In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Summary

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

Abstract

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-β). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Introduction

Cerebrospinal fluid (CSF) bathes the brain and spinal cord and is involved in maintaining homeostasis, supplying nutrients, and regulating intracranial pressure¹. CSF in the subarachnoid space enters the brain through a network of perivascular spaces (PVS) surrounding cortical pial arteries and then flows down along penetrating arterioles². Once in the parenchyma, CSF exchanges with interstitial fluid (ISF), carrying harmful metabolites such as amyloid-β (Aβ) and tau protein aggregates out of the brain through low resistance white matter tracts and perivenous spaces^2,

Protocol

All experiments were approved by the University Committee on Animal Resources (UCAR, Protocol No. 2011-023) at the University of Rochester and performed according to the NIH Guide for the Care and Use of Laboratory Animals.

1. Preparing the cisterna magna cannula, head plate, and head holder

1. Sterilize all surgical instruments and head plates before surgery.
   NOTE: Fluorescent tracers are delivered directly into CSF via a cisterna magna cannulation. For detailed instructions on this procedure, please refer to Xavier et al⁹.
2. Briefly, using a needle driver, break the tip of a 30G x 12.7 mm (&....

Results

CSF influx is imaged on an epifluorescent macroscope (Figure 1A), which allows for mesoscopic imaging of CSF tracer transport in the murine cortex. The whole-skull head plate permits the visualization of the rostral nasal bones, both frontal and parietal bones in the center, and the rostral portion of the interparietal bone caudally (Figure 1B). During imaging, the nasofrontal, sagittal, coronal, and lambdoid sutures can be readily identified (

Discussion

We have described a detailed protocol for performing transcranial CSF imaging in live mice using commercially-available fluorescent macroscopes and tracers. This technique is simple and minimally-invasive, yet quantitative. In vivo imaging correlates well with sensitive methods such as liquid scintillation counting of radio-labeled tracers including ³H-dextran and ¹⁴C-inulin after CM delivery, and with ex vivo coronal section quantification^10,18

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Bupivacaine HCl			University of Rochester Vivarium
100 µL Gastight Syringe Model 1710 TLL, PTFE Luer Lock	Hamilton Company	81020
A-M Systems Dental Cement Powder	Fisher Scientific	NC9991371
Carprofen			University of Rochester Vivarium
Chlorhexidine	Prevantics	B10800
CMOS Camera	Hammamatsu	ORCA Flash 4.0
Head Plate	University of Rochester	No catalog #	Custom made at the machine shop at the University of Rochester
High-Temperature Cautery	Bovie Medical Corporation	AA01
Insta-set Accelerator	Bob Smith Industries	BSI-151
Isoflurane - Fluriso	Vet One	502017	University of Rochester Vivarium
Ketamine			Strong Memorial Hospital Pharmacy
Krazy Glue	Elmer's Products, Inc	No catalog #, see link in comments	https://www.amazon.com/Krazy-Glue-KG48348MR-Advance-Multicolor/dp/B000BKO6DG
Micropore Surgical tape	Fisher Scientific	19-027-761
Paraformaldehyde	Sigma-aldrich	P6148
PE10 - Polyethylene .011" x .024" per ft., 100 ft. continuous	Braintree Scientific	PE10 100 FT
Pump 11 Elite Infusion Only Dual Syringe	Harvard Apparatus	70-4501
PURALUBE VET OINTMENT	Dechra
Puritan PurSwab Cotton Tipped Cleaning Sticks	Fisher Scientific	22-029-553
Research Macro Zoom Microscope	Olympus	MVX10
Simple Head Holder Plate (for mice)	Narishige International USA Inc	MAG-1
Single-use Needles, BD Medical	VWR	BD305106
Sterile Alcohol Prep Pads	Fisher Scientific	22-363-750
Tunable LED	PRIOR Lumen 1600-LED
Xylazine			University of Rochester Vivarium