Personalized Needles for Microinjections in the Rodent Brain

Summary

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

Abstract

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

Introduction

Microinjections have been used for a long time for the delivery of pharmacologically active compounds to modulate neuronal activity in specific brain areas. In addition, they have been used to inject toxins near particular neuronal populations, to mimic neurodegenerative events characteristic of certain diseases, for example 6-hydroxy-dopamine in the nigrostriatal dopamine system to mimic Parkinson's disease^1,2 or the immunotoxin 192 IgG saporin to lesion the cholinergic system³. More recently, microinjection procedures have been used to deliver viral vectors or cells grafts for gen....

Protocol

All experimental protocols were approved by the University of Ferrara Ethical Committee for Animal Experimentation and by the Italian Ministry of Health. The ARRIVE (Animal Research: Reporting In Vivo Experiments¹¹) guidelines have been followed.

1. Preparation of Quartz Needles

1. Clean and sterilize the quartz capillaries (see Table of Materials) by placing them for 5 min in distilled water, 5 min in ethanol 99%, and 5 min in diethyl-ether.
2. Leave capillaries under the hood for at least 1 h to let them dry completely.
3. Prepare the needles using a laser puller according to....

Results

We compared damage induced by direct microinjection in the rat dorsal hippocampus and striatum using a quartz needle (60 µM external diameter tip; one 20 µM diameter side hole; type C, Figure 1) compared with two classic, 26 G blunt end and 30 G bevel edge stainless steel needles. To this aim, we injected 2 µL of aCSF in the right and left dorsal hippocampus and striatum using respectively the quartz and the steel needle and, 48 h after injection, we evaluate.......

Discussion

The technique described in this article fulfills the needs outlined in the Introduction for optimizing microinjections that are performed for various purposes¹². The needles described here reduce damage to a minimum, essentially non-detectable level; at variance with glass needles (that are prone to bend), quartz needles are rigid and ensure a reliable hit of the desired brain region even in deep coordinates. Moreover, the side hole ensures delivery of the solution even if the tip.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Quartz capillaries	Sutter Instruments, Novato, CA USA	Q100-50-10	Without filament
Puller	Sutter	P2000
Micropipette storage jar	World Precision Instruments (WPI), Sarasota, FL, USA	E210
Laser microdissector	Leica Microsystems, Wetzlar, Germany	LMD6500
Hamilton syringe	Hamilton ILS Innovative Labor Systeme GmbH, StŸtzerbach, Germany	19138-U
Microinfusion pump	Univentor, Zejtun, Malta	Model 864
Manual microinjection pump kit	WPI	Item#: MMP-KIT	Kit allowing for micropipettes to be securely mounted to the stereotactic frame
Precision Drill	Proxxon	28510 MicroMot 50/E	Ball bearing drive shaft with variable speed
Artficial Cerebral Spinal Fluid	Tocris	3525
Needles 26 G Blunt and 30 G Bevel	Hamilton	26 G Blunt: 19138-U 30 G Bevel: 20757
Microtome	Leica, Germany	LEICA RM212RT