Gel-Mediated Electrospray Assembly of Silica Supraparticles for Sustained Drug Delivery.

Supraparticles (SPs) composed of smaller colloidal particles provide a platform for the long-term, controlled release of therapeutics in biomedical applications. However, current synthesis methods used to achieve high drug loading and those involving biocompatible materials are often tedious and low throughput, thereby limiting the translation of SPs to diverse applications. Herein, we present a simple, effective, and automatable alginate-mediated electrospray technique for the assembly of robust spherical silica SPs (Si-SPs) for long-term (>4 months) drug delivery. The Si-SPs are composed of either porous or nonporous primary Si particles within a decomposable alginate matrix. The size and shape of the Si-SPs can be tailored by controlling the concentrations of alginate and silica primary particles used and key electrospraying parameters, such as flow rate, voltage, and collector distance. Furthermore, the performance (including drug loading kinetics, loading capacity, loading efficiency, and drug release) of the Si-SPs can be tuned by changing the porosity of the primary particles and through the retention or removal (via calcination) of the alginate matrix. The structure and morphology of the Si-SPs were characterized by electron microscopy, dynamic light scattering, N2 adsorption-desorption analysis, and X-ray photoelectron spectroscopy. The cytotoxicity and degradability of the Si-SPs were also examined. Drug loading kinetics and loading capacity for six different types of Si-SPs, using a model protein drug (fluorescently labeled lysozyme), demonstrate that Si-SPs prepared from primary silica particles with large pores can load significant amounts of lysozyme (∼10 µg per SP) and exhibit sustained, long-term release of more than 150 days. Our experiments show that Si-SPs can be produced through a gel-mediated electrospray technique that is robust and automatable (important for clinical translation and commercialization) and that they present a promising platform for long-term drug delivery.

Safety Studies for a 44-Channel Suprachoroidal Retinal Prosthesis: A Chronic Passive Study.

Purpose: Following successful clinical outcomes of the prototype suprachoroidal retinal prosthesis, Bionic Vision Australia has developed an upgraded 44-channel suprachoroidal retinal prosthesis to provide a wider field of view and more phosphenes. The aim was to evaluate the preclinical passive safety characteristics of the upgraded electrode array. Methods: Ten normal-sighted felines were unilaterally implanted with an array containing platinum electrodes (44 stimulating and 2 returns) on a silicone carrier near the area centralis. Clinical assessments (color fundus photos, optical coherence tomography, full-field electroretinography, intraocular pressure) were performed under anesthesia prior to surgery, and longitudinally for up to 20 weeks. Histopathology grading of fibrosis and inflammation was performed in two animals at 13 to 15 weeks. Results: Eight animals showed safe electrode array insertion (good retinal health) and good conformability of the array to the retinal curvature. Eight animals demonstrated good mechanical stability of the array with only minor (<2 disc diameters) lateral movement. Four cases of surgical or stability complications occurred due to (1) bulged choroid during surgery, (2) hemorrhage from a systemic bleeding disorder, (3) infection, and (4) partial erosion of thin posterior sclera. There was no change in retinal structure or function (other than that seen at surgery) at endpoint. Histopathology showed a mild foreign body response. Electrodes were intact on electrode array removal. Conclusions: The 44-channel suprachoroidal electrode array has an acceptable passive safety profile to proceed to clinical trial. The safety profile is expected to improve in human studies, as the complications seen are specific to limitations (anatomic differences) with the feline model.

Stimulation of a Suprachoroidal Retinal Prosthesis Drives Cortical Responses in a Feline Model of Retinal Degeneration.

PURPOSE: Retinal prostheses have emerged as a promising technology to restore vision in patients with severe photoreceptor degeneration. To better understand how neural degeneration affects the efficacy of electronic implants, we investigated the function of a suprachoroidal retinal implant in a feline model. METHODS: Unilateral retinal degeneration was induced in four adult felines by intravitreal injection of adenosine triphosphate (ATP). Twelve weeks post injection, animals received suprachoroidal electrode array implants in each eye, and responses to electrical stimulation were obtained using multiunit recordings from the visual cortex. Histologic measurements of neural and glial changes in the retina at the implant site were correlated with cortical thresholds from individual stimulating electrodes. RESULTS: Adenosine triphosphate-injected eyes displayed changes consistent with mid-to-late stage retinal degeneration and remodeling. A significant increase in electrical charge was required to induce a cortical response from stimulation of the degenerated retina compared to that in the fellow control eye. Spatial and temporal characteristics of the electrically evoked cortical responses were no different between eyes. Individual electrode thresholds varied in both the control and the ATP-injected eyes and were correlated with ganglion cell density. In ATP-injected eyes, cortical threshold was also independently correlated with an increase in the extent of retinal gliosis. CONCLUSIONS: These data suggest that even when ganglion cell density remains unaffected, glial changes in the retina following degeneration can influence the efficacy of suprachoroidal electrical stimulation. A better understanding of how glial change impacts retinal prosthesis function may help to further the optimization of retinal implants.

Retinal Changes in an ATP-Induced Model of Retinal Degeneration.

In rodents and felines, intravitreal administration of adenosine triphosphate (ATP) has been shown to induce photoreceptor death providing a tractable model of retinal degeneration in these species. This study investigated the long term effects of photoreceptor loss in an ATP induced feline model of retinal degeneration. Six normal sighted felines were unilaterally blinded using intravitreal ATP injections and assessed using electroretinography (ERG) and optical coherence tomography (OCT). At 30 h (n = 3) or 12 weeks (n = 3) post-injection, the animals were euthanized and the eyes enucleated. Retinae were sectioned and labeled using immunohistochemistry for markers of cell death, neural remodeling and gliosis. Ongoing cell death and retinal degeneration was observed in the outer retina at both 30 h and 12 weeks following unilateral ATP injection. Markers of mid to late-stage retinal remodeling such as cell displacement and aberrant neurite growth were observed in the inner retina at 12 weeks post-injection. Ganglion cells appeared to remain intact in ATP injected eyes. Müller cell gliosis was observed throughout the inner and outer retina, in some parts completely enveloping and/or displacing the surviving neural tissue. Our data suggests that the ATP injected feline retina continues to undergo progressive retinal degeneration and exhibits abnormalities consistent with a description of retinal remodeling commonly seen in other models of retinal degeneration. These findings validate the use of intravitreal ATP injection in feline as a large animal model of retinal degeneration which may aid in development of therapies aiming to restore visual function after photoreceptor degeneration.

Safety and efficacy of explanting or replacing suprachoroidal electrode arrays in a feline model.

BACKGROUND: A key requirement for retinal prostheses is the ability for safe removal or replacement. We examined whether suprachoroidal electrode arrays can be removed or replaced after implantation. METHODS: Suprachoroidal electrode arrays were unilaterally implanted into 13 adult felines. After 1 month, arrays were surgically explanted (n = 6), replaced (n = 5) or undisturbed (n = 2). The retina was assessed periodically using fundus photography and optical coherence tomography. Three months after the initial implantation, the function of replaced or undisturbed arrays was assessed by measuring the responses of the visual cortex to retinal electrical stimulation. The histopathology of tissues surrounding the implant was examined. RESULTS: Array explantation or replacement was successful in all cases. Fundus photography showed localized disruption to the tapetum lucidum near the implant's tip in seven subjects following implantation. Although optical coherence tomography showed localized retinal changes, there were no widespread statistically significant differences in the thickness of the retinal layers or choroid. The distance between the electrodes and retina increased after device replacement but returned to control values within eight weeks (P < 0.03). Staphylomas developed near the scleral wound in five animals after device explantation. Device replacement did not alter the cortical evoked potential threshold. Histopathology showed localized outer nuclear layer thinning, tapetal disruption and pseudo-rosette formation, but the overall retinal morphology was preserved. CONCLUSIONS: It is feasible to remove or replace conformable medical grade silicone electrode arrays implanted suprachoroidally. The scleral wound requires careful closure to minimize the risk of staphylomas.

ATP-induced photoreceptor death in a feline model of retinal degeneration.

PURPOSE: To develop and characterize a feline model of retinal degeneration induced by intravitreal injection of adenosine triphosphate (ATP). METHODS: Nineteen normally sighted adult cats received 100 µL intravitreal injections of ATP with a final concentration of 11, 22, or 55 mM at the retina. Four animals were euthanized 30 hours after injection and retinal sections examined for apoptosis using a TUNEL cell death assay. In the remaining animals, structural and functional changes were characterized over a 3-month period using a combination of electroretinography (ERG) and optical coherence tomography (OCT). RESULTS: Using a TUNEL cell death assay, we detected widespread photoreceptor death 30 hours after injection with 55 mM intravitreal ATP. All concentrations of ATP caused loss of retinal function and gross changes in retinal structure within 2 weeks of injection. Intravitreal injection of ATP led to a rapid loss of rod photoreceptor function and a gradual loss of cone photoreceptor function within 3 months. Outer nuclear layer thickness was globally reduced by 3 months, with the inner nuclear layer including the retinal nerve fiber layer remaining intact. Structural abnormalities were observed, including focal retinal detachment with evidence of both intravitreal and intraretinal inflammation in some eyes. CONCLUSIONS: Development of an ATP-induced feline model of retinal degeneration provides a rapid and effective large-eyed animal model for research into vision restoration.

Development of a surgical procedure for implantation of a prototype suprachoroidal retinal prosthesis.

BACKGROUND: Current surgical techniques for retinal prosthetic implantation require long and complicated surgery, which can increase the risk of complications and adverse outcomes. METHOD: The suprachoroidal position is known to be an easier location to access surgically, and so this study aimed to develop a surgical procedure for implanting a prototype suprachoroidal retinal prosthesis. The array implantation procedure was developed in 14 enucleated eyes. A full-thickness scleral incision was made parallel to the intermuscular septum and superotemporal to the lateral rectus muscle. A pocket was created in the suprachoroidal space, and the moulded electrode array was inserted. The scleral incision was closed and scleral anchor point sutured. In 9 of the 14 eyes examined, the device insertion was obstructed by the posterior ciliary neurovascular bundle. Subsequently, the position of this neurovascular bundle in 10 eyes was characterized. Implantation and lead routing procedure was then developed in six human cadavers. The array was tunnelled forward from behind the pinna to the orbit. Next, a lateral canthotomy was made. Lead fixation was established by creating an orbitotomy drilled in the frontal process of the zygomatic bone. The lateral rectus muscle was detached, and implantation was carried out. Finally, pinna to lateral canthus measurements were taken on 61 patients in order to determine optimal lead length. RESULTS: These results identified potential anatomical obstructions and informed the anatomical fitting of the suprachoroidal retinal prosthesis. CONCLUSION: As a result of this work, a straightforward surgical approach for accurate anatomical suprachoroidal array and lead placement was developed for clinical application.

A wide-field suprachoroidal retinal prosthesis is stable and well tolerated following chronic implantation.

PURPOSE: The safety of chronic implantation of a retinal prosthesis in the suprachoroidal space has not been established. This study aimed to determine the safety of a wide-field suprachoroidal electrode array following chronic implantation using histopathologic techniques and electroretinography. METHODS: A platinum electrode array in a wide silicone substrate was implanted unilaterally in the suprachoroidal space in adult cats (n = 7). The lead and connector were tunneled out of the orbit and positioned subcutaneously. Postsurgical recovery was assessed using fundus photography and electroretinography (ERG). Following 3 months of passive implantation, the animals were terminated and the eyes assessed for the pathologic response to implantation. RESULTS: The implant was mechanically stable in the suprachoroidal space during the course of the study. The implanted eye showed a transient increase in ERG response amplitude at 2 weeks, which returned to normal by 3 months. Pigmentary changes were observed at the distal end of the implant, near the optic disc. Histopathologic assessment revealed a largely intact retina and a thin fibrous capsule around the suprachoroidal implant cavity. The foreign body response was minimal, with sporadic presence of macrophages and no active inflammation. All implanted eyes were negative for bacterial or fungal infections. A midgrade granuloma and thick fibrous buildup surrounded the extraocular cable. Scleral closure was maintained in six of seven eyes. There were no staphylomas or choroidal incarceration. CONCLUSIONS: A wide-field retinal prosthesis was stable and well tolerated during long-term suprachoroidal implantation in a cat model. The surgical approach was reproducible and overall safe.

Challenges for stem cells to functionally repair the damaged auditory nerve.

INTRODUCTION: In the auditory system, a specialized subset of sensory neurons are responsible for correctly relaying precise pitch and temporal cues to the brain. In individuals with severe-to-profound sensorineural hearing impairment these sensory auditory neurons can be directly stimulated by a cochlear implant, which restores sound input to the brainstem after the loss of hair cells. This neural prosthesis therefore depends on a residual population of functional neurons in order to function effectively. AREAS COVERED: In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, the benefits derived from a cochlear implant may be minimal. One way in which to restore function to the auditory nerve is to replace these lost neurons using differentiated stem cells, thus re-establishing the neural circuit required for cochlear implant function. Such a therapy relies on producing an appropriate population of electrophysiologically functional neurons from stem cells, and on these cells integrating and reconnecting in an appropriate manner in the deaf cochlea. EXPERT OPINION: Here we review progress in the field to date, including some of the key functional features that stem cell-derived neurons would need to possess and how these might be enhanced using electrical stimulation from a cochlear implant.

Hydrogel limits stem cell dispersal in the deaf cochlea: implications for cochlear implants.

Auditory neurons provide the critical link between a cochlear implant and the brain in deaf individuals, therefore their preservation and/or regeneration is important for optimal performance of this neural prosthesis. In cases where auditory neurons are significantly depleted, stem cells (SCs) may be used to replace the lost population of neurons, thereby re-establishing the critical link between the periphery (implant) and the brain. For such a therapy to be therapeutically viable, SCs must be differentiated into neurons, retained at their delivery site and damage caused to the residual auditory neurons minimized. Here we describe the transplantation of SC-derived neurons into the deaf cochlea, using a peptide hydrogel to limit their dispersal. The described approach illustrates that SCs can be delivered to and are retained within the basal turn of the cochlea, without a significant loss of endogenous auditory neurons. In addition, the tissue response elicited from this surgical approach was restricted to the surgical site and did not extend beyond the cochlear basal turn. Overall, this approach illustrates the feasibility of targeted cell delivery into the mammalian cochlea using hydrogel, which may be useful for future cell-based transplantation strategies, for combined treatment with a cochlear implant to restore function.

Nanoporous peptide particles for encapsulating and releasing neurotrophic factors in an animal model of neurodegeneration.

Neurotrophin-BDNF can be effectively encapsulated in nanoporous poly(L-glutamic acid) particles prepared via mesoporous silica templating. The loaded BDNF can be released in a sustained manner with retained biological activity. Animal experiments demonstrate the released BDNF can efficiently rescue the auditory neurons (as indicated by the arrows) in the cochlea of guinea pigs with sensorineural hearing loss.

Neurotrophin gene therapy for sustained neural preservation after deafness.

The cochlear implant provides auditory cues to profoundly deaf patients by electrically stimulating the residual spiral ganglion neurons. These neurons, however, undergo progressive degeneration after hearing loss, marked initially by peripheral fibre retraction and ultimately culminating in cell death. This research aims to use gene therapy techniques to both hold and reverse this degeneration by providing a sustained and localised source of neurotrophins to the deafened cochlea. Adenoviral vectors containing green fluorescent protein, with or without neurotrophin-3 and brain derived neurotrophic factor, were injected into the lower basal turn of scala media of guinea pigs ototoxically deafened one week prior to intervention. This single injection resulted in localised and sustained gene expression, principally in the supporting cells within the organ of Corti. Guinea pigs treated with adenoviral neurotrophin-gene therapy had greater neuronal survival compared to contralateral non-treated cochleae when examined at 7 and 11 weeks post injection. Moreover; there was evidence of directed peripheral fibre regrowth towards cells expressing neurotrophin genes after both treatment periods. These data suggest that neurotrophin-gene therapy can provide sustained protection of spiral ganglion neurons and peripheral fibres after hearing loss.

Development of a surgical approach for a wide-view suprachoroidal retinal prosthesis: evaluation of implantation trauma.

BACKGROUND: Our research goal is to develop a safe, reproducible surgical approach for implantation of a wide-field retinal stimulating array. The aim of this study was to evaluate the pathological response to acute implantation of a functional prototype electrode array in the suprachoroidal space. METHODS: The surgical techniques to implant a 72 platinum electrode array fabricated on 8 × 13 × 0.4 mm polyimide and silicone substrate were developed in a pilot study in anesthetized cats. For the main study, nine eyes were implanted in vivo and unoperated eyes were used as controls. Surgery consisted of a temporal approach with a full-thickness scleral incision 5 mm posterior to the limbus. A suprachoroidal "pocket" was created, the electrode array inserted to sit beneath the area centralis, and placement was confirmed visually. The eyes were collected subsequently for histopathology. RESULTS: The array was consistently inserted into the suprachoroidal space beneath the area centralis in nine eyes. There was a significant hemorrhage in two cases where implantation was complicated by choroidal congestion. Retinal folding occurred only when the array tip was within 2.6 mm of the optic disc (p < 0.01). There was choroidal incarceration at the incision in six eyes and scleral distortion at the array edges in five. No cases were found where the implant breached the retina, choroid, or sclera. CONCLUSIONS: A large stimulation array can be reliably inserted into the suprachoroidal space without trauma to the neuroretina. These findings suggest that this is an appropriate surgical approach for the placement of an electrode array for use in retinal stimulation.

The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection.

A cochlear implant can restore hearing function by electrically exciting spiral ganglion neurons (SGNs) in the deaf cochlea. However, following deafness SGNs undergo progressive degeneration ultimately leading to their death. One significant cause of SGN degeneration is the loss of neurotrophic support that is normally provided by cells within the organ of Corti (OC). The administration of exogenous neurotrophins (NTs) can protect SGNs from degeneration but the effects are short-lived once the source of NTs has been exhausted. NT gene therapy, whereby cells within the cochlea are transfected with genes enabling them to produce NTs, is one strategy for providing a cellular source of NTs that may provide long-term support for SGNs. As the SGNs normally innervate sensory cells within the OC, targeting residual OC cells for gene therapy in the deaf cochlea may provide a source of NTs for SGN protection and targeted regrowth of their peripheral fibers. However, the continual degeneration of the OC over extended periods of deafness may deplete the cellular targets for NT gene therapy and hence limit the effectiveness of this method in preventing SGN loss. This study examined the effects of deafness duration on the efficacy of NT gene therapy in preventing SGN loss in guinea pigs that were systemically deafened with aminoglycosides. Adenoviral vectors containing green fluorescent protein (GFP) with or without genes for Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT3) were injected into the scala media (SM) compartment of cochleae that had been deafened for one, four or eight weeks prior to the viral injection. The results showed that viral transfection of cells within the SM was still possible even after severe degeneration of the OC. Supporting cells (pillar and Deiters' cells), cells within the stria vascularis, the spiral ligament, endosteal cells lining the scala compartments and interdental cells in the spiral limbus were transfected. However, the level of transfection was remarkably lower following longer durations of deafness. There was a significant increase in SGN survival in the entire basal turn for cochleae that received NT gene therapy compared to the untreated contralateral control cochleae for the one week deaf group. In the four week deaf group significant SGN survival was observed in the lower basal turn only. There was no increase in SGN survival for the eight week deaf group in any cochlear region. These findings indicated that the efficacy of NT gene therapy diminished with increasing durations of deafness leading to reduced benefits in terms of SGN protection. Clinically, there remains a window of opportunity in which NT gene therapy can provide ongoing trophic support for SGNs.

Pneumococcal meningitis post-cochlear implantation: potential routes of infection and pathophysiology.

OBJECTIVE: This review describes the current concept of pneumococcal meningitis in cochlear implant recipients based on recent laboratory studies. It examines possible routes of Streptococcus pneumoniae infection to the meninges in cochlear implant recipients. It also provides insights into fundamental questions concerning the pathophysiology of pneumococcal meningitis in implant recipients. DATA SOURCES: Medline/PubMed database; English articles after 1960. Search terms: cochlear implants, meningitis, pneumococcus, streptococcus pneumonia. REVIEW METHODS: Narrative review. All articles relating to post-implant meningitis without any restriction in study designs were assessed and information extracted. RESULTS: The incidence of pneumococcal meningitis in cochlear implant recipients is greater than that of an age-matched cohort in the general population. Based on the current clinical literature, it is difficult to determine whether cochlear implantation per se increases the risk of meningitis in subjects with no existing risk factors for acquiring the disease. As this question cannot be answered in humans, the study of implant-related infection must involve the use of laboratory animals in order for the research findings to be applicable to a clinical situation. The laboratory research demonstrated the routes of infection and the effects of the cochlear implant in lowering the threshold for pneumococcal meningitis. CONCLUSION: The laboratory data complement the existing clinical data on the risk of pneumococcal meningitis post-cochlear implantation.

Pneumococcal meningitis post-cochlear implantation: preventative measures.

OBJECTIVE: Both clinical data and laboratory studies demonstrated the risk of pneumococcal meningitis post-cochlear implantation. This review examines strategies to prevent post-implant meningitis. DATA SOURCES: Medline/PubMed database; English articles after 1980. Search terms: cochlear implants, pneumococcus meningitis, streptococcus pneumonia, immunization, prevention. REVIEW METHODS: Narrative review. All articles relating to post-implant meningitis without any restriction in study designs were assessed and information extracted. RESULTS: The presence of inner ear trauma as a result of surgical technique or cochlear implant electrode array design was associated with a higher risk of post-implant meningitis. Laboratory data demonstrated the effectiveness of pneumococcal vaccination in preventing meningitis induced via the hematogenous route of infection. Fibrous sealing around the electrode array at the cochleostomy site, and the use of antibiotic-coated electrode array reduced the risk of meningitis induced via an otogenic route. CONCLUSION: The recent scientific data support the U.S. Food and Drug Administration recommendation of pneumococcal vaccination for the prevention of meningitis in implant recipients. Nontraumatic cochlear implant design, surgical technique, and an adequate fibrous seal around the cochleostomy site further reduce the risk of meningitis.

Effects of localized neurotrophin gene expression on spiral ganglion neuron resprouting in the deafened cochlea.

A cochlear implant may be used to electrically stimulate spiral ganglion neurons (SGNs) in people with severe sensorineural hearing loss (SNHL). However, these neurons progressively degenerate after SNHL due to loss of neurotrophins normally supplied by sensory hair cells (HCs). Experimentally, exogenous neurotrophin administration prevents SGN degeneration but can also result in abnormal resprouting of their peripheral fibers. This study aimed to create a target-derived neurotrophin source to increase neuron survival and redirect fiber resprouting following SNHL. Adenoviral (Ad) vectors expressing green fluorescent protein (GFP) alone or in combination with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT3) were injected into the cochlear scala tympani or scala media of guinea-pigs (GPs) deafened via aminoglycosides for 1 week. After 3 weeks, cochleae were examined for gene expression, neuron survival, and the projection of peripheral fibers in response to gene expression. Injection of vectors into the scala media resulted in more localized gene expression than scala tympani injection with gene expression consistently observed within the partially degenerated organ of Corti. There was also greater neuron survival and evidence of localized fiber responses to neurotrophin-expressing cells within the organ of Corti from scala media injections (P < 0.05), a first step in promoting organized resprouting of auditory peripheral fibers via gene therapy.

Promoting neurite outgrowth from spiral ganglion neuron explants using polypyrrole/BDNF-coated electrodes.

Release of neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF) from hair cells in the cochlea is essential for the survival of spiral ganglion neurons (SGNs). Loss of hair cells associated with a sensorineural hearing loss therefore results in degeneration of SGNs, potentially reducing the performance of a cochlear implant. Exogenous replacement of either or both neurotrophins protects SGNs from degeneration after deafness. We previously incorporated NT3 into the conducting polymer polypyrrole (Ppy) synthesized with para-toluene sulfonate (pTS) to investigate whether Ppy/pTS/NT3-coated cochlear implant electrodes could provide both neurotrophic support and electrical stimulation for SGNs. Enhanced and controlled release of NT3 was achieved when Ppy/pTS/NT3-coated electrodes were subjected to electrical stimulation. Here we describe the release dynamics and biological properties of Ppy/pTS with incorporated BDNF. Release studies demonstrated slow passive diffusion of BDNF from Ppy/pTS/BDNF, with electrical stimulation significantly enhancing BDNF release over 7 days. A 3-day SGN explant assay found that neurite outgrowth from explants was 12.3-fold greater when polymers contained BDNF (p < 0.001), although electrical stimulation did not increase neurite outgrowth further. The versatility of Ppy to store and release neurotrophins, conduct electrical charge, and act as a substrate for nerve-electrode interactions is discussed for specialized applications such as cochlear implants.

A protocol for cryoembedding the adult guinea pig cochlea for fluorescence immunohistology.

Green fluorescent protein (GFP) has been used extensively to label cells in vitro and to track them following their transplantation in vivo. During our studies using the mouse embryonic stem cell line R1 B5-EGFP, we observed variable levels of fluorescence intensity of the GFP within these transfected cells. The variable fluorescence of this protein coupled with the innately autofluorescent nature of several structures within the cochlea collectively made the in vivo identification of these transplanted stem cells difficult. We have modified previously published protocols to enable the discrimination of an authentic GFP signal from autofluorescence in the adult guinea pig cochlea using fluorescence-based immunohistochemistry. The protocol described can also be used to label tissues of the cochlea using a chromogen, such as 3,3'-diaminobenzidine tetrahydrochloride (DAB). Moreover, the described method gives excellent preservation of structural morphology making the tissues useful for both morphological and quantitative studies in combination with robust immunohistochemistry in the adult guinea pig cochlea.

Cochlear implants stimulate activity-dependent CREB pathway in the deaf auditory cortex: implications for molecular plasticity induced by neural prosthetic devices.

Neural activity modulates the maturation of synapses and their organization into functional circuits by regulating activity-dependent signaling pathways. Phosphorylation of cyclic AMP/Ca(2+)-responsive element-binding protein (CREB) is widely accepted as a stimulus-inducible event driven by calcium influx into depolarized neurons. In turn, phosphorylated CREB (pCREB) activates the transcription of brain-derived neurotrophic factor (BDNF), which is needed for synaptic transmission and long-term potentiation. We examined how these molecular events are influenced by sensorineural hearing loss and long-term reactivation via cochlear implants. Sensorineural hearing loss reduced the expression of pCREB and BDNF. In contrast, deafened animals subject to long-term, unilateral intracochlear electrical stimulation exhibited an increased expression of pCREB and BDNF in the contralateral auditory cortical neurons, relative to ipsilateral ones. These changes induced by cochlear implants are further accompanied by the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, which has been implicated in long-lasting forms of synaptic plasticity. Because CREB and BDNF are critical modulators of synaptic plasticity, our data describe for the first time possible molecular candidate genes, which are altered in the auditory cortex, following cochlear implantation. These findings provide insights into adaptive, molecular mechanisms recruited by the brain upon functional electrical stimulation by neural prosthetic devices.