Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

Summary

This study demonstrates delivery of a repetitive traumatic brain injury to mice and simultaneous implantation of a cranial window for subsequent intravital imaging of a neuron-expressed EGFP using two-photon microscopy.

Abstract

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

Introduction

Traumatic brain injury (TBI), which can result from sports injuries, vehicle collisions, and military combat, is a worldwide health concern. TBI can lead to physiological, cognitive, and behavioral deficits, and lifelong disability or mortality^1,2. TBI severity can be classified as mild, moderate, and severe, the vast majority being mild TBI (75%-90%)³. It is increasingly recognized that TBI, particularly repetitive occurrences of TBI, can promote neuronal degeneration and serve as risk factors for several neurodegenerative diseases, including Alzheimer's disease (AD), amyotrop....

Protocol

All the animal related protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council (US) Committee. The protocols were approved by the Institutional Animal Care and Use Committee of University of Massachusetts Chan Medical School (UMMS) (Permit Number 202100057). In brief, as shown in the schematic of study (Figure 1), the animal receives a virus injection, a TBI, a window implantation, and then intravital imaging in a time sequence.

NOTE: Commercial terms have been removed. Please refer to the Table of Materials for....

Results

As proof of concept for this protocol, viral particles expressing AAV-Syn1-EGFP were injected into the brain cortex of male TDP-43^Q331K/Q331K mice (C57BL/6J background)¹⁹ at the age of 3 months. It is noted that wild-type C57BL/6J animals can also be used, however this study was carried out in TDP-43^Q331K/Q331K mice because the laboratory is focused on neurodegenerative disease research. A TBI surgery was performed 4 weeks after AAV injection. Within the same surgical setting.......

Discussion

In this study, AAV injection, TBI administration, and a headpost with cranial window implantation were combined for longitudinal imaging analysis of EGFP-labeled neurons within the mouse brain cortex (layers IV and V) to observe the effects of TBI on cortical neurons. This study notes that the TBI site chosen here, above the hippocampus, provides a relatively flat and broad surface for implantation of the cranial window. Conversely, the skull is relatively narrow anterior to this site, and therefore it is difficult to en.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adjustable Precision Applicator Brushes	Parkell	S379
BD insulin syringe	BD	NDC/HRI#08290-3284-38	5/16" x 31G
Betadine	Purdue	NDC67618-151-17	including 7.5% povidone iodine
Buprenorphine	PAR Pharmaceutical	NDC 42023-179-05
Cefazolin	HIKMA Pharmaceutical	NDC 0143-9924-90
Ceramic Mixing Dish	Parkell	SKU: S387	For dental cement preparation
Cotton Tipped Applicators	ZORO	catlog #: G9531702
Catalyst	Parkell	S371	full name: "C" Universal TBB Catalyst
Dental cement powder	Parkell	S396	Radiopaque L-Powder for C&B Metabond
Dental drill	Foredom	H.MH-130
Dental drill controller	Foredom	HP4-310
Dexamethasone	Phoenix	NDC 57319-519-05
EF4 carbide bit	Microcopy	Lot# C150113	Head Dia/Lgth/mm 1.0/4.2
Ethonal	Fisher Scientific	04355223EA	75%
FG1/4 carbide bit	Microcopy	Lot# C150413	Head Dia/Lgth/mm 0.5/0.4
FG4 carbide bit	Microcopy	Lot# C150309	Head Dia/Lgth/mm 1.4/1.1
Headpost	N/A	N/A	Custom-manufactured
Heating apparatus	CWE	TC-1000 Mouse	equiped with the stereotaxic instrument and be used while operating surgery
Heating blanket	CVS pharmacy	E12107	extra heating device and be used after surgery
Isoflurane	Pivetal	NDC 46066-755-03
Isoflurane induction chamber	Vetequip	89012-688	induction chamber for short
Isoflurane volatilizing machine	Vetequip	911103
Isoflurane volatilizing machine holder	Vetequip	901801
Leica surgical microscope	Leica	LEICA 10450243
Lubricant ophthalmic ointment	Picetal	NDC 46066-753-55
Marker pen	Delasco	SMP-BK
Meloxicam	Norbrook	NDC 55529-040-10
Microinjection pump and its controller	World Precision Instruments	micro4 and UMP3
Microliter syringe	Hamilton	Hamilton 80014	1701 RN, 10 μL gauge for syringe and 32 gauge for needle, 2 in, point style 3
Mosquito forceps	CAROLINA	Item #:625314	Stainless Steel, Curved, 5 in
Depilatory agent	McKesson Corporation	N/A	Nair Hair Aloe & Lanolin Hair Removal Lotion
Microscope 1	Nikon	SMZ745	Nikon microscope for cranial window preparation
Microscope 2	Zeiss	LSM 7 MP	two-photon microscope
Multiphoton laser	Coherent	Chameleon Ultra II, Model: MRU X1, VERDI 18W	laser for two-photon microscopy
Non-absorbable surgical suture	Harvard Apparatus	catlog# 59-6860	6-0, with round needle
Norland Optical Adhesive 81	Norland Products	NOA 81
No-Snag Needle Holder	CAROLINA	Item #: 567912
Quick base liquid	Parkell	S398	"B" Quick Base For C&B Metabond
Regular scissor 1	Eurostat	eurostat es5-300
Regular scissor 2	World Precision Instruments	No. 501759-G
Round cover glass 1	Warner instruments	CS-5R Cat# 64-0700	for 5 mm of diameter
Round cover glass 2	Warner instruments	CS-3R Cat# 64-0720	for 3 mm of diameter
Rubber rings	Orings-Online	Item # OO-014-70-50	O-Rings
Saline	Bioworld	L19102411PR
Spring scissor 1	World Precision Instruments	No. 91500-09	tip straight
Spring scissor 2	World Precision Instruments	No. 91501-09	tip curved
Stereotaxic platform	KOPF	Model 900LS
Super glue	Henkel	Item #: 1647358
surgical Caliper	World Precision Instruments	No. 501200
Surgical forceps 1	ELECTRON MICROSCOPY SCIENCES	Catlog# 0508-5/45-PO	style 5/45, curved
Surgical forceps 2	ELECTRON MICROSCOPY SCIENCES	catlog# 0103-5-PO	style 5, straight
Surgical forceps 3	ELECTRON MICROSCOPY SCIENCES	catlog# 72912
Surgical forceps 4	ELECTRON MICROSCOPY SCIENCES	Catlog# 0508-5/45-PO	style 5/45, curved
Surgical gauze	ZORO	catlog #: G0593801
Surgical lamp	Leica	Leica KL300 LED
UV box	Spectrolinker	XL-1000	also called UV crosslinker
Vaporguard	Vetequip	931401
Vetbond Tissue Adhesive	3M Animal Care	Part Number:014006