that overlays a layer of oleic acid on the surface of the iron oxide nanoparticles (POA@SPIONs). POA@SPIONs is a promising T2-negative contrast agent that is detectable within the inner ear by MRI [123]. Functionalization of POA@SPIONs can be performed that make it a target for inflammatory cytokines in the inner ear; however, they were found not to enter the inner ear efficiently after the transtympanic injection [106]. Another novel, highly hydrophilic, anti-aggregative super-paramagnetic maghemite (γ-Fe2O3) nanoparticle (NP) was developed using ceric ammonium nitrate (CAN)-mediated oxidation of starting magnetite (Fe3O4) NPs (CAN-γ-Fe2O3 NPs), which were highly stable aqueous suspensions/ferrofluids due to a unique ultrasound-mediated doping process of the Fe3O4 NP surface using lanthanide Ce3/4+ cations [124]. Zou et al. [125] have also demonstrated that the novel CAN-γ-Fe2O3 NPs is a strong T2 MRI contrast agent that penetrates both round and oval windows, and has potential applications in molecular imaging of the inner ear [125].
Fig. 7. Super-paramagnetic iron oxide nanoparticles (SPION) contrasted inner ear in a rat. The SPION administered into the perilymph will extinguish the signal from the perilymph and only endolymphatic spaces are visible on MRI. Reprinted with permission of Europ J nanomed [122]. Cochlea, vestibule and the semicircular canals are shown.
By developing a novel nanomaterial to be used as contrasting agent, for example, encapsulation of metals and metal clusters in fullerenes (endohedral metallofullerenes) opens additional vistas for inner ear imaging [126–128]. The carbon cage has inherent advantages because of its high stability and characteristic resistance to any potential metabolic cage-opening process. This prevents the release of toxic metal ions from endohedral metallofullerenes into surrounding tissue, serum, and other biologic components [126]. Water-soluble endohedral gadolinium-lutetium fullerene is generating considerable interest because of the possibility of using these novel nanomaterials as both MRI and MDCT imaging contrast agents. It is possible that specific molecular MRI and MDCT imaging can be performed after single injection of the targetable dual contrast agent in future.
Targeted Contrast Agents
Contrast agents to enhance or darken fluid or tissue signals help to visualize regions of interest, and efforts are now being made to create biological tags using these agents for molecular imaging at the level of cellular processes. Aimed to visualize liposome NPs in the inner ear, GdC-encapsulated liposomes were developed and distribution of the NPs in the cochlea was detected in vivo using MRI after either intracochlear injection or intratympanic injection [129–131]. These studies open a window in specific visualization of inner ear pathology using MRI. GdC-encapsulated liposomes pass through both the oval and round windows and were not toxic in in vivo experiments. Potential molecular imaging in the inner ear using the novel CAN-γ-Fe2O3 NPs was also demonstrated in an animal study [132]. The novel NPs are especially useful for molecular imaging of the inner ear to detect molecular changes in pathological conditions.
Microtomography
In CT, the cochlear partition and soft tissue as membranes are not adequately visualized [10, 11]. The gray levels in a CT slice image correspond to X-ray attenuation, which reflects the proportion of X-rays scattered or absorbed as they pass through each voxel, and is affected by the density and composition of the material being imaged. Non-destructive X-ray μCT has proven its utility in 3D assessment of mineralized and soft tissue morphology [133, 134]. The cochlear partition and the basilar membrane could not be distinguished and reconstructed with µCT [134]. Recently, Poznyakovskiy et al. [135] presented an algorithm for cochlear segmentation, which resulted in the reconstruction of scala tympani. µCT has been engaged in middle and inner ear imaging of animals and implicated to be a useful tool to trace the distribution of drugs in the inner ear. However, it can only be used for ex vivo imaging due to the extremely high dose of exposure and the close imaging distance which is only suitable for the head [136]. The contrast-enhanced μCT methodology is further developed for ex vivo cochlear imaging [28]. It can demonstrate the position of Reissner’s membrane and basilar membrane if a contrast agent is used [136]. Figure 8 demonstrates CI electrode imaged with μCT. However, μCT produces extremely high radiation dose and in present form cannot be applied in humans.
Recently, this technique has been advanced in animal experiments by revealing the inner ear compartment with simultaneous 9T MRI scanner and μCT [2]. The combined MRI-µCT imaging techniques were complementary, and provided high-resolution dynamic and static visualization of the morphological features of the normal mouse inner ear structures.
Fig. 8. Imaging of CI electrode with µCT, a shows the basal electrodes stimulating areas close to the round window, b shows the first and second tip electrodes aimed to stimulate the apex of the cochlea.
High-Resolution CARS
Raman spectroscopy is a powerful tool to generate a characteristic signature of specific tissues and operates by detecting energy with the molecular bond vibration of incident photons. The process results in non-elastically scattered light, also known as Raman scattering [137]. Raman spectroscopy is capable of discerning molecular pathology of differential proliferative middle ear lesions and may help in the assessment of borders of the pathological process to improve the surgical outcome of the middle ear diseases [7, 138–141]. CARS occurs when a target molecule is irradiated using 2 laser beams simultaneously at different frequencies, a pump beam and a Stokes beam. When the difference between the higher frequency (pump beam) and the lower frequency (Stokes beam) equals the vibrational frequency of the target bond of the molecule, a CARS signal is generated [139–
