- Email: [email protected]
Effect of postmortem autolysis on Na,K-ATPase activity and antigenicity in the gerbil cochlea
ELSEVIER
Hearing Research 89 (1995) 14-20
Effect of postmortem autolysis on Na,K-ATPase activity and antigenicity in the gerbil cochlea Daniel A. Vincent Jr. a,*, Michael Anne Gratton a,1, Brendan J. Smyth b, Bradley A. Schulte b a Department of Otolaryngology and Communicative Sciences, Medical Unit,ersity of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA b Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA
Received 7 October 1994; revised 8 May 1995; accepted 16 May 1995
Abstract
Alterations in the enzymatic activity and antigenicity of Na,K-ATPase as well as changes in cochlear morphology were assessed in gerbil inner ears harvested at selected time intervals up to 18 h postmortem. Na,K-ATPase activity was assayed biochemically in one cochlea from each animal and the other cochlea was fixed and embedded in paraffin for evaluation by light microscopy. Na,K-ATPase antigenicity was assessed by immunostaining with a broad-spectrum antiserum reactive with all known isoforms of the enzyme, and structural preservation was evaluated on adjacent sections stained with hematoxylin and eosin. The results showed a downward trend in enzymatic activity of Na,K-ATPase in lateral wall tissues within 1 h of death. In contrast, Na,K-ATPase immunoreactivity was fairly well preserved with postmortem fixation delays up to 12 h, despite the considerable structural degradation of cochlear tissues which began 2-3 h postmortem. It is concluded that under controlled environmental conditions, cochleas collected up to 4 h postmortem are suitable for morphological and immunohistochemical study of Na,K-ATPase by light microscopy. Cochleas collected more than 5 h postmortem were useful only for relatively gross immunohistochemical studies. It is suggested that cochleas intended for biochemical assays of Na,K-ATPase and probably most other enzymes should be collected within 1 h of death. Keywords: Na,K-ATPase; Cochlea; Autolysis; Immunohistochemistry;Antigenicity; Gerbil
1. Introduction
The study of cochlear histopathology depends inherently on adequate preservation of tissues after death of the host. As a result of postmortem autolysis, a delay in tissue fixation can seriously compromise structural preservation. Previous investigators have described the effect of delayed fixation for light microscopic (LM) study of human and rodent cochleas (Fernandez, 1958; Rutledge, 1969), but no study using LM has determined a consistent maximum delay that allows useful analysis despite interference from autolytic degradation. For ultrastructural analysis, the 'acceptable' postmortem delay prior to fixation has ranged from 15 min (Comis et al., 1990) to 10 h (Bredberg et al., 1972). Preservation of potential target antigens is critical to the
* Corresponding author: Tel.: (803) 792-7977; Fax: (803) 792-7736. 1 Reprint requests: Tel.: (803) 792-7977; Fax: (803) 79247736. 0378-5955/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0378-5955(95)00117-4
success of immunologic studies in tissues harvested from any source. Recent interest in immune-mediated hearing loss has made immunologic study of the cochlea a priority. However, the issue of 'inner ear antigen' stability during postmortem autolysis has not been specifically addressed. Such information is necessary to draw reasonable conclusions about the pathologic significance of any particular antigen identified, especially if cochlear antigens are sought in material harvested after uncontrolled postmortem delays. This problem is compounded by the inherent difficulty of rapidly obtaining and fixing human temporal bones. No previous study has been devoted specifically to examining changes in antigenicity and in functional integrity of individual proteins resulting from autolytic digestion associated with delayed fixation. The purpose of the present study was to investigate postmortem changes in antigenicity and in enzymatic activity of a specific inner ear protein in an animal model under controlled laboratory conditions. The transport enzyme Na,K-ATPase was chosen as a model protein to
D,4. VincentJr. et al. / Hearing Research 89 (1995) 14-20
study autolytic changes. This enzyme has been shown to be abundant in the cochlear lateral wall and minimally present in the organ of Corti using both immunohistochemical techniques (Schulte and Adams, 1989) and a highly sensitive biochemical assay to quantify Na,K-ATPase activity in subregions of individual cochleas (Gratton et al., 1995). The activity of the enzyme is dependent on its structure and orientation in the plasmalemma, and should correlate to some extent with the degree of autolysis. The biochemical assay was used to gauge functional degradation of the enzyme during the postmortem period. Light microscopy was used to monitor morphological alterations and changes in immunoreactivity of Na,K-ATPase resulting from delayed fixation. A preliminary report of this work was presented at the 16th Midwinter Meeting of the Association for Research in Otolaryngology (Vincent et al., 1993).
2. Methods 2.1. Animals and tissue collection
Thirty-nine Mongolian gerbils (average age: 6.5 months; range: 4.5-8.0 months) were studied. All animals were born and raised in an acoustically controlled environment to minimize otoacoustic trauma (Mills et al., 1990). This age range was used since Na,K-ATPase immunoreactivity (Schulte and Adams, 1989) and enzyme activity (Gratton et al., 1995) remain stable in the gerbil cochlea across these ages. The care and use of animals was approved by the Medical University of South Carolina Animal Review Committee under NIH Grant DC 00713. The animals were assigned randomly to groups with postmortem intervals of 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, or 18 h (11 groups). Each animal was killed by lethal injection of urethane (2.0 g / k g body weight i.p.). The time of respiratory arrest was noted and the animal was left intact at room temperature ( = 25°C) for the assigned time interval. The animal was then decapitated, the temporal bones were quickly excised from the skull, and the middle ear bullae were opened to expose the cochlea. One ear from each animal was quick-frozen for biochemical analysis, and the other was fixed for morphologic or immunohistochemical study (see below). 2.2. Determination of Na, K-ATPase activity Tissue preparation Temporal bones allocated for the Na,K-ATPase assay were immediately immersed in liquid nitrogen and stored at - 70°C. They were lyophilized ( - 70°C, 100 Mt) for 24 h (FTS Systems, Stone Ridge, NY), and then stored over dessicant at - 1 0 ° C until dissection. Microdissection (Hawkins and Johnsson, 1976) was performed under a high-power dissecting microscope (Zeiss STEMI SR, 32 X
15
) at room temperature. The otic capsule was thinned with a diamond burr, and the remaining bone was broken away. The lateral wall (including stria vascularis and spiral ligament) and organ of Corti tissues were collected separately in 1.5 ml of micro-centrifuge tubes. The two tubes were returned to a vial over dessicant and stored at - 10°C until assayed. Na, K-ATPase activity assay Enzyme specific activity in lateral wall and organ of Corti specimens was measured using an assay modified from the methods of Forbush (1983) and Johnson et al. (1986), as described in detail by Gratton et al. (1995). Briefly, each dissected sample was dounce homogenized prior to the assay. As a referent for enzyme activity determination, the protein concentration in each sample was measured, using a modified commercial colorimetric assay (Bicinchoninic (BCA) Enhanced Protein Assay, Pierce Chemical Company, Rockford, IL). The modified assay is accurate for protein concentrations between 5 and 250 /xg/ml, which has previously been shown to be an appropriate range for microdissected cochlear tissues (Gratton et al., 1995). Na,K-ATPase-specific activity was determined by measuring the liberation of inorganic phosphate (Pi) from ATP by the enzyme. The details of the colorimetric assay adapted for this purpose are available in Gratton et al. (1995). The fraction of the total liberated Pi that was inhibitable by ouabain was attributed to Na,K-ATPase activity, taking advantage of its relative sensitivity to ouabain inhibition compared to other ATPase enzymes. Specific activity of Na,K-ATPase was expressed as /xmol P i / m g protein/h, corrected for background Pi using heatdeactivated controls. Lack of sufficient tissue amounts precluded multiple measures of enzyme activity on the same cochlea. Therefore, the specific activity of Na,K-ATPase in samples of cultured human kidney proximal tubule cells (Hazen-Martin et al., 1993) was measured concurrently in each run of the assay to determine intra- and inter-assay variance. The stability of Na,K-ATPase activity in this culture line has previously been demonstrated for this application (Gratton et al., 1995). Briefly, the cells were isolated from kidneys deemed inappropriate for transplant for technical reasons, and homogenate preparations of the cells were prepared as described elsewhere (Hazen-Martin et al., 1993). The enzyme assay was performed as described above. The cochlear specific activities were corrected for the variation in the kidney controls when an analysis of covariance (ANCOVA) was significant ( P < 0.05), to permit pooling of data from assays performed on each of three different dates. Statistical methods Statistical analysis of the data included Student's paired t test to assess the difference between lateral wall and
16
D.A. Vincent Jr. et al. /Hearing Research 89 (1995) 14-20
organ of Corti activities with postmortem delay, since the activity of these regions in each ear was measured. Simple regression was applied to determine whether a significant change in activity occurred with postmortem delay for each cochlear region measured. Significance of the regression slope was tested by an analysis of variance (ANOVA). For further analysis of the rate of decline of activity, the data were grouped by postmortem delay interval and an ANOVA was applied to assess differences between groups. A multiple comparison test was applied when an ANOVA was significant to compare the differences between groups, with significance indicated by Fisher's least-significant difference (LSD). For all tests performed, the level of significance was set at 0.05. Statistical analysis was performed using StatView II TM software (Abacus Concepts) on Macintosh hardware.
normal goat serum (NGS) for 30 min, rinsed with P B S NGS, and incubated overnight at 4°C with the Na,KATPase antiserum diluted 1:2000. The sections were rinsed in PBS-NGS, flooded with biotinylated goat anti-rabbit IgG (1:400) for 30 min, rinsed again, and incubated in Vectastain ABC reagent for 30 min (Vector Laboratories, Burlingame, CA). Sites of bound antibody were visualized by development in 3,3'-diaminobenzidine-H20 2 substrate medium. The degree of immunostaining intensity was judged subjectively as higher, lower, or the same as sections not subjected to postmortem fixation delay.
2.3. Morphology and immunohistochemistry
Na,K-ATPase activity in the organ of Corti was much lower than that in the lateral wall (Fig. 1). The ratio of lateral wall to organ of Corti activity in tissue analyzed with no postmortem delay was approximately 15, comparable to our previous data (Gratton et al., 1995). There was a significant difference between lateral wall and organ of Corti enzyme activity at 0 h postmortem (Student's paired t test, P = 0.003, n = 5), which was maintained through all postmortem intervals (paired t test, P = 0.0001, n--39). The cultured kidney control activity showed minimal variability between the three assays (6.43 ___0.32 /zmol P i / m g protein/h) with a coefficient of variation of 5.06%. The effect of the variability in the kidney control on measurement of Na,K-ATPase-specific activity in each region of the cochlea was significant by an ANCOVA only in lateral wall tissues ( F = 10.91, P = 0.002). For organ of Corti tissues, the effect was not significant ( F = 1.09, P = 0.423).
Tissue preparation Temporal bones were prepared for LM study as previously described by Schulte and Schmiedt (1992). After removal of the stapes, the cochleas were perfused through a round-window perforation with 1 ml of 10% formalin in normal saline containing 0.5% zinc dichromate (pH adjusted to 5.0 just prior to use), immersed in the same fixative for 30 min, then rinsed with 0.1 M phosphatebuffered saline (PBS). They were decalcified in 0.35 M 4SS EDTA for 24 h, then dehydrated through a graded ethanol series (70%, 2 h; 80%, 2 h; 95%, 2 × 2 h; 100%, 3 × 1 h). They were cleared in Histoclear (2 × 1 h; National Diagnostics, Manville, N J), and embedded in Paraplast Plus (2 × 1 h at 58°C; Curtin Matheson, Marietta, GA).
Morphology Paraffin-embedded cochleas were serially sectioned (4 /zm) and every 25th section was stained with hematoxylin and eosin. Morphologic preservation of tissue subregions was assessed by LM. Attention was directed particularly to the lateral wall, organ of Corti, and the spiral ganglion cells.
3. Results
3.1. Na, K-ATPase activity
70 60 ' "~' .~
so-
A
A Lateral Wall • Orean of Corti
A
,_~'~ ~
lmmunohistochemistry Immunostaining on selected sections was performed as described previously (Schulte and Adams, 1989) using a rabbit polyclonal antiserum raised against Na,K-ATPase isolated from bovine brain cortex. This antiserum (31b) has been shown to react with all known a- and /3-subunit isoforms of Na,K-ATPase (Hieber et al., 1989) and has been used extensively by this laboratory. It was generously provided by Dr. George Siegel of the Department of Neurology, University of Michigan, Ann Arbor, MI. Tissue sections were deparaffinized, rehydrated, and treated with methanolic-H20 2 to inactivate endogenous peroxidases. They were then equilibrated in PBS and 1.0%
[
A
30'
A
20
z
0.
l',il,|flle • ~
~~
II ~
•
v
IN
"; 1'0fi1:11'61'020
Postmortem Delay (hrs)
Fig. 1. Specific activity of N a , K - A T P a s e in cochlear tissues is plotted as a function of p o s t m o r t e m delay prior to tissue collection. Simple linear regression revealed a significant decrease in lateral wall N a , K - A T P a s e specific activity ( r 2 = 0.23, slope = -1.095, P = 0.002, n = 39), but not in o r g a n of Corti e n z y m e activity ( r 2 = 0.01, P = 0.454, n = 39) after death•
D.A. Vincent Jr. et al• // Hearing Research 89 (1995) 14-20
Organ of Corti Na,K-ATPase activity did not decline significantly postmortem (F1,37 =0•49, ANOVA, P = 0•486)• In contrast, enzyme activity in the lateral wall
" ~,:~ ~/'~U~
;." •
~!!~iii!il¸
17
decreased significantly after death, as shown in Fig. 1 (F1,37 = 11.29, ANOVA, P = 0.002)• Notable in Fig. 1 is the large spread of lateral wall Na,K-ATPase activity for
!iiii~:~ ~
....
2
ao
•-
)
i
?
' 0,
:..,y.
9
o-
.
o.
I
,
.,
k~
Fig. 2. Typical sections from the middle turn of gerbil cochleas fixed after 1 h (a,b), 3 h (c,d), or 12 h (e,f) postmortem delays• Sections were stained with hematoxylin and eosin (a,c,e), or with a polyclonal antiserum against Na,K-ATPase (b,d,f). Morphological preservation in the cochlea harvested 1 h postmortem is similar to that in cochleas fixed immediately after death, and the cochlea fixed 3 h postmortem shows only minimal effects of autolysis with haziness of cellular borders• At 12 h postmortem, there is marked loss of cellular definition and the stria has separated from the lateral wall• The intensity of immunostaining for the Na,K-ATPase complex is maintained fairly well despite loss of the underlying morphology with longer postmortem fixation delay. Original magnification is 100 × .
D.A. Vincent Jr. et al. / Hearing Research 89 (1995) 14-20
18
Table 1 Rate of decline in lateral wall Na,K-ATPase-specific activity with postmortem delay Multiple comparisons Group Postmortem delay (h) n Mean activity ± SD Significance * ( P < 0.05)
1 0-1 11 39.09 + 9.82 a
2 2-4 10 32.11 + 12.42 a,b
3 5-10 10 22.20 + 12.80 b
ANOVA: significant between all groups (F3,35 = 4.92, * Same letter denotes no significant difference.
4 12-18 8 24.06 + 8.81 b P = 0.006).
each postmortem interval group. The variability in enzyme activity did not correlate with gender, age or sibling status, and most probably relates more to the quality of the dissection than to any other factor (Gratton et al., 1995). Changes in Na,K-ATPase activity after death were further examined by apportioning the data into four groups of approximately equal size defined by postmortem delay intervals (Table 1). Again, no significant differences between groups were demonstrated for enzyme activity in the organ of Corti (F3,35 = 0.47, ANOVA, P = 0.703). Lateral wall Na,K-ATPase activity, on the other hand, was found to differ significantly between groups (F3,35 = 4.92, ANOVA, P = 0.006), as shown in Table 1. Multiple comparisons among the four groups indicated significant differences in lateral wall enzyme activity between group 1 (0-1 h delay) and group 3 (5-10 h) (Fisher LSD 9.91), and between group 1 and group 4 (12-18 h) (Fisher LSD 10.54). A downward trend in lateral wall Na,K-ATPasespecific activity was observed in the first 2 h postmortem (Fig. 1). An attempt was made to analyze this decline statistically by dividing the activity data into smaller postmortem interval groups for ANOVA and multiple comparisons as above. However, because of the small number of samples within each group when further subdivided, meaningful analysis was not possible.
3.2. Morphology and Immunohistochemistry Morphology and immunostaining characteristics were assessed at the LM level in cochleas fixed at 0, 1, 3, 5, and 12 h postmortem. Tissue preservation in cochleas fixed I h after death (Fig. 2a) was comparable to that seen in cochleas fixed immediately postmortem. By 3 h postmortem, early indications of deterioration were observed (Fig. 2c). Cellular borders in the organ of Corti and stria vascularis became indistinct, though hair cell nuclei were visible and the stria remained well-defined on the lateral wall. At 5 h after death and beyond, there was increasing loss of structural definition both in the organ of Corti and in the stria vascularis, including separation of the stria from the lateral wall (Fig. 2e). The connective tissue structures of the membranous cochlea were more resistant to autolytic degradation, with
little change noted in the basilar membrane, tectorial membrane, spiral limbus, suprastrial cells, or fibrocytes in the spiral ligament. The spiral ganglion cells showed only minor morphologic change at 3 h postmortem (Fig. 2c), but showed a marked loss of structural integrity at 5 and 12 h after death (Fig. 2e). Antigenicity of Na,K-ATPase as evidenced by immunostaining was not notably affected by postmortem fixation delays up to 3 h (Fig. 2b,d). A decrease in immunostaining intensity was apparent in some sites with a 12 h delay in fixation (Fig. 2f). However, this may be related more to alterations in structure than to decreased antigenicity. Localization of the enzyme was unchanged, with staining seen at the basolateral surface of strial marginal cells (despite separation from the lateral wall), in type II and suprastrial fibrocytes, and in satellite cells surrounding degenerating neurons (Fig. 2f).
4. Discussion It is difficult, if not impossible, to obtain human temporal bones within minutes of death. In fact, the best source of such specimens is often the forensic pathology suite, where a delay in fixation of several hours is routine. This study was initiated to ascertain the potential usefulness of human temporal bones collected at uncontrolled times after death for immunologic, immunohistochemical, and functional studies of various types of hearing loss. To this end, changes in the activity and antigenicity of a key transport enzyme and concurrent changes in cochlear morphology due to in situ postmortem autolysis were examined in a gerbil model under controlled conditions. The enzymatic activity of Na,K-ATPase was shown to decline in the first few hours after death. The decrease in biochemical activity may result from a number of processes occurring during the postmortem period. One likely mechanism is degradation of Na,K-ATPase by endogenous proteases. The most probable site of degradation is the a-subunit, since the /3-subunit is heavily glycosylated and resists proteolysis despite its extracellular exposure (Jorgensen and Farley, 1988). In addition, the ATP catalytic site of the Na,K-ATPase complex is located in the a-subunit on the cytoplasmic face of the plasmalemma, and exposure to specific proteases is known to inhibit its function. For example, trypsin inhibits enzyme activity by cleaving the a-subunit in exposed regions of transmembrane loops, with the precise sites of cleavage determined by the conformational state of the enzyme (Jorgensen and Farley, 1988). Common lysosomal enzymes also have been shown to inhibit Na,K-ATPase function, particularly when the enzyme is in an oxidized state, as occurs during ischemia-reperfusion injury. Zolotarjova et al. (1994) investigated the effects of endogenous proteolytic enzymes on all three a-subunit isoforms of Na,K-ATPase, both in the native state and after exposure to partially reduced
D.A. Vincent Jr. et al. / H e a r i n g Research 89 (1995) 14-20
oxygen metabolites by pretreatment with H202. In all cases, the oxidized forms were more sensitive to inhibition by lysosomal cathepsin D and the ubiquitous Ca2+-depen dent intracellular calpains (m-calpain and /z-calpain). This finding supports the theory that oxidatively damaged Na,K-ATPase may preferentially undergo endocytosis and lysosomal degradation as tissue subjected to ischemia attempts to repair itself (Zolotarjova et al., 1994). While the data in the present study only allow limited conjecture concerning the precise means by which Na,KATPase activity is lost during necrosis, it does demonstrate a gross decline in enzyme function postmortem. Statistical analysis revealed that the decline becomes significant 2 - 4 h after death. However, a decrease in activity was observed in the 1st hour after death, but it did not reach statistical significance most likely owing to the small sample size at each postmortem interval (Fig. 1). Because of this early trend, we conclude that biochemical assay of Na,K-ATPase, and possibly other enzymes as well, should ideally be performed on tissues collected and preserved within the 1st hour after death. The decrease in enzymatic activity of Na,K-ATPase was not reflected by a similar loss in its antigenicity. The polyclonal antiserum used in this study has been shown to react with most (if not all) of the known subunit isoforms of Na,K-ATPase (O/1--O~3, ~1' ~2 )' and most probably reacts with several different epitopes in each. Successful immunostaining for Na,K-ATPase by LM was demonstrated with fixation delays up to 12 h postmortem. With fixation delays up to 5 h, neither the distribution of immunoreactive Na,K-ATPase nor the intensity of immunostaining changed appreciably from that reported in inner ears fixed immediately after death (Schulte and Adams, 1989). This finding cannot be generalized to other antigens, since resistance or susceptibility to postmortem autolysis is certain to vary among epitopes. In any case, loss of morphological detail precludes high-resolution immunostaining studies in the organ of Corti and stria vascularis of cochleas unprotected by fixation longer than 3-5 h postmortem. Marked morphologic changes were not detectable by routine LM on hematoxylin/eosin-stained sections of gerbil cochleas prior to 3 h postmortem. The earliest morphologic changes occurred in the stria vascularis and the outer hair cells. This is consistent with the work of Matschinsky and Thalmann (1967) and Thalmann et al. (1972), who investigated the sensitivity of cochlear energy reserves to ischemic insult. Their data showed that the metabolically active stria vascularis was the first cochlear region to consume its energy reserve following an ischemic insult, with marked depletion of energy stores in the first few minutes of ischemia. The organ of Corti was demonstrated to have a much more ample energy reserve for its metabolic rate, and was more resistant to ischemia (Thalmann et al., 1972); however, it still showed much more metabolic activity than supporting tissues. It is probable that deple-
19
tion of energy stores in the absence of respiration and circulation leaves the stria vascularis and organ of Corti susceptible to degradation during the first stages of tissue necrosis. The relative absence of early postmortem morphologic changes observed at the LM level in the supporting fibrocytes and bony capsule possibly reflects the resistance of these structures to ischemia and autolysis by virtue of their low metabolic demand. This study was undertaken in light of recent interest in immune-mediated hearing loss. To our knowledge, it is the first study to examine concurrently the antigenic and functional properties of a particular inner ear protein under conditions of postmortem autolysis. Such information could be of considerable value in any study of cochlear function or structure in which postmortem delay in temporal bone collection is likely. These findings emphasize the fact that antigenicity and functional structure are not inseparably linked.
Acknowledgements The authors wish to thank Dr. George Siegel for his gift of the antiserum against Na,K-ATPase, and Dr. Debra Hazen-Martin (Department of Pathology and Laboratory Medicine, Medical University of South Carolina) for providing cultured human kidney proximal tubule cells for our assay control. We also thank Dr. Fu-Shing Lee for his assistance with statistical analysis, Ms. Sharon Munyer for her help in preparation of histologic sections, and Ms. Nancy Smythe for her technical assistance in preparation of the photomicrographs. This study was supported in part by NIH Grants DC 00713 and DC 00422.
References Bredberg, G., Ades, H.W., and Engstrom, H. (1972) Scanning electron microscopy of the normal and pathologically altered organ of Corti. Acta Otolaryngol. (Stockh). Suppl. 301. Comis, S.D., Osborne, M.P., O'Connell, J., and Johnson, A.P. (1990) The importance of early fixation in preservation of human cochlear and vestibular sensory hair bundles. Acta Otolaryngol. (Stockh). 109, 361-368. Fernandez, C. (1958) Postmortem changes and artifacts in human temporal bones. Laryngoscope 68, 1586-1615. Forbush, B. (1983) Assay of Na, K-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin. Anal. Biochem. 128, 159-163. Gratton, M.A., Smyth, B.J., Schulte, B.A., and Vincent, D.A. (1995) Na,K-ATPase activity decreases in the cochlear lateral wall of quietaged gerbils. Hearing Res. 83, 43-50. Hawkins, J.E., and Johnsson, L.-G. (1976) Microdissection and surface preparations of the inner ear. In: C.A. Smith and J.A. Vernon (Eds.), Handbook of Auditory and Vestibular Research Methods, CC Thomas, Springfield, IL, pp. 5-52. Hazen-Martin, D.J., Todd, J.H., Sens, M.A., Khan, W., Bylander, J.E., Smyth, B.J., and Sens, D.A. (1993) Electrical and freeze-fracture
20
D.A. Vincent Jr. et aL // Hearing Research 89 (1995) 14-20
analysis of the effects of ionic cadmium on cell membranes of human proximal tubule cells. Environ. Hlth Pers. 101,510-516. Hieber, V., Siegel, G.J., Desmond, T., Liu, J.L.-H., and Ernst, S.A. (1989) Na,K-ATPase: comparison of the cellular localization of alpha-subunit mRNA and polypeptide in mouse cerebellum, retina, and kidney. J. Neurosci. Res. 23, 9-20. Johnson, J.P., Jones, D., and Wiesmann, W.P. (1986) Hormonal regulation of Na,K-ATPase in cultured epithelial cells. Am. J. Physiol. 251, C186-C190. Jorgensen, P.L., and Farley, R.A. (1988) Proteolytic cleavage as a tool for studying structure and conformation of pure membrane-bound Na,KATPase. Methods Enzymol. 156, 291-301. Matschinsky, F.M., and Thalmann, R. (1967) Quantitative histochemistry of microscopic structures of the cochlea. II. Ischemic alterations of levels of glycolytic intermediates and cofactors in the organ of Corti and stria vascularis. Ann. Otol. 76, 638-647. Mills, J.H., Schmiedt, R.A., and Kulish, L.F. (1990) Age-related changes in auditory potentials of mongolian gerbil. Hearing Res. 46, 201-210. Rutledge, L.J. (1969) Histologic study of the perfused human temporal bone. Laryngoscope 79, 2104-2125.
Schulte, B.A., and Adams, J.C. (1989) Distribution of immunoreactive Na,K-ATPase in gerbil cochlea. J. Histochem. Cytochem. 37, 127134. Schulte, B.A., and Schmiedt, R.A. (1992) Lateral wall Na,K-ATPase and endocochlear potentials decline with age in quiet-reared gerbils. Hearing Res. 61, 35-46. Thalmann, R., Miyoshi, T., and Thalmann, I. (1972) The influence of ischemia upon the energy reserves of inner ear tissues. Laryngoscope 82, 2249-2272. Vincent, D.A., Smyth, B.J., Winston, D., Gratton, M.A., and Schulte, B.A. (1993) Effect of postmortem autolysis on protein structure and Na, K-ATPase activity in lateral wall and organ of Corti from individual cochleas. Abstr. Assoc. Res. Otolaryngol. 16, 137. Zolotarjova, N., Ho, C., Mellgren, R.L., Askari, A., and Huang, W. (1994) Different sensitivities of native and oxidized forms of Na,KATPase to intracellular proteinases. Biochem. Biophys. Acta 1192, 125-131.
Hearing Research 89 (1995) 14-20
Effect of postmortem autolysis on Na,K-ATPase activity and antigenicity in the gerbil cochlea Daniel A. Vincent Jr. a,*, Michael Anne Gratton a,1, Brendan J. Smyth b, Bradley A. Schulte b a Department of Otolaryngology and Communicative Sciences, Medical Unit,ersity of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA b Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA
Received 7 October 1994; revised 8 May 1995; accepted 16 May 1995
Abstract
Alterations in the enzymatic activity and antigenicity of Na,K-ATPase as well as changes in cochlear morphology were assessed in gerbil inner ears harvested at selected time intervals up to 18 h postmortem. Na,K-ATPase activity was assayed biochemically in one cochlea from each animal and the other cochlea was fixed and embedded in paraffin for evaluation by light microscopy. Na,K-ATPase antigenicity was assessed by immunostaining with a broad-spectrum antiserum reactive with all known isoforms of the enzyme, and structural preservation was evaluated on adjacent sections stained with hematoxylin and eosin. The results showed a downward trend in enzymatic activity of Na,K-ATPase in lateral wall tissues within 1 h of death. In contrast, Na,K-ATPase immunoreactivity was fairly well preserved with postmortem fixation delays up to 12 h, despite the considerable structural degradation of cochlear tissues which began 2-3 h postmortem. It is concluded that under controlled environmental conditions, cochleas collected up to 4 h postmortem are suitable for morphological and immunohistochemical study of Na,K-ATPase by light microscopy. Cochleas collected more than 5 h postmortem were useful only for relatively gross immunohistochemical studies. It is suggested that cochleas intended for biochemical assays of Na,K-ATPase and probably most other enzymes should be collected within 1 h of death. Keywords: Na,K-ATPase; Cochlea; Autolysis; Immunohistochemistry;Antigenicity; Gerbil
1. Introduction
The study of cochlear histopathology depends inherently on adequate preservation of tissues after death of the host. As a result of postmortem autolysis, a delay in tissue fixation can seriously compromise structural preservation. Previous investigators have described the effect of delayed fixation for light microscopic (LM) study of human and rodent cochleas (Fernandez, 1958; Rutledge, 1969), but no study using LM has determined a consistent maximum delay that allows useful analysis despite interference from autolytic degradation. For ultrastructural analysis, the 'acceptable' postmortem delay prior to fixation has ranged from 15 min (Comis et al., 1990) to 10 h (Bredberg et al., 1972). Preservation of potential target antigens is critical to the
* Corresponding author: Tel.: (803) 792-7977; Fax: (803) 792-7736. 1 Reprint requests: Tel.: (803) 792-7977; Fax: (803) 79247736. 0378-5955/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0378-5955(95)00117-4
success of immunologic studies in tissues harvested from any source. Recent interest in immune-mediated hearing loss has made immunologic study of the cochlea a priority. However, the issue of 'inner ear antigen' stability during postmortem autolysis has not been specifically addressed. Such information is necessary to draw reasonable conclusions about the pathologic significance of any particular antigen identified, especially if cochlear antigens are sought in material harvested after uncontrolled postmortem delays. This problem is compounded by the inherent difficulty of rapidly obtaining and fixing human temporal bones. No previous study has been devoted specifically to examining changes in antigenicity and in functional integrity of individual proteins resulting from autolytic digestion associated with delayed fixation. The purpose of the present study was to investigate postmortem changes in antigenicity and in enzymatic activity of a specific inner ear protein in an animal model under controlled laboratory conditions. The transport enzyme Na,K-ATPase was chosen as a model protein to
D,4. VincentJr. et al. / Hearing Research 89 (1995) 14-20
study autolytic changes. This enzyme has been shown to be abundant in the cochlear lateral wall and minimally present in the organ of Corti using both immunohistochemical techniques (Schulte and Adams, 1989) and a highly sensitive biochemical assay to quantify Na,K-ATPase activity in subregions of individual cochleas (Gratton et al., 1995). The activity of the enzyme is dependent on its structure and orientation in the plasmalemma, and should correlate to some extent with the degree of autolysis. The biochemical assay was used to gauge functional degradation of the enzyme during the postmortem period. Light microscopy was used to monitor morphological alterations and changes in immunoreactivity of Na,K-ATPase resulting from delayed fixation. A preliminary report of this work was presented at the 16th Midwinter Meeting of the Association for Research in Otolaryngology (Vincent et al., 1993).
2. Methods 2.1. Animals and tissue collection
Thirty-nine Mongolian gerbils (average age: 6.5 months; range: 4.5-8.0 months) were studied. All animals were born and raised in an acoustically controlled environment to minimize otoacoustic trauma (Mills et al., 1990). This age range was used since Na,K-ATPase immunoreactivity (Schulte and Adams, 1989) and enzyme activity (Gratton et al., 1995) remain stable in the gerbil cochlea across these ages. The care and use of animals was approved by the Medical University of South Carolina Animal Review Committee under NIH Grant DC 00713. The animals were assigned randomly to groups with postmortem intervals of 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, or 18 h (11 groups). Each animal was killed by lethal injection of urethane (2.0 g / k g body weight i.p.). The time of respiratory arrest was noted and the animal was left intact at room temperature ( = 25°C) for the assigned time interval. The animal was then decapitated, the temporal bones were quickly excised from the skull, and the middle ear bullae were opened to expose the cochlea. One ear from each animal was quick-frozen for biochemical analysis, and the other was fixed for morphologic or immunohistochemical study (see below). 2.2. Determination of Na, K-ATPase activity Tissue preparation Temporal bones allocated for the Na,K-ATPase assay were immediately immersed in liquid nitrogen and stored at - 70°C. They were lyophilized ( - 70°C, 100 Mt) for 24 h (FTS Systems, Stone Ridge, NY), and then stored over dessicant at - 1 0 ° C until dissection. Microdissection (Hawkins and Johnsson, 1976) was performed under a high-power dissecting microscope (Zeiss STEMI SR, 32 X
15
) at room temperature. The otic capsule was thinned with a diamond burr, and the remaining bone was broken away. The lateral wall (including stria vascularis and spiral ligament) and organ of Corti tissues were collected separately in 1.5 ml of micro-centrifuge tubes. The two tubes were returned to a vial over dessicant and stored at - 10°C until assayed. Na, K-ATPase activity assay Enzyme specific activity in lateral wall and organ of Corti specimens was measured using an assay modified from the methods of Forbush (1983) and Johnson et al. (1986), as described in detail by Gratton et al. (1995). Briefly, each dissected sample was dounce homogenized prior to the assay. As a referent for enzyme activity determination, the protein concentration in each sample was measured, using a modified commercial colorimetric assay (Bicinchoninic (BCA) Enhanced Protein Assay, Pierce Chemical Company, Rockford, IL). The modified assay is accurate for protein concentrations between 5 and 250 /xg/ml, which has previously been shown to be an appropriate range for microdissected cochlear tissues (Gratton et al., 1995). Na,K-ATPase-specific activity was determined by measuring the liberation of inorganic phosphate (Pi) from ATP by the enzyme. The details of the colorimetric assay adapted for this purpose are available in Gratton et al. (1995). The fraction of the total liberated Pi that was inhibitable by ouabain was attributed to Na,K-ATPase activity, taking advantage of its relative sensitivity to ouabain inhibition compared to other ATPase enzymes. Specific activity of Na,K-ATPase was expressed as /xmol P i / m g protein/h, corrected for background Pi using heatdeactivated controls. Lack of sufficient tissue amounts precluded multiple measures of enzyme activity on the same cochlea. Therefore, the specific activity of Na,K-ATPase in samples of cultured human kidney proximal tubule cells (Hazen-Martin et al., 1993) was measured concurrently in each run of the assay to determine intra- and inter-assay variance. The stability of Na,K-ATPase activity in this culture line has previously been demonstrated for this application (Gratton et al., 1995). Briefly, the cells were isolated from kidneys deemed inappropriate for transplant for technical reasons, and homogenate preparations of the cells were prepared as described elsewhere (Hazen-Martin et al., 1993). The enzyme assay was performed as described above. The cochlear specific activities were corrected for the variation in the kidney controls when an analysis of covariance (ANCOVA) was significant ( P < 0.05), to permit pooling of data from assays performed on each of three different dates. Statistical methods Statistical analysis of the data included Student's paired t test to assess the difference between lateral wall and
16
D.A. Vincent Jr. et al. /Hearing Research 89 (1995) 14-20
organ of Corti activities with postmortem delay, since the activity of these regions in each ear was measured. Simple regression was applied to determine whether a significant change in activity occurred with postmortem delay for each cochlear region measured. Significance of the regression slope was tested by an analysis of variance (ANOVA). For further analysis of the rate of decline of activity, the data were grouped by postmortem delay interval and an ANOVA was applied to assess differences between groups. A multiple comparison test was applied when an ANOVA was significant to compare the differences between groups, with significance indicated by Fisher's least-significant difference (LSD). For all tests performed, the level of significance was set at 0.05. Statistical analysis was performed using StatView II TM software (Abacus Concepts) on Macintosh hardware.
normal goat serum (NGS) for 30 min, rinsed with P B S NGS, and incubated overnight at 4°C with the Na,KATPase antiserum diluted 1:2000. The sections were rinsed in PBS-NGS, flooded with biotinylated goat anti-rabbit IgG (1:400) for 30 min, rinsed again, and incubated in Vectastain ABC reagent for 30 min (Vector Laboratories, Burlingame, CA). Sites of bound antibody were visualized by development in 3,3'-diaminobenzidine-H20 2 substrate medium. The degree of immunostaining intensity was judged subjectively as higher, lower, or the same as sections not subjected to postmortem fixation delay.
2.3. Morphology and immunohistochemistry
Na,K-ATPase activity in the organ of Corti was much lower than that in the lateral wall (Fig. 1). The ratio of lateral wall to organ of Corti activity in tissue analyzed with no postmortem delay was approximately 15, comparable to our previous data (Gratton et al., 1995). There was a significant difference between lateral wall and organ of Corti enzyme activity at 0 h postmortem (Student's paired t test, P = 0.003, n = 5), which was maintained through all postmortem intervals (paired t test, P = 0.0001, n--39). The cultured kidney control activity showed minimal variability between the three assays (6.43 ___0.32 /zmol P i / m g protein/h) with a coefficient of variation of 5.06%. The effect of the variability in the kidney control on measurement of Na,K-ATPase-specific activity in each region of the cochlea was significant by an ANCOVA only in lateral wall tissues ( F = 10.91, P = 0.002). For organ of Corti tissues, the effect was not significant ( F = 1.09, P = 0.423).
Tissue preparation Temporal bones were prepared for LM study as previously described by Schulte and Schmiedt (1992). After removal of the stapes, the cochleas were perfused through a round-window perforation with 1 ml of 10% formalin in normal saline containing 0.5% zinc dichromate (pH adjusted to 5.0 just prior to use), immersed in the same fixative for 30 min, then rinsed with 0.1 M phosphatebuffered saline (PBS). They were decalcified in 0.35 M 4SS EDTA for 24 h, then dehydrated through a graded ethanol series (70%, 2 h; 80%, 2 h; 95%, 2 × 2 h; 100%, 3 × 1 h). They were cleared in Histoclear (2 × 1 h; National Diagnostics, Manville, N J), and embedded in Paraplast Plus (2 × 1 h at 58°C; Curtin Matheson, Marietta, GA).
Morphology Paraffin-embedded cochleas were serially sectioned (4 /zm) and every 25th section was stained with hematoxylin and eosin. Morphologic preservation of tissue subregions was assessed by LM. Attention was directed particularly to the lateral wall, organ of Corti, and the spiral ganglion cells.
3. Results
3.1. Na, K-ATPase activity
70 60 ' "~' .~
so-
A
A Lateral Wall • Orean of Corti
A
,_~'~ ~
lmmunohistochemistry Immunostaining on selected sections was performed as described previously (Schulte and Adams, 1989) using a rabbit polyclonal antiserum raised against Na,K-ATPase isolated from bovine brain cortex. This antiserum (31b) has been shown to react with all known a- and /3-subunit isoforms of Na,K-ATPase (Hieber et al., 1989) and has been used extensively by this laboratory. It was generously provided by Dr. George Siegel of the Department of Neurology, University of Michigan, Ann Arbor, MI. Tissue sections were deparaffinized, rehydrated, and treated with methanolic-H20 2 to inactivate endogenous peroxidases. They were then equilibrated in PBS and 1.0%
[
A
30'
A
20
z
0.
l',il,|flle • ~
~~
II ~
•
v
IN
"; 1'0fi1:11'61'020
Postmortem Delay (hrs)
Fig. 1. Specific activity of N a , K - A T P a s e in cochlear tissues is plotted as a function of p o s t m o r t e m delay prior to tissue collection. Simple linear regression revealed a significant decrease in lateral wall N a , K - A T P a s e specific activity ( r 2 = 0.23, slope = -1.095, P = 0.002, n = 39), but not in o r g a n of Corti e n z y m e activity ( r 2 = 0.01, P = 0.454, n = 39) after death•
D.A. Vincent Jr. et al• // Hearing Research 89 (1995) 14-20
Organ of Corti Na,K-ATPase activity did not decline significantly postmortem (F1,37 =0•49, ANOVA, P = 0•486)• In contrast, enzyme activity in the lateral wall
" ~,:~ ~/'~U~
;." •
~!!~iii!il¸
17
decreased significantly after death, as shown in Fig. 1 (F1,37 = 11.29, ANOVA, P = 0.002)• Notable in Fig. 1 is the large spread of lateral wall Na,K-ATPase activity for
!iiii~:~ ~
....
2
ao
•-
)
i
?
' 0,
:..,y.
9
o-
.
o.
I
,
.,
k~
Fig. 2. Typical sections from the middle turn of gerbil cochleas fixed after 1 h (a,b), 3 h (c,d), or 12 h (e,f) postmortem delays• Sections were stained with hematoxylin and eosin (a,c,e), or with a polyclonal antiserum against Na,K-ATPase (b,d,f). Morphological preservation in the cochlea harvested 1 h postmortem is similar to that in cochleas fixed immediately after death, and the cochlea fixed 3 h postmortem shows only minimal effects of autolysis with haziness of cellular borders• At 12 h postmortem, there is marked loss of cellular definition and the stria has separated from the lateral wall• The intensity of immunostaining for the Na,K-ATPase complex is maintained fairly well despite loss of the underlying morphology with longer postmortem fixation delay. Original magnification is 100 × .
D.A. Vincent Jr. et al. / Hearing Research 89 (1995) 14-20
18
Table 1 Rate of decline in lateral wall Na,K-ATPase-specific activity with postmortem delay Multiple comparisons Group Postmortem delay (h) n Mean activity ± SD Significance * ( P < 0.05)
1 0-1 11 39.09 + 9.82 a
2 2-4 10 32.11 + 12.42 a,b
3 5-10 10 22.20 + 12.80 b
ANOVA: significant between all groups (F3,35 = 4.92, * Same letter denotes no significant difference.
4 12-18 8 24.06 + 8.81 b P = 0.006).
each postmortem interval group. The variability in enzyme activity did not correlate with gender, age or sibling status, and most probably relates more to the quality of the dissection than to any other factor (Gratton et al., 1995). Changes in Na,K-ATPase activity after death were further examined by apportioning the data into four groups of approximately equal size defined by postmortem delay intervals (Table 1). Again, no significant differences between groups were demonstrated for enzyme activity in the organ of Corti (F3,35 = 0.47, ANOVA, P = 0.703). Lateral wall Na,K-ATPase activity, on the other hand, was found to differ significantly between groups (F3,35 = 4.92, ANOVA, P = 0.006), as shown in Table 1. Multiple comparisons among the four groups indicated significant differences in lateral wall enzyme activity between group 1 (0-1 h delay) and group 3 (5-10 h) (Fisher LSD 9.91), and between group 1 and group 4 (12-18 h) (Fisher LSD 10.54). A downward trend in lateral wall Na,K-ATPasespecific activity was observed in the first 2 h postmortem (Fig. 1). An attempt was made to analyze this decline statistically by dividing the activity data into smaller postmortem interval groups for ANOVA and multiple comparisons as above. However, because of the small number of samples within each group when further subdivided, meaningful analysis was not possible.
3.2. Morphology and Immunohistochemistry Morphology and immunostaining characteristics were assessed at the LM level in cochleas fixed at 0, 1, 3, 5, and 12 h postmortem. Tissue preservation in cochleas fixed I h after death (Fig. 2a) was comparable to that seen in cochleas fixed immediately postmortem. By 3 h postmortem, early indications of deterioration were observed (Fig. 2c). Cellular borders in the organ of Corti and stria vascularis became indistinct, though hair cell nuclei were visible and the stria remained well-defined on the lateral wall. At 5 h after death and beyond, there was increasing loss of structural definition both in the organ of Corti and in the stria vascularis, including separation of the stria from the lateral wall (Fig. 2e). The connective tissue structures of the membranous cochlea were more resistant to autolytic degradation, with
little change noted in the basilar membrane, tectorial membrane, spiral limbus, suprastrial cells, or fibrocytes in the spiral ligament. The spiral ganglion cells showed only minor morphologic change at 3 h postmortem (Fig. 2c), but showed a marked loss of structural integrity at 5 and 12 h after death (Fig. 2e). Antigenicity of Na,K-ATPase as evidenced by immunostaining was not notably affected by postmortem fixation delays up to 3 h (Fig. 2b,d). A decrease in immunostaining intensity was apparent in some sites with a 12 h delay in fixation (Fig. 2f). However, this may be related more to alterations in structure than to decreased antigenicity. Localization of the enzyme was unchanged, with staining seen at the basolateral surface of strial marginal cells (despite separation from the lateral wall), in type II and suprastrial fibrocytes, and in satellite cells surrounding degenerating neurons (Fig. 2f).
4. Discussion It is difficult, if not impossible, to obtain human temporal bones within minutes of death. In fact, the best source of such specimens is often the forensic pathology suite, where a delay in fixation of several hours is routine. This study was initiated to ascertain the potential usefulness of human temporal bones collected at uncontrolled times after death for immunologic, immunohistochemical, and functional studies of various types of hearing loss. To this end, changes in the activity and antigenicity of a key transport enzyme and concurrent changes in cochlear morphology due to in situ postmortem autolysis were examined in a gerbil model under controlled conditions. The enzymatic activity of Na,K-ATPase was shown to decline in the first few hours after death. The decrease in biochemical activity may result from a number of processes occurring during the postmortem period. One likely mechanism is degradation of Na,K-ATPase by endogenous proteases. The most probable site of degradation is the a-subunit, since the /3-subunit is heavily glycosylated and resists proteolysis despite its extracellular exposure (Jorgensen and Farley, 1988). In addition, the ATP catalytic site of the Na,K-ATPase complex is located in the a-subunit on the cytoplasmic face of the plasmalemma, and exposure to specific proteases is known to inhibit its function. For example, trypsin inhibits enzyme activity by cleaving the a-subunit in exposed regions of transmembrane loops, with the precise sites of cleavage determined by the conformational state of the enzyme (Jorgensen and Farley, 1988). Common lysosomal enzymes also have been shown to inhibit Na,K-ATPase function, particularly when the enzyme is in an oxidized state, as occurs during ischemia-reperfusion injury. Zolotarjova et al. (1994) investigated the effects of endogenous proteolytic enzymes on all three a-subunit isoforms of Na,K-ATPase, both in the native state and after exposure to partially reduced
D.A. Vincent Jr. et al. / H e a r i n g Research 89 (1995) 14-20
oxygen metabolites by pretreatment with H202. In all cases, the oxidized forms were more sensitive to inhibition by lysosomal cathepsin D and the ubiquitous Ca2+-depen dent intracellular calpains (m-calpain and /z-calpain). This finding supports the theory that oxidatively damaged Na,K-ATPase may preferentially undergo endocytosis and lysosomal degradation as tissue subjected to ischemia attempts to repair itself (Zolotarjova et al., 1994). While the data in the present study only allow limited conjecture concerning the precise means by which Na,KATPase activity is lost during necrosis, it does demonstrate a gross decline in enzyme function postmortem. Statistical analysis revealed that the decline becomes significant 2 - 4 h after death. However, a decrease in activity was observed in the 1st hour after death, but it did not reach statistical significance most likely owing to the small sample size at each postmortem interval (Fig. 1). Because of this early trend, we conclude that biochemical assay of Na,K-ATPase, and possibly other enzymes as well, should ideally be performed on tissues collected and preserved within the 1st hour after death. The decrease in enzymatic activity of Na,K-ATPase was not reflected by a similar loss in its antigenicity. The polyclonal antiserum used in this study has been shown to react with most (if not all) of the known subunit isoforms of Na,K-ATPase (O/1--O~3, ~1' ~2 )' and most probably reacts with several different epitopes in each. Successful immunostaining for Na,K-ATPase by LM was demonstrated with fixation delays up to 12 h postmortem. With fixation delays up to 5 h, neither the distribution of immunoreactive Na,K-ATPase nor the intensity of immunostaining changed appreciably from that reported in inner ears fixed immediately after death (Schulte and Adams, 1989). This finding cannot be generalized to other antigens, since resistance or susceptibility to postmortem autolysis is certain to vary among epitopes. In any case, loss of morphological detail precludes high-resolution immunostaining studies in the organ of Corti and stria vascularis of cochleas unprotected by fixation longer than 3-5 h postmortem. Marked morphologic changes were not detectable by routine LM on hematoxylin/eosin-stained sections of gerbil cochleas prior to 3 h postmortem. The earliest morphologic changes occurred in the stria vascularis and the outer hair cells. This is consistent with the work of Matschinsky and Thalmann (1967) and Thalmann et al. (1972), who investigated the sensitivity of cochlear energy reserves to ischemic insult. Their data showed that the metabolically active stria vascularis was the first cochlear region to consume its energy reserve following an ischemic insult, with marked depletion of energy stores in the first few minutes of ischemia. The organ of Corti was demonstrated to have a much more ample energy reserve for its metabolic rate, and was more resistant to ischemia (Thalmann et al., 1972); however, it still showed much more metabolic activity than supporting tissues. It is probable that deple-
19
tion of energy stores in the absence of respiration and circulation leaves the stria vascularis and organ of Corti susceptible to degradation during the first stages of tissue necrosis. The relative absence of early postmortem morphologic changes observed at the LM level in the supporting fibrocytes and bony capsule possibly reflects the resistance of these structures to ischemia and autolysis by virtue of their low metabolic demand. This study was undertaken in light of recent interest in immune-mediated hearing loss. To our knowledge, it is the first study to examine concurrently the antigenic and functional properties of a particular inner ear protein under conditions of postmortem autolysis. Such information could be of considerable value in any study of cochlear function or structure in which postmortem delay in temporal bone collection is likely. These findings emphasize the fact that antigenicity and functional structure are not inseparably linked.
Acknowledgements The authors wish to thank Dr. George Siegel for his gift of the antiserum against Na,K-ATPase, and Dr. Debra Hazen-Martin (Department of Pathology and Laboratory Medicine, Medical University of South Carolina) for providing cultured human kidney proximal tubule cells for our assay control. We also thank Dr. Fu-Shing Lee for his assistance with statistical analysis, Ms. Sharon Munyer for her help in preparation of histologic sections, and Ms. Nancy Smythe for her technical assistance in preparation of the photomicrographs. This study was supported in part by NIH Grants DC 00713 and DC 00422.
References Bredberg, G., Ades, H.W., and Engstrom, H. (1972) Scanning electron microscopy of the normal and pathologically altered organ of Corti. Acta Otolaryngol. (Stockh). Suppl. 301. Comis, S.D., Osborne, M.P., O'Connell, J., and Johnson, A.P. (1990) The importance of early fixation in preservation of human cochlear and vestibular sensory hair bundles. Acta Otolaryngol. (Stockh). 109, 361-368. Fernandez, C. (1958) Postmortem changes and artifacts in human temporal bones. Laryngoscope 68, 1586-1615. Forbush, B. (1983) Assay of Na, K-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin. Anal. Biochem. 128, 159-163. Gratton, M.A., Smyth, B.J., Schulte, B.A., and Vincent, D.A. (1995) Na,K-ATPase activity decreases in the cochlear lateral wall of quietaged gerbils. Hearing Res. 83, 43-50. Hawkins, J.E., and Johnsson, L.-G. (1976) Microdissection and surface preparations of the inner ear. In: C.A. Smith and J.A. Vernon (Eds.), Handbook of Auditory and Vestibular Research Methods, CC Thomas, Springfield, IL, pp. 5-52. Hazen-Martin, D.J., Todd, J.H., Sens, M.A., Khan, W., Bylander, J.E., Smyth, B.J., and Sens, D.A. (1993) Electrical and freeze-fracture
20
D.A. Vincent Jr. et aL // Hearing Research 89 (1995) 14-20
analysis of the effects of ionic cadmium on cell membranes of human proximal tubule cells. Environ. Hlth Pers. 101,510-516. Hieber, V., Siegel, G.J., Desmond, T., Liu, J.L.-H., and Ernst, S.A. (1989) Na,K-ATPase: comparison of the cellular localization of alpha-subunit mRNA and polypeptide in mouse cerebellum, retina, and kidney. J. Neurosci. Res. 23, 9-20. Johnson, J.P., Jones, D., and Wiesmann, W.P. (1986) Hormonal regulation of Na,K-ATPase in cultured epithelial cells. Am. J. Physiol. 251, C186-C190. Jorgensen, P.L., and Farley, R.A. (1988) Proteolytic cleavage as a tool for studying structure and conformation of pure membrane-bound Na,KATPase. Methods Enzymol. 156, 291-301. Matschinsky, F.M., and Thalmann, R. (1967) Quantitative histochemistry of microscopic structures of the cochlea. II. Ischemic alterations of levels of glycolytic intermediates and cofactors in the organ of Corti and stria vascularis. Ann. Otol. 76, 638-647. Mills, J.H., Schmiedt, R.A., and Kulish, L.F. (1990) Age-related changes in auditory potentials of mongolian gerbil. Hearing Res. 46, 201-210. Rutledge, L.J. (1969) Histologic study of the perfused human temporal bone. Laryngoscope 79, 2104-2125.
Schulte, B.A., and Adams, J.C. (1989) Distribution of immunoreactive Na,K-ATPase in gerbil cochlea. J. Histochem. Cytochem. 37, 127134. Schulte, B.A., and Schmiedt, R.A. (1992) Lateral wall Na,K-ATPase and endocochlear potentials decline with age in quiet-reared gerbils. Hearing Res. 61, 35-46. Thalmann, R., Miyoshi, T., and Thalmann, I. (1972) The influence of ischemia upon the energy reserves of inner ear tissues. Laryngoscope 82, 2249-2272. Vincent, D.A., Smyth, B.J., Winston, D., Gratton, M.A., and Schulte, B.A. (1993) Effect of postmortem autolysis on protein structure and Na, K-ATPase activity in lateral wall and organ of Corti from individual cochleas. Abstr. Assoc. Res. Otolaryngol. 16, 137. Zolotarjova, N., Ho, C., Mellgren, R.L., Askari, A., and Huang, W. (1994) Different sensitivities of native and oxidized forms of Na,KATPase to intracellular proteinases. Biochem. Biophys. Acta 1192, 125-131.