Where is perilymph manufactured

Despite this difficulty we could track dye in each configuration but cannot compare absolute intensity values between ages for this experiment. In contrast to PSCC injections, visual inspection of RWM injections, ex vivo , show dye accumulation at the cochlea base with less dye found in middle turn and virtually no dye seen at the apex. Figure 2. The regions of interest where the intensity was measured at apex , middle , and base for each experiment are shown in yellow in the last picture of each row s.

The X -axis provides time snapshots and the Y -axis the specific group being injected. ROIs are demarcated in the last column of Figure 2. Average intensities in the apex, middle, and base were measured from time 0 starting the injection until 34 min after starting the infusion. Video examples for each condition are presented in the Supplementary Material S1 — S4. The graphs in Figures 3A—L present data analyzed from the movies Supplementary Videos for individual animals selected as those with parameters closest to the mean values presented in Figure 4.

Lower intensity I values indicate darker regions, meaning the dye is reaching that area. We interpret intensity changes as equivalent to changes in dye levels. These data suggest more dye entry for adult compared to neonatal animals for both injection sites and also that dye reaches the apex more readily for PSCC injections than for RWM injections.

Figure 3. Injection time was from 0 to 3 min. Graphs A,D,G,J show the raw intensity measurements in the apex squares , middle circles , and base triangles. Graphs B,E,H,K present intensity changes in the same regions at each time point with respect to the initial values at 0 min. Graphs C,F,I,L show the percentage of intensity change at each time point, in respect to I max , during injection. M Intensity changes 3 min after starting the pump in respect to the intensity values at 0 min I 0.

Asterisks indicate levels of significant change in intensity values between 3 and 0 min at each cochlear region. N Intensity changes from 4 min time point when the micropipette was removed until 30 min later.

Asterisks indicate levels of significant change in intensity values between 34 and 4 min at each cochlear region. Boxes represent standard deviations of the mean. Pair-sample t -test in panels M,N were used for calculating the p -values. Figure 4. Analysis of normalized intensity changes in the cochlea during the injection period 0—3 min summary from data in Figure 3.

In each plot N is neonatal and A is adult. Apical is represented by squares, middle by the circles, and base by triangles. Open symbols represent data from the individual examples presented in Figures 3A—L.

Graph A describes the measured parameters presented in panels B,C. B Onset time and C steepest slope obtained from individual animals as shown in the third column of Figure 3. In panels B and C statistical comparisons are done on pooled data showed with horizontal lines. Two-sample t -test in panels B,C were used for calculating the p -values. A slight increase in the intensity value was observed at the end of infusion indicated by the arrow in Figure 3A , correlating with the time when the glass micropipette was removed from the injection site.

This increase was not observed in RWM injected animals, likely because the limited amount of dye present at apex and middle ROIs did not allow for the dye reduction detection. The lack of change in the basal region is not due to a lack of dye and perhaps suggests a difference due to injection site.

A summary of maximal changes monitored at the end of the injection from the imaging angle shown in Figure 2 shows a base to apex gradient for PSCC injected neonates and RWM injected animals Figure 3M. The relative changes associated with PSCC injection were greater than RWM injections Figure 3M ; however, the orientation of cochlea during in vivo imaging provides a better view for monitoring the dye progression in PSCC injection compared to the RWM so we performed additional experiments to obtain more equivalent views see Figures 5 , 6 for more direct comparisons at different orientations.

Figure 5. The contralateral cochleae of neonatal and adult mice are also presented for comparisons C,H. Figure 6. The contralateral cochleae of neonatal and adult mice were used as control non-injected cochleae. Error bars represent standard errors. Two-sample t -test was used for calculating the p -values between groups. The majority of PSCC mice showed a reduction in dye accumulation, as indicated by positive values at 30 min post injection Figure 3N.

RWM injected mice showed a continued increase in dye accumulation as indicated by negative values meaning more dye present Figure 3N. These data support the idea that PSCC had uniform distribution early with later time points reflecting diffusion out of the cochlea. In contrast, RWM injections showed less dye within the cochlea; the existing dye distributed more uniformly over the following 30 min.

To evaluate the kinetic differences between modes of injection independent of absolute concentrations we normalized data to the time point immediately before termination of the injection Figures 3C,F,I,L. The presented examples demonstrate time delays and rate differences in dye progression between modes of delivery and age. The dramatic differences in dye level are not included in these plots but rather simply an indication of the timing differences, summaries of which are presented in Figure 4.

The measured parameters are depicted in Figure 4A. The onset time is defined as the first time point where the steepest rate of change for dye accumulation occurs Figure 4A. Figure 4B summarizes changes in onset time between groups. A delay in onset time was observed between regions for neonatal animals, being most delayed in the apex, regardless of delivery mode. Adult animals did not show intracochlear differences in onset time. The age difference may simply represent the change in size of the inner ear reducing resistance to flow in adult mice.

To compare rates of change we simply used the steepest slope as described in Figure 4A , for each experimental group. Given that these measurements are during perfusion a common slope is predicted that basically relates injection rate to cochlea properties.

Reductions in this rate suggest that flow is bifurcating or that different resistances are encountered. That is, if the dye splits into flow in multiple directions the rate will be reduced for either pathway. Results of this analysis are shown in Figure 4C. The PSCC injected adult mice had steeper slopes compared to the neonatal ones; but no significant difference in the slope was observed between the neonatal and adult mice injected through the RWM.

The slope difference between ages is likely a result of a reduced resistance to flow in the adult animal. The steeper slope with PSCC injections suggests higher levels of dye are entering the cochlea from this site of injection at each time unit. All biological paths from the PSCC injection lead through the cochlea while the RWM injection can bifurcate to the cochlea aqueduct prior to distributing through the entire cochlea.

This simple difference likely accounts for the difference in dye distributions for the two injection sites. A problem with the in vivo imaging is that orientation of the cochlea makes it more difficult to assess distribution with RWM injections. To further assess diffusion post injection and to better visualize dye distributions throughout the cochlea a separate set of experiments was performed where the brains and both cochleae were obtained at 5- and min post injection for each experimental group.

This approach allows us to evaluate dye distribution ex vivo within the cochlea from angles that were not accessible in the in vivo images.

No dye was detected in any contralateral cochleae. To better assess the dye pathway with RWM injections and to investigate uniformity of distribution, images were obtained from three perspectives at 5 min post injection Figures 5K—P. In contrast, RWM injections show dye in the basal areas with less distribution to apical regions. In addition, these data suggest that dye intensity differences may in part be due to the dye going elsewhere. This conclusion is supported also by the reduced steepest slopes described in Figure 4.

Immediate dissection and inspection of cochlea following the end of PSCC injection about 5 min post injection show all cochlea regions were significantly darker than non-injected ones, at both ages Figures 6A,C.

In contrast, RWM injected animals showed a steep gradient where only base in adults and base and middle in neonates were significantly darker than the non-injected cochleae at the 5-min time point Figures 6A,C. One hour after injection, dye levels in the PSCC injected neonatal cochleae were significantly reduced in all regions compared to 5 min after injection Figures 6B,E , see asterisks. No significant difference in the cochlear dye levels was observed in RWM injected neonatal Figures 6A,B,F or adult Figures 6C,D,F for 1 h compared to 5 min after injection, suggesting dilution was not happening with this mode of injection at either age.

For simplicity, the control data from neonatal and adult mice are combined. The dye distribution in neonatal animals presented a gradient from base to apex, after PSCC injection at the 5-min time point Figure 6E but not at the min time point.

This gradient did not exist in the PSCC injected adults at either time points. A steep gradient in dye distribution at both 5 and 60 min was observed after RWM injection in both neonatal and adult mice Figure 6F.

One possibility is that part of the dye injected through the RWM travels through the cochlea aqueduct which is located very close to the injection site. In contrast, the PSCC injection site is at the opposite end of the cochlear perilymphatic space, so that during the injection perilymph will be pushed through the cochlea aqueduct while dye enters the cochlea. We inspected the brains of injected animals as a proxy for dye traveling through the cochlea aqueduct.

Brain images from each group of injected animals are shown in Figure 7. In PSCC injected adult mice, no dye was detected in the brain 5 min after injection, but small traces of trypan blue were observed in four out of seven adult mice 1 h after injection, suggesting travel through the cochlea aqueduct post injection. Thus, these data are consistent with the hypothesis that RWM injections lose dye through the cochlea aqueduct more readily than PSCC injections.

Figure 7. Representative brain pictures of the neonatal and adult mice injected with trypan blue through PSCC or RWM, 5 and 60 min after injection. Number of evaluated brains in each group of experiments is indicated. The arrows point to the regions where the dye was observed.

It has previously been suggested that the chemical composition of the injected compound can affect distribution within the ear Nomura, ; Salt and Plontke, In order to investigate the potential variations in compounds progression in the cochlea following the same mode of delivery and using the same injection parameters, two other compounds were tested.

The results are shown in Figure 8. Methylene blue presented very similar to trypan blue. One hour after injecting methylene blue into the PSCC of P1 mice, no dye was visible through the skin, and after dissection, no trace of dye was detected in the brain or contralateral cochlea Figure 8F. However, GTTR distribution was starkly different. Three hours after the injection, the drug was still visible in different parts of the body through the skin Figure 8B.

One hour after the injection, the drug was visible through the whole cochlea, and most of the inner and outer hair cells Figure 8D. For this level of distribution, the GTTR must be able to access the blood supply by crossing membranes within the inner ear. These experiments highlighted the fact that in addition to the injection parameters e. Figure 8. The bright field and fluorescent images are shown in left and right panels, respectively. Otic capsule and SCCs are shown in the top.

Organ of corti is shown in the bottom. Fiori, M. Permeation of calcium through purified connexin 26 hemichannels.

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Otol Neurotol. Goycoolea MV: Clinical aspects of round window membrane permeability under normal and pathological conditions. Acta Otolaryngol. Download references. You can also search for this author in PubMed Google Scholar. Correspondence to Jing Zou. JZ participated in the design of the study and performed the MRI measurement. RS and SR participated to the design of the liposomes and prepared the liposomes. DP participated in MRI measurements.

PK supervised the design and preparation of liposomes. IP supervised the study. All authors have read and approved the final manuscript. Reprints and Permissions. Zou, J. Manufacturing and in vivo inner ear visualization of MRI traceable liposome nanoparticles encapsulating gadolinium.

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Download PDF. Abstract Background Treatment of inner ear diseases remains a problem because of limited passage through the blood-inner ear barriers and lack of control with the delivery of treatment agents by intravenous or oral administration.

The ear is organized into three different anatomical structures: the outer, middle, and inner ear. The outer ear consists of the pinna, external auditory canal, and tympanic membrane and is responsible for the transmission of sound waves from the external environment. Vibrations are transmitted from the malleus through the incus to the stapes, which is in contact with the cochlear oval window.

The inner ear is located within the bony labyrinth of the temporal bone and contains the cochlea, semicircular canals, utricle, and saccule. These organs make up the membranous labyrinth that is within the bony labyrinth, separated only by perilymph. The membranous labyrinth contains a fluid known as endolymph, which plays a vital role in the excitation of hair cells responsible for sound and vestibular transmission.

The cochlea is a spiral-shaped fluid-filled organ located within the cochlear duct of the inner ear. The cochlea contains three distinct anatomic compartments: the scala vestibuli, scala media also referred to as the cochlear duct , and scala tympani.

The scala vestibuli and scala tympani both contain perilymph and surround the scala media, which contains endolymph. The endolymph within the scala media originates from cerebrospinal fluid CSF and is secreted by the stria vascularis, which is a network of capillaries located in the spiral ligament. The perilymph in the scala vestibuli originates from blood plasma, whereas the perilymph in the scala tympani comes from CSF. Endolymph and perilymph vary significantly in their concentration of ions, which is essential to the overall function of the cochlea.

Endolymph is rich in potassium and low in sodium and calcium, whereas perilymph is rich in sodium and low in potassium and calcium. This difference in concentration allows for a positive endocochlear potential. The difference in concentration of potassium ions among the three fluid compartments within the cochlea enables proper transduction of current along with the hair cells.

Vibration from the stapes gets transmitted through the oval window, which is an opening into the inner ear through which the middle and inner ear communicate. Vibrations across the oval window initiate a perilymph wave that propagates along the scala vestibuli, with high frequency sounds dissipating earlier at the base of the cochlea and low-frequency sounds dissipating later towards the apex of the cochlea.

The perilymphatic wave terminates at the round window, another point at which the middle ear communicates with the inner ear. In contrast to the oval window, the round window does not articulate with the stapes. Rather, the round window membrane is located inferomedial to the oval window and functions to counteract the fluid shift created in the cochlea. The presence of the round window allows for fluid to move more freely through the cochlea, thereby improving sound transmission.

As vibration transmits across the oval window, perilymph gets pushed towards the cochlear apex, which causes the scala media to become compressed. Within the scala media, there is a tectorial membrane that sits atop the organ of Corti. The compression of the scala media causes the tectorial membrane to change the position of cells within the organ of Corti. The organ of Corti is located within the scala media and is responsible for converting mechanical forces into electrical impulses.

It contains inner and outer hair cells that are arranged tonotopically throughout the cochlea to help distinguish between sounds of varying frequencies. The hair cells have projections known as stereocilia and kinocilia that are in contact with the tectorial membrane. Vibrations transmitted to the tectorial membrane cause displacement of stereocilia, leading to the displacement of the adjacent kinocilia.

Movement of the kinocilia triggers depolarization of the hair cell, leading to an influx of calcium and the release of specific neurotransmitters that act at the cochlear ganglion. This activity produces an action potential that is propagated along the cochlear nerve and along auditory pathways, where it eventually reaches the cochlear nuclei located in the brainstem.

The inner ear also contains the vestibular organs that are responsible for balance and position. The vestibular organs include the semicircular canals, utricle, and saccule. To understand the anatomy of the vestibular organs, it is helpful to separate the vestibular organs based on their specific functions.

The semicircular canals, including their ampullas, are responsible for angular acceleration rotational movement of the head , whereas the utricle and saccule are involved in linear acceleration. There are three semicircular canals; anterior, posterior, and lateral. Each semicircular canal is located in a different plane x,y, and z and connects to the utricle via an ampulla, which is a widening of the canal. Within the ampulla, there are sensory epithelia known as cristae that contain projections of hair cells.

Above the hair cells and cristae, there is a gelatinous cupula. As the head rotates in various directions, endolymph flowing through the semicircular canals displaces the gelatinous cupula that rests above the cristae leading to excitation of the hair cells embedded within the cristae.

The hair cells become depolarized or hyperpolarized depending on the direction in which endolymph flows. The utricle and saccule each contain a macula, which is the fundamental end-organ the equivalent of the crista within the ampulla described in the previous section involved in detecting linear acceleration. The utricle is involved in longitudinal acceleration, whereas the saccule is involved in acceleration along the vertical axis.