Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.
Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.
Despite recent advances in obstetrics, the problem of NBPP caused by stretch injury to the BP complex continues to be a global health burden, with an incidence of 1.5 cases per 1,000 live births1,2. Associated risk factors can be maternal (i.e., excessive weight, maternal diabetes, uterine abnormalities, history of BP paralysis), fetal (i.e., fetal macrosomia), or birth-related (i.e., shoulder dystocia, prolonged labor, assisted delivery with forceps or vacuum extractors, breech presentation3). While these complications are unavoidable in certain circumstances, prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of the neonatal BP when subjected to stretch.
Reported biomechanical studies on the BP have used adult animals and human cadaveric tissue and show significant discrepancies4,5,6,7,8,9,10,11,12,13,14,15. Clinical relevancy of biomechanical properties of the complex BP tissue warrants a neonatal animal model as well as an in vivo biomechanical testing approach. Furthermore, limitations with studying BP stretch injury in complicated real-world delivery scenarios increases the reliance on computer models that provide methods that allows investigation of the effects of various delivery complications and techniques. The key to clinical relevance of these models is their biofidelity (human-like response). Available computational models by Gonik et al.16 and Grimm et al.17 rely on rabbit and rat nerve tissue but not neonatal BP tissue. Performing in vivo biomechanical testing in a clinically relevant neonatal animal model can fill the critical gap of unavailable neonatal BP data.
The current study describes an in vivo mechanical testing device and procedure to conduct biomechanical testing in 3-5 day-old male Yorkshire neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads during failure. The camera system also allows monitoring of the failure location during rupture. Overall, the system allows for detailed biomechanical characterization of the neonatal BP when subjected to stretch, thereby providing the BP’s threshold strains and stresses for mechanical failure in vivo. The data obtained can further improve human-like behavior (biofidelity) of the existing computational models that are designed to investigate the effects of exogenous and endogenous forces on BP stretch in delivery scenarios associated with NBPP.
Institutional Animal Care and Use Committee at Drexel University approved all procedures (#20704).
1. Animal Arrival and Acclimation
2. Day of Experiment
3. Induction and Maintenance of Anesthesia
4. Monitoring and Care
5. Brachial Plexus Surgery
6. Biomechanical Testing
A representative load-time plot and strains from four segments of BP plexus (between four markers) are shown in Figure 5 and Figure 6, respectively. The obtained failure load of 8.3 N at 35% average failure strain reports the biomechanical responses of neonatal BP when subjected to stretch. Some regions of the nerve undergo higher strains than others, indicative of non-uniform injury along the length of the nerve. The camera data allows reporting the location of failure being proximal to the foramen.
Figure 1: Details of in vivo mechanical testing device including the actuator, load cell, and clamp. Please click here to view a larger version of this figure.
Figure 2: Markers placed over the BP segments to record strains sustained by the tissue during stretch. Please click here to view a larger version of this figure.
Figure 3: Steps for data acquisition using graphical user interface. Please click here to view a larger version of this figure.
Figure 4: Marker tracking and strain analysis details. Test videos saved in AVI format are imported in the tracking software. Strain between each marker and the first and last markers are obtained as detailed. An average of between markers strains is used to report the failure strains. An example of nerve stretch with three markers and the calculated average strain-time plot are shown here, with reported failure strains of 43%. Please click here to view a larger version of this figure.
Figure 5: Maximum load reported during failure. Load cell attached to the actuator acquires the load data during stretch. The data are used to obtain a load-time plot as shown. Please click here to view a larger version of this figure.
Figure 6: Strains reported in four different segments of the stretched plexus. Strains are calculated between each marker and compared against average strains obtained from all four segments (between each of the two adjacent markers). Some regions of the nerve undergo higher strains than others and the average strains indicative of non-uniform injury along the length of the nerve. Please click here to view a larger version of this figure.
Available literature on the biomechanical responses of stretch on the BP tissue exhibit a wide range of threshold values as well as methodological discrepancies4,6,8,18,19,20,21,22,23. Variations in published results could be due to differences in the tissue processing (e.g., fixed vs. unfixed tissue), methodological differences in measuring elongation, and differences in species used. Moreover, these data are obtained from adult animals or human cadavers and not neonates. Ethical reasons make it difficult to obtain mechanical data from live human neonates, so large animal models that have anatomical similarities to humans may be used instead. Piglets serve as an animal model that has already been used in BP-related studies6,24.
The proposed methods and set-up allow for measuring the in vivo biomechanical response of neonatal BP in a large animal model, offering an understanding of injury mechanism during BP stretch. While the testing protocol and set-up is robust, it offers some limitations (i.e., slips occurring during mechanical testing, loss of marker visibility during testing, movement of the entire body when testing until failure occurs). While slips occur during testing, ensuring proper clamping can minimize slippage. Adding padding can further secure the tissue and avoid slips. Clamps can also be easily substituted with other different types of clamps as needed. Loss of marker visibility occurs in less than 2% cases and are inevitable. Securing the animal torso while testing may require a securing rig. Since the set-up allows tracking of the insertion movement through a camera system, it accounts for any animal movements during testing. An additional limitation of the system is its ability to provide a camera view live through a separate program, thereby limiting live camera view during testing. This can be improved in the future by integrating a live camera view into the program that is currently used to run the test.
In summary, NBPP is a significant injury with life-long sequelae for many individuals. Unfortunately, over the last three decades there has not been a decrease in the rate of its occurrence, despite increased technological development and training of obstetricians. This lack of a decrease in occurrence may directly be attributed to the limitations in developing preventative strategies that minimize the occurrence of NBPP. Preventative strategies cannot be explored until a detailed understanding of the injury mechanism at all levels (i.e., mechanical, functional, and histological) becomes available. No method to date has been reported to measure in vivo BP strains in a neonatal large animal model, and the current study is the first to offer a protocol that further explores physiological and functional changes in neonatal BP tissue post-stretch. By performing tests at various strains, injury threshold values for functional and structural injuries in the neonatal brachial plexus can be reported.
The authors have nothing to disclose.
Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number R15HD093024 and by the National Science Foundation CAREER Award Number 1752513.
Omega Subminature Tension & Compression Load Cell | Omega | LCM201-200N | 200N load cell |
Basler acA640-120uc camera | Basler | acA640-120uc | |
Feedback Linear Actuator | Progressive Automations | PA-14P | 10" stroke, 150lb force, 15mm/s speed |
Motion Tracking Software | Kinovea | N/A | Open Source |
Proramming Software – MATLAB | Mathworks | N/A | version 2018A |
Surgical instruments | |||
Forceps | Fine Science Tools Inc | 11006-12 and 11027-12 or 11506-12 | |
Hemostats | Fine Science Tools Inc | 13009-12 | |
Scissors | Fine Science Tools Inc | 14094-11 or 14060-09 |