Cisterna Magna Injection Approach for the Treatment of Leptomeningeal Metastatic Brain Tumors

Summary

This protocol presents a straightforward procedure that enables direct intrathecal delivery of agents to cerebrospinal fluid without operative damage to the skull or brain parenchyma. This approach provides a solution to deliver molecules that could not otherwise cross the blood-brain barrier or blood-cerebrospinal fluid barrier to treat leptomeningeal metastatic disease.

Abstract

Leptomeningeal metastases (LM) in the setting of pediatric brain tumors are associated with particularly poor prognosis. Current therapies for pediatric brain tumors include surgery, radiation, and chemotherapy. However, LM are not amenable to surgical resection and frequently do not respond to radiation. Moreover, most chemotherapeutic drugs are unable to cross the blood-brain barrier or blood-cerebrospinal fluid (CSF) barrier to reach LM. Here, we describe a mouse model of pediatric brain tumors that develop LM and an easy, reproducible approach to deliver therapeutic agents to treat LM. We intrathecally injected a CSF tracer via the cisterna magna in mice. After a single injection, the CSF tracer influx was observed along the leptomeninges throughout the entire central nervous system and persisted for at least 6 hours. Daily injection did not result in apparent toxicity or impact animal weight. The results of this study highlight the utility of a mouse model of metastatic pediatric brain tumors as well as the potential application of a repeated intrathecal injection technique to deliver therapeutic agents to treat metastatic brain tumors.

Introduction

Leptomeningeal metastases (LM) are common in patients with advanced brain cancer^1,2,3, breast cancer^4,5, lung cancer⁶, melanoma^7,8, and leukemia⁹. The presence of LM uniformly confers a poor prognosis and negatively impacts patient quality of life. Because meaningful surgical resection is impossible due to the diffuse nature of LM, the standard of care for leptomeningeal metastatic disease involves radiation and ....

Protocol

All animal procedures were performed according to the NIH guidelines and were approved by the Research Animal Facility Committee, Children's Research Institute (Protocol #30351).

1. Generation of a mouse model of metastatic medulloblastoma

NOTE: Because this is a recovery surgery, procedures should be performed under sterile conditions. All surgical tools should be sterilized by autoclaving or with a hot bead sterilizer.

1. Stereotactic implantation of .......

Discussion

In this report, we present a detailed protocol to generate a metastatic brain tumor mouse model and perform intrathecal injection into the cisterna magna. This injection approach provides a straightforward method to deliver labeled molecules to CSF compartments. By injecting CSF tracer, we observed a tracer influx into different regions of the brain and spinal cord (see Figure 2). Agents with differing molecular sizes may have distinct diffusion properties; molecules that are smaller in size.......

Materials

Name	Company	Catalog Number	Comments
33-gauge Neuros syringe, 10uL	Hamilton Company	65460-05	10uL glass syringe with 33-gauge beveled needle and stopper
4% Paraformaldehyde solution in PBS	Alfa Aeser	J19943	4% PFA
5 µL Microliter Syringe Model 7105 KH, Knurled Hub Needle, 24 gauge, 2.75 in, point style 3	Hamilton Company	88000	Microliter syringe
aCSF	Tocris Bioscience (Fisher Scientific)	35-252-5ML	artificial cerebrospinal fluid
Bard-Parker Sterile Disposable Scalpels #10	VWR	89176-380	Scalpels
Buprenorphine HCL injectable, 0.3MG/ML, C3	Covetrus	059122
Butler Schein Paralube Non-antibiotic opthalmic ointment	Fisher Scientific	NC1865385	eye ointment
Cryostat	Leica	CM1950
DAPI, FluoroPure grade	Thermo Fisher Scientific	D21490
Deltaphase Isothermal Pad	Braintree Scientific	BP-PAD	heating pad
Digital 3-axes Manipulator Arm, left	Stoelting	51904	Manipulator
Digital Conversion for Stoelting Manipulator Arm, left	Stoelting	51914	Digital Display for manipulator
Digital Just for Mouse Stereotaxic	Stoelting	51730D	Stereotaxic frame
DPBS, no calcium, no magnesium	Thermo Fisher Scientific	14190250	Phosphate buffered saline
Dumont #5 Forceps	Fine Science Tools	11295-00	forceps
Dumont #7 Forceps	Fine Science Tools	11297-00	forceps
Ethanol	Fisher Scientific	04-355-226
Extra Narrow Scissors	Fine Science Tools	14088-10	scissors
Fisherbrand Sterile Alcohol Prep Pad	Fisher Scientific	22-363-750
Fluorescein isothiocyanate–dextran	Sigma Aldrich	FD500S	FITC dextran 500
Fluorescence microscope	Olympus	BX53
Fluorescence stereo microscope	Leica	M205 FA	Dissection microscope
Gas Anesthesia Mask for Stereotaxis	Stoelting	51609M
Germinator 500	Fisher Healthcare	NC9956482	Hot bead sterilizer
Graefe Forceps, 10.2 cm	Stoelting	52102-60P	forceps
Heat Lamp with table clamp	Braintree Scientific	HL-1 120V
Induction chamber, anesthesia accessories, small animlal, 5.25 liter	Medvet International	93805107	induction chamber
Isoflurane solution	Covetrus	029405
IVIS Lumina III In Vivo Imaging System	Perkin Elmer	CLS136334	IVIS Imager
Liquivet (Rapid) Tissue Adhesive	World Precision Instruments	504561	Tissue adhesive
Neurocult stem cell medium and proliferation supplement	Stem Cell Technologies	Cat# 05751	cell culture medium
Quintessential Stereotaxic Injector (QSI)	Stoelting	53311	electronic pump for injection
Restrainer & Split Shield	Braintree Scientific	MHS2-HE set	Restrainer
RS 2000 Biological System Irradiator	Rad Source Technologies		Irradiator
Sterile Cotton Tipped Applicators	Medline Industries	MDS202000
Sucrose	VWR International	EM-8510
Surgical Clipper, Cordless, Rechargeable w/#40 blade	Stoelting	51465	Electric shaver
Tissue-Plus OCT Compound	Fisher Scientific	23-730-571	Tissue embedding medium
Stereomicroscope	Leica	M60
XenoLight D-Luciferin Potassium Salt	Perkin Elmer	1227799	Luciferin