Use of Cerebral Open-Flow Microperfusion for the Longitudinal Collection of Interstitial Fluid in an Animal Model of Glioblastoma

Summary

In vivo, intercellular communications underpinning many central nervous system diseases, such as glioblastoma (GBM), are notoriously difficult to measure and characterize. Here, we describe procedures of cerebral open-flow microperfusion (cOFM) that can be employed to sample interstitial fluid components in a longitudinal animal model of GBM.

Abstract

Diseases of the central nervous system (CNS) manifest through a complex, dynamic network of intercellular communication. Glioblastoma (GBM), the most common and aggressive primary brain tumor, is emblematic of this complexity, with its rapid progression and dismal prognosis measured in months rather than years. GBM's biology is driven by intricate signaling molecule exchanges between neoplastic and stromal cells, underpinning aggressive disease progression. Here, we describe a detailed cerebral open-flow microperfusion (cOFM) protocol in GBM mouse models, enabling real-time longitudinal monitoring of tumor microenvironment dynamics within the interstitial fluid (ISF). Our approach describes the implantation of durable guide cannula head-mounts, their use for intracerebral glioma engraftment directly through the guide, and the collection of high-fidelity cOFM samples for metabolomic and proteomic LC-MS analyses. Crucially, cOFM overcomes the molecular size limitations of traditional microdialysis. Beyond GBM, cOFM methodology promises transformative insights into a spectrum of CNS disorders, including neurodegenerative, epileptic, and neuropsychiatric conditions, through its capacity to provide etiological and treatment-responsive biomarkers within their respective animal models.

Introduction

Investigating central nervous system (CNS) diseases is notoriously challenging due to the complex, multimodal intercellular signaling mediated by the interstitial fluid (ISF)^1,2,3,4. This complexity is particularly pronounced in brain tumors, such as glioblastoma (GBM), where intercellular signals are conveyed between both neoplastic and stromal cells^5,6,7,8,9. In many cases, the ....

Protocol

All animal procedures within this protocol are approved by the Institutional Animal Care and Use Committee (IACUC) at Northwestern University (protocol ID: IS00021383) and follow National Institutes of Health (NIH) Guidelines. This protocol was developed for use in C57BL/6J mice, both male and female, with a minimum age of 8-10 weeks. The murine glioma cell line used in this protocol, CT2A, was provided by the Seyfried Lab at Boston University and was cultured and prepared following sterile tissue culture conditions.

1. Surgical planning and preparation

1. Animal information: For cOFM experiments, use mice that are a minimum of 8-1....

Results

Various evaluative measures to determine experimental success can occur at intermediate or culminating experimental stages, depending on the specific nature of the underlying pathology and investigational animal model. For examinations into GBM biology, bioluminescent imaging (BLI) can be used as an intermediate evaluative measure for the efficacy of cOFM guide-mediated tumor cell engraftment (Figure 4A). However, this is used for simple dichotomous present or absent tumor evaluations, given.......

Discussion

The cOFM procedures described in this protocol hold the potential to revolutionize the study of CNS diseases through their ability to longitudinally sample endogenous components of the ISF of pathologic tissue. However, the investigational success of employing this methodology relies upon the proper performance of several critical steps. Of these, the correct and secure placement of the cOFM guide cannula is one of the most important. Improper surgical procedures or attachment strategies can result in the dislodgement of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.7 mm Drill bit	Fine Science Tools	19008-07	Burr hole drilling
1 to 5 mL syringe	BD	#309646	Recirculated perfusate sample retreival
18 G needle	BD	#305195	Recirculated perfusate sample retreival
30G needle	BD Ultra-Fine	328438	Puncturing dura
4-0 vicryl sutures	Dynarex	9130	Closing surgical would
Alcohol wipes	FisherBrand	22-363-750	Surgical site skin disinfection
Anchor screws	BASi	MD-1310	cOFM head-mount placement
Artificial cerebral spinal fluid (aCSF)	BASi (Joanneum)	MD-2400	Sampling ISF
cOFM dummy insert	BASi (Joanneum)	cOFM-D-#	cOFM head-mount placement
cOFM guide	BASi (Joanneum)	cOFM-GD-#-#	cOFM head-mount placement
cOFM guide holder	BASi (Joanneum)	OFM-STM-1	cOFM head-mount placement
cOFM infusion insert	BASi (Joanneum)	cOFM-I-#	Cell injection
cOFM locking wedge	BASi (Joanneum)	cOFM-LOCK	cOFM head-mount placement
cOFM sampling insert	BASi (Joanneum)	cOFM-S-X	Sampling ISF
Cotton-tipped applicators	FisherBrane	22363160	Fluid absorption, applying hydrogen peroxide
CT2A mouse glioma cell line	Seyfried Lab - Boston University	NA	Engrafted tumor cells
Dental cementing agent	IvoClar	595953US	cOFM head-mount placement
Dental curing light	Henry Schein	5700253	cOFM head-mount placement
Depilatory cream	Veet	3116875	Surgical site hair removal
Electric shaver	Wahl Clipper Corp.	8841	Surgical site hair removal
Fine tipped forceps	Fine Science Tools	5SF	Incision, anchor screw placement
Flanged tubing connectors	BASi (Joanneum)	MD-1510	Cell injection
Fraction collection vials	BASi (Joanneum)	MF-5281	Fractionated sample storage
Gastight glass syringe (25 μL)	Hamilton	80465	Cell injection
Hydrogen peroxide (1-3%)	Walgreens	NDC: 0363-0268-32	Skull surface cleaning
Insulin syringes (0.3 to 1.0 mL) with 31G needles	BD Ultra-Fine	328438	Anesthetic and analgesic administration
Microcentrifuge tubes	Basix	02-682-002	Recirculated perfusate sample retreival
Microdrill and stereotactic holder	Harvard Apparatus	75-1874	Burr hole drilling
Mouse jacket	Lomir	MJ 02	Animal connection to rotating cage system
OFM low-bind tubing	BASi (Joanneum)	OFM-PP2-100-LB	Sampling ISF
OFM-Pump - Microperfusion pump	BASi (Joanneum)	MPP102-PC	Sampling ISF
Ophthalmic Ointment	Dechra	211-38	Peri-sugrical eye lubrication
Passivated metal sampling needle	BASi (Joanneum)	MW-2310	Fractionated sample storage
Perfusate bag	BASi (Joanneum)	OFM-BAG	Sampling ISF
Physiologic saline solution	ICU Medical	0990-7983-03	Skull surface cleaning, aid in burr hole drilling
Povidone-iodine swabs	PDI Healthcare	SKU B40600	Surgical site skin disinfection
Probe holder	Stoelting	51633
Refrigerated fraction collector	BASi (Joanneum)	MD-1201	Fractionated sample storage
Rotating cage system	BASi (Joanneum)	AMD-R Raturn	Automated housing for awake cOFM sampling
Scalpel blades and handles	Fisher Scientific	22079693	Incision
Screw driver			cOFM head-mount placement
Self-etching dental adhesive	Tokuyama		Skull surface priming for cementing agent
Silicone push cap for collection vials	BASi (Joanneum)	MF-5283	Fractionated sample storage
Sterotactic frame	Stoelting	51725