Adeno-Associated Viral Gene Therapy for Retinal Disorders - Gene Delivery and Therapy for Neurological Disorders - page 224

holding the superior or inferior rectus muscle with a notched
forceps. Virus is drawn up via a 10 mm 34-gauge needle (Hamilton;
Hamilton AG) mounted on a 5 ʼ l syringe (Hamilton 65 RN,

Hamilton AG). 1-2 l of virus can be injected into the eye, depend-

ing on the age of the mouse and mode of delivery (subretinal ver-
sus intravitreal). We have also found that drawing up 1

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l of air,
prior to the vector, is a useful adjunct since a visualization of air
following the injection clarifi es whether the vector has been deliv-
ered subretinally or intravitreally.
The needle is inserted tangentially through the sclera directly
into the vitreous cavity for intravitreal injections, taking care to
avoid the large crystalline lens in the mouse eye. For subretinal
injection, the needle is carefully tunneled between the RPE and
retina until the whole bevel is visible and the virus injected.
Complete subretinal or intravitreal delivery can be confi rmed by
direct visualization of fl uid and the air bubble. After injection, the
needle is left in position for an additional 20-30 s and then with-
drawn quickly to minimize refl ux and to allow self-sealing of the
scleral tunnel. Different syringes and needles should be used for
different viruses, and between individual injections, the needle
should be fl ushed with sterile water.

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6.2 Confocal
Scanning Laser
Ophthalmoscopy

In recent years, new methods of retinal imaging such as confocal
scanning laser ophthalmoscopy (cSLO; Spectralis HRA, Heidelberg
Engineering) have been developed for clinical use. This allows a
noninvasive assessment of fundus autofl uorescence, the main
source of which in humans is an accumulation of lipofuscin, a
by-product of the visual cycle. This same equipment can be used to
assess fundus autofl uorescence in small laboratory animals. This is
particularly useful for the assessment of the accumulation of auto-
fl uorescent pigment such as A2E in the ABCA4 −/− mouse or AAV
transgene expression if a fl uorescent pigment such as GFP is
included in the expression cassette (Fig. 8 ). Imaging is usually

Fig. 8 Confocal scanning laser ophthalmoscopy in vivo imaging of injected mice showing ( a ) infrared fundal
image, ( b ) fl uorescence image, and ( c ) composite demonstrating widespread retinal transduction