of the plunger is removed from the plunger and glued to the impactor tip with super glue. (E) As shown, the impactor tip has a diameter of 3 mm. (F) The actuator with the impactor tip is screwed on well (but not excessively tight) and positioned vertically when fully assembled.
*Important notes:* 1) It is crucial to avoid over tightening the impactor tip, as it can result in damage to actuator integrity. 2) IACUC recommends that piston tip is disinfected with 70% ethanol before a CHI for each mouse.
Embroidery Frame Equipment for Assembly
Figure 3. Embroidery Frame Equipment for Assembly
The tools required for assembling the embroidery frame on which the mouse is positioned for a CHI.
• Aluminum foil
• Rulers and a marker: to mark the X-axis, Y-axis, and the target site on the aluminum foil
• Scissors to cut the aluminum foil
• Embroidery frame
• Embroidery hoop
• 3D-printed pins
Embroidery hoop and frame assembly
Figure 4. Embroidery hoop and frame assembly
(A) Disassemble the embroidery hoop by loosening the screw at the top of the outer ring to allow disengagement of the inner hoop. (B, C) Relieve the pressure on the outer ring by pulling the top of the outer frame away from the midline, then release the inner hoop forward with finger pressure. (D) Position the inner hoop on a pre-cut sheet of aluminum foil on a flat surface with the matte side facing down (E) Wrap the edges of the foil around the inner embroidery hoop so that the foil is stretched tight. (F) Turn the inner hoop with the foil over so the flat surface faces up. (G) Take the frame of the outer hoop, expand the outer edge of the frame, and then push the bottom end of the inner hoop into the bottom grooves of the outer frame. (H, I) Push the top end of the inner ring into the outer frame until it is securely in place, checking that the tin foil remains taut. Then tighten the screw at the top to lock the frames in place. (J, K) Mount the foil-loaded embroidery hoop into the embroidery frame, taking care not to deform the tin foil surface. (L) Use a ruler to label the Y-axis on the foil with a marker. The grooves on the embroidery frame can be used as guides (M) Repeat this step to label the X-axis. (N) A loaded and labeled stereotactic support. (O, R) Use a ruler to mark the intended target for impact. In the example shown, the target site is marked at 3 mm to the right of the Y-axis and 3 mm below the X-axis with the “top” end facing the experimenter. This coordinate is used for single CHI (sCHI) and on the first CHI of a repeated CHI (rCHI) group. (Q) Example for coordinates used during the repeated CHI, where the X-axis remains at 3mm and the Y-axis is moved to 5mm. (P) Complete stereotactic assembly with clips mounted at the corners to stabilize the embroidery hoop on the base.
CHI Preparation
Figure 5. CHI Preparation
(A) Turn on the heat table to warm the anesthesia induction chamber and recovery chamber to 37°C. (B-D) Turn on the Impact controller (The switch is in the back of the instrument). Set the Impact controller on the front panel with age-specific parameters as shown in the table on panel E. Note that the velocity is adjusted by turning the large black control dial to the desired velocity shown in the digital display (C), while the dwell time is selected on the timer as shown in panel (D). (F) Mount the pre-marked embroidery hoop & frame on the stereotactic apparatus (Figure 1). Adjust the X- and Y- coordinates on the stereotaxic manipulator so that the tip of the impactor is positioned above the pre-marked target. (G) Manually retract the actuator arm by turning the knob controlling the Z coordinate counter-clockwise (moving the impactor tip upward) on the stereotaxic apparatus so there is sufficient space to position the mouse between the tip of the impactor and the foil (About 2 inches). (H) Reminder: Make sure the impactor tip is secured but not overly tight before beginning the CHI procedure.
CHI Procedure
Figure 6. CHI Procedure
Note: There are slight deviations in the SOP based on the age of mice. Images show procedure for pups. (Pups wake up fast after exposure to isoflurane: work quickly). Adult variation: See Appendix 6.
(A) Weigh the mouse. Juvenile mice should be excluded if they weigh less than 5.9g. (B) Turn on the air pump and set the air flow rate to 1 L/min. (C) Deliver 2.5% isoflurane into the induction chamber. (D) Transfer the mouse into the induction chamber. Juveniles (P17) should remain in the induction chamber for exactly 5 minutes, while adults (8- or 12-months-old) are anesthetized for 3 minutes in the induction chamber, then exposed to isoflurane for 2 additional minutes while on the stereotactic base by a nose cone via plastic tubing (see example in Appendix 6). (E) Quickly remove the mouse from the induction chamber and transfer to the aluminum foil under the impactor tip, placing the mouse on its ventral side with its head facing the experimenter. Make sure the mouse limbs are spread out away from the body. (E) Align the head so the X-axis on the foil transects the middle of both ears when viewed from above. The nose to tail axis should be aligned with the vertical Y-axis line on the foil (E) The impactor tip should be on the left side of the head of the mouse, behind the eyes. (F) Once the mouse is in position, set the Impactor control knob to Extend by pressing down the left toggle switch of the controller. (G) This will lower the impactor so that it comes close to the head of the mouse but does not touch it. There should be a gap between the impactor tip and the head of the mouse. In panel G this is illustrated by the gap between the red line at the bottom of the impactor tip and the blue line at the head of the mouse. Important caveat: In some impactor control models, triggering the Extend knob will not lower the impactor from the start position; The knob should be triggered regardless. (H) Lower the Actuator by turning the Z-axis knob on the stereotaxic apparatus until the impactor touches the head. The piston should press against the head, but the animal’s head should not move (fur should be compressed). Note that the dashed red line drawn at the end of the impactor tip in the previous panel now overlaps the dashed blue horizontal line drawn at the head of the mouse. (I) Flip the left toggle switch to Retract. (J) This will cause the impactor tip to automatically retract away from the head. (K) Lower the actuator again by the age-appropriate depth (P17 pups: 3mm. adults: 5mm), by turning the Z-plane knob on the stereotaxic apparatus clockwise. (L) The actuator is lowered 3mm in the example shown. Initially 8- on the main scale (blue) aligns with -10 on the Vernier scale (red). The Z-axis knob on the stereotaxic apparatus is turned clockwise (represented by red arrow), lowering the Vernier scale 3 mm down such that the 7.7- on the main scale now aligns with the -10 marking on the Vernier scale. For a description on how to read a Vernier scale please see (https://www.wpieurope.com/solutions/micromanipulators-knowledgebase/line-up-reading-a-vernier-scale.aspx). (M, N) When ready, press down the Impact toggle switch on the right to trigger the impact. This will release the impactor tip driving it onto the desired location on the head; resulting in a CHI with a dwell time of 100 milli-seconds. Important note: pups are impacted as fast as possible since they are removed from isoflurane exposure by removal from the induction chamber exactly at 5 minutes. By contrast, adults are exposed to isoflurane via an anesthesia mask when positioned on the stereotaxic frame. Although all mice are exposed to a total of 5 minutes of isoflurane, adults require at least 10 seconds without isoflurane exposure before CHI administration. (O) Immediately turn the left toggle switch to the Off position and start the timer to measure recovery time. Take notes on the following parameters on the Experiment Data Sheet (Appendix #4 or #5) at CHI: 1) whether the animal’s head rotated from the impact, and 2) whether the animal experienced apnea: the length of time it ceased breathing. These details can be video recorded, reviewed, and archived for future reference. Videos should capture the injury with the camera positioned in front of the mouse.
Recovery
Figure 7. Recovery
(A) Lay the mouse on its right side in the recovery chamber that rests on a heating block pre-heated to 37°C and make sure the timer was started. (B) Take notes on the time it takes for the mouse to B) “Right” and “Explore” – Righting can be defined as the time it takes for the animal to flip from its side to all
four limbs, while Exploring is the amount of time it takes for the animal to re-gain consciousness, walk around, and ambulate. (C) Re-adjust the piston and ensure it is tight for the next mouse. Reminder: IACUC recommends that the plunger is disinfected with 70% ethanol before a CHI is performed on thee next
mouse.
Table 1. Typical righting & exploration times
*Important note: Pups that take over 30 minutes to recover should be excluded from the study. Return the animal to its home cage after it starts exploring.
APPENDICES
• Appendix 1: SOP Record Log
• Appendix 2: Embroidery Frame – 3D Printing Instructions
• Appendix 3: Clips for securing Stereotactic Frame – 3D Printing Instructions
• Appendix 4: CHI Experiment Data Sheet (single CHI)
• Appendix 5: CHI Experiment Data Sheet (repeated CHI)
• Appendix 6: CHI procedure: Adult-specific CHI modification
Protocol references
Clément, T., Lee, J. B., Ichkova, A., Rodriguez-Grande, B., Fournier, M. L., Aussudre, J., Ogier, M., Haddad, E., Canini, F., Koehl, M., et al. (2020). Juvenile mild traumatic brain injury elicits distinct spatiotemporal astrocyte responses. Glia 68, 528-542.
Dubois, C. J., Yan, J., Obenaus, A. and Badaut, J. (2025). A Pediatric Concussion Model in Mice: Closed Head Injury with Long-Term Disorders (CHILD). Journal of visualized experiments : JoVE.
Flurkey, K., Mcurrer, J. and Harrison, D. (2007). Mouse Models in Aging Research (2nd ed. edn). p.637-672. Amsterdam ; Boston :: Elsevier,.
Ichkova, A., Rodriguez-Grande, B., Zub, E., Saudi, A., Fournier, M. L., Aussudre, J., Sicard, P., Obenaus, A., Marchi, N. and Badaut, J. (2020).Early cerebrovascular and long-term neurological modifications ensue following juvenile mild traumatic brain injury in male mice. Neurobiology of disease 141, 104952.
Leyba, K., Paiyabhroma, N., Salvas, J. P., Damen, F. W. Janvier, A., Zub, E., Bernis, C., Rouland, R., Dubois, C. J., Badaut, J., et al. (2023). Neurovascular hypoxia after mild traumatic brain injury in juvenile mice correlates with heart-brain dysfunctions in adulthood. Acta physiologica (Oxford, England) 238, e13933.
Obenaus, A., Rodriguez-Grande, B., Lee, J. B., Dubois, C. J., Fournier, M. L., Cador, M., Caille, S. and Badaut, J. (2023). A single mild juvenile TBI in male mice leads to regional brain tissue abnormalities at 12 months of age that correlate with cognitive impairment at the middle age. Acta neuropathologica communications11, 32.
Rodriguez-Grande, B., Obenaus, A., Ichkova, A., Aussudre, J., Bessy, T., Barse, E., Hiba, B., Catheline, G., Barrière, G. and Badaut, J. (2018). Gliovascular changes precede white matter damage and long-term disorders in juvenile mild closed head injury. Glia 66, 1663-1677
Charles W. Scouten’s article on the Leica instrument and
what determines TBI severity: