Novel 3D-Printed Hollow Microneedles Facilitate Safe, Reliable, and Informative Sampling of Perilymph from Guinea Pigs

Abstract

Background:

Inner ear diagnostics is limited by the inability to atraumatically obtain samples of inner ear fluid. The round window membrane (RWM) is an attractive portal for accessing perilymph samples as it has been shown to heal within one week after the introduction of microperforations. A 1 μL volume of perilymph is adequate for proteome analysis, yet the total volume of perilymph within the scala tympani of the guinea pig is limited to less than 5 μL. This study investigates the safety and reliability of a novel hollow microneedle device to aspirate perilymph samples adequate for proteomic analysis.

Methods:

The guinea pig RWM was accessed via a postauricular surgical approach. 3D-printed hollow microneedles with an outer diameter of 100 μm and an inner diameter of 35 μm were used to perforate the RWM and aspirate 1 μL of perilymph. Two perilymph samples were analyzed by liquid chromatography-mass spectrometry-based quantitative proteomics as part of a preliminary study. Hearing was assessed before and after aspiration using compound action potential (CAP) and distortion product otoacoustic emissions (DPOAE). RWMs were harvested 72 h after aspiration and evaluated for healing using confocal microscopy.

Results:

There was no permanent damage to hearing at 72 h after perforation as assessed by CAP (n=7) and DPOAE (n=8), and all perforations healed completely within 72 h (n=8). In the two samples of perilymph analyzed, 620 proteins were detected, including the inner ear protein cochlin, widely recognized as a perilymph marker.

Conclusion:

Hollow microneedles can facilitate aspiration of perilymph across the RWM at a quality and volume adequate for proteomic analysis without causing permanent anatomic or physiologic dysfunction. Microneedles can mediate safe and effective intracochlear sampling and show great promise for inner ear diagnostics.

1. INTRODUCTION

Inner ear diagnostics is limited by the inability to atraumatically obtain samples of cochlear fluid, owing to the inaccessibility of the cochlea. The cochlea is an anatomically complex fluid-filled structure encased within the otic capsule, the densest portion of the temporal bone. Recent evidence suggests tremendous potential of perilymph testing using various modalities, including transcriptomic and proteomic analyses, to contribute to diagnostics of inner ear disease (1–3). Protein concentrations are elevated in the perilymph of those with vestibular schwannomas (4), and microRNA expression regulating certain gene pathways in the perilymph may be upregulated in the setting of otosclerosis (3). Multiplex protein arrays have been used for analysis of inflammatory markers within the perilymph (5). Despite the diagnostic value of perilymph, perilymph sampling from the inner ear for such analyses is not routinely conducted as it remains traumatic with currently available technologies.

The round window membrane (RWM), a thin semipermeable layer of tissue, represents a communication between the inner ear and middle ear space and is the only portal for accessing the perilymphatic space (6,7) from the middle ear without perforating bone. In patients undergoing cochlear implantation, glass capillary tubes have been used for intraoperative perilymph sampling via the RWM with preservation of residual hearing (5,8). Our laboratory has developed microneedles based on the mechanical properties of the RWM (9–12). Solid microneedles have been shown to increase the rate of diffusion across the RWM (13,14). Dual wedge needles fabricated using electrical discharge machining within our laboratory facilitated the aspiration of perilymph from guinea pig cochleae in vitro (11). The average major and minor axes of the perforations created by this microneedle were 344 μm and 143 μm, respectively. A microneedle of this design was also tested in fresh frozen human temporal bones by an independent research group (15), using a silver/silver chloride-coated tip to detect the moment of penetration (16). This manufacturing technique created perforations of undesirable sizes, due to inadequate precision inherent in the technique. Large perforations can release inner ear pressure, leading to cerebrospinal fluid (CSF) entry through the cochlear aqueduct and efflux of perilymph through the perforation (17). The microneedles utilized in the current work were completely redesigned with a different manufacturing strategy. An additive manufacturing technique, two-photon polymerization lithography (2PP), was used to direct-write the microneedles. As a result, the taper angle, length, sharpness, and diameter of the needles, as well as the circular cross-sectional shape, could be easily controlled. Solid microneedles fabricated using this technique caused less damage when used to introduce perforations into the RWM, in vitro and in vivo (9,18). Resulting perforations had a mean major axis length of 95.9 μm and mean minor axis length of 25.4 μm in vitro, and microperforations introduced in vivo healed within one week without functional consequences.

Flexibility in design allowed for the creation of similar hollow microneedles that can introduce perforations in the RWM and facilitate the sampling of microliter amounts of perilymph (Figure 1). Rapid prototyping afforded by 2PP allowed for rapid iteration of microneedle geometry to experimentally optimize and finalize a design that best suited the challenges of the experiment. 2PP has also been used to manufacture hollow microneedles for transdermal sampling (19,20).

Figure 1. Hollow surgical microneedle designed for perilymph sampling, with a needle length of 435 μm.

Section A-A shows the larger 139μm lumen diameter in the base where the microneedle mates with the 30G stainless steel syringe needle. Section B-B shows the smaller 35 μm lumen diameter that runs through the needle shaft, which has an outer diameter of 100 μm.

Herein we present the use of 2PP 3D-printed ultra-sharp hollow microneedles for aspiration of microliter volumes of perilymph from guinea pigs for proteomic analysis. We first demonstrate that these microneedles facilitate aspiration of perilymph across the RWM without lasting anatomic damage to the RWM and no significant functional consequences on hearing. We then present the results of a preliminary proteomics study on perilymph aspirated using this technique.

2. MATERIALS AND METHODS

2.1. Microneedle Design and Fabrication

Computer-aided design of microneedles was done using SolidWorks software (Dassault Systems SolidWorks Corporation, Concord, NH, USA). The design choices are shown in the detailed sketch in Figure 1. The outer geometry of the microneedle, which will interact with the RWM, was designed to be identical to that of the solid microneedle demonstrated to be safe in vivo (18). The shaft of the microneedle is 435 μm long, a conservative length chosen to avoid damage of the basilar membrane during perforation. The cone-and-cylinder shape was chosen for axisymmetry, which renders the experiment independent of needle orientation with respect to the RWM.

The lumen of the needle, with a circular cross-section, is designed such that the cross-sectional area changes throughout the needle. It extends throughout the length of the needle shaft, where its size is limited by the outer diameter of the shaft (Section B-B, Figure 1). It widens at the base of the needle, where the outer diameter is greater, to decrease fluidic resistance, which scales inversely with the fourth order of the diameter (Section A-A, Figure 1). At the tip of the needle, the lumen curves from the center of the shaft and opens at the side of the microneedle. This design was chosen to maximize the bending stiffness of the geometry by keeping the hollow region as close to the central axis as possible, while preserving the tip geometry. All these features were enabled by the additive manufacturing techniques employed and would have been very difficult to achieve using conventional methods.

The diameter of the lumen extending through the shaft of the needle (Section B-B, Figure 1) was initially set at 30 μm. Initial in-vitro testing, in which artificial perilymph was aspirated from a petri dish, demonstrated an insufficient volumetric flow rate. It was hypothesized that the hydrodynamic resistance within the 30 μm diameter was too great to sustain the prescribed flow. Hence, the diameter was increased to 35 μm. This 16% change in diameter, according to Poiseuille’s law (21),

$$R=\frac{8\eta L}{\pi r^4}$$ (1)

where R is hydrodynamic resistance, η is the dynamic viscosity of the fluid, L is lumen length, and r is lumen radius, resulted in a 46% decrease in hydrodynamic resistance, and aspirations were successfully carried out with the larger lumen.

Stereolithography files were generated and parsed using the Describe software (Nanoscribe Gmbh, Karlsruhe, Germany), with a slicing distance of 1 μm and laser intensity of 80%. Microneedles were fabricated using 2PP by Photonic Professional GT system (Nanoscribe GmbH, Karlsruhe, Germany) using photoresist IP-S (Nanoscribe Gmbh, Karlsruhe, Germany). The microneedle was then secured onto the tip of a 2-inch long, 30-gauge, blunt small hub removable needle (Hamilton Company, Reno, NV, USA) using epoxy glue (Gorilla Glue Company, Cincinnati, OH, USA) and gas sterilized with ethylene oxide. Figure 2 shows a cross-section computer rendering of the design of the hollow ultra-sharp microneedle, alongside a scanning electron microscope image of the actual needle, presenting a side-by-side comparison of the design and the outcome.

Figure 2. Hollow surgical microneedle designed for perilymph sampling (outer diameter: 100 μm, inner diameter: 35 μm).

A: 3D rendering of a hollow microneedle section, bisected to show lumen. B: SEM image of microneedle mounted on a 30-gauge syringe needle.

2.2. Animals and Surgery

All animal procedures described in this study were reviewed and approved by the Columbia University Institutional Animal Care and Use Committee. Eight juvenile guinea pigs of either sex weighing between 150 and 300 grams were obtained from a commercial vendor (Charles River Laboratories, Inc., Wilmington, MA, USA). All guinea pigs underwent the aspiration procedure detailed below.

Guinea pigs were anesthetized with isoflurane gas (3.0% for induction and 1.0–3.0% for maintenance). Lidocaine was injected subcutaneously for local anesthesia. Buprenorphine sustained release (0.1 mg/kg) and meloxicam (0.5 mg/kg) were administered prior to surgery for post-operative analgesia.

To reduce head motion from breath cycles, the guinea pig’s head was fixed using a modular 3D-printed head holder with 2 pointed screws placed anterior to the external auditory meatus and posterior to the orbit without piercing the skin (22). All surgeries were performed on the right ear. The guinea pig RWM was accessed via a postauricular surgical approach. A 1 cm postauricular incision was made using a scalpel. Blunt dissection was then used to expose the bulla. A 1 mm diameter drill tip fixed onto a Stryker S2 πDrive drill (Stryker, Kalamazoo, MI, USA) was used to create a small opening. Forceps were used to remove additional bone and enlarge the opening to a 2–3 mm diameter for optimal visualization of the RWM. A hollow microneedle, mounted onto the tip of a 2-inch long, 30-gauge, blunt small hub removable needle (Hamilton Company, Reno, NV, USA), was secured onto a 10 μL Gastight Hamilton syringe (Model 1701 RN, Hamilton Company, Reno, NV, USA). The syringe was mounted onto a UMP3 UltraMicroPump (World Precision Instruments, Sarasota, FL, USA), and the pump was fixed to a micromanipulator. Using the micromanipulator, the mounted hollow microneedle was slowly introduced into the surgical opening and advanced toward the RWM. The RWM was perforated and 1 μL of perilymph was aspirated from the cochlea across the RWM over the course of 45 seconds using the UltraMicroPump. Perforation of the RWM was confirmed either by visualization of the perforation or a small efflux of perilymph into the middle ear side of the RWM upon withdrawal of the microneedle.

The sample was ejected into a 0.5 mL LoBind Microcentrifuge tube (Eppendorf, Hamburg, Germany) containing 2 μL of 1% protease cocktail inhibitor solution (P8340, Sigma Aldrich, St. Louis, Missouri, USA) in liquid chromatography/mass spectrometry grade water. The sample was spun down using a desktop centrifuge and stored at −80°C. The animals were sacrificed with pentobarbital overdose 72 h after aspiration. Temporal bones were harvested using blunt dissection and the RWM was then fixed for 1 h in 10% formalin and stored in PBS.

2.3. Audiometric Testing

Audiometric testing was conducted using methods previously described (18,23,24). Under anesthesia, compound action potential (CAP) was used to assess changes to hearing before RWM perforation and immediately prior to harvesting at 72 h after the procedure (n=7). Distortion product otoacoustic emissions (DPOAE) were used to assess changes to hearing before and after RWM perforation and immediately prior to harvesting at 72 h after the procedure (n=8). As these are survival experiments, CAP was not measured immediately after RWM perforation to reduce anesthesia time. Only one animal underwent a CAP measurement immediately after RWM perforation.

CAP measures auditory nerve activity by recording the synchronous activity of all the individual unit action potentials near the RWM after sound stimulation. In determining the CAP, tone pips were played to the ear and neural responses were measured with a silver electrode placed at the base of the cochlea. The reference electrode was placed subcutaneously 5–10 mm away from the incision site, and the ground electrode was placed between the shoulders as a reference. The CAP response was measured using an AC amplifier with a first order high pass filter and a second order low pass filter, with a pass band of ~200 Hz to 4 kHz. A Tucker Davis Technologies System (Tucker Davis Technologies Inc., Alachua, FL, USA) driving a Radio Shack dynamic speaker, connected in a closed-field configuration to the ear canal was used to generate sound stimulation. Sound calibration was performed within the ear canal using a Sokolich ultrasonic probe microphone. The CAP stimulus was composed of a 3 ms tone pip of variable frequency presented every 12 ms, with alternating polarity to eliminate the linear component of the cochlear microphonics from the averaged responses. CAP responses were collected for 18 frequencies ranging from 0.5 to 40 kHz. Stimulus intensity was steadily increased in 5 dB increments to determine a hearing threshold. The threshold was defined as the lowest stimulus level that evoked a recognizable response curve. Experimenters were blinded to previous threshold measurements to minimize bias and error. CAP threshold shifts were considered significantly greater than zero at a p<0.05 using two-tailed paired t-tests.

DPOAE are responses the cochlea produces when it is stimulated concurrently by two pure tone frequencies and is used to measure outer hair cell health and to evaluate hearing loss. To measure DPOAE, an ear tube containing a speaker and a low-noise Sokolich ultrasonic probe microphone was placed into the ear canal. The speaker played sound stimuli at sound pressure levels of 70 dB SPL with a fixed frequency ratio of f2/f1 = 1.2 at 1 kHz increments between 1 kHz and 32 kHz. The microphone detected resulting distortion products from the ear. A DPOAE at 2f1 – f2 that is 3 dB above the noise floor level was identified as a positive response.

2.4. Confocal Microscopy of RWMs

Confocal microscopy was used to evaluate RWMs for healing. Fixed RWMs were soaked in 1 mM rhodamine B, a fluorescent stain selective for elastic tissue, for 5 min and then rinsed with PBS and soaked in PBS for 15 min. A Nikon A1R laser-scanning confocal microscope (Nikon Instruments, Melville, NY, USA) was used to acquire z-stack images through each RWM. Images were collected using a Nikon 10x Plan Apo VC (0.45 NA) objective with a 1.0x zoom, providing a pixel size of 1.2 by 1.2 μm, and a 5μm step size. Samples were scanned with a 561 nm laser line with a pixel dwell time of 1.1 μs, and emitted light from 570 to 620 nm was allowed to pass to the detector. Maximum intensity projection images were generated from z-stacks using NIS-Elements (Nikon) to visualize the surface of the membrane and examine for perforation closure.

2.5. Scanning Electron Microscopy Imaging

Scanning electron microscopy (Zeiss, Oberkochen, Germany) imaging was used to examine the microneedles for integrity after use.

2.6. Proteomics Analysis

Two perilymph samples (1 μL) were stored at −80°C and then analyzed by liquid chromatography-mass spectrometry-based quantitative proteomics. The samples were purified with a methanol chloroform protein precipitation as previously described (25). Briefly, methanol and chloroform were added to the perilymph in a 4:4:1 volume ratio of perilymph:methanol:chloroform. Samples were centrifuged for 5 min at 3,000 × g and three additional methanol washes were performed to remove lipids and other potentially contaminating compounds. Pelleted proteins were allowed to air dry briefly followed by resuspension in a solubilization buffer (8M urea, 3 mM DTT, 100 mM ammonium bicarbonate in liquid chromatography/mass spectrometry grade water) and reduced with 5.5 mM dithiothreitol at room temperature for 40 min. Proteins were alkylated with 15 mM iodoacetamide in the dark at room temperature for 40 min. Samples were diluted five-fold in 100 mM ammonium bicarbonate and then digested using sequencing grade trypsin (Promega V511) at room temperature for 16 h. The resulting digest was acidified to ~ pH 1 with trifluoroacetic acid, kept on ice for 15 min to precipitate lipids, and centrifuged for 15 min at 20,000 × g. Supernatants were transferred to Nestgroup C18 Macrospin columns (Southborough, MA). The peptides were eluted with 40 % acetonitrile, lyophilized in an autosampler vial, and rehydrated in 3 % acetonitrile with 0.1 % formic acid. Peptides were analyzed by liquid chromatography/mass spectrometry as described previously (26).

Raw data files Spectra were processed with Proteome Discoverer V. 1.4.1.14 (Thermo Scientific) to exported Mascot generic format files which were then were searched with Mascot server (v2.5.1, Matrix Science Ltd., London, UK) against a database derived from UniProt release 2019_10, published November 13, 2019. This database included 25,731 sequences 14,311,265 residues of reviewed and unreviewed Cavia porcellus sequences and isoforms. These Cavia porcellus sequences were from reference proteome # up000005447. The database also included porcine trypsin, human keratins and lab contaminants. Search parameters included fixed modification on Cys (carbamidomethyl), and variable modification of oxidation (Met) with mass accuracy limits of 10 ppm for MS and 0.02 Da for MS/MS. Semiquantitative protein abundances were represented by emPAI scores (27). All mass spectrometry raw data files generated in this work have been deposited in an international public repository (MassIVE proteomics repository at https://massive.ucsd.edu/).

3. RESULTS

3.1. Anatomic and Functional Consequences of Perilymph Aspiration

All microneedles remained intact and without breakage after the perforation. Scanning electron microscope images of the hollow microneedles showed minimal to no bending at the tip (Figure 3). As determined by confocal microscopy at 72 h, all eight RWMs demonstrated complete closure of the perforation by 72 h (Figure 4).

Figure 3. Scanning electron microscope images of a microneedle after use during surgery at 134X magnification (A) and at 1540X magnification (B).

The microneedles did not break, and there was minimal to no bending at the tip.

Figure 4. Confocal image of the round window membrane, harvested at 72 h after in vivo perforation, at 10x magnification.

Area of perforation is indicated by arrow. Perforations healed within 72 h with minimal to no scarring (n=8).

Out of the eight guinea pigs that underwent the aspiration procedure, the CAP data of one guinea pig was not included for analysis because of events unrelated to aspiration. For this animal, a dental wick used to dry out of the middle ear space disrupted the ossicles prior to the final 72-h CAP measurement. However, a CAP measurement obtained immediately after aspiration for this animal showed no hearing loss, and the DPOAE obtained at 72 h prior to ossicle disruption remained well above the noise floor level.

Of the seven guinea pigs that underwent all CAP measurements successfully, there was no significant CAP threshold change at 72 h after perforation compared to baseline, and p-values of the two-tailed paired t-tests were greater than 0.05 for all tested frequencies (Figure 5). For all eight animals, the DPOAE remained well above noise floor immediately after perforation and at 72 h post-perforation (Figure 6). When comparing the DPOAE signals at 0 h post-perforation to baseline using the two-tailed paired t-test, there were significant changes at two frequencies (worsening at 3 kHz and improvement at 29 kHz). When comparing the DPOAE signals at 72 h post-perforation to baseline using the two-tailed paired t-test, there were significant changes at seven frequencies (worsening at 5 kHz and improvement at 9 kHz, 11 kHz, 12 kHz, 14 kHz, and 15 kHz).

Figure 5. Mean CAP threshold shifts at 72 h compared to baseline, for all tested frequencies.

The shaded area is bounded by the upper and lower limits of the 95% confidence interval. There was no significant shift in CAP measured at 72 h after perforation, for all 18 frequencies (n=7).

Figure 6. Distortion product otoacoustic emissions (DPOAE) at the frequency 2f1 – f2 in response to a 70 dB stimulus averaged over all survival experiments at 0 h after perforation (A, n=8) and 72 h after perforation (B, n=8).

Solid gray lines show the average noise for the experiments; Solid blue lines show the mean measured baseline DPOAE signal; and dotted red lines show mean DPOAE signal at 0 h (A) or 72 h (B) post-perforation. Shaded areas are bounded by the upper and lower limits of the 95% confidence interval for the average noise, and mean DPOAE signals. All DPOAE signals remained out of noise. When comparing the DPOAE signals at 0 h post-perforation to baseline using the two-tailed paired t-test, there were significant changes at two frequencies (worsening at 3 kHz and improvement at 29 kHz). When comparing the DPOAE signals at 72 h post-perforation to baseline using the two-tailed paired t-test, there were significant changes at seven frequencies (worsening at 5 kHz and improvement at 9 kHz, 11 kHz, 12 kHz, 14 kHz, and 15 kHz). See Figure 6 online for colors.

3.2. Characterizing the Guinea Pig Proteome

Perilymph from two guinea pigs were included for proteomic analysis in this preliminary perilymph proteomic study. Identifications were returned for 620 guinea pig proteins with a 1% false discovery rate for peptide matches (Table S1).

Data were analyzed with the PANTHER ontology tool (http://www.pantherdb.org/) using gene names (Table S1) searched against a Mus musculus background (28). Figure 7 shows the number of proteins per functional category and the distribution of these functional categories, accounting for protein abundances. Among these 620 proteins, the most common functional categories are metabolite interconversion enzymes (82 proteins), cytoskeletal proteins (57 proteins), protein-binding activity modulators (42 proteins), and proteases (35 proteins).

Figure 7. Composition of guinea pig perilymph proteome, by functional categories.

The 620 gene names were searched against the mouse gene list in PANTHER (http://www/pantherdb.org) to determine the distribution of proteins across functional classes. The fold-enrichment, PANTHER protein class, and the number of proteins within each class are presented.

The subcellular location of each protein was also annotated using GO annotations from UniProt. Of all detected proteins, 42% are from an unknown location; 22% are from multiple locations; 16% are from an extracellular location; and 13% are from the cytoplasm (Figure 8).

Figure 8. Subcellular locations GO annotations from UniProt for Cavia porcellus.

The “extracellular” category is represented by annotations that contain “extracellular”. The “cytoplasm” category is represented by annotations that contain “cytosol” or “cytoplasm”. The “plasma membrane” category is represented by annotations that contain “cell surface”, “plasma membrane”, or “synapse”. The “nucleus” category is represented by annotations that contain “nucleoplasm”, “nucleus”, or “nucleosome”.

At the individual level, preproalbumin, globin A1, transthyretin, Globin C1, SERPIN domain-containing protein, and fructose-bisphosphate aldolase were among the most abundant. The well-known inner-ear protein cochlin (A0A2C9F1F1_CAVPO) was identified based on 4.5 unique peptides, with 10% sequence coverage, and an emPAI abundance 0.61.

4. DISCUSSION

In this study, hollow surgical microneedles safely facilitated the aspiration of 1 μL of perilymph from the guinea pig scala tympani through the RWM. While liquid chromatography/mass spectrometry enables detection of minute amounts of protein in as little as 1 μL (8), the volume of the guinea pig scala tympani is only 4.8 μL (29). Our laboratory previously showed that perforation of the RWM with a 100 μm diameter microneedle did not cause lasting anatomic or functional damage (18). In the current study, after introduction of a hollow 100 μm diameter microneedle into the RWM, we further aspirated 1 μL of perilymph from the cochlea over the course of 45 seconds without lasting impact on hearing or damage to the RWM. Of note, the human scala tympani has a volume of 29.2 μL (29), and aspiration of perilymph in a controlled manner using 2PP 3D-printed hollow microneedles would be expected to yield similarly safe results.

The ultra-sharp tip and 100 μm outer diameter of our microneedle are properties that contribute to small hole size, permitting complete perforation closure within 72 h. However, a small outer diameter accordingly limits the inner diameter. A larger inner diameter is desirable for sustaining flow and limiting clogging and biofouling. Indeed, in early in vitro testing, we found that a 30-μm lumen diameter did not support our prescribed 1μL/min flow rate. Hydrodynamic resistance was sufficiently high to impede flow, and the compliance of the microneedle-syringe-pump system led to a lag between the syringe pump and the microneedle, resulting in significantly lengthened aspiration times. Increasing the lumen diameter to 35 μm reduced the hydrodynamic resistance by 46%, and this reduction was adequate to support a 1 μL/min flow. Performing this change in design could have been a major hurdle using traditional manufacturing methods, but usage of 2PP enabled this change to be trivial. Of note, we did not observe any clogging during our experiments in which the surgical microneedles were used once and discarded. Our 3D-printed microneedles have an in-plane resolution of <100nm, generating a smooth surface and preventing any significant fouling.

Upon perforation of a guinea pig RWM, these ultra-sharp surgical microneedles showed minimal to no bending at the tip. However, the human RWM is considerably thicker and stronger than the guinea pig RWM (12,30). Our laboratory has determined a microneedle geometry for perforation of the human RWM using 2PP, and our hollow microneedles could easily be adapted to meet these geometric requirements (10). More recently, we have used a new hybrid manufacturing method called two-photon templated electrodeposition, to produce metallic microneedles that are biocompatible (31). This method similarly affords tremendous flexibility in design and is a promising candidate for the manufacture of precision microneedles for use in humans. While a postauricular approach was used in this study, the flexibility conferred by the additive manufacturing technique enables the microneedles to be fixed onto a variety of tools, with the goal of in-office use through the external ear canal.

In animals, proteomic analysis of the inner ear thus far have required the harvesting of cochlear tissue involving sacrifice of the animals (32) or sampling of perilymph using traumatic methods (33,34). Thalmann et al. (1992) utilized a cochleostomy for perilymph sampling (33), and Palmer et al. (2018) dissected the cochlea from the skull, removing up to 10 μL of perilymph with a syringe (34). Our use of 2PP 3D-printed microneedles represents the first non-traumatic perilymph sampling method successfully used in survival experiments to facilitate perilymph analyses. Furthermore, the absence of cochlear damage and complete RWM perforation closure, makes possible repeated perilymph sampling should it be necessary. Within human subjects, proteomic analyses of perilymph have been conducted in cases where there are clinical indications for breaching the cochlea, such as in patients receiving a cochlear implant or in patients undergoing procedures for transotic and transcochlear resection of meningiomas or vestibular schwannomas (1,8). In these reports, sampling was achieved using a glass pipette or a 28-gauge needle that created a large hole in the RWM prior to surgical opening of the cochlea. These methods would not be suitable for subjects in whom surgical opening of the cochlea was not clinically warranted. Furthermore, glass capillary tubes are brittle and cannot aspirate a predetermined amount of perilymph. In contrast, 2PP 3D-printed microneedles are significantly less brittle than glass and can aspirate at a fixed amount. 2PP 3D-printed hollow microneedle mediated diagnostic aspiration of perilymph via the RWM is less traumatic and could be considered in humans.

Within 1μl of guinea pig perilymph, 620 proteins were identified, including the inner ear protein cochlin, a widely recognized perilymph marker (Table S1) (35). Using GO annotations from UniProt, the subcellular location of each protein was determined. While the subcellular location of the majority of proteins was unknown or multiple, 16% of proteins were from an extracellular location, representing the next largest category, as expected. However, a considerable number of proteins (13%) were determined to have a subcellular location within the cytoplasm.

A list of 39 perilymph proteins was previously reported for guinea pig (34). Proteins found in six out of eight samples were listed and blood proteins such as hemoglobin were deleted (34). As the cochlea was dissected from the skull prior to removal of perilymph, the authors also acknowledge a possibility of some protein degradation post mortem. 32 of the 39 protein which had associated gene names or protein accessions number were present on our list while the remaining seven did not have gene names or protein accessions number for comparison.

In the guinea pig, patency of the cochlear aqueduct and its proximity to RWM aspiration site raise concern about contamination of perilymph with CSF (36). This concern is lessened by the finding that total protein content in guinea pig perilymph is 12.6 times that in CSF, thus minor contamination from CSF is unlikely to affect proteomic results (33). Alternatively, Salt et al. reported perilymph sampling from cochlear apex cochleostomy in guinea pigs to avoid CSF contamination; however, this method is inherently traumatic and is not suitable for diagnostic purposes in humans (37). In our study, we limited the volume of perilymph aspirated to 1 μL to minimize CSF contamination. While potential CSF contamination remains a limitation in guinea pigs, it is less concerning in humans in whom the scala tympani has a greater volume (29) and there is limited mixture of CSF and perilymph (38).

Blood contamination of the sample is also likely due to the presence of blood vessels on the RWM, although on gross visual inspection, our aspirated samples were of a clear color without any red tint. Notably, recognized plasma proteins, including globin A1, were among the proteins detected. Nevertheless, in past proteomic studies of guinea pig and rat perilymph, hemoglobin was detected but removed from analysis, and it was noted that even small amounts of hemoglobin will be detected due to its high concentration in red blood cells (34,39). Moreover, given the high abundance of cochlin, the samples likely contain adequate perilymph for diagnostic value. Other proteins of interest include HSP70 and 90, proteins that are modulated by steroid administration (32,40,41). While this preliminary study included only two perilymph samples for proteomic analysis, techniques in this study may be used to investigate the impact of steroid therapy and modes of delivery on the cochlear proteome in a larger study. This study focused on one mode of perilymph analysis, namely proteomic analysis, but microneedle-mediated aspiration could also conceivably facilitate electrochemical and RNA analyses of perilymph for diagnostic purposes.

5. CONCLUSION

In this study, we demonstrated that 2PP 3D-printed hollow microneedles can facilitate aspiration of perilymph across the RWM at microliter volumes without causing permanent anatomic or physiologic dysfunction. Using these ultra-sharp hollow surgical microneedles, we successfully facilitated the sampling of perilymph for proteomic analyses in a series of survival experiments. In this preliminary proteomic study of microneedle-aspirated perilymph, we demonstrate the informative value of safe perilymph sampling. The ability of microneedles to mediate safe and effective intracochlear sampling in the guinea pig suggests great promise for their potential application for inner ear diagnostics in humans.