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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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, rAAVrh.8, and rAAVrh.101,2,3,4,5,6.

Different CNS diseases require different AAV delivery strategies due to the various affected CNS regions and cell types. The CNS regions and cell types that AAV can transduce varies depending on the serotype as well as delivery method. For example, rAAVrh10 has been shown to transduce predominantly astrocytes when delivered by systemic intravenous injection (IV), whereas it transduced both neurons and glia when delivered by intrathecal injection4,7. Additionally, parenchyma injection resulted in local transduction to the vicinity of the injection site, whereas injection into the cerebrospinal fluid (CSF) through intraventricular or intrathecal injection resulted in widespread CNS transduction8. Studies have also demonstrated therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration9,10,11. In diseases that affect broad areas of the CNS such as ALS, intrathecal injection into the CSF has been shown to cover most areas that are afflicted by the disease with a lower dose, compared to a systemic delivery method4,10. Recent studies have also shown that lumbar puncture can be used to inject AAV in mouse models for ALS, which avoids potential injuries associated with laminectomy and intrathecal catheterization4.

Experimental direct lumbar puncture was first used to deliver agents, especially anesthetics, to the spinal cord for analgesia and anesthesia in 188512,13. In this report, we illustrate the lumbar puncture IT injection method in adult mice with the aid of 1% lidocaine hydrochloride, a local amide-derived anesthetic, in the injection solution to evaluate and monitor injection quality. Successful injections were marked by lidocaine-induced transient paralysis, whereas failed injections did not show this behavior. We classified the level of transient weakness as one of five grades to help predict the injection efficiency. Finally, we show that the rAAVrh10 transduction level may be predicted by the grade of paralysis. Therefore, this intrathecal AAV delivery method can be used to enhance AAV-mediated gene-delivery for experimental therapy of CNS diseases.

Protocol

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 water and vortex gently. Increase the volume to a total of 10 mL with sterile water.
  2. 1.2 Filter the stock solution through 0.2-micron filters. Aliquot the stock solution, 1 mL per 1.5 mL microcentrifuge tube,and seal it with a sealing film. Store at 4 °C.

2. Direct Intrathecal AAV Delivery in Awake Mice

  1. Wipe the work area using sterile gauze with 70% ethanol and prepare the required supplies as mentioned in Table 1.
  2. Prepare 100 µL of AAVrh.10/1% lidocaine hydrochloride complex by adding 95 µL of rAAVrh10 stock solution (5 x 1012 genome copies/mL) into a sterile 200 µL microcentrifuge tube, and add 5 µL of 20% lidocaine hydrochloride stock solution. Mix well by pipetting up and down. Then, store the virus solution on ice (4 °C).
    NOTE: 8 µL per mouse4 will be used.
  3. Preparing the syringe with AAV solution for injection
    1. Assemble a 25 µL Hamilton syringe with a 27 G needle and align the beveled tip of the needle with the volumetric scale on the syringe.
    2. Draw 8 µL with 4 x 1010 genome copies of the virus solution into the syringe gently.Make sure to remove air bubbles.
  4. Preparing the mouse
    NOTE:
    Male or female FVB/NJ mice (30–70 days old) were used in this study. IT injection was operated in the hood.
    1. Sterilize the work area in a hood with 70% ethanol. Put the awake mouse (male or female, 30–70 days old, 13–20 g weight) on a bedpiece in a prone position in the hood. Cover the upper body with sterile gauze to calm the mouse and avoid being bitten.
    2. Fix the animal by gripping appropriately and firmly on its pelvic girdle with a thumb on one side and forefinger/middle finger on the other side. Keep the skin between bilateral pelvic girdles taut with the thumb and forefinger. Hold gently on the upper body of the animal with the palm.
    3. Shave the fur on its back between the bilateral pelvic girdles, then sterilize the skin surface with an iodide-based scrub and 70% ethanol.
  5. Intrathecal Injection12
    1. Feel the intervertebral space along the middle line between the bilateral pelvic girdles with a thumb or forefinger of the other hand and press an indentation with a fingernail to indicate the L5-L6 intervertebral space (locate the injection site).
    2. Rotate the base of the tail slightly and gently to indicate the midline of the spine. Adjust the needle bevel towards the head of the animal before injection (mentioned in step 2.3.1).
    3. Make sure that the animals are fixed firmly and align the needle along the midline of spine.
    4. Insert the needle gently and vertically (or tilt slightly 70–80°) in the intersection of indentation and keep the syringe in a central sagittal plane. Reduce the angle to approximately 30° slowly when it connects the bone, then slip the needle into the intervertebral space.
      NOTE: An evident sudden tail flick is a sign of successful entry into the intradural space. Once the needle enters the intervertebral space, the needle tip will feel firmly clamped. The 27 G needle used in this study is suited for IT delivery in mice but not rats.
    5. Inject the vector solution(mentioned in step 2.2). Start the timer and inject 8 µL of the vector solution at a speed of 1 µL/4 s. Retain the needle approximately 1 min after finishing delivery. Withdraw the needle with gentle rotation to avoid leaking.
    6. Score the transient weakness of the mouse limbs immediately after delivery to evaluate the injection quality4.
      NOTE: The standard is as follows4. Score 0: no weakness; score 1: minor weakness of the hind limbs without gait abnormality; score 2: moderate weakness of the hind limbs with obvious gait abnormality; score 3: complete paralysis of the hind limbs; score 4: complete paralysis of the hind limbs, shortness of breath, and moderate weakness of the fore limbs; and score 5: complete paralysis of all four limbs and evident shortness of breath.
    7. Move the mouse back to the cage for recovery from paralysis.
  6. Clean-up
    1. Flush the syringe with 1 mL sterile water. Sort laboratory supplies and collect all non-disposable materials for autoclaved sterilization. Clean the bench with 70% ethanol.

3. Tissue Preparation for Immunohistochemical Staining

  1. Tissue collection
    1. Anesthetize mice at 21 days post-injection with 3% chloral hydrate (0.1 mL/10 g) deeply by intraperitoneal injection.
    2. Perfuse transcardially with 20 mL of ice-cold 0.01 M PBS (NaCl 147 mM; NaH2PO4 1.9 mM; K2HPO4 8.1 mM, pH 7.4) firstly, then 4% ice-cold paraformaldehyde (in 0.01 M PBS) with pump (10 mL/min for 1 min, then 5 mL/min for 9 min).
      CAUTION: Paraformaldehyde is carcinogenic and toxic. Handle it only in the fume hood while wearing gloves.
  2. Dissection of the spinal cord and brain
    1. Fix the limbs and head of each animal in a prone position on a foam box cover with syringe needles, then strip and remove the skin from the head to sacrum with scissors.
    2. Clip the skull between eyes, cut alongside the middle route of the skull and horizontal line upon the cerebellum, then open the skull to each side.
    3. Lift the occipital bone with tweezers and open the spinal canal bilaterally with ophthalmic scissors. Cut off the ribs on both sides and remove the upper half of vertebrae carefully.
    4. Lift the brain with curved tweezers and sever the nerves of the skull base, then dissect out the whole brain and spinal cord carefully. Post-fix the tissues in 4% paraformaldehyde for 24 h.
  3. Preparation of tissue slices
    1. Cryoprotect the brain and cervical and lumbar spinal cord in 30% sucrose solution overnight at 4 °C. Embed the tissue in optimum cutting temperature (OCT) compound and freeze fast with liquid nitrogen.
    2. Cut the tissue at 25 µm using a cryostat and store the frozen sections in 0.01 M PBS at 4 °C for use .

4. Immunohistochemistry

  1. Pretreat free-floating sections in 1% H2O2 for 10 min, then wash in PBS for 10 min. Incubate in a blocking solution containing 5% serum and 0.3% non-ionic detergent in PBS for 1 h.
  2. Incubate the slices with corresponding primary antibodies overnight at 4 °C. Wash the sections in PBST (0.2% Tween 20 in PBS) for 30 min (3 times for 10 min each).
  3. Incubate the slices with corresponding biotin-secondary antibody at room temperature for 1 h. Wash in PBST for 30 min (3 times for 10 min each).
  4. Incubate the sections with affinity biotin peroxidase complex for 40 min, and stain with achromogenic agent. Mount the sections onto slides and dry properly.
  5. Soak the slides in anhydrous ethanol for 5 min and xylene for 10 min, then seal the slides with a mounting medium. Finally, image the slides with a microscope equipped with a charge-coupled device (CCD) at 100x, 200x, and 400x magnifications.

Results

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...

Discussion

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...

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

NameCompanyCatalog NumberComments
FVB/NJ miceCharles River Laboratories China
Lidocaine hydrochloride monohydrateHEOWNS73-78-9
AAVViral Vector Core of the Gene Therapy Center at University of Massachusetts Medical School
25 µL  Hamilton syringe/27-30 G needleGASTIGHT1702
O.C.T compondSAKURA4583
H 2O 2SHUI HUAN PAI170401
Goat serumSolarbioS9070
Triton X-100LIFE SCIENCEST8200
Rabbit anti-GFPLife techG103621:333 dilution
The second antibody (goat-anti rabbit)Jackson Immuno Research111-005-1441:1,000 dilution
VECTASTAIN ABC REAGENTVector LabPK-6100
ImmPACT DAB Peroxidase Substrate KitVector LabSK-4105
Mounting medium for fluorescence with DAPIVectorshieldH-1200
NaClYong Da Chemical
NaH2PO4·2H2OYong Da Chemical
Na2HPO4·12H2OYong Da Chemical
ParaformaldehydeYong Da Chemical307699
Adhesion Microscope SlidesCITOGLAS1708325 mm x 75 mm
SUPER-SLIP MICRO-GLASElectro Microscopy Siences72236-6024 mm x 60 mm
15 mL Centrifuge tubeCORNING430790
96 well cell culture clusterCoster3599
24 well cell culture clusterCoster3524
70% EthanolWEN ZHI
GauzeWei AN051711128 cm x 10 cm x 12 cm
1 mL syringeHong Da
MicrotubesPlasmed
Micropipet eppendorf
Peppet tipsRainin
Centirifugeeppendorf5427R
RegeratorHaierBCD-539WT
FilterMILLEX GPR4PA42342
PumpLongerPumpBT-100-2J/YZ1515X
MicroscopeOlympusBX53
Freezing-microtomeLeicaCM1520

References

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  3. Yang, B., et al. Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10. Molecular Therapy. 22 (7), 1299-1309 (2014).
  4. Guo, Y., et al. A Single Injection of Recombinant Adeno-Associated Virus into the Lumbar Cistern Delivers Transgene Expression Throughout the Whole Spinal Cord. Molecular Neurobiology. 53 (5), 3235-3248 (2016).
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  7. Tanguy, Y., et al. Systemic AAVrh10 provides higher transgene expression than AAV9 in the brain and the spinal cord of neonatal mice. Frontiers in Molecular Neuroscience. 8, 36 (2015).
  8. Federic, T., et al. Robust spinal motor neuron transduction following intrathecal delivery of AAV9 in pigs. Gene Therapy. 19, 852-859 (2012).
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  10. Li, D., et al. Slow Intrathecal Injection of rAAVrh10 Enhances its Transduction of Spinal Cord and Therapeutic Efficacy in a Mutant SOD1 Model of ALS. Neuroscience. 365, 192-205 (2017).
  11. Borel, F., et al. Therapeutic rAAVrh10 Mediated SOD1 Silencing in Adult SOD1(G93A) Mice and Nonhuman Primates. Human Gene Therapy. 27 (1), 19-31 (2016).
  12. Fairbanks, C. A. Spinal delivery of analgesics in experimental models of pain and analgesia. Advanced Drug Delivery Reviews. 55 (8), 1007-1041 (2003).
  13. Hylden, J. L., Wilcox, G. L. Intrathecal morphine in mice: a new technique. European Journal of Pharmacology. 67, 313-316 (1980).
  14. Wang, H., et al. Widespread spinal cord transduction by intrathecal injection of rAAV delivers efficacious RNAi therapy for amyotrophic lateral sclerosis. Human Molecular Genetics. 23 (3), 668-681 (2014).
  15. Wang, Y., et al. scAAV9-VEGF prolongs the survival of transgenic ALS mice by promoting activation of M2 microglia and PI3K/Akt pathway. Brain Research. 1648, 1-10 (2016).

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