No funding was received.

This protocol was approved by the local ethics committee in accordance with the Helsinki Declaration (Reg. No. A2019-0214). Informed consent was obtained from all participants. In the presented case involving a child, written informed consent was obtained from both parents.
NOTE: Fluorescein is a fluorescent dye that emits green light (520-530 nm) when stimulated with blue light (of wavelength ~485 nm). It is used for visualization of the tear film in ophthalmology. Its intrathecal use is off-label and based on an individual therapeutic agreement. Fluorescein-Natrium at a 10% concentration is available and regularly used in fluorescein angiography of the ocular fundus in ophthalmology.
1. Preparation for surgery
<ol>
	<li>Obtain audiological testing, high resolution computed tomography (HRCT), and magnetic resonance imaging (MRI) of the skull base.</li>
	<li>Obtain written, informed consent from the patient regarding off-label use of fluorescein.</li>
	<li>Perform a lumbar puncture and place a lumbar drain for later application of the dye.</li>
	<li>Use fluorescein sodium (10% concentration). For lumbar application, dilute the fluorescein to 5% concentration with water.</li>
	<li>Apply 0.1 mL of the dilution per 10 kg of body weight, with a maximum of 1 mL, intrathecally.</li>
	<li>Perform the application immediately before surgery to achieve intraoperative visualization. 
	NOTE: When applying the method to children, the application should be performed under general anesthesia.</li>
	<li>Perform general anesthesia according to the patient&#39;s individual clinical history and risk factors and according to the decision of the anesthesiologist.</li>
	<li>Cover the microscope with a blue light filter to visualize the fluorescein, or use a fully digital microscope.</li>
	<li>Use sterile draping to cover the patient.</li>
</ol>
2. Surgery
<ol>
	<li>For the transmastoid approach, perform a mastoidectomy and posterior tympanotomy.</li>
	<li>Expand the surgery according to the expected origin of the leak.</li>
	<li>As a first landmark, expose the dura to the middle cranial fossa.</li>
	<li>Thin the posterior canal wall of the outer ear canal.</li>
	<li>Expose the short incus process in the antrum and identify the lateral semicircular canal.</li>
	<li>Expose the chorda facial angle at which the chorda tympani leaves the facial nerve.</li>
	<li>Open the facial recess. Leave the facial nerve with a bony coverage.</li>
	<li>Access the middle ear through a posterior tympanotomy. The origin of the fluid leak can easily be identified in the stapes footplate (Figure 2).</li>
	<li>Use fascia from the temporal muscle and absorbable collagen fibrin sealant patches to seal the CSF leak until no more fluid leaves the footplate. The stapes suprastructure stabilizes the sealant.</li>
</ol>

<ol>
	<li>Gacek, R. R., Gacek, M. R., Tart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+spontaneous+cerebrospinal+fluid+otorrhea:+diagnosis+and+management.">Adult spontaneous cerebrospinal fluid otorrhea: diagnosis and management.</a> The American Journal of Otology. 20 (6), 770-776 (1999).</li><li>Schraven, S. P., Bisdas, S., Wagner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronous+spontaneous+cerebrospinal+fluid+leaks+in+the+nose+and+ear.">Synchronous spontaneous cerebrospinal fluid leaks in the nose and ear.</a> The Journal of Laryngology and Otology. 126 (11), 1186-1188 (2012).</li><li>Raza, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+and+specificity+of+intrathecal+fluorescein+and+white+light+excitation+for+detecting+intraoperative+cerebrospinal+fluid+leak+in+endoscopic+skull+base+surgery:+a+prospective+study.">Sensitivity and specificity of intrathecal fluorescein and white light excitation for detecting intraoperative cerebrospinal fluid leak in endoscopic skull base surgery: a prospective study.</a> Journal of Neurosurgery. 124 (3), 621-626 (2016).</li><li>Englhard, A. S., Volgger, V., Leunig, A., Messmer, C. S., Ledderose, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+nasal+cerebrospinal+fluid+leaks:+management+of+24+patients+over+11+years.">Spontaneous nasal cerebrospinal fluid leaks: management of 24 patients over 11 years.</a> European Archives of Oto-rhino-laryngology. 275 (10), 2487-2494 (2018).</li><li>Sieskiewicz, A., Lyson, T., Rogowski, M., Mariak, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+management+of+cerebrospinal+fluid+rhinorhea.">Endoscopic management of cerebrospinal fluid rhinorhea.</a> The Polish Otolaryngology. 63 (4), 343-347 (2009).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+topical+intranasal+fluorescein+in+endoscopic+endonasal+repair+of+cerebrospinal+fluid+rhinorrhea.">The use of topical intranasal fluorescein in endoscopic endonasal repair of cerebrospinal fluid rhinorrhea.</a> Surgical Neurology. 72 (4), 341-345 (2009).</li><li>Javadi, S. A. H., Samimi, H., Naderi, F., Shirani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+low-+dose+intrathecal+fluorescein+in+endoscopic+repair+of+cerebrospinal+fluid+rhinorrhea.">The use of low- dose intrathecal fluorescein in endoscopic repair of cerebrospinal fluid rhinorrhea.</a> Archives of Iranian Medicine. 16 (5), 264-266 (2013).</li><li>Cheng, E., Grande, D., Leonetti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+spontaneous+temporal+bone+cerebrospinal+fluid+leak:+A+30-year+experience.">Management of spontaneous temporal bone cerebrospinal fluid leak: A 30-year experience.</a> American Journal of Otolaryngology. 40 (1), 97-100 (2019).</li><li>Tandon, S., Singh, S., Sharma, S., Lahiri, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Intrathecal+Fluorescein+in+Recurrent+Meningitis+after+Cochlear+Implantation.">Use of Intrathecal Fluorescein in Recurrent Meningitis after Cochlear Implantation.</a> Iranian Journal of Otorhinolaryngology. 28 (86), 221-226 (2016).</li><li>Warnecke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+relevance+of+beta2-transferrin+for+the+detection+of+cerebrospinal+fluid+fistulas.">Diagnostic relevance of beta2-transferrin for the detection of cerebrospinal fluid fistulas.</a> Archives of Otolaryngology - Head &amp; Neck Surgery. 130 (10), 1178-1184 (2004).</li><li>Keerl, R., Weber, R. K., Draf, W., Wienke, A., Schaefer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sodium+fluorescein+solution+for+detection+of+cerebrospinal+fluid+fistulas:+an+analysis+of+420+administrations+and+reported+complications+in+Europe+and+the+United+States.">Use of sodium fluorescein solution for detection of cerebrospinal fluid fistulas: an analysis of 420 administrations and reported complications in Europe and the United States.</a> The Laryngoscope. 114 (2), 266-272 (2004).</li><li>Keerl, R., Weber, R. K., Draf, W., Radziwill, R., Wienke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+of+lumbar+administration+of+5%+sodium+fluorescein+solution+for+detection+of+cerebrospinal+fluid+fistula.">Complications of lumbar administration of 5% sodium fluorescein solution for detection of cerebrospinal fluid fistula.</a> Laryngo-rhino-otologie. 82 (12), 833-838 (2003).</li><li>Harley, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+transnasal+repair+of+two+cases+of+spontaneous+cerebrospinal+fluid+fistula+in+the+foramen+rotundum.">Endoscopic transnasal repair of two cases of spontaneous cerebrospinal fluid fistula in the foramen rotundum.</a> Journal of Clinical Neuroscience: Official Journal of the Neurosurgical Society of Australia. 59, 350-352 (2019).</li><li>Wolf, G., Greistorfer, K., Stammberger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+detection+of+cerebrospinal+fluid+fistulas+with+a+fluorescence+technique.+Report+of+experiences+with+over+925+cases.">Endoscopic detection of cerebrospinal fluid fistulas with a fluorescence technique. Report of experiences with over 925 cases.</a> Laryngo-rhino-otologie. 76 (10), 588-594 (1997).</li></ol>

The authors declare no financial disclosures.

While many reports concerning CSF leaks focus on transnasal endoscopic approaches in skull base surgery, a standardized method of visualizing CSF leaks of the temporal bone via microscopy5,6,7 is lacking. This protocol describes an adaption of existing literature reviews for use in microscopic ear surgery.
The application of fluorescein has been described as a safe method in skull base surgery and use...

CSF leaks can be caused by trauma, preexist congenitally, or appear spontaneously. Clinically, they appear via otoliquorrhea or rhinoliquorrhea and can be confirmed by positive &#946;-transferrin secretion1,2. In cases of CSF leaks, reliable detection of its origin is needed to seal the leak sufficiently and prevent complications, such as meningitis.
Intrathecal fluorescein application (IFA) has been known to be highly sensitive in detecting CSF leaks after neurosurgical skull base operations3. However, there is no common consensus on its exact applications (e.g., concentration, amount of fluid, addition of other drugs such as dexamethasone)3,4. A majority of cases describe transnasal endoscopic skull base surgery, while a standardized method of visualizing CSF leaks in the temporal bone via microscopy5,6,7 is missing. These leaks frequently occur at multiple localizations and bear the risk of recurrence if their whole extent is not visualized during surgery8. Specifically, a transmastoid approach only has been found to bear the risk of recurrent leaks8.
This protocol presents the use of intrathecal administered fluorescein via a transmastoid approach. A clinical case of bilateral congenital ear malformation (right ear: cochlear aplasia, left ear: incomplete partition type I), in which minor trauma led to perforation of the left stapes footplate in the middle ear, is also presented. A similar case has only been reported once9.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluorescein ALCON 10% injection solution</td>
			<td>NOVARTIS</td>
			<td>1467007</td>
			<td></td>
		</tr>
		<tr>
			<td>External lumbar drain catheter Silverline</td>
			<td>Spiegelberg</td>
			<td>ELD33.010.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Otologic instruments (round knifes, hooks, curette, sickle knife, microscissors and microforceps)</td>
			<td>Spiggle und Theis</td>
			<td></td>
			<td>No further specification; instruments can vary between the clinics and their use depends on the surgeon&#39;s preferation</td>
		</tr>
		<tr>
			<td>Surgical microscope ARRISCOPE 1.0</td>
			<td>ARRI Medicals</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td>No further specification; material of the standard equipment of the operating clinic can be used</td>
		</tr>
		<tr>
			<td>Water to rinse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antifog solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton pads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cottonoid pledges</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gown</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mask</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suction tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blade</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

intrathecal application of a fluorescent dye for the identification of cerebrospinal fluid leaks in cochlear malformation

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such as meningitis. A method is presented here using intrathecal administered fluorescein in a clinical case of bilateral congenital ear malformation. A fluorescent dye is administered intrathecally to achieve intraoperative visualization of CSF leaks. The dye is applied 20 min before surgery, and concentration of 5% is used. Per every 10 kg of body weight, 0.1 mL of the fluid is applied intrathecally. The fluorescein is visualized using a fully digital microscope. The origin of the fluid leak is identified in the stapes footplate. During primary surgery, it is sealed, and cochlea implantation is performed for hearing restoration. In this specific case, 6 weeks later, the implant was explanted due to acute meningitis, and the electrode array was left as a spacer. Postoperatively, in the aural smear, &#946;-transferrin was detected. During a revision mastoidectomy, dislocated coverage of the leak was found. The stapes was removed and oval window sealed. Five days after revision surgery, no &#946;-transferrin was detected in the aural smear. During the revision of cochlea implantation 6 months later, intact coverage of the oval niche was observed. Thus, intrathecal fluorescein application proves to be a reliable tool for the detection of CSF leaks. It facilitates the orientation in malformations and complicated or unknown surgical situs. In the literature, its use is described for CSF fistulas in endonasal surgery but is rarely described in skull base and mastoid surgeries. The method has been used successfully in several cases with CSF leaks, and the results confirm the feasibility of safely accessing the origin of the leak.

In the presented case, minor trauma led to a CSF leak from the nose in a 10 month-old child. Magnetic resonance imaging (MRI) revealed a bilateral temporal bone malformation with aplasia of the right cochlea and dilation of the left cochlea and vestibule with absent interscalar septum identical to an incomplete partition type one. Accordingly, brainstem-evoked audiometry was performed (Figure 1) and confirmed bilateral deafness.
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Intrathecal Application of a Fluorescent Dye for the Identification of Cerebrospinal Fluid Leaks in Cochlear Malformation

Intrathecally applied fluorescein is used to achieve intraoperative visualization of CSF leaks. This protocol describes a lumbar puncture, the application of 5% fluorescein, and intraoperative visualization using a fully digital microscope.

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such ...

intrathecal-application-fluorescent-dye-for-identification

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8.0K Views.  University Medical Center Rostock. Intrathecally applied fluorescein is used to achieve intraoperative visualization of CSF leaks. This protocol describes a lumbar puncture, the application of 5% fluorescein, and intraoperative visualization using a fully digital microscope.

Video: Intrathecal Application of a Fluorescent Dye for the Identification of Cerebrospinal Fluid Leaks in Cochlear Malformation

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Cochlear Implantation in the Guinea Pig

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception outcomes, candidacy criteria have been expanded over the last few decades. This includes patients with substantial residual hearing that benefit from electrical and acoustical stimulation of the same ear, which makes hearing preservation during cochlear implantation an important issue. Electrode impedances and the related issue of energy consumption is another major research field, as progress in this area could pave the way for fully implantable auditory prostheses. To address these issues in a systematic way, adequate animal models are essential. Therefore, the goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address various research questions. Due to its large tympanic bulla, which allows easy surgical access to the inner ear, as well as its hearing range which is relatively similar to the hearing range of humans, the guinea pig is a commonly used species in auditory research. Cochlear implantation in the guinea pig is performed via a retroauricular approach. Through the bullostomy a cochleostomy is drilled and the cochlear implant electrode is inserted into the scala tympani. This electrode can then be used for electrical stimulation, determination of electrode impedances and the measurement of compound action potentials of the auditory nerve. In addition to these applications, cochlear implant electrodes can also be used as drug delivery devices, if a topical delivery of pharmaceutical agents to the cells or fluids of the inner ear is intended.

The goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address a multitude of research questions. Potential applications include the evaluation of pharmaceutical interventions or electrical stimulation for beneficial effects on hearing thresholds or electrode impedances.

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

Robotic Cochlear Implantation for Direct Cochlear Access

Robot-assisted systems offer great potential for gentler and more precise cochlear implantation. In this article, we provide a comprehensive overview of the clinical workflow for robotic cochlear implantation using a robotic system specifically developed for a minimally invasive, direct cochlear access. The clinical workflow involves experts from various disciplines and requires training to ensure a smooth and safe procedure. The protocol briefly summarizes the history of robotic cochlear implantation. The clinical sequence is explained in detail, beginning with the assessment of patient eligibility and covering surgical preparation, preoperative planning with the special planning software, drilling of the middle ear access, intraoperative imaging to confirm the trajectory, milling of the inner ear access, insertion of the electrode array, and implant management. The steps that require special attention are discussed. As an example, the postoperative outcome of robotic cochlear implantation in a patient with advanced otosclerosis is presented. Finally, the procedure is discussed in the context of the authors' experience.

Robotic cochlear implantation is a procedure for minimally invasive inner ear access. Compared to conventional surgery, robotic cochlear implantation involves additional steps that need to be carried out in the operating room. In this article we give a description of the procedure and highlight the important aspects of robotic cochlear implantation.

Robot-assisted systems offer great potential for gentler and more precise cochlear implantation. In this article, we provide a comprehensive overview of ...

The Miniature Pig: A Large Animal Model for Cochlear Implant Research

Cochlear implants (CI) are the most effective method to treat people with severe-to-profound sensorineural hearing loss. Although CIs are used worldwide, no standard model exists for investigating the electrophysiology and histopathology in patients or animal models with a CI or for evaluating new models of electrode arrays. A large animal model with cochlea characteristics similar to those of humans may provide a research and evaluation platform for advanced and modified arrays before their use in humans. 
To this end, we established standard CI methods with Bama mini-pigs, whose inner ear anatomy is highly similar to that of humans. Arrays designed for human use were implanted into the mini pig cochlea through a round window membrane, and a surgical approach followed that was similar to that used for human CI recipients. Array insertion was followed by evoked compound action potential (ECAP) measurements to evaluate the function of the auditory nerve. This study describes the preparation of the animal, surgical steps, array insertion, and intraoperative electrophysiological measurements. 
The results indicated that the same CI used for humans could be easily implanted in mini-pigs via a standardized surgical approach and yielded similar electrophysiological outcomes as measured in human CI recipients. Mini-pigs could be a valuable animal model to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches before applying them to human beings.

Miniature pigs (mini-pigs) are an ideal large animal model for research into cochlear implants. Cochlear implantation surgery in mini-pigs can be utilized to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches in a living system similar to human beings.

Cochlear implants (CI) are the most effective method to treat people with severe-to-profound sensorineural hearing loss. Although CIs are used worldwide, ...

Facile Preparation and Photoactivation of Prodrug-Dye Nanoassemblies

Self-assembly is a simple yet reliable method for constructing nanoscale drug delivery systems. Photoactivatable prodrugs enable controllable drug release from nanocarriers at target sites modulated by light irradiation. In this protocol, a facile method for fabricating photoactivatable prodrug-dye nanoparticles via molecular self-assembly is presented. The procedures for prodrug synthesis, nanoparticle fabrication, physical characterization of the nanoassembly, photocleavage demonstration, and in vitro cytotoxicity verification are described in detail. A photocleavable boron-dipyrromethene-chlorambucil (BC) prodrug was first synthesized. BC and a near-infrared dye, IR-783, at an optimized ratio, could self-assemble into nanoparticles (IR783/BC NPs). The synthesized nanoparticles had an average size of 87.22 nm and a surface charge of -29.8 mV. The nanoparticles disassembled upon light irradiation, which could be observed by transmission electronic microscopy. The photocleavage of BC was completed within 10 min, with a 22% recovery efficiency for chlorambucil. The nanoparticles displayed enhanced cytotoxicity under light irradiation at 530 nm compared with the non-irradiated nanoparticles and irradiated free BC prodrug. This protocol provides a reference&#160;for the construction and evaluation of photoresponsive drug delivery systems.

This protocol describes the fabrication and characterization of a photoresponsive prodrug-dye nanoassembly. The methodology for drug release from the nanoparticles by light-triggered disassembly, including the light irradiation setup, is explicitly described. The drugs released from the nanoparticles following light irradiation exhibited excellent anti-proliferation effects on human colorectal tumor cells.

Self-assembly is a simple yet reliable method for constructing nanoscale drug delivery systems. Photoactivatable prodrugs enable controllable drug release ...

Establishment of the KOA Model in Rabbits Using the Modified Videman Method

Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of people with KOA. Among several pathogenic factors, the biomechanical imbalance of the knee joint is one of the main causes of KOA. Acupotomology believes that restoring the mechanical balance of the knee joint is the key to treating KOA. Clinical studies have shown that acupotomy can effectively reduce pain and improve knee mobility by reducing adhesion, contracture of soft tissues, and stress concentration points in muscles and tendons around the knee joint. 
In this protocol, we used the modified Videman method to establish a KOA model by immobilizing the left hindlimb in a straight position. We have outlined the method of operation and the precautions related to acupotomy in detail and evaluated the efficacy of acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" through the detection of the mechanical properties of quadriceps femoris and tendon, as well as cartilage mechanics and morphology. The results show that acupotomy has a protective effect on cartilage by adjusting the mechanical properties of the soft tissues around the knee joint, improving the cartilage stress environment, and delaying cartilage degeneration.

Author Spotlight: Investigating the Mechanism of Action of Acupotomy in Treating Knee Osteoarthritis

In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...