Fluid Percussion Injury

Introduction

The fluid percussion injury (FPI) model is a widely used study model for traumatic brain injury. FPI model produces an injury through a craniectomy by applying a momentary fluid pressure pulse on to the exposed dura. This pulse is created by a pendulum striking to the piston of the fluid reservoir. The percussion briefly displaces the neuronal tissue and deforms it. Among the TBI models, FPI is the most established and commonly used TBI model to evaluate mixed, focal and diffused brain injury. It is reproducible and standardized to provide control over the injury parameters. FPI recapitulates injuries observed in humans, making it suitable for the exploration of novel therapeutics for clinical translation (Alder et al. 2011).

History

Fluid percussion model was initially developed for use in cat and rabbit and was later adapted for rat and then modified to create traumatic brain injury over a single hemisphere in rodents. Initially, the fluid-percussion technique employed a fluid-filled column to strike the exposed dura of an animal. Denny-Brown and Russell attempted to generate a generalized loading to the brain rather than the focal injury by applying a brief, localized pressure pulse to the dura of a cat. A decade later, Gurdjian applied a similar technique on mongrel dogs, by applying compressed air in place of the fluid to rapidly raise the intracranial pressure for concussion. In the late 1980s, the fluid-percussion injury model enabled the researchers to study possible behavioral outcomes to perform large-scale studies of experimental therapeutics. Fluid percussion was later adapted for use in ferrets, pigs, and smaller animals, providing the means of studying TBI in experimentally and genetically altered animals. The fluid percussion injury (FPI) model has since become the most well-characterized and extensively used model of experimental TBI (Lyeth, 2016).

Principle

In the FPI model, the injury is inflicted by a pendulum striking to the piston of a fluid reservoir fluid to generate a fluid pressure pulse on the intact dura through a craniotomy. The craniotomy is created either centrally around the midline between bregma and lambda, or laterally over the surface of parietal bone between bregma and lambda. The percussion briefly displaces and deforms the brain tissue, and the severity of the injury depends on the strength of the pulse. FPI models present the conditions of clinical TBI without skull fracture. FPI can efficiently replicate intracranial hemorrhage, brain swelling, and progressive grey matter damage.

Apparatus

The fluid-percussion device consists of a cylindrical fluid reservoir (60 cm long and 4.5 cm in diameter) bounded at one end by a cork-covered piston mounted on O-rings. And on the other, the cylinder is attached with a 2 cm long metal housing connected to a 5 mm tube. This tube is attached to a male Leur-Loc fitting to the female Leur-Loc fitting that has been implanted over the exposed parietal cortex of the rat. The severity and the extent of the injury are regulated by varying the height of the pendulum, resulting in variations in extracranial pressure pulses. These pressure pulses are then measured extracranially by a transducer at the time of injury, recorded on a storage oscilloscope, and photographed with an attached camera (Vink., McIntosh., Weiner.., & Faden, 1987).

Protocols

1. Anesthetize the animal and place it on the stereotaxic frame.
2. Shave the head and inject local anesthesia under the scalp. Make a midline incision to open the scalp and retract the skin to expose the skull bone. Pull the scalp using hemostatic forceps.
3. Clean the skull from connective tissue and periosteum with the help of Dumont forceps.
4. Perform craniotomy after identifying the midline and bregma sutures as reference points. Using trephine, perform craniotomy carefully, and make sure not to rupture the dura mater.
5. Make a hole for the anchor screw with the help of a drill without going through the bone. Attach the screw to the skull.
6. Place the trauma coupling in the craniotomy and fix it using the tissue adhesive.
7. Pour dental acrylic around the trauma coupling. Include the anchor screw in the cast. Let the dental cement set properly.
8. Wean the subject off anesthesia enough for it to regain the pinch reflex in the paws.
9. Place the animal on the FPI device and perform the impact.
10. Place the animal on a heated pad and record the time till the animal regains the pinch reflex in the contralateral paws.
11. Replace the bone piece and suture the scalp after fixing it with tissue adhesive.
12. Return the animal to a recovery cage after t has regained the pinch reflex.

Alternate protocol (Katz. & Molina, 2018)

Craniotomy

1. Carefully wipe the operating field with a sterile drape and open the surgery pack onto the sterile field.
2. Weigh the rat and record its weight.
3. Anesthetize the rat with 3-4% isoflurane for 5 minutes in the induction chamber.
4. Shave the hair from behind the ears to the middle of the snout.
5. Return the animal to the induction chamber for 2-3 minutes.
6. Move the rat to the stereotactic frame and redirect isoflurane through the nose holder on the stereotactic frame. Maintain the anesthesia induction at 2-3% throughout the surgery.
7. With the help of the incisor bar and ear bars, stabilize the head in the stereotaxic frame.
8. Apply a small amount of eye lubricant to each eye.
9. Scrub and clean the surgical area with Betadine followed by Isopropyl alcohol.

Note: Repeat this step three times.

10. Using a #22 scalpel blade, cut the skin (~3 cm) from between the eyes and over the cranial crown.
11. Keep the overlying skin and muscle aside to expose the cranium and pull back each side with curved hemostats.
12. With the help of sterile cotton-tipped applicators, swab the skull to remove the membrane.
13. Using a bone scraper, gently fix the skull in both directions to make the surface suitable for dental acrylic and super glue.
14. Place a 2 x 2 piece of gauze over the skull and leave it for a few minutes to dry the skull completely.
15. Hand drill one hole posterior to lambda and place an anchor screw to secure the dental acrylic.
16. Carefully fix the screw, making certain not to contact the dura.
17. On the stereotaxic frame, locate the bregma and position the left arm of the frame between the center of the bregma and the lateral suture.
18. Move the left arm 2mm posterior (back) to the bregma, and 3mm lateral (left) to the midline by moving the dials on the frame.
19. Mark the skull to indicate the site of the drill hole.
20. Gently drill a circular track with the markings from step 18 as its center.
21. Continue drilling until the skull flap depresses with even light force by forceps.
22. Remove the circular skull flap.
23. Wipe the skull with sterile gauze to remove any bone fragments and make sure that the skull is entirely dry.
24. Introduce the head cannula (make sure that it is completely dry) to the craniotomy hole until it is directly above the dura.
25. Fit the cannula in the craniotomy hole warmly.
26. Once the cannula is aligned directly over the drill hole, close the perimeter by applying super glue.
27. Let the glue dry for approximately 5 minutes.
28. Fill the cannula with sterile saline to prevent drying.
29. Mix equal parts of dental acrylic and jet liquid in a small tray. The amount needed should cover the exposed skull and surround the cannula.
30. Draw the acrylic mixture using 1 ml syringe and apply one layer of dental acrylic glue. Note: Allow the first layer to dry before applying the second layer.
31. Once the dental acrylic is dry, apply the final layer of glue around the cannula to seal the cannula and the dental acrylic.
32. Cap the cannula to keep the dura moist.
33. Remove the animal from the frame and place it in a clean cage for observations until the animal regains consciousness.

TBI induction

1. Set the FPI device and fill the chamber with sterile water.
2. Attach a 25 mL syringe filled with sterile water to the center port and remove any bubbles. Then, fill the chamber after use.
3. Before the use, make sure that the FPI and the high-pressure tubing are filled with sterile water.
4. Turn ON the pressure transducer amplifier connected to the FPI device.
5. Calibrate the instrument by adjusting the 100 PSI calibration switch. A two-point calibration can be achieved by setting the pressure in pounds per square inch (PSI), with the pressure tubing open. Set your value to zero in data acquisition software; to set the 100 level, switch ON the 100 PSI calibration switch and set it to 100 in your data acquisition software.
6. To complete the setup, turn the data acquisition ON. Release the pendulum with the Luer-lok end of the tubing closed to deliver the pulses.
7. Adjust the pendulum angle to increase or decrease the pulse intensity. The standard angle of the pendulum’s starting position for a mild to moderate TBI is approximately 18-20o.
8. Place the rat in 4-5% isoflurane anesthesia chamber (pre-charged) until a surgical anesthesia plane is reached.
9. Place the rat on the stereotaxic frame next to the FPI and clean the hub of any clotted blood and then fill it with sterile saline.
10. Attach the tubing of the FPI with a male Luer-lok to the female Luer-lok fitting of the cannula hub.
11. Turn OFF the isoflurane, once a normal breathing pattern resumes but before the animal regains complete consciousness (~1-2 min).
12. Release the pendulum to create a single pulse of injury.

Note: Do not induce the injury while the animal is too anesthetized as it may lead to severe respiratory depression and ultimately, death.

13. Start the timer at the time of injury to record the apnea and righting reflex.
14. Soon after the injury, place the animal on the right side and allow it to recover.
    • For apnea, record the time taken by the animal to take its first breath after injury.
    • For righting reflex, observe the time taken by the animal to right itself on all four paws.
15. Record the respiratory rate following the injury.
16. Return the animal to its cage once it has the right to all four paws.

Confirm the injury severity by observing the behavioral changes in the animals post-injury.

Applications

Studying traumatic brain injury (Kabadi et al. 2010)

The fluid-percussion injury model (FPI) is one of the oldest and most widely used models of experimentally induced TBI. Both central and lateral variations of the model have been used in brain injury research. The FPI model was developed initially for use in larger species and was then adapted to smaller laboratory animals and rodents. The fluid-percussion injury model has been applied to study open and closed head TBI, graded injuries, and the long-term deficits and epilepsy following TBI and concussion.

A well-suited model for post-traumatic epilepsy (Hameed., et al., 2014)

Traumatic brain injury is one of the leading causes of acquired epilepsy. Lateral fluid percussion injury (LFPI) has become a widely used and well-characterized model for studying rodent traumatic brain injury and post-traumatic epilepsy. For this, anesthetized male Long-Evans rats were subjected to fluid percussion. LFPI resulted in nonlethal focal cortical injury in the test subjects. The procedure efficiently created post-traumatic seizures and regional gliosis. Also, apoptotic cell death was confined to the perilesional cortex, and chronic pathologic changes such as ipsilesional ventriculomegaly were observed in the injury model. It was concluded that the LFPI method is a valuable research model that has the advantage of a shorter experiment turnaround and reduced anesthetic exposure.

Assessment of anticonvulsant drugs (Echegoyen., Armstrong., Morgan., & Soltesz., 2009)

The study was conducted to investigate the effects of anticonvulsant drugs on hyper-excitability and prophylaxis in post-traumatic epilepsy. The clinical trials using anti-convulsant drugs have not presented any long-term anti-epileptogenic effects. In the study, a single, rapid post-traumatic application of the pro-convulsant cannabinoid type-1 (CB1) receptor antagonist SR141716A was used to investigate its effect on seizure susceptibility in rats. The results suggested that anti-convulsants could be used to prevent the long-term increase in seizure susceptibility, a likely condition of post-traumatic epilepsy.  Also, a seizure-enhancing drug could disrupt the epileptogenic process if applied timely. The study has validated the usefulness of the fluid percussion injury model in evaluating the efficacy of potential anti-epileptogenic drugs.

Evaluating the therapeutic effects of neurotrophic factors (Sinson., Voddi., & McIntosh., 1995)

Lateral fluid-percussion brain injury efficiently presents cognitive deficits, motor dysfunction, and selective hippocampal cell loss in test animals. Neurotrophic factors have therapeutic potential to treat neurodegenerative diseases. Also, nerve growth factor (NGF) has shown neuroprotective behavior in models of excitotoxicity. This study was conducted to evaluate the neuroprotective efficacy of intracerebral NGF infusion in fluid percussion injury model of traumatic brain injury. For this, male Sprague-Dawley rats received a lateral fluid percussion brain injury. NGF was infused through a mini osmotic pump in the experimental group. NFG infusions continued until the animal was killed at 72 hours, one week, or two weeks following injury. Then, the animals were evaluated for cognitive dysfunction and regional neuronal cell loss. The results suggested that NGF administration, in the acute, post-traumatic period following fluid-percussion brain injury, improves post-traumatic cognitive deficits in rats. Also, the study presented the fluid percussion injury model as a powerful tool to assess the neuroprotective functions of neurotrophic factors.

Strengths and limitations

• The fluid percussion injury model has become the most commonly used and well-characterized model to study experimental traumatic brain injury.
• Using FPI, reproducible levels of injury severity could be achieved by adjusting the pendulum height that decides the force of the fluid pressure pulse to deliver the injury.
• FPI has been primarily used for the identification of TBI-induced cellular and molecular changes and to evaluate potential therapies for post-traumatic epilepsy and traumatic brain injury.
• The lateral fluid percussion injury model provides construct validity in that it efficiently recreates the etiological processes of TBI in humans.
• Despite the widespread applications of the FPI model, there can be problems with its variability, as the pressure wave generation is highly sensitive to operational factors.

References

1. G. Sinson., M. Voddi., & McIntosh., K. T. (1995). Nerve growth factor administration attenuates cognitive but not neurobehavioral motor dysfunction or hippocampal cell loss following fluid-percussion brain injury in rats. J Neurochem, 65(5), 2209-16.
2. J. Alder., W. Fujioka., J. Lifshitz., P. D. Crockett., & Thakker-Varia., S. (2011). Lateral fluid percussion: model of traumatic brain injury in mice. J Vis Exp, 22(54).
3. J. Echegoyen., C. Armstrong., J. R. Morgan., & Soltesz., I. (2009). Single application of a CB1 receptor antagonist rapidly following head injury prevents long-term hyperexcitability in a rat model. Epilepsy Res, 85(1), 123-7.
4. Lyeth, G. B. (2016). Historical Review of the Fluid-Percussion TBI Model. Front Neurol, 7(217).
5. Q. M. Hameed., S. G. Goodrich., C. S. Dhamne., A. Amandusson., H. T. Hsieh., D. Mou., . . . Rotenberg., A. (2014). A rapid lateral fluid percussion injury rodent model of traumatic brain injury and post-traumatic epilepsy. Neuroreport, 25(7), 532-6.
6. R. Vink., K. T. McIntosh., Weiner.., W. M., & Faden, I. A. (1987). Effects of traumatic brain injury on cerebral high-energy phosphates and pH: a 31P magnetic resonance spectroscopy study. J Cereb Blood Flow Metab, 7(5), 563-71.
7. S. P. Katz., & Molina, E. P. (2018). A Lateral Fluid Percussion Injury Model for Studying Traumatic Brain Injury in Rats. Methods Mol Biol, 1717, 27-36.
8. V. S. Kabadi., D. G. Hilton., A. B. Stoica., N. D. Zapple., & Faden., I. A. (2010). Fluid-percussion-induced traumatic brain injury model in rats. Nat Protoc, 5(9), 1552-63.