Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery
March 8th, 2024
The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.
Our group employs an interdisciplined approach to involve genomic discovery and functional modeling to study genetics and epigenetic basis of neurodevelopmental disorder. We perform molecular, neurophysiological and neurobehavioral analysis using patient-induced pluripotent stem cells or iPSC in the mutant mice to dissecting the pathophysiology of neurodevelopmental disorder. We aim to develop gene editing based treatment for this disease.
Intracerebroventricular, also called ICV injection, is only method to directly deliver treatment to the central nervous system of neonatal mice. However, it has to penetrate through the cerebral cortex. The intrathecal IT injection is a commonly employed procedure in clinics serving as an effective means to administrator treatments directly into the central nervous system.
However, intrathecal injection in neonatal pups can pose significant technical challenges due to their small size and fragile nature. We're trying to develop an efficient and reliable delivery method to test the efficacy and gene editing in mouse brain that is non-viral and non-nanoparticle. Compared to conventional ICV injection, intrathecal injection presents a significant lower risk as they avoid the need for directly penetration through the cerebral cortex.
This advantage minimize potential damage to regional cortical tissue and the surrounding nerves. Intrathecal injections also enable at least a fivefold increase in the administrable volume of medications, so a single injection and greatly enhance the visibility of repeated administrations. To begin, move the pups to a separate cage away from the dam while handling them.
Before beginning the injection procedure, using the dissecting microscope, confirm the intervertebral space, or at a minimum the midline of the spinal canal of the pup. Load a 25 or 10 microliter syringe with 10 microliters of the desired injection substance like the drug formulation, virus preparation, or control artificial spinal fluid, et cetera. Once the animal is fully anesthetized as confirmed by reduced or absent body movement, gently positioned the pups under the microscope.
With the left index finger and thumb, carefully palpate the intervertebral space along the midline, situated between the bilateral pelvic girdles. Gently rotate the base of the tail slightly to help identify the midline of the spine. Carefully insert the needle, tilting it to an angle of 70 to 80 degrees at the point where the indentation intersects while ensuring it remains aligned with the central sagittal plane.
As the needle makes contact with the bone, gradually decrease the angle to approximately 30 degrees and advance the needle about two millimeters into the intervertebral space. Slowly inject up to 10 microliters volume within 50 to 60 seconds. Keep the needle in place for 10 to 20 seconds after the injection is complete.
Withdraw the needle with a gentle rotation to avoid leaking. Post the injection, place the pup on a heating pad for 10 to 15 minutes to allow the pup to recover fully and rewarm. Delivering intrathecal injection of fast green in wild type neonates immediately resulted in the widespread distribution of the administered solution.
YFP reporter mice showed widespread YFP expression across the entire brain 10 days after intrathecal injection of CRISPR-Cas9 based gene editing.
Overview
This study presents a detailed protocol for performing intrathecal injections in neonatal mice, facilitating gene editing and drug delivery directly into the central nervous system. The technique aims to overcome challenges related to the fragile nature of neonatal pups and to improve treatment reliability without damaging surrounding tissues.
Key Study Components
Area of Science
- Neuroscience
- Gene Editing
- Developmental Biology
Background
- Neurodevelopmental disorders can benefit from targeted gene editing.
- Intrathecal injections provide a less invasive alternative to intracerebroventricular methods.
- The procedure is particularly suited for neonatal mice due to their small size and fragility.
- Effective delivery methods are crucial for testing gene editing efficacy in animal models.
Purpose of Study
- To develop a reliable protocol for administering treatments directly into the central nervous system.
- To enhance the safety and efficacy of gene editing approaches in mouse models of neurodevelopmental disorders.
- To investigate the distribution and effects of injected substances post-administration.
Methods Used
- Intrathecal injection technique using neonatal mice as the biological model.
- Substances such as drug formulations and CRISPR-Cas9 preparations were explored for direct spinal administration.
- Precision is critical: syringes with specific volumes are used, with careful anatomical identification required.
- Post-injection care and recovery processes for the pups are emphasized.
Main Results
- Successful delivery and distribution of solutions in the neonatal mouse brain were confirmed post-injection.
- Widespread expression of YFP reporter in response to gene editing was observed after intrathecal injection.
- The method showed significantly increased administrable volumes compared to traditional methods.
- Lower risk of damaging cortical tissues enhances the accuracy of gene delivery.
Conclusions
- The study demonstrates an innovative and effective technique for drug delivery and gene editing in neonatal models.
- This approach allows for safer interventions in the study of neurodevelopmental disorders.
- It highlights the importance of precise anatomical targeting in improving the efficacy of central nervous system therapies.
