Summary

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

Published: July 28, 2022
doi:

Summary

The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.

Abstract

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

Introduction

Orthopedic trauma accounts for 25% of all injuries sustained by nearly 500 million people each year worldwide1,2,3. Orthopedic trauma can be associated with complex muscle, bone, nerve, and connective tissue damage, necessitating hospitalization and surgery to ensure adequate recovery3,4. Acute and chronic pain after orthopedic trauma can result in significant physical, psychological, and financial burdens that affect a patient's quality of life1,4. Additionally, orthopedic surgery to stabilize and fix fractures is also associated with severe acute and chronic post-surgical pain5,6,7,8,9.

The mechanisms underlying acute and chronic trauma-related pain need to be better understood to develop better treatments. To achieve this, reliable, reproducible, and clinically relevant preclinical models are required. Since most animal models of orthopedic trauma did not involve simultaneous injury to multiple tissue types (bone, muscle, nerves), they were not truly representative of human complex orthopedic trauma, for example, trauma after falls, motor vehicle crashes, or war-related injuries10,11. Therefore, we developed the tibial fracture-pin mouse model to examine the major manifestations of such injury, including bone and muscle tissue damage and acute and chronic pain11. The tibial fracture-pin model consists of a unilateral open tibial fracture with IMN internal fixation and simultaneous TA muscle injury. Histological sections of the TA show injury to the muscle in which dense fibrosis develops with associated loss of large, mature muscle fibers as early as 2 weeks post injury. Moreover, the fracture callus is apparent on microcomputer tomography (microCT) 4 weeks post injury and continues to undergo remodeling11.

Various reflexive and nonreflexive behavior assays can be used to evaluate the sensory and affective components of pain in the tibial fracture-pin model. For example, one can use the Von Frey filaments to demonstrate mechanical hypersensitivity in this model. In fact, mice develop long-lasting mechanical hypersensitivity in the ipsilateral hind paw after tibial fracture-pin surgery11. Another particularly useful behavioral paradigm is the hot plate assay, which traditionally measures the latency to paw withdrawal to a thermal stimulus. While this assay has been used for decades12, truly a gold standard in preclinical pain research, measuring reflexive behavior alone has its limitations13. As a result, we have developed a modified hot plate paradigm that can capture elements of both reflexive and nonreflexive responses in the setting of a thermal stimulus14.

This modified hot plate assay determines the initial response latency as in the original hot plate test and an extended observation period to record additional nocifensive behaviors. By categorizing these extended behaviors into distinct categories (flinching, licking, guarding, jumping), the nonreflexive response to the thermal stimulus can be captured. Flinching is the rapid removal of the paw and/or splaying of digits, but the limb is quickly returned to the hot plate. Licking and biting of the hind and front paws are both defined as licking for analysis. Guarding is the continued raising of the limb beyond when afferent nociceptive information ends. Finally, jumping is the removal of all four limbs from the hot plate surface. These behaviors can be analyzed individually and grouped together with special care to still note the initial response latency.

Protocol

All methods used while conducting this research were performed in compliance and with approval by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC #33114) in accordance with American Veterinary Medical Association guidelines and the International Association for the Study of Pain. Mice (C57BL/6J, 9-11 weeks old upon arrival, 11-12 weeks old at study initiation) were housed 2-5 per cage and maintained on a 12 h light/dark cycle in a temperature-controlled environment with ad libitum a…

Representative Results

The tibial fracture-pin orthotrauma model reproduces the bone, muscle, and pain-like behaviors seen in complex human injury. As shown in Figure 1C, the tibial fracture heals over time, forming a callus at the fracture site that is still seen at 4 weeks post injury. As a result of the lateral approach with the bone saw described above (step 3.5), the tibialis anterior muscle is injured, becoming extensively fibrotic, as seen by increased collagen deposition throughout the tissue (<strong clas…

Discussion

Critical steps within the protocol
It is crucial to maintain sterile conditions throughout the surgery. Moreover, proper animal care before, during, and after the surgery is paramount for the successful generation of the model. As mentioned earlier in the protocol, when performing the surgery, fracture the bone from the lateral side to ensure muscle injury. Take care not to fracture the tibia too low (below the advised junction between the middle and distal thirds of the tibia) because this will af…

Disclosures

The authors have nothing to disclose.

Acknowledgements

GM is supported by an NDSEG Graduate Fellowship and a Stanford Bio-X Honorary Graduate Fellowship. VLT is supported by NIH NIGMS grant #GM137906 and the Rita Allen Foundation.

Materials

27 G needles Medsitis 305136 https://medsitis.com/products/bd-precisionglide-27-g-x-1-1-4-hypodermic-needles-305136?variant=39724583299
5-0 suture esuture SN5668 https://www.esutures.com/product/0-in-date/2-/132-/16552-medtronic-monosof-black-18-p-11-cutting-SN5668/
Alcohol swabs Amazon B00VS4F91W https://www.amazon.com/Dynarex-Alcohol-Prep-Sterile-Medium/dp/B00VS4F91W
Alternative drill bits Rio Grande 341602 https://www.riogrande.com/product/BuschTungstenVanadiumRoundBur
Set0314mm/341602
Bone saw drill attachment Amazon B07DSXR3NY https://www.amazon.com/dp/B07DSXR3NY
Buprenorphine Fidelis Pharmaceuticals https://ethiqaxr.com/ordering/
Ceramic implant (alternative to pin) RISystem RIS.221.103 https://risystem.com/platefixation/mousescrew
Chux (Absorbent Underpad) Fisher Scientific NC0059881 https://www.fishersci.com/shop/products/underpad-17×24-chux-300-cs/nc0059881#?keyword=true
C57BL/6J mice The Jackson Laboratory Jax #00664 https://www.jax.org/strain/000664
Cotton swabs Uline S-18991 https://www.uline.com/Product/Detail/S-18991/First-Aid/Cotton-Tipped-Applicators-Industrial-6
Cutting pliers Amazon B076XYVS6Y https://www.amazon.com/iExcell-Diagonal-Cutting-Nippers-Chrome-Vanadium/dp/B076XYVS6Y
Drill Chewy 129044 https://www.chewy.com/dremel-cordless-dog-cat-rotary-nail/dp/156127
Drill bits Amazon B00HVIGSX2 https://www.amazon.com/Universal-Diamond-Dremel-Rotary-Tool/dp/B00HVIGSX2
Electric shaver Kent Scientific CL9990-KIT https://www.kentscientific.com/products/trimmer-combo-kit/
Eye lube Amazon B07H2NLCX5 https://www.amazon.com/OptixCare-Lube-Plus-Hyaluron-Horses/dp/B07H2NLCX5
Gauze pads 2" x 2" Amazon B07GHDTB53 https://www.amazon.com/Covidien-Curity-Sterile-Peel-Back-Package/dp/B07GHDTB53
Gauze pads 4" x 4" Amazon B00KJ6YFTC https://www.amazon.com/Covidien-6309-Curity-Gauze-Pads/dp/B00KJ6YFTC
High definition video camera The Imaging Source DFK 22AUC03 https://www.theimagingsource.com/products/industrial-cameras/usb-2.0-color/dfk22auc03/?adsdyn&gclid=Cj0KCQiA3-yQBhD3ARIsAHuHT64uIIlImBvh_
toh-3GFSgBcL_fRc1gQTDyXlqDEa
Qu4n2_VbWEiRuIaAiueEALw_wcB
Inhalational anesthesia system Kent Scientific https://www.kentscientific.com/products/vaporizer-with-vetflo-single-channel-anesthesia-stand/
Iodine solution Amazon B005FR7XIK https://www.amazon.com/Dynarex-Povidone-Iodine-Scrub-Solution/dp/B005FR7XIK
Iodine swab sticks Amazon B001V9QKMG https://www.amazon.com/POVIDONE-IODINE-SWAB-1202-25Box/dp/B001V9QKMG
Isoflurane California pet pharmacy https://www.californiapetpharmacy.com/fluriso-isoflurane-250ml.html
NCH Prism Software https://www.nchsoftware.com/prism/index.html
Plastic Cylinder Amazon B08R5KM5B6 https://www.amazon.com/FixtureDisplays-Acrylic-Diameter-Thickness-15140-8-NPF/dp/B08R5KM5B6
Saline Fisher Scientific NC9054335 https://www.fishersci.com/shop/products/saline-injection-0-9-10ml/NC9054335
Scalpel Fisher Scientific 12-000-162 https://www.fishersci.com/shop/products/high-precision-10-style-scalpel-blade/12000162#?keyword=
Scalpel handle Amazon B0056ZX1R8 https://www.amazon.com/Swann-Morton-Scalpel-Handle-blades/dp/B0056ZX1R8
Thermal place preference apparatus BIOSEB BIO-T2CT https://www.bioseb.com/en/pain-thermal-allodynia-hyperalgesia/897-thermal-place-preference-2-temperatures-choice-nociception-test.html

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Cite This Article
Muwanga, G. P. B., Siliezar-Doyle, J., Ortiz, A. A., Kaslow, J., Haight, E. S., Tawfik, V. L. The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury. J. Vis. Exp. (185), e63590, doi:10.3791/63590 (2022).

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