Rapid hearing loss and outer hair cell loss in kanamycin- and furosemide-treated adult mice
To optimize the use of AAV vectors for therapeutic treatment of hair cell loss, it was important to compare virus-mediated gene transduction in normal and damaged ears of adult mice. For deafening, we employed a recently published method of rapid hair cell degeneration that used a combination of a single dose of aminoglycoside following by a single dose of a loop diuretic delivered intraperitoneally into adult CBA/CaJ mice.45,49,50
Auditory brainstem responses (ABRs) were measured at 3 days, 7 days, 1 month, or 3–5 months after treatment (). A significant ABR threshold shift (about 40–70 dB SPL across all tested frequencies) was revealed as early as 3 days after treatment. No signs of functional recovery were seen at 3–5 months after treatment, which was the longest recovery period examined.
Figure 1 Hair cell degeneration and severe hearing loss induced by kanamycin and furosemide (K+F) treatment in adult CBA/CaJ mice. (a) Mean ABR thresholds in mice tested at 3 days, 7 days, 1 month and 3–5 months post-deafening. Data are expressed as mean (more ...)
Drug-treated mice were examined at 7 days, 1 month and 3 months to evaluate the extent of hair cell destruction. A loss of OHCs was seen in the entire basal turn and most of the apical turn of the examined ears (n=19) (). Only a small number of OHCs remained in the extreme apex. The majority of IHCs were intact but a scattered loss of IHCs was observed in the basal turns of treated mice. These results correspond with previous studies.45,49,50
Most importantly, relatively normal architecture of supporting cells was seen 1–3 months after treatment (). Therefore, this deafened model is ideal for testing the transduction efficiency of AAV viruses in surviving supporting cells in the context of nearly complete OHC loss.
Scala media inoculation with limited surgical trauma
The organ of Corti is a delicate epithelium located on the basilar membrane, an acellular structure suspended across a fluid-filled space deep within the temporal bone. Furthermore, delivery of virus directly to the apical surfaces of the sensory epithelium requires a breach of the scala media, a tubular compartment filled with potassium-rich endolymphatic fluid. Excessive leakage of endolymph at the site of injection will degrade the transepithelial endocochlear potential that is an essential driving force for effective mechanosensory transduction. To better preserve the integrity of both the basilar membrane and the organ of Corti, we established a surgical approach to deliver AAV vectors into the scala media through the lateral wall (). The bulla was surgically exposed and prepared to permit access to the basal turn of the mouse ear. This approach allows direct visualization of the bony lateral wall of the scala media and the richly vascularized spiral ligament of the stria vascularis that lies just beneath. We were able to minimize injection trauma by optimizing the delivered volume and speed of injection through glass micropipettes using a Nanoliter Microinjection System. Although injection trauma was minimal, 7% of animal subjects showed significant leakage of endolymph at the injection site and thus were not included in the study.
Figure 2 AAV inoculation into the scala media (SM) of normal and deafened adult mice with minor surgical trauma. (a) A post-auricular surgical approach for delivering AAV vectors into the adult mouse cochlea using a glass micropipette. (b) Surgical approach to (more ...)
Physiological assessments were used to evaluate whether the surgical procedure itself was potentially damaging to the inner ear. show that no significant ABR threshold shifts were seen at 4 to 22.6 kHz in a normal mouse 7 days after AAV inoculation. However, a 30–40 dB SPL shift in the ABR threshold did appear at higher frequencies (32, 40 and 45.2 kHz), suggesting a cochlear injury at the extreme basal turn adjacent to the typical injection site. In contrast, deafened mice, which were already missing most OHCs and some IHCs in the basal turn (data not shown), demonstrated no additional high-frequency ABR threshold shifts in response to virus inoculation ()
Transduction efficiency of five AAV serotypes in the normal and deafened mouse ears
Five types of AAV-GFP vectors (serotypes 1, 2, 5, 6, and 8) were delivered to normal or deafened adult mice. Although histological analysis revealed that many cell types could express GFP following AAV inoculation, IHCs were by far the most commonly transfected ( and ). For a semi-quantitative evaluation of the relative transduction efficiency of AAVs under different experimental conditions, we counted IHC expression in two different ways. First, the number of mice showing any GFP expression in IHCs was counted relative to the total number of mice inoculated. Second, the number of mice with more than 100 GFP+ IHCs was counted and compared to the total number of mice inoculated. Both of these measures can be found in for the full set of inoculated animals.
Figure 3 AAV-GFP transduction in hair cells of normal adult mice. (a) AAV-GFP transduction in IHC and OHC areas of the apical turn of a mouse 7 days after virus inoculation. (b) Higher magnification view of the GFP+ cells in the apical turn shown in (a). (c) GFP (more ...)
Figure 4 AAV-GFP transduction in IHCs of deafened mouse ears. (a) AAV-GFP transduction of IHCs (arrows) in the cochlea of a deafened mouse 7 days after virus inoculation with serotype 2 vector. (b) AAV-GFP transduction in an IHC (arrowhead) of the apical turn (more ...)
Transduction efficiency of AAV-GFP vectors in the mouse cochleae.
The epithelium of a deafened animal is likely to differ from that of a normal animal, and these changes might influence the number or types of cells infected under the two conditions. Furthermore, an acutely deafened organ of Corti may not necessarily be infected with similar efficiency to that which is chronically deafened. To examine the effects of post-deafening recovery time, AAV was delivered into the scala media of the deafened cochlea following either short term (3–7 days, acutely-deafened) or long term (1–6 month, chronically-deafened) recovery periods after kanamycin and furosemide treatment. There is a trend showing that AAV transduction is more effective in acutely-deafened mice than chronically-deafened mice (). About 87% (32/37) of the acutely-deafened mice expressed GFP protein, as compared to 68% (17/25) of the chronically-deafened mice. This compares to 88% (35/39) of normal animals that were transduced with GFP. However, these numbers do not take into account the rather large differences in infection efficiency across viral serotypes.
For each AAV serotype, we initially examined 3–4 animals per group. The overall transduction efficiency showed that GFP transduction was best achieved with serotypes 2 and 8, secondarily with serotype 6 and lastly with serotypes 1 and 5. The initial results led us to focus on serotypes 2 and 8 to further examine the effect of survival time after virus inoculation on the transduction efficiency ( and ). The transduction patterns of AAV vectors in sensory cells of normal and deafened ears were also characterized using vector serotypes 2 and 8 ( and ).
Transduction efficiency of serotypes 2 and 8 vectors with variable inoculation time.
Figure 5 A small number of SCs transduced with AAV-GFP vectors. (a–c) Double labeling for GFP (green) and Sox2 (red) in a deafened mouse inoculated with serotype 2. This mouse was allowed to recover for 7 days after drug delivery and before virus inoculation. (more ...)
Figure 6 AAV-GFP transduction in the sensory epithelia of the vestibular organs in normal and deafened mice for serotype 8. (a) GFP+ cells in the HC and SC layers of the utricular macula in a normal mouse one month after virus inoculation. (b) GFP expression in (more ...)
The effect of recovery period on transduction efficiency of AAV was compared for serotypes 2 and 8 at seven days and one month after surgery (). The percentage of inoculated mice showing any GFP expression in IHCs ranges from 88–100%, with no clear advantage of one serotype over the other using this measure. The percentage of inoculated mice with >100 GFP+ IHCs shows a large range from 41–64%, with the highest percentage seen at one month for serotype 2. Qualitatively, there was no clear trend towards a larger number of GFP-expressing cells per ear with increased recovery time.
AAV transduction in the auditory organ of normal and deafened mice
For all five AAV serotypes, IHCs were the most effectively transduced cell type in the organ of Corti. Robust expression of GFPin IHCs was seen in both normal and deafened mice, especially for serotypes 2 and 8 ( and ). shows a deafened mouse with near complete IHC transduction 7 days after serotype 2 virus inoculation. OHCs were also shown to express GFP in the normal ear (). However, robust GFP+ OHCs were only seen in the apical turns of four normal mice (out of 25 mice that were inoculated with serotype 2 and 8 viruses).
A small number of GFP+
SCs were seen in normal and deafened mice for serotypes 2, 6 and 8 (, , ). Sox2 was used as a marker to identify SCs. A previous study has indicated that Sox2 can be expressed in the nuclei of all cochlear SC subtypes including Deiters’, Hensen’s, inner and outer pillars, inner phalangeal, and border cells.45
reveals that some of the GFP+
cells were co-labeled with Sox2. However, there are some GFP+
cells in the SC region (outer pillar cell region) of the deafened mice that were not stained for Sox2 (). It is possible that these GFP+
cells are fibroblasts or macrophages that appeared in response to the loss of cochlear OHCs.
AAV transduction in the vestibular organs of normal and deafened mice
We were also interested in whether scala media inoculation could yield GFP gene transduction in vestibular organs. The examination of frozen sections of the inner ears of normal and deafened mice revealed GFP expression in both a subset of HCs and SCs of the sensory epithelia of vestibular organs including the macula and ampulla (). Oesterle et al45
reported that Sox2 protein was expressed in SCs and type II HCs of both the striolar and extra-striolar regions. We found that a majority of the GFP+
HCs were co-labeled with Sox2, suggesting these cells are type II HCs (). GFP expression patterns were similar in normal and deafened ears. A few GFP+
cells were also seen within the stromal region of the vestibular organs (data not shown).
AAV transduction in the auditory nerve and cochlear lateral wall of normal and deafened mice
Scala media inoculation can also produce efficient GFP transduction in cells within Rosenthal’s canal, although this region was not specifically targeted. Based upon their morphological features, GFP+ cells within Rosenthal’s canal are mostly glial cells (). The proportion of normal and deafened animals showing GFP expression as a function of viral serotype is shown in . Transduction was best achieved with serotype 8. GFP+ auditory nerve cells were seen in 60% of inoculated mice for serotype 8, 43% for serotype 1, 42% for serotype 3, 38% for serotype 6 and 22% for serotype 5. Overall, we observed a trend towards higher transduction efficiency in deafened ears as compared to normal ears.
AAV-GFP transduction in the auditory nerve of a normal mouse.
Transduction efficiency of AAV-GFP vectors in the auditory nerves of the mouse cochleae.
GFP+ cells were also seen in the spiral ligament of normal and deafened mice that received serotypes 2 and 8 viruses (). In most cases, GFP was expressed in the outer sulcus epithelial cells as well as type II and IV fibrocytes of the spiral ligament. GFP+ fibrocytes were mostly located in the basal turns of both normal and deafened mice. No GFP+ cells were seen in the stria vascularis of the specimens examined.
Figure 8 AAV-GFP transduction in the cochlear lateral wall of normal and deafened mice. (a, b) GFP+ cells in the spiral ligament of the basal turn of a normal mouse one month after virus inoculation with serotype 8. These GFP+ cells are located among the outer (more ...)