Morphometry and autoradiography of early hypertrophic changes in the ventricular myocardium of adult rat. A light microscopic study.

Quantitative autoradiographic localization of newly synthesized proteins was studied in the ventricular myocardium of adult rats 2 hours after the intravenous injection of 3H-leucine and 20 hours after the induction of hypertrophy by constriction of the abdominal aorta. Equal area samples of tissue from experimental and sham-operated controls were examined for differential grain counts and morphometric measurements by light microscopy. In comparison with control levels, the hypertrophic hearts show a 20 per cent increase in the average cross-sectional area of myocytes and a 43 per cent increase in the incorporation of amino acid into proteins. The higher concentration of labeled proteins is confined mainly to cardiac muscle cells, which show a 90 per cent increase in grain counts per cell cross-section, whereas no significant change is observed over the interstitium. A test of the specificity of the autoradiographic technique shows the expected reduction in tissue labeling following cycloheximide inhibition of protein synthesis, indicating that there is no significant amount of nonspecific binding of free amino acid during the paraformaldehyde-glutaraldehyde fixation procedure.     

Microwave irradiation as a form of fixation for light and electron microscopy

Microwave irradiation was used to fix a wide range of surgical and autopsy specimens as well as tissue from freshly killed rats. Although there appeared to be varying optimum temperatures of fixation for different tissues, from a practical standpoint, the heating to 58 degrees C of tissues submerged in normal saline resulted in fixation of a quality comparable with that produced by conventional fixation to 10 per cent formalin. Microwave irradiation applied to tissues which had up to 1 h prior immersion in 10 per cent formalin also produced excellent preservation of cytomorphology, the time required for microwave fixation being no more than 150 s. This form of rapid heat fixation had no deleterious effects on special stains, sectioned as well as control fixed blocks and produced less shrinkage artefact than conventional formalin fixation. Immunocytochemical staining for more stable cytoplasmic antigens revealed no significant difference between microwave fixation compared with formalin fixation. The more labile membrane associated antigens of lymphocytes, however, were not preserved by either method of fixation. Microwave fixation was also found to be applicable to electron microscopy. Tissue samples immersed in 2.5 per cent glutaraldehyde and fixed in 90 s by irradiation to 50 degrees C showed excellent preservation of ultrastructural morphology.     

Absolute morphometric study of myocardial hypertrophy in experimental hypertension. I. Determination of myocyte size.

A method for determining the mean absolute volume of a specific population of cells within a tissue is described and applied to the measurement of endocardial and epicardial myocytes in the left ventricle of normal and hypertensive rats. The technique, based on nuclear counts per unit area in tissue slices of different known thicknesses, measures the mean cell volume per nucleus independent of previously unknown nuclear dimensions and systematic counting errors. Duplicate determinations, demonstrating reproducibility, were made in mutually perpendicular longitudinal and transverse sections of the myocardium. Combining these light microscopic measurements with electron microscopic data enabled the evaluation of the mean diameter and length of the cylindrical myocyte nuclei showing those in the epicardial cells to be significantly longer than the nuclei in endocardial cells. It was estimated that 2 to 4 per cent of ventricular myocytes are binucleate. After 1 to 4 weeks of hypertension, induced by constriction of the left renal artery, endocardial myocytes were enlarged 21 per cent, from 10,370 +/- 410 to 12,520 +/- 490 cu. micrometer., while epicardial myocytes showed a 37 per cent hypertrophy, from 12,600 +/- 1,600 to 17,300 +/- 1,100 cu. micrometer. The availability of a reliable determination of cell volume will make possible the interpretation of much biochemical, functional, and morphometric data at the whole cell level.     

A comparison of the effects of three widely used glutaraldehyde fixatives on cellular volume and structure. A TEM, SEM, Volumetric and Cytochemical Study.

The effects of three widely used glutaraldehyde-based fixatives on cellular volume and structure have been studied utilizing TEM, SEM, time-lapse micrography during the fixation procedure, volumetry and demonstration of the lysosomal enzyme acid phosphatase. The cells used were in vitro cultivated human glia and glioma cells and suspensions of isolated rat liver parenchymal cells. The fixatives compared were the following: 2 per cent glutaraldehyde (GA) in 0.1 M Na-cacodylate-HCL buffer (cac) with 0.1 M sucrose (pH 7.2); total osmolality (T) 510 mOsmol; vehicle osmolality (V) 300 mOsm, 2 per cent GA in 0.1 M cac (pH 7.2; T = 410 mOsmol; V = 200 mOsmol) and 1.5 per cent GA in 0.067 M cac with 0.033 M sucrose (pH 7.2; T = 320 mOsmol; V = 170 mOsmol). It was found that the fixative with a vehicle osmolality of 300 mOsmol gave results which were interpreted as ideal while the two fixatives were hypotonic vehicles resulted in changes which were easily demonstrated during volumetry, time-lapse micrography, SEM and cytochemistry. However, the differences observed in the TEM were less obvious and difficult to interpret, the major alternations being changes in the configuration of the ER in the liver cells. In conclusion, our findings show that even small variations in the composition of a glutaraldehyde fixative can result in structural changes which do not correspond to the functional morphology of a living cell. Such changes make correct interpretation of micrographs difficult.     

Delay in fixation does not affect the immunoreactivity of proliferating cell nuclear antigen (PCNA).

The effect of delayed fixation on the immunoreactivity of proliferating cell nuclear antigen (PCNA) was investigated using eight breast carcinomas. Topologically shuffled samples of each tumour were immersed in fixative at times of 0.5, 1, 2, 4, 6, 18, and 24 h after surgical removal. In addition to a PCNA index (percentage of positive cells per 1200 tumour cells), a semi-quantitative PCNA grading system was used, based on estimates of more than or less than 50 per cent of positive tumour cells at each time interval. The PCNA index of six tumours increased by a mean of 10 per cent with a fixation delay of 24 h. The PCNA grade of all eight tumours showed no change with delayed fixation.     

Validation of cell volume determination for stereological studies of adrenocortical cells: Comparison of values from semifine sections with those of dissociated zona fasciculata cells

The average cell volume of rat adrenocortical zona fasciculata cells, determined using three different stains on semifine sections, was compared to that of dissociated, unfixed zona fasciculata cells. The maximum number of nuclei per area (N_A) was obtained by counting nuclear profiles in semifine sections from nonosmicated adrenocortical tissue stained with toluidine blue. In osmicated adrenocortical tissue on the other hand, fewer nuclear profiles were seen and more of the small size classes were missing from the distribution of profiles. The skewness of the nuclear-profile distribution was much greater in osmicated than in nonosmicated tissue, reflecting the missing small profiles. Although the average diameter of nuclei (D) in nonosmicated tissue was smaller than that for osmicated tissue, profiles were in all likelihood more readily identified because they stained quite intensely. Three different methods for correcting nuclear-size profile distribution and determining nuclei per volume (N_V) (Giger-Riedwyl, Cruz-Orive, and Weibel-Gomez) were generally in agreement for nonsmicated tissue. Considerable shrinkage occurred during preparation for electron microscopy, with the greater change occurring in nonosmicated tissue. Cell volume in osmicated tissue corrected for shrinkage varied considerably among the different methods, producing significantly greater values for cell volume than those in nonosmicated tissue. The cell volume of nonosmicated tissue, corrected for shrinkage during processing and for missing small nuclear profiles, did not differ significantly from that of freshly dissociated zona fasciculata cells. The one exception was nonosmicated tissue stained by Feulgen-methylene blue and corrected for missing small nuclear profiles by the method of Cruz-Orive. Use of nonosmicated adrenocortical tissue in stereological studies with an appropriate correction for shrinkage for each experiment is recommended for determining cell volume, although osmicated tissue should also be included to facilitate identification of intracellular membranes when electron microscopy is required.     

An assessment of the distortion of arteries due to sectioning in endomyocardial biopsios

Arteries are usually studied morphometrically after pressurized fixation and resin embedding. These procedures are impracticable when dealing with diagnostic biopsies. The accuracy of arterial morphometry is determined partly by the degree of tissue distortion during section preparation. The axial ratios of 7340 arteries were measured in 353 endomyocardial biopsies from 23 patients and then compared with those expected from mathematical modelling. An excess of elliptical arteries was found. The distribution of orientation of the long axes of these best fitted a simulated 10 per cent linear distortion in the direction of microtomy. In conclusion, these results suggest that although there is some tissue distortion during sectioning, useful data may be obtained from morphometry of arteries in routinely processed endomyocardial biopsies. © 1997 John Wiley & Sons, Ltd.     

Terbutaline-induced triphasic changes in volume of rat alveolar type II cells: the role of cAMP

Changes in the volume of rat alveolar type II cells (AT-II cells) induced by terbutaline, a beta(2)-agonist, were measured using video-enhanced contrast microscopy. The changes consisted of three phases: initial cell shrinkage, cell swelling, and gradual cell shrinkage. The initial cell shrinkage was Ca(2+)-dependent and was inhibited by quinine (a K+ channel blocker). The subsequent cell swelling was cAMP-dependent and was inhibited by amiloride (a Na+ channel blocker). The final cell shrinkage was cAMP-dependent and was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, a Cl- channel blocker). Thus, terbutaline-induced cell volume changes were regulated by both Ca2+ and cAMP. Accumulation of cAMP alone, however, induced the Ca2+ -dependent cell shrinkage of AT-II cells and H-89 (a PKA inhibitor) inhibited terbutaline-induced cell volume changes. This suggests that cAMP accumulation stimulates the Ca2+ signal during terbutaline stimulation. In conclusion, terbutaline stimulates not only Na+ influx, but also K+ and Cl- release mediated via cAMP accumulation in rat AT-II cells, which induces the triphasic cell volume changes.     

Osmometric and water-transporting properties of guinea pig cardiac myocytes

To elucidate the mechanism of water flux across heart cell membranes, osmotically induced volume changes and sarcolemmal water permeability were evaluated in isolated guinea pig ventricular myocytes by videomicroscopic measurements of cell surface dimensions. Superfusion with anisosmotic solution (0.5-4 times normal osmolality) caused a rapid (lt;3 min to new steady state) and reversible cell swelling or shrinkage mainly because of proportional changes in cell width and thickness. The van't Hoff relationship between relative cell volume and the reciprocal of relative osmolality was linear and predicted an apparent osmotically dead space of approximately 35% cell volume. The osmotic water permeability coefficient (P(f)) measured from the time course of cell swelling/shrinkage was approximately 22 microm.s(-1) at 35 degrees C. Arrhenius activation energy (E(a)), a measure of the energy barrier to water flux, was approximately 3.8 kcal.mol(-1) between 11 and 35 degrees C; this value is equivalent to E(a) for free-water diffusion in bulk solution ( approximately 4 kcal.mol(-1)). Treatment with 0.1 mM Hg(2+), a sulfhydryl-oxidizing reagent, reduced P(f) by approximately 90%, and the sulfhydryl-reducing reagent dithiothreitol (10 mM) antagonized the inhibitory action of Hg(2+). E(a) measured from Hg(2+)-treated myocytes (12.3 kcal.mol(-1)) was in the range of that for diffusional water movement through the lipid bilayers (>10 kcal.mol(-1)). Although the observed P(f) is small in magnitude, both the low E(a) and the sulfhydryl-related modifications of P(f) are characteristic of channel-mediated water transport. These data suggest that water channels form a major conduit for water crossing the sarcolemma of guinea-pig heart cells.     

A quantitative study of lamina densa alterations in hamster cheek pouch carcinogenesis

Lesions produced by topical application of 0.5 per cent. DMBA to the hamster cheek pouch epithelium were classified as hyperplasia, dysplasia and carcinoma groups using strict histological criteria. Untreated epithelium served as a control. Tissue samples from five animals in each group were processed for electron microscopy and electron micrographs from the epithelialconnective tissue junction were obtained from 5 blocks per animal. The micrographs were subjected to stereological intersection counting to determine the relative surface (S_SLD,BM) of lamina densa which was in normal relationship to the basal cell plasma membranes. Quantitative results indicated a progressive loss of lamina densa during carcinogenesis and this was accompanied by the extrusion of pseudopodia from the basal cells through the gaps. The pseudopodia were frequently related to peripheral cytoplasmic microfilaments. Quantitative data confirmed the progressive nature of this loss, with values for S_SLD,BM being of the order of 98 per cent., 88 per cent., 76 per cernt. and 42 per cent. for normal epithelium and for the hyperplastic, dysplastic and carcinomatous lesions respectively. The loss of lamina densa is discussed in relation to the specificity of the response and to the development of features indicative of motility in transforming cells.     

Thermal stability of reconstituted collagen fibrils. Shrinkage characteristics upon in vitro maturation

Thermal stability measured as area shrinkage without tension during heating was determined for membranes of collagen fibrils reconstituted from solutions of highly purified rat skin collagen. Shrinkage in per cent of area at 25 °C and shrinkage temperature were quantitated in a standardized way and determined as a function of in vitro maturation time from 11 to 104 days after aggregation for the collagen membranes. Similar to reports on intact rat skin, shrinkage temperature remained constant and shrinkage per cent declined with a rate decreasing with time during maturation. Solubility in water at 80 °C for 2 hours was 95–96% and remained unchanged for the maturation time (about 2 months) studied. The decreased shrinkage reflecting a lower degree of collapse is ascribed to an increasing thermal stability of the membranes during maturation. Development of heat-stable bonds in the reconstituted collagen fibrils is taken to be amenable to this increased stability. Similarity in changes of shrinkage characteristics during in vivo and in vitro maturation indicates that maturation changes in reconstituted collagen fibrils reflect those occurring in intact collagen during in vivo aging.     

Cell-volume regulation by swelling-activated chloride current in guinea-pig ventricular myocytes

The cell-volume regulation by swelling-activated Cl- current (I(Cl,swell)) was studied in guinea pig ventricular myocytes, using a microscopic video-image analysis. We have previously shown that in ventricular cells depolarized in high-K+ ([K+]o>45 mM) solution, an activation of the cyclic AMP-dependent Cl- current (I(Cl,cAMP)) leads to cell swelling. We first investigated the mechanism underlying the I(Cl,cAMP)-independent recovery (shrinkage) of the swollen cells. They shrank when the membrane potential (Vm) was made negative to the equilibrium potential of Cl- (ECl) by lowering [K+]o or [Cl-]o in the high-K+ solution. This shrinkage was attenuated by the inhibitors (DIDS, glibenclamide, furosemide) of swelling-activated Cl- current (I(Cl,swell)). These findings suggested an involvement of I(Cl,swell) in the observed isosmotic cell shrinkage. On the other hand, an application of hyposmotic (70% of control) solution to the cells at normal [K+]o (ECl>Vm) induced a cell swelling, and the swollen cells underwent a slight but definite spontaneous cell shrinkage during hyposmotic challenge, indicating the operation of the mechanism of regulatory volume decrease (RVD). This RVD was pronounced at low [Cl-]o, at which ECl was much more positive than Vm. On the contrary, when the hyposmotic solution was applied to the cells at high [K+]o, at which ECl was negative to Vm, the cells swelled vigorously and monotonically without showing RVD, the swelling being much greater than that seen at normal [K+]o. Both the RVD at normal [K+]o and the extra cell swelling at high [K+]o were suppressed by DIDS. These results suggest that I(Cl,swell) activated by cell swelling can shrink or inflate the cardiac cells under hyposmotic as well as isosmotic conditions, depending on Vm and ECl.     

The effects of preparative procedures for scanning electron microscopy on the size of isolated lymphocytes

The diameter of lymphocytes, isolated from a sample of peripheral blood by Ficoll-Hypaque sedimentation, was determined after eight different steps in the preparative procedure for scanning electron microscopy (SEM). There was a 43% total reduction in mean cellular diameter of the lymphocytes studied, the diameter dropping from 8.4 μm prior to fixation, to a final diameter of 4.8 μm in the cells examined under the scanning electron microscope. The bulk of this shrinkage occurred during critical-point drying. In view of this finding, caution should be exercised in describing detailed cell surface morphology by SEM when similar preparative procedures are employed.     

Two different mechanisms underlie reversible, intrinsic optical signals in rat hippocampal slices

Intrinsic optical signals (IOSs) induced by synaptic stimulation and moderate hypotonic swelling in brain tissue slices consist of reduced light scattering and are usually attributed to cell swelling. During spreading depression (SD), however, light-scattering increases even though SD has been shown to cause strong cell swelling. To understand this phenomenon, we recorded extracellular voltage, light transmission (LT), which is inversely related to light scattering, and interstitial volume (ISV) simultaneously from the same site (stratum radiatum of CA1) in both interface and submerged hippocampal slices. As expected, moderate lowering of bath osmolarity caused concentration-dependent shrinkage of ISV and increase in LT, while increased osmolarity induced opposite changes in both variables. During severe hypotonia, however, after an initial increase of LT, the direction of the IOS reversed to a progressive decrease in spite of continuing ISV shrinkage. SD caused by hypotonia, by microinjection of high-K(+) solution, or by hypoxia, was associated with a pronounced LT decrease, during which ISV shrinkage indicated maximal cell swelling. If most of the extracellular Cl(-) was substituted by the impermeant anion methylsulfate and also in strongly hypertonic medium, the SD-related decrease in LT was suppressed and replaced by a monotonic increase. Nevertheless, the degree of ISV shrinkage was similar in low and in normal Cl(-) conditions. The optical signals and ISV changes were qualitatively identical in interface and submerged slices. We conclude that there are at least two mechanisms that underlie reversible optical responses in hippocampal slices. The first mechanism underlies light-scattering decrease (hence enhancing LT) when ISV shrinks (cell swelling) under synaptic stimulation and mild hypotonia. Similarly, as result of this mechanism, expansion of ISV (cell shrinkage) during mild hypertonia leads to an increased light scattering (and decreased LT). Thus optical signals associated with this first mechanism show expected cell-volume changes and are linked to either cell swelling or shrinkage. A different mechanism causes the light-scattering increase (leading to a LT decrease) during severe hypotonia and various forms of SD but with a severely decreased ISV. This second mechanism may be due to organelle swelling or dendritic beading but not to cell-volume increase. These two mechanisms can summate, indicating that they are independent in origin. Suppression of the SD-related light-scattering increase by lowering [Cl(-)](o) or severe hypertonia unmasks the underlying swelling-related scattering decrease. The simultaneous IOS and ISV measurements clearly distinguish these two mechanisms of optical signal generation.     

Alterations to subplasmalemmal leptomeres in adult canine myocytes during total in vitro ischemia

Published evidence suggests that ischemia-induced cell swelling renders myocytes vulnerable to plasmalemmal disruption and consequent cell death. Alterations to the myocyte cytoskeleton may be involved in the pathogenesis of this plasmalemmal injury. One putative cytoskeletal structure in cardiac muscle that has received little consideration is the subplasmalemmal network of periodic densities with linking microfilaments termed leptomeres or leptofibrils. We demonstrate these structures in dog heart papillary muscle and describe the improvement in their definition brought about by tissue fixation at 37 degrees C in 2% glutaraldehyde with addition of 0.05 M lysine-HCl, followed by brief postfixation with osmium tetroxide. Alterations to leptomeres during ischemic injury were examined in myocardium subjected to total in vitro ischemia for 30-180 min at 37 degrees C. Leptomeres showed little morphological alteration during the first 90-120 min, after which leptomere periodic densities (striae) increased in size, from 10-20 to 50-80 nm, and were more densely stained. The leptomeres eventually (150-180 minutes) lose definition. The course of these alterations coincided with the appearance of ultrastructural evidence of irreversible ischemic injury to the myocytes.     

The bovine pericardial xenograft: I. Effect of fixation in aldehydes without constraint on the tensile viscoelastic properties of bovine pericardium

Tensile testing of tissue strips has been used to examine the effect of simple fixation in glutaraldehyde and formaldehyde on the viscoelastic properties of bovine pericardium. To assess tissue anisotropy, tissue strips were cut at 0 degree, 30 degrees, 60 degrees, and 90 degrees relative to the base-to-apex direction. Fresh anterior pericardium was modestly anisotropic, being least extensible in the base-to-apex direction; however, fixation removed this anisotropy. Fixation also produced a marked change in the response of the material to initial cyclic loading during preconditioning. Overall extensibility of the fixed material was significantly greater than that for the fresh tissue, consistent with a 10.7% shrinkage in aldehydes calculated from strain at fracture data. Reductions in stress relaxation and creep after fixation were noted as well, consistent with intrafibrillar crosslinking. Cyclic hysteresis and ultimate tensile strength were unaffected. Since the observed changes in the stress-strain response were largely attributable to shrinkage, control of shrinkage by physical means would allow for engineering modification of bovine pericardial mechanics for controlled anisotropy.     

Differential effects of tissue processing on human embryonic and fetal skin

To most accurately evaluate quantitative data from studies of developing epidermis, the effects of tissue processing on human embryonic and fetal skin (8-20 weeks gestational age) were examined using two different techniques: 1) EDTA-separated epidermal sheets that were briefly fixed in 2% paraformaldehyde, processed through Permount infiltration, and prepared as whole mounts on glass slides, and 2) skin that was fixed in Karnovsky's fixative and embedded in Epon. Based on en face measurements of surface area before and after tissue processing, both procedures caused differential, age-dependent shrinkage. However, the trend of increasing shrinkage was inversely related to increasing age in the paraformaldehyde-fixed epidermal sheets (y = 57.14 + 1.26x, where x = gestational age in weeks and y = % of original surface area), but directly correlated with aging in the Karnovsky-fixed skin (y = 955.62 - 232.77x + 20.38x2). Shrinkage of epidermal sheets occurred during the dehydration and clearing steps, whereas most of the dimensional changes in whole skin took place during fixation in Karnovsky's. These differences are probably due to greater cross-linking of proteins and longer fixation time in the more concentrated and fast-acting Karnovsky's, as well as the influence of increasing quantities of fibrous proteins in the dermis of whole skin.     

Autopsy sampling and elemental analysis: Errors arising from post-mortem changes

The implication of post-mortem changes such as cell swelling, imbibition and autolysis on the elemental composition of body organs has been studied in rats. Liver has been chosen as an example. Retaining the liver inside the intact dead body for different periods of time at ambient temperature induced significant changes in its weight due to post-mortem tissue degeneration. Livers from animals that were frozen at ?15°C also showed significant decrease in weight when they were thawed on the third day. The effect of these changes on the concentrations of various elements depended on the association of the elements with extracellular fluid and intracellular components. For example, concentration of K⁺ was affected more by the lysis of the cell and sustained losses up to 30 per cent, in relation to the control values, while the total content was reduced by more than 40 per cent. as a result of both lysis and tissue liquefaction. For Na⁺, differences ranging from +10 to ?20 per cent. in concentrations and +20 to ?40 per cent. in total content were observed which could be explained by the movement of fluid in and out of the organ. Among trace elements, variations observed for iron ranged from ?20 to +40 per cent., while both copper and zinc were found to fluctuate between ?20 and +20 per cent. The loss observed in the total content in the liver for the five trace elements studied (Cu, Fe, Mn, Rb and Zn) was found to be about 20 to 40 per cent.     

Absolute morphometric study of myocardial hypertrophy in experimental hypertension. II. Ultrastructure of myocytes and interstitium.

The left ventricular myocardium of normal and hypertensive rats has been characterized morphometrically in the endocardial and epicardial zones. Compared to the epicardial regions, the normal endocardial regions contain 30 per cent more myocytes, 27 per cent less interstitial space, 48 per cent less capillary volume, 17 per cent less capillary surface, and the same capillary length per unit tissue volume. In terms of both the relative and absolute volumes and surface areas of their organelles, the cytoplasmic composition of normal endocardial and epicardial myocytes is nearly identical. After 14 weeks of hypertension, induced by constriction of the left renal artery, left ventricular weight is increased by 30 per cent, wall thickness by 42 per cent. The number of myocytes and the total length of capillaries remain constant. The epicardial region enlarged 37 per cent with proportional increases of myocyte and interstitial volumes. In contrast, the endocardial enlargement was only 26 per cent, comprised of 21 per cent hypertrophy of myocytes and a 55 per cent increase in interstitial components. Expansion of capillary lumina accounted for much of the interstitial enlargement throughout the myocardium. Hypertrophy of myocytes is 76 per cent greater in the epicardial region and is accompanied by a reduced mitochondria to myofibril ratio and disproportionately large increases (2- to 3-fold) in both smooth endoplasmic reticulum and T-system volume and surface area. On a cellular basis the absolute morphometric characteristics of myocytes from hypertensive rats are significantly different from normal, and significant differences occur between the inner and outer layers of the myocardium for practically every cytoplasmic component.     

Glutaraldehyde induces cell shape changes in isolated outer hair cells from the inner ear

Individual isolated outer hair cells (OHCs) from the cochlea were maintained in a collagen gel and viewed in the light microscope. They were observed during fixation and processing for transmission electron microscopy and individual cells were selected for observation in the electron microscope. Application of glutaraldehyde at several concentrations caused OHCs to become shorter. Shrinkage occurred during dehydration but there was no further change during infiltration with the epoxy resin. Ultrastructural analysis of isolated cells fixed with glutaraldehyde and postfixed with osmium tetroxide showed that these cells were similar to cells fixed in the intact cochlea. The glutaraldehyde-induced cell shape change is similar to the shortening seen in intact OHCs in response to the application of solutions containing high potassium or caffeine. Application of glutaraldehyde to cells pretreated with potassium or caffeine caused further shortening. Glutaraldehyde-induced cell shape change was not blocked by the application of tetracaine, which did prevent potassium-induced and caffeine-induced shortening. Glutaraldehyde-induced cell shape change was not stopped by short treatment with N-ethylmaleimide, which did inhibit potassium-induced shortening. Results from these experiments suggest that the glutaraldehyde-induced OHC shape change is not caused by an effect on the membrane or by calcium activation of a contractile response. Shortening may be caused by shrinkage due to cross-linking of proteins.