Journal of Microscopy and Ultrastructure

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 7  |  Issue : 3  |  Page : 117--123

Estimation of volume of stria vascularis and the length of its capillaries in the human cochlea


Sree Vani Poorna Pillutla, Charanjeet Kaur, Tara Sankar Roy, Tony George Jacob 
 Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India

Correspondence Address:
Dr. Tony George Jacob
Department of Anatomy, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029
India

Abstract

Background: The stria vascularis (SV) is a vascularized epithelium that secretes endolymph and is located on the lateral wall of the membranous cochlea. The capillaries of SV directly influence the composition of the endolymph and hence the generation of impulses by the hair-cells that are auditory receptors and thus affect hearing. Therefore, the real morphology of the SV would be very important for understanding the hearing system. There are few reliable reports of the morphology of the human SV. Aims and Objectives: In this research, we have estimated the volume of the SV and total length of strial capillaries in the apical, middle and basal turns of the human cochlea by updated stereological techniques. Methods: The point-counting Cavalieri's method and hemispherical volume probes were applied on stained, 40 μm-thick serial sections of five celloidin-embedded, decalcified cochleae. Results: The mean age of persons at the time of death was 51 ± 15.25 years, the mean volume of the SV was 0.56 ± 0.054 mm3 and the mean length of the SV capillaries was 289.08 ± 72.96 mm. We also estimated the same parameters with different stereological parameters, probes and in differently stained sections and checked the relationship and limits of agreement between different methods by paired T-test and Bland-Altman plot. We found agreement in our results. Conclusion: We provide reliable baseline data on the real morphology of the human SV.



How to cite this article:
Poorna Pillutla SV, Kaur C, Roy TS, Jacob TG. Estimation of volume of stria vascularis and the length of its capillaries in the human cochlea.J Microsc Ultrastruct 2019;7:117-123


How to cite this URL:
Poorna Pillutla SV, Kaur C, Roy TS, Jacob TG. Estimation of volume of stria vascularis and the length of its capillaries in the human cochlea. J Microsc Ultrastruct [serial online] 2019 [cited 2019 Oct 18 ];7:117-123
Available from: http://www.jmau.org/text.asp?2019/7/3/117/264773


Full Text



 Introduction



Stria vascularis (SV) is a highly vascular epithelium found in the lateral wall of the membranous labyrinth that is situated within the bony canal of the cochlea.[1] The SV secretes endolymph through a process of active pumping of ions such as Na+, K+, and Cl− and the passive diffusion of water molecules. Endolymph bathes the hair cells of the organ of Corti, which is the auditory receptor, and helps to maintain the positive endocochlear potential.[1] The secretion of endolymph (in quantity and quality) is directly related to the morphology of the SV, i.e., the total volume of the SV and also the total length of capillaries within the SV.[2] Thus, the morphology of the SV would affect the functioning of the auditory receptors because the hair cells of the organ of Corti are extremely sensitive to ionic changes in their environment, and the composition of the endolymph can be altered by changes in the morphology of the SV.[2] These changes over time can affect the quantity and quality of hearing.[3] Metabolic presbycusis (age-related hearing loss) is associated with strial atrophy and decreases in the number of strial capillaries.[3] Previous investigators have reported changes in the spiral ligament with aging[4] and concluded that its atrophy preceded the degenerative changes in the SV. Another group of investigators[5] using design-based stereology confirmed these findings. However, Jorgensen[5] did not find any correlation between the degree of strial atrophy and degeneration of the organ of Corti. Therefore, there is continuing debate regarding the true morphology of the SV, its capillaries, and the consequences of these changes on hearing. In such a scenario, it becomes important to provide reliable, baseline morphological data on the SV that can be used to compare future studies that may explore the causes of metabolic presbycusis in humans. A reliable morphological tool is a design-based stereology that estimates morphometric parameters independent of considerations of shape and size of the region of interest. We have already used this method in our laboratory to study aging and development of different components of the auditory pathway.[6],[7],[8],[9],[10],[11] In this study, we have used modern stereological methods to provide reliable, baseline morphological data on the human SV.

 Materials and Methods



Specimen collection and tissue preparation

We collected cadaveric, human temporal bones in accordance with the protocol approved by the Institute Ethics Committee (Ref no. IESC/T-438/26.08.2015) and according to the guidelines of the Helsinki Declaration from the mortuary after obtaining written consent from the legal representatives. All the donors were male, died of various causes without any head injury. The donors were 31, 45, 49, 58, and 72 years old at the time of death [Table 1]. The bones were fixed by immersion in 4% buffered paraformaldehyde (0.1 M phosphate buffer, pH 7.4), decalcified in 10% EDTA solution, dehydrated in ascending grades of alcohol, impregnated with celloidin, and embedded in 8% celloidin [Supplementary Figure 1] [SUPPORTING:1]. The celloidin blocks were sectioned serially on a sliding microtome, at 40 μm thickness, and every tenth section (starting with a random number between 1 and 10), containing the SV, was stained with hematoxylin and eosin (H and E), as per the standard protocol.[12] A series of every tenth section starting with a different random number in the sample obtained from a 31-year-old individual was stained by Masson's trichrome method as per the standard protocol.[12]{Table 1}

Stereological estimates

Every tenth, systematically and randomly selected section (the first section was selected using a random number series between 1 and 10, both numbers included), stained and observed under the Olympus BX61 research microscope attached to a computer, motorized stage controller (LUDL, Germany) and video camera (MBF Biosciences, CX 9000). The live images of the cochlear serial sections were analyzed using the StereoInvestigator software (MicroBrightField Inc., VT, USA), and the volume of SV and the total length of capillaries within it were calculated by the Cavalieri estimator and hemisphere probes, respectively. Before applying the probes, we first identified the turns of the cochlea under the 2X objective lens. The cochlea having the form of a conical helix; in horizontal sections, the basal turn (BT) appeared to be the largest, followed by the middle turn (MT) and apical turn (AT) in size and distance from the base of the cochlea [Figure 1]. The turns of the cochlea appeared as either one or two cross-sections or a single tangential section. Unlike Kurata et al.,[3] we did not differentiate between the upper and lower BTs. Next, the boundaries of the SV were identified as described previously[13] [Figure 2].{Figure 1}{Figure 2}

Estimation of volume of stria vascularis by Cavalieri probe

Every tenth section of the series was selected, and contours of all the areas of SV were marked under low magnification (×2 objective lens). Each section contained from one to six areas of SV. Under higher magnification (×40 objective lens), differently colored lines were used to mark out the contours of the SV in the BT, MT, and AT [Supplementary Figure 2] [SUPPORTING:2]. The Cavalieri probe was separately applied to the contours, and the grid points falling within contours were painted using different markers for the three turns of the cochlea [Supplementary Figure 3] [SUPPORTING:3]. The total volume of SV, in each turn, was calculated using the formula described before.[14],[15]

Estimation of total capillary length by hemispherical probe

The hemisphere probe was applied to estimate the length of capillaries within the SV of the cochlea, in every tenth section. After the contours of the SV in the different turns of the cochlea were drawn as described above, a hemispherical probe with a radius of 35 μm was applied within each contour, within a grid of 70 μm by 70 μm. We applied a guard zone of 2 μm on the upper and lower sides of the section. Sampling sites were generated by the software as a part of systematic random sampling workflow. The sections were then scanned through the whole thickness, and markers applied wherever the spline (line passing through the center of the capillary) of the capillaries intersected the probe [Supplementary Figure 4] [SUPPORTING:4].[15] All the intercepts from every tenth section were counted, and the length of capillaries was calculated using the following formula for the hemispherical probe:

Where,

n: Number of sections used (I = 1 to n)

Ii: Intersections counted

v: Volume (“grid-X”× “grid-Y”× section thickness)

a: Surface area of the sphere

ssf: Section sampling fraction

The volume of SV was also estimated by the hemispherical probe as a part of the reference volume in estimating the total length of capillaries (calculated from the Vref according to the formula Vref= number of hemispherical probes × grid area × height of the section).

We used the paired t-test and the Bland–Altman plots to compare the estimated total length of capillaries visualized by two different staining techniques, i.e., Masson's trichrome and H and E in sections obtained from the 31-year-old individual. We also used these statistical tests to compare the estimates of SV capillary length obtained using slightly different parameters for the hemispherical probe [parameters have been tabulated; [Table 2].{Table 2}

The coefficient of error was calculated as follows:

TotalVar =s2 +VARSRS

Variance due to systematic random sampling,

Statistical analysis

The data are represented as individual values in mm for length and mm/mm3 for length density and mm3 for volume. A Q-Q plot was made to verify if the data of volume estimation of the SV obtained by the Spaceball and Cavalieri probes distributed normally. Further, paired t-test and tests of agreement (Bland–Altman plot) between estimates of volume were obtained from the Cavalieri probe and the hemispherical probe. We used Microsoft Office Excel 2007 (Microsoft Corporation, California, USA) and SPSS version 17 (IBM, New York, USA) for statistical analysis, where required.

 Results



Morphology of the stria vascularis

We studied the cochlea and its parts in mid-modiolar sections [Figure 1].

The SV was a stratified cuboidal epithelium in the lateral wall of the scala media [Figure 2] and [Figure 3]. It was made of three cellular layers – marginal, basal, and intermediate. Capillaries were seen among the epithelial cells [Figure 4]. Marginal cells were the outermost layer of cuboidal cells [Figure 4]. Basal cells were found at the interface between spiral ligament and SV and had one to three cell layers. Their nuclei were predominantly heterochromatic, were flattened, and were observed to lie parallel to the apical surface of marginal cells. Like the marginal cells, some basal cells contained brown pigment granules in their cytoplasm [Figure 4] and [Figure 5]. Intermediate cells were found lying between marginal and basal cell layers. Their nuclei were round, centrally placed within the cells, darker, and smaller than those of the marginal cells. Their cytoplasm formed a thin layer around their nuclei and was thrown into processes that enclosed the surrounding intercellular spaces. Some intermediate cells also contained brown pigment granules in their cytoplasm [Figure 5], which were also seen in their cytoplasmic processes, extending toward neighboring cells. The capillaries of the SV were branches of the blood vessels found in the spiral ligament and the SP. Both longitudinal and cross-sectional profiles of capillaries with a single layer of endothelial cells were found within the intermediate layer. Red and white blood corpuscles were visible within most of the sections of capillaries.{Figure 3}{Figure 4}{Figure 5}

Volume of stria vascularis

The total volume of the SV in the five samples has been tabulated [Table 3]. The volumes of the individual turns of the cochlea in each sample have also been tabulated [Table 4]. The volume of SV was also estimated by the hemispherical probe as a part of the reference volume in estimating the total length of capillaries. The volumes of SV, calculated by these two methods in individual samples, were compared, plotted on a Q-Q plot [Figure 6] which gave a straight line (slope r2 = 0.99). The limits of agreement were also calculated by Bland–Altman test [Figure 7]. Further, there was no significant difference between the estimated volume of the SV by Cavalieri and the hemispherical volume probe (paired t-test, P = 0.622).{Table 3}{Table 4}{Figure 6}{Figure 7}

Length of capillaries in stria vascularis

The total length of capillaries in the SV of the five samples was estimated by the hemispherical probe and has been tabulated [Table 2]. The length of capillaries in the individual turns of the cochlea for each sample has also been tabulated [Table 2] and compared. In brief, the length of the SV capillaries decreased from base to apex in all the samples. We observed that the length of the SV capillaries of the BT was approximately 2–3 times that of the MT and 4–7 times that of the AT. We also observed that the ratio of the lengths of the SV capillaries in the MT to AT ranged from 1.3 to 2 times that of the AT. The total length of the capillaries in SV was also calculated in sections stained with Masson's trichrome in the sample obtained from the 31-year-old individual, as described above. The total length of SV capillaries in different turns of the cochlea in these three sets of serial sections of this sample was also estimated and tabulated [Table 2]. There was no significant difference between the length of the SV capillaries estimated in the AT, MT, and BT of the cochlea in the sections stained by either H and E or Masson's trichrome method (Student's paired t-test, P = 0.406) or by different grids (70 μm × 70 μm versus 80 μm × 80 μm, P = 0.304) and different stains and grid sizes (H and E, 70 μm × 70 μm versus Masson's trichrome, 80 μm × 80 μm, P = 0.646) [Table 5]. Bland–Altman plots also revealed good agreement between the mean of the estimates of the length of capillary of the SV in H and E and Masson's trichrome-stained sections and in H and E-stained sections and different grid sizes [Figure 8] and [Figure 9]. We have also calculated the length density of the SV capillaries in each turn of the cochlea [Table 6].{Table 5}{Figure 8}{Figure 9}{Table 6}

 Discussion



In this study, we estimated its volume and the total length of capillaries of the human SV in five cochleae and also separately in the AT, MT, and BT.

Design-based stereology provides estimates of number, length, area, and volume of an object of interest without making assumptions regarding the geometry of the object of interest, and hence, it eliminates potential sources of bias and systematic errors[15],[16] that occur commonly with conventional morphometric techniques. In this study, we have used two isotropic probes – Cavalieri estimator and the hemispherical probe – for volume and length estimations, respectively. We have previously used the grid point-counting probe to estimate the volume of the developing inferior colliculus and the adult cochlear nucleus.[6],[7],[8]

Capillaries in the SV, which have a regular radial arrangement, as determined in corrosion casts,[17] do not display such features in histological sections of the cochlea because they are tortuous and could be sectioned in a plane independent of the long axis of the capillary, thus behaving as anisotropic elements. Thus, to estimate the total length of the capillaries, we utilized an isotropic hemispherical probe. We had the option of using a two-dimensional probe, like a cycloid, to estimate the length of the capillaries; however, the values obtained would have varied according to the angle of sectioning. This would then require specific mathematical corrections. Therefore, we used the three-dimensional hemispherical probe that is isotropic in nature and thus does not require isotropic sections for its application.[18]

In our study, the volume of SV obtained by Cavalieri method was in agreement to those obtained by hemispherical probe in all the samples [Table 3] and [Figure 6]. The length of the strial capillaries also decreased from the BT to the AT. The BT is the largest coil of the helical cochlea and in our study had the maximum volume of SV, and hence, as expected, it also had the maximum length of strial capillaries within it while the AT being the smallest had a lower volume of SV and the least length of capillaries.

It has been seen that the capillary length decreases from the BT to AT in cochleae of gerbils.[19] In this study, we did not use immunohistochemical techniques to identify the capillary endothelial cells. However, we used another staining technique to identify the RBCs and connective tissue – the Masson's trichrome staining technique. We ran the hemispherical probe, with the same parameters, in the sample obtained from the 31-year-old individual and observed that the length of the strial capillaries was almost the same in both methods of identifying capillaries. This, thus, validated our estimates of the total length of the SV capillaries [Table 5] even though neither of the techniques is the gold standard for the identification of a blood vessel. Santi et al.[13] reported that the capillary density in the different turns of the cochlea of chinchilla remains constant. We found that the length densities of the BT and MT in 72-year-old samples were 406.85 mm/mm3 and 363.66 mm/mm3, respectively, while that of the AT were 484.15 mm/mm3 [Table 6] and that the length of the strial capillaries and volume of SV were 120.45 mm and 0.30 mm3, 53.48 mm and 0.15 mm3, and 25.97 mm and 0.05 mm3 in the BT, MT, and AT, respectively, of the same sample [Table 2] and [Table 4]. Hence, on reviewing [Table 2], [Table 4], and 6, we can conclude that length density is not a reliable indicator of the status of strial vasculature. It is better to estimate the total length of the capillaries.

Here, we have standardized a morphometric procedure of studying the SV in human cochlea, and we would like to extend this study in a larger sample set to draw definitive conclusions related to the changes in the SV and its correlation with age-related hearing loss.

Acknowledgments

The authors would like to thank:

The skills – Laboratory in the Department of Neurosurgery, where the authors dissected the human temporal bones to obtain the cochleaProf. D. N. Bhardwaj of the Department of Forensic Medicine and Toxicology, All India Institute of Medical Sciences, New Delhi, for facilitating collection of temporal bones from the mortuary.

Financial support and sponsorship

This study was undertaken as part of the dissertation work of Dr. Sree Vani and was thus funded by departmental funds.

Conflicts of interest

There are no conflicts of interest.

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