Auditory Research publications

Objective: This study aims to establish an economically viable and easily accessible adult animal model for optogenetic activation of auditory neurons using adeno-associated viruses (AAVs) carrying ChR2(H134R) to explore the potential of cochlear optogenetics as a hearing restoration technology. Methods: Healthy adult guinea pigs were used in the experiments. The viral vector AAV2/8-ChR2(H134R)-hSyn-eYFP was administered to the right cochlea via the round window membrane. The confocal microscopy and reverse transcription polymerase chain reaction (RT-PCR) were utilized to analyze the ChR2(H134R) expression localized to spiral ganglion neurons (SGNs). The auditory pathway activation was assessed by recording the optical compound action potential (oCAP) and acoustic compound action potential (aCAP) at various laser intensities. Results: The ChR2(H134R)-eYFP expression was confirmed in 90% of the tested animals, localized to the SGNs of the injected ear. Higher mRNA levels of ChR2(H134R) and eYFP were observed in the injected ear compared to the non-injected ear, while actin (Actb) mRNA levels were not significantly different. The oCAP was successfully elicited by a 470 nm blue light laser stimulus, with similar amplitudes and latency periods to those of aCAPs when the oCAP was evoked by 5.80 mW blue light and the aCAP was evoked by a 40 dB SPL click. The amplitudes of oCAPs increased with increasing laser intensity. Conclusion: This study demonstrates the viability of optogenetic activation of the auditory system in adult guinea pigs through the transduction of AAV-ChR2(H134R) in SGNs. Cochlear optogenetics demonstrates potential as a hearing restoration technology, providing a basis for further clinical research and opening new avenues for investigation.

This study aimed to characterize maturation of the binaural intelligibility level difference (BILD) for school-age children in the context of speech-in-speech recognition. Children (5–17 years) and adults completed an adaptive, open-set sentence recognition task in a two-talker masker in two binaural conditions: (1) target and masker speech in-phase across the two ears, and (2) target speech presented 180° out-of-phase across the two ears and masker speech presented in-phase. Estimates of the BILD, computed as the difference score between the two binaural conditions, were mature by ten years of age, consistent with previous data on the BILD for speech-in-noise recognition.

Cross-modal responses are common in primary sensory cortex, yet a recent study showed that signals in primary visual cortex (V1) once labeled multisensory were instead more closely related to face movements. To what extent can multisensory processing in other modalities also be explained by face movements? We measured face movements and neural activity in awake mouse primary auditory cortex (A1) and V1 in response to visual and auditory stimulation as well as during spontaneous activity. We find that visual stimuli rarely evoke face movements, A1 responses to visual input remain robust even in the absence of face movement, and optogenetic silencing of V1 reduces A1 visual responses. Taken together, these results confirm that cross-modal responses in the auditory system are primarily sensory, not state driven, in origin.

Humans live in an environment that contains rich auditory stimuli, which must be processed efficiently. The entrainment of neural oscillations to acoustic inputs may support the processing of simple and complex sounds. However, the characteristics of this entrainment process have been shown to be inconsistent across species and experimental paradigms. It is imperative to establish whether neural activity in response to speech is a result of combination of simple evoked responses or of entrainment of neural oscillations in human participants. In this study, 12 participants with intracranial electrodes listened to natural speech and neural entrainment as evidenced by oscillatory activity persisting beyond the evoked responses was assessed. Neural activity was recorded from 165 contacts in Heschl's gyrus and superior temporal gyrus. First, acoustic edges in the speech envelope induced coherence between speech and auditory cortex activity. Further, entrainment in the theta-alpha band outlasted the acoustic stimulation. This activity exceeded what could be expected from a simple evoked response. These findings suggest that speech has the potential to entrain neural oscillations in the human auditory cortex.

There are two broad modes of information transfer in the brain: the labeled line model, where neurons relay inputs they receive, and the mixed tuning model, where neurons transform different inputs. In the visual pathway, information transfer between retinal ganglion cells (RGCs) and dorsal lateral geniculate nucleus (dLGN) neurons is viewed as a labeled line. However, recent work in mice demonstrated that different RGC types, encoding distinct visual features, converge onto a dLGN neuron, raising the question of how the dLGN transforms visual information. Using optogenetics, we activated distinct RGC populations and measured dLGN neuron spiking in vivo. We found that visual response properties of strongly driven dLGN neurons largely match properties of the activated RGC population. While in vitro dual-opsin experiments demonstrate that strong functional convergence from distinct RGC types does occur at modest frequencies, our data largely support a labeled line model of retinogeniculate information transfer in mice.

Introduction The posterior tail of the striatum receives dense inputs from sensory regions of cortex and thalamus, as well as midbrain dopaminergic innervation, providing a neural substrate for associative sensory learning. Previously, we have demonstrated that developmental hearing loss is associated with aberrant physiological states in striatal medium spiny neurons (MSNs). Methods Here we directly investigated auditory associative learning impairments in the striatum of adult Mongolian gerbils that underwent transient developmental hearing loss or sham hearing loss during the critical period of auditory development. We used electrophysiology to reveal significant changes to neuronal population responses in vivo and intrinsic and synaptic properties to medium spiny neurons in vitro as animals learned an appetitive “Go/No-Go” auditory discrimination task. For in vivo experiments a 64-channel electrode was implanted in the auditory region of the posterior tail of the striatum and neuronal recordings were carried out as animals learned the task. For in vitro experiments, corticostriatal slice preparations were made from animals on each day of training. Results In naïve animals from both groups there was limited to no phase locking to either auditory stimulus in vivo , and long term depression resulted from theta burst stimulation in vitro . Furthermore, intrinsic and synaptic properties in normal hearing animals were unaffected; however, the hearing loss group continued to show lowered synaptic inhibition, synaptic hyperexcitation, and suppressed intrinsic excitability in the hearing loss group. Starting around day 3–4 in both groups, the emergence of striatal medium spiny neuron phase locking to the auditory conditioning stimuli was observed in vivo . This occurred contemporaneous to an increased probability of theta burst induced LTP during MSN whole cell recording in vitro , and acquisition of the task as the correct rejection response significantly increased in the behaving animals. During the acquisition phase MSNs in the normal hearing group showed a significant decrease in synaptic inhibition and increase in synaptic excitation with no change to intrinsic excitability, while the MSNs in the hearing loss group showed a significant increase in synaptic inhibition, reduction of synaptic hyper excitability, and compensatory changes to intrinsic excitability that supported normal action potential generation. In both groups, synaptic properties were resolved to similar level of E/I balance that could be part of a conserved learning state. Discussion These changes to the intrinsic and synaptic properties likely support LTP induction in vivo and the strengthening of synapses between auditory inputs and MSNs that facilitate neuronal phase locking. These findings have significant implications for our understanding of striatal resilience to sensory impairments in early life, in addition to establishing a granular understanding of the striatal circuit changes that support reward driven stimulus–response learning.

Deep Brain Stimulation (DBS) of the superolateral medial forebrain bundle has shown promising long-term anti-depressant effects in treatment-resistant depression patients, although the mechanisms are not clear. The study explored medial forebrain bundle DBS mediated modulation of central noradrenaline transmission in a rodent depression model, the Flinders Sensitive Line (FSL), and in controls, Sprague Dawley (SD) rats. In vivo noradrenergic signaling in the prefrontal cortex (PFC) and nucleus accumbens (NAc) and ultrasonic vocalization were monitored during unilateral mfb-DBS across diverse stimulation parameters. The fiber amount, myelination status, and the activation of ascending projected noradrenergic cell groups (A1, A2, A6) were quantified. Moreover, stimulation induced changes in the parvalbumin-mediated feedforward microcircuitry and neuron activation at PFC and NAc were assessed. FSL rats showed decrease in NA fibers in mfb. Stimulation increased PFC noradrenergic signaling similarly across both groups compared to baseline, but in the NAc, the FSLs had notably higher signaling compared with SDs. FSLs demonstrated more positive affective ultrasonic vocalizations post-DBS than SDs. Brainstem nuclei A1 and A2 had similar noradrenergic neuron density across the experimental groups, and mfb DBS increased neuronal activation in both groups. FSLs had fewer noradrenergic neurons in the A6 nuclei, fewer unmyelinated noradrenergic fibers traversing the mfb, and decreased parvalbumin interneuron activity in both PFC and NAc. DBS normalized parvalbumin interneuron activity in the FSL rats. The study proposes that mfb DBS, via the modulation of the central NA system and the GABAergic inhibitory control of neural excitability, likely contributes to the anti-depressant therapeutic mechanisms reported in both clinical and experimental studies.

Mutations in MYO7A , the gene encoding the unconventional myosin 7a, cause hereditary deafness in mice and humans. In the cochlea, MYO7A is present in the sensory hair cells from embryonic stages of development, and plays a critical role in the development and maintenance of the mechanosensitive hair bundles composed of actin‐rich stereocilia. Shaker‐1 mutant mice ( Myo7a Sh1/Sh1 ), the murine model of Usher 1B syndrome, exhibit a progressive loss of the stereocilia, subsequent degeneration of the sensory epithelium and ultimately profound deafness. In addition to the hair bundle defects, we found that the shaker‐1 mutation prevented both inner hair cells (IHCs) and outer hair cells (OHCs) from acquiring their fully mature basolateral current profile. Delivering exogenous Myo7a to newborn Myo7a Sh1/Sh1 mice using dual‐adeno‐associated virus 8 (AAV8)‐ Myo7a or dual‐AAV9‐PhP.eB‐ Myo7a , which primarily target IHCs, led to a substantial rescue of their hair bundle structure. The rescued bundles regained their ability to generate mechanoelectrical transducer (MET) currents in response to fluid jet displacement. Although the average MET current was smaller than in control IHCs, the normal resting open probability of the MET channel was fully restored. The IHCs of the treated cochlea also regained a mature basolateral membrane current profile. Functionally, rescue of the IHC structure and function, but not that of OHCs, leads to an average improvement of 20–30 dB in hearing thresholds across most frequencies. These results support dual AAV‐induced gene replacement therapy as an effective strategy to recover hair‐cell function in Myo7a Sh1/Sh1 mice. image Key points Shaker‐1 mutant mice ( Myo7a Sh1/Sh1 ), which carry a mutation in the unconventional myosin MYO7A and are the murine model of Usher 1B syndrome, become profoundly deaf at 1 month of age or soon after. In the mammalian cochlea, MYO7A is expressed in the hair cells, including within their actin‐rich stereociliary bundles. We show that hair cells of Myo7a Sh1/Sh1 mice progressively lose their transducing stereocilia and mechanoelectrical transduction, and fail to acquire their fully mature basolateral current profile. Delivering exogenous Myo7a to newborn Myo7a Sh1/Sh1 mice using dual‐adeno‐associated virus (AAVs) led to a substantial rescue of the bundle structure and function of inner hair cells, including mechanoelectrical transduction. This functional rescue led to a 20–30 dB improvement in hearing thresholds across most frequencies. These results support dual AAV‐induced gene replacement therapy as an effective strategy to recover the hair‐cell function in Myo7a Sh1/Sh1 mice.

Layer 6 corticothalamic neurons (CTs) provide strong feedback input that is crucial to perception and cognition in normal and pathological states; however, the synaptic properties of this input remain largely unknown, especially in pathology. Here, we examined the synaptic properties of CT axon terminals in the medial geniculate body (MGB), the auditory thalamus, in normal hearing male and female mice and in a mouse model of noise-induced hearing loss (NIHL), also in male and female mice. In normal hearing mice, we found that the amplitude of CT-evoked excitatory postsynaptic current (EPSC) to the core-type ventral subdivision of the auditory thalamus (MGv), which mainly conveys rapid sensory information, is larger compared with the amplitude of CT-evoked EPSC to the matrix-type dorsal subdivision of the auditory thalamus (MGd), which likely conveys higher-order internal state information. This is due to higher axonal density and/or axonal recruitment in CT→MGv compared with CT→MGd synapses. After noise trauma, we observed enhanced short-term facilitation in CT→MGd but not CT→MGv synapses. Our findings reveal a previously unknown mechanism of short-term synaptic plasticity after NIHL via which CTs enhance the throughput of matrix-type thalamus, likely to improve perceptual recovery via higher-order contextual modulation.

Cisplatin is a widely used and effective anticancer drug. However, it causes permanent sensorineural hearing loss in over 50% of treated patients. There are no FDA-approved therapies to prevent cisplatin-induced hearing loss (CIHL) in adults, highlighting a critical unmet clinical need. Previous studies suggest that statins, commonly prescribed cholesterol-lowering drugs, are associated with reduced incidence and severity of cisplatin-induced hearing loss. Statins are primarily used to lower cholesterol, but they also exert several pleiotropic effects, making the mechanism(s) underlying this protection unclear. Here we examine whether reduced plasma cholesterol confers protection against CIHL independent of statin treatment. We utilized mice lacking serine protease proprotein convertase subtilisin/kexin type 9 ( Pcsk9 knockout (KO) mice) as a genetic model of reduced plasma cholesterol. We find that Pcsk9 KO mice are protected against cisplatin-induced hearing loss, as reflected by significantly lower ABR and DPOAE threshold shifts relative to wild type (WT) mice following treatment. Histological analyses confirmed preservation of cochlear outer hair cells (OHCs) in Pcsk9 KO mice treated with cisplatin, whereas WT mice showed significant OHC loss in the high-frequency cochlear regions. Finally, hearing loss positively correlated with baseline plasma cholesterol levels. Together our data demonstrate that systemic cholesterol reduction provides significant protection against CIHL, and they suggest that the protective effect of statins against CIHL is mediated by cholesterol reduction.

Speech comprehension, especially in the presence of background sounds, allegedly declines as a consequence of noise-induced hearing loss. However, the connection between noise overexposure and deteriorated speech-in-noise perception despite normal audiometric thresholds (hidden hearing loss) is not yet clear. This study investigates speech-in-noise discrimination in young-adult Mongolian gerbils before and after an acoustic trauma to examine the link between noise exposure and speech-in-noise perception. Nine young-adult gerbils were trained to discriminate a deviant consonant-vowel-consonant combination (CVC) or vowel-consonant-vowel combination (VCV) in a sequence of CVC or VCV standards, respectively. The logatomes were spoken by different speakers and masked by a steady-state speech-shaped noise. After the gerbils obtained the behavioral baseline data, they underwent an acoustic trauma and participated in the behavioral experiments again. Applying multidimensional scaling, response latencies were used to generate perceptual maps reflecting the gerbils’ internal representations of the sounds pre- and post-trauma. To evaluate how the discrimination of vowels and consonants was altered after noise exposure, changes in response latencies between phoneme pairs were investigated in relation to their articulatory features. Numbers of intact inner hair cell synapses were counted, and auditory brainstem responses were measured to assess peripheral auditory function. Perceptual maps of vowels and consonants were very similar before and after noise exposure. Interestingly, the gerbils’ overall vowel discrimination ability was improved after the acoustic trauma, even though the gerbils suffered from noise-induced hearing loss with a temporary threshold shift for frequencies above 4 kHz. In contrast, there were only minor changes in the gerbils’ consonant discrimination ability. Moreover, noise exposure showed a differential influence on response latencies for vowel and consonant discriminations depending on the articulatory features. Altogether, the results show that an acoustic trauma followed by a temporary threshold shift is not necessarily linked to speech-in-noise perception difficulties associated with hidden hearing loss.

Peripheral damage drives auditory cortex (ACtx) plasticity, but the underlying synaptic and cellular mechanisms remain poorly understood. We used a combination of in vitro slice electrophysiology, optogenetics, and in vivo two-photon imaging to investigate layer 5 extratelencephalic (ET) and layer 6 corticothalamic (CT) neuronal plasticity in mice, following noise-induced hearing loss (NIHL). Thalamocortical input was initially balanced between CTs and ETs but shifted to CT-dominant 1 day post-NIHL and then normalized by day 7. This transient shift was accompanied by increased quantal size and suprathreshold excitability in CTs, with minimal changes in ETs. In vivo, CTs exhibited persistent elevation in sound intensity thresholds, while ETs showed a transient shift in frequency tuning and reduced high-frequency responsiveness that recovered within a week. These findings reveal distinct, cell type–specific plasticity mechanisms in deep-layer ACtx neurons following peripheral damage and highlight potential targets for treating hearing loss–related disorders such as tinnitus and hyperacusis.

The neural representations of acoustic features that differ in the location or timbre of the emitter elicit similar perceptions, suggesting the existence of a robust stimulus‐response function between complex sounds and the activity of neural populations at all stages of the auditory system. This hypothesis is tested by decoding a random sound stream, using spike trains from a biophysical model of the auditory nerve and from large‐scale recordings in the inferior colliculus, the auditory thalamus, and the auditory cortex of awake mice. At the level of individual neurons, the reliability of temporal and rate codes is found to decrease along the ascending auditory pathways. Rate coding is progressively favored with increasing independence of neuron frequency tuning. Firing in the auditory cortex is found to be synergistic, whereas that in subcortical areas is more redundant. Finally, combinatorial codes involving neural firing and neural silence within neuron pairs are shown to efficiently encode sound information, particularly in the auditory cortex. Overall, these findings reveal a progressive transformation of the neural code from an individual, redundant, and temporal code at the periphery to a more distributed rate‐based code in the auditory cortex.

Genetic mutations cause hereditary deafness, in which mutations in the POU4 transcription factor 3 gene (POU4F3) lead to autosomal dominant non-syndromic deafness 15 (DFNA15), for which no effective clinical treatment currently exists. Gene editing holds promise for precisely repairing mutated nucleotides, thus offering a potential cure for hereditary hearing loss. Here, we establish a Pou4f3WT/Q113* mutant mouse model mimicking DFNA15. We develop and screen adenine base editors (ABEs) targeting the Pou4f3Q113* allele by fusing diverse adenine deaminases to Cas9 we discovered before. SchABE8e accomplishes highly precise and efficient editing (up to 48.5%) at sgRNA3 in vitro. Neonatal Pou4f3WT/Q113* mice are treated via synthetic AAV (Anc80L65)-delivered SchABE8e-sgRNA3, resulting in near-complete hearing recovery, with the effect persisting for at least four months. Biosafety analyses further support the feasibility of base editing, providing a therapeutic strategy for DFNA15.

We developed an extended reciprocity theorem approach to model neuron signals which handles heterogeneous dielectric environments and arbitrary electrode shapes, and use it to study analytically the single fiber action potential. We then established a semi-analytic model that also uses hybrid electromagnetic-electrophysiological simulations to model evoked compound action potential (eCAP) signals from multi-fascicular nerves populated with heterogeneous fiber populations. For validation, model predictions were compared with cuff electrode recordings of activity induced by vagus nerve stimulation in in vivo porcine experiments. The semi-analytic model produces signals that approximate the shape and amplitude of in vivo measurements. It can account for the important variation in the recorded eCAP due to changes in the shape and placement of the stimulus and recording electrodes. We find that partially activated fascicles contribute particularly to the signal, as eCAP contributions from smoothly varying fiber calibers in fully activated ones partially cancel. As a result, eCAP magnitude does not depend monotonically on the stimulation current and recruitment level. Our method can be used to rapidly assess new stimulation and recording setups involving complex nerves and neurovascular bundles, e.g., to maximize signal information content, for closed-loop control in bioelectronic medicine applications, and potentially to non-destructively reconstruct structural and functional nerve topologies through inverse problem solving. In a proof-of-concept study, we demonstrate that parameter optimization can recover the ground-truth distribution of fiber diameters in a simplifed variant of our model.

The locus coeruleus–norepinephrine (LC-NE) system regulates arousal and awakening; however, it remains unclear whether the LC does this in a global or circuit-specific manner. We hypothesized that sensory-evoked awakenings are predominantly regulated by specific LC-NE efferent pathways. Anatomical, physiological, and functional modularities of LC-NE pathways involving the mouse basal forebrain (BF) and pontine reticular nucleus (PRN) were tested. We found partial anatomical segregation between the LC → PRN and LC → BF circuits. Extracellular NE dynamics in BF and PRN exhibited distinct sound-evoked activation during sleep, including a fast sound-evoked NE peak specific to PRN. Causal optogenetic interrogation of LC efferent pathways, by retro-channelrhodopsin (ChR2) activation or Platynereis dumerilii ciliary opsin (PdCO) silencing of synapses in target regions, revealed a role for early LC → PRN activity in driving arousal and sound-evoked awakenings. Together, our results uncover a role for early LC-NE PRN activity in connecting sensory and arousal pathways and establish LC heterogeneity in regulating arousal.

Noise‐induced hearing loss (NIHL), caused by irreversible cochlear hair cell (HC) damage, lacks effective therapies due to a limited understanding of endogenous protective mechanisms. The echolocating bats exhibit natural resistance to intense noise, and this suggested novel insights into methods to protect against NIHL. Here, through comparative transcriptomic analysis of noise‐exposed cochleae from the eastern bent‐winged bats ( Miniopterus fuliginosus ) and mice, the specific transcriptional dynamics in noise‐resistant Miniopterus fuliginosus are revealed, thus highlighting potential mechanisms for preventing cochlear damage that mouse models cannot replicate, with Hras emerging as the most significant hub upregulator. Functional validation in mice demonstrates that HC‐specific Hras overexpression significantly attenuates noise‐induced HC death, synaptic loss, and auditory threshold shifts. Mechanistically, Hras confers protection by activating the PI3K/Akt signaling pathway, a critical pro‐survival cascade. The findings further disentangle the mechanisms of cochlear resistance to intense noise in echolocating bats and suggest that targeting Hras expression may be a potential therapeutic intervention against NIHL.

We investigated the auditory responses of anesthetized rhesus macaques to complex natural sounds using functional magnetic resonance imaging (fMRI) and a near-awake anesthesia protocol combining low-dose sevoflurane and dexmedetomidine. Eleven animals were scanned while listening to macaque vocalizations and non-vocal sounds. Robust activation was observed in primary and belt auditory cortices as well as subcortical structures, indicating preserved auditory responsiveness under anesthesia. However, unlike in awake animals, selective responses to vocalizations in anterior temporal voice areas (aTVAs) were absent. Instead, vocalization sensitivity emerged in the ventral premotor cortex, prefrontal cortex, and posterior middle temporal gyrus, regions associated with the dorsal auditory stream and multisensory integration. These findings suggest that while anesthesia preserves basic auditory processing, it modulates higher-order cortical responses involved in voice perception. Anesthetized fMRI thus offers a valuable tool for large-scale studies of primary auditory functions, while it must be approached with caution when studying higher-order auditory functions.

Early sensory experience can exert lasting perceptual consequences. For example, a brief period of auditory deprivation early in life can lead to persistent spatial hearing deficits. Some forms of hearing loss (i.e., conductive; CHL) can distort acoustical cues needed for spatial hearing, which depend on inputs from both ears. We hypothesize that asymmetric acoustic input during development disrupts auditory circuits that integrate binaural information. Here, we identify prolonged maturation of the binaural auditory brainstem in the guinea pig by tracking auditory evoked potentials across development. Using this age range, we induce a reversible unilateral CHL and ask whether behavioral and neural maturation are disrupted. We find that developmental CHL is associated with alterations in a brainstem readout of binaural function, an effect that was not observed in a separate cohort with adult-onset CHL. Startle-based behavioral measures suggest that Early CHL animals exhibit reduced spatial resolution for high-frequency sound sources. Finally, single-unit recordings of auditory midbrain neurons reveal significantly poorer neural acuity to a sound location cue that largely depends on high-frequency sounds. Thus, these findings show that unilateral deprivation can disrupt developing auditory circuits that integrate binaural information and may give rise to lingering spatial hearing deficits.

The treatment of inner and middle ear diseases remains a significant challenge, often requiring surgical intervention as the only option. In this study, we investigated porphysomes, self-assembled porphyrin-based nanoparticles, as a minimally invasive drug delivery platform for inner ear applications and as photothermal agents for cholesteatoma ablation. Three porphysome formulations were evaluated: parent porphysomes (PS), porphyrin-stabilized nanoemulsions (nPS), and EDTA-lipid incorporated porphysomes (ePS). Rats received intratympanic injections of each formulation, and fluorescence imaging performed 1 h postinjection demonstrated concentration-dependent inner ear penetration for all formulations. Notably, no signs of ototoxicity were observed based on histological and functional assessments of hearing and balance. Among ePS-treated rats, the 250 and 1,000 μM groups showed significantly higher inner ear fluorescence compared with the 50 μM group. Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) tests were performed at baseline and 2 and 6 weeks postinjection. No significant threshold shifts were detected in PS and ePS groups compared with controls. In contrast, nPS-treated rats exhibited a significant ABR threshold elevation at 6 weeks ( p < 0.02). At 6 weeks, minor yet statistically significant DPOAE threshold differences were observed at 16 kHz (PS group) and 32 kHz (all porphysome groups); however, all shifts remained below 0 dB, indicating no functional hearing loss. Vestibular assessments, including swim and beam tests, revealed no significant impairments. Upon laser activation, all porphysome formulations induced substantial temperature elevation (Δ T = 31 ± 3.76°C, p < 0.05) and histological burn effects in cholesteatoma tissues. These findings support the potential of porphysomes as a safe, minimally invasive drug delivery system and photothermal agent for the treatment of inner and middle ear disorders.