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Study on neural stem cell transplantation into natural rat cochlea via round window
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American Journal of Otolaryngology–Head and Neck Medicine and Surgery 30 (2009) 8 – 16 www.elsevier.com/locate/amjoto
Study on neural stem cell transplantation into natural rat cochlea via round window Yong Fu, MD, PhD a,b , Shenqing Wang, MD a , Yingpeng Liu, MD b , Jianting Wang, MD, PhD c , Guopeng Wang, MD b , Qingguo Chen, MD b , Shusheng Gong, MD, PhD b,d,⁎ a
Department of Otorhinolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hang Zhou, People's Republic of China b Department of Otorhinolaryngology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China c Department of Otorhinolaryngology Head and Neck Surgery, Beijing, Chaoyang Hospital, Capital Medical University, Beijing, People's Republic of China d Department of Otorhinolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Key Laboratory of OtorhinolaryngologyHead & Neck Surgery (CMU), Ministry of Education, Beijing, People's Republic of China Received 3 October 2007
Abstract
Objective: The aim of the study is to investigate the survival of neural stem cells (NSCs) in normal rat cochlea and their potential effect on auditory function and cochlea structures via round window transplantation. Methods: In comparison with the normal rats without any transplantation (group III), normal rat cochleae were transplanted with NSCs infected with adenovirus carrying green fluorescence protein (GFP) gene (group I) or the artificial perilymph (group II) via round windows. Auditory functions were monitored by thresholds of auditory brain stem responses (ABRs); the cochlea structures were examined by hematoxylin and eosin staining; survivals of implanted NSCs were determined by the expression of GFP; survivals of hair cells were accessed by whole mount preparation; and ultrastructures of hair cells were examined by scanning electron microscopy. Result: There were significant differences in the click-ABR thresholds in rats among all 3 groups neither at pretransplantation nor at posttransplantation; there were no significant differences in these values before and after transplantation in the same rats from each group. After transplantation, the cochlea structures were normal in both group I and group II. Grafted NSCs were visualized by the GFP expression in every turn of the cochlea in all animals of group I. There were no significant differences in the losses of outer hair cells (OHCs) among 3 groups. The inner hair cells and most OHCs were normal in every turns of cochleae of all groups. Conclusion: Neural stem cells survived in normal rat cochlea after transplantation via round window and showed no obvious effects on auditory functions and inner ear pathologic examination of the rat cochlea. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Hearing loss in mammals is irreversible because the lost hair cells (HCs) cannot be replaced by cell division and regeneration from endogenous cells in the inner ear epithelia. The past year, stem cells were introduced into the search for ⁎ Corresponding author. Department of Otorhinolaryngology Head and Neck Surgery, Beijing, Tongren Hospital, Capital Medical University. Beijing 100730, People's Republic of China. Tel.: +86 13911068366. E-mail address: [email protected] (S. Gong). 0196-0709/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.amjoto.2007.12.006
new approaches to HC regeneration in mammals. A major advance in the use of stem cells for the replacement of inner ear cells came with the recent discoveries [1-5] that neural stem cells (NSCs) survived and differentiated into neurons and astrocytes after transplantation into normal or damaged rat cochleae via different approaches. However, the changes in the auditory function and the cochlea morphology after NSC transplantation had not been studied in details. Previously, a recombinant adenovirus-expressing green fluorescence protein GFP (Ad-GFP) was successfully constructed from this laboratory, as described by Fu et al
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[6]. We showed that the efficiency of Ad-GFP infection into NSCs isolated from fetal rat hippocampus was high, and the infected NSCs were able to divide and self-renew, as well as sustain the expression of GFP. In this study, primary NSCs infected with Ad-GFP were transplanted into natural rat cochleae via round window, the cell survival was examined by the expression of GFP reporter, and the changes in the auditory function and cochlea morphology were also investigated to provide additional information for stem cellbased cell therapy for the damaged inner ear. 2. Materials and methods 2.1. Experimental rats All animal experiments were approved by Tongji Medical College of Hua-Zhong University of Science and Technology (Wu Han, People's Republic of China) and were performed using accepted veterinary standards. Animals were obtained from the experimental animal center of Tongji Medical College. At the beginning of the experiment, 45 Sprague-Dawley rats weighed 200 to 250 g were selected, and all had normal Preyer's reflexes. Animals were assigned randomly to 1 of 3 groups: group I, NSC implantation group (experimental group [n = 15]); group II, artificial perilymph implantation group (control group [n = 15]); and group III, nonimplantation group (normal rat group [n = 15]). Before any procedure, clickauditory brainstem response (ABR) was recorded and calibrated to decibel sound pressure level (dBSPL) peak-topeak-equivalent according to the method by Mitchell et al [7] to determine the baseline hearing threshold. 2.2. Preparation of NSCs infected with Ad-GFP Neural stem cells infected with Ad-GFP were prepared as described by Fu et al [6,8]. Briefly, the hippocampal tissues from rat embryos were isolated and transferred to neurosphere culture medium (DMEM/F12 supplemented with 2% B27, 20 ng/mL of basic fibroblast growth factor, 20 ng/mL epidermal growth factor). On day 3, primary spheres were collected, spun down, resuspended in neurosphere culture medium, and completely dissociated by pipetting. The obtained cell suspension was continued in the same culture condition. On day 6, secondary NSC spheres were infected by Ad-GFP that was constructed in our laboratory. On day 5 after infection, NSCs were centrifuged and rinsed twice by fresh culture medium, then completely dissociated by pipetting into cell suspension and stored at 4°C for transplantation at a cell density of 1 × 106 cells/mL. 2.3. Implantation of NSCs into rat cochlea via the round window Neural stem cells infected by Ad-GFP were transplanted into cochleae of rats in group I, artificial perilymph was similarly injected into cochleae of rats in group II; the animals in group III were not under surgery. Artificial fluid of perilymph composed of 125 mmol/L NaCl, 3.5 mmol/L
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KCl, 1.20 mmol/L MgCl, 0.75 mmol/L NaH2PO4, 25 mmol/L NaHCO3, and 5 mmol/L glucose was filtrated and stored at 4°C. For implantations, the left ears of rats from group I and group II were processed under general anesthesia (ketamine, 4 mg/100 g body weight, and xylazine, 1 mg/100 g body weight intramuscular) and antibiotic protection (chloramphenicol, 30 mg/kg subcutaneous). Briefly, the left postauricular region was shaved and sterilized with 70% ethanol. The animal was then placed on a heating pad (37°C), the left bulla was exposed by the postauricular approach, and the basal turn and the round window membrane of the cochlea were identified. A 20-μL microsyringe (Exmire microsyringe, Ito Corporation, Fuji, Shizuoka, Japan) was inserted into round window; 5 μL perilymph was drawn out and then 10 μL cell suspension (group I) or artificial perilymph (group II) was injected slowly at the speed of 1 μL/min into the scala tympani at the basal cochlear turn. The needle of the microsyringe was kept in the inner ear for several minutes to prevent the NSCs and the culture medium from flowing out. A small piece of fascia was then placed over the round window, and the incision was closed with sutures. 2.4. Postimplantation evaluations of experimental rats On day 14 after surgery, click-ABR was again recorded under general anesthesia to determine any postsurgical threshold shift in group I and group II, as described above. Click-ABR was recorded at the same time for rats in group III. Then, 5 animals from each group were used to observe the structure of cochlea and assess the survival of NSCs by tracking the expression of GFP in the cochlea; another 5 animals from each group were processed for scanning electronic microscopy (SEM); and the last 5 animals from each group were used for surface preparation of whole organ of Corti of the cochlea. 2.4.1. Tissue processing The animals were perfused intracardially with normal saline, followed by 4% paraformaldehyde in phosphate buffered saline. The left temporal bones were removed and immersion fixed in the same fixative at 4°C for 4 hours. After decalcification with 0.1 mol/L EDTA for 24 hours at 4°C, 10-mm cryostat sections of the temporal bones were prepared. The sections were then mounted on α-aminopropyltriethoxysilane-coated slide glasses. 2.4.2. Hematoxylin and eosin staining Midmodiolus sections from each animal were provided for histologic analysis by hematoxylin and eosin staining. Briefly, after stained by hematoxylin, slides were disposed by hydrochloric acid, ethanol, and lithium carbonate; stained by eosin; dehydrated by graded ethanol solutions; cleared by xylene; and embedded by the natural gum. 2.4.3. Observing under fluorescent microscope Neighboring midmodiolus sections were used to view the expression of GFP as indication of the survival of
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Fig. 1. Survival of NSCs and expression of GFP in the cochlea. Grafted NSCs were visualized by GFP expression in every turn of the cochlea in all animals of group I (original magnification ×40 [A]; ×200 [B-C]; ×400 [D]). The expression of GFP was not found in animals of group II (original magnification ×100 [E]).
NSCs under a Zeiss fluorescent microscope equipped with a digital camera (Spot RT, Diagnostic Instrument or Polaroid DMC le, Sterling Heights, MI). 2.4.4. Accessing numbers of HCs To count the cell number and determine the loss of HCs, another 5 animals from each group were processed for silver nitrate staining and surface preparation of whole organ of Corti. Briefly, the left cochleae were exposed. After the round window and the oval window were opened and a small hole was then made in the apex of the cochlea using a 26-gauge needle, about 5 mL of 0.5% silver nitrate solution was gently perfused into perilymph from the hole. After immersed in 0.5% silver nitrate solution for 15 minutes, the cochleae
was rinsed by distilled water, perfused by 10% formalin and then immersed for 4 hours. The cochleae were exposed in the sun until the cochleae presented tawny. Under microTable 1 Average thresholds (dBSPL) of click-ABR Group
Pretransplantation
Posttransplantation
I II III
31.00 ± 3.87 30.67 ± 4.17 30.83 ± 3.28
33.00 ± 4.14 32.67 ± 4.17 32.75 ± 4.21
Before implantation, the click-ABR threshold had no significant difference in rats of all 3 groups. There were also no significant difference between pretransplantation and posttransplantation in each group and no significant difference after transplantation among all groups (P N .05).
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scope, the whole basilar membrane was separated and divided into 3 parts by different turns. After mounted on slides containing 50% glycerol, the stained specimens were observed. Then, the number of HCs was counted by the method of Ding et al [9]. 2.4.5. Scanning electronic microscopy To observe the morphologies of HCs and the stereocilia, 5 animals from each group were processed for SEM. Briefly, the bullae were quickly removed, and the cochleae were perfused gently with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.2; 4°C). After being stored in the same fixative for 24 hours, the cochleae were decalcified with 10% EDTA for 4 days and then postfixed with 2% osmic tetroxide in 0.1 mol/L phosphate buffered saline. The basilar membrane was microdissected and dehydrated through serially graded ethanol solutions ending at 100%. Specimens were rinsed in acetone and dried at a critical point in liquid carbon dioxide. They were then coated with gold sputter to a depth of approximately 25 nm and examined on a field emission of SEM (SEM-800) using accelerating voltage of 25 kV. 2.5. Statistic analysis The data were analyzed using Sigma Stat statistical software (SPSS/Jandel Scientif Software, Chicago, IL). Differences between presurgery thresholds and postsurgery thresholds of each group were analyzed using paired t tests. Further analyses were performed to compare presurgery thresholds or postsurgery thresholds among 3 groups using analysis of variance. Differences of the loss of HCs from each turn in each group and differences of the total loss of HCs in each group were analyzed using analysis of variance. In all cases, a value of P less than .05 was considered statistically significant. 3. Results There were no mortalities. Animals did not develop head tilts nor lose weights after surgery. There were no signs of wound infection, middle ear infection, vestibular dysfunction, or any other pathologic malfunction caused by the surgical procedure in any animal. The injection sites were completely sealed at day 5 after injection. After decapitation, normal middle ear cavities were found in all animals. No blood effusion was found inside the cochlear space in any animal, indicating that no major trauma was caused by the inoculation procedure itself. 3.1. Survival of implanted NSCs and expression of GFP Grafted NSCs were identified in the inner ear of each specimen by the expression of GFP. Grafted NSCs were found in every turn of the cochlea in all the rats from group I Fig. 2. Histologic examination of cochlea from experiment animals (×100). Hematoxylin and eosin staining of cochlea sections indicated the intact and normal structures from all experimental animals (A [group I]; B [group II]; C [group III]).
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(Fig. 1A). Most grafted NSCs were found as a cluster of cells in the perilymphatic spaces (Fig. 1B-D) in which some cells attached to the walls of scala tympani and scala vestibuli, some cells attached to the vestibular membrane or existed within the vestibular membrane, and some cells attached to the basilar membrane in scala tympani. Some grafted NSCs were found in the spiral ganglions (Fig. 1C). Quite a few of grafted NSCs were observed in endolymphatic spaces attached to the tectorial membrane (Fig. 1D). No grafted cells were found in the organ of Corti and spiral ligament of the cochlea. The expression of GFP was not found in any rat of group II (Fig. 1E). 3.2. Measurement of ABR threshold There were significant differences in the click-ABR thresholds in rats among all 3 groups neither at pretransplantation nor at posttransplantation; there were no significant differences in these values before and after transplantation in the same rats from each group. These results were recorded in Table 1. 3.3. Histologic examination of rat cochlea after hematoxylin and eosin staining In rats of all the 3 groups, the result of histologic examination of cochleae showed intact and normal cochlea, including the structures of organ of Corti, spiral ligament, and spiral ganglions of cochlea (Fig. 2). Most grafted cells in group I were found as a cluster of cells in the perilymphatic spaces (Fig. 2A). 3.4. Accessing HCs and their losses The structure of basilar membranes of the basal turns was intact and normal with stereocilia of HCs in order. There were dispersive losses of outer hair cells (OHCs) in all basal turns of cochlea of every group (Fig. 3A, B, and C). The OHC loss increased gradually from the basal turn to the apex turn, but the total losses from all groups were all less than 1%. Among all groups, neither the OHC losses of each turn nor the total losses had significant differences. The stereocilia of OHC3 (OHCs in the third row) of the apex turn were out of order, and the structure of some stereocilia was illegible (Fig. 4A, B, and C). There was not evident loss of inner hair cells (IHCs) in all turns of cochlea of every group. The numbers of HCs were recorded in Table 2, and the losses of HCs were recorded in Table 3. 3.5. Scanning electron microscopy examination of HCs There were losses of OHCs in every turn of cochlea from rats of all groups. Normal stereocilia and cuticular plate of most OHCs existed in the basal turn where the arrangement Fig. 3. Whole mount preparation for accessing HCs and their losses (the basal turn ×200). The basilar membranes from all animal groups were intact and normal, with the stereocilia of HCs in order, and the dispersive loss of OHCs (arrow) in the area. OHC1 indicates OHCs in the first row.
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“V”model (Fig. 5D). Normal stereocilia and cuticular plate of IHCs existed in every turn of cochlea of rats in all groups, and no losses of IHCs were found.
4. Discussion
Fig. 4. Whole mount preparation for accessing HCs and their losses (the apex turn ×200). The stereocilia of OHC3 were out of order, and the structure of some stereocilia was illegible. The lost number of OHCs (arrow) in the apex turn became more than those in the basal turn.
of stereocilia of OHCs presented “V” model (Fig. 5A-C). But the arrangement of stereocilias of OHC2 and OHC3 (OHCs in the second and third row) were relatively in disorder in the apex turn, where the arrangement became cluster shape from
Neural stem cells have been reported to be capable of selfrenewal and differentiation into the major cell types of the neural tissue, including neurons, astrocytes, and oligodendrocytes [10,11]. Thus, it is possible to differentiate NSCs into cells of functional auditory system via transplantation. Previous studies have shown that adult rat hippocampusderived NSCs grafted into the newborn rat inner ear survived in the cochlear cavity [1]. In addition, fetal mouse NSCs derived from the dorsal telencephalon have been shown to have the potential to survive and differentiate in the normal adult mouse inner ear, and NSC-derived cells can survive in the perilymphatic space for at least 28 days, but no grafted cells are found in endolymphatic space [2]. In another study [3], when the recipient mouse were injured by neomycin, grafted cells migrated beyond the bony wall that separates the endolymphatic space from the perilymphatic space and survived in both the perilymphatic and endolymphatic spaces. Some of these grafted cells even migrated into the labyrinth membrane, sensory epithelia, spiral limbs, and modiolus. In the cochlea injured by cisplatin [4], grafted NSCs survived and differentiated in midmodiolus and spiral ganglions. It has also been reported that [5] implanted cells attached to both the osseous Rosenthal canal and to the close proximity of the organ of Corti in the scala tympani. Moreover, implanted NSCs were also observed outside fluidfilled compartments and some even located along the auditory nerve fibers projecting to the hearing organ. The implanted cells were predominantly found 1 and 2 weeks after transplantation in normal-hearing animals and mostly differentiated into astrocytes. Together, these findings provide strong evidences that NSC transplantation may be used as an alternative for cell therapy for HC restoration. In these experiments, however, locations where implanted cells survived were different, and the changes of hearing functions or cochlea morphologies have not been investigated in details. There are 2 commonly used approaches for introducing foreign active materials into the cochlear fluid—inoculation into the scala tympani through the round window membrane or through a cochleostomy [12–14]. Round window injection has been considered less invasive because it preserves the natural structure and produces less local bleeding and less inflammatory reaction. In addition, it requires a shorter time and easier procedure to operate [12,14]. Our study showed that the structures of cochleae of all the rats after round window implantation were intact and normal; the morphologies for the organ of Corti, spiral ligament, and spiral ganglions of cochlea were all normal without red cells and exudation inside the cochlear space.
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Table 2 The number of HC of each turn of every group Group
I II III
The basal turn
The second turn
The apex turn
OHCs
IHCs
OHCs
IHCs
OHCs
IHCs
1867 ± 7.55 1870 ± 5 1865 ± 5
550 ± 2.52 543 ± 7.64 545 ± 5
1141 ± 6.56 1137 ± 7.64 1145 ± 5
280 ± 4.73 280 ± 5 278 ± 4.51
658 ± 12.58 658 ± 10.41 652 ± 7.64
183 ± 5 180 ± 5 182 ± 3
The number of OHCs in natural rat is 3662 ± 11.55; the number of IHCs in natural rat is 1005 ± 6.43.
This indicated that transplantation surgery through round window was not invasive. Using this technique, the absence of a significant and conspicuous perilymph leakage is intriguing, considering that the fluid is incompressible. One (or more) of the after mechanisms may account for the ability to inoculate the cochlea without leakage as follows: (1) the increased pressure leads to perilymph displacement via the cochlear aqueduct; (2) the stapes and round window may both be transiently distended to accommodate the positive pressure; or (3) endolymph may be compressed into the endolymphatic duct and sac, thereby transiently reducing the volume of scala media. In addition, because the injected volume (10 μL) was larger than the total volume of perilymph in rats [15], 5 μL perilymph was removed slowly before the injection of 10 μL cell suspension into the scala tympani slowly at the speed of 1 μL/min to inject more cell suspension and avoid a mechanical trauma to the cochlea that would injure or cause degeneration of HCs. In this study, NSCs infected by Ad-GFP were transplanted into normal rat cochlea via round window and were investigated for their survival, the changes of hearing function, and cochlea morphology in the host animals. Using round window approach, on day 14 after transplantation, grafted NSCs were identified in the inner ear of each specimen by the expression of GFP. Grafted NSCs were found in every turn of the cochlea in all the experimental animals of group I and existed not only in scala tympani but also in scala vestibuli, suggesting that the grafted cells might have moved significant distances along the cochlea, possibly after the flow of perilymph from the transplantation site in scala tympani through helicotrema to scala vestibuli [16]. Some cells attached to the vestibular membrane or existed within the vestibular membrane, quite a few of grafted NSCs were observed in endolymphatic spaces attached to the tectorial membrane. Because the vestibular membrane separates the endolymphatic space from the perilymphatic space, grafted cells attached to the vestibular membrane or existed within the vestibular membrane could migrate from perilymphatic space into endolymphatic space through the vestibular membrane. In our study, a fewer grafted NSCs were observed in endolymphatic spaces as compared with the study of Tateya [3]. Since the study of Tateya [3], NSCs were grafted into the cochlea of mice injured by neomycin so the injury of sensory epithelia caused by aminoglycoside toxicity might
have promoted integration of grafted NSCs into the sensory epithelia of the inner ear. More important, it was shown that NSCs also migrated from the implantation site within the fluid-filled scala tympani toward spiral ganglion neurons through the bony modiolus, also observed in the study of Hu [5]. This result suggested that adult NSCs indeed possessed the capacity to migrate to functionally important structures after implantation into mature inner ear. It has been suggested that the implanted cells were directed by the release of growth factors from the host neural tissues and migrated by means of the minute holes passing from scala tympani through the bone toward the Rosenthal canal [5]. None of grafted cells was found in the organ of Corti and spiral ligament of the cochlea. Although grated cells were not found in the organ of Corti, the surviving cells attached to the basilar membrane in scala tympani and to the bony walls of scala tympani and scala vestibuli. Because these were the sites closer to growth factors or essential nutritional supplements [17,18], lacking of some of the essential growth factors or nutrition may be one reason for the lack of grated cells in the spiral ligament of the cochlea. Before surgery, the click-ABR thresholds were similar in all animal groups, which indicated that the experimental condition was equal. There were no significant differences in ABR thresholds before and after transplantation in both group I and group II, which was consistent with previous studies [14,19,20]. In those studies, almost complete preservation of the cochlear structure and ABR thresholds were also shown after fluid application to the scala tympani via round window. There was also lack of significant difference in ABR thresholds in the 3 groups after transplantation, which indicated that embryonic NSC transplantation into cochlea through round window had no obvious effects on auditory Table 3 The loss of OHCs Group The basal turn loss rate
The second turn The apex turn loss rate loss rate
The total loss rate (%)
I II III
0.44 ± 0.09 0.47 ± 0.05 0.49 ± 0.05
0.53 ± 0.02 0.55 ± 0.03 0.54 ± 0.03
0.23 ± 0.06 0.24 ± 0.03 0.23 ± 0.03
1.52 ± 0.03 1.52 ± 0.13 1.53 ± 0.02
The loss of OHCs increased gradually from the basal turns to the apex turns, but the total losses were all less than 1% from all groups. Both the OHC loss in each turn and the total losses in each group had no significant differences among rats in 3 groups (P N .05).
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Fig. 5. Scanning electron microscopy examination of HCs. There were dispersive losses of OHCs (⁎) in each turn of cochleae from all groups and normal stereocilia and cuticular plate of most OHCs in the basal turns. (D) The arrangement of stereocilia of OHC2 and OHC3 was relatively in disorder in the apex turn.
function of the normal rat. However, as we had assessed hearing thresholds with click-ABR, predominantly representing the frequency region around 8 kHz, we cannot rule out high frequency hearing loss. The total number of OHCs was 3662 ± 11.55 in the cochlea of the natural rats, and the total number of IHCs was 1005 ± 6.43. There was dispersive loss of OHCs in all basal turns of cochlea of every group. The loss of OHCs increased
gradually from the basal turn to the apex turn, but the total losses of all groups were all less than 1%. These results were consistent with previously correlative studies by others [19,20]. In addition, there were no significant differences in HC losses of each turn in every group or the total losses among these 3 groups. The IHCs were normal in all turns of cochleae from all groups. These results indicated that the losses of OHCs were natural loss and not related to the
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surgery. Examination by SEM also showed that stereocilia and cuticular plate of IHCs and most OHCs were normal in each turn of cochlea of every group, and there were no losses of IHCs, which indicated that NSC transplantation into cochlea through round window had no obvious effects on ultrastructures of HCs of the normal rat. This experiment was only a primary study on NSC transplantation into cochlea via round window. Extended observation periods and more detailed physiologic measures will be necessary to examine long-term effects. To realize stem cell-based cell therapies for the damaged inner ear in the future, it will be necessary to examine the final fate of grafted cells, to increase the differentiation toward neurons from the grafted cells, to promote migration of the cells into the sensory epithelia, and even to transfer into HCs. Together, the present findings show that the GFP-labeled embryonic NSCs survived in normal rat cochlea after transplantation and expressed GFP efficiently. Although the implanted NSCs showed no obvious effect to auditory function and inner ear pathologic finding of rat cochlea, our study provided useful information for stem cell-based cell therapies. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 30672308) and the Medical Science Research Fund of Zhejiang Province, China (No. 2008A056). References [1] Ito J, Kojima K, Kawaguchi S. Survival of neural stem cells in cochlea. Acta Otolaryngol 2001;121(2):140-2. [2] Iguchi F, Nakagawa T, Tateya I, et al. Trophic support of mouse inner ear by neural stem cell transplantation. Neuroreport 2003;14:77-80. [3] Tateya I, Nakagawa T, Iguchi F, et al. Fate of neural stem cells grafted into injured inner ears of mice. Neuroreport 2003;14(13): 1677-81.
[4] Tamura T, Nakagawa T, Iguchi F, et al. Transplantation of neural stem cells into the modiolus of mouse cochleae injured by cisplatin. Acta Otolaryngol 2004;551:65-8. [5] Hu Z, Wei D, Johansson CB, et al. Survival and neural differentiation of adult neural stem cells transplanted into the mature inner ear. Exp Cell Res 2005;302(1):40-7. [6] Fu Y, Gong SS, Liu YP, et al. An experimental study of effects of recombinant adenovirus with GFP transfer into neural stem cells. J Audiol Speech Pathol 2007;15(5):379-82. [7] Mitchell A, Miller JM, Finger PA, et al. Effects of chronic high-rate electrical stimulation on the cochlea and eighth nerve in the deafened guinea pig. Hear Res 1997;105:30-43. [8] Fu Y, Gong SS, Xue QH, et al. Culture identification and label of embryonic rat neural stem cells. J Clin Otorhinolaryngol Head Neck Surg 2007;21(4):172-5. [9] Ding DL, Li M, Jiang SC. Morphology of the inner ear [M]. Heilongjiang. Haerbing (China): Science and Technology Publishing House; 2001. p. 24-8. [10] Clarke DL, Johansson CB, Wilbertz J, et al. Generalized potential of adult neural stem cells. Science 2000;288:1660-3. [11] Gage FH. Mammalian neural stem cells. Science 2000;287:1433-8. [12] Raphael Y, Frisancho JC, Roessler BJ. Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo. Neurosci Lett 1996;207: 137-41. [13] Lalwani A, Walsh B, Reilly P, et al. Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus. Gene Ther 1998;5:277-81. [14] Stöver T, Yagi M, Raphael Y. Cochlear gene transfer: round window versus cochleostomy inoculation. Hear Res 1999;136:124-30. [15] Thorne M, Salt AN, De Mott JE, et al. Cochlear fluid space dimensions for six species derived from reconstructions of three-dimensional magnetic resonance images. Laryngoscope 1999;109:1661-8. [16] Salt AN, Ma Y. Quantification of solute entry into cochlear perilymph through the round window membrane. Hear Res 2001; 154:88-97. [17] Rubel EW, Fritzsch B. Auditory system development: primary auditory neurons and their targets. Annu Rev Neurosci 2002;25:51-101. [18] Qun LX, Pirvola U, Saarma M, et al. Neurotrophic factors in the auditory periphery. Ann N Y Acad Sci 1999;884:292-304. [19] Stöver T, Yagi M, Raphael Y. Transduction of the contralateral ear after adenovirus-mediated cochlear gene transfer. Gene Ther 2000;7: 377-83. [20] Yu ZL, Han DM, Lin C, et al. Experimental study of transgenic expression in inner ear. Chin J Otorhinolaryngol 2003;38(5):340-2.
American Journal of Otolaryngology–Head and Neck Medicine and Surgery 30 (2009) 8 – 16 www.elsevier.com/locate/amjoto
Study on neural stem cell transplantation into natural rat cochlea via round window Yong Fu, MD, PhD a,b , Shenqing Wang, MD a , Yingpeng Liu, MD b , Jianting Wang, MD, PhD c , Guopeng Wang, MD b , Qingguo Chen, MD b , Shusheng Gong, MD, PhD b,d,⁎ a
Department of Otorhinolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hang Zhou, People's Republic of China b Department of Otorhinolaryngology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China c Department of Otorhinolaryngology Head and Neck Surgery, Beijing, Chaoyang Hospital, Capital Medical University, Beijing, People's Republic of China d Department of Otorhinolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Key Laboratory of OtorhinolaryngologyHead & Neck Surgery (CMU), Ministry of Education, Beijing, People's Republic of China Received 3 October 2007
Abstract
Objective: The aim of the study is to investigate the survival of neural stem cells (NSCs) in normal rat cochlea and their potential effect on auditory function and cochlea structures via round window transplantation. Methods: In comparison with the normal rats without any transplantation (group III), normal rat cochleae were transplanted with NSCs infected with adenovirus carrying green fluorescence protein (GFP) gene (group I) or the artificial perilymph (group II) via round windows. Auditory functions were monitored by thresholds of auditory brain stem responses (ABRs); the cochlea structures were examined by hematoxylin and eosin staining; survivals of implanted NSCs were determined by the expression of GFP; survivals of hair cells were accessed by whole mount preparation; and ultrastructures of hair cells were examined by scanning electron microscopy. Result: There were significant differences in the click-ABR thresholds in rats among all 3 groups neither at pretransplantation nor at posttransplantation; there were no significant differences in these values before and after transplantation in the same rats from each group. After transplantation, the cochlea structures were normal in both group I and group II. Grafted NSCs were visualized by the GFP expression in every turn of the cochlea in all animals of group I. There were no significant differences in the losses of outer hair cells (OHCs) among 3 groups. The inner hair cells and most OHCs were normal in every turns of cochleae of all groups. Conclusion: Neural stem cells survived in normal rat cochlea after transplantation via round window and showed no obvious effects on auditory functions and inner ear pathologic examination of the rat cochlea. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Hearing loss in mammals is irreversible because the lost hair cells (HCs) cannot be replaced by cell division and regeneration from endogenous cells in the inner ear epithelia. The past year, stem cells were introduced into the search for ⁎ Corresponding author. Department of Otorhinolaryngology Head and Neck Surgery, Beijing, Tongren Hospital, Capital Medical University. Beijing 100730, People's Republic of China. Tel.: +86 13911068366. E-mail address: [email protected] (S. Gong). 0196-0709/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.amjoto.2007.12.006
new approaches to HC regeneration in mammals. A major advance in the use of stem cells for the replacement of inner ear cells came with the recent discoveries [1-5] that neural stem cells (NSCs) survived and differentiated into neurons and astrocytes after transplantation into normal or damaged rat cochleae via different approaches. However, the changes in the auditory function and the cochlea morphology after NSC transplantation had not been studied in details. Previously, a recombinant adenovirus-expressing green fluorescence protein GFP (Ad-GFP) was successfully constructed from this laboratory, as described by Fu et al
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[6]. We showed that the efficiency of Ad-GFP infection into NSCs isolated from fetal rat hippocampus was high, and the infected NSCs were able to divide and self-renew, as well as sustain the expression of GFP. In this study, primary NSCs infected with Ad-GFP were transplanted into natural rat cochleae via round window, the cell survival was examined by the expression of GFP reporter, and the changes in the auditory function and cochlea morphology were also investigated to provide additional information for stem cellbased cell therapy for the damaged inner ear. 2. Materials and methods 2.1. Experimental rats All animal experiments were approved by Tongji Medical College of Hua-Zhong University of Science and Technology (Wu Han, People's Republic of China) and were performed using accepted veterinary standards. Animals were obtained from the experimental animal center of Tongji Medical College. At the beginning of the experiment, 45 Sprague-Dawley rats weighed 200 to 250 g were selected, and all had normal Preyer's reflexes. Animals were assigned randomly to 1 of 3 groups: group I, NSC implantation group (experimental group [n = 15]); group II, artificial perilymph implantation group (control group [n = 15]); and group III, nonimplantation group (normal rat group [n = 15]). Before any procedure, clickauditory brainstem response (ABR) was recorded and calibrated to decibel sound pressure level (dBSPL) peak-topeak-equivalent according to the method by Mitchell et al [7] to determine the baseline hearing threshold. 2.2. Preparation of NSCs infected with Ad-GFP Neural stem cells infected with Ad-GFP were prepared as described by Fu et al [6,8]. Briefly, the hippocampal tissues from rat embryos were isolated and transferred to neurosphere culture medium (DMEM/F12 supplemented with 2% B27, 20 ng/mL of basic fibroblast growth factor, 20 ng/mL epidermal growth factor). On day 3, primary spheres were collected, spun down, resuspended in neurosphere culture medium, and completely dissociated by pipetting. The obtained cell suspension was continued in the same culture condition. On day 6, secondary NSC spheres were infected by Ad-GFP that was constructed in our laboratory. On day 5 after infection, NSCs were centrifuged and rinsed twice by fresh culture medium, then completely dissociated by pipetting into cell suspension and stored at 4°C for transplantation at a cell density of 1 × 106 cells/mL. 2.3. Implantation of NSCs into rat cochlea via the round window Neural stem cells infected by Ad-GFP were transplanted into cochleae of rats in group I, artificial perilymph was similarly injected into cochleae of rats in group II; the animals in group III were not under surgery. Artificial fluid of perilymph composed of 125 mmol/L NaCl, 3.5 mmol/L
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KCl, 1.20 mmol/L MgCl, 0.75 mmol/L NaH2PO4, 25 mmol/L NaHCO3, and 5 mmol/L glucose was filtrated and stored at 4°C. For implantations, the left ears of rats from group I and group II were processed under general anesthesia (ketamine, 4 mg/100 g body weight, and xylazine, 1 mg/100 g body weight intramuscular) and antibiotic protection (chloramphenicol, 30 mg/kg subcutaneous). Briefly, the left postauricular region was shaved and sterilized with 70% ethanol. The animal was then placed on a heating pad (37°C), the left bulla was exposed by the postauricular approach, and the basal turn and the round window membrane of the cochlea were identified. A 20-μL microsyringe (Exmire microsyringe, Ito Corporation, Fuji, Shizuoka, Japan) was inserted into round window; 5 μL perilymph was drawn out and then 10 μL cell suspension (group I) or artificial perilymph (group II) was injected slowly at the speed of 1 μL/min into the scala tympani at the basal cochlear turn. The needle of the microsyringe was kept in the inner ear for several minutes to prevent the NSCs and the culture medium from flowing out. A small piece of fascia was then placed over the round window, and the incision was closed with sutures. 2.4. Postimplantation evaluations of experimental rats On day 14 after surgery, click-ABR was again recorded under general anesthesia to determine any postsurgical threshold shift in group I and group II, as described above. Click-ABR was recorded at the same time for rats in group III. Then, 5 animals from each group were used to observe the structure of cochlea and assess the survival of NSCs by tracking the expression of GFP in the cochlea; another 5 animals from each group were processed for scanning electronic microscopy (SEM); and the last 5 animals from each group were used for surface preparation of whole organ of Corti of the cochlea. 2.4.1. Tissue processing The animals were perfused intracardially with normal saline, followed by 4% paraformaldehyde in phosphate buffered saline. The left temporal bones were removed and immersion fixed in the same fixative at 4°C for 4 hours. After decalcification with 0.1 mol/L EDTA for 24 hours at 4°C, 10-mm cryostat sections of the temporal bones were prepared. The sections were then mounted on α-aminopropyltriethoxysilane-coated slide glasses. 2.4.2. Hematoxylin and eosin staining Midmodiolus sections from each animal were provided for histologic analysis by hematoxylin and eosin staining. Briefly, after stained by hematoxylin, slides were disposed by hydrochloric acid, ethanol, and lithium carbonate; stained by eosin; dehydrated by graded ethanol solutions; cleared by xylene; and embedded by the natural gum. 2.4.3. Observing under fluorescent microscope Neighboring midmodiolus sections were used to view the expression of GFP as indication of the survival of
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Fig. 1. Survival of NSCs and expression of GFP in the cochlea. Grafted NSCs were visualized by GFP expression in every turn of the cochlea in all animals of group I (original magnification ×40 [A]; ×200 [B-C]; ×400 [D]). The expression of GFP was not found in animals of group II (original magnification ×100 [E]).
NSCs under a Zeiss fluorescent microscope equipped with a digital camera (Spot RT, Diagnostic Instrument or Polaroid DMC le, Sterling Heights, MI). 2.4.4. Accessing numbers of HCs To count the cell number and determine the loss of HCs, another 5 animals from each group were processed for silver nitrate staining and surface preparation of whole organ of Corti. Briefly, the left cochleae were exposed. After the round window and the oval window were opened and a small hole was then made in the apex of the cochlea using a 26-gauge needle, about 5 mL of 0.5% silver nitrate solution was gently perfused into perilymph from the hole. After immersed in 0.5% silver nitrate solution for 15 minutes, the cochleae
was rinsed by distilled water, perfused by 10% formalin and then immersed for 4 hours. The cochleae were exposed in the sun until the cochleae presented tawny. Under microTable 1 Average thresholds (dBSPL) of click-ABR Group
Pretransplantation
Posttransplantation
I II III
31.00 ± 3.87 30.67 ± 4.17 30.83 ± 3.28
33.00 ± 4.14 32.67 ± 4.17 32.75 ± 4.21
Before implantation, the click-ABR threshold had no significant difference in rats of all 3 groups. There were also no significant difference between pretransplantation and posttransplantation in each group and no significant difference after transplantation among all groups (P N .05).
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scope, the whole basilar membrane was separated and divided into 3 parts by different turns. After mounted on slides containing 50% glycerol, the stained specimens were observed. Then, the number of HCs was counted by the method of Ding et al [9]. 2.4.5. Scanning electronic microscopy To observe the morphologies of HCs and the stereocilia, 5 animals from each group were processed for SEM. Briefly, the bullae were quickly removed, and the cochleae were perfused gently with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.2; 4°C). After being stored in the same fixative for 24 hours, the cochleae were decalcified with 10% EDTA for 4 days and then postfixed with 2% osmic tetroxide in 0.1 mol/L phosphate buffered saline. The basilar membrane was microdissected and dehydrated through serially graded ethanol solutions ending at 100%. Specimens were rinsed in acetone and dried at a critical point in liquid carbon dioxide. They were then coated with gold sputter to a depth of approximately 25 nm and examined on a field emission of SEM (SEM-800) using accelerating voltage of 25 kV. 2.5. Statistic analysis The data were analyzed using Sigma Stat statistical software (SPSS/Jandel Scientif Software, Chicago, IL). Differences between presurgery thresholds and postsurgery thresholds of each group were analyzed using paired t tests. Further analyses were performed to compare presurgery thresholds or postsurgery thresholds among 3 groups using analysis of variance. Differences of the loss of HCs from each turn in each group and differences of the total loss of HCs in each group were analyzed using analysis of variance. In all cases, a value of P less than .05 was considered statistically significant. 3. Results There were no mortalities. Animals did not develop head tilts nor lose weights after surgery. There were no signs of wound infection, middle ear infection, vestibular dysfunction, or any other pathologic malfunction caused by the surgical procedure in any animal. The injection sites were completely sealed at day 5 after injection. After decapitation, normal middle ear cavities were found in all animals. No blood effusion was found inside the cochlear space in any animal, indicating that no major trauma was caused by the inoculation procedure itself. 3.1. Survival of implanted NSCs and expression of GFP Grafted NSCs were identified in the inner ear of each specimen by the expression of GFP. Grafted NSCs were found in every turn of the cochlea in all the rats from group I Fig. 2. Histologic examination of cochlea from experiment animals (×100). Hematoxylin and eosin staining of cochlea sections indicated the intact and normal structures from all experimental animals (A [group I]; B [group II]; C [group III]).
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(Fig. 1A). Most grafted NSCs were found as a cluster of cells in the perilymphatic spaces (Fig. 1B-D) in which some cells attached to the walls of scala tympani and scala vestibuli, some cells attached to the vestibular membrane or existed within the vestibular membrane, and some cells attached to the basilar membrane in scala tympani. Some grafted NSCs were found in the spiral ganglions (Fig. 1C). Quite a few of grafted NSCs were observed in endolymphatic spaces attached to the tectorial membrane (Fig. 1D). No grafted cells were found in the organ of Corti and spiral ligament of the cochlea. The expression of GFP was not found in any rat of group II (Fig. 1E). 3.2. Measurement of ABR threshold There were significant differences in the click-ABR thresholds in rats among all 3 groups neither at pretransplantation nor at posttransplantation; there were no significant differences in these values before and after transplantation in the same rats from each group. These results were recorded in Table 1. 3.3. Histologic examination of rat cochlea after hematoxylin and eosin staining In rats of all the 3 groups, the result of histologic examination of cochleae showed intact and normal cochlea, including the structures of organ of Corti, spiral ligament, and spiral ganglions of cochlea (Fig. 2). Most grafted cells in group I were found as a cluster of cells in the perilymphatic spaces (Fig. 2A). 3.4. Accessing HCs and their losses The structure of basilar membranes of the basal turns was intact and normal with stereocilia of HCs in order. There were dispersive losses of outer hair cells (OHCs) in all basal turns of cochlea of every group (Fig. 3A, B, and C). The OHC loss increased gradually from the basal turn to the apex turn, but the total losses from all groups were all less than 1%. Among all groups, neither the OHC losses of each turn nor the total losses had significant differences. The stereocilia of OHC3 (OHCs in the third row) of the apex turn were out of order, and the structure of some stereocilia was illegible (Fig. 4A, B, and C). There was not evident loss of inner hair cells (IHCs) in all turns of cochlea of every group. The numbers of HCs were recorded in Table 2, and the losses of HCs were recorded in Table 3. 3.5. Scanning electron microscopy examination of HCs There were losses of OHCs in every turn of cochlea from rats of all groups. Normal stereocilia and cuticular plate of most OHCs existed in the basal turn where the arrangement Fig. 3. Whole mount preparation for accessing HCs and their losses (the basal turn ×200). The basilar membranes from all animal groups were intact and normal, with the stereocilia of HCs in order, and the dispersive loss of OHCs (arrow) in the area. OHC1 indicates OHCs in the first row.
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“V”model (Fig. 5D). Normal stereocilia and cuticular plate of IHCs existed in every turn of cochlea of rats in all groups, and no losses of IHCs were found.
4. Discussion
Fig. 4. Whole mount preparation for accessing HCs and their losses (the apex turn ×200). The stereocilia of OHC3 were out of order, and the structure of some stereocilia was illegible. The lost number of OHCs (arrow) in the apex turn became more than those in the basal turn.
of stereocilia of OHCs presented “V” model (Fig. 5A-C). But the arrangement of stereocilias of OHC2 and OHC3 (OHCs in the second and third row) were relatively in disorder in the apex turn, where the arrangement became cluster shape from
Neural stem cells have been reported to be capable of selfrenewal and differentiation into the major cell types of the neural tissue, including neurons, astrocytes, and oligodendrocytes [10,11]. Thus, it is possible to differentiate NSCs into cells of functional auditory system via transplantation. Previous studies have shown that adult rat hippocampusderived NSCs grafted into the newborn rat inner ear survived in the cochlear cavity [1]. In addition, fetal mouse NSCs derived from the dorsal telencephalon have been shown to have the potential to survive and differentiate in the normal adult mouse inner ear, and NSC-derived cells can survive in the perilymphatic space for at least 28 days, but no grafted cells are found in endolymphatic space [2]. In another study [3], when the recipient mouse were injured by neomycin, grafted cells migrated beyond the bony wall that separates the endolymphatic space from the perilymphatic space and survived in both the perilymphatic and endolymphatic spaces. Some of these grafted cells even migrated into the labyrinth membrane, sensory epithelia, spiral limbs, and modiolus. In the cochlea injured by cisplatin [4], grafted NSCs survived and differentiated in midmodiolus and spiral ganglions. It has also been reported that [5] implanted cells attached to both the osseous Rosenthal canal and to the close proximity of the organ of Corti in the scala tympani. Moreover, implanted NSCs were also observed outside fluidfilled compartments and some even located along the auditory nerve fibers projecting to the hearing organ. The implanted cells were predominantly found 1 and 2 weeks after transplantation in normal-hearing animals and mostly differentiated into astrocytes. Together, these findings provide strong evidences that NSC transplantation may be used as an alternative for cell therapy for HC restoration. In these experiments, however, locations where implanted cells survived were different, and the changes of hearing functions or cochlea morphologies have not been investigated in details. There are 2 commonly used approaches for introducing foreign active materials into the cochlear fluid—inoculation into the scala tympani through the round window membrane or through a cochleostomy [12–14]. Round window injection has been considered less invasive because it preserves the natural structure and produces less local bleeding and less inflammatory reaction. In addition, it requires a shorter time and easier procedure to operate [12,14]. Our study showed that the structures of cochleae of all the rats after round window implantation were intact and normal; the morphologies for the organ of Corti, spiral ligament, and spiral ganglions of cochlea were all normal without red cells and exudation inside the cochlear space.
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Table 2 The number of HC of each turn of every group Group
I II III
The basal turn
The second turn
The apex turn
OHCs
IHCs
OHCs
IHCs
OHCs
IHCs
1867 ± 7.55 1870 ± 5 1865 ± 5
550 ± 2.52 543 ± 7.64 545 ± 5
1141 ± 6.56 1137 ± 7.64 1145 ± 5
280 ± 4.73 280 ± 5 278 ± 4.51
658 ± 12.58 658 ± 10.41 652 ± 7.64
183 ± 5 180 ± 5 182 ± 3
The number of OHCs in natural rat is 3662 ± 11.55; the number of IHCs in natural rat is 1005 ± 6.43.
This indicated that transplantation surgery through round window was not invasive. Using this technique, the absence of a significant and conspicuous perilymph leakage is intriguing, considering that the fluid is incompressible. One (or more) of the after mechanisms may account for the ability to inoculate the cochlea without leakage as follows: (1) the increased pressure leads to perilymph displacement via the cochlear aqueduct; (2) the stapes and round window may both be transiently distended to accommodate the positive pressure; or (3) endolymph may be compressed into the endolymphatic duct and sac, thereby transiently reducing the volume of scala media. In addition, because the injected volume (10 μL) was larger than the total volume of perilymph in rats [15], 5 μL perilymph was removed slowly before the injection of 10 μL cell suspension into the scala tympani slowly at the speed of 1 μL/min to inject more cell suspension and avoid a mechanical trauma to the cochlea that would injure or cause degeneration of HCs. In this study, NSCs infected by Ad-GFP were transplanted into normal rat cochlea via round window and were investigated for their survival, the changes of hearing function, and cochlea morphology in the host animals. Using round window approach, on day 14 after transplantation, grafted NSCs were identified in the inner ear of each specimen by the expression of GFP. Grafted NSCs were found in every turn of the cochlea in all the experimental animals of group I and existed not only in scala tympani but also in scala vestibuli, suggesting that the grafted cells might have moved significant distances along the cochlea, possibly after the flow of perilymph from the transplantation site in scala tympani through helicotrema to scala vestibuli [16]. Some cells attached to the vestibular membrane or existed within the vestibular membrane, quite a few of grafted NSCs were observed in endolymphatic spaces attached to the tectorial membrane. Because the vestibular membrane separates the endolymphatic space from the perilymphatic space, grafted cells attached to the vestibular membrane or existed within the vestibular membrane could migrate from perilymphatic space into endolymphatic space through the vestibular membrane. In our study, a fewer grafted NSCs were observed in endolymphatic spaces as compared with the study of Tateya [3]. Since the study of Tateya [3], NSCs were grafted into the cochlea of mice injured by neomycin so the injury of sensory epithelia caused by aminoglycoside toxicity might
have promoted integration of grafted NSCs into the sensory epithelia of the inner ear. More important, it was shown that NSCs also migrated from the implantation site within the fluid-filled scala tympani toward spiral ganglion neurons through the bony modiolus, also observed in the study of Hu [5]. This result suggested that adult NSCs indeed possessed the capacity to migrate to functionally important structures after implantation into mature inner ear. It has been suggested that the implanted cells were directed by the release of growth factors from the host neural tissues and migrated by means of the minute holes passing from scala tympani through the bone toward the Rosenthal canal [5]. None of grafted cells was found in the organ of Corti and spiral ligament of the cochlea. Although grated cells were not found in the organ of Corti, the surviving cells attached to the basilar membrane in scala tympani and to the bony walls of scala tympani and scala vestibuli. Because these were the sites closer to growth factors or essential nutritional supplements [17,18], lacking of some of the essential growth factors or nutrition may be one reason for the lack of grated cells in the spiral ligament of the cochlea. Before surgery, the click-ABR thresholds were similar in all animal groups, which indicated that the experimental condition was equal. There were no significant differences in ABR thresholds before and after transplantation in both group I and group II, which was consistent with previous studies [14,19,20]. In those studies, almost complete preservation of the cochlear structure and ABR thresholds were also shown after fluid application to the scala tympani via round window. There was also lack of significant difference in ABR thresholds in the 3 groups after transplantation, which indicated that embryonic NSC transplantation into cochlea through round window had no obvious effects on auditory Table 3 The loss of OHCs Group The basal turn loss rate
The second turn The apex turn loss rate loss rate
The total loss rate (%)
I II III
0.44 ± 0.09 0.47 ± 0.05 0.49 ± 0.05
0.53 ± 0.02 0.55 ± 0.03 0.54 ± 0.03
0.23 ± 0.06 0.24 ± 0.03 0.23 ± 0.03
1.52 ± 0.03 1.52 ± 0.13 1.53 ± 0.02
The loss of OHCs increased gradually from the basal turns to the apex turns, but the total losses were all less than 1% from all groups. Both the OHC loss in each turn and the total losses in each group had no significant differences among rats in 3 groups (P N .05).
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Fig. 5. Scanning electron microscopy examination of HCs. There were dispersive losses of OHCs (⁎) in each turn of cochleae from all groups and normal stereocilia and cuticular plate of most OHCs in the basal turns. (D) The arrangement of stereocilia of OHC2 and OHC3 was relatively in disorder in the apex turn.
function of the normal rat. However, as we had assessed hearing thresholds with click-ABR, predominantly representing the frequency region around 8 kHz, we cannot rule out high frequency hearing loss. The total number of OHCs was 3662 ± 11.55 in the cochlea of the natural rats, and the total number of IHCs was 1005 ± 6.43. There was dispersive loss of OHCs in all basal turns of cochlea of every group. The loss of OHCs increased
gradually from the basal turn to the apex turn, but the total losses of all groups were all less than 1%. These results were consistent with previously correlative studies by others [19,20]. In addition, there were no significant differences in HC losses of each turn in every group or the total losses among these 3 groups. The IHCs were normal in all turns of cochleae from all groups. These results indicated that the losses of OHCs were natural loss and not related to the
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surgery. Examination by SEM also showed that stereocilia and cuticular plate of IHCs and most OHCs were normal in each turn of cochlea of every group, and there were no losses of IHCs, which indicated that NSC transplantation into cochlea through round window had no obvious effects on ultrastructures of HCs of the normal rat. This experiment was only a primary study on NSC transplantation into cochlea via round window. Extended observation periods and more detailed physiologic measures will be necessary to examine long-term effects. To realize stem cell-based cell therapies for the damaged inner ear in the future, it will be necessary to examine the final fate of grafted cells, to increase the differentiation toward neurons from the grafted cells, to promote migration of the cells into the sensory epithelia, and even to transfer into HCs. Together, the present findings show that the GFP-labeled embryonic NSCs survived in normal rat cochlea after transplantation and expressed GFP efficiently. Although the implanted NSCs showed no obvious effect to auditory function and inner ear pathologic finding of rat cochlea, our study provided useful information for stem cell-based cell therapies. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 30672308) and the Medical Science Research Fund of Zhejiang Province, China (No. 2008A056). References [1] Ito J, Kojima K, Kawaguchi S. Survival of neural stem cells in cochlea. Acta Otolaryngol 2001;121(2):140-2. [2] Iguchi F, Nakagawa T, Tateya I, et al. Trophic support of mouse inner ear by neural stem cell transplantation. Neuroreport 2003;14:77-80. [3] Tateya I, Nakagawa T, Iguchi F, et al. Fate of neural stem cells grafted into injured inner ears of mice. Neuroreport 2003;14(13): 1677-81.
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