A simplified and reproducible protocol for percutaneous lumbar intrathecal injection in rats

Intrathecal injection is widely used in preclinical research to deliver compounds directly into the cerebrospinal fluid, bypassing the blood-brain barrier. However, current percutaneous lumbar puncture protocols vary considerably in equipment and procedural detail, limiting reproducibility across laboratories.

We present a standardized, step-by-step protocol for percutaneous lumbar intrathecal injection in isoflurane-anesthetized rats using a single-use insulin syringe with a permanently attached fine-gauge needle. The protocol includes animal positioning with a custom 3D-printed scaffold, identification of the L5-L6 intervertebral space, injection technique, and validation of intrathecal delivery.

The protocol enables reliable intrathecal administration of volumes up to 50µL without surgical incision or catheterization. Successful intrathecal delivery was confirmed by Evans blue dye distribution throughout the spinal cord.

Compared with previously published protocols, this method combines a minimally invasive percutaneous approach with a low-dead-space integrated syringe-needle system, standardized animal positioning, and openly available 3D-printable scaffold, providing a practical and reproducible workflow.

This protocol provides a simple, reproducible, and refined method for low-volume intrathecal injection in rats, supporting reliable compound administration and promoting reproducibility and refinement in preclinical research.