Cell transplantation to the auditory nerve and cochlear duct

We have developed a technique to deliver cells to the inner ear without injuring the membranes that seal the endolymphatic and perilymphatic chambers. The integrity of these membranes is essential for normal hearing, and the technique should significantly reduce surgical trauma during cell transplantation. Embryonic stem cells transplanted at the internal auditory meatal portion of an atrophic auditory nerve migrated extensively along it. Four-five weeks after transplantation, the cells were found not only throughout the auditory nerve, but also in Rosenthal's canal and the scala media, the most distal portion of the auditory nervous system where the hair cells reside. Migration of the transplanted cells was more extensive following damage to the auditory nerve. In the undamaged nerve, migration was more limited, but the cells showed more signs of neuronal differentiation. This highlights an important balance between tissue damage and the potential for repair.     

An animal experimental model of auditory neuropathy induced in rats by auditory nerve compression

Several animal models of auditory neuropathy (AN) have been produced by employing pharmacological agents to damage auditory neurons or hair cells selectively. The specificity of pharmacological lesions is generally assessed by observation of visible structural damage but it is difficult to localize the delivery, which could lead to functional side effects in other anatomical structures. Although genetic analyses of human AN patients have provided important information on the pathophysiology of AN, specific genetic defects have not been fully correlated with functional deficits in the auditory nervous system. To address this problem, we compressed rat auditory nerves to assess neural degeneration for up to 35 weeks. The method produced a good model of auditory neuropathy, including profound deterioration of the auditory brainstem response and preservation of both cochlear microphonics and distortion product otoacoustic emissions. Histological examination revealed that in spite of profound degeneration of the auditory nerve, the hair cells remained intact. The model provides a complementary alternative to those based on pharmacological lesions and genetic analyses of AN patients and should allow analysis of the pathophysiology of auditory neuropathy with less risk of the results being confounded by unknown deficits in other cell types.     

Central migration of neuronal tissue and embryonic stem cells following transplantation along the adult auditory nerve

The regeneration of the auditory nerve remains a challenge in restoring hearing. An interesting approach would be to use a cell replacement therapy with the potential to establish connections from the inner ear to the central auditory system. This hypothesis was tested by xenografted (mouse to rat) implantation of embryonic dorsal root ganglion (DRG) neurons and embryonic stem (ES) cells along the auditory nerve in the adult host. DRG neurons were obtained at embryonic day 13-14 in transgenic animals expressing enhanced green fluorescence protein (EGFP). For embryonic stem cells, a tau-GFP ES cell line was used as a donor. The fibers of the auditory nerve in the adult rat were transected through the modiolus at the first cochlear turn, and the biological implants were transplanted into the transection. The transplanted DRG neurons and ES cells survived for a postoperative survival time ranging from 3 to 9 weeks, verified by EGFP/GFP fluorescence, and neurofilament or TUJ1 immunostaining. At 9 weeks following implantation, the implanted DRG neurons were found to have migrated along the auditory nerve in the internal meatus. At the same postoperative time, the ES cells had migrated into the brain stem close to the ventral cochlear nucleus. The results demonstrate not only the survival and migration of xenografted DRG neurons and stem cells along the adult auditory nerve but also the feasibility of a cell replacement therapy in the degenerated auditory system.     

The maturation of the auditory brainstem response compared to peripheral nerve conduction velocity in preterm and full-term infants

The maturation of the auditory brainstem response in preterm and full-term infants is compared with that of nerve conduction velocity. There is a linear relationship between wave I latency, the peripheral component of the response, and nerve conduction velocity, but the negative correlation is not high. A poor negative correlation exists between the I-V interval, an index of brainstem transmission, and nerve conduction velocity. The factors governing the maturation of central transmission along the auditory pathway in the brainstem are not related to myelination of peripheral nerves. Abnormal nerve conduction velocities are not related to any particular abnormal brainstem response in a stable external environment.     

Differentiated adipose-derived stem cells promote myelination and enhance functional recovery in a rat model of chronic denervation

Transplantation of autologous Schwann cells (SCs) is a promising approach for treating various peripheral nerve disorders, including chronic denervation. However, given their drawbacks, such as invasive biopsy and lengthy culture in vitro, alternative cell sources would be needed. Adipose-derived stem cells (ASCs) are a candidate, and in this study rat ASCs transdifferentiated into a SC phenotype (dASC) cocultured with dorsal root ganglion neurons were shown to associate with neurites and to express myelin basic protein (MBP)-positive myelin protein. Furthermore, dASCs transplanted into a chronically denervated rat common peroneal nerve survived for at least for 10 weeks, maintaining their differentiated state. Immunohistochemical analysis revealed that transplanted dASCs associated with regenerating axons, forming MBP-/protein zero-positive myelin sheaths. The cell survival and myelin expression assessed by double labelling with S100 and glial fibrillary acidic protein were similar between the dASC- and SC-transplanted nerves. Importantly, transplantation of dASCs resulted in dramatically improved motor functional recovery and nerve regeneration, with a level comparable to, or even superior to, transplantation of SCs. In conclusion, dASCs are capable of myelinating axons in vivo and enhancing functional outcome after chronic denervation.     

Hereditary auditory, vestibular, motor, and sensory neuropathy in a Slovenian Roma (Gypsy) kindred.

Members of a Roma (Gypsy) family with hereditary motor and sensory peripheral neuropathy (HMSN) and concomitant auditory and vestibular cranial neuropathies were identified in Kocevje, Slovenia. The illness begins in childhood with a severe and progressive motor disability and the deafness is delayed until the second decade. There are no symptoms of vestibular dysfunction. The family structure is consistent with an autosomal recessive pattern of inheritance and the genetic locus for the disorder is linked to the same region of chromosome 8q24 as other Roma families with HMSN and deafness from Lom, Bulgaria (HMSN-Lom). The present study shows that the deafness is caused by a neuropathy of the auditory nerve with preserved measures of cochlear outer hair cell function (otoacoustic emissions and cochlear microphonics) but absent neural components of auditory brainstem potentials. The hearing loss affects speech comprehension out of proportion to the pure tone loss. Vestibular testing showed absence of caloric responses. Physiological and neuropathological studies of peripheral nerves were compatible with the nerve disorder contemporaneously affecting Schwann cells and axons resulting in both slowed nerve conduction and axonal loss. Genetic linkage studies suggest a refinement of the 8q24 critical region containing the HMSN-Lom locus that affects peripheral motor and sensory nerves as well as the cranial auditory and vestibular nerves.     

Behavior of Schwann cells from Trembler mouse unmyelinated fibers transplanted into myelinated nerves

In the peripheral nerves of Trembler mice, Schwann cells produce little or no myelin and continue to multiply beyond the normal neonatal period. However, in the unmyelinated (Remak) fibers of these mutants, Schwann cell morphology and multiplication are normal. To determine if such phenotypically normal Schwann cells would show the Trembler abnormalities when challenged to form myelin in nerve grafts, an unmyelinated nerve, the cervical sympathetic trunk, was transplanted into the richly myelinated sural nerve in a series of normal host animals. Two months after transplantation, regenerated grafts, composed of normal host axons and Trembler cervical sympathetic trunk Schwann cells, showed the characteristic Trembler abnormalities of myelination and multiplication whereas Schwann cells from normal cervical sympathetic trunks myelinated the host axons normally. Thus, the Trembler mutation appears to affect Schwann cell differentiation at a specific phase, the formation of myelin. This primary expression of the Schwann cell abnormality initiates a cycle of secondary effects that include demyelination, Schwann cell multiplication, and attempted remyelination.     

Human amnion tissue injected with human umbilical cord mesenchymal stem cells repairs damaged sciatic nerves in rats

Human umbilical cord mesenchymal stem cells,incorporated into an amnion carrier tubes,were assessed for nerve regeneration potential in a rat nerve defect model.Damaged nerves were exposed to human amnion carriers containing either human umbilical cord mesenchymal stem cell (cell transplantation group)or saline(control group).At 8,12,16 and 20 weeks after cell implantation,the sciatic functional index was higher in the cell transplantation group compared with the control group.Furthermore,electrophysiological examination showed that threshold stimulus and maximum stimulus intensity gradually decreased while compound action potential amplitude gradually increased.Hematoxylin-eosin staining showed that regenerating nerve fibers were arranged in nerve tracts in the cell transplantation group and connective tissue between nerve tracts and amnion tissue reduced over time.Gastrocnemius muscle cell diameter,wet weight and restoration ratio were increased.These data indicate that transplanted human umbilical cord mesenchymal stem cells,using the amnion tube connection method,promote restoration of damaged sciatic nerves in rats.     

Transplantation of primary satellite cells improves properties of reinnervated skeletal muscles

Skeletal muscle demonstrates a force deficit after repair of injured peripheral nerves. We tested the hypothesis that transplantation of satellite cells into reinnervated rabbit tibialis anterior (TA) muscles improves their properties. Adult rabbits underwent transection and immediate suture of the common peroneal nerve. In order to provide an environment favorable for cell transplantation, TA were then made to degenerate by cardiotoxin injection, either immediately or after a 2-month delay, which is sufficient for muscle reinnervation. In both cases, the injured TA were transplanted with cultured satellite cells 5 days after induction of muscle degeneration. When cells were transferred immediately after nerve repair, drastic morphological and functional muscle alterations were observed. However, when the muscles were allowed to become reinnervated before cell transplantation, muscles were heavier and developed a significantly higher maximal force compared to denervated-reinnervated muscles. Thus, application of the cell therapy protocol improved properties of denervated muscles only when they were allowed to become innervated. These results, which represent the application of cell therapy to improve force recovery of reinnervated muscles, will be of significant interest in certain clinical contexts, particularly after immediate or delayed muscle reinnervation.     

嗅神经鞘细胞和自体坐骨神经移植修复受损视神经

背景：有研究已初步证明了甲泼尼龙对受损视网膜节细胞的早期保护作用。 目的：观察甲泼尼龙联合嗅神经鞘细胞和自体坐骨神经移植大鼠受损视神经纤维及其髓鞘的形态学及计数变化。 设计、时间及地点：随机对照动物实验，于2005-09/2006-03于北京大学医学部神经解剖实验室完成。 材料：选取成年雄性SD大鼠40只，取其中4只做形态学正常对照。 方法：其余36只大鼠按随机数字表法分为6组，即视神经损伤模型组、甲泼尼龙组、嗅神经鞘细胞移植组、嗅神经鞘细胞移植+甲泼尼龙组、坐骨神经移植组、坐骨神经移植+甲泼尼龙组，每组6只。 主要观察指标：取视神经，制作冰冻切片，移植后14，21 d分别从各组切片中随机抽取10张载玻片，对生物素标记的葡聚糖胺染色的视神经纤维及甲苯胺蓝染色的视神经髓鞘进行形态学观察，电镜下对视神经纤维髓鞘进行计数。 结果：顺行追踪剂生物素标记的葡聚糖胺在视神经中的染色结果主要表现为嗅神经鞘细胞移植+甲泼尼龙组、坐骨神经移植+甲泼尼龙组，与视神经损伤模型组相比，神经纤维排列较整齐，发生中晚期溃变的纤维比例较小。移植后14，21 d，甲泼尼龙组视神经纤维髓鞘数量均显著高于视神经损伤模型组(P < 0.05，0.01)。坐骨神经移植+甲泼尼龙组均显著高于坐骨神经移植组(P < 0.05)。嗅神经鞘细胞移植+甲泼尼龙组均显著高于嗅神经鞘细胞移植组(P < 0.05)。 结论：形态学结果表明嗅神经鞘细胞和自体坐骨神经移植与甲泼尼龙的联合作用能激发并增强受损中枢神经的自我保护及修复作用，对视神经纤维及其髓鞘溃变速度有延缓作用。     

Morphological consequences of acoustic trauma on cochlear hair cells and the auditory nerve

Aims: Hearing loss is the most common form of sensory impairment in humans. Short impulses of a high intensity noise can trigger sudden hearing loss, which is generally irreversible and associated with structural tissue damage of the cochlea and auditory nerve. It is well established that myelination is essential for the rapid propagation of action potentials along axons, and that Schwann cells are responsible for myelin sheath production in the peripheral nervous system. In the cochlea, spiral ganglion neuron axons are myelinated by Schwann cells. This myelin contributes to axonal protection and allows for efficient action potential transmission along the auditory nerve. For this reason, here we studie the morphological changes on cochlear hair cells and myelin sheaths of the auditory nerve, directly linked to hearing impairment induced by acoustic trauma.

Material and methods: To study the auditory functions, auditory brainstem responses and distortion products were measured at baseline, 2 days, and 21 days after trauma in rats. Then, scanning and transmission electron microscopy techniques were performed to analyze cochleae and the auditory nerve at 21 days after trauma.

Results: We observed that acoustic trauma induced cochlear outer hair cell loss and fusion of inner hair cell stereocilia. We also observed an axonal loss and myelin sheath disorganization of the auditory nerve.

Conclusions: These data confirm that a strong acoustic trauma induced histological changes in the cochlea and auditory nerve, leading to permanent hearing loss.     

Effect of epithelial stem cell transplantation on noise-induced hearing loss in adult mice

Noise trauma in mammals can result in damage to multiple epithelial cochlear cell types, producing permanent hearing loss. Here we investigate whether epithelial stem cell transplantation can ameliorate noise-induced hearing loss in mice. Epithelial stem/progenitor cells isolated from adult mouse tongue displayed extensive proliferation in vitro as well as positive immunolabelling for the epithelial stem cell marker p63. To examine the functional effects of cochlear transplantation of these cells, mice were exposed to noise trauma and the cells were transplanted via a lateral wall cochleostomy 2 days post-trauma. Changes in auditory function were assessed by determining auditory brainstem response (ABR) threshold shifts 4 weeks after stem cell transplantation or sham surgery. Stem/progenitor cell transplantation resulted in a significantly reduced permanent ABR threshold shift for click stimuli compared to sham-injected mice, as corroborated using two distinct analyses. Cell fate analyses revealed stem/progenitor cell survival and integration into suprastrial regions of the spiral ligament. These results suggest that transplantation of adult epithelial stem/progenitor cells can attenuate the ototoxic effects of noise trauma in a mammalian model of noise-induced hearing loss.     

Mechanisms of intraoperative brainstem auditory evoked potential changes.

Brainstem auditory evoked potential (BAEP) changes during intraoperative monitoring may reflect damage to or potentially reversible dysfunction of the ear, the eighth nerve, or the brainstem auditory pathways up to the level of the mesencephalon. They may also be caused by other physiologic mechanisms such as anesthesia, hypothermia, and acoustic masking from drilling noise, or they may result from technical factors that prevent proper stimulus delivery or recording of an evoked potential that is actually present. Cochlear ischemia or infarction resulting from compromise of the internal auditory artery and inner ear damage during temporal bone drilling will affect all BAEP components, including wave I. Direct mechanical or thermal trauma to the eighth nerve will delay, attenuate, and possibly eliminate waves III and V, but wave I, which is generated at the cochlear end of the eighth nerve, may be preserved. During scraping of tumor off the eighth nerve, force applied in an ear-toward-brainstem direction can avulse the fragile fibers of the distal eighth nerve at the area cribrosa. Prolonging the I-to-III interpeak interval during retraction of the cerebellum and brainstem reflects stretching of the eighth nerve, and is often reversible. Vasospasm within the eighth nerve can cause similar, potentially reversible BAEP changes. Damage to the brainstem auditory pathways at or below the level of the mesencephalon will delay and attenuate or eliminate wave V. Wave III is affected similarly if the damage is at or caudal to the region of the superior olivary complex. These BAEP changes may reflect direct mechanical or thermal damage to the brainstem, brainstem compression, or ischemia or infarction resulting from vascular compromise. During BAEP monitoring, examination of the pattern of BAEP changes, analysis of their correlation with surgical maneuvers, and investigation for possible contributory technical factors can help to determine the cause of the BAEP changes and provide the appropriate information to the rest of the surgical team.     

Axonal regeneration of retinal ganglion cells in the cat geniculocortical pathway

The optic nerve of anesthetized cats was completely cut and the autologous sciatic nerve was transplanted. Sixty days later some populations of retinal ganglion cells were shown to regenerate the axon with retrograde HRP labeling. We verified that ganglion cells that had projected to the lateral geniculate nucleus (LGN) were able to regenerate through the transplant with a double-labeling method: diI was injected into the LGN prior to the transplantation, and dextran-fluorescein was injected into the graft after axonal regeneration. Intracellular injection of HRP into regenerating ganglion cells in an in vitro preparation revealed that the two major cell types projecting to the LGN, and β, regenerated axons and showed normal dendritic morphology.     

Postoperative facial and vestibular nerve palsy: experimental study of its pathophysiological mechanisms

The 7th and 8th cranial nerves were shifted in the cerebellopontine (CP) angle of dogs by cerebellar retractions that were similar to those performed in humans with monitoring of auditory evoked brainstem responses (ABR). Postoperatively, the vestibular, facial nerves, and brainstem were histologically examined. Caudal-to-rostral shifts of the nerves could induce vestibular and/or facial nerve damages. The most vulnerable portion of the vestibular nerve was located between the vestibular ganglions and the area vestibularis-the most lateral end of the internal auditory canal. This indicated that due to traction force derived from surgical interventions, the nerves and vessels were avulsed at the fundus of the internal auditory canal. The vestibular nerve may be potentially injured more easily and frequently than the cochlear and facial nerves in retromastoid craniectomies with lateral decubitus position in humans. Direct injuries of the facial nerves in the CP angles were not observed in this study. It was elucidated that the facial nerve was usually injured in the facial canal proximal to the geniculate ganglion due to traction force derived from manipulations in the CP angle. It is likely that as facial nerve edema progresses postoperatively, the facial nerve is gradually compressed within the narrow labyrinthine portion of the facial canal. This may be the cause of delayed postoperative facial nerve palsy. The importance to recognize how not only cochlear but also vestibular and facial nerve are injured by the usual manipulations in the CP angle is stressed.     

神经干细胞与神经生长因子对豚鼠损伤内耳保护效果的比较

背景：研究证明神经干细胞在体外能自然分泌一定量的神经生长因子，因此若将神经干细胞移植入内耳，可作为神经生长因子源源不断的供体。 目的：比较神经干细胞内耳移植和神经生长因子肌肉注射两种方式对豚鼠损伤内耳的保护效果。 设计：随机对照动物实验。 材料：新生24 h豚鼠5只，用于脑海马神经干细胞的制备。 方法：①动物实验：健康豚鼠40只，随机分为6组：正常对照组4只，不施加任何处理因素；单纯耳蜗打孔组8只，仅施以耳蜗底转骨壁打孔术；单纯神经干细胞移植组8只，经耳蜗底转骨壁打孔后，向内耳注入Brdu标记的神经干细胞；致聋模型组4只，按4 mg/kg体质量连续3 d腹腔注射顺铂建立耳聋模型；致聋后神经干细胞移植组8只，先造成耳聋模型，而后经耳蜗底转骨壁打孔向内耳注入Brdu标记的神经干细胞；致聋后神经生长因子移植组8只，先造成耳聋模型，然后肌肉注射神经生长因子。移植后14，28 d测定纯音听阈值，各处死4只用于耳蜗基底膜四唑氮蓝铺片、苏木精-伊红染色、Brdu免疫组化染色、神经生长因子免疫组化染色。②RT-PCR检测实验：健康豚鼠30只，随机分为4组：正常对照组8只、致聋模型组8只、致聋后神经干细胞移植组5只、致聋后神经生长因子移植组9只，干预措施同上。移植后28 d RT-PCR检测各组动物耳蜗组织神经生长因子mRNA的表达。 结果：与正常对照组比较，移植后14，28 d致聋模型组、致聋后神经干细胞移植组、致聋后神经生长因子移植组的纯音听阈值均显著升高（P < 0.05），但后2组升高幅度明显低于致聋模型组（P < 0.05），且毛细胞排列整齐，无缺失、核溶解、碎裂等现象，螺旋神经节细胞的结构及形态基本接近正常对照组。移植后7 d，耳蜗底转的移植位点、鼓阶蜗轴侧周围及基底膜下方均可见大量BrdU阳性细胞，血管纹、鼓唇和蜗神经纤维神经生长因子免疫组化染色均呈阳性表达，28 d时致聋后神经干细胞移植组染色程度强于致聋后神经生长因子移植组。RT-PCR检测耳蜗神经生长因子mRNA的表达与免疫组化染色结果基本一致。 结论：内耳移植神经干细胞和肌肉注射神经生长因子对豚鼠损伤内耳的毛细胞、螺旋神经节以及听力保护作用基本相似，但免疫组化及RT-PCR结果表明前者耳蜗内分布的神经生长因子较后者更为密集。     

Repair of a myelin lesion by Schwann cells transplanted in the adult mouse spinal cord

In multiple sclerosis and experimental demyelination, oligodendrocytes and Schwann cells are able to repair myelin lesions of the central nervous system. However, spontaneous myelin repair is often insufficient. Several approaches to enhance remyelination have been considered and transplantation of myelin-forming cells has been proposed as one of them. In this paper, we present results which confirm the ability of transplanted Schwann cells to remyelinate an induced lesion of the spinal cord. Schwann cells were either purified Schwann cells isolated from 1–day-old rat sciatic nerves, or immortalized Schwann cells (MSC80) arising from a purified culture of 7-day-old mouse sciatic nerves. They were transplanted into or at a distance from a lysolecithin-induced lesion of the Shiverer spinal cord. Labelling of the Schwann cells with the fluorochrome Hoechst 33342 enabled us to trace them after transplantation in their host and evaluate their ability to reach and to repair the demyelinated lesion. Using the Hoechst-Shiverer model, we show that when transplanted in the lesion, cultured Schwann cells, even immortalized, are able to remyelinate such a lesion efficiently. In addition, when transplanted at a distance from the lesion, they are able to reach and repair the lesion in time frames which allow them to complete actively with host oligodendrocytes.     

Schwann cell remyelination is restricted to astrocyte-deficient areas after transplantation into demyelinated adult rat brain

The ability to generate large numbers of Schwann cells from a peripheral nerve biopsy makes them potential candidates for the clinical application of cell transplantation to enhance remyelination in human demyelinating disease. Transplant-derived Schwann cell remyelination has previously been demonstrated in the spinal cord but not for demyelinated axons in the brain, a more likely site for initial clinical intervention. We have transplanted Schwann cells from male neonatal rat sciatic nerves into ethidium bromide-induced areas of demyelination in the deep cerebellar white matter of adult female rats. The extent of Schwann cell remyelination 28 days after transplantation was significantly increased in lesions that received direct injections of Schwann cells compared with non-transplanted lesions. Using in situ hybridisation to identify the rat Y chromosome, transplanted male cells were found to co-localise with the P0 immunoreactive area of Schwann cell remyelination. Combined immunohistochemistry and in situ hybridisation confirmed that many remyelinating Schwann cells were transplant-derived. P0 immunoreactivity and transplanted male cells were found in GFAP-negative, astrocyte-free areas. Transplanted Schwann cells were not identified outside of transplanted lesions, nor did they did not contribute to remyelination of a lesion at a distance from the site of transplantation. Our findings indicate that demyelinated axons in the adult brain can be remyelinated by transplanted Schwann cells but that migration and remyelination are restricted to areas from which astrocytes are absent.