Development of a Neonatal Rat Model for Brachial Plexus Birth Injury

Summary

This protocol presents a step-by-step instruction to create a neonatal brachial plexus injury in rats to be used as a model in brachial plexus birth injury research.

Abstract

Studying brachial plexus birth injuries (BPBI) has been challenging due to heterogeneous clinical presentations and limited reliable animal models. To that end, we have developed a surgical protocol that reproducibly induces a pan-plexal neonatal brachial plexus injury in 5-day-old Sprague-Dawley rat pups. In this article, step-by-step instructions are provided for approaching the brachial plexus in a rat pup. The technique was performed on 15 Sprague Dawley rat pups. Using a dorsal approach under operative microscopy, a 5 mm dorsal incision was made starting at the base of the skull towards the level of C7-T1 vertebral transition. Blunt dissection is carried down to the trapezius muscle, followed by dissection through the underlying rhomboid and levator scapulae muscles. After identifying anatomical landmarks such as the spine and the scapula, the superior angle of the scapula was gently retracted to visualize the brachial plexus. The brachial plexus was localized and cauterized, evidenced by observable twitches in the upper extremity. To prevent nerve regeneration, a small block of an absorbable gelatin sponge was placed between the proximal and distal ends of the injured nerves. This procedure was successfully performed on all 15 rat pups with 87% survival. Postoperative and at sacrifice, all surviving rats demonstrated complete pan-plexal injuries, validating this model as effective and reproducible for research in brachial plexus birth injuries.

Introduction

Brachial plexus birth injury (BPBI) is a traumatic nerve injury that occurs during delivery. Traction forces during a complicated delivery, often due to shoulder dystocia, prolonged labor, or fetal macrosomia, result in weakness or paralysis in the affected arm¹. The incidence ranges from 1-2 per 1,000 live births^2,3,4. While most babies recover full function, 30% have residual symptoms as they grow into adulthood, such as functional limitations, limb length inequalities, deformities, and joint contractures^5,6,7,8.

The BPBI model demonstrates different injury patterns with implications for treatment and prognosis. Upper plexus injuries (C5-C6, Erb palsy) are the most common and predominantly involve rupture rather than avulsions⁹. In contrast, pan-plexal injuries (C5-T1) frequently demonstrate a mixed pattern of cranial root ruptures and caudal avulsions¹⁰. Avulsions are preganglionic injuries that disrupt the dorsal root ganglion with no capacity for spontaneous recovery¹¹.

Current clinical management strategies range from conservative physical therapy to early microsurgical nerve reconstruction, depending on the severity of the injury^12,13. Patients often need secondary surgeries, including soft-tissue releases, tendon transfers, and osteotomies, to further improve motor function, quality of life, and secondary musculoskeletal sequelae. However, the effectiveness of these early and late interventions varies^14,15,16. Moreover, the altered musculoskeletal development contributes to functional limitations, limb length inequalities, and other deformities, not currently amenable to treatment, highlighting the need for further research^17,18. Therefore, there remains a critical need for a reliable, feasible, and reproducible animal model to investigate these injuries.

Rodent models are commonly used in brachial plexus injury models, with mice the most common choice due to their cost-effectiveness and ease of handling. However, creating a reproducible neonatal brachial plexus injury can be anatomically challenging due to their small size. Large animal models such as rabbits, pigs, and primates offer several advantages for model creation, but at a significantly higher cost¹⁹. Rats, on the other hand, are larger and more genetically similar to humans than mice, while remaining substantially more cost-effective and accessible than larger animal models. Therefore, there remains a need for a feasible and reproducible animal to investigate BPBI. This study describes a step-by-step pan-plexal BPBI creation model in neonatal rats that can be used to study injury mechanisms, treatment interventions, and short- and long-term sequelae.

Protocol

All animal procedures were performed with approval of the Institutional Animal Care and Use Committee (IACUC A0007335). The technique was performed in 15 Sprague-Dawley rat pups to assess feasibility and long-term functional outcomes.

1. Animal husbandry

1. House a multiparous pregnant Sprague Dawley rat (gestational age: 14 days) in a breeder box (1354 cm²) containing cellulose paper-based bedding, enriched with nesting material and cage enrichments; maintain room temperature at 21 °C with relative humidity at 28% and a 12:12 h light/dark cycle. Provide rat chow and water ad libitum.
2. Clean cages weekly; leave the cage undisturbed for 48 h after delivery of the pups. To promote habituation and reduce stress, provide auditory reassurance through gentle handling and speech. During late gestation, ensure the dam is habituated to the investigator through daily gentle handling and olfactory conditioning by placing ethanol and betadine-scented cotton balls into the dam's cage.
3. After delivery of the pups, place treats in the cage without disturbing the dam for 48 h or opening the cage.

2. Material preparation

1. Ensure all required surgical instruments are prepared before the surgery (see the Table of Materials). Sterilize all surgical supplies using a steam autoclave at 121 °C for 1 h.
2. Create a surgical bed out of expanded polystyrene. (Figure 1)

3. Surgical preparation

1. Perform surgeries on rat pups at an age of 5 days old [range between 3 and 9 days].
2. Induce anesthesia by placing the rat in an induction chamber with 2% isoflurane in 2 L/min oxygen until the medium-to-deep anesthetic stage is achieved. Confirm adequate anesthesia with a gentle toe pinch using forceps. Place the rat pup on a prewarmed heating pad. Maintain anesthesia on 1.5-2% isoflurane in oxygen and monitor respiratory rate continuously.
3. Determine the appropriate dosage of analgesia based on the pup's weight. Administer buprenorphine extended release (1 mg/kg s.c.) to manage pain.
4. Prepare an aseptic surgical site by applying three alternating scrubs of betadine and ethanol.
5. Position the anesthetized pup in a prone position on the premade surgical bed with the nose and mouth placed securely in the isoflurane vaporizer nose cone (Figure 1B).
6. To minimize movement during the procedure, tape the head of the rat pup onto the isoflurane vaporizer and cover the lower part of its body, just below the upper extremities, withfoil.

4. Surgical approach to the brachial plexus

1. Create a 5 mm skin incision using a No. 11 blade, beginning at the base of the skull and extending caudally to the C7-T1 vertebral transition (Figure 1C).
2. Carefully cauterize superficial arteries while avoiding skin edges.
3. Perform blunt dissection until the trapezius muscle is reached.
4. Dissect through the trapezius and underlying rhomboid and levator scapulae muscles using cauterization. Place the cautery forceps perpendicular to the tissue to avoid thermal injury to the skin edges.
5. Gently retract the superior angle of the scapula to expose the brachial plexus. Identify and isolate the brachial plexus via blunt dissection (Figure 1D and Figure 2A).
6. Cauterize the identified nerves of the brachial plexus using a bipolar electrocautery set at 15, and confirm successful injury by observing upper extremity muscle twitches.
7. Place a small piece of an absorbable gelatin sponge (approximately 2 mm by 2 mm), between the proximal and distal segments of the brachial plexus to prevent nerve regeneration (Figure 2B).
8. Close the skin using a 7-0 absorbable suture in a continuous suture pattern and apply a veterinary tissue adhesive to prevent wound dehiscence (Figure 3).

5. Postoperative management

1. Place the rat pup on a heating pad with nesting material from the rat dam's cage (Figure 4). Monitor the respiratory rate and movements of the rat pup until it returns to normal respiratory patterns.
2. To prevent maternal rejection, rub the rat pup gently with the home-cage nesting material before placing it back with the dam.
3. Inspect rat pup 3 times a day for the first 48 h post surgery. If signs of pain or absence of a milk spot are observed, administer supplemental analgesia (buprenorphine 0.05 mg/kg s.c.).
4. Closely monitor for postoperative complications, including wound dehiscence, wound infection, reduced limb sensation with potential automutilation, and maternal cannibalism.
5. Weigh the rat pup daily. If the rat pup exhibits weight loss or no weight gain for 2 consecutive days, consider it a failure to thrive and proceed with euthanasia by an injection of pentobarbital (390 mg/mL) intraperitoneal.
6. Assess motor function 1 day post surgery and follow up motor function weekly using a limb movement scale: 0 = absent function, 1 = partial function, 2 = normal symmetric function.

Results

The effectiveness of the surgical technique can be evaluated by the absence of movement in the affected limb, the presence of contractures, reduced range of motion, and, upon sacrifice, the degree of muscle atrophy, limb shortening, and morphological bone and muscle differences compared to the unaffected limb. A total of 15 rat pups were included in this study. During the survival period, two of the rat pups failed to thrive postoperatively due to poor weight gain. Self-mutilation was observed in 2 out of the 13 survivin...

Discussion

This protocol details the development of a brachial plexus injury in a neonatal rat to conduct research on the short-term and long-term outcomes of BPBI, explore treatment options and understand sequela.

The use of a noninvasive rodent model to study brachial plexus injury has been previously described; however, these models have not been specifically adapted to replicate a specific type of nerve injury^21,22. By focusing on postnatal d...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL Syringe Luer-Lok Tip	BD	309628
7-0 Coated Vicryl (Polyglactin 910)	Ethicon	J576G
70% isopropylalcohol	Vet-One	13985-598-01
Absorable gelatin sponge	Surgifoam	ETH1972
Betadine Solution 5% Povidone-Iodine Antiseptic Microbicide	PBS Animal Health	11205
Nesting material	Bed 'r Nest	BRN8SR
Bipolar Cords	ASSI	ASSI.ATK26426
Buprenorphine (Base 0.5 mg/mL) Extended Release	Wedgewood connect	49426-0517-1
Cage enrichments	Twist 'n Rich	10003949
Cellulose based bedding	Alpha Dri	NC2193839
Expanded polystyrene (Styrofoam)	Fisher	22-130-407
FREER skin hook	Freer	MI2152
Gillies Skin Hook Semi-sharp	DTR	GHB2001
Isoflurane Auto-Flow Anesthesia Machine	E-Z Systems	EZ-190F
Isoflurane, USP	Patterson Veterinary	1403-704-06
Jewelers Bipolar Forceps Non-Stick, 11 cm, straight pointed tip, 0.25 mm, tip diameter	ASSI	ASSI.BPNS11223
Micro Scissors Bayonet Style	ASSI	ASSI.SNS21
Microscope (surgical)	Leica	M525 F40
Microsurgical Dumont #4 forceps Dumont Swiss made 1708-4TM-PO	Ambler Surgical	31-906
Microsurgical needle holder	ASSI	B-14-8
Nose Cone, 9mm, Mouse, length 2cm	VetEquip	921609
Pentobarbital-euthanasia vial (390 mg/mL) 250 mL	Fatal plus	9373
Plastic food wrap foil	Press 'n Seal	Press'n-70-1-no3
Single-use Needles, 30 G	BD	14-821-13A
Small Animal Heated Operating table (adjustable)	Peco Services Ltd	69023
Stainless Steel Blade No. 11	Bard Parker	371211
Sterile towel drape	Dynarex Corporation	4410
Tissue adhesive	Vetbond 3M	NC9259532
Wood Handled Cotton Swabs and Applicators	Fisher	22-363-157