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.
