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 patholo....

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....

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