Physiologically the middle ear, containing the three ossicles, serves primarily to

Outer Ear

The outer ear is the portion of the ear that can be seen by casual inspection. It consists of the pinna (what we generally call the ‘ear’), which is attached to a bowl-shaped structure called the concha. The concha ends at the ear canal, most correctly called the external auditory meatus. The ear canal ends at the eardrum (the tympanic membrane), which serves as the boundary between the outer and middle ears. The outer ear amplifies the level of some sounds due to its physical properties. Having two ears (one on each side of the head) means that sounds arriving at the ears may differ in level or timing – cues used by the brain to determine where the sound is coming from.

Outer and Middle Ear

The outer ear, including the pinna, concha, and the ear canal to the ear drum, does not undergo any age-related change that significantly affects auditory perception in everyday life. Two outer ear conditions that may affect hearing testing are cerumen build up and collapsing ear canals. Build up of cerumen, or ear wax, may impede the perception of sound and possibly cause tinnitus, or ringing in the ears; however, cerumen can be safely removed by a qualified health professional. The cartilage of the outer ear loses rigidity, with the consequence that the ear canal may collapse if pressure is applied to the pinna. Collapsing canals occur in about a third of older adults. The occlusion produced when the ear canal collapses impedes the transmission of high-frequency sounds such that thresholds measured using circum-aural earphones may over-estimate the degree of hearing loss. This testing artifact can be avoided by using insert earphones. A tester can easily identify a collapsing ear canal by pressing on the pinna with a finger and observing a change in the shape of the opening of the ear canal.

The middle ear cavity, from the ear drum to the inner ear, is normally air-filled and contains a chain of three ossicles. The middle ear structures transduce the air-borne acoustical sound vibrations arriving at the ear drum into mechanical vibrations that are relayed to the fluid-filled inner ear. Some older adults have hearing loss resulting from middle ear damage that developed earlier in their lifetime; however, the middle ear does not undergo any changes specifically related to age that significantly affect auditory perception in everyday life or during hearing testing.

Abstract

The mammalian outer, middle, and inner ears have different embryonic origins and evolved at different times in the vertebrate lineage. The outer ear is derived from first and second branchial arch ectoderm and mesoderm, the middle ear ossicles are derived from neural crest mesenchymal cells that invade the first and second branchial arches, whereas the inner ear and its associated vestibule-acoustic (VIIIth) ganglion are derived from the otic placode. In this chapter, we discuss recent findings in the development of these structures and describe the contributions of members of a Forkhead transcription factor family, the Foxi family to their formation. Foxi transcription factors are critical for formation of the otic placode, survival of the branchial arch neural crest, and developmental remodeling of the branchial arch ectoderm.

External Ear

At 6 weeks the outer ears start developing from folds on the front neck area of the embryo. A set of six auricular hillocks or bumps arises for each ear. These hillocks are visible as early features of the adult ear. The ear canal forms at 5 weeks, and at about 12 weeks a plate forms at the inner end of the canal. This plate remains until about 7 months, when it dissolves and the remaining tissue forms the tympanic membrane or eardrum. The outer ear and ear canal continue to grow longer after birth, reaching adult sizeat about 9 years. Failure of the outer ear to develop at all is called anotia, limited growth is called microtia, while partial or complete closure of the ear canal is called atresia.

Timing and Position of Otic NCC Emigration

The middle and outer ear develop from fusion and remodeling of the first and second pharyngeal arches, which are filled with NCC, while the inner ear arises from the otic vesicle located initially directly posterior to the dorsal portion of the second arch NCC stream. Study of middle ear ossicle development predates the identification of NCC. The formation of the middle ear ossicles from the first and second pharyngeal arches was described initially by Carl Reichert in his seminal comparative anatomical study of pig, bird, and frog nearly two centuries ago [1]. The outer ear pinna and ear canal also derive from the first and second pharyngeal arches and the cleft between them, an ontogeny defined by morphological analysis of a variety of vertebrate embryos, including human [2,3].

The first and second pharyngeal arches that are developmentally remodeled to form the middle and outer ear are filled with NCC that migrate from defined axial levels of the midbrain and hindbrain. The organization and origin of NCC migrating to the arches were defined based on classic lineage tracing experiments, initially in amphibian and subsequently avian and cultured mouse embryos [4–9]. In chick and mouse, the mesenchyme of the first pharyngeal arch (mandibular arch) is populated by NCC that emerge from the posterior midbrain and from the anterior hindbrain at the level of rhombomere 1 and 2 (R1 and R2). First arch NCC contributing to ear development are primarily those located in the posterior portion of the arch, originating from R1–R2. The second pharyngeal arch (hyoid arch) is populated by NCC that emigrate from the neural tube primarily at the level of R4, with a very minor contribution from R3 and R5. Thus, the middle ear and outer ear, which are known to develop by remodeling of the first and second pharyngeal arches, derive from NCC that fill those arches—namely, NCC originating from a region of the neural tube spanning from the posterior midbrain to R4.

It is well accepted that the middle and outer ear develop from the first and second pharyngeal arches and that NCC populate those arches. Less noted is the contribution of NCC to the inner ear, which is often described as arising entirely from the otic vesicle. The otic vesicle forms directly adjacent to the proximal portion of the second arch NCC stream and NCC contribute several cell types to the developing inner ear. Lineage tracing studies of avian embryos demonstrate that glial support Schwann cells of the cochleovestibular nerve are derived from NCC emigrating from the rostral myelencephalon [10], presumably the region corresponding to R4. Quail-chick chimeric analysis indicates a portion of the bony capsule that surrounds the membranous labyrinth of the inner ear is derived from NCC, and transgenic lineage tracing in mice demonstrates that that these NCC originate from R4 [7,11]. With respect to inner ear NCC derivatives, arguably the most important is a population of melanocytes of the stria vascularis in the cochlea, which are essential for inner ear function. The axial origin of the inner ear melanocyte progenitor NCC has yet to be defined.

The timing of emigration of otic NCC has been determined by lineage tracing studies primarily in chick and mouse. In chick, tracing of NCC derivatives by timed radiolabeling or by quail-chick chimeric transplants indicates that the first and second arch NCC streams emerge from the neural tube between stages 9 and 11 (7 somites to 13 somites) [12]. In mouse, NCC emerge from the neural tube to enter the first arch stream as early as the 1–2 somite stage and continue through the 13 somite stage, that is, from embryonic day 8.0 (E8.0) through E9.0 [5,13]. This interval corresponds to Carnegie stage 9–10, or week 3–4 of human embryo development (clinical gestational age 5–6 weeks from last ovulation). The stages of emergence and early migration of otic NCC precede morphological formation of the first and second pharyngeal arches, and also precede morphological formation of the otic placodes.

The timing and migratory route of the NCC melanocyte progenitors populating the cochlear stria vascularis are not known. The migration characteristics have, however, been established for NCC melanoblast progenitors of the dermis of the trunk. In chick and mouse, some of the NCC melanoblast progenitors destined to form skin melanocytes of the body migrate along a dorsolateral pathway [14]. Other trunk dermis melanocytes arise from Schwann cell NCC progenitors, which migrate along a more ventral route [15]. The dorsolateral migrating trunk melanoblasts emerge approximately 24 h later than the corresponding ventrally migrating NCC that give rise to peripheral nerves [16]. In the head region of the mouse, dorsal migrating dermal melanoblast NCC are observed at the 25–26 somite stage [17]. Based on the migration characteristics of dermal trunk melanocytes, it may be speculated that the melanocytes of the inner ear may arise from NCC migrating with the cochleovestibular nerve Schwann progenitors, or may arrive in a later wave of migration of melanoblast progenitor NCC following a more dorsal route, or both.

In addition to lineage tracing analysis, the timing of otic NCC development has also been inferred from the temporal interval when these cells are sensitive to perturbation. Exposure of mouse or primate embryos to retinoic acid disrupts formation or migration of first and second arch NCC populations and causes malformations of the middle ear bones [18,19]. Administration of this teratogen at precisely timed intervals relative to the onset of pregnancy defines a discreet temporal window when middle ear development is sensitive to disruption. From these experiments it can be inferred that an interval within mouse embryonic day 8, specifically, E8 plus 4–5 h [18], up to E8.5 [20] is the critical period when the formation, migration, or development of NCC destined for the middle ear may be disrupted. This temporal window corresponds with the gestational time window of 2–5 weeks when excessive retinoids cause teratogenic ear phenotypes in humans [21].

THE PALPATION OF THE AURICLE

Palpation of the outer ear can be carried out by anyone. Even the patient can learn to find the sensitive points of his auricle easily and massage them for relieving, for example, musculoskeletal pain.

Palpation can be carried out with the patient lying down, which is the ideal position for inducing maximum relaxation; it can also be done with the patient in the sitting or standing position. For all these positions it is best if the therapist places himself behind the patient and presses both ears simultaneously and symmetrically between the thumb and the index finger. The therapist should regulate the pressure according to the patient's sensitivity: certain people react only to strong pinching, whereas others react to the slightest compression. The whole pinna should be explored, taking care to follow all of the cartilaginous irregularities. When such investigation is rendered impossible through the anatomy, for example at the level of the concha, one may press bilaterally with the index fingers, being careful to do this in an identical fashion.

Identifying a painful point at palpation is a good start in the examination of a patient but there are several aspects to be considered before proposing a diagnostic hypothesis. The first issue to clarify is the exact meaning of the identified area: for example we do not know if the area is the expression of an acute problem or of a chronic, recurrent disorder. Another possibility is that the painful point could represent a symptom hitherto not manifested.

Sometimes a persistent sensitivity of the auricle may indicate a functional disorder: in this case it is important to verify possible tenderness of the auricular zones such as the concha, the incisura intertragica, the ear lobe, etc. which are more often related to visceral, endocrinal and psychosomatic disorders. It is obvious that Chinese acupuncturists give a lot of importance to these areas since traditional Chinese medicine (TCM) is able to reveal dysfunctions before they manifest.

Another important aspect is the variability in the number of detected areas and their relation to the tender points of the auricle to which to apply therapy. The number of painful points identified by thumb and index finger palpation is very variable and depends on several factors such as the general sensitivity of the subject, the number of symptoms and districts involved, the intake of drugs, etc. I examined 92 consecutive patients from my general practice and randomly alternated palpation and the PPT with a commercially available pressure-probe of 250 g maximal pressure (Sedatelec). The reason for the random application was to exclude any possible mutual interference between the two methods. The number of painful areas, however, ranged from 0 to 21 (average 5.1) but was not significantly different from the average of tender points (average 5.7). Each area could contain from 1 to 4 sensitive points, but what is interesting in my research is that only in 55.7% of the identified areas could I isolate at least 1 tender point.

The meaning of this observation is that for every case the therapist should try to localize the points hidden in each tender area at palpation; however, in about half of the cases he will not detect any point at all. It is possible, however, that a repeated examination of the area with the pressure-probe or the application of an instrument with higher maximal pressure (for example 400 g) may finally detect one or more tender points.

1 Introduction

The obesity epidemic causing the increase in metabolic disorders has spurred scientific approaches to understand the origin of fat tissue and its expansion during ontogenetic development. To dissect the molecular signals and metabolic pathways that govern adipogenesis, different cell culture systems have been developed. Immortalized murine cell lines (3T3-L1, 3T3-F442A, ob1771, and OP9), multipotent murine embryonic cell line C3H10T1/2, and primary culture model (stromal-vascular fraction of fat depots) have brought advances in our understanding of the cascade of molecular events during the adipogenic differentiation (Green & Kehinde, 1975; Lee et al., 2013; Park et al., 2008; Wolins et al., 2006). Major progress in in vitro techniques has facilitated the isolation and culture of stem cells/progenitor cells from adult tissues including adipose tissues (Gimble & Guilak, 2003; Pittenger et al., 1999; Toma et al., 2001; Zuk et al., 2001). However, it is well established that different adipose depots from the same subject vary in adipogenic capacities/function. Several studies have demonstrated that such differences are linked to depot-derived preadipocytes and their capacity for adipogenesis (Caserta et al., 2001; Gesta et al., 2006; Tchkonia et al., 2005; Tchkonia et al., 2007). It has also been observed that the expression of adipocyte genes, lipid synthesis, lipolysis, and production of secreted proteins differ between preadipocytes from different fat depots (Bastelica et al., 2002; Berman, Nicklas, Rogus, Dennis, & Goldberg, 1998; Van Harmelen et al., 1998). Additionally, stem cells/progenitor cells that reside in adipose tissues are potentially committed to the adipocyte lineage. Summarizing, it is important to develop easily accessible, primary, unbiased/uncommitted cell culture system that will facilitate the elucidation of the earliest steps in mesenchymal stem cell commitment and adipogenic differentiation.

Ear mesenchymal stem cells (EMSC), a primary culture model of mesenchymal stem cells, are capable of readily differentiating into the four main lineages: adipocytes, osteocytes, chondrocytes, and spontaneously contracting myocytes (Gawronska-Kozak, 2004; Gawronska-Kozak, Manuel, & Prpic, 2007; Rim, Mynatt, & Gawronska-Kozak, 2005). This culture system promises to provide a model for the analysis of the early molecular events controlling stem cell commitment to the adipocyte lineage and its subsequent adipogenic differentiation (Rim et al., 2005).

EMSC were initially isolated from mice that have the capacity for regenerative healing of ear punches (Hsd: Nude-nu; Gawronska-Kozak, 2004). Follow-up studies revealed that EMSC populate external ears of all studied strains of mice: C57BL/6 J, FVB, aP2-agouti, and BAP-agouti transgenic mice (Gawronska-Kozak, 2004), Sfrp5 mutant (Mori et al., 2012), and rats (Sart, Schneider, & Agathos, 2009, 2010) regardless of their regenerative ability. Although currently we do not know whether EMSC participate in regeneration processes in vivo, their potential to differentiate into four lineages in vitro makes them a useful tool for the study of cell lineage-specific emergence and lineage differentiation. One of the enormous advantages of the EMSC model is that in addition to accessing the entire external ear collected postmortem, small pieces of ear tissue (ear punches) collected from live adult mice provide sufficient numbers of EMSC to isolate, culture, and differentiate (Gawronska-Kozak et al., 2007). This noninvasive surgical procedure for obtaining EMSC from live animals allows the simultaneous conduct of in vivo and in vitro studies on the same set of animals. Additionally, EMSC are an excellent alternative to mouse embryonic fibroblasts that can be collected from transgenic and KO mice in order to study commitment/differentiation towards adipogenesis, chondrogenesis, myogenesis, and osteogenesis as well as cellular metabolisms of lineage-differentiated EMSC. Recent data from Eilertsen lab emphasize the profound utility of EMSC as a superb source of cells for pluripotent stem cell (iPS) induction (Gao et al., 2013). The pluripotency of mouse EMSC-derived miPS, reprogrammed by overexpressing the four pluripotency factors Oct4, Klf4, Sox2, and c-Myc, was confirmed in in vitro and in vivo (teratoma formation in nude mice) studies. The data indicate that mEMSC-derived iPS share functional characteristics with (embryonic stem cell) ES cell clones (Gao et al., 2013).

Conductive Hearing Loss

Any dysfunction in the outer or middle ear is termed a conductive hearing impairment. (The outer ear refers to the ear canal [external auditory meatus], the cartilaginous tube that runs from the ear itself [the pinna] to the tympanic membrane). The consequence of a conductive loss is a general attenuation of the loudness of the sounds one hears. The most common, and easily treatable, cause of a conductive impairment is a plugging of the ear canal by an excess accumulation of cerumen (ear wax). More serious is a conductive loss due to restricted movement of the ossicles themselves, whether due to inflammation or infection in the middle ear (otitis media), or an age-related stiffening of the ossicles. The integrity of ossicle movement in the middle ear can be measured using tympanometry, a relatively non-invasive procedure in which the eardrum, and hence the ossicles, are set in motion by a controlled burst of air pressure, with the measured strength of the pressure return serving as an index of the conductance properties of the ossicles (Fowler & Shanks, 2002). Available medical and surgical treatments can often ameliorate this type of loss.

II.A. Outer and Middle Ears

As sound travels to the outer ear, it passes over the torso, head, and especially the pinna. All these structures attenuate and delay the passage of the sound to the outer ear, and the attenuation and delay depend on the interaction between the size of the structures and the wavelength of the sound, such that high-frequency sounds are affected more than low-frequency sounds. These transformations alter the spectrum of the originating sound and are called head-related transfer functions (HRTFs). HRTFs are significant for sound localization.

The outer ear canal has a resonant frequency near 4000 Hz (i.e., standing waves exist within the outer ear), and as such sounds with frequency components near 4000 Hz are more intense in the ear canal than are other frequency components. The middle ear ossicles (bones) provide further enhancement in sound level in the region of 2000–5000 Hz. All these increases in sound level are necessary if air pressure is to produce an effective vibration within the inner ear. The inner ear is a fluid-filled space containing neural structures and their support. These inner ear structures offer significant impedance to the transmission of vibration from the air-filled outer ear to the inner ear. The resonance of the outer ear and the increases provided by the ossicular chain provide an impedance match between air and the fluids and structures of the inner ear so that over a significant portion of the audible range of hearing, changes in air pressure impinging on the auditory system are efficiently transmitted to the inner ear with no loss in sound level. Damage to the ossicular chain leads to significant hearing loss because of the loss of this crucial impedance matching function.