Neural and behavioral markers of inhibitory control predict symptom improvement during internet-delivered cognitive behavioral therapy for depression.

Poor inhibitory control contributes to deficits in emotion regulation, which are often targeted by treatments for major depressive disorder (MDD), including cognitive behavioral therapy (CBT). Brain regions that contribute to inhibitory control and emotion regulation overlap; thus, inhibitory control might relate to response to CBT. In this study, we examined whether baseline inhibitory control and resting state functional connectivity (rsFC) within overlapping emotion regulation-inhibitory control regions predicted treatment response to internet-based CBT (iCBT). Participants with MDD were randomly assigned to iCBT (N = 30) or a monitored attention control (MAC) condition (N = 30). Elastic net regression was used to predict post-treatment Patient Health Questionnaire-9 (PHQ-9) scores from baseline variables, including demographic variables, PHQ-9 scores, Flanker effects (interference, sequential dependency, post-error slowing), and rsFC between the dorsal anterior cingulate cortex, bilateral anterior insula (AI), and right temporoparietal junction (TPJ). Essential prognostic predictor variables retained in the elastic net regression included treatment group, gender, Flanker interference response time (RT), right AI-TPJ rsFC, and left AI-right AI rsFC. Prescriptive predictor variables retained included interactions between treatment group and baseline PHQ-9 scores, age, gender, Flanker RT, sequential dependency effects on accuracy, post-error accuracy, right AI-TPJ rsFC, and left AI-right AI rsFC. Inhibitory control and rsFC within inhibitory control-emotion regulation regions predicted reduced symptom severity following iCBT, and these effects were stronger in the iCBT group than in the MAC group. These findings contribute to a growing literature indicating that stronger inhibitory control at baseline predicts better outcomes to psychotherapy, including iCBT.

Circulating PACAP levels are associated with altered imaging measures of entorhinal cortex neurite density in posttraumatic stress disorder.

Introduction: Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates plasticity in brain systems underlying arousal and memory and is associated with posttraumatic stress disorder (PTSD). Research in animal models suggests that PACAP modulates entorhinal cortex (EC) input to the hippocampus, contributing to impaired contextual fear conditioning. In PTSD, PACAP is associated with higher activity of the amygdala to threat stimuli and lower functional connectivity of the amygdala and hippocampus. However, PACAP-affiliated structural alterations of these regions have not been investigated in PTSD. Here, we examined whether peripheral PACAP levels were associated with neuronal morphology of the amygdala and hippocampus (primary analyses), and EC (secondary) using Neurite Orientation Dispersion and Density Imaging.Methods: Sixty-four (44 female) adults (19 to 54 years old) with DSM-5 Criterion A trauma exposure completed the Clinician-Administered PTSD Scale (CAPS-5), a blood draw, and magnetic resonance imaging. PACAP38 radioimmunoassay was performed and T1-weighted and multi-shell diffusion-weighted images were acquired. Neurite Density Index (NDI) and Orientation Dispersion Index (ODI) were quantified in the amygdala, hippocampus, and EC. CAPS-5 total score and anxious arousal score were used to test for clinical associations with brain structure.Results: Higher PACAP levels were associated with greater EC NDI (ß = 0.0099, q = 0.032) and lower EC ODI (ß = -0.0073, q = 0.047), and not hippocampal or amygdala measures. Neither EC NDI nor ODI was associated with clinical measures.Conclusions: Circulating PACAP levels were associated with altered neuronal density of the EC but not the hippocampus or amygdala. These findings strengthen evidence that PACAP may impact arousal-associated memory circuits in PTSD.

PACAP was associated with altered entorhinal cortex neurite density in PTSD.PACAP was not associated with altered neurite density in amygdala or hippocampus.PACAP may impact arousal-associated memory circuits.

Examining State Licensing Requirements for Select Master's-Level Behavioral Health Providers for Children.

OBJECTIVE: The authors examined licensing requirements for select children's behavioral health care providers. METHODS: Statutes and regulations as of October 2021 were reviewed for licensed clinical social workers, licensed professional counselors, and licensed marriage and family therapists for all 50 U.S. states and the District of Columbia. RESULTS: All jurisdictions had laws regarding postgraduate training and license portability. No jurisdiction included language about specialized postgraduate training related to serving children and families or cultural competence. Other policies that related to the structure, composition, and authority of licensing boards varied across states and licensure types. CONCLUSIONS: In their efforts to address barriers to licensure, expand the workforce, and ensure that children have access to high-quality and culturally responsive care, states could consider their statutes and regulations.

Hampshire Sheep as a Large-Animal Model for Cochlear Implantation.

BACKGROUND: Sheep have been proposed as a large-animal model for studying cochlear implantation. However, prior sheep studies report that the facial nerve (FN) obscures the round window membrane (RWM), requiring FN sacrifice or a retrofacial opening to access the middle-ear cavity posterior to the FN for cochlear implantation. We investigated surgical access to the RWM in Hampshire sheep compared to Suffolk-Dorset sheep and the feasibility of Hampshire sheep for cochlear implantation via a facial recess approach. METHODS: Sixteen temporal bones from cadaveric sheep heads (ten Hampshire and six Suffolk-Dorset) were dissected to gain surgical access to the RWM via an extended facial recess approach. RWM visibility was graded using St. Thomas' Hospital (STH) classification. Cochlear implant (CI) electrode array insertion was performed in two Hampshire specimens. Micro-CT scans were obtained for each temporal bone, with confirmation of appropriate electrode array placement and segmentation of the inner ear structures. RESULTS: Visibility of the RWM on average was 83% in Hampshire specimens and 59% in Suffolk-Dorset specimens (p = 0.0262). Hampshire RWM visibility was Type I (100% visibility) for three specimens and Type IIa (> 50% visibility) for seven specimens. Suffolk-Dorset RWM visibility was Type IIa for four specimens and Type IIb (< 50% visibility) for two specimens. FN appeared to course more anterolaterally in Suffolk-Dorset specimens. Micro-CT confirmed appropriate CI electrode array placement in the scala tympani without apparent basilar membrane rupture. CONCLUSIONS: Hampshire sheep appear to be a suitable large-animal model for CI electrode insertion via an extended facial recess approach without sacrificing the FN. In this small sample, Hampshire specimens had improved RWM visibility compared to Suffolk-Dorset. Thus, Hampshire sheep may be superior to other breeds for ease of cochlear implantation, with FN and facial recess anatomy more similar to humans.

"I Missed School to Take Care of Someone Else": Middle and High School Students' Caregiving Responsibilities as a Reason for Absenteeism.

BACKGROUND: Middle and high school students who are involved in caregiving for aging, chronically ill, and/or disabled family members report more learning challenges compared to their non-caregiving peers. However, little is known about how many students miss school to take care of someone else, and which students are most likely to have this experience. Such knowledge could reveal an important, largely unrecognized reason for school absences and educational disparities. METHODS: Our research-practice partnership surveyed middle-and-high schoolers across Rhode Island public schools in 2022. RESULTS: Among 55,746 students (45% White non-Latinx; 21% Latinx; 45% girls), 13.80% reported they had missed school to take care of someone else, with up to 35% in some districts. Students who missed school for caregiving were disproportionately girls, non-binary, transgender, or preferred not to report gender, older youth, and from historically marginalized racial and ethnic groups, and from urban districts. CONCLUSIONS: Children's experiences caregiving for others may be an important and overlooked contributor to absenteeism and achievement gaps, especially in urban areas. We suggest school policies to better serve these students.

Smaller total and subregional cerebellar volumes in posttraumatic stress disorder: a mega-analysis by the ENIGMA-PGC PTSD workgroup.

Although the cerebellum contributes to higher-order cognitive and emotional functions relevant to posttraumatic stress disorder (PTSD), prior research on cerebellar volume in PTSD is scant, particularly when considering subregions that differentially map on to motor, cognitive, and affective functions. In a sample of 4215 adults (PTSD n = 1642; Control n = 2573) across 40 sites from the ENIGMA-PGC PTSD working group, we employed a new state-of-the-art deep-learning based approach for automatic cerebellar parcellation to obtain volumetric estimates for the total cerebellum and 28 subregions. Linear mixed effects models controlling for age, gender, intracranial volume, and site were used to compare cerebellum volumes in PTSD compared to healthy controls (88% trauma-exposed). PTSD was associated with significant grey and white matter reductions of the cerebellum. Compared to controls, people with PTSD demonstrated smaller total cerebellum volume, as well as reduced volume in subregions primarily within the posterior lobe (lobule VIIB, crus II), vermis (VI, VIII), flocculonodular lobe (lobule X), and corpus medullare (all p-FDR < 0.05). Effects of PTSD on volume were consistent, and generally more robust, when examining symptom severity rather than diagnostic status. These findings implicate regionally specific cerebellar volumetric differences in the pathophysiology of PTSD. The cerebellum appears to play an important role in higher-order cognitive and emotional processes, far beyond its historical association with vestibulomotor function. Further examination of the cerebellum in trauma-related psychopathology will help to clarify how cerebellar structure and function may disrupt cognitive and affective processes at the center of translational models for PTSD.

An Implantable Piezofilm Middle Ear Microphone: Performance in Human Cadaveric Temporal Bones.

PURPOSE: One of the major reasons that totally implantable cochlear microphones are not readily available is the lack of good implantable microphones. An implantable microphone has the potential to provide a range of benefits over external microphones for cochlear implant users including the filtering ability of the outer ear, cosmetics, and usability in all situations. This paper presents results from experiments in human cadaveric ears of a piezofilm microphone concept under development as a possible component of a future implantable microphone system for use with cochlear implants. This microphone is referred to here as a drum microphone (DrumMic) that senses the robust and predictable motion of the umbo, the tip of the malleus. METHODS: The performance was measured by five DrumMics inserted in four different human cadaveric temporal bones. Sensitivity, linearity, bandwidth, and equivalent input noise were measured during these experiments using a sound stimulus and measurement setup. RESULTS: The sensitivity of the DrumMics was found to be tightly clustered across different microphones and ears despite differences in umbo and middle ear anatomy. The DrumMics were shown to behave linearly across a large dynamic range (46 dB SPL to 100 dB SPL) across a wide bandwidth (100 Hz to 8 kHz). The equivalent input noise (over a bandwidth of 0.1-10 kHz) of the DrumMic and amplifier referenced to the ear canal was measured to be about 54 dB SPL in the temporal bone experiment and estimated to be 46 dB SPL after accounting for the pressure gain of the outer ear. CONCLUSION: The results demonstrate that the DrumMic behaves robustly across ears and fabrication. The equivalent input noise performance (related to the lowest level of sound measurable) was shown to approach that of commercial hearing aid microphones. To advance this demonstration of the DrumMic concept to a future prototype implantable in humans, work on encapsulation, biocompatibility, and connectorization will be required.

Regional differences in cochlear nonlinearity across the basal organ of Corti of gerbil: Regional differences in cochlear nonlinearity.

Auditory sensation is based in nanoscale vibration of the sensory tissue of the cochlea, the organ of Corti complex (OCC). Motion within the OCC is now observable due to optical coherence tomography. In a previous study (Cooper et al., 2018), the region that includes the electro-motile outer hair cells (OHC) and Deiters cells (DC) was observed to move with larger amplitude than the basilar membrane (BM) and surrounding regions and was termed the "hotspot." In addition to this quantitative distinction, the hotspot moved qualitatively differently than the BM, in that its motion scaled nonlinearly with stimulus level at all frequencies, evincing sub-BF activity. Sub-BF activity enhances non-BF motion; thus the frequency tuning of the OHC/DC region was reduced relative to the BM. In this work we further explore the motion of the gerbil basal OCC and find that regions that lack significant sub-BF activity include the BM, the medial and lateral OCC, and the reticular lamina (RL) region. The observation that the RL region does not move actively sub-BF (already observed in Cho and Puria 2022), suggests that hair cell stereocilia are not exposed to sub-BF activity in the cochlear base. The observation that the lateral and RL regions move approximately linearly sub-BF indicates that linear forces dominate non-linear OHC-based forces on these components at sub-BF frequencies. A complex difference analysis was performed to reveal the internal motion of the OHC/DC region and showed that amplitude structure and phase shifts in the directly measured OHC/DC motion emerge due to the internal OHC/DC motion destructively interfering with BM motion.

Physiologic Effects of Microneedle-Mediated Intracochlear Dexamethasone Injection in the Guinea Pig.

OBJECTIVES: Oral or intratympanic corticosteroids are commonly used to treat sudden sensorineural hearing loss (SSHL), tinnitus, and Meniere disease. Direct intracochlear delivery has been proposed to overcome the variability in bioavailability and efficacy of systemic or middle ear delivery. In this study, we aim to characterize the physiologic consequences of microneedle-mediated direct intracochlear injection of dexamethasone through the round window membrane (RWM). METHODS: In Hartley guinea pigs (n = 5), a post-auricular incision followed by bullostomy was made to access the round window membrane. Using 100 µm diameter hollow microneedles, 1.0 µl of 10 mg/ml dexamethasone was injected through the RWM over 1 min. Compound action potential (CAP) and distortion product otoacoustic action emissions (DPOAE) were measured before perforation, at 1 h, and at 5 h following injection. CAP hearing thresholds were measured from 0.5 to 40 kHz, and DPOAE f2 frequencies ranged from 1.0 and 32 kHz. Repeated measures ANOVA followed by pairwise t-tests were used for statistical analysis. RESULTS: ANOVA identified significant CAP threshold shifts at four frequencies (4, 16, 36, and 40 kHz) and differences in DPOAE at 1 frequency (6 kHz). Paired t-tests revealed differences between the pre-perforation and 1 h time point. By 5 h post injection, both CAP hearing thresholds and DPOAE recover and are not significantly different from baseline thresholds. CONCLUSION: Direct intracochlear delivery of dexamethasone via microneedles results in temporary shifts in hearing thresholds that resolve by 5 hours, thus supporting microneedle technology for the treatment of inner ear disorders. LEVEL OF EVIDENCE: NA Laryngoscope, 134:388-392, 2024.

Anhedonia and Delay Discounting: Differing Patterns of Brain-Behavior Relationships in Healthy Control Participants Versus Individuals With Posttraumatic Stress Disorder.

BACKGROUND: Anhedonia may contribute to individual differences in delay discounting (DD). In prior work, we found that higher anhedonia was associated with shallower DD in healthy control (HC) participants but steeper DD in individuals with posttraumatic stress disorder (PTSD). In this study, we aimed to directly compare the relationship between anhedonia and DD across groups and to identify functional brain correlates of this interaction. METHODS: Participants (HC group: n = 23, DSM-5 PTSD group: n = 23) completed a questionnaire assessing anhedonia (Snaith-Hamilton Pleasure Scale [SHAPS]), task-based functional magnetic resonance imaging of decision making including DD, and resting-state functional magnetic resonance imaging. Task-based activity and resting-state functional connectivity were evaluated in reward-related regions that have also been implicated in PTSD (nucleus accumbens [NAcc], right anterior insula). RESULTS: Higher SHAPS scores were associated with steeper DD in PTSD, but there was no relationship between DD and SHAPS in the HC group. There was a significant group-by-SHAPS interaction for NAcc activity, t31 = 2.92, p = .007: Greater NAcc activity when immediate rewards were chosen was associated with higher SHAPS in the PTSD group but lower SHAPS in the HC group. In resting-state functional connectivity, there was a group-by-SHAPS interaction between the NAcc seed and right parietal and frontal pole clusters. CONCLUSIONS: These results extend prior findings that anhedonia is associated with steeper DD in PTSD and demonstrate that this behavioral finding occurs in the context of NAcc hyperactivity to immediate rewards and hyperconnectivity in anhedonic individuals with PTSD.

"Caregiving Youth" and the Patchwork History of Recognition in the United States.

This article examines the U.S. legislative and policy landscape and its historical and contemporary recognition of young people as caregivers and their importance to public health, both as care providers and as a category of special concern for overall wellbeing. Drawing on feminist geographies of health to situate a historical analysis, we aim to answer two key questions: First, what is the history of recognition of caregiving youth in key moments of federal action to address family caregiving needs? Second, how might we use this history to better understand and analyze the patchwork geography of caregiving youth recognition in the U.S. and other countries that similarly lack formal national policy recognition to improve and enhance public health? We use the term patchwork to describe how federal recognition of caregiving youth in broader debates about public health is uneven across both time and space, and contingent upon civil society, non-profit organizations, and researchers working in and with geographically bound communities. Our results illustrate how a focus on the relationships of recognition, both in the past and the present and at local and national scales, reveals a different perspective on caregiving youth in the U.S. with a much more complex history than previously identified. The article describes how relationships established in the absence of federal policy or legislation are sometimes directed towards building more formal recognition, and other times with the goal of changing practices in a specific location.

A frame and a hotspot in cochlear mechanics.

Auditory sensation is based in nanoscale vibration of the sensory tissue of the cochlea, the organ of Corti complex (OCC). Motion within the OCC is now observable due to optical coherence tomography. In the cochlear base, in response to sound stimulation, the region that includes the electro-motile outer hair cells (OHC) was observed to move with larger amplitude than the basilar membrane (BM) and surrounding regions. The intense motion is based in active cell mechanics, and the region was termed the "hotspot" (Cooper et al., 2018, Nature comm). In addition to this quantitative distinction, the hotspot moved qualitatively differently than the BM, in that its motion scaled nonlinearly with stimulus level at all frequencies, evincing sub-BF activity. Sub-BF activity enhances non-BF motion; thus the frequency tuning of the hotspot was reduced relative to the BM. Regions that did not exhibit sub-BF activity are here defined as the OCC "frame". By this definition the frame includes the BM, the medial and lateral OCC, and most significantly, the reticular lamina (RL). The frame concept groups the majority OCC as a structure that is largely shielded from sub-BF activity. This shielding, and how it is achieved, are key to the active frequency tuning of the cochlea. The observation that the RL does not move actively sub-BF indicates that hair cell stereocilia are not exposed to sub-BF activity. A complex difference analysis reveals the motion of the hotspot relative to the frame.

Sheep as a Large-Animal Model for Otology Research: Temporal Bone Extraction and Transmastoid Facial Recess Surgical Approach.

PURPOSE: Sheep are used as a large-animal model for otology research and can be used to study implantable hearing devices. However, a method for temporal bone extraction in sheep, which enables various experiments, has not been described, and literature on middle ear access is limited. We describe a method for temporal bone extraction and an extended facial recess surgical approach to the middle ear in sheep. METHODS: Ten temporal bones from five Hampshire sheep head cadavers were extracted using an oscillating saw. After craniotomy and removal of the brain, a coronal cut was made at the posterior aspect of the orbit followed by a midsagittal cut of the occipital bone and disarticulation of the atlanto-occipital joint. Temporal bones were surgically prepared with an extended facial recess approach. Micro-CT scans of each temporal bone were obtained, and anatomic dimensions were measured. RESULTS: Temporal bone extraction was successful in 10/10 temporal bones. Extended facial recess approach exposed the malleus, incus, stapes, and round window while preserving the facial nerve, with the following surgical considerations: minimally pneumatized mastoid; tegmen (superior limit of mastoid cavity) is low-lying and sits below temporal artery; chorda tympani sacrificed to optimize middle ear exposure; incus buttress does not obscure view of middle ear. Distance between the superior aspect of external auditory canal and tegmen was 2.7 (SD 0.9) mm. CONCLUSION: We identified anatomic landmarks for temporal bone extraction and describe an extended facial recess approach in sheep that exposes the ossicles and round window. This approach is feasible for studying implantable hearing devices.

Circulating PACAP levels are associated with altered imaging measures of entorhinal cortex neurite density in posttraumatic stress disorder.

Background: Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates plasticity in brain systems underlying arousal and memory and is associated with posttraumatic stress disorder (PTSD). Research in animal models suggests that PACAP modulates entorhinal cortex (EC) input to the hippocampus, contributing to impaired contextual fear conditioning. In PTSD, PACAP is associated with higher activity of the amygdala to threat stimuli and lower functional connectivity of the amygdala and hippocampus. However, PACAP-affiliated structural alterations of these regions have not been reported. Here, we examined whether peripheral PACAP levels were associated with neuronal morphology of the amygdala and hippocampus (primary analysis), and EC (secondary analysis) using Neurite Orientation Dispersion and Density Imaging. Methods: Sixty-four (44 female) adults (19 to 54 years old) with DSM-5 Criterion A trauma exposure completed the Clinician-Administered PTSD Scale (CAPS-5), a blood draw, and magnetic resonance imaging. PACAP38 radioimmunoassay was performed and T1-weighted and multi-shell diffusion- weighted images were acquired. Neurite Density Index (NDI) and Orientation Dispersion Index (ODI) were quantified in the amygdala, hippocampus, and EC. CAPS-5 total score and anxious arousal score were used to test for clinical associations with brain structure. Results: Higher PACAP levels in blood were associated with greater EC NDI (ß=0.31, q=0.034) and lower EC ODI (ß=-0.30, q=0.042) and not hippocampal or amygdala measures. Neither EC NDI nor ODI was associated with clinical measures. Conclusions: Circulating PACAP levels were associated with altered neuronal density of the EC but not hippocampus or amygdala. These findings strengthen evidence that PACAP may impact arousal- associated memory circuits.

Microneedles Facilitate Small-Volume Intracochlear Delivery Without Physiologic Injury in Guinea Pigs.

HYPOTHESIS: Microneedle-mediated intracochlear injection through the round window membrane (RWM) will facilitate intracochlear delivery, not affect hearing, and allow for full reconstitution of the RWM within 48 hours. BACKGROUND: We have developed polymeric microneedles that allow for in vivo perforation of the guinea pig RWM and aspiration of perilymph for diagnostic analysis, with full reconstitution of the RWM within 48 to 72 hours. In this study, we investigate the ability of microneedles to deliver precise volumes of therapeutics into the cochlea and assess the subsequent consequences on hearing. METHODS: Volumes of 1.0, 2.5, or 5.0 µL of artificial perilymph were injected into the cochlea at a rate of 1 µL/min. Compound action potential (CAP) and distortion product otoacoustic emission were performed to assess for hearing loss (HL), and confocal microscopy was used to evaluate the RWM for residual scarring or inflammation. To evaluate the distribution of agents within the cochlea after microneedle-mediated injection, 1.0 µL of FM 1-43 FX was injected into the cochlea, followed by whole mount cochlear dissection and confocal microscopy. RESULTS: Direct intracochlear injection of 1.0 µL of artificial perilymph in vivo , corresponding to about 20% of the scala tympani volume, was safe and did not result in HL. However, injection of 2.5 or 5.0 µL of artificial perilymph into the cochlea produced statistically significant high-frequency HL persisting 48 hours postperforation. Assessment of RWMs 48 hours after perforation revealed no inflammatory changes or residual scarring. FM 1-43 FX injection resulted in distribution of the agent predominantly in the basal and middle turns. CONCLUSION: Microneedle-mediated intracochlear delivery of small volumes relative to the volume of the scala tympani is feasible, safe, and does not cause HL in guinea pigs; however, injection of large volumes induces high-frequency HL. Injection of small volumes of a fluorescent agent across the RWM resulted in significant distribution within the basal turn, less distribution in the middle turn, and almost none in the apical turn. Microneedle-mediated intracochlear injection, along with our previously developed intracochlear aspiration, opens the pathway for precision inner ear medicine.

Reconstruction of transverse-longitudinal vibrations in the organ of Corti complex via optical coherence tomography.

Optical coherence tomography (OCT) is a common modality for measuring vibrations within the organ of Corti complex (OCC) in vivo. OCT's uniaxial nature leads to limitations that complicate the interpretation of data from cochlear mechanics experiments. The relationship between the optical axis (axis of motion measurement) and anatomically relevant axes in the cochlea varies across experiments, and generally is not known. This leads to characteristically different motion measurements taken from the same structure at different orientations. We present a method that can reconstruct two-dimensional (2-D) motion of intra-OCC structures in the cochlea's longitudinal-transverse plane. The method requires only a single, unmodified OCT system, and does not require any prior knowledge of precise structural locations or measurement angles. It uses the cochlea's traveling wave to register points between measurements taken at multiple viewing angles. We use this method to reconstruct 2-D motion at the outer hair cell/Deiters cell junction in the gerbil base, and show that reconstructed transverse motion resembles directly measured transverse motion, thus validating the method. The technique clarifies the interpretation of OCT measurements, enhancing their utility in probing the micromechanics of the cochlea.

Anatomic, physiologic, and proteomic consequences of repeated microneedle-mediated perforations of the round window membrane.

BACKGROUND: We have developed 3D-printed microneedle technology for diagnostic aspiration of perilymph and intracochlear delivery of therapeutic agents. Single microneedle-mediated round window membrane (RWM) perforation does not cause hearing loss, heals within 48-72 h, and yields sufficient perilymph for proteomic analysis. In this study, we investigate the anatomic, physiologic, and proteomic consequences of repeated microneedle-mediated perforations of the same RWM at different timepoints. METHODS: 100-µm-diameter hollow microneedles were fabricated using two-photon polymerization (2PP) lithography. The tympanic bullae of Hartley guinea pigs (n = 8) were opened with adequate exposure of the RWM. Distortion product otoacoustic emissions (DPOAE) and compound action potential (CAP) were recorded to assess hearing. The hollow microneedle was introduced into the bulla and the RWM was perforated; 1 µL of perilymph was aspirated from the cochlea over the course of 45 s. 72 h later, the above procedure was repeated with aspiration of an additional 1 µL of perilymph. 72 h after the second perforation, RWMs were harvested for confocal imaging. Perilymph proteomic analysis was completed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Two perforations and aspirations were performed in 8 guinea pigs. In six, CAP, DPOAE, and proteomic analysis were obtained; in one, only CAP and DPOAE results were obtained; and in one, only proteomics results were obtained. Hearing tests demonstrated mild hearing loss at 1-4 kHz and 28 kHz, most consistent with conductive hearing loss. Confocal microscopy demonstrated complete healing of all perforations with full reconstitution of the RWM. Perilymph proteomic analysis identified 1855 proteins across 14 samples. The inner ear protein cochlin was observed in all samples, indicating successful aspiration of perilymph. Non-adjusted paired t-tests with p < 0.01 revealed significant changes in 13 of 1855 identified proteins (0.7%) between the first and second aspirations. CONCLUSIONS: We demonstrate that repeated microneedle perforation of the RWM is feasible, allows for complete healing of the RWM, and minimally changes the proteomic expression profile. Thus, microneedle-mediated repeated aspirations in a single animal can be used to monitor the response to inner ear treatments over time.