Abstract
Purpose
The purposes of the current study were to assess the feasibility of post-auricular microinjection of lentiviruses carrying enhanced green fluorescent protein (EGFP) into the scala media through cochleostomies in rats, determine the expression of viral gene in the cochlea, and record the post-operative changes in the number and auditory function of cochlear hair cells (HCs).
Methods
Healthy rats were randomly divided into two groups. The left ears of the animals in group I were injected with lentivirus carrying EGFP (n = 10) via scala media lateral wall cochleostomies, and the left ears of the animals in group II were similarly injected with artificial endolymph (n = 10). Prior to and 30 days post-injection, auditory function was assessed with click-auditory brainstem response (ABR) testing, EGFP expression was determined with cochlear frozen sections under fluorescence microscopy, and survival of HCs was estimated based on whole mount preparations.
Results
Thirty days after surgery, click-ABR testing revealed that there were significant differences in the auditory function, EGFP expression, and survival of HCs in the left ears before and after surgery in the same rats from each group. In group I, EGFP was noted in the strial marginal cells of the scala media, the organ of Corti, spiral nerves, and spiral ganglion cells.
Conclusion
Lentiviruses were successfully introduced into the scala media through cochleostomies in rats, and the EGFP reporter gene was efficiently expressed in the organ of Corti, spiral nerves, and spiral ganglion cells.
1
Introduction
In adult mammals, the cochlear sensory epithelium and nerve tissues cannot be repaired or regenerated after serious injury . One of the greatest challenges in the treatment of inner ear disorders is to find a cure for the hearing loss that is caused by the loss of cochlear hair cells (HCs) or spiral ganglion neurons. Gene therapy can be used to protect the loss of HCs or to stimulate HC regeneration . Gene therapy for inner ear disorders has developed along two major lines (HCs or sensory regeneration; and gene therapy for genetic deafness). To date, a majority of the research involving inner ear disorders has focused on the methodology of effective cochlear gene transfer or specific applications, such as HC regeneration. Future success in cochlear gene therapy will rely upon improved targeted gene delivery and appropriate timing and length of transgene delivery .
Two methods can be used to drive protein expression in specific cell types (cell-specific promoters and specific viral subtypes). Non-viral vectors have low transduction efficiency and poor transgene expression patterns, which limits usefulness . To date, the following vectors have been tested for cochlear gene delivery: adenovirus, adeno-associated viruses (AAV); herpes simplex virus; vaccinia virus; retrovirus; helper-dependent adenovirus; and lentivirus . Lentivirus vectors are potential gene transfer vectors because of the natural ability of lentivirus to infect dividing and non-dividing cells; however, compared to other virus vectors , research on lentivirus delivery in mammalian cochleas is limited .
The following two approaches are commonly used for introducing foreign active materials into the cochlear fluid: inoculation into the scala tympani through the round window membrane; and via cochleostomy . Round window injection might be considered less invasive and can be performed in a shorter period of time; however, the efficiency of transgene expression is lower than observed with a cochleostomy . Alternative cochleostomy techniques include injecting foreign active materials via a lateral wall cochleostomy into the scala media, scala tympani, and scala vestibuli compartments of the cochlea .
To provide future experimental support for cochlear gene therapy using lentiviral vectors, we microinjected lentiviruses carrying EGFP into the scala media through cochleostomies in rats using carefully designed post-auricular open surgery, and determined the efficiency of viral gene expression in the cochlea and the post-operative changes in the number and auditory function of inner and outer hair cells (IHCs and OHCs, respectively).
2
Materials and methods
2.1
Materials
Lentivirus carrying EGFP (titer = 2 × 10 8 TU/mL) was purchased from Shanghai Telebio Biomedical (Shanghai, China) . Artificial endolymph was prepared as previously described ; the final preparation contained 1 mM NaCl, 126 mM KCl, 325 mM KHCO, 0.025 mM MgCl 2 , 0.025 mM CaCl 2 , and 1.4 mM K 2 HPO 4.
2.2
Animal grouping
All animal experiments were approved by the Tongji Medical College of Hua-Zhong University of Science and Technology and were performed using accepted veterinary standards.
Twenty healthy male SD rats with normal ear reflexes and binaural hearing functions, weighing 250–300 g, were obtained from the Experimental Animal Center of Tongji Medical College. Animals were randomly divided into two groups, as follows: group I (n = 10) were microinjected with lentiviruses carrying the EGFP gene through cochleostomies into the scala media; and group II (n = 10) were similarly microinjected with artificial endolymph. The left ear from each rat was microinjected, and the opposite ear was used as a normal control.
2.3
Surgical procedures
Rats were processed under general anesthesia (ketamine [40 mg/kg body weight] and chlorpromazine [15 mg/kg body weight] intramuscular [im]ly). The body temperature was maintained between 36 °C and 38 °C using a heating pad. After sterile procedures, the left ear was situated in a face-up position. The skin and subcutaneous tissues were incised and opened as an arc along the post-auricular sulcus from the 4:00-to-7:00 position. The platysma was incised, opened, and expanded with a home-made incisor, and the sternocleidomastoid muscle was exposed by blunt dissection. Then sternocleidomastoid muscle was elevated until the facial nerves and the posterior belly of the digastric muscle were exposed ( Fig. 1 A ). The posterior belly of the digastric muscle was retracted after continuous dissection backward the triangular area enclosed by the facial nerve and the digastric muscle was dissected, the covered soft tissue and fascia were removed, and the lateral wall of the bulla was exposed ( Fig. 1 B). A hole approximately 1.5 mm in diameter was drilled in the dorsal bulla to expose the stapes, round window niche, and the cochlear lateral wall ( Fig. 1 C). An orifice was opened as far as possible away from the stapedial arteries in the pigmented areas on the lateral wall of the cochlea at the basal turn. The bone of the scala media lateral wall was punctured with an edged needle, then the soft tissue membrane was broken with a fine needle until a small amount of blood and clear lymph fluid was discharged. A glass microelectrode fixed on a microinjection system (531IOV and220v; Stoelting Corporation, USA) was placed into the hole, then 5 μl of virus or artificial endolymph was slowly injected into the cochlea over 5 min ( Fig. 1 D). After the injection, the hole was covered with autologous small platysma and dental cement. The middle ear cavity was disinfected with iodophor. The hole on the surface of the bulla was closed with dental cement and the overlying incision was closed with suture. The rats were returned to their cages after awakening from anesthesia, and treated with penicillin (a single im 200 000 IU injection daily for 3 days).
2.4
Click- auditory brainstem response (ABR) testing
The click-ABR threshold was recorded before and 30 days after surgery in all animals. Animals were anesthetized with ketamine (40 mg/kg im) and wintermin (20 mg/kg im) before ABR measurement. A Nicolet Compass system was used with the following parameters: 10 ms duration; repeat rate, 11.1 /sec; filtering band, 150–2000 Hz; superposition, 1000 times; and stimulation sound, a 0.1 ms broad band click. A recording electrode was inserted into the scalp in the middle of the two ears. A reference electrode was inserted below the left ear and a ground electrode was inserted contralaterally. The threshold was defined as the lowest decibel sound pressure level (dBSPL) of the stimulus at which a positive waveform in the evoked response tracing was evident, and was repeated at least one time.
2.5
Sample preparation
Thirty days after surgery all animals under intracardial perfusion with normal saline under anaesthesia, followed by 10% formalin in phosphate buffer. The temporal bone was removed and immersed in 10% formalin in phosphate buffer at 4 °C for 24 h. After decalcification with 10% EDTA at 4 °C for 4 days, the sample was rinsed with 0.1 M PBS.
2.6
Assessing the number of HCs
Four animals from each group were prepared for cochlear surface preparation and cochleogram evaluations as described previously . Briefly, after fixation and decalcification, the cochlear tissue was stained with Harris’ hematoxylin solution. The cochlear basilar membrane was removed using microdissection, and placed on glass as a flat surface preparation, mounted in glycerin, and cover slipped. The cochlear surface preparation was examined with a microscope (Zeiss Standard, 400X magnification, and the number of missing IHCs and OHCs were determined at 0.24-mm intervals along the entire basilar membrane. The HCs were counted as present if the cell body and cuticular plate were intact. Data were entered into a computer to obtain a cochleogram showing the percent IHCs and OHCs loss as a function of the percent total distance from the apex; the cochleograms were based on the laboratory norms of the HC density for young normal rats. The percent distance from the apex was related to the frequency using a frequency-place map. The mean cochleograms were computed for each treatment group as described previously .
2.7
Fluorescence microscopy
The temporal bones from the remaining 6 animals in each group were routinely prepared for cochlear frozen sections to detect the expression of EGFP in the cochleas as described previously . Briefly, the samples were dehydrated with 20% sucrose and embedded with OCT. Ten-mm cryostat sections of the temporal bones were prepared. Tissue sections were then mounted with a cover slip and sealed in 20% glycerol. Sections were observed under a Zeiss fluorescent microscope equipped with a digital camera (Spot RT; Diagnostic Instrument or Polaroid DMC le, Sterling Heights, MI, USA). The samples were kept in the dark throughout the process.
2.8
Statistical analysis
All data were analyzed with SPSS16.0 statistical software. The results are expressed as X (−) ± SD. The ABR thresholds between groups were compared with a paired t-test. Differences in the numbers of HCs between groups were analyzed using analysis of variance (ANOVA). In all cases, a P value < 0.05 was considered statistically significant.
2
Materials and methods
2.1
Materials
Lentivirus carrying EGFP (titer = 2 × 10 8 TU/mL) was purchased from Shanghai Telebio Biomedical (Shanghai, China) . Artificial endolymph was prepared as previously described ; the final preparation contained 1 mM NaCl, 126 mM KCl, 325 mM KHCO, 0.025 mM MgCl 2 , 0.025 mM CaCl 2 , and 1.4 mM K 2 HPO 4.
2.2
Animal grouping
All animal experiments were approved by the Tongji Medical College of Hua-Zhong University of Science and Technology and were performed using accepted veterinary standards.
Twenty healthy male SD rats with normal ear reflexes and binaural hearing functions, weighing 250–300 g, were obtained from the Experimental Animal Center of Tongji Medical College. Animals were randomly divided into two groups, as follows: group I (n = 10) were microinjected with lentiviruses carrying the EGFP gene through cochleostomies into the scala media; and group II (n = 10) were similarly microinjected with artificial endolymph. The left ear from each rat was microinjected, and the opposite ear was used as a normal control.
2.3
Surgical procedures
Rats were processed under general anesthesia (ketamine [40 mg/kg body weight] and chlorpromazine [15 mg/kg body weight] intramuscular [im]ly). The body temperature was maintained between 36 °C and 38 °C using a heating pad. After sterile procedures, the left ear was situated in a face-up position. The skin and subcutaneous tissues were incised and opened as an arc along the post-auricular sulcus from the 4:00-to-7:00 position. The platysma was incised, opened, and expanded with a home-made incisor, and the sternocleidomastoid muscle was exposed by blunt dissection. Then sternocleidomastoid muscle was elevated until the facial nerves and the posterior belly of the digastric muscle were exposed ( Fig. 1 A ). The posterior belly of the digastric muscle was retracted after continuous dissection backward the triangular area enclosed by the facial nerve and the digastric muscle was dissected, the covered soft tissue and fascia were removed, and the lateral wall of the bulla was exposed ( Fig. 1 B). A hole approximately 1.5 mm in diameter was drilled in the dorsal bulla to expose the stapes, round window niche, and the cochlear lateral wall ( Fig. 1 C). An orifice was opened as far as possible away from the stapedial arteries in the pigmented areas on the lateral wall of the cochlea at the basal turn. The bone of the scala media lateral wall was punctured with an edged needle, then the soft tissue membrane was broken with a fine needle until a small amount of blood and clear lymph fluid was discharged. A glass microelectrode fixed on a microinjection system (531IOV and220v; Stoelting Corporation, USA) was placed into the hole, then 5 μl of virus or artificial endolymph was slowly injected into the cochlea over 5 min ( Fig. 1 D). After the injection, the hole was covered with autologous small platysma and dental cement. The middle ear cavity was disinfected with iodophor. The hole on the surface of the bulla was closed with dental cement and the overlying incision was closed with suture. The rats were returned to their cages after awakening from anesthesia, and treated with penicillin (a single im 200 000 IU injection daily for 3 days).
