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 births 1,2 . 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 presentation 3 ). 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 discrepancies 4,5,6,7,8,9,10,11,12,13,14,15 . 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.
1. Monitor the depth of anesthesia by confirming the absence of canthal reflex and absence of withdrawal response to toe pinch. 2. Perform continuous monitoring of physiological parameters during anesthesia and throughout the experiment, which includes arterial blood pressure, electrocardiography (ECG), end-tidal CO 2 , pulse oximetry, and body temperature. 3. Monitor blood gases and blood sugars every 0.5-1 h and give intravenous fluids (50% dextrose and 50% normal saline) to animals anesthetized longer than 1 h at ~100 cc/kg/day, as needed, to ensure euglycemia. 4. Monitor the animal's anesthetic plane closely and frequently. Provide analgesia and/or increase inhalant anesthesia. 5. Maintain the animal at normal oxygen tension by controlling the ventilator parameters and drug dosages as needed to ensure normoxia, then place the animal on a temperature-regulated circulating water blanket such that normal body temperature is maintained at 39 °C for the duration of the experiment.
1. Place the animal in a supine position on the operating table after proper anesthesia as described in section 3, with the upper limb in abduction, exposing the axillary region. 2. Use any surgical drape to cover the animal. Use clean but non-sterile techniques. 3. Expose the brachial plexus complex on both sides of the spine by making a midline incision (using a #10 blade) over the skin and fascia overlying the trachea, down to the upper third of the sternum, corresponding to spine levels between C3-T3. 4. Extrapolate the incision using the forceps and hemostat horizontally on each side from the suprasternal notch along the edge of the clavicle to the upper arm, while sparing the cephalic and basilic veins. 5. Release the superior and inferior flaps by blunt dissection using scissors and forceps, allowing access to the cervical and thoracic regions of the brachial plexus, respectively. 6. Identify the axis (C2) and first rib at the T1. Using these landmarks, identify the lower three cervical (C6-C8) and first thoracic (T1) spinal vertebral foramen, then examine the plexus carefully to locate bifurcations of the divisions (M shape) to achieve exposure. 7. Label (using nerve loops) the brachial plexus regions above these bifurcations closer to the spine as root/trunk and label those below these bifurcations as chord followed by the nerve, which are located closer to the arm.
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.
Available literature on the biomechanical responses of stretch on the BP tissue exhibit a wide range of threshold values as well as methodological discrepancies . 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 studies 6,24 .
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.