The authors have declared that no competing interests exist.
Conceived and designed the experiments: SP JG MSE AS-R DJK XOB. Performed the experiments: SP JG XZ DG RB JB GRW. Analyzed the data: SP JG RB JWC GRW MSE AS-R DJK XOB. Contributed reagents/materials/analysis tools: SP JG JB DG MSE AS-R DJK XOB. Wrote the paper: SP JG JWC AS-R DJK XOB.
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder due to mutations in either TSC1 or TSC2 that affects many organs with hamartomas and tumors. TSC-associated brain lesions include subependymal nodules, subependymal giant cell astrocytomas and tubers. Neurologic manifestations in TSC comprise a high frequency of mental retardation and developmental disorders including autism, as well as epilepsy. Here, we describe a new mouse model of TSC brain lesions in which complete loss of
Tuberous sclerosis complex (TSC) is a genetic disorder affecting about 1 in 6,000 newborns caused by inactivating mutations in
Although there is clear evidence that loss of a single allele of
A number of mouse models of TSC brain disease have been generated using conditional alleles of either
In this study we aimed to recreate the stochastic nature of
AAV vector plasmid, AAV-CBA-Cre-BGHpA was derived from the plasmid AAV-CBA-EGFP-W
AAVrh8 and AAV1 serotype vectors were produced by transient co-transfection of 293T cells by calcium phosphate precipitation of vector plasmids (AAV-CBA-Cre or AAV-CBA-GFP-W), adenoviral helper plasmid pFΔ6 and a plasmid encoding for the AAVrh8 cap (pAR-rh8) or AAV1 cap gene (pXR1), as previously described
Experimental research protocols were approved by the Institutional Animal Care and Use Committee (IACUC) for the Massachusetts General Hospital (MGH) following the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals. Experiments were performed on
For vector injections, on the day of birth (P0), neonates were cryo-anesthetized and injected with 2 µl of viral vector into each cerebral lateral ventricle with a glass micropipette (70–100 µm diameter at the tip) using a Narishige IM300 microinjector at a rate of 2.4 psi/sec (Narshige International, East Meadow, NY, USA). The viral vector solution consisted of either 2×1010 g.c. per 2 µl or 2×109 g.c. per 2 µl. Mice were then placed on a warming pad and returned to their mothers after regaining normal color and full activity typical of newborn mice. Mice were euthanized when they showed a weight loss of >15%, greatly reduced movement or other signs of distress.
Mice were sacrificed using CO2 generated from dry ice and brains were rapidly frozen in 2-methyl-butane/dry ice bath. Coronal serial sections were cut to a thickness of 10 µm using a cryostat, directly mounted on glass slides and stored at −80°C. Slides were fixed in 0.25% glutaraldehyde in PBS (pH 7.4) for 20 min, stained in X-gal solution (Gold Biotechnology, St. Louis, MO, USA) overnight at 37°C and counterstained with Nuclear Fast Red solution (Vector Laboratories, Burlingame, CA, USA).
For standard histologymouse brains were prepared after euthanasia with CO2by immediate removal of brains and 2–4 days of fixation in Bouin’s solution (VWR International, Radnor, PA). Following paraffin embedding, 5 µm sections were cut and stained with either Haematoxylin and Eosin (H&E) or were used for IHC. IHC was performed after deparaffinization and re-hydration steps and antigen retrieval in citrate buffer (pH 6) using the EnVision System (Dako, Carpinteria, CA, USA) or HistoMouse-Plus kit (Invitrogen, Carlsbad, CA, USA), per manufacturer’s instruction. pS6 antibody (#2211) and TSC2 antibody (#4308) were from Cell Signaling (Danvers, MA, USA).
For DCX, GFAP, NeuN and GPNMB staining, sections were deparaffinized in xylenes followed by re-hydration in decreasing ethanol concentrations. Endogenous peroxidase was blocked with 0.5% hydrogen peroxide, then tissues were washed in PBS. Heat-induced antigen retrieval was performed using sodium citrate 10 mM, pH 6.0, in a 95°C water bath (20 min for GPNMB and 30 min for DCX). Tissues were blocked in a 10% normal goat serum (GPNMB) or 10% normal horse serum (GFAP, DCX, NeuN) then incubated overnight with primary antibody. Dilutions were: 1∶300 rabbit polyclonal GPNMB (Lifespan Biosciences, Seattle, WA, USA) LS-C80662/28556, 1∶250 goat polyclonal DCX (Santa Cruz Biotechnologies, Santa Cruz, CA, USA, sc-8066), 1∶400 mouse monoclonal NeuN (MAB377, EMD Millipore) and 1∶800 mouse monoclonal GFAP (Sigma, St. Louis, MO, USA, C9205). Tissues were incubated for 30 min with secondary antibody 1∶250 biotinylated goat anti-rabbit (Vector Laboratories, BA-1000), 1∶250 biotinylated horse anti-goat (Vector Laboratories, BA-9500) and 1∶250 and 1∶400 biotinylated horse anti-mouse (Vector Laboratories, BA-2000), washed in PBS and incubated with avidin-biotin complex (Vector Laboratories, PK-6100) for 30 min. Staining was performed with 3,3′-diaminobenzidine (DAB) in H2O2 (Vector Laboratories), then counterstained with haematoxylin, dehydrated and coverslipped.
Mice were sacrificed at 1 month of age by transcardiac perfusion with PBS followed by ice-cold 4% paraformaldehyde in PBS. Brains were dissected and post-fixed for 4 hrs at 4°C, followed by overnight incubation in 30% sucrose in PBS at 4°C and were embedded in tissue freezing medium (Tissue-Tek O.C.T compound, Sakura Finetek Inc., Torrance, CA, USA). Coronal serial sections were cut to a thickness of 10 µm and directly mounted on glass slides. Sections were stained for the neuronal marker, 1∶1000 mouse monoclonal NeuN (MAB377, EMD Millipore) or glial marker, 1∶500 mouse monoclonal anti-GFAP (Clone G-A-5 Cy3 conjugate, Sigma), or for pS6 - 1∶1000 pS6 rabbit antibody (Cell Signaling) in 0.1% Tween-20 in PBS overnight at 4°C, washed in PBS 3×10 min, incubated for 30 min with 1∶1000 Alexa 488-conjugated goat anti-mouse secondary antibody (Life Technologies, Grand Island, NY, USA) or 1∶1000 Alexa 546-conjugated anti-rabbit secondary antibody (Life Technologies) in 0.1% Tween-20 in PBS. After another 3×10 min washes in PBS, sections were counterstained with 4,6 diamidino-2-phenylindole (DAPI, Sigma) for 5 min, washed in PBS and coverslipped.
In NeuN immunofluorescence stained brains, the widest diameter of NeuN+ cells in cortex was measured using Metavue software (Molecular Devices, Sunnyvale, CA, USA) for 180 randomly selected cells in the cortex just above the lateral ventricles from 3 animals in each group. In NeuN immunostained brain, the widest diameter of NeuN+ cells in cortex was measured using photoshop software (Adobe) for 30 randomly selected cells in the cortex just above the lateral ventricles from 3 animals in each group.
The mice were scanned using a T2-weighted TurboRARE-3D (TE = 43 ms and TR = 1200 ms) sequence for a 192×256×192 matrix and a voxel size of 0.0976×0.130×1.302 mm after reconstruction on a Bruker Pharmascan 4.7 tesla magnet using a Bruker mouse brain coil (Model T8118, Bruker Scientific Instruments, Billerica, MA USA). Regions of interest (ROI) of the brains were automatically segmented using a house-built Matlab program by searching for the largest connected region of a given threshold. These ROI’s were manually modified using Amira software (Amira, Burlington, MA, USA) to check for any anomalies in the auto-segment routine. An additional ROI was manually drawn to represent the normal brain tissue and, utilizing a region-based threshold that 3 standard deviations above the mean normal brain value, the CSF was segmented. This segmented region was manually modified by removing any spurious voxels above this threshold around the edges of the brain that were due to partial volume effect.
All analyses of survival curves (Chi square test) and brain ventricular volumes (t test) were performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). The p values depicted are statistically significant.
We initially evaluated the survival of
Survival is shown for
Brains obtained from
In
Since the above mice demonstrated high level and global brain recombination, we also injected two
(A) Co-immunostaining for GFAP and NeuN in AAVrh8-CBA-Cre injected
To achieve a milder phenotype than in the
Death was determined by the point at which animals were in severe distress at which time they were euthanized.
Extensive histological analysis was carried out on
Brains obtained from
Immunostaining for NeuN (counterstained with haematoxylin) shows that neurons above the lateral ventricles in (A) AAV1-CBA-Cre injected
To assess astrocyte transduction, brains of AAV1-CBA-Cre and AAV1-CBA-GFP neonatal injected
Injection of AAV1-CBA-Cre (N = 4) or AAV1-CBA-GFP (N = 2) 2×1010 g.c. per 2 µl into each ventricle was carried out on P0 and MR images were evaluated on P30. (A) Measurement of ventricle size in voxels (each voxel = 0.0976×0.130×1.302 mm).Difference between groups is significant (p<0.044). (B) Measurement of brain parenchyma (excluding ventricle volume) in voxels. Difference between groups, p<0.23, not significant. Measurements were made by observer blinded to genotype.
AAV1-CBA-Cre injected mouse brains (see Fig. 8 legend) showed abnormalities associated with the ventricular regions. (A) Multiple apparent subependymal nodules (arrowheads) were seen in the ventricles in the brains of two AAV1-CBA-Cre injected mice. First two images are from the same mouse. (B) Ventricles also appeared to have thickening of the ependymal lining (arrowheads; two left panels). (C) None of these abnormalities were observed in the control vector (AAV1-CBA-GFP)-injected brains.
(A) Three months after ICV vector injection into
Coronal sections of brains of AAV1-CBA-GFP and AAV1-CBA-Cre injected
In order to identify the phenotype of the abnormal ventricular structures in AAV1-CBA-Cre injected
In this study we have generated a new model of TSC brain disease by exogenous injection of Cre expressing AAV virus at birth to pups that are homozygous for the conditional allele of
Subependymal nodules (SENs) are seen in the great majority of TSC children. The potential for growth of these lesions has led to formal guidelines that recommend frequent periodic screening by MRI in TSC children and young adults. Further, a recent report indicated that about one third of SENs were observed to grow over a 4-year period postnatally
Although rapamycin and related drugs represent a major breakthrough in the therapy of TSC tumors that develop at multiple sites
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Histological analysis was carried out on
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We thank Ms. Suzanne McDavitt for skilled editorial assistance, Michelle Forrestall Lee, the Medical Photographer in Pathology Media Lab, MGH for her assistance in acquiring images, Mei Huan Lin and Shelby Parton for assistance with MLPA analysis.