Citation: Morell, M.; Rojas, L.;Haulena, M.; Busse, B.; Siebert, U.;Shadwick, R.E.; Raverty, S.A.Selective Inner Hair Cell Loss in aNeonate Harbor Seal (Phoca vitulina).Animals 2022, 12, 180. https://doi.org/10.3390/ani12020180Academic Editor: Catarina EiraReceived: 22 December 2021Accepted: 7 January 2022Published: 12 January 2022Publisher’s Note: MDPI stays neutralwith regard to jurisdictional claims inpublished maps and institutional affil-iations.Copyright: © 2022 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).animalsArticleSelective Inner Hair Cell Loss in a Neonate Harbor Seal(Phoca vitulina)Maria Morell 1,2,* , Laura Rojas 3 , Martin Haulena 4, Björn Busse 5, Ursula Siebert 1 , Robert E. Shadwick 2and Stephen A. Raverty 2,61 Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover,Foundation, 25761 Büsum, Germany; ursula.siebert@tiho-hannover.de2 Zoology Department, The University of British Columbia, Vancouver, BC V6T 1Z4, Canada;shadwick@zoology.ubc.ca3 Faculty of Veterinary Medicine and Zootechnics, National Autonomous University of Mexico,Av. Universidad 3000, Delegación Coyoacán, Mexico City 04510, Mexico; laura.fmvz@gmail.com4 Vancouver Aquarium Marine Science Center, Vancouver, BC V6G 3E2, Canada; martin.haulena@ocean.org5 Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf,22529 Hamburg, Germany; b.busse@uke.de6 Animal Health Center, Ministry of Agriculture, Abbotsford, BC V3G 2M3, Canada; stephen.raverty@gov.bc.ca* Correspondence: maria.morell@tiho-hannover.deSimple Summary: Congenital hearing loss (i.e., hearing impairment present at birth) is recognized inhumans and other terrestrial species, but there is a lack of information on congenital malformationsand associated hearing loss in pinnipeds (seals, sea lions, and walruses). Baseline knowledge onmarine mammal inner ear malformations is essential to differentiate between congenital and acquiredabnormalities, which may be caused by infectious agents, age, or anthropogenic interactions, such asnoise exposure. Analysis of the cochlea of a neonate harbor seal (Phoca vitulina) revealed bilateralloss of inner hair cells (sensory cells responsible for transducing the auditory signal) while the outerhair cells (sensory cells responsible for sound amplification and frequency selectivity and sensitivity)were intact. The selective inner hair cell loss (up to 84.6% of loss) was more severe in the basal turn,where the high frequencies are encoded. Potential causes and consequences are discussed. This is thefirst report of a case of selective inner hair cell loss in a marine mammal neonate, likely congenital.Abstract: Congenital hearing loss is recognized in humans and other terrestrial species. However,there is a lack of information on its prevalence or pathophysiology in pinnipeds. It is importantto have baseline knowledge on marine mammal malformations in the inner ear, to differentiatebetween congenital and acquired abnormalities, which may be caused by infectious pathogens, age,or anthropogenic interactions, such as noise exposure. Ultrastructural evaluation of the cochlea of aneonate harbor seal (Phoca vitulina) by scanning electron microscopy revealed bilateral loss of innerhair cells with intact outer hair cells. The selective inner hair cell loss was more severe in the basalturn, where high-frequency sounds are encoded. The loss of inner hair cells started around 40% awayfrom the apex or tip of the spiral, reaching a maximum loss of 84.6% of hair cells at 80–85% of thelength from the apex. Potential etiologies and consequences are discussed. This is believed to be thefirst case report of selective inner hair cell loss in a marine mammal neonate, likely congenital.Keywords: congenital hearing loss; organ of Corti; marine mammals; pinnipeds; scanning electronmicroscopy; hair cell loss1. IntroductionProfound congenital hearing loss (i.e., hearing impairment present at birth) is presentin 1–3 children out of 1000 [1,2]. Around 50 to 60% of cases of congenital hearing lossare due to a genetic etiology, while the remainder may be attributed to environmentalAnimals 2022, 12, 180. https://doi.org/10.3390/ani12020180 https://www.mdpi.com/journal/animalsAnimals 2022, 12, 180 2 of 10factors, including noise exposure, ototoxic drug exposure, and protozoal, bacterial, or viralinfections [3,4]. Genetic mechanisms of congenital hearing loss are divided into syndromic(when hearing loss occurs along with a variety of other malformations) or non-syndromic(when hearing loss is the only apparent abnormality, which accounts for approximately 70%of cases of genetic-related hearing loss) [5,6]. In humans, half of all the non-genetic causes ofcongenital hearing loss are attributed to infectious pathogens, including Toxoplasma gondii,rubella, cytomegalovirus, herpes, and syphilis infections. Within these infectious agents,congenital cytomegalovirus is the most common cause of non-hereditary sensorineuralhearing loss in childhood [2].The organ of Corti (hearing organ) in mammals is formed by sensory cells that aretypically arranged in one row of inner hair cells (IHCs) and three parallel rows of outerhair cells (OHCs). While OHCs amplify the incoming signal and are responsible forfrequency sensitivity and selectivity, IHCs transduce the mechanical sound stimulationinto the release of neurotransmitters onto the afferent auditory nerve fibers that conductthe auditory information to the brainstem. In mammals, low frequencies are encoded inthe apex (apical region or tip of the spiral), and the high frequencies are encoded in thebase of the cochlea, closer to the stapes.Structural alterations can occur as a consequence of severe noise exposure, includingloss of entire hair cells, alterations in stereocilia, nuclei karyorrhexis and karyopycknosis,and degeneration of type I innervation, among others [7,8]. Following cochlear hair cellapoptosis, neighboring supporting cells initiate the elimination of the hair cell, leavinga distinct “scar.” This scarring process results in the simultaneous expansion of the sup-porting cells and sealing of the reticular lamina [9]. The presence of scars among hair cellrows is an important criterion that can be used to assess a possible history of noise-inducedhearing loss [10]. However, potential lesions due to noise exposure and other environ-mental factors in stranded marine mammals can be confused with hair cell loss due tocongenital malformations.Therefore, it is imperative to develop baseline information on the pathogenesis andprevalence of congenital hearing loss in marine mammals, to further differentiate amongcongenital or acquired lesions, such as infectious pathogens or anthropogenic interactionsassociated with noise overexposure. Clinical and pathologic examination of neonatesprovides the optimal information on congenital hearing loss since it is less likely that theyhave been exposed to any agent that might cause hair cell damage after birth.Congenital diseases previously reported in harbor seals include cleft palate, cleft lips,cardiac defects, hydronephrosis, hiatal hernia, scoliosis, arthrogryposis, lens triplication,macroglossia, anorectal malformation and vaginal artesia, brain or cranial malformations,dwarfism, intestinal atresia, and neuroglial heterotopia [11–17]. However, no descriptionsof inner ear congenital malformations in harbor seals or other marine mammals havebeen documented. Herein, we present the index case of a harbor seal neonate with innerear lesions.2. Materials and MethodsIn British Columbia, distressed or abandoned harbor seal pups are reported to theVancouver Aquarium Marine Mammal Rescue (MMR) or British Columbia Marine MammalStranding Response Network as part of the general protocol. Depending on the locationand resources, trained volunteers and experienced staff are mobilized to recover animalsand transport them for rehabilitation at the MMR. On arrival, animals are triaged andclinically assessed by either an Animal Health Technologist or Veterinarian with experiencein marine mammal health. After an initial evaluation, animals are placed individuallyin large totes and maintained on a milk formula herring-based diet for 3–4 weeks, thenweaned. The enclosures are cleaned, and the animals are clinically assessed daily.On 15 July 2014, a male harbor seal (PV 1475) was admitted to MMR with a history ofpossible maternal loss or abandonment. The animal was assessed and stabilized. On August9, the pup was observed to be quiet with a retracted third eyelid, and congested ulcers wereAnimals 2022, 12, 180 3 of 10noted in the oral cavity. Lip smacking was observed, and an antiemetic was administered.The condition of this animal deteriorated, and on August 10, bloody diarrhea with bloodynasal discharge and dyspnea with open mouth breathing were observed. The animal didnot improve after a course of Dexamethasone 5 [5 mg/mL, manufactured by Vetoquinol(Lavaltrie, QC, Canada), dosage administered 0.2 mg/kg], Ceftriaxone Sodium [100 mg/mLreconstituted, manufactured by Sandoz (Boucherville, QC, Canada), dosage administered20 mg/kg], ceftiofur crystalline free acid Excede [200 mg/mL, manufactured by Zoetis(Kirkland, QC, Canada), dosage administered 7 mg/kg] and benzylpenicillin procaine andbenzylpenicillin benzathine suspension Duplocillin LA [dosage administered 1.0 mL ofpenicillin solution by intramuscular injection, i.e., 150,000 iu of each/mL, manufacturedby Merck (Kirkland, QC, Canada)]. Due to a poor prognosis, the pup was humanelyeuthanized and presented for necropsy. The animal was approximately 1 month of agewhen it died, with a weight of 9.0 kg and a total length of 58 cm.An extensive post-mortem examination was conducted following international proto-cols [18,19] at the Animal Health Center, Abbotsford, British Columbia.2.1. Inner Ear AnalysisThe head was removed, and the inner ears were collected at the University of BritishColumbia (UBC) within 4.25 h post-mortem. The skull was opened with a hand saw toextract the brain, and the occipital bone was removed with a chisel T-Shape (Virchowskull breaker) post-mortem from the occipitomastoid suture (Figure 1a). The ear bones(periotic and tympanic) were separated and extracted from the squamosal bone using achisel T-shape (Figure 1b), and the inner ears were perfused perilymphatically with 2.5%glutaraldehyde in 0.1M cacodylate buffer (Figure 1c), changed the media into 0.1M cacody-late buffer the following day, and subsequently processed for ultrastructural evaluation,following a previously optimized protocol for marine mammals [10,20–22].Animals 2022, 12, 180  3  of  10  perience in marine mammal health. After an initial evaluation, animals are placed indi‐vidually in large totes and maintained on a milk formula herring‐based diet for 3–4 weeks, then weaned. The enclosures are cleaned, and the animals are clinically assessed daily. On 15 July 2014, a male harbor seal (PV 1475) was admitted to MMR with a history of possible maternal loss or abandonment. The animal was assessed and stabilized. On August 9, the pup was observed to be quiet with a retracted third eyelid, and congested ulcers were noted in the oral cavity. Lip smacking was observed, and an antiemetic was administered. The condition of this animal deteriorated, and on August 10, bloody diarrhea with bloody nasal discharge and dyspnea with open mouth breathing were observed. The animal did not im‐prove after a course of Dexamethasone 5 [5 mg/mL, manufactured by Vetoquinol (Lavaltrie, QC, Canada), dosage administered 0.2 mg/kg], Ceftriaxone Sodium  [100 mg/mL  reconsti‐tuted, manufactured by Sandoz (Boucherville, QC, Can da), dosage administer d 20 mg/kg], ceftiofur crystalline  free acid Excede  [200 mg/mL, manufactured by Zoetis  (Kirkland, QC, Canada), dosage administered 7 mg/kg] and benzylpenicillin procaine and benzylpenicillin benzathine suspension Duplocillin LA [dosage administered 1.0 mL of penicillin solution by intramuscular injection, i.e., 150,000 iu of ea /mL, manufacture  by Merck (Kirkland, QC, Canada)]. Due to a poor prognosis, the pup was humanely euthanized and presented for nec‐ropsy. The animal was approximately 1 month of age when it died, with a weight of 9.0 kg and a total length of 58 cm. An extensive post‐mortem examinati n was conducted following inter ational pro‐tocols [18,19] at the Animal Health Center, Abbotsford, British Columbia. 2.1. Inner Ear Analysis The head was removed, and the inner ears were collected at the University of British Columbia (UBC) within 4.25 h post‐mortem. The skull was opened with a hand saw to extract the brain, and the occipital bone was removed with a chisel T‐Shape (Virchow skull breaker) post‐mortem from the occipitomastoid suture (Figure 1a). The ear bones (periotic and tympanic) were separated and  xtracted from the squamosal bone using a chisel T‐shape (Figure 1b), and the inn r e rs were p rfus  perilymph tically with 2.5% glutaral‐dehyde  in 0.1M cacodylate buffer (Figure 1c), changed the media  into 0.1M cacodylate buffer the following day, and subsequently processed for ultrastructural evaluation, fol‐lowing a previously optimized protocol for  arine ma mals [10,20–22].  Figure 1. (a) Skull after the extraction of the brain with the location of the ears (asterisks). The dotted line indicates the position of the occipitomastoid suture, where the chisel is placed to remove the occipital bone. (b) Separation of the periotic from the tympanic bone by first placing the chisel in the location highlighted with the double arrow, and collection of the periotic bone by sectioning the squamosal bone through the dotted line. (c) The final step of the perilymphatic perfusion through the oval window with fixative, after extracting the stapes and perforating the round and oval win‐dow membranes with a small needle. 2.2. Scanning Electron Microscopy (SEM) The periotic bones (surrounding the cochlea) were decalcified with 14% Ethylenedi‐aminetetraacetic acid (EDTA) tetrasodium salt, changing the media every 7–15 days for 47 and 192 days (right and left ears, respectively). Both cochleae were dissected to remove Figure 1. (a) Skull after the extraction of the brain with the location of the ears (asterisks). The dottedline indicates the position of the occi ito astoid sut re, where the chis l is placed to remove theoccipital bone. (b) Separation of the iotic from the tympanic bone y first placing the hisel inthe location hi lighted with the do l ar ow, and collection of the periotic bone by sectioning thesquamosal bone through the dotted line. (c) The final step of the perilymphatic perfusion through theoval window with fixative, after extracting the stapes and perforating the round and oval windowmembranes with a small needle.2.2. Scanning Electron Microscopy (SEM)The periotic bones (surrounding the cochlea) were decalcified with 14% Ethylene-diaminetetraacetic acid (EDTA) tetrasodium salt, changing the media every 7–15 daysfor 47 and 192 days (right and left ears, respectively). Both cochleae were dissected toremove the bone, vestibular wall, Reissner and tectorial membranes, and dehydrated withincreasing concentrations of ethanol.The right cochlea was critical point dried (Supercritical Autosamdri 815B, Tousimis),coated with platinum/palladium, and imaged with a Hitachi S-4700 SEM at the UBCAnimals 2022, 12, 180 4 of 10Bioimaging Facility, Canada. The left cochlea was critical point dried (Bal-Tec CPD030),coated with gold, and imaged with a Zeiss Crossbeam 340 FIB-SEM at the UniversityMedical Center Hamburg-Eppendorf, Germany. The brightness and contrast of imageswere adjusted in Adobe (San Jose, CA, USA) Photoshop® 2021.2.3. Characterization of the LesionsThe cochlear length was measured with ImageJ® software (https://imagej.nih.gov/ij/index.html accessed on 19 April 2021) from SEM micrographs at the level of the limitbetween the first row of OHCs and the inner pillar cells. A total of 33 micrographswere used for the calculation of the cochlear spiral from the left ear. Specific pointson each image were identified to achieve an accurate consecutive delineation of length.Contiguous measurements avoided overlapping or gaps in the calculation of the structure(see Girdlestone and colleagues [23]).Once the cochlear length was measured, it was possible to identify equidistant loca-tions every 5% along the cochlear spiral. Due to damage in the end of the base or hookregion, the locations were evaluated up to 88.6% from the apex. Counting of IHCs wasperformed at 5% increments to determine the number of IHCs present and absent. Inaddition, to confirm the number of absent IHCs at each location, the length of the cuticularplate of the IHCs was measured and averaged every 10% of the cochlear spiral. To illustratethe results, location 5% represents the counting of IHCs from 0 to 5%, location 10% from5.01 to 10%, onwards.3. Results3.1. Post-Mortem ExaminationThe animal presented for necropsy in moderate body and good post mortem condition.Morphologic diagnoses included marked bronchopneumonia, with transmural vasculitisand atelectasis, multifocal necrotizing adrenocortical adenitis with intralesional inclusionsconsistent with phocid herpesvirus infection, hepatocellular hemosiderosis, splenic ex-tramedullary hematopoiesis, and renal congestion. No bacteria were recovered from thelung, but light Pseudomonas aeruginosa was cultured from the spleen, with moderate mixedgrowth of Staphylococcus sp., Corynebacterium sp., Psychrobacter sp., Escherichia coli, andEnterococcus sp. isolated from a lymph node. Based on the nature of the bacterial isolatesand histopathology (vasculitis and pneumonia), the Pseudomonas aeruginosa was consideredsignificant. The lack of more significant growth from the lung was attributed to ante-mortem antimicrobial administration. Molecular studies of pooled tissues (striated muscle,diaphragm, heart, and liver) [24] did not detect Apicomplexa, including T. gondii.3.2. Inner Ear AnalysisUltrastructural evaluation of the organ of Corti revealed selective IHC loss throughoutthe cochlear spiral (Figure 2). While the OHCs were present forming three and oftenscattered four rows (Figure 2c), there was a loss of IHCs, which was more severe towardsthe base of the cochlea. The loss of IHCs was determined by the detection of scars, resultingfrom the overgrowth of adjoining, supporting cells (orange arrows in Figure 2).The left cochlea was better dissected and exposed than the right. The cochlear lengthwas 27.19 mm. In the left ear, because the basilar membrane was artefactually folded, thesensory cells of the organ of Corti from the hook region (88.6% to 99% from the apex) couldnot be assessed. However, the rest of the cochlea was well preserved, with some signsof post-mortem decomposition due to delay between the death of the individual and thefixation of the inner ear. The number of IHCs present and absent were counted every 5% ofthe cochlear length. There was little loss of IHCs in the apical region, up to 35% from theapex, and an increasing trend of IHC loss towards the base of up to 84.6% loss of IHCs at80 to 85% of the apex (Figure 3). The exposed areas of the hook region featured a similarpattern of IHC loss. However, in the first 50 µm of the hook, IHCs were present, with thethree first IHCs arranged in two rows (Figure 2e).Animals 2022, 12, 180 5 of 10Animals 2022, 12, 180  5  of  10  5% of the cochlear length. There was little loss of IHCs in the apical region, up to 35% from the apex, and an increasing trend of IHC loss towards the base of up to 84.6% loss of IHCs at 80 to 85% of the apex (Figure 3). The exposed areas of the hook region featured a similar pattern of IHC loss. However, in the first 50 μm of the hook, IHCs were present, with the three first IHCs arranged in two rows (Figure 2e).  Figure 2. Scanning electron microscopy images of the organ of Corti of the left ear along the cochlear spiral, at 30% (a), 45% (b), 65% (c), and 80% (d) distances from the apex. Note that while the outer hair cells (OHCs) are present forming three (and sometimes four) rows, there is a loss of inner hair cells (IHCs, highlighted with orange arrows), with  increasing severity towards the base. (e) First IHCs of the hook. The three first IHCs are arranged in two rows. The undersurface of the tectorial membrane shows the imprints where the stereocilia of OHCs are inserted. The right cochlea was well preserved, especially in the region of the apical and mid‐dle turns. However, there was a dissection and processing artifact that hampered the ul‐trastructural evaluation of the reticular lamina of the sensory epithelium in the majority Figure 2. Scanning electron icroscopy i ages of the organ of Corti of the left ear along the cochlearspiral, at 30% (a), 45% (b), 65% (c), and 80% (d) distances from the apex. Note that while the outerhair cells (OHCs) are present forming three (and sometimes four) rows, there is a loss of inner haircells (IHCs, highlighted with orange arrows), with increasing severity towards the base. (e) FirstIHCs of the hook. The three first IHCs are arranged in two rows. The undersurface of the tectorialmembrane shows the imprints where the stereocilia of OHCs are inserted.The right cochlea was well preserved, especially in the region of the apical andmiddle turns. However, there was a dissection and processing artifact that hampered theultrastructural evaluation of the reticular lamina of the sensory epithelium in the majorityof the basal turn. In those locations where the organ of Corti was visible, there was acomparable distribution of IHCs loss as in the left cochlea, while the OHCs appearedmorphologically intact. However, as the regions of the base where the sensory cells werevisible were limited, there was insufficient exposure of the right ear to confirm comparableseverity in the bilateral loss of IHCs.Animals 2022, 12, 180 6 of 10Animals 2022, 12, 180  6  of  10  of the basal turn. In those locations where the organ of Corti was visible, there was a com‐parable distribution of IHCs loss as in the left cochlea, while the OHCs appeared morpho‐logically intact. However, as the regions of the base where the sensory cells were visible were limited, there was insufficient exposure of the right ear to confirm comparable se‐verity in the bilateral loss of IHCs.  Figure 3. Loss of inner hair cells along the left cochlear spiral, represented in percentage from the apex. The number of inner hair cells was calculated for each 5% increment of the cochlear length. 4. Discussion Cochlear ultrastructural analysis of a neonate harbor seal showed an extremely rare pattern of selective loss of IHCs, which was particularly severe in the basal turn (Figures 2 and 3). Since this individual was very young, probably around one month old, it is likely that the IHC loss was congenital. In most cases of sensorineural hearing loss in terrestrial mammals, either due to noise exposure [25], ototoxic drugs exposure [26], or genetic anomalies, the loss of IHCs is asso‐ciated with significant or complete loss of OHCs. Thus, OHCs tend to be the most vulner‐able elements in the inner ear, and selective loss of IHCs is highly uncommon. Conversely, systemic administration of  the anti‐neoplastic drug carboplatin  in  the chinchilla, damages IHCs, leaving the OHCs morphologically intact [27]. In other species (e.g., guinea pig), carboplatin causes loss of both IHCs and OHCs [28]. Review of medical records for this seal confirmed that no carboplatin or other ototoxic drugs were adminis‐trated. Many cases of congenital hearing  loss are due  to viral  infections during different stages of  fetal development  (see review by Karimi‐Boroujeni and colleagues  [29]). Alt‐hough congenital cytomegalovirus is the leading non‐genetic cause of sensorineural hear‐ing loss in children [30–32], herpes simplex virus and rubella virus infections are also de‐tected  [29]. Loss of OHCs was described  in an  infant with congenital cytomegalovirus infection [33]. In addition, loss of IHCs and OHCs was observed in murine cytomegalovi‐rus‐infected mice, despite spiral ganglion cells and perilymphatic epithelial cells, but not hair cells, were sites of viral infection [34]. Schachtele and colleagues suggested that OHCs were more susceptible than IHCs to murine cytomegalovirus infection. Similarly, guinea pigs inoculated with herpes simplex virus showed a marked loss of OHCs, while changes in the IHCs were less apparent [35]. In humans, congenital rubella virus infection leads to bilateral sensorineural hearing loss through apoptosis in the stria vascularis and the organ .4. DiscussionCochlear ultrastructural analysis of a neonate harbor seal showed an extremelyrare pattern of selective loss of IHCs, which was particularly severe in the basal turn(Figures 2 and 3). Since this individual was very young, probably around one month old, itis likely that the IHC loss was congenital.In most cases of sensorineural hearing loss in terrestrial mammals, either due tonoise exposure [25], ototoxic drugs exposure [26], or genetic anomalies, the loss of IHCsis associated with significant or complete loss of OHCs. Thus, OHCs tend to be the mostvulnerable elements in the inner ear, and selective loss of IHCs is highly uncommon.Conversely, systemic administration of the anti-neoplastic drug carboplatin in the chin-chilla, damages IHCs, leaving the OHCs morphologically intact [27]. In other species (e.g.,guinea pig), carboplatin causes loss of both IHCs and OHCs [28]. Review of medical recordsfor this seal confirmed that no carboplatin or other ototoxic drugs were administrated.Many cases of congenital hearing loss are due to viral infections during differentstages of fetal development (see review by Karimi-Boroujeni and colleagues [29]). Althoughcongenital cytomegalovirus is the leading non-genetic cause of sensorineural hearing lossin children [30–32], herpes simplex virus and rubella virus infections are also detected [29].Loss of OHCs was described in an infant with congenital cytomegalovirus infection [33].In addition, loss of IHCs and OHCs was observed in murine cytomegalovirus-infectedmice, despite spiral ganglion cells and perilymphatic epithelial cells, but not hair cells,were sites of viral infection [34]. Schachtele and colleagues suggested that OHCs weremore susceptible than IHCs to murine cytomegalovirus infection. Similarly, guinea pigsinoculated with herpes simplex virus showed a marked loss of OHCs, while changes inthe IHCs were less apparent [35]. In humans, congenital rubella virus infection leads tobilateral sensorineural hearing loss through apoptosis in the stria vascularis and the organof Corti [36], but both IHCs and OHCs were degenerated [37]. Cytomegalovirus and herpessimplex virus belong to the family Herpesviridae. Since this seal pup had lesions consistentwith phocid herpesvirus infection, we considered the possibility that the ultrastructuralfeatures found in the inner ear of this individual were correlated with herpesvirus infection.However, selective IHC loss was not previously described with a congenital viral infection,making a viral etiology unlikely.Selective IHC loss was reported in the Bronx waltzer mutant mice (bv/bv), wherethe IHCs were either absent or abnormally haired, but the OHCs appeared normal [38].Animals 2022, 12, 180 7 of 10Selective IHC loss was also shown in a mutant mouse with targeted deletion of high-affinitythiamine transporter gene SLC19A2 [39]. However, the IHC loss was observed throughoutthe cochlear spiral in the Bronx waltzer mutant mice [38], or more severe in the upperbasal than the apical turn [40], and starting in the apical turn of the cochlea in the thiamineSlc19a2-null mice [39], but not specifically or with higher severity in the base of the cochlea.The phenotypes resulting from the two mutations reported in mice are not consistent withthe pathological pattern found in this harbor seal.Connexin 26 mutation (encoded by the GJB2 gene) is considered the most commoncause for non-syndromic hereditary deafness [41,42]. In this disorder, the OHCs were morevulnerable than IHCs [43]. Therefore, it is unlikely that the pathogenesis observed in ourindividual might be due to the mutation of Connexin 26, one of the most common birthdefects in humans.Auditory neuropathy spectrum disorder refers to several hearing dysfunctions char-acterized by compromised signal processing along the auditory nerve or by deficienttransmission of this signal to the auditory nerve by the presynaptic IHCs with normalfunction of OHCs (see review by De Siati and colleagues [44]). There is a wide rangeof localization of anatomic sites of impaired function, ranging from the region of IHCssynapses to the auditory neural fibers [45,46]. “Auditory synaptopathy” is the term usedfor auditory neuropathy spectrum disorder due to a defective or poorly functioning IHCsribbon synapse, and the term “auditory neuropathy” when they are due to the dysfunctionof neural fibers [46,47]. Cases of selective IHC loss were also reported nine-fold higherin premature infants in comparison to full-term infants [48]. Amatuzzi and colleaguesproposed that a common cause of non-genetic auditory neuropathy spectrum disorder(called “auditory neuropathy” by the authors) can be a selective loss of IHCs rather thanprimary damage to the cochlear nerve.On initial presentation to the MMR, this harbor seal neonate was not deemed prema-ture. In addition, since the dissection of the cochlea prepared for SEM was optimized toimage the sensory cells of the organ of Corti, it was not possible to evaluate if there wasa degeneration of type I afferent neurons, precluding evaluation for potential auditoryneuropathy spectrum disorder.In summary, based on the review of human and laboratory studies, there are noapparent precedents that may account for the pattern of IHC loss observed in the harborseal of our study. However, it is possible that harbor seals have unique hereditary diseases,distinct to humans and reported rodent models.Since the disposition of the first sensory cells in the extreme hook in mammals can bevariable [49], the finding of the three first IHCs disposed of in two rows (Figure 2e) in thisharbor seal is documented but possibly not relevant.Losses of up to 70% of IHCs and 50% of cochlear neurons were associated with amoderate elevation of hearing threshold (by an average of 20 dB SPL) in Bronx waltzermutant mice [40]. As a result, for sufficient threshold hearing, not all IHCs might berequired, particularly if the OHCs are present and functional [40]. In addition, carboplatin-induced IHC loss (ranging from 40 to 80%) in chinchillas had little effect on thresholds inquiet surroundings, but thresholds increased significantly when tested in the presence ofbroadband or narrowband noise [50]. Consequently, IHC loss or dysfunction may play asignificant role in hearing-in-noise independent of OHC integrity, and these deficits maybe present even when thresholds in quiet are within normal limits. Despite the harbor sealhad other severe pathologies, it is likely that the lesions in the organ of Corti would havealso caused difficulties for its survival.This study highlights the importance to have baseline knowledge on “natural” con-genital malformation of the hearing apparatus of marine mammals to be able to furtherdifferentiate from potential damage caused by exposure to factors (including noise) thatthe individuals might encounter during their lifetime.Animals 2022, 12, 180 8 of 105. ConclusionsThis is the first study to report a case of selective IHC loss in a neonate marine mammal,likely congenital.Author Contributions: Conceptualization, M.M.; validation, U.S., S.A.R.; formal analysis, M.M., L.R.and S.A.R.; investigation, M.M., L.R. and S.A.R.; methodology: M.M., resources, M.H., B.B.; datacuration, M.M. and L.R.; writing—original draft preparation, M.M., L.R., M.H., B.B., U.S., R.E.S. andS.A.R.; writing–review and editing, M.M., L.R., M.H., B.B., U.S., R.E.S. and S.A.R.; visualization,M.M. and L.R.; supervision, U.S., R.E.S. and S.A.R.; project administration, U.S. and R.E.S.; fundingacquisition, M.M., U.S. and R.E.S. All authors have read and agreed to the published version ofthe manuscript.Funding: This study was funded by the Natural Sciences and Engineering Research Council (NSERC)of Canada Discovery and Accelerator grants RGPAS 446012-13 and RGPAN 312039-13 and as part ofthe SATURN project, which has received funding from the European Union’s Horizon 2020 researchand innovation programme under grant agreement No 101006443. This publication was supportedby Deutsche Forschungsgemeinschaft and University of Veterinary Medicine Hannover, Foundation,within the funding program Open Access Publishing.Institutional Review Board Statement: Ethical or animal welfare approval for the research were notrequired since the study was performed post mortem on a harbor seal, humanely euthanized as partof the veterinary care standard procedure at our Marine Mammal Rescue Centre.Informed Consent Statement: Not applicable.Data Availability Statement: The original contributions presented in the study are included in thearticle. Further inquiries can be directed to the corresponding author.Acknowledgments: We would like to thank the personnel of the Vancouver Aquarium MarineMammal Rescue who took care of this individual, the personnel of the Animal Health Center whowere involved in the necropsy and subsequent post-mortem examinations, and Derrick Horne(UBC Bioimaging Facility) and Imke Fiedler (University Medical Center Hamburg-Eppendorf) fortechnical assistance.Conflicts of Interest: The authors declare no conflict of interest.References1. Smith, R.; Bale, J.; White, K. Sensorineural hearing loss in children. Lancet 2005, 365, 879–890. [CrossRef]2. Belcher, R.; Virgin, F.; Duis, J.; Wootten, C. Genetic and non-genetic workup for pediatric congenital hearing loss. Front. Pediatr.2021, 9, 536730. [CrossRef]3. Morton, N.E. Genetic epidemiology of hearing impairment. Ann. N. Y. Acad. Sci. USA 1991, 630, 16–31. [CrossRef]4. Raymond, M.; Walker, E.; Dave, I.; Dedhia, K. 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