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Neuroscience

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Published: February 15th, 2019

DOI:

10.3791/58565

1Department of Neurology, Second Hospital of Hebei Medical University, 2No.2 Middle School of Shijiazhuang, 3Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 4Key Laboratory of Hebei Neurology
* These authors contributed equally

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS. Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

AAV can mediate long-term and widespread gene expression in the CNS transduction with few side effects, and therefore has become one of the most promising vehicles for gene therapy to treat CNS diseases including amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Alzheimer's disease (AD), lysosomal storage diseases (LSD), Gaucher disease (GD), and neuronal ceroid lipofuscinosis (NCL)1. Presently, more than 100 AAV serotypes have been isolated from humans and animals. Among these, at least 12 have been used in preclinical and clinical trials, including the most commonly used gene vectors such as AAV1, 2, 4, 5, 6, 8, 9, rAAVr....

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FVB/NJ mice were bred in the animal facility of Key Laboratory of Hebei Neurology. All mouse experiments were approved by the Second Hospital of Hebei Medical University Ethics Committee and carried out according to the regulations of laboratory animal management promulgated by the Ministry of Science and Technology of the People's Republic of China.

1. Preparation of 20% Lidocaine Hydrochloride Stock Solution

  1. Weigh 2 g of lidocaine hydrochloride. Add 5–6 mL of sterile wa.......

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Mice showed different degrees of transient weakness right after IT injection of AAV solution in 1% lidocaine hydrochloride due to various quality of intrathecal injection. According to the semi-quantitative 5-grade scoring system we have established, we tested the transduction patterns of AAV in mice with different degrees of lidocaine-induced limb weakness (score 0, n = 2; score 1, n = 1; score 4, n = 4; score 5, n = 3). EGFP immunostaining of spinal cords showed either no or little tran.......

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Technically, there are several critical steps during the IT injection in awake mice. First, proper gesture and firm control of the mice throughout the entire operation is a prerequisite for successful delivery. Second, the most difficult point is feeling the intervertebral space with the needle tip, as it is necessary not to insert too deeply without resistance or insert forcibly under strong resistance in the case of injuring the animals or bending the needle tip. Third, although the transient paralysis due to lidocaine.......

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This work was funded by a grant from HEBEI Provincial Department of Human Resources and Social Security (CY201605) and a grant from Natural Science Foundation of Hebei Province (H2017206101), and we are very grateful to Dr. for Guangping Gao, who provided the AAV for this study.

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Name Company Catalog Number Comments
FVB/NJ mice Charles River Laboratories China
Lidocaine hydrochloride monohydrate HEOWNS 73-78-9
AAV Viral Vector Core of the Gene Therapy Center at University of Massachusetts Medical School
25µL  Hamilton syringe/27-30g needle GASTIGHT 1702
O.C.T compond SAKURA 4583
H 2O 2 SHUI HUAN PAI 170401
Goat serum Solarbio S9070
Triton X-100 LIFE SCIENCES T8200
Rabbit anti-GFP Life tech G10362 1:333 dilution
The second antibody (goat-anti rabbit) Jackson Immuno Research 111-005-144 1:1000 dilution
VECTASTAIN ABC REAGENT Vector Lab PK-6100
ImmPACT DAB Peroxidase Substrate Kit Vector Lab SK-4105
Mounting medium for fluorescence with DAPI Vectorshield H-1200
NaCl Yong Da Chemical
NaH2PO4·2H2O Yong Da Chemical
Na2HPO4·12H2O Yong Da Chemical
Paraformaldehyde Yong Da Chemical 307699
Adhesion Microscope Slides CITOGLAS 17083 25*75 mm
SUPER-SLIP MICRO-GLAS Electro Microscopy Siences 72236-60 24*60 mm
15 ml Centrifuge tube CORNING 430790
96 well cell culture cluster Coster 3599
24 well cell culture cluster Coster 3524
70% Ethanol WEN ZHI
Gauze Wei AN 05171112 8cm*10cm*12cm
1mL syringe Hong Da
Microtubes Plasmed
Micropipet  eppendorf
Peppet tips Rainin
Centirifuge eppendorf 5427R
Regerator Haier BCD-539WT
Filter MILLEX GP R4PA42342
Pump LongerPump BT-100-2J/YZ1515X
Microscope Olympus BX53
Freezing-microtome Leica CM1520

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