Adult mouse inner ear is more susceptible to hearing loss than neonatal inner ear for inner ear gene delivery
Posterior semicircular canal approach (PSC) has been shown to be a safe and effective surgical approach for inner ear gene delivery in the neonatal mouse inner ear17,20. In addition, some studies have also shown that it can be safely implemented in the adult mouse inner ear15,21. However, our initial attempts at using the PSC approach for gene delivery in the adult mouse inner ear showed significant ABR threshold elevation in many mice (Fig. 1a). Therefore, we decided to investigate the implementation of PSC approach in adult mice more carefully. The three main surgical steps in the PSC approach are, 1) fenestration of PSC, 2) insertion of injection tubing into the PSC, and 3) injection of fluid into the PSC (Fig. 1b). We decided to examine each of these surgical steps involved with PSC gene delivery to see if we could refine this surgical technique to minimize trauma to the adult mouse inner ear.
The duration of perilymphatic leakage after PSC fenestration negatively affects hearing in adult mouse inner ear
We first examined the effect of PSC fenestration on the adult mouse inner ear. The fenestration of PSC is performed using a small 27-gauge needle to expose the canal lumen. Observation of perilymphatic leakage is used as confirmation for successful access to the PSC lumen. In this experiment, we performed PSC fenestration on adult CBA/J mice. The PSC fenestration was left open for several minutes to allow for perilymphatic leakage to occur, and then sealed off using a muscle plug. We found that some mice developed significant hearing loss after PSC fenestration while others didn’t (Fig. 2a). We decided to examine the PSC fenestration more closely by timing the duration of PSC opening and perilymphatic leakage. We separated our animals into three groups based on various durations of PSC opening and perilymphatic leakage: 2, 5, and 10 minutes. We found that mice with 2-minute and 5-minute PSC opening had minimal ABR threshold elevation compared with non-surgery control mice. However, mice in the 10-minute PSC opening group had significant ABR threshold elevation compared to the non-surgery control mice (Fig. 2b; p=0.0153 for 4kHz, p=0.0006 for 8kHz, p=0.0082 for 16kHz, and p=0.0010 for 32kHz, t-test). This indicates that prolonged PSC opening time and perilymphatic leakage can adversely affect the hearing outcome in adult mouse inner ear.
Tube insertion into the PSC had no effect on hearing in the adult mouse inner ear
Next, we compared ABR thresholds in mice with or without insertion of injection tubing into the PSC to determine whether this surgical step would adversely affect auditory function. It is important to remember that mice undergoing tube insertion will have to undergo PSC fenestration. Therefore, this surgical step cannot be evaluated on its own, but must be evaluated after the PSC fenestration has been created. The PSC opening time was kept below 5 minutes to minimize perilymphatic leakage. We found that there was no significant difference in ABR thresholds between mice that underwent tube insertion and non-surgery control mice when the PSC opening was kept below 5 minutes (Fig. 3).
The rate of injection has significant effect on hearing in the adult mouse inner ear
Lastly, we evaluated the effect of injection rate in the adult mouse inner ear via the PSC. Again, it is important to remember that in order for mice to be injected with gene therapy, the PSC must be fenestrated first, and then the injection tubing must be inserted into the PSC lumen in order for the injection to take place. In neonatal mice, we have shown previously that we could deliver approximately 1 µl of fluid volume within a span of 30 seconds into the inner ear without any ABR threshold elevation compared to non-surgery control mice18. However, when we used the same injection rate in adult mice, significant ABR threshold elevation was observed (Fig. 4a). Substantial IHC and OHC loss was found throughout the cochlear turns (Fig. 4b). In the basal turn of the cochlea, all IHCs and OHCs were damaged, suggesting the hearing loss observed resulted from hair cell damage after injection (Fig. 4c).
Therefore, we decided to examine the effect of injection rate in the adult mouse inner ear more carefully. The micro-injector that we use allows us to set the fluid volume per injection (e.g. 13.8 nl, 27.6 nl, 46 nl, etc.), and the injection interval can be spaced out as determined by the investigators. We assessed the following three different injection regimens: 72 injections of 13.8 nl per injection every 10 sec (for a total volume of 993.6 nl), 36 injections of 27.6 nl per injection every 10 sec (for a total volume of 993.6 nl), and 20 injections of 46 nl per injection every 10 sec (for a total volume of 920 nl). We found that there was no significant difference in the average ABR thresholds between mice in the 13.8 nl per injection and 27.6 nl per injection groups compared to non-surgery control mice (Fig. 4d). However, mice in the 46 nl per injection group exhibited significantly higher ABR thresholds compared to non-surgery control mice (Fig. 4d). The differences in ABR threshold were significant at all tested frequencies except 4kHz ( p=0.0641 for 4kHz, p=0.0027 for 8kHz, p=0.0258 for 16kHz, and p=0.0318 for 32kHz, t-test).
AAV2.7m8 transduced adult cochlear hair cells with high efficiency
We previously showed that AAV2.7m8 is a powerful viral vector for gene delivery in the neonatal mouse inner ear18. However, it has been shown that AAV transduction efficiency can be different between neonatal and adult mouse inner ears16,21,22. Therefore, we assessed the transduction pattern and efficiency of AAV2.7m8 in the adult mouse inner ear using our newly refined PSC approach. When 1 µl of AAV2.7m8 was delivered via PSC approach by 72 injections of 13.8 nl per injection every 10 sec, IHC and OHC transduction rates were 65.3±10.1 and 37.9±7.4% in the apical turn, 69.2±10.8 and 35.2±7.7% in the middle turn, and 40.3±7.8 and 10.8±6.3% in the basal turn of the cochlea (Fig. 5a). Even though the overall transduction rate is lower compared to our previous study in neonatal mouse inner ears, there were some adult mice that had very high rates of IHC and OHC transduction, comparable to neonatal ears. The reduction in overall IHC and OHC transduction rates in the adult mouse inner ear is likely due to the increased technical challenge with adult mouse inner ear gene delivery compared to neonatal ears.
To determine whether overall injection volume affects transduction efficiency, we injected some mice with ~2 µl of AAV2.7m8-GFP using 72 injections of 27.6 nl per injection every 10 sec (Fig. 5b). IHC and OHC transduction efficiencies with 2 µl were 91.2±0.9% and 86.3±4.3% in the apical turn, 91.0±2.5% and 56.9±12.7% in the middle turn, and 83.7±6.3% and 21.6±9.8% in the basal turn of the cochlea (Fig. 5c). When compared with 1 µl injection, the overall transduction rate was higher across the cochlear turns, and the difference in transduction rate was significant in the basal turn of the cochlea for IHCs, and the apical turn of the cochlea for OHCs. This indicates that AAV2.7m8 is capable of transducing cochlear IHCs and OHCs at high levels, and the transduction efficiency in adult mice increases in a dose-dependent manner.
Hearing is preserved in adult mouse inner ear after 2 µl injection using the PSC approach
Even though AAV2.7m8 is capable of transducing cochlear IHCs and OHCs at high levels in the adult mouse inner ear, we had to increase the total injection volume to 2 µl in order to match the transduction efficiency seen in the neonatal mice. Since the adult mouse inner ear is more vulnerable to surgical manipulation and injection volume, we assessed whether a 2 µl injection would have any effect on auditory function in these animals. We found that neither 1 µl nor 2 µl injection volume caused significant ABR threshold shift compared to non-surgery control mice, as long as the injection parameters were kept below 27.6 nl every 10 seconds. We also found that the average ABR threshold was not significantly different between mice injected with 1 µl and 2 µl (Fig. 6). These results demonstrate that up to 2 µl of fluid volume can be safely injected into the adult mouse inner ear using the PSC approach without causing any significant ABR threshold elevation.