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We compared adeno-associated virus (AAV) serotypes for expression levels of green fluorescent protein (GFP) in the adult rat hippocampus by biophotonic imaging. Preparations of AAV serotypes 8, 9, Rh10, and Rh43 incorporating cytomegalovirus (CMV) promoter–driven GFP were purified by a CsCl method. Neither AAV Rh10 nor AAV Rh43 produced greater levels of GFP than AAV8, which was used as a reference. For AAV9, there was an increase relative to AAV8. The CsCl-purified AAV8 displayed an astroglial transduction pattern in contrast to the expected neuronal expression of other AAVs. After preparing the same CMV-GFP plasmid in AAV8 with an iodixanol purification method, the expected neuronal pattern resulted. The astroglial expression with the CsCl AAV8 was probably due to relatively high levels of protein impurities. We compared the CMV promoter with the CMV/chicken β-actin (CBA) promoter in the context of AAV8, both prepared by iodixanol, and found the CBA promoter to produce stronger GFP expression. At two doses of vectors optimized for serotype, promoter and purification, we did not observe serotype differences among AAV8, AAV9, or AAV Rh10. The purification method can therefore impact the transduction pattern as well as the results when comparing serotype strengths.
There are many adeno-associated virus (AAV) serotypes.1,2 We investigated several in this study for efficiency of high expression levels, in order to decide which one to use for our research on neurodegenerative diseases in rat brain models.3 Comparative data on brain gene transfer with AAV serotypes may also be useful for ongoing clinical trials using AAV for neurological and neurodegenerative diseases.4 There are a number of studies comparing AAV serotypes in the brain, consistently showing improved gene transfer with newer serotypes as compared to the first serotype that was well characterized, AAV2.5–12 We found that AAV8 was a strong vector for hippocampus relative to AAV2 or AAV5,8 while all these serotypes led to transgene expression that was largely limited to neurons, consistent with earlier studies (reviewed in ref. 13). We proceeded to compare the robust AAV8 with some newer serotypes in this study. AAV8 is also an efficient vector for liver14 or muscle15 and AAV9 has recently been shown to transduce cardiac tissue efficiently, to a greater extent than AAV8.16,17 There is evidence for enhancement of gene transfer with either AAV9 or AAV Rh10 (AAV10) compared to AAV8 in certain mouse brain regions,18 and for AAV10 relative to AAV8 in the rat brain.19 We studied the relative pattern of expression for AAV8, AAV9, AAV10 in rat hippocampus. The biophotonic method that we used is rapid and quantitative for green fluorescent protein (GFP) and shows a two-dimensional spread of expression while imaging the entire brain at once.8 These results were confirmed with GFP Western blots of rat hippocampus. GFP expression in hippocampal astroglia was surprisingly observed in the case of CsCl-purified AAV8. We investigated if the astroglial expression was due to serotype, or alternatively, due to the purity of the vector stock, looking at CsCl or iodixanol purified vectors. We compared two promoters, the cytomegalovirus (CMV) promoter and the CMV/chicken β-actin (CBA) promoter, for their relative strengths and the neuronal/ glial transduction pattern. AAV8, AAV9, or AAV10 vectors with the CBA promoter and purified by iodixanol, were tested at two doses in order to choose the strongest serotype using an optimized promoter and purification method.
We received a vector set from the University of Pennsylvania Vector Core laboratory for AAV serotypes 8, 9, 10, Rh 43 (AAV43). This set uses the CMV promoter/woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to drive GFP expression, and these AAVs were purified by a CsCl-gradient method. These stocks had a visible green tint, which suggested there was residual GFP generated during packaging that was unsuccessfully removed by CsCl. It is not unusual to observe GFP in such preparations but still use them in experiments in liver and muscle (G.P. Gao, Gene Therapy Program, University of Pennsylvania, personal communication, 20 March 2007); so even though these four stocks had contaminating GFP, we compared them as a set relative to each other.
The AAVs were added to human embryonic kidney 293-T cells and they all led to increase in GFP from 1 to 5 days. The GFP was not observed during the early intervals, so this shows that the contaminating GFP in the preps was not producing the signal. With equal vector genome (vg) concentrations added, the AAV8 produced the brightest GFP and AAV43 the least, with AAV9 and AAV10 being similar to each other (Supplementary Figure S1). The AAV8 was brighter throughout the 5 days as expressed by shorter automatic camera exposure times. The batches of CMV-GFP AAV8, 9, 10, 43 purified by CsCl all resulted in visibly lower cell densities relative to untreated samples when 1–5 µl of the AAVs were added to a 24-well with 1 × 105 cells. The cytotoxicity was pronounced with the batch of AAV8 (Supplementary Figure S2).
Iodixanol-purified vectors were prepared at our laboratory. The same GFP plasmid used above (CMV/WPRE), obtained from the University of Pennsylvania Vector Core, was used to make AAV8 iodixanol vector. Alternatively, a CBA/WPRE GFP plasmid was packaged into AAV8 and purified by iodixanol. This GFP plasmid and the iodixanol preparation of the AAV8 were the same as before.8 Based on the preliminary results we observed with the AAV9 or AAV10 CsCl vectors, we made CBA AAV9 or AAV10 iodixanol vectors so as to optimize serotype, promoter and purification. These stocks were all well tolerated in the 293-T cells with no sign of cell loss with up to 40 µl added per 24 well with 1 × 105 cells, the highest volume tested (Supplementary Figure S2), in contrast to the CsCl vectors above. The relative strengths of the iodixanol-purified vectors for GFP expression in 293-T cells were AAV8 > AAV9 = AAV10 using the CBA plasmid, which was the same pattern for the AAV8, 9, 10 CsCl-purified CMV-GFP vectors above. Camera exposure times were always shorter with the AAV8. However, in terms of the fraction of visibly green cells, the iodixanol AAV8, 9, and 10 vectors all produced 100% transduction in vitro, with the percentage of green cells from AAV9 or 10 approaching that of AAV8 within 4–5 days. The CMV promoter was more efficient than CBA in vitro with AAV8 iodixanol vectors (Supplementary Figure S3).
We injected the CMV/CsCl-purified AAV vectors into the dorsal hippocampus at a fixed dose of 1.28 × 1011 vg. Four weeks later, brains were prepped for biophotonic imaging (Figure 1). The GFP luminescent signal was quantified for both intensity (photons/second) and spread (cm2) as per the IVIS system manufacturer’s instructions. We observed a trend for greater intensity with either AAV9 or AAV10 relative to either AAV8 or AAV43 with eight rats per serotype group. In the analysis of variance, there was a significant serotype effect (F3,31 = 3.35, P < 0.05), although no individual serotype comparisons were significant in Bonferroni’s multiple comparison post test; this was a surprise because on average the intensity was 3.3-fold greater for AAV9 relative to AAV8 (Table 1), which did result in a difference in an individual t test (P < 0.01). For the comparison of spread, there were individual group differences. The serotype effect for spread was significant (F3,31 = 9.25, P < 0.0005) and in the Bonferroni post test, AAV9 was greater than AAV8 (P < 0.05) and AAV43 (P < 0.001), and AAV10 was greater than AAV43 (P < 0.05). The spread of the GFP luminescent area above the whole brain was 2.5-fold greater for AAV9 as compared to AAV8 (Table 1). While there were discernible individual serotype differences for spread but not intensity in the analysis of variances, the two values correlated (r = 0.86, P < 0.0001, N = 32).
The same vectors were compared for GFP levels on Western blots (Figure 2) at the same vector dose and expression interval as above. The GFP signal was normalized to the housekeeping gene product glyceraldehyde-3-phosphate dehydrogenase for comparisons and AAV9, 10, or 43 were directly compared to AAV8 on separate blots. While we observed a considerable degree of variability in the AAV8 group (Figure 2a), the normalized GFP signal was significantly greater for AAV9 relative to AAV8 by 92% (P < 0.05, t-test, N = 5–7/serotype). The ~2-fold difference on Westerns matched the 2.5-fold greater area observed with AAV9 relative to AAV8 with biophotonic imaging. GFP levels from AAV10 (Figure 2b) or AAV43 (Figure 2c) were similar to AAV8 on Westerns.
We prepared the same CMV/WPRE-GFP expression plasmid as used above and purified it by using iodixanol; we then compared it to the CBA/WPRE-GFP plasmid, both packaged into AAV8 and purified the same way (Figure 1b). Both AAV8 promoter vectors were injected at a dose of 7.8 × 109 vg and the expression interval was 3 weeks. Despite an average 14-fold greater intensity with the CBA promoter there was no significant difference due to variability, although there was a significant 4.6-fold greater spread in the CBA group (Table 1; P < 0.005, t-test). The 293-T cell culture model did not predict in vivo relative to either serotype strength or to promoter strength, consistent with observations by others.10
We then compared AAV8, 9, 10 at two vector doses using the optimized CBA promoter and iodixanol purification (Figure 1c). We analyzed the data by means of serotype-by-dose two-way analysis of variances and found a significant dose effect for photons/sec (F2,23 = 15.36, P < 0.001) and for area (F2,23 = 27.24, P < 0.0001), although no serotype effect or interaction for either comparison was found. Individual comparisons by t-test yielded a dose effect on photons/second for AAV9 or AAV10 (P < 0.03) and on area for AAV8, AAV9, or AAV10 (P < 0.03). While no serotype differences were found in the analysis of variances, we noticed a trend for more consistent and efficient expression with the low-dose AAV9 relative to the low-dose AAV10 or AAV8: there was a difference between AAV9 and AAV10 in individual t-tests for either photons/second (P < 0.002) or area (P < 0.01), and no differences between AAV8 and AAV10 at the low dose.
When we viewed GFP epifluorescence on sections, the results with the CMV-GFP AAV8 vector purified by CsCl were surprising, as this vector did not produce the usual neuronal pattern of AAV transduction. Previous studies with AAV1, 2, 5, 8 in rat hippocampus all showed an almost exclusive neuronal transduction, as we observed in this study except for the CsCl-purified CMV-GFP AAV8 (Figure 3). With the latter vector, large GFP positive cells were observed in the stratum radiatum of hippocampus, a position where we normally do not see transduced cells, and the cells had non-neuronal morphology (Figure 3a; more examples of non-neuronal morphology and positions in Supplementary Figure S4). In contrast, CMV-AAV9 (Figure 3b), CMV-AAV10 (Figure 3c), CMV-GFP AAV43 (Figure 3d) all produced results in the expected transduction pattern, by incorporating cells with neuronal morphology, mainly in the positions of dentate gyrus granule cells, hilus, CA1 pyramidal cells, stratum oriens. The overall strength of the GFP signal on sections matched the Western and biophotonic data, with AAV9 appearing brighter relative to AAV8 and even more so relative to AAV43. All of the vectors were efficient in transducing the dentate gyrus and hilus (the lower part of Figure 3a–d) and AAV9 had the most propensity for transduction of CA1 pyramidal neurons (upper part of Figure 3b) with this vector set.
We tested if the non-neuronal pattern in Figure 3a was due to the purification method. The vectors used in Figure 3a–d were purified by CsCl. When we packaged the same CMV-GFP plasmid in AAV8 as in Figure 3a and then purified it by iodixonal, the results suggested that purification method was the most important factor, as this vector showed the usual neuronal transduction pattern in the dentate gyrus (Figure 3e). Using the CBA promoter rather than CMV, GFP expression was stronger with AAV8 (Figure 3f), AAV9 (Figure 3g), and AAV10 (Figure 3h), incorporating efficient neuronal transduction of both dentate gyrus and CA1 pyramidal cells. While the purification method is probably important for the different patterns found between Figure 3a and e, it is worth noting that AAV9, 10, 43 in Figure 3b–d were also purified by CsCl and showed mainly a neuronal pattern.
For the CsCl-purified vector stocks from Figure 3a–d, we confirmed neuronal and astroglial transduction patterns with antibodies for NeuN and glial fibrillary acidic protein (GFAP), respectively. It was clear that the AAV8 transduced non-neuronal cells, i.e., GFP cells, did not co-localize with NeuN (Figure 4a and b). There were substantial numbers of cells positive for both GFP and GFAP in the AAV8 group (Figure 4c–e). While the non-neuronal and astroglial transduction were clear with this vector, so was an upregulation of GFAP staining, or astrogliosis (Figure 4d). For example, there was less GFAP staining in the injected area with the AAV9 (Figure 4f) and this general pattern was observed throughout the study. The expression from the CsCl-purified AAV9 showed the normal neuronal pattern (Figure 4g) and co-localized with NeuN (Figure 4h). The enhanced astroglial staining and astroglial transduction with the AAV8 vector could be related to serotype since the promoter and purification method remained the same for this set. However, the purification method was important too as the AAV8 CMV-GFP that was purified by iodixanol displayed the usual neuronal pattern (Figure 3e), as did the AAV8 CBA-GFP iodixanol vector (Figure 3f).
All of the vector injections caused an astrocytic scar along the needle track as did diluent vehicle injections. Diluent and iodixanol-purified AAV9 are shown in Figure 5. The scar was generally more intense and widespread over ~6 (300 µm) sections for AAV injections compared to vehicle, where the scar was usually observed on ~3 sections (150 µm). The batch of CsCl-purified AAV8, however, produced a greater and more widespread increase in GFAP away from the needle track, which overlapped with the spread of GFP expression. For example, the batch of CsCl-purified AAV8 led to more GFAP staining than the similarly prepared AAV9 (Figure 4), and the latter was like all the rest of the vectors used causing some elevation of GFAP around the needle track as observed with the iodixanol-purified AAV9 (Figure 5).
Although not quantified, the batch of the CMV-GFP CsCl-purified AAV8 appeared to uniquely induce astroglial transduction as well as astrogliosis, as the GFAP staining induced from the other vectors used, was more limited to the needle track. Instances of cells that appeared to be non-neuronal, either with astroglial shape or positioned in white matter, occurred in all of the vector groups, but rarely, whereas propensity for astroglial transduction was efficient with the batch of the CsCl-purified CMV-GFP AAV8. To confirm that the unusual non-neuronal pattern of GFP expression in the hippocampus with the CsCl AAV8 was due to gene transfer and not potential uptake of the residual GFP that was present, we injected a rat injected the same way and viewed the tissue 1 day later and found no GFP signal on sections. Because the unusual non-neuronal transduction pattern was only observed with one of the four batches of the CsCl-purified AAVs, the AAV8, we obtained and tested a second batch of the CsCl-purified AAV8. We observed a similar non-neuronal transduction pattern with the second batch of AAV8, consistent with the results from the initial batch tested. Two out of five batches of the CsCl-purified AAVs resulted in an unexpected non-neuronal transduction pattern, both times with CsCl-purified AAV8.
Because the purification method appeared to be more important in determining the astroglial expression in Figure 4 than either promoter or serotype effects, we evaluated the relative levels of proteins present in the preps. We viewed the proteins by Ponceau S staining of a transferred gel that was directly loaded with AAVs at a fixed load of 1.3 × 1011 vg (Figure 6a). Overall, there were more proteins in the CsCl preps. Among the CsCl vectors, the AAV8 prep had comparatively the highest levels of proteins, with protein bands barely detectable for the iodixanol preps. We ran GFP immunoblots for the directly loaded AAVs because the CsCl preps had a visible green tint, and all of the CsCl preps had detectable levels of GFP on Western blots (Figure 6b). The CsCl AAV8 had comparatively the highest level of residual GFP, while no GFP at all was detected in iodixanol preps, with all serotypes tested up to 2.5 × 1011 vg. Despite the clear differences between the CsCl and the iodixanol preps for overall proteins and GFP, immunoblots for AAV capsid proteins showed bands of consistent sizes (~60–90 kd) and stoichiometry (1:1:20) for VP1, VP2, VP3 proteins20 for both the CsCl and the iodixanol preps (Figure 6c). The B1 capsid antibody appeared to detect AAV8, AAV9, AAV10, or AAV43 equally well and AAV9 capsid proteins consistently ran at smaller sizes relative to the other serotypes.
The AAV9 and AAV10 vectors incorporating the strong CBA promoter and purified by iodixanol produced robust widespread GFP expression that was well targeted to the hippocampus (Figure 7 and Supplementary Figure S5). Both vectors resulted in efficient transduction of the dentate gyrus granule layers to a more posterior extent than found in previous studies. The AAV9 led to more efficient transduction of the CA1 pyramidal neuron layer compared to the AAV10, consistently throughout the study as shown in Figure 7. However, pyramidal neuron transduction with AAV10 was detected better with a GFP immunostaining method (Supplementary Figure S4c) than with GFP fluorescence. While not quantified, viewing sections for GFP and comparing these two serotypes suggested that the AAV9 transduced more cells and that the intraneuronal expression levels derived from AAV10 were higher, with brighter GFP in fewer transduced cells. The GFP-labeled anterograde projections in the contralateral hippocampi appeared similar with either serotype (Figure 7d and h), which may be consistent with a balance between number of cells transduced and intraneuonal levels. Examples of retrogradely transduced cells in distal sites that innervate the injected hippocampus (i.e., GFP expressing cell bodies in entorhinal cortex, medial septum, contralateral hippocampus), were found in all of the vector groups used in the study including the CsCl vectors. We did not notice any promoter, serotype, or purification effect on the frequency of retrograde transduction, other than that stronger expression at the injection site correlated with more distal GFP cells. The entorhinal cortex is shown in Supplementary Figure S4d after a hippocampal injection of AAV10 purified by iodixanol. Consistent with Reimsnider et al.,12 the weaker expression in distal areas was detected better with GFP immunostaining than with epifluorescence.
AAV9 and AAV10 are efficient gene transfer vectors for the adult rat hippocampus as previously observed in mice.18 Using a similar set of vectors with the same GFP expression cassette and CsCl purification, Cearley and Wolfe18 reported more transduced cells with AAV9 or 10 relative to AAV8 in mouse hippocampus or thalamus, using an in situ hybridization detection method. However the method they used appears to have been run under saturating conditions. In this study in rats (>10 per serotype), with equivalent dosing, we quantified GFP levels by two methods under non-saturating conditions. The rat data corroborated with that of mice18 in terms of AAV9 producing more GFP than AAV8 by both biophotonic imaging and Western blot methods. There was also some evidence of enhancement with AAV10 as it produced a greater spread of GFP on biophotonic imaging than did AAV43, while there was no difference between AAV8 and AAV43, which is also consistent with a study in rat striatum comparing AAV8 and AAV10.19 In contrast to Cearley and Wolfe,18 we found efficient astroglial transduction with the AAV8 from the CsCl/CMV-GFP vector set, and we did not observe a unique all-or-none property of AAV9 for transduction of cells distal to the injection site. However, there was the tendency to observe more GFP expression in distal perikarya with AAV9, which we hypothesize is due to the general correlation we found with stronger proximal expression linked with stronger distal expression, rather than a unique property of AAV9 for retrograde axonal transport.
It was surprising that the CMV-GFP AAV8, purified by CsCl, transduced astroglia in the hippocampus with high efficiency. This was probably due to the relatively high amounts of proteins, including GFP, in this prep. The potential effect of serotype was initially suggested, as similar vectors for AAV9, AAV10, or AAV43 did not show this pattern. The overall protein levels are probably more important than serotype because the CsCl AAV8 had the most extra protein and was the only one to show this pattern, and when the CMV-GFP AAV8 was more successfully purified by iodixanol, astroglial expression was not observed, as was the case with the CBA-GFP AAV8 purified by iodixanol. We hypothesize that the CsCl purification method has a greater likelihood for carryover of non-AAV proteins, which can affect the transduction pattern; we observed a non-neuronal transduction pattern in two of five CsCl-purified batches tested, both of the two being batches of AAV8. While the influence of the promoter cannot be ruled out, a promoter effect may not be as important in conferring the astroglial expression; this is because Harding et al. showed astroglial expression for AAV8 with the CBA promoter and purified by CsCl.10
As the vector that led to expression in astrocytes in vivo had a pronounced cytotoxic effect in vitro, rat brain tissue injury with the CsCl-purified CMV-GFP AAV8 could have been an underlying factor. This vector produced a noticeably more widespread and intense upregulation of GFAP than the astroglial scars produced by either vehicle or other AAV injections purified by either CsCl or iodixanol. In a study using the GFAP promoter in the context of AAV2 in the rat spinal cord, the expression was predominantly neuronal despite the aim for astroglial specificity.21 However, the combination of the GFAP/AAV2 with deliberate physical injury, resulted in a greater propensity for glial expression.22 We therefore hypothesize that AAV serotypes (at least AAV2 and AAV8) infect astroglia and that the transgene expression cassettte is activated only with injury and astrogliosis. The hypothesis is consistent with transduction of cultured astrocytes by AAV2 or AAV8.8,22
A careful study by Reimsnider et al. tracked the micro- and astroglial response to rat brain (striatum) injections of CBA promoter/iodixanol AAV vectors for GFP at 4 days, 1 week, 2 weeks, 4 weeks and 9 months post-gene transfer.12 For AAV1 at a dose of 1.2 × 1011 vg, there was increasing microglia detection for up to 2 weeks and increasing astroglia detection up to 4 weeks. In 9 months, detection for both was back to basal levels. Lower doses of AAVs (AAV2, AAV5, AAV8) caused less micro- and astrogliosis. While the goal of our study was to measure GFP levels in 3–4 weeks, we would predict that the time-course of astrogliosis with the CBA promoter/iodixanol vectors was increasing up to 3–4 weeks (AAV9 at 4 weeks, dose of 7.8 × 109 vg shown in Figure 5) and would attenuate by 9 months, based on Reimsnider et al.12 In fairness, the vectors used in Reimsnider et al.12 used iodixanol followed by the chromatography purification method which results in better purification23 and therefore possibly less astrogliosis than we observed in 4 weeks with our CBA/iodixanol vectors. With respect to the pronounced astrogliosis with the CsCl AAV8 vector (Figure 4), we only studied this for 4 weeks and therefore cannot predict the time-course.
With optimized AAVs (CBA promoter/iodixanol purified), similar widespread and robust neuronal GFP expression resulted with AAV8, AAV9, or AAV10, without pronounced astrogliosis or excessive needle track damage. Because the high expression levels achieved could mask serotype differences, we applied them at a threefold lower dose, which did result in dose-dependent expression although not a significant effect of serotype. However, consistent with the results given above and those of Cearley and Wolfe,18 when using CsCl-purified CMV-GFP vectors18 there was a trend for enhanced expression with AAV9 relative to AAV8 at the low dose. One of the limitations of the AAV vector system for nervous system gene transfer is the inability to transduce glia. Here we showed relatively efficient astroglial transduction with a preparation of AAV8 that had relatively high levels of proteins, including residual GFP generated during packaging. Admittedly the analysis does not address a specific factor that could be applied for astroglial targeting, although achieving astroglial expression could be of value for ailments affecting astroglia, such as astrocytoma,24 amyotrophic lateral sclerosis25 or Alzheimer’s disease.26 AAV vectors from different laboratories can differ in several parameters such as capsid, promoter and purification, which can make direct comparisons difficult. The results suggest that CsCl purification compared to iodixanol purification has a greater probability for residual non-AAV proteins, such as the GFP generated during packaging. We hypothesize that the extra proteins can affect the neuronal transduction pattern, although three of the five batches of CsCl-purified AAVs produced the expected neuronal pattern. The highly efficient neuronal gene transfer with sucessfully purified AAV8, AAV9, or AAV10 will be useful for basic proof of principle studies, such as those in animal models of neurodegenerative diseases.3
A set of vector stocks for AAV8, AAV9, AAV10, and AAV43 was made by the University of Pennsylvania Vector Core. The expression cassette incorporated the AAV2 terminal repeats flanking the CMV promoter-GFP-WPRE-bovine growth hormone polyadenylation sequences (TR2-CMV-GFP-WPRE-pA-TR2). The purification method for this set used three-step CsCl gradients and desalting and concentrating on Amicon Ultra 15 (100K) filter units from Millipore (Billerica, MA). Vector genome copies were quantified by a real-time PCR method.18 Alternatively, vectors were made at our laboratory using iodixanol (OptiPrep; Greiner Bio-One, Longwood, FL) gradient centrifugation followed by washing and concentrating on Amicon filter units, sterilizing with Millipore Millex-GV syringe filters (Billerica, MA), and titering for genome copies by dot-blot.3,8 Vectors were aliquoted and stored frozen. The above-mentioned GFP expression cassette was obtained from the University of Pennsylvania Vector Core and packaged into AAV8 and purified by the iodixanol method. We packaged another expression cassette which we have used repeatedly before:3,8 AAV2 terminal repeats flanking the CBA promoter-GFP-WPRE-bovine growth hormone polyadenylation sequences (TR2-CBA-GFP-WPRE-pA-TR2), into AAV8, 9, or 10, and purified by the iodixanol method. Helper plasmids used in packaging were the same in the case of either preparation method,2,18 also obtained from the University of Pennsylvania Vector Core. Vector preps used in the study ranged from 1 × 1012 to 4 × 1013 vgs/ml and equal dose comparisons were made by normalizing titers with the diluent (lactated Ringer’s solution, Baxter, Deerfield, IL).
Male Sprague-Dawley rats (3-months old, from Harlan, Indianapolis, IN) were anesthetized with a cocktail of 3 ml xylazine (20 mg/ml, from Butler, Columbus, OH), 3 ml ketamine (100 mg/ml, from Fort Dodge Animal Health, Fort Dodge, IA), and 1 ml acepromazine (10 mg/ml, from Boerhinger Ingelheim, St. Joseph, MO) administered intramuscularly at a dose of 1 ml/kg. Viral stocks or lactated ringer’s vehicle were injected through a 27 gauge cannula connected via 26 gauge internal diameter polyethylene tubing to a 10 µl Hamilton syringe mounted to a microinjection pump (CMA/Microdialysis, North Chelmsford, MA) at a rate of 0.2 µl/min. The stereotaxic injection coordinates for hippocampus were 3.6 mm Bregma, 2.0 mm lateral, 3.5, 2.8 mm ventral with 3 µl injected at each depth.27 The needle remained in place at each injection site for 1 additional min before the cannula was moved/removed slowly. The skin was sutured, and the animal was placed on a heating pad until it began to recover from the surgery, before being returned to its individual cage. All animal care and procedures were in accordance with Institutional Animal Care and Use Committee and National Institutes of Health guidelines.
The dorsal hippocampus was dissected and the soluble fraction was prepared in radioimmunoprecipitation assay buffer (1% Nonidet-P40/0.5% sodium deoxycholate/0.1% sodium dodecyl sulfate polyacrylamide gel electrophoresis) with protease inhibitors (Halt protease inhibitor cocktail kit from Pierce, Rockford, IL) by Dounce homogenization and centrifugation. Samples were normalized for protein content by Bradford assay and subjected to 12% sodium dodecyl sulfate/polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA). The primary antibodies for immunoblots were GFP monoclonal from Chemicon (Temecula, CA; 1:1000), glyceraldehyde-3-phosphate dehydrogenase monoclonal from Ambion (Austin, TX; 1:1000), or AAV capsid monoclonal antibody B1 from Meridian (Saco, ME; 1: 1000). Secondary antibody and ECL reagents were from Amersham (Buckinghamshire, UK). The resulting GFP and glyceraldehyde-3-phosphate dehydrogenase bands were on the linear range and compared with ScionImage (Frederick, MD) software and optical units were compared by student t-tests. Polyvinylidene fluoride membranes were also stained for proteins with 0.2% Ponceau S (Sigma, St. Louis, MO)/1% acetic acid.
Anesthetized animals were perfused with phosphate-buffered saline (PBS), followed by cold 4% paraformaldehyde in PBS. The brain was removed and immersed in fixative overnight at 4 °C. Brains were equilibrated in a cryoprotectant solution of 30% sucrose/PBS at 4 °C. Coronal sections (50 µm thick) were cut on a sliding microtome with a freezing stage. Antigen detection was conducted on free-floating sections. Primary antibody incubations were carried out overnight at 4 °C on a shaking platform. For immunoperoxidase staining, endogenous peroxidase activity was quenched with 0.1% H2O2/PBS for 10 min. The sections were washed in PBS and incubated for 5 min in 0.3% Triton X-100/PBS, and washed before applying the primary antibody. Primary antibodies for immunostaining included: NeuN monclonal, GFAP polyclonal (both from Chemicon, Temecula, CA, and used at dilutions of 1:500) and GFP polyclonal from Molecular Probes/Invitrogen (Carlsbad, CA, 1:100,000). Biotinylated secondary antibodies for peroxidase staining were from DAKO Cytomation (Carpinteria, CA; 1:2000), and incubated for 1 hour at room temperature. The sections were washed with PBS and labeled with horseradish peroxidase-conjugated Extravidin (Sigma, St. Louis, MO; 1:2000) for 30 minutes at room temperature. The chromogen was diaminobenzidine (0.67 mg; Sigma, St. Louis, MO) in 0.3% H202, 80 mM sodium acetate buffer containing 8 mM imidazole and 2% NiSO4. After mounting on slides, the sections were dehydrated in a series of alcohol and xylene and coverslipped with Eukitt (Electron Microscopy Sciences, Hatfield, PA). For immunofluorescence, sections were incubated in primary antibody overnight, washed and incubated with Cy3-conjugated secondary antibodies (Jackson ImmunoResearch, 1:300) for 2 hours, followed by 4´,6-diamidino- 2-phenylindole counterstaining (1 µg/ml), washes and coverslipping with glycerol/gelatin (Sigma, St. Louis, MO).
Rats were anesthetized as above and perfused with 100 ml PBS. The brains were then extracted and stored in PBS. The order in which brains were harvested equalized the average time between extraction and imaging session for all the groups. Within 30 minutes, the brains were placed in the Xenogen (Alameda, CA) IVIS 100/XFO-12 apparatus and imaged with the GFP filter set. Exposures were constant (0.5 second/f-stop 16) and viewed with a constant min/max range. The luminescent region of interest above the hippocampus was encircled and quantified for photons/second and area.
We are grateful to the Vector Core of the Gene Therapy Program at the University of Pennsylvania for AAV8, 9, 10, 43 vector stocks, the CMV-GFP plasmid and the helper plasmids for AAV8, 9, 10. We thank Richard Zweig for critiquing the manuscript. NIH/NINDS R01 NS048450 and The Society for Progressive Supranuclear Palsy supported the work.