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, 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.
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
2. Direct Intrathecal AAV Delivery in Awake Mice
3. Tissue Preparation for Immunohistochemical Staining
4. Immunohistochemistry
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 transduction in the lumbar spinal cord of mice scoring 0, slightly enhanced transduction in mice scoring 1, and strong and widespread transductions in mice scoring 4 or 5 (Figure 1A). We quantified the GFP staining intensity of those mice displaying various degrees of transient limb weakness (Figure 1B) and concluded that the severity of weakness after injection correlated closely with the extent of spinal cord transduction.
We further explored the detailed transduction profile of rAAVrh10 in the whole CNS, and noticed that full length of the spinal cord and wide areas of the brain were well transduced in well-injected mice which scored 4 or 5. In the brain, robust EGFP signals were detected in olfactory bulb (Figure 2A), dorsolateral prefrontal cortex (Figure 2B), dentate gyrus and CA3 zone of hippocampus (Figures 2C and 2D), cerebellar cortex (Figure 2E), and marginal areas of brainstem including facial nucleus (Figure 2F), choroid plexus, and ependymal epithelial cells (Figure 2G). However, fewer EGFP-positive cells were detected in deep regions of the brain. In the spinal cord and ventral and dorsal horns, the ventral effluent motor axons and dorsal affluent sensory axons were strongly GFP-positive. Motor neurons in the anterior horns were strongly transduced in different levels of the spinal cord (Figures 2H-2J). Moreover, GFP-positive neurons in the cortex including pyramidal cells were detected (Figures 3A and 3B). Various glial cell types including microglia, astrocytes and oligodendrocytes, were also found to be EGFP-positive (Figures 3C-3E).
Figure 1: Lidocaine-induced weakness extent predicts transduction efficiency. AAV with 1% lidocaine or PBS (control) was injected by direct IT injection. Mice were sacrificed and examined for GFP expression by immunohistochemistry 3 weeks later (score 0, n = 2; score 1, n = 1; score 4, n = 4; score 5, n = 3). (A) GFP staining of cervical (CSC) and lumbar spinal cord (LSC) sections is shown. (B) GFP staining intensity in both LSC and CSC was directly correlated with the degree of transient weakness. Each mark represents values (mean ± SD) from one mouse. This figure has been adapted from a previous publication4. Please click here to view a larger version of this figure.
Figure 2: Widespread transduction of rAAVrh10 in the brain and spinal cord. (A) Olfactory bulb; (B) cortex; (C) dentate gyrus4; and (D) CA3 of hippocampus; (E) cerebellar cortex; (F) facial nucleus; (G) lateral ventricle; (H) cervical anterior horn4; (I) thoracic anterior horn4; and (J) lumbar anterior horn4. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 3: Transduction of various cell types in the brain after direct intrathecal rAAVrh10 injection. (A) Pyramidal cells; (B) multipolar neuron; (C) microglial cell; (D) astrocyte; and (E) oligodendrocyte. Scale bars = 100 µm. Please click here to view a larger version of this figure.
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 provides an objective indicator for IT injection quality, more practice is needed to achieve consistent and successful results.
In this report, we have developed a direct intrathecal injection method in awake mice for delivery of AAV, in which lidocaine serves as an indicator for the extent of IT injection success and as a predictor for efficiency of gene therapy. Experimental direct lumbar puncture was first used to deliver agents, especially anesthetics, to the spinal cord for analgesia and anesthesia, and it has been highly recommended in gene therapy for CNS diseases. Given the difficulty of IT injection in smaller animals like mice, we combined the two applications of direct lumbar puncture and chose local anesthetics (lidocaine, which has been used in clinics widely as an objective indicator of injection quality by evaluating transient and restorable paralysis). Additionally, we defined a standard to predict delivery efficiency of AAV through paralysis levels and confirmed this by immunostaining. We demonstrated that the well-injected animals had higher levels of rAAVrh10-EGFP transduction in the CNS in adult mice.
Compared with the previous intrathecal delivery method involving deep anesthesia of the animal and intrathecal catheterization with laminectomy14,15, our current method has several advantages. First, the simple lumbar puncture procedure can be completed within a few minutes for each animal, whereas the previous procedure takes ~1 h per animal. Second, the current method does not employ anesthesia and surgery, and therefore reduces the risk of injury4. Third, by addition of 1% lidocaine hydrochloride to the AAV solution, we established a five-point scoring system to rank transient paralysis following the injection and proved that the degree of weakness induced by lidocaine can be used to predict the extent of CNS transduction by each injection. Our data demonstrated that the well-injected animals have high levels of rAAVrh10-EGFP transduction in the CNS of adult mice. The transduction is also widespread to a similar extent of the earlier method involving laminectomy and intrathecal catheterization. Compared with existing IT puncture methods in awake mice, we provide an objective indicator of injection quality by using lidocaine and avoid the blindness to failed injection and subsequent interference in therapeutic efficacy.
Taken together, the current intrathecal delivery containing 1% lidocaine is a promising method in experimental therapies for CNS diseases by delivering genes or drugs in mice. Furthermore, it is a practical and convenient approach to practice IT injection in small animals such as mice.
The authors have nothing to disclose.
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.
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 |