Therapeutic Effects Of AAV-medicated MicroRNA Delivery In ALS Mice Between Different Intrathecal Injection Speeds

Objective: Amyotrophic lateral sclerosis(ALS)is a fatal neurodegenerative disease characterized by progressive loss of motor neurons in spinal cord,brain stem,and cortex.SOD1 is the first ALS causative gene and its mutation has been the leading cause of familial cases.Although precisely how mutant SOD1(m SOD1)causes ALS remains unknown,silencing of m SOD1 induced by AAV proves to be a promising therapeutic approach.However,optimal delivery method to achieve the highest transduction efficiency and most widespread gene delivery in the entire central nervous system has been constantly explored.Here,on the basis of our previously defined,efficient intrathecal(IT)delivery method,we first examined the influence of injection speed on transduction profile in the central nervous system(CNS),as well as the transduction intensity at different periods after IT delivery of r AAVrh.10-GFP-ami R-SOD1 in SOD1-G93 A mice.Then the corresponding therapeutic effects were further explored.Methods:1 Intrathecal(IT)delivery and experimental groups1.1 Intrathecal(IT)delivery Direct lumbar puncture was carried out in SOD1-G93 A mice at 30 days of age.Eight microliters of PBS or r AAVrh.10-GFP-ami R-SOD1 vector(3×1012GC/ml)16 with 1% lidocaine hydrochloride was intrathecally injected.The AAV vector treated group was further divided into two subgroups according to the different injection speed,which is low speed of 8?l in 8min(8?l/8min)or high speed of 8ul in 30s(8?l/30s).Scoring of transient weakness of the mouse limbs was used to evaluate whether the injection is successful or not.Score 0 meant no weakness,score 1 indicated minor weakness of hind limbs,score 2 moderate weakness of hind limbs with gait abnormality and single hind limb paralysis,score 3 paralysis of both hind limbs,score 4paralysis of hind limbs,breath hardly and moderate weakness of fore limbs,score 5 paralysis of all four limbs.Only successfully injected mice were analyzed in this study(score 4-5).1.2 Experimental groups Male and female SOD1 transgenic mice with Gly93 Ala mutation were divided into three groups respectively: the low-speed group(r AAVrh.10-GFP-ami R-SOD1,8?l/8min),the high-speed group(r AAVrh.10-GFP-ami RSOD1,8?l/30s)and the control group(PBS,8?l/8min ? 8?l/30s),and according to our observation,injection speed had no significant influence on the survival time of the mice treated with PBS,so we compared with one control group.2 Behavioral observation and weight measurement To monitor the disease progression,all animals were inspected daily for signs of motor deficit and weighed per week,starting at 30 days of age.Wight was measured once a week before 90 days,and twice a week later,and the measurement was conducted during the same time.And the limb footprint was recorded at 110 d,120 d,and 130 d respectively.Disease onset was determined when the following criteria were met: two continuous weight losses were observed after the animals reached their peak body weight.The end stage was defined when the animals can no longer right themselves within30 s after being placed on their backs or sides.Mice were sacrificed at different time points and tissues were harvested for histological processes or western blot analysis.3 Histological processing and immunostaining3.1 Histological processing Three or four SOD1-G93 A mice were sacrificed at different time points,6 weeks after injection,around onset age(about 120 days of age),or till end stage(135-144 days of age).Mice were deeply anesthetized and transcardially perfused with PBS followed by fixation buffer containing 4%paraformaldehyde in PBS.Brain,spinal cord with roots were dissected carefully and post-fixed for approximately 24 h in the same fixative.Brain and spinal cord were cryoprotected in 30% sucrose for 24 h,and then embedded in OCT compound.Twenty-five micron frozen sections were cut using a Leica cryostat.And the spinal nerve roots of L4-L5 were preserved in PBS for use.Moreover,Three or four mice around onset age(about 120 days)each group were dealt with 2.5% glutaraldehyde,then the spinal nerve roots of L4-L5 were preserved in 4% glutaraldehyde for toluidine blue staining.3.2 Immunohistochemistry Free-floating sections were pre-treated in 1% H2O2 and then washed in PBS.After incubation in blocking solution containing 5% goat serum and0.3% Triton X-100 in PBS,slices were incubated in primary antibodies in the blocking solution at 4°C overnight.The primary antibodies used were as follows: anti-GFP,anti Iba-1,anti-GFAP,anti-Neu N,and anti-APC.Following incubation,sections were washed in PBST(0.2% Tween 20 in PBS),and then incubated with corresponding biotin-secondary antibody.After washing,sections were incubated in the VECTASTAIN ABC REAGENT,and stained using DAB.The sections were then mounted onto slides and dried properly.After soaking in xylene,the slides were sealed with mounting medium and photographed with Olympus microscope.For the quantification of motor neuron numbers,Neu N-positive cells,located in the ventral horn with an identifiable nucleus and a cellular diameter larger than 20?m were counted.3.3 Immunofluorescence For immunofluorescence processing,spinal cord sections or the whole mount of roots were pre-treated in 1% Triton X-100 in PBS,after washed in PBS tissues were incubated with blocking solution.Primary antibodies,including anti-GFP and anti-h SOD1,anti Iba-1,anti-GFAP,mouse anti-Neu N,and anti-APC,in the blocking solution were added to the tissues and incubated at 4°C overnight.Following the incubation,sections or roots were washed,and incubated in the appropriate secondary antibody.After washing in PBS,sections and roots were mounted with mounting medium containing DAPI and sealed with nail polish.Images were taken with an Olympus FV1000 confocal microscopy.3.4 Toluidine blue staining For toluidine blue staining,ventral roots were dissected after transcardial perfusion of SOD-G93 A mice at about 120 days of age and post-fixed in 4%glutaraldehyde for 24 h.After washing in phosphate buffer,roots and nerves were incubated in 1% osmium tetroxide,dehydrated through graded acetones,and embedded in epoxy resin Epon 812.Transversal semithin sections(1?m)were cut and placed on the slides,and oven dried.The slides were stained with 1% toluidine blue solution,rinsed with running water,dehydrated and mounted.As for the quantification of axons,integrated image of ventral roots were captured with Olympus microscope and were counted using IPP 6.0 software.More than three slices from three mice in each group were analyzed.Those nerve fibers with a lightly stained axon and homogenously dark myelin sheath were classified as normal myelinated nerve fibers.4 Western Blotting Total protein was extracted from frozen tissues of end-stage mouse(140-147 days of age),including spinal cord and brain using a protein extraction kit.The lysates of each sample(50?g protein)were separated in12% SDS–PAGE gels,and transferred to PVDF membranes.The membranes were then probed overnight at 4°C with different primary antibodies with primary antibodies,including anti-GFP,anti-SOD1,and anti-GAPDH.The bands of interest were detected using an Odyssey Infrared Imaging System,and GAPDH was used as internal control.5 Statistical analysis All data were presented as mean ± SEM,all statistical analysis were conducted using SPSS 18.0 software.p < 0.05 was considered statistically significant.Ages of onset and survival rates were analyzed using Kaplan–Meier analysis.Comparison among multiple groups was analyzed using the One-way ANOVA followed by a Student–Newman–Keuls test or Dunn T test.Independent-sample T Test was used for comparison between two groups.Results:1 r AAVrh.10 conducts sustained transduction throughout the spinal cord following intrathecal delivery in SOD1-G93 A mice and displayed a higher efficacy at a lower injection speed.2 Quick injection leads to a superior transgene expression in brain.3 Low-speed intrathecal r AAVrh.10-GFP-ami R-SOD1 delivery achieves a higher transduction in spinal nerve roots and protects ventral roots better.4 Low-speed intrathecal injection of r AAVrh.10-GFP-ami R-SOD1 delays disease onset and extends survival of SOD1-G93 A mice.5 r AAVrh.10-GFP-ami R-SOD1 transduces neurons and glial cells,and silences the expression of mutant SOD1 in vivo.6 Low-speed intrathecal r AAVrh.10-GFP-ami R-SOD1 delivery reduces gliosis and protects motor neurons in the lumbar anterior horn of SOD1-G93 A mice.Conclusion: Intrathecal injection of rAAVrh.10-GFP-ami R-SOD1 leads to widespread transduction throughout the CNS in the whole lifespan of ALS mice and shows therapeutic effects on SOD1-G93 A mice.Interestingly,the transduction profiles and therapeutic effects varies according to different injection speeds,suggesting the transiently elevated CSF pressure plays a role in the diffusion of AAV vectors.