# Perilymph sampling in Mongolian gerbil, technical note and procedure evaluation.

Claire Olivier, Cyril Beck, Mickaël Risoud, Pierre-Emmanuel Lemesre, Juergen Siepmann, Florence Siepmann, Jérémy Vérin, Jean-François Goossens, Mostafa Kouach, Antonino Bongiovanni, et al.

► To cite this version:

Claire Olivier, Cyril Beck, Mickaël Risoud, Pierre-Emmanuel Lemesre, Juergen Siepmann, et al. Perilymph sampling in Mongolian gerbil, technical note and procedure evaluation. Hearing Research, 2025, Hearing Research, 458, pp.109188. 10.1016/j.heares.2025.109188. hal-04965672

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Research Paper

# Perilymph sampling in Mongolian gerbil, technical note and procedure evaluation.

Claire Olivier<sup>a,b,*</sup>, Cyril Beck<sup>a,b</sup>, Michaël Risoud<sup>a,b</sup>, Pierre Emmanuel Lemesre<sup>a,b</sup>, Juergen Siepmann<sup>b</sup>, Florence Siepmann<sup>b</sup>, Jeremy Vérin<sup>b</sup>, Jean-François Goossens<sup>c</sup>, Mostafa Kouach<sup>c</sup>, Antonino Bongiovanni<sup>d</sup>, Thomas Hubert<sup>e</sup>, Meryem Tardivel<sup>d</sup>, Christophe Vincent<sup>a</sup>, Philippine Toulemonde<sup>a,b</sup>

ARTICLE INFO

Keywords:

Mongolian gerbil (Meriones unguiculatus)

Inner ear

Pharmacokinetics

Perilymph sampling

Surgical technique

ABSTRACT

**Objective:** The aim of this study is to detail and evaluate the surgical procedure for perilymph sampling from the cochlear apex in the Mongolian gerbil.

**Design:** Perilymph sampling from the cochlear apex was performed one to three time in 12 male gerbils aged 8 to 12 months via the submandibular route. 11 of them were previously implanted with intracochlear implants loaded with dexamethasone and placed in the scala tympani, the 12th was used to collect control samples. The procedure was performed under general gas anesthesia, with head support, by a submandibular approach. After opening the bulla and sacrificing the external and middle ears, the cochlea was exposed and perilymph sampled. Macroscopic features of perilymph samples were reported before an HPLC-MS/MS assay to detect and quantify dexamethasone. Some cochleae were then harvested, immunostained and cleared to study fibrosis formation along the labyrinth and evaluate the consequences of multiple sampling procedures.

**Results:** The perilymph sampling procedure was technically difficult and required experimenter training. The procedure was well tolerated by the animals. One drawback of this technique in our gerbil model was the need to sacrifice the external ear, eardrum and ossicular chain to allow sufficient exposure for a quality procedure. Our technique enabled the collection of perilymph samples of the desired volume. Most of our samples were contaminated by bleeding from the otic capsule bone secondary to the cochleostomy. Nevertheless, HPLC-MS/MS analyses which successfully found dexamethasone in the perilymph of implanted gerbils, confirmed that our method was suitable for pharmacokinetic analyses. The occurrence of areas of fibrosis in the labyrinth after a sampling procedure compromises the quality and reliability of subsequent samples from the same animal.

**Conclusion:** The Mongolian gerbil is an animal model that can be used to collect perilymph from the cochlear apex to perform pharmacokinetic studies. The number of samples taken from a single animal must be limited to one.

# 1. Introduction and rationale of the study

The administration of therapeutics to the inner ear is a major challenge in the field of medical otology today. Indeed, many chronic (Meniere's disease) or acute (sudden deafness, iatrogenic ototoxicity)

pathologies can be treated by local administration of active ingredients. In current practice, this is achieved by intratympanic injections (Marshak et al., 2014; Ng et al., 2015; SFORL, 2016). While this route of administration improves the bioavailability of molecules in the perilymph and limits the adverse effects that occur with systemic

<footnote><sup>a</sup> CHU Lille, Department of Otology and Neurotology, F-59000 Lille, France; Univ. Lille, France</footnote>

<footnote><sup>b</sup> Univ. Lille, Inserm, CHU Lille, U1008, F-59000 Lille, France</footnote>

<footnote><sup>c</sup> Univ. Lille, CHU Lille, ULR 7365 - PSM-GRITA - Plateau de Spectrométrie de Masse - Groupe de Recherche sur les formes Injectables et les Technologies Associées, F-59000 Lille, France</footnote>

<footnote><sup>d</sup> Univ. Lille, BioImaging Center Lille-Nord de France (BICeL), Lille, France</footnote>

<footnote><sup>e</sup> Univ. Lille 2, CHU Lille, Experimental Resources Platform, Animal Housing Facility, F-59000 Lille, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: claire.olivier@chu-lille.fr (C. Olivier).</footnote>
administration, it does not allow stable perilymph concentrations to be maintained over time (Delaney et al., 2023; Salt and Plontke, 2018).

The development of galenic forms enabling prolonged and stable release of active ingredients over time in the inner ear could improve management of these pathologies (Bedulho Das Lages et al., 2024; Dash et al., 2022; Delaney et al., 2023). To achieve this, the pharmacokinetic characteristics of these devices must be assessed (Sawamura et al., 2021).

The analysis of perilymph samples enables us to characterize the diffusion kinetics of molecules of interest in the inner ear, thanks to current assay techniques (liquid chromatography coupled with mass spectrometry, for example) (Sawamura et al., 2021). The steps involved in collecting perilymph from the cochlear apex after approaching the tympanic bulla are documented in the literature for guinea pigs (Plontke et al., 2008; Salt et al., 2006).

The Mongolian gerbil (*Meriones unguiculatus*) is a small rodent model commonly used in otology research due to the characteristics of its auditory spectrum, which is broader in the low frequencies than in other rodent species, bringing it closer to humans' one (Batchelder et al., 2012; Müller, 1996; Otto and Jrge, 2012). It is an advantageous model for otological surgery, thanks to its enlarged tympanic bulla, which makes it easily accessible (Risoud et al., 2016). Our team has already used the Mongolian gerbil as a study model to characterize the pharmacokinetic properties of a silicone implant loaded with dexamethasone and implanted in the oval window (Sircoglou et al., 2015).

The main goal of this study was to describe and evaluate the technique of perilymph sampling at the cochlear apex in the Mongolian gerbil via the submandibular route. Our secondary objective was to evaluate the consequences of multiple sampling procedures on the cochlea.

## 2. Materials and methods

### 2.1. Study design

12 Mongolian gerbils of 8 to 12 months old were either or not implanted with intracochlear devices loaded with 20 % dexamethasone in the scala tympani. They then underwent perilymph sampling according to the procedure detailed below from one to three times per animal. After the perilymph sampling period, animals were sacrificed to harvest cochleae and evaluate multiple perilymph samplings consequences. Perilymph samples were assayed to measure dexamethasone concentration and verify the procedure was suitable for pharmacokinetic analyses.

### 2.2. Intracochlear devices (implants)

Gerbils were bilaterally implanted with two different experimental cylindrical implants designed for the treatment of chronic otologic diseases. These implants were designed to fit the gerbil's inner ear and were all 2 mm long and 300 µm in diameter (theoretical volume 0.14 µL).

- 5 gerbils were implanted with a non-absorbable implant made of ethylene-vinyle-acetate (EVA) polymer matrix (DuPont Elvax EVA 40 W, Dow, Geneva, Switzerland) loaded with 20 % of dexamethasone (Discovery Fine Chemicals, Dorset, UK) (Bedulho Das Lages et al., 2024).

- 6 gerbils were implanted with an absorbable implant made of poly-lactic-co-glycolic (PLGA) polymer matrix (Resomer RG503H, Evonik, Dermstadt, Germany) loaded with 20 % of dexamethasone (Discovery Fine Chemicals, Dorset, UK).

- 1 gerbil wasn't implanted.

### 2.3. Surgical procedures

* Anesthesia, peri- and post-operative monitoring.

Surgical procedures were performed under general anesthesia using a MINITAG veterinary gas anesthesia station (Tem Sega, Pessac, France) for induction (gas mixture of air (2L/min) and isoflurane (5 %)) and maintenance (gas mixture of air (0.8L/min) and isoflurane (1.5–2 %)). An injection of Buprenorphine (Bupaq 0.3mg/ml; Virbac, Carros, France) at a dosage of 0.1mg/kg was performed 45 min before incision. Surgical areas were shaved, and eye protection was applied. Each intervention was followed by twice-daily behavioral monitoring for 72 h, then daily for the remainder of the follow-up period. Animals were weighed regularly during the follow-up period. Operative scars were monitored daily without any additional care.

* Implantation surgery.

11 gerbils were implanted bilaterally. The entire surgical procedure was performed under binocular loupe (Euromex microscopes, Arnhem, Netherlands). The animal was positioned in lateral decubitus position. Implantation was performed by a post-auricular approach (Risoud et al., 2016). The implant was placed in the scala tympani after opening the round window membrane. The round window niche was then closed with autologous tissue plasty secured with a layer of biological glue to prevent any perilymph leakage after surgery. The list of equipment used for surgical procedures is available in appendix 1.

* Perilymph sampling surgery.

All implanted gerbils were used to collect perilymph from the right cochleae from one to three times. A precise chronology of perilymph sampling in implanted gerbils is provided in appendix 2. The non-implanted animal underwent bilateral perilymph sampling to collect control samples.

The animal was positioned supine on the stereotaxis table adapter (Stoelting Mouse/Neonatal rat adaptor; Fisherscientific, Pittsburgh, Pennsylvania, USA). The head was secured using foam-sided ear bars, gently clamped at cheek level, under the zygoma on each side. The installation is shown in Fig. 1. The entire surgical procedure was performed under binocular loupe (Euromex microscopes, Arnhem, Netherlands).

Fig. 2 shows the surgical steps of perilymph sampling. A paramedian "J" incision was made from the chin to the supra-clavicular region (Fig. 2-A). An incision was then made in subcutaneus muscles following the same path as the skin incision. Lifting the subcutaneus muscles flap revealed fatty tissue and lymph nodes, which were dissected to the supra-clavicular region (Fig. 2-B). Lowering the fat flap revealed the submandibular gland (whose pedicle vertically blocked access to the

<figure><img src="image_3.png" /><figcaption>Fig. 1. Workstation, material and installation. A: 1. Binocular loupe; 2. Light source; 3. Stereotaxis table mouse adapter; 4. Ear bars; 5. Compress rolls; 6. Holding belt; 7. Heating plate; 8. Conical mask; 9. Gas circulation circuit; 10. Surgical equipment; 11. Cotton buds; 12. Cotton pellets; 13. Betadine; 14. Suture thread; 15. Trocar 21 G; 16. Cyanoacrylate glue; 17. Kwik-Cast silicone; 18. Mixing tips; 19. Microcapillary tube; 20. Pipette bulb; 21. 2 mL vial (containing micro-insert). B: 1. Stereotaxis table mouse adapter; 2. Ear bars; 3. Compresses; 4. Animal holding belt; 5. Conical mask.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Surgical technique for perilymph sampling. Right ear. A - skin incision; B - exposure of the cervical fat flap (*); C - exposure of the masseter (M) and digastric (D) muscles, submaxillary gland (SG) and its vascular pedicle, after lowering of the fat flap (*); D - vascular pedicles to ligature; E - exposure of the digastric muscle: anterior belly (Da), intermediate tendon and posterior belly (Dp); F - section of the intermediate tendon (T) of the digastric muscle; G - exposure of the tympanic bulla (B) and hypoglossal nerve (XII); H - opening of the auditory bulla; I - exposure of the bulla contents: external auditory canal (C) and cochlea (+); J - exposure of the cochlea after external auditory canal, eardrum, malleus and incus removal. The apex is pointed by the white arrow. Round window (Rw), oval window (Ow), stapedial artery (A); K - exposure of the auditory bulla after building the silicone cup (S) around the apical turn of the cochlea; L - perilymph sampling after cochleostomy, using the micro-capillary (the leakage of perilymph through the hole is shown schematically). Orientation: ↓out; ←top.</figcaption></figure>

underlying structures), as well as the anterior belly of the digastric muscle and its tendon located medially to the masseter muscle (Fig. 2-C). The vascular pedicle of the submandibular gland was tied with Vicryl 5-0 suture (Fig. 2-D). Dissection of the fat flap continued in depth to detach it from the mandibular angle laterally and upwards, from the anterior edge of the sternocleidomastoid muscle laterally and downwards, and from the posterior belly of the digastric muscle medi- ally (Fig. 2-E). Dissection of the fat flap had to be cautious, as the in- ternal jugular vein runs its cervical course through the flap, following the anterior edge of the sternocleidomastoid muscle. At this stage, the animal's tympanic bulla could be located by following the posterior belly of the digastric muscle to its insertion (since the insertion of this muscle lies on the tympanic bulla). The intermediate tendon of the digastric muscle was then sectioned (Fig. 2-F). Section had to be cautious and gradual, to respect the hypoglossal nerve which runs along the deep side of the muscle. The posterior belly of the digastric muscle was carefully dissected down to its insertion on the tympanic bulla (Fig. 2-G). Lowering the cervical fat flap and the posterior belly of the digastric muscle after tendon section allowed correct exposure of the tympanic bulla. The bulla was opened thanks to a bone puncture on its convex part. The remaining bulla bone wall was then removed with forceps (Fig. 2-H), exposing the middle ear and the proximal part of the external auditory canal overhanging the cochlea (Fig. 2-I). To properly expose the cochlea, the portion of the external auditory canal that enters the bulla and the eardrum were sacrificed. Careful interruption of the ossicular chain was required to avoid perilymphatic leakage through dislocation of the stapes plate. After this stage, the cochlea was fully visible (Fig. 2-J). The next step was to make a silicone cup inside the bulla, surrounding the cochlear apex. A thin layer of cyanoacrylate glue was applied to the surface of the cochlea and to the epitympanic and mesotympanic walls of the bulla, to enable silicone adhesion. The sili- cone, which is hydrophobic, enabled the perilymph to be collected after cochleostomy, so that any cochlear fluid that escaped was contained and collected (Fig. 2-K). Once the silicone had been applied and dried, the scala tympani was pierced at the apical turn of the cochlea. After cochleostomy, the perilymph was collected from the silicone cup

surrounding the apex using the microcapillary tube (Fig. 2-L). The capillary used (3μL microcapillary; MicroCaps; Drummond, USA) was graduated with a digital caliper (0.01 mm accuracy), measuring 10.67 mm from one end under binocular loupe. The length (L) of the capillary to be filled to obtain 1 μL of perilymph was calculated from the cylinder volume formula (1μL = L*π*r²; r being internal radius of the capillary) using the internal diameter of the capillary which was known to be 0.3454mm:

$$L = \frac{1}{\pi * r^2} = \frac{1}{\pi * \left( \frac{0.3454}{2} \right)^2} \sim 10.67 \text{ mm}$$

Graduation of the capillary allowed to collect the desired amount of perilymph, in this case one microliter. The perilymph was then trans- ferred to the insert using the pipette bulb, and the insert placed in a labeled vial. The liquid was then stored at -80 °C, until analysis. To prevent persistent perilymphatic leakage after sampling, a plasty of autologous tissue (fat, muscle or connective tissue) was harvested from surrounding tissues, placed tightly over the cochleostomy and held in place with a layer of biological surgical glue (Tisseel). The approach was then sutured in 3 planes.

The entire surgical procedure is illustrated in the attached video (appendix 3). The list of equipment used for surgical procedures is available in appendix 1.

## 2.4. Perilymph samples evaluation and assay

Immediately after sampling, the length of the fluid-filled capillary was measured under binocular loupe using a digital caliper to calculate the exact volume of perilymph collected. The macroscopic aspect of perilymph was also reported.

All collected samples were stored at -80 °C after sampling until the day of analysis. After defrosting, they were diluted 30-fold with a 50:50 mixture of pure water (Purelab, Veolia Water Solution & Technologies) and acetonitrile (FisherScientific, Illkirch, France) containing dexamethasone internal standard (IS) (Dexaméthasone-4,6alpha,21,21-d4; Cluzeau info labo, Sainte Foy la Grande, France). Dexamethasone signal was measured, in each sample, by high performance liquid chroma- tography coupled with tandem mass spectrometry (HPLC-MS/MS: CLHP SHIMADZU UFLC-XR; Shimadzu, Kyoto, Japan; Kinetex EVO C18 2.6 μm, 100 × 2,1 mm; Phenomenex, Le Pecq, France; AB SCIEX QTRAP 5500; AB SCEIX, Framingham, USA). The concentration of dexametha- sone was then obtained by relating the ratio of the areas under the curve (AUC) of the dexamethasone and internal standard signals (AUC ana- lyte/AUC IS) to the previously edited calibration curve, after correcting for the dilution factor and averaging the values obtained from 2 in- jections. Dexamethasone limits of detection and quantification were respectively 0.4ng/mL and 2ng/mL. Samples collected from the non-implanted gerbil were used as negative control samples.

## 2.5. Microscopic study of cochlea

At the end of the follow-up, gerbils were sacrificed by lethal injection of pentobarbital (Doléthal; Vétoquinol S.A, Lure, France) followed by an intracardiac perfusion of heparinized serum (5UI/mL; Panpharma, Luitré-Dompierre, France) and then paraformaldehyde 4 % (Thermo- Scientific, Illkirch, France). The samples were prepared according to a 29-day protocol including steps of fixation, microdissection, decalcifi- cation, permeabilization, blocking, fluorescent immunolabeling, dehy- dration and finally clearing in ethyl cinnamate solution.

Six cochlea were harvested to study fibrosis formation in the laby- rinth in reaction to the perilymph sampling procedure which involves a cochleostomy. The antibodies used for fibrosis immunostaining were, first an anti-alpha-SMA antibody (Monoclonal mouse IgG2a anti-alpha- SMA; Sigma-Aldrich, Lezennes, France; concentration 1:400) secondary labelled by a goat anti-mouse antibody (goat anti-mouse IgG2A antibody
+ AlexaFluor 488; Invitrogen, Villebon-sur-Yvette, France; concentration 1:500).

Intact, cleared and immunostained cochleae were then observed using lightsheet microscopy (UltraMicroscope Blaze; MiltenyiBiotec, Paris, France) and images were processed using Imaris software (Imaris 10.2; Oxford Instruments, Abingdon-on-Thames; United-Kingdom). To quantify the volume of fibrosis formed within the labyrinth, cochleae acquired by light-sheet microscopy were studied in axial sections by a single experimenter. The immunolabelled fibrous tissue present in the labyrinth was manually contoured every 50 sections (every 0.1 mm), from base to apex over the entire height of the cochlea, using the software's "surface" function. This contouring produced a volume corresponding to the volume of the fibrous tissue, within which the voxels were automatically counted. The "statistics" function then converted the number of voxels counted into a volume measurement rendered in µL.

## 2.6. Statistical analyses

Continuous numerical variables were described in terms of mean and standard deviation. Categorical variables were described in terms of frequencies and percentages. Comparisons of proportions derived from 2 × 2 contingency tables were performed using Fisher's exact test.

# 3. Results

## 3.1. Perilymph sampling procedure

Ten gerbils underwent perilymph sampling using the procedure described above by a single trained operator. The installation of the animal and the anesthesia procedure described above resulted in good tolerance of the animals and an absence of general complications, except for one gerbil which did not tolerate the anesthesia during the second sampling and died at the end of the procedure. No feeding difficulties were detected in the post-operative period, despite the digastric muscle section-suture procedure.

It should be noted that two gerbils (different from the ten gerbils mentioned in the previous paragraph) died during or shortly after the operation due to spinal lesions explained by traumatic cervical rotation, justifying the absolute necessity of using a stereotaxis adapter to hold the head during surgery.

The submandibular approach allowed good exposure of the tympanic bulla and access to the cochlear apex. This good exposure couldn't be obtained if the external auditory canal and the ossicular chain were preserved.

The procedure could be repeated for a second or even third sampling, with the drawback of a more difficult surgical approach.

## 3.2. Perilymph samples quality (macroscopic features)

23 perilymph samples were collected. The average volume of liquid collected in the capillary, calculated by measuring the length of capillary filled during sampling, was 1.04 µL (standard deviation 0.23). This result was near our objective of 1µL.

Blood contamination occurred in 74 % of samples (slightly pinkish appearance of the liquid, which nevertheless remained transparent - an example is shown in the appended video). Samples considered uncontaminated were completely transparent. As no significant bleeding of surrounding tissue was observed in the silicone cup during the sampling procedure, we assumed that this contamination originated from the cochlear bone, which may bleed slightly during a cochleostomy. There was no statistical difference between the proportion of samples contaminated during a first sampling (69 % of 13 samples) or a repeat surgery (second or third sampling; 80 % of 10 samples) (p = 0.66).

## 3.3. Perilymph samples assay (HPLC-MS/MS)

Perilymph samples were analyzed to determine the pharmacokinetics of the two implants, by tracking dexamethasone concentrations over time. Dexamethasone was detectable and measurable in our samples, enabling us to determine its release profile in the perilymph. Dexamethasone concentrations ranged from 1450 to 48 ng/mL (over 54 days) and from 472 to 0 ng/mL (over 40 days) in the perilymph of gerbils implanted respectively with dexamethasone-loaded EVA and PLGA implants. Analysis of samples taken from the non-implanted gerbil revealed no signal corresponding to dexamethasone.

Detailed data on the pharmacokinetics of these implants are beyond the scope of this study and will be the subject of future publications.

## 3.4. Microscopic study of cochleae (lightsheet microscopy of immunostained cleared cochleae)

Six right cochleae were harvested from gerbils that died during surgery or were sacrificed by lethal injection to study fibrosis formation (Fig. 3):

- For one of them, immediately after the 1st sampling surgery to study the distribution of fibrosis formation since the implantation (died during surgery of an intraoperative complication). No fibrosis was detected in this cochlea.

- For one of them, immediately after the 2nd sampling surgery to study the distribution of fibrosis formation since the 1st sampling (died during surgery of complications of anesthesia). Fibrosis volume was there measured to 0.01 µL (Fig. 3-A).

- For three of them, 6 to 21 days after the 2nd sampling surgery to study the distribution of fibrosis formation since the 2nd sampling. Fibrosis volumes ranged there from 0.17 to 2.9 µL (Fig. 3-B).

- For one of them, 12 days after the 3rd sampling surgery to study the distribution of fibrosis formation since the 3rd sampling. Fibrosis volume was there measured to 0.21 µL (Fig. 3-C).

All cochleae that underwent cochleostomy for perilymph sampling showed fibrous tissue in the labyrinthine spaces after different healing times, even after a single procedure. This phenomenon could take the form of full-blown ossificans labyrinthitis after repeated sampling procedures (Fig. 3-B).

# 4. Discussion

The perilymph sampling technique from the apex of a rodent cochlea is documented in the literature for guinea pigs (Salt et al., 2006). The procedure used here, once the cochlea has been exposed, is identical to the one developed by Salt, Hale and Plontke (Salt et al., 2006). The approach to the cochlear apex in our gerbil model has been adapted from the submandibular approaches described in the rat (Qiu et al., 2007). The post-auricular route, in both gerbil (Risoud et al., 2016) and rat (Qiu et al., 2007), does not allow visualization of the entire cochlea (it only gives access to the round window) and is therefore unsuitable for perilymph sampling at the cochlear apex.

Perilymph sampling in the Mongolian gerbil is a difficult procedure which required training for the experimenter to be properly mastered.

The procedure was well tolerated by the animals. The use of the stereotaxis table's mouse adapter with special head fixation during the procedure (using ear bars) was essential. This prevented traumatic cervical rotation movements, which could cause damage to the cervical spine and spinal cord, leading to death.

One of the major drawbacks of this technique was the need to sacrifice parts of the gerbils' ear which is certain to impair hearing function. Indeed, in this study, to optimally expose the cochlea, gerbils external auditory canal, eardrum and ossicular chain were sacrificed. The shaping of the silicone cup was crucial to succeed in sampling. The
<figure><img src="image_5.png" /><figcaption>Fig. 3. Fibrosis repartition in cleared cochlea after perilymph sampling surgery. 3D reconstructions of cochlea collected from gerbils which underwent perilymph sampling surgery. A – after one procedure; B – after two procedures; C – after three procedures. Measured fibrosis volumes (in µL) are indicated for each cochlea.</figcaption></figure>

silicone had to be carefully laid out to form a watertight cup to avoid any loss of perilymph into the bulla, but without covering the apical turn of the cochlea, which formed the bottom of the cup. Respecting the apical turn of the cochlea enabled the cochleostomy to be performed at the location chosen by the experimenter. This precision work required the widest possible exposure of the cochlea, even more so as the Kwik-Cast silicone mixing tips were bulky and difficult to handle in this restricted space. Tests previously carried out without removing gerbils' external ear and ossicular chain had also confirmed that the exposure obtained keeping these structures was too imprecise to guarantee quality sampling.

The detection of dexamethasone in the samples taken from gerbils implanted in scala tympani (with devices loaded with dexamethasone), the absence of any signal detected in the control samples, using an assay method recognized for its specificity (HPLC-MS/MS) (Sawamura et al., 2021) and the cochleostomy site chosen by the experimenter after exposure of the cochlear apex, allow us to confirm that the fluid collected was indeed perilymph.

To ensure that only perilymph was collected, and to prevent dilution of the sample by cerebrospinal fluid flowing through the cochlear aqueduct into the scala tympani (Salt et al., 2006, 2003), the sample volume had to be smaller than the volume of the Mongolian gerbil scala tympani (Thorne et al., 1999). We therefore chose to limit the sample volume to 1 microliter. Prior graduation of the microcapillary tube enabled us to sample a precise volume of perilymph (1.04±0.23 µL), near our objective of 1 µL, which is of vital importance for pharmacokinetic analyses.

The microcapillary chosen (3µL microcapillary; MicroCaps; Drummond, USA) had a small internal diameter, which reduced the margin of error on the volume of perilymph sampled during the procedure compared with larger capillaries. Indeed, a one-millimeter variation in the length of the liquid-filled capillary (ΔL) corresponded to a variation in the volume (ΔV) sampled of around 0.09 µL, which was still

acceptable given the total volume of perilymph contained in the gerbil's scala tympani (Thorne et al., 1999):

$$ \Delta V = \pi * r^2 * \Delta L = \pi * \left( \frac{0.3454}{2} \right)^2 * \pm 1 \sim \pm 0.09\mu L $$

Bloody contamination of the samples, although frequent (74 %), did not compromise perilymph analysis in our study. This contamination was probably due to bleeding from the otic capsule secondary to the cochleostomy. Studies carried out on other animal models used to collect perilymph from the apex, by cochleostomy, do not detail the macroscopic appearance of the samples obtained (Hahn et al., 2012; Liu et al., 2015; Salt et al., 2006). As a result, we do not know whether this phenomenon is specific to the gerbil, whose otic capsule is richly vascularized (Hutson et al., 2021). Nevertheless, these findings call for reconsideration of the cochleostomy technique used here. In our study, cochleostomies were performed using a Rosen hook with a 0.3 mm diameter tip (Pouret Medical, Clichy, France). In the article presenting the technique of perilymph sampling in guinea pigs, Salt et al. (Salt et al., 2006) reported using a similar instrument, but the diameter of cochleostomies performed was measured between 50 and 100 µm. In our study, the size of the cochleostomies was probably larger, although no precise measurements were made. A large cochleostomy could indeed damage the stria vascularis - which is the most vascularized part of the lateral wall (Hutson et al., 2021) - leading to increased bleeding. This may explain the hemorrhagic nature of most of our samples and this is certainly a point that should be corrected to improve the technique presented here.

Comparison of the concentrations of active ingredients obtained in a contaminated sample and a clear sample at identical times would certainly enable us to specify the extent of this contamination, which here was not quantifiable. Unfortunately, the design of our study and the samples available did not allow us to carry out this analysis.

In the literature, perilymph samples collected after cochleostomy are
usually taken only once from each animal (Hahn et al., 2012; Liu et al., 2015). However, to reduce the number of animals used - which is a growing concern in the scientific community (French national committee for ethical reflection on animal experimentation and French National commission for the protection of animals used for scientific purposes, 2023) - we wanted to assess the feasibility of repeated surgeries to take several samples from the same animal and the reliability of the samples obtained. Performing multiple sampling on one animal were technically possible, but riskier and the difficulty of the procedure increased with each sample, due to tissue healing in the approach and the more hemorrhagic nature of dissection, which increased exposure difficulties. Results of the immunostained cochlea study showed the formation of labyrinthine fibrosis at the apex in all cases after cochleostomy, from the first sampling. In gerbils that had undergone multiple sampling, these lesions could take the form of genuine ossificans labyrinthitis. This raises the question of the impact of these tissue changes on cochlear pharmacokinetics. In addition, fibrosis, by reducing the liquid volume of the scala tympani, could increase the risk of cerebrospinal fluid contamination of samples (Salt et al., 2003). Indeed, as the cochlear aqueduct opens at the base of the scala tympani, any removal of the perilymph it contains - whose volume in gerbils is around 1 µL (Thorne et al., 1999) - results in an influx of cerebrospinal fluid to replace it (Salt et al., 2006, 2003). However, if fibrosis forms in this space after an initial sampling, the volume of fluid it can contain is reduced. When a second sample is taken, there is therefore a risk that the microliter sampled will contain both a proportion of perilymph (fluid that has remained in the scala in contact with the implant since the first sample, and whose volume corresponds to the volume of the scala amputated by fibrosis, i.e. <1 µL) and a quantity of cerebrospinal fluid flowing through the cochlear aqueduct. This probably results in a dilution of the sample taken, in a proportion that could not be quantified at the time of sampling. It would therefore be advisable to limit the number of perilymph samples taken to one per animal.

## 5. Conclusion

In conclusion, our team has demonstrated the feasibility of perilymph sampling in the Mongolian gerbil, a relevant animal model for the evaluation of otoprotective drugs pharmacokinetics. This is a difficult technique, requiring rigorous set-up and operator training to master the technique, guarantee reproducibility and avoid intra- and post-operative complications. The number of samples taken from a single animal must be limited to one.

## Funding

This project has received funding from the French National Research Agency (ANR-15-CE19-0014-01) and from the Interreg 2 Seas programme co-funded by the European Regional Development Fund under subsidy contract "Site Drug 2S07-033".

## Institutional review board statement

The study was approved by the regional committee for experimental animal care and use (CEEA Nord-Pas de Calais n_75, France) listed under protocol #38,692. Studies were performed in agreement with the standard guidelines of the French Ministry of Agriculture, according to the EU Directive 2010/63/EU for the protection of animals used for scientific purposes.

## CRediT authorship contribution statement

Claire Olivier: Writing - review & editing, Writing - original draft, Methodology, Investigation, Data curation, Conceptualization. **Cyril Beck:** Methodology, Conceptualization. **Michaël Risoud:** Supervision, Methodology, Conceptualization. **Pierre Emmanuel Lemesre:**

Methodology, Conceptualization. **Juergen Siepmann:** Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization. **Florence Siepmann:** Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization. **Jeremy Vérin:** Resources, Methodology. **Jean-François Goossens:** Resources, Methodology. **Mostafa Kouach:** Resources, Methodology. **Antonino Bongiovanni:** Supervision, Resources. **Thomas Hubert:** Supervision, Resources. **Meryem Tardivel:** Supervision, Resources, Methodology, Conceptualization. **Christophe Vincent:** Validation, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. **Philippine Toulemonde:** Writing - review & editing, Validation, Supervision, Project administration, Methodology, Investigation, Conceptualization.

## Declaration of competing interest

The authors declare no conflict of interest. Funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

## Acknowledgments

The authors would like to thank Martin Fourdrinier, Franck Stevendart and Arnold Dive from DHURE as well as U1172 INSERM Team for technical support.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.heares.2025.109188.

## Data availability

Data will be made available on request.

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