Fixation of Ultrasound Transducers for Spinal Cord Neuromodulation in Mice

Summary

This protocol describes an ultrasound transducer fixation system specifically designed for ultrasound stimulation of the spinal cord in awake mice. The method employs a fixation system to ensure accurate positioning of the ultrasound transducer while simultaneously allowing the experimental subjects to move freely without compromising the precision of stimulation.

Abstract

Focused ultrasound (FUS) has emerged as an innovative non-invasive neuromodulation technology demonstrating significant potential for spinal cord neural regulation. However, achieving stable transducer fixation along the spinal column presents notable technical challenges. Existing immobilization methods often require prolonged animal anesthesia or demonstrate inadequate adhesion reliability. These critical constraints underscore the urgent need for developing a novel fixation paradigm that ensures both operational stability and physiological compatibility. This protocol introduces an innovative ultrasound transducer fixation system for stimulation of the spinal cord in awake, freely moving mice. Establishing a reliable method for precise ultrasound transducer placement on the vertebral column is key for neurostimulation experiments. This method integrates three phases: surgical exposure of the T13 vertebral body, precision grinding of the spinous process to create a planar contact interface, and secure adhesive bonding of the metal base and collimator to the vertebral column using denture base material and adhesive mixture. It allows natural motor behaviors through unrestricted movement and ensures stable positioning with minimal spatial variation between sessions, facilitating investigation of ultrasound-mediated spinal cord interventions in conscious animal models. It is particularly relevant to studies requiring behavioral assessments in spinal injury repair, blood-spinal cord barrier modulation, and chronic pain mechanisms. By addressing a critical technical challenge in awake-animal neuromodulation research, this protocol offers a stable transducer-spinal interface without movement restriction, paving the way for more accurate and reliable experimental outcomes.

Introduction

The spinal cord is a crucial hub for integrating information between the central and peripheral nervous systems, making it valuable for clinical research in areas such as pain signal transduction, motor neural network reconstruction, and neuroplasticity regulation. Focused ultrasound (FUS), an emerging non-invasive neuromodulation technology, has become an important means of spinal cord neural regulation. With its physical characteristics of deep tissue penetration, millimeter-level spatial resolution, and controllable energy output parameters, its application extends from basic neuro-mechanism research to preclinical translation studies of various spinal cord pathological models.

Research shows that FUS can regulate spinal cord neural circuits in two ways: either by inhibiting or by activating them through thermal and mechanical effects. It is also characterized by its temporal reversibility and safety for tissues^1,2,3. In the study of spinal cord injury models, FUS has improved the motor ability of rats by modulating inflammatory responses and promoting neural repair^4,5,6. Furthermore, FUS has been shown to open the blood-spinal cord barrier and enhance local drug delivery efficiency through a microbubble-mediated cavitation effect^7,8. In the field of neuropathic pain, Liao et al. have confirmed that FUS effectively alleviates neuropathic pain manifestations in rats by regulating KCC2 expression and inhibiting the CaMKIV KCC2 pathway⁹. Song et al. demonstrated that low-intensity tsFUS applied at the lumbar spinal cord significantly reduced mechanical allodynia and suppressed microglial activation in a rat chronic constriction injury model¹⁰. Achieving precise spinal cord modulation with FUS depends on the spatial stability of the ultrasound energy delivery system. The current fixation strategies are mainly divided into two categories: 1) Rigid fixation method: It achieves precise positioning by using a stereotactic frame, but requires continuous anesthesia, which suppresses the neural activity of the experimental subjects and affects the validity of behavioral assessments^1,2,3,4,10; 2) Flexible fixation method: It allows for research on conscious subjects by using an elastic belt device, but due to the large mobility of the spine, there are displacement errors and transducer detachment^6,8,9. In addition, the geometric heterogeneity of the vertebral column's lamina leads to a significant attenuation of ultrasound energy, reducing the efficiency of neural modulation^7,8.

This study innovatively proposes an implantable focused ultrasound transducer fixation scheme for spinal cord neural modulation research in conscious, freely moving mice. This protocol uses a minimally invasive surgical approach to integrate the metal base, collimator, and vertebral column, supports the repeated wearing of the ultrasound transducer, ensures the consistency of the ultrasound target, and keeps the ultrasound energy attenuation through the lamina within an acceptable range. The developed methodology addresses the critical limitations of existing approaches, thereby enabling more reliable and reproducible spinal cord neuromodulation experiments in awake, behaving animals.Key parameters include a 2.5 cm midline incision at T13, stainless steel metal base fixation, and a 3:1 volume ratio of denture base material to cyanoacrylate adhesive. Limitations include moderate surgical invasiveness and operator-dependent precision in bone surface preparation.

Protocol

All animal procedures described in the present study were approved by the Institutional Ethical Committee of Animal Experimentation of Shenzhen Institutes of Advanced Technology (certificate number: SIAT-IACUC-200721-YGS-NLL-A1313). All experiments used male C57BL/6J mice (8 weeks old). Mice weighed 18-22 g at the time of surgery.

1. Anesthesia induction and maintenance (5 ± 1 min)

1. Pre-charge the anesthesia circuit for 5 min.
2. Set the induction chamber to deliver 4% isoflurane in medical air at a flow rate of 2 L/min. Administer carprofen (5 mg/kg, SC) as preoperative analgesia.
3. Switch to facemask ventilation after loss of the righting reflex.
4. Adjust the vaporizer to maintain a concentration of 1.5-2% isoflurane.
5. Maintain mouse body temperature using a warming blanketset to 37 °C, with a target range of 36-38 °C throughout anesthesia.
6. Apply Terramycin ophthalmic ointment.

2. Positioning (1 ± 1 min)

1. Use an adjustable stereotactic adapter for the mouse.
2. Prone position the mouse with spinal alignment to maintain spinal physiological curvature.
3. Secure the head of the mouse using ear bars.
4. Position limbs symmetrically.

3. Surgical field preparation (1 ± 1 min)

1. Use an electric clipper (blade gap 0.1 mm) to remove hair from the surgical site.
2. Ensure that the depilation area is approximately 3 cm × 1 cm.
3. Perform three passes of clipping in the opposite direction of hair growth.

4. Incision and approach (2 ± 1 min)

1. Disinfect the surgical area with iodophor 3 times (30 s intervals) followed by alcohol each time. Confirm the depth of anesthesia via a toe-pinch response.
2. Palpate the last rib (attached to T13 vertebra in mice) and use it as
   the primary landmark. Create a 2.5 cm incision at the T13 vertebral level along
   the dorsal midline using a scalpel.
3. Sequentially dissect the subcutaneous fascia.
4. Expose the paraspinal muscles through the anatomical planes.

5. Paraspinal muscle dissection (5 ± 1 min)

1. Set the surgical microscope magnification to 5x for optimal visualization of paraspinal muscles and vertebral structures.
2. Perform sharp dissection of paraspinal muscles using micro-scissors.
3. Resect dorsal and lateral paraspinal muscles completely while preserving the ventral muscle layers.
4. Manage bleeding with absorbable hemostatic gauze.

6. Metal base installation (1 ± 1 min)

1. Mount the fixation rod (plastic) to the stereotactic adapter.
2. Mount the custom metal base (stainless steel, 0.28 g) at the rod's distal end (Figure 1A).
3. Position the custom metal base bilaterally at the T13 spinal transverse processes.
4. Verify coupling stability.

7. Bone interface preparation (2 ± 1 min)

1. Remove residual muscle tissue from the lamina and spinous processes under the surgical microscope.
2. Achieve complete hemostasis with sterile cotton pledgets compression.
3. Create a planar contact surface using a precision bone drill with continuous saline irrigation. Operate the drill at a speed of 5,000-10,000 rpm to minimize heat generation. Wear an eye shield and a mask during drilling.
4. Remove bone dust with saline-moistened cotton swabs.

8. Fixation system sealing (3 ± 1 min)

1. Mix denture base materials and cyanoacrylate adhesive (502 glue) at a 3:1 volume ratio. Wear nitrile gloves and safety goggles to prevent skin/eye contact.
2. Attach the ultrasonic collimator (plastic, 0.6 g, Figure 1B) and metal base to the vertebral column using the adhesive mixture (Figure 1C).
3. Allow the adhesive mixture to cure under controlled ambient conditions (24 ± 1 °C, 40-60% relative humidity). Maintain static positioning for 60 s until visual confirmation of complete solidification (loss of surface gloss and formation of a rigid interface).
4. Separate the metal base from the plastic fixation rod.
5. Fixation stability verification: Apply a gentle rotational force to the metal base in the horizontal plane. Observe the collimator-vertebra interface under 5x magnification for any displacement (<0.1 mm tolerance). If movement exceeds tolerance, reapply adhesive mixture (step 8.2) and repeat curing (step 8.3).

9. Wound closure (1 ± 1 min)

1. Align wound edges to fully encapsulate the metal base.
2. Perform interrupted suturing with 4-0 nylon sutures.
3. Wipe the incision with iodophor.
4. Clear residual blood/bone debris within the collimator's laminar exposure site and pack with sterile cotton for sealing and hemostasis.

10. Postoperative management

1. Thermoregulation: Maintain the mouse on a warming blanket (37°C) until full recovery of ambulation (typically 2-3 h post-anesthesia).
2. Postoperative antibiotic prophylaxis: Administer cefazolin sodium (50 mg/kg, dissolved in 0.9% saline to a concentration of 50 mg/mL) via subcutaneous injection into the dorsal neck region immediately after surgery.
3. Maintain wound hygiene. Clean the wound using a saline-moistened cotton swab once daily for the first 3 days and perform daily inspection of the surgical site for signs of infection, dehiscence, or inflammation.
4. Postoperative analgesia: Administer carprofen (5 mg/kg, subcutaneously) every 12 h for 3 days post-surgery¹¹.
   NOTE: Figure 1D shows the fixation system implanted in a live mouse post-recovery.

11. Hazardous waste disposal

1. Collect bone dust and blood-contaminated materials in leak-proof biohazard bags labeled with "Biohazard" symbols for transfer to a licensed medical waste disposal facility.
2. Place uncured denture base material and 502 glue residues in sealed chemical-resistant containers labeled "Chemical Waste", and store them in a designated ventilated cabinet for disposal via chemical waste management protocols.
3. Discard needles, scalpel blades, and bone drills in puncture-resistant sharps containers marked with "Biohazard" labels.

12. Safety warnings

1. When handling denture-base materials, avoid direct skin contact by wearing nitrile gloves and safety goggles, and perform mixing and curing in a well-ventilated fume hood to minimize inhalation of volatile monomers.
2. Dispose of residual material as chemical waste per institutional guidelines. For 502 Cyanoacrylate adhesives, prevent skin or eye contact and rinse immediately with water for 15 min if exposure occurs, seeking medical help. Use them in ventilated areas, store them in cool, dry places away from ignition sources, and avoid contact with cotton or wool fabrics.
3. When using precision drills, secure the handpiece, wear a face shield and surgical mask, and irrigate the drilling site with saline to prevent thermal injury.

Results

Open field test
The open field test was designed to evaluate potential behavioral changes in mice following surgery (Figure 2A). A standard open field chamber of 40 cm × 40 cm was employed to ensure sufficient activity space for the mice while maintaining relatively consistent environmental conditions and minimizing external interference. The experiment consisted of two phases: pre-surgery and 7 days post-surgery. In each phase, a 10-min video recording was made to docum...

Discussion

This study presents a novel vertebral coupling protocol that facilitates precise ultrasound neuromodulation of the spinal cord in awake, freely behaving mice. Unlike conventional fixation approaches, this methodology overcomes the limitations of conventional fixation approaches. Unlike rigid stereotactic frames needing continuous anesthesia (which suppresses neural activity and complicates behavioral assessments¹⁰) and elastic belts prone to displacement errors from spinal movement

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anesthesia Induction Chamber	Shenzhen RWD	https://www.rwdls.com/product-solutions/life-sciences/modeling/anesthesia-parts
Cyanoacrylate Adhesive (502 Glue)	Beijing Chemical Works	https://item.taobao.com/item.htm? detail_redpacket_pop=true&id=736 869038961<k2=1752156592779 mdbp47qqp4vyv57mq156e&ns=1& priceTId=214780251752156543206 3926e1996&query=502%E8%83% B6%E6%B0%B4%20%E5%8C%97 %E4%BA%AC%E5%8C%96%E5% B7%A5%E5%8E%82&skuId=52584 34238219
Denture base materials	New Century Dental	https://www.sncdental.com/product-item-11.html
Fixation rod			Custom
Isoflurane	Shenzhen RWD	https://www.rwdls.com/product-solutions/life-sciences/modeling/anesthesia-parts
Metal base			Custom
Microinstrument	Shenzhen Zongshi Scientific Instruments Co., Ltd.
Precision drill	Shenzhen RWD	https://www.rwdls.com/product-solutions/life-sciences/modeling/auxiliary-equipment
Small Animal Multi-Channel Anesthesia System	Shenzhen RWD	https://www.rwdls.com/product-solutions/life-sciences/modeling/anesthesia/data_66.html
Stereotactic Adapter for Mice and Juvenile Rats	Changsha Mayue	https://item.taobao.com/item.htm?abbucket=14&detail_redpacket_pop =true&id=651346369142<k2=1752 157416717wlsrozz5uroehtbq5ycim4 &ns=1&priceTId=214781ce1752157 3520498589e101a&query=%E8%84 %91%E7%AB%8B%E4%BD%93%E5%AE% 9A%E4%BD%8D%E4%BB%AA%20% E9%95%BF%E6%B2%99%E8%BF% 88%E8%B6%8A&skuId=4891677573 207&spm=a21n57.1.hoverItem.14& utparam=%7B%22aplus_abtest% 22%3A%22a478799868a3178d0dd 433b763909e16%22%7D&xxc= taobaoSearch
Surgical microscope	Shenzhen Zongshi Scientific Instruments Co., Ltd.	https://www.rwdls.com/product-solutions/life-sciences/modeling/auxiliary-equipment
Surgical suture (Prolene)	Ethicon (Johnson & Johnson)	https://item.taobao.com/item.htm?abbucket=14&detail_redpacket_pop =true&id=774292384846<k2= 1752157506907c0321finubb5 rpkub6av6n&ns=1&priceTId= 214781ce1752157493734800 4e101a&query=%E5%BC%B A%E7%94%9F%E7%BC%9D %E7%BA%BF&skuId=594224 1331046&spm=a21n57.1.hoverI
Terramycin Ophthalmic Ointment	Pfizer	https://theterramycin.com/terramycin-ophthalmic-ointment/
Ultrasonic collimator			Custom