Induction of heat shock protein in interdental cells by hyperthermia ANN M. THOMPSON, PhD, and J. GAIL NEELY, MD, FACS, Oklahoma City, Oklahoma, and St. Louis, Missouri
The effect of hyperthermia on Induction of the 72 kllodalton (kDa) heat shock protein (HSP72) was examined In Interdental cells of the guinea pig cochlea. After being lmmersed In a water bath of either normal body temperature (370 C, control condition) or 430 C (hyperthermic condition), animals were killed either 0, 1, 2, 6, or 18 hours later. Cochlear sections were Incubated with a monoclonal antibody raised against HSP72 and relative staining densities were quantified with a light microscopic Image analysis system. Optical densities of the Interdental cell region of animals receiving hyper· thermla treatment were significantly greater than those of animals In the control group. Further analysis revealed that levels of HSP72 Immunoreactivity began Increasing by 1hour after hyperthermia and continued to Increase thereafter, to reach maximal levels at 6 hours.Themaximal levels were maintained for the rest of the experlment-18 hours. The results Indicate that hyperthermia leads to an Increase In the synthesis of HSP72 In guinea pig Interdental cells. (OTOLARYNGOL HEAD NECK SURG 1992;107:769,)
Hyperthermia is one of several stressful stimuli that can trigger a number of cellular mechanisms, collectively known as the heat stress or shock response. 1-3 One facet of the response is an increase in the synthesis of a group of proteins known as heat stress or shock proteins. Synthesis of the 72 kDa heat shock protein (HSP72), in particular, is readily induced by stress, although its presence in a cell is not dependent upon stressful conditions. For example, cells of both monkeys and human beings have been reported to synthesize HSP72 under normal conditions." Furthermore, HSP72 has been found in various tissues of normal rats including the bladder, adrenals, and esophagus," and in ependymal cells lining the ventricles of normal gerbil brains." HSP72 has also been found in the cochlea of normal guinea pigs.?:"
From the Department of Otorhinolaryngology (Dr. Thompson), The University of Oklahoma Health Sciences Center, and the Department of Otolaryngology (Dr. Neely), Washington University School of Medicine. Supported by The Deafness Research Foundation. Presented at the Fifteenth Midwinter Research Meeting of the Association for Research in Otolaryngology. St. Petersburg. Fla.• Feb. 2-6, 1992. Received for publication March 2. 1992; revision received May 27, 1992; accepted June 9, 1992. Reprint requests: Ann M. Thompson, PhD. Department of Otorhinolaryngology, The University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City. OK 73190. 23/10/41098
The presence of HSP72 in normal cells suggests that it may playa role in the normal metabolic activities of the cells in which it is found. For example, in normal cells, HSP72 is intricately involved in the process of protein maturation." Besides its role in the normal cell, HSP72 also appears to serve a protective function in stressed cells. For example, hyperthermia, with concomitant induction of HSP72, has been shown to protect the retina from subsequent light damage. 10 Because HSP72 induction occurs in response to various stressful stimuli and may be associated with protection, our laboratory has been examining HSP72 in the mammalian cochlea. In an earlier study, we found that HSP72 immunoreactivity is present in a number of cell types in the normal guinea pig cochlea, including the interdental cell." Interdental cells form the endolymphatic surface of the spiral limbus and secrete and maintain the integrity of the tectorial membrane.":" Whether involved with normal metabolic processes or serving a protective function, we expect that HSP72 will be synthesized in interdental cells in response to hyperthermia. The goal of the present study was to determine whether hyperthermia induces the synthesis of HSP72 in interdental cells. Determination that HSP72 is induced by hyperthermia may serve as a basis for future investigations of a possible therapeutic role of HSP72, not only in interdental cells, but in other cell types of the cochlea, and its potential use as a tool for studies of cochlear responses to stressful stimuli such as noise, hypoxia, or drugs.
769
OtolaryngologyHead and Neck Surgery
770 THOMPSON and NEELY
METHODS AND MATERIAL
Cochlear Processing
Guinea pigs were exposed to either hyperthermic or control conditions and, after various recovery times, were perfused. The cochleae were harvested and processed for the immunohistochemical detection of HSP72. The cochlear sections were image-analyzed to detect differences in densities of immunostaining. Twenty pigmented guinea pigs were obtained from Richglo (El Campo, Texas) and were kept in their home cages for at least 1 week after delivery from the vendor before use. All animals used exhibited a positive Preyer's reflex bilaterally. Two separate, but otherwise identical, experiments were done; for each experiment, ten animals were run in five matched pairs, with one control and one hyperthermia-treated animal per pair. For each matched pair, the animals were treated at the same time and day with the appropriate condition (i.e., control or hyperthermia), The two animals of each pair had the same recovery time and, therefore, were killed and perfused the same day and time. Also for each matched pair, the cochleae were treated in the same manner and decalcified in the same container simultaneously. Likewise, sections from those cochleae were immunohistochemically processed simultaneously. (All animal procedures were approved by the Institutional Animal Care and Use Committee and were in compliance with federal and local regulations concerning humane use of animals in research).
Pairs of animals were overdosed with 100 mg/kg sodium pentobarbital intraperitoneally and perfused at 0, 1, 2, 6, or 18 hours after the water bath procedure (n = one animal for each recovery time condition in each experiment). Zero time was defined as the time at which the animal was removed from the 370 C water bath; recovery time was defined as the time elapsed after the zero time until perfusion (0, 1, 2, 6, and 18 hours). Each animal was transcardially perfused, first with phosphate-buffered saline (PBS), and then with cold fixative-4% parafonnaldehyde in 0.1 mol/L phosphate buffer (PB). The temporal bones were quickly removed and the bone surrounding the cochlea was trimmed. To aid in penetration of the fixative, the footplate of the stapes was removed from the oval window, the round window membrane was punctured, and a small hole was made in the cupula of the apex. The cochlea was then post-fixed for 2 hours, decalcified with EDTA, IS and cryoprotected, all at 40 C. Matched pairs were processed simultaneously in the same container. After the cochleae sank in 30% sucrose, they were embedded (OCT compound) and quick-frozen in liquid nitrogen. Frozen sections (12 microns thick) were cut along the midmodiolar plane of each cochlea with a cryostat (Hacker Instruments, Fairfield, N.J.) and collected on ProbeOn glass slides (Fisher Scientific, Springfield, N.J.). The sections were stored at - 800 C until immunohistochemically processed.
Hyperthermia Treatment
The animals were given 35 mg/kg ketamine hydrochloride (Ketamine) and 3.5 mg/kg xylazine hydrochloride (Rompun) intramuscularly and a rectal thermistor probe was inserted to measure core body temperature. Hyperthermia was produced by submerging the body of the guinea pig in a water bath maintained at 43 0 C. Animals were kept in the water bath for 15 minutes after their core body temperature reached 43 0 C. On average, it took 17 minutes for the core body temperature to increase from baseline (mean, 37.2 0 C) to 430 C. After hyperthermia, the animals were removed from the 43 0 C water bath and placed in a 370 C water bath until the core body temperature returned to normal (about 15 minutes). The control animal was submerged similarly, but in a 370 C water bath, in which it remained until the hyperthermia-treated animal was removed from the second water bath. Thus, both animals received a water bath for the same length of time, the only difference being the temperature of fhe first bath.
Immunohistochemistry
For the immunohistochemistry, the sections were treated with 3% H20 2 for 5 minutes, rinsed with PBS, and blocked with normal horse serum (1.5% + 0.1 % Triton-X in PBS) for 20 minutes. They were then incubated in mouse monoclonal anti-HSP72 (l: 10,000; StressGen) overnight at 4 0 C. Normal mouse serum was substituted for the monoclonal antibody in sections serving as immunohistochemical controls. All sections were processed identically from this point on. They were processed according to the standard ABC procedure (Vector Labs), with diaminobenzidine hydrochloride (DAB) as the chromogen. The sections were then dehydrated, coverslipped, and viewed and photographed with an Olympus Vanox AH2 light microscope equipped with differential interference contrast optics. Sections from matched pairs were processed simultaneously. No staining was observed in immunohistochemical control sections.
Volume 107 Number 6 Part 1 December 1992
Induction of heat shock protein In Interdental cells by hyperthermia
Data Analysis
For quantification of the relative amounts of HSP72 , the optical density of the interdental cell region of the spiral limbus (n = 550 and 677 interdental cell regions for the two experiments) was determined with a computerized image analysis system (Microcomp, Southern Micro Instruments, Atlanta, Ga.) attached to the microscope. Comparisons of the optical density values were made between treatments (control and hyperthermic) and analyzed with a two-way analysis of variance with an a priori significance level set at p < 0.01 (CSS:Statistica, StatSoft, Inc., Tulsa, Okla.). Comparisons among recovery times (0, I, 2, 6, and 18 hours) between groups, were analyzed with Sheffe's post hoc tests with the built-in significance level of p < 0.05. Comparisons of optical density values were also made between right and left cochleae and between turns of the cochlea; since the differences were not significant, all sections were pooled together as if they were samples from a homogeneous population. RESULTS
Figure I shows the means and standard errors of the optical densities of the interdental cell areas for the first experiment; the mean optical density in the control group was 0.0228 (SE = 0.0017) and in the hyper.thermic group, 0.0446 (SE = 0.0016)-a statistically significant difference (F = 98.83; df = 1530; p < 0.01). The difference between the control and hyperthermic groups of the second experiment was also significant. The mean optical densities of the interdental cell areas for the control and hyperthermic pairs at each recovery time for the first experiment are shown in Fig. 2. In comparisons between control and hyperthermic animals at each recovery time, there was no significant difference in the mean opticai densities of the interdental cell areas at 0 and 1 hour. However, the mean optical density significantly increased over control at all other times after the hyperthermia treatment (i.e., 2, 6, and 18 hour recovery times-p < 0.05). With the same analysis for the second experiment, the mean optical density was significantly increased over control at I, 6, and 18 hours after hyperthermia treatment. For both experiments, the percent increase of mean optical density for the hyperthermic animals, as compared to the control animals, was calculated for each recovery time; the results for both experiments were combined. Figure 3 shows the combined values of the
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Fig. 1. Increase of the relative amount of HSP72 Immunoreactivity In Interdental cells of hyperthermia-treated, as compared to control, guinea pigs, The histogram Is of the means and standard errors of 550 optical density measurements taken . from cochlear sections at all recovery times measured of five control and five hyperthermia-treated animals, * Indicates the difference was statistically significant (p < 0,01),
percent increase (jilled circles) with the best-fit simple curve (solid line). The graph illustrates that after hyperthermia, the optical density measurements began increasing rapidly, and by 6 hours, had reached a maximal level. The maximal level was maintained for 18 hours, the longest recovery time included in the study. Especially with the longer recovery times, the increase in HSP72 immunoreactivity in interdental cells of hyperthermic animals, as compared to matched control animals, was large enough to be detected without benefit of the image analysis system. Figure 4 illustrates this point with photomicrographs of sections from a matched pair of animals (A. control; B, hyperthermic) whose recovery time was 18 hours. The increase in the density of immunostaining is evident in the row of interdental cells (arrowheads) of the hyperthermic animal (B).
DISCUSSION
Because the optical density measurements indicate relative levels of HSP72 immunoreactivity, the results suggest that in guinea pig interdental cells, HSP72 synthesis is induced in response to hyperthermia; the increase of HSP72 immunoreactivity over control can be detected as early as 2 hours after hyperthermia. The
772
OtolaryngologyHead and Neck Surgery
THOMPSON and NEELY
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Recovery Time (hours) Fig. 2. Time course of the relative amount of HSP72 ImmunoreactivitY In guinea pig Interdental cells after control and hyperthermia treatments. The means and standard errors of the optical density measurements are plotted, indicating an Increase of HSP72 Immunoreactivity at 1, 2, 6, and 18 hours In the hyperthermia-treated group greater than In the control animals. The asterisks Identify points of significant differences (p< 0.05). Open circles, control; dark circles, hyperthermia-treated.
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Volume 107 Number 6 Part 1 December 1992
Induction of heat shock protein in interdental cells by hyperthermia
increase continues until 6 hours after treatment, when maximal levels of HSP72 are reached. Maximal levels are then maintained for at least 18 more hours. The increase in the amount of HSP72 immunoreactivity after hyperthermia is thought to be a result of its accumulation, subsequent to activation of heat stress genes, that occurs relatively early in the stress response' and, whereas the changes in levels of HSP72 mRNA parallel the changes in body temperature during treatment." the changes in levels of HSP72 itself do not. Levels of HSP72 are stable in the recovery period after stress and the subsequently accumulated HSP72 can be detected with the immunohistochemical technique. I? The present data support the conclusions that the protein accumulates and its levels are stable for at least 18 hours after hyperthermia. The initial rapid increase (by 2 hours) of HSP72 immunoreactivity observed in the present study is also in agreement with other studies that have shown that, in mammalian systems, HSP72 accumulates rapidly after stress associated with hyperthermia' and hypoxia-ischemia. 1M Many investigators using heat to trigger HSP72 synthesis typically use normal animals kept at room temperature as controls; in the present study, the control animals were restrained and then submerged in a water bath maintained at the average core body temperature of a guinea pig. We decided that a closely matched . design was necessary because it is not known what influences the synthesis of HSP72 in interdental cells of the normal guinea pig cochlea and we hoped to rule out as many confounding factors as possible that may induce HSP72 synthesis, accumulation, and degradation. The control treatment itself may have stressed the animals; it is anticipated that the differences in the amounts of HSP72 immunoreactivity of normal, completely nonstressed guinea pigs, as compared with the hyperthermic guinea pigs, will be even greater than those observed in the present study between the controltreated and the hyperthermic animals. In a previous article,' we reported the location of HSP72 in the cochlea of normal guinea pigs. In that report, we referred to the staining as HSP70 immunoreactivity, alluding to the general class of inducible heat shock proteins with masses of approximately 70 kDa. In fact, the antibody used in that study, as well as in the current study, was raised against a heat shock protein of 72 kDa. The cell types that were immunoreactive in the first study were the same cell types that were immunoreactive in the present study. And even after hyperthermia, no additional cell types, other than those previously described, contained HSP72 immunoreactivity.
773
Fig. 4. Photomicrographs Illustrate the Increase of HSP72 Immunoreactlvl1y In interdental cells as a result of hyperthermia. Sections were harvested from A, a control animal and 8, a matched experimental (hyperthermia-treated) animal. Both animals had a recovery time of 18 hours; the temporal bones from which these sections were cut were processed In the same container simultaneously and. likewise.the sections were Immunohistochemlcally processed simultaneously. The arrowheads indicate the row of interdental cells. Bar = 20 microns.
The present study showed that hyperthermia induces HSP72 in interdental cells. The finding suggests that, although present normally, HSP72, because its synthesis increases in response to stress, may have a special role in the stressed interdental cell. Further studies will be needed to determine whether that function is protective, as suggested for other cell types and systems. We can speculate that interdental cells may have a builtin protective mechanism that is initiated in response to stress and reinforces continued maintenance of the tectorial membrane and, therefore, may help preserve hearing. In situ hybridization experiments are planned to examine the expression of HSP72 to further confirm its synthesis in response to hyperthermia in the guinea pig
OtolaryngologyHead and Neck Surgery
774 THOMPSON and NEELY
cochlea. Furthermore, we are currently analyzing levels of immunoreactivity in the organ of Corti, in the same sections analyzed in the present study, to evaluate the effects of hyperthermia on levels of HSP72 immunoreactivity in celIs of that cochlear region. The induction of HSP72 may be a tool to detect traumatic events in the abnormal cochlea, and cells containing HSP72 immunoreactivity may indicate areas of vulnerability. 18.19 And, as shown in other systems, future studies will be useful to determine a protective or repair function of HSP72 in the cochlea. We wish to thank Glenn C. Thompson, PhD, for his critical review of the manuscript and Kevin Brewer for his expert technical assistance. REFERENCES
I. Schlesinger MJ. Heat shock proteins. The search for functions. J Cell Bioi 1986;103:321-5. 2. Lindquist S, CraigEA. The heatshockproteins. AnnuRevGenet 1988;22:631-77. 3. Nover L. Heat shock response. Boca Raton, Fla.: CRC Press, Inc., 1991. 4. Welch WJ, Garrels 11, Thomas GP, Lin J-C, Feramisco JR. Biochemical characterization of the mammalian stress proteins and identification of two stress proteinsas glucose-and calcium ionophore-regulated proteins. J Bioi Chern 1983;258:7102-11. 5. Currie RW, White FP. Characterization of the synthesis and accumulation of a 71-kilodalton protein induced in rat tissues after hyperthermia. Can J Biochem Cell Bioi 1983;61:438-46. 6. Vass K, WelchWJ, Nowak TS Jr. Localization of 70-kDastress proteininduction in gerbilbrainafterischemia. ActaNeuropathol 1988;77:128-35. 7. NeelyJG, Thompson AM, GowerDJ. Detection and localization of heat shock protein70 in the normalguineapig cochlea. Hearing Res 1991 ;52:403-6.
8. Kim HN, Dechesne CJ, Vu TD, Nowak TS, Wenthold RJ. Expression of heat shock protein, HSP70, in the guinea pig and rat cochlea after hyperthermia. ARO Abstracts 1991;14:74. 9. Beckmann RP, Mizzen LA, Welch WJ. Interaction of HSP70 with newly synthesized proteins: implication for protein folding and assembly. Science 1990;248:850-4. 10. Barbe MF, Tytell M, Gower DJ, WelchWJ. Hyperthermia protects against light damage in the rat retina. Science 1988;241:1817-20. II. Smith CA. Capillary areas of the cochlea in the guinea pig. Laryngoscope 1951 ;61:1073-95. 12. Lim DJ. Morphology and function of the interdental cell. J LaryngolOtol 1970;84:1241-56. 13. Voldrich L. Morphology and function of the epithelium of the limbus spiralis cochleae. Acta Otolaryngol 1967;63:503-14. 14. KimuraRS, Nye CL, SouthardRE. Normal and pathologic features of the limbus spiralis and its functional significance. Am J Otolaryngol 1990; II :99-111. 15. Mori S, Sawai T, Teshima T, Kyogoku MJ. A new decalcifying technique for immunohistochemical studies of calcified tissue, especially applicable to cell surface marker demonstration. J Histochem Cytochem 1988;36:111-4. 16. Brown IR, Rush SJ. Expression of heat shock genes (HSP70) in the mammalian brain: distinguishing constitutively expressed and hyperthermia-inducible mRNA species. J Neurosci Res 1990;25: 14-9. 17. Craig EA. The heat shock response. CRC Crit Rev Biochem 1985;18:239-80. 18. Ferriero D, Soberano HQ, Simon RP, Sharp FR. Hypoxiaischemia induces heat shock protein-like (HSP72) immunoreactivity in neonatal rat brain. Dev Brain Res 1990;53:145-50. 19. Brown IR. Induction of heat shock (stress) genes in the mammalian brain by hyperthermia and other traumatic events: a current perspective. J Neurosci Res 1990;27:247-55.
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The effect of hyperthermia on Induction of the 72 kllodalton (kDa) heat shock protein (HSP72) was examined In Interdental cells of the guinea pig cochlea. After being lmmersed In a water bath of either normal body temperature (370 C, control condition) or 430 C (hyperthermic condition), animals were killed either 0, 1, 2, 6, or 18 hours later. Cochlear sections were Incubated with a monoclonal antibody raised against HSP72 and relative staining densities were quantified with a light microscopic Image analysis system. Optical densities of the Interdental cell region of animals receiving hyper· thermla treatment were significantly greater than those of animals In the control group. Further analysis revealed that levels of HSP72 Immunoreactivity began Increasing by 1hour after hyperthermia and continued to Increase thereafter, to reach maximal levels at 6 hours.Themaximal levels were maintained for the rest of the experlment-18 hours. The results Indicate that hyperthermia leads to an Increase In the synthesis of HSP72 In guinea pig Interdental cells. (OTOLARYNGOL HEAD NECK SURG 1992;107:769,)
Hyperthermia is one of several stressful stimuli that can trigger a number of cellular mechanisms, collectively known as the heat stress or shock response. 1-3 One facet of the response is an increase in the synthesis of a group of proteins known as heat stress or shock proteins. Synthesis of the 72 kDa heat shock protein (HSP72), in particular, is readily induced by stress, although its presence in a cell is not dependent upon stressful conditions. For example, cells of both monkeys and human beings have been reported to synthesize HSP72 under normal conditions." Furthermore, HSP72 has been found in various tissues of normal rats including the bladder, adrenals, and esophagus," and in ependymal cells lining the ventricles of normal gerbil brains." HSP72 has also been found in the cochlea of normal guinea pigs.?:"
From the Department of Otorhinolaryngology (Dr. Thompson), The University of Oklahoma Health Sciences Center, and the Department of Otolaryngology (Dr. Neely), Washington University School of Medicine. Supported by The Deafness Research Foundation. Presented at the Fifteenth Midwinter Research Meeting of the Association for Research in Otolaryngology. St. Petersburg. Fla.• Feb. 2-6, 1992. Received for publication March 2. 1992; revision received May 27, 1992; accepted June 9, 1992. Reprint requests: Ann M. Thompson, PhD. Department of Otorhinolaryngology, The University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City. OK 73190. 23/10/41098
The presence of HSP72 in normal cells suggests that it may playa role in the normal metabolic activities of the cells in which it is found. For example, in normal cells, HSP72 is intricately involved in the process of protein maturation." Besides its role in the normal cell, HSP72 also appears to serve a protective function in stressed cells. For example, hyperthermia, with concomitant induction of HSP72, has been shown to protect the retina from subsequent light damage. 10 Because HSP72 induction occurs in response to various stressful stimuli and may be associated with protection, our laboratory has been examining HSP72 in the mammalian cochlea. In an earlier study, we found that HSP72 immunoreactivity is present in a number of cell types in the normal guinea pig cochlea, including the interdental cell." Interdental cells form the endolymphatic surface of the spiral limbus and secrete and maintain the integrity of the tectorial membrane.":" Whether involved with normal metabolic processes or serving a protective function, we expect that HSP72 will be synthesized in interdental cells in response to hyperthermia. The goal of the present study was to determine whether hyperthermia induces the synthesis of HSP72 in interdental cells. Determination that HSP72 is induced by hyperthermia may serve as a basis for future investigations of a possible therapeutic role of HSP72, not only in interdental cells, but in other cell types of the cochlea, and its potential use as a tool for studies of cochlear responses to stressful stimuli such as noise, hypoxia, or drugs.
769
OtolaryngologyHead and Neck Surgery
770 THOMPSON and NEELY
METHODS AND MATERIAL
Cochlear Processing
Guinea pigs were exposed to either hyperthermic or control conditions and, after various recovery times, were perfused. The cochleae were harvested and processed for the immunohistochemical detection of HSP72. The cochlear sections were image-analyzed to detect differences in densities of immunostaining. Twenty pigmented guinea pigs were obtained from Richglo (El Campo, Texas) and were kept in their home cages for at least 1 week after delivery from the vendor before use. All animals used exhibited a positive Preyer's reflex bilaterally. Two separate, but otherwise identical, experiments were done; for each experiment, ten animals were run in five matched pairs, with one control and one hyperthermia-treated animal per pair. For each matched pair, the animals were treated at the same time and day with the appropriate condition (i.e., control or hyperthermia), The two animals of each pair had the same recovery time and, therefore, were killed and perfused the same day and time. Also for each matched pair, the cochleae were treated in the same manner and decalcified in the same container simultaneously. Likewise, sections from those cochleae were immunohistochemically processed simultaneously. (All animal procedures were approved by the Institutional Animal Care and Use Committee and were in compliance with federal and local regulations concerning humane use of animals in research).
Pairs of animals were overdosed with 100 mg/kg sodium pentobarbital intraperitoneally and perfused at 0, 1, 2, 6, or 18 hours after the water bath procedure (n = one animal for each recovery time condition in each experiment). Zero time was defined as the time at which the animal was removed from the 370 C water bath; recovery time was defined as the time elapsed after the zero time until perfusion (0, 1, 2, 6, and 18 hours). Each animal was transcardially perfused, first with phosphate-buffered saline (PBS), and then with cold fixative-4% parafonnaldehyde in 0.1 mol/L phosphate buffer (PB). The temporal bones were quickly removed and the bone surrounding the cochlea was trimmed. To aid in penetration of the fixative, the footplate of the stapes was removed from the oval window, the round window membrane was punctured, and a small hole was made in the cupula of the apex. The cochlea was then post-fixed for 2 hours, decalcified with EDTA, IS and cryoprotected, all at 40 C. Matched pairs were processed simultaneously in the same container. After the cochleae sank in 30% sucrose, they were embedded (OCT compound) and quick-frozen in liquid nitrogen. Frozen sections (12 microns thick) were cut along the midmodiolar plane of each cochlea with a cryostat (Hacker Instruments, Fairfield, N.J.) and collected on ProbeOn glass slides (Fisher Scientific, Springfield, N.J.). The sections were stored at - 800 C until immunohistochemically processed.
Hyperthermia Treatment
The animals were given 35 mg/kg ketamine hydrochloride (Ketamine) and 3.5 mg/kg xylazine hydrochloride (Rompun) intramuscularly and a rectal thermistor probe was inserted to measure core body temperature. Hyperthermia was produced by submerging the body of the guinea pig in a water bath maintained at 43 0 C. Animals were kept in the water bath for 15 minutes after their core body temperature reached 43 0 C. On average, it took 17 minutes for the core body temperature to increase from baseline (mean, 37.2 0 C) to 430 C. After hyperthermia, the animals were removed from the 43 0 C water bath and placed in a 370 C water bath until the core body temperature returned to normal (about 15 minutes). The control animal was submerged similarly, but in a 370 C water bath, in which it remained until the hyperthermia-treated animal was removed from the second water bath. Thus, both animals received a water bath for the same length of time, the only difference being the temperature of fhe first bath.
Immunohistochemistry
For the immunohistochemistry, the sections were treated with 3% H20 2 for 5 minutes, rinsed with PBS, and blocked with normal horse serum (1.5% + 0.1 % Triton-X in PBS) for 20 minutes. They were then incubated in mouse monoclonal anti-HSP72 (l: 10,000; StressGen) overnight at 4 0 C. Normal mouse serum was substituted for the monoclonal antibody in sections serving as immunohistochemical controls. All sections were processed identically from this point on. They were processed according to the standard ABC procedure (Vector Labs), with diaminobenzidine hydrochloride (DAB) as the chromogen. The sections were then dehydrated, coverslipped, and viewed and photographed with an Olympus Vanox AH2 light microscope equipped with differential interference contrast optics. Sections from matched pairs were processed simultaneously. No staining was observed in immunohistochemical control sections.
Volume 107 Number 6 Part 1 December 1992
Induction of heat shock protein In Interdental cells by hyperthermia
Data Analysis
For quantification of the relative amounts of HSP72 , the optical density of the interdental cell region of the spiral limbus (n = 550 and 677 interdental cell regions for the two experiments) was determined with a computerized image analysis system (Microcomp, Southern Micro Instruments, Atlanta, Ga.) attached to the microscope. Comparisons of the optical density values were made between treatments (control and hyperthermic) and analyzed with a two-way analysis of variance with an a priori significance level set at p < 0.01 (CSS:Statistica, StatSoft, Inc., Tulsa, Okla.). Comparisons among recovery times (0, I, 2, 6, and 18 hours) between groups, were analyzed with Sheffe's post hoc tests with the built-in significance level of p < 0.05. Comparisons of optical density values were also made between right and left cochleae and between turns of the cochlea; since the differences were not significant, all sections were pooled together as if they were samples from a homogeneous population. RESULTS
Figure I shows the means and standard errors of the optical densities of the interdental cell areas for the first experiment; the mean optical density in the control group was 0.0228 (SE = 0.0017) and in the hyper.thermic group, 0.0446 (SE = 0.0016)-a statistically significant difference (F = 98.83; df = 1530; p < 0.01). The difference between the control and hyperthermic groups of the second experiment was also significant. The mean optical densities of the interdental cell areas for the control and hyperthermic pairs at each recovery time for the first experiment are shown in Fig. 2. In comparisons between control and hyperthermic animals at each recovery time, there was no significant difference in the mean opticai densities of the interdental cell areas at 0 and 1 hour. However, the mean optical density significantly increased over control at all other times after the hyperthermia treatment (i.e., 2, 6, and 18 hour recovery times-p < 0.05). With the same analysis for the second experiment, the mean optical density was significantly increased over control at I, 6, and 18 hours after hyperthermia treatment. For both experiments, the percent increase of mean optical density for the hyperthermic animals, as compared to the control animals, was calculated for each recovery time; the results for both experiments were combined. Figure 3 shows the combined values of the
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Hyperthermia
Fig. 1. Increase of the relative amount of HSP72 Immunoreactivity In Interdental cells of hyperthermia-treated, as compared to control, guinea pigs, The histogram Is of the means and standard errors of 550 optical density measurements taken . from cochlear sections at all recovery times measured of five control and five hyperthermia-treated animals, * Indicates the difference was statistically significant (p < 0,01),
percent increase (jilled circles) with the best-fit simple curve (solid line). The graph illustrates that after hyperthermia, the optical density measurements began increasing rapidly, and by 6 hours, had reached a maximal level. The maximal level was maintained for 18 hours, the longest recovery time included in the study. Especially with the longer recovery times, the increase in HSP72 immunoreactivity in interdental cells of hyperthermic animals, as compared to matched control animals, was large enough to be detected without benefit of the image analysis system. Figure 4 illustrates this point with photomicrographs of sections from a matched pair of animals (A. control; B, hyperthermic) whose recovery time was 18 hours. The increase in the density of immunostaining is evident in the row of interdental cells (arrowheads) of the hyperthermic animal (B).
DISCUSSION
Because the optical density measurements indicate relative levels of HSP72 immunoreactivity, the results suggest that in guinea pig interdental cells, HSP72 synthesis is induced in response to hyperthermia; the increase of HSP72 immunoreactivity over control can be detected as early as 2 hours after hyperthermia. The
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OtolaryngologyHead and Neck Surgery
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Recovery Time (hours) Fig. 2. Time course of the relative amount of HSP72 ImmunoreactivitY In guinea pig Interdental cells after control and hyperthermia treatments. The means and standard errors of the optical density measurements are plotted, indicating an Increase of HSP72 Immunoreactivity at 1, 2, 6, and 18 hours In the hyperthermia-treated group greater than In the control animals. The asterisks Identify points of significant differences (p< 0.05). Open circles, control; dark circles, hyperthermia-treated.
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Volume 107 Number 6 Part 1 December 1992
Induction of heat shock protein in interdental cells by hyperthermia
increase continues until 6 hours after treatment, when maximal levels of HSP72 are reached. Maximal levels are then maintained for at least 18 more hours. The increase in the amount of HSP72 immunoreactivity after hyperthermia is thought to be a result of its accumulation, subsequent to activation of heat stress genes, that occurs relatively early in the stress response' and, whereas the changes in levels of HSP72 mRNA parallel the changes in body temperature during treatment." the changes in levels of HSP72 itself do not. Levels of HSP72 are stable in the recovery period after stress and the subsequently accumulated HSP72 can be detected with the immunohistochemical technique. I? The present data support the conclusions that the protein accumulates and its levels are stable for at least 18 hours after hyperthermia. The initial rapid increase (by 2 hours) of HSP72 immunoreactivity observed in the present study is also in agreement with other studies that have shown that, in mammalian systems, HSP72 accumulates rapidly after stress associated with hyperthermia' and hypoxia-ischemia. 1M Many investigators using heat to trigger HSP72 synthesis typically use normal animals kept at room temperature as controls; in the present study, the control animals were restrained and then submerged in a water bath maintained at the average core body temperature of a guinea pig. We decided that a closely matched . design was necessary because it is not known what influences the synthesis of HSP72 in interdental cells of the normal guinea pig cochlea and we hoped to rule out as many confounding factors as possible that may induce HSP72 synthesis, accumulation, and degradation. The control treatment itself may have stressed the animals; it is anticipated that the differences in the amounts of HSP72 immunoreactivity of normal, completely nonstressed guinea pigs, as compared with the hyperthermic guinea pigs, will be even greater than those observed in the present study between the controltreated and the hyperthermic animals. In a previous article,' we reported the location of HSP72 in the cochlea of normal guinea pigs. In that report, we referred to the staining as HSP70 immunoreactivity, alluding to the general class of inducible heat shock proteins with masses of approximately 70 kDa. In fact, the antibody used in that study, as well as in the current study, was raised against a heat shock protein of 72 kDa. The cell types that were immunoreactive in the first study were the same cell types that were immunoreactive in the present study. And even after hyperthermia, no additional cell types, other than those previously described, contained HSP72 immunoreactivity.
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Fig. 4. Photomicrographs Illustrate the Increase of HSP72 Immunoreactlvl1y In interdental cells as a result of hyperthermia. Sections were harvested from A, a control animal and 8, a matched experimental (hyperthermia-treated) animal. Both animals had a recovery time of 18 hours; the temporal bones from which these sections were cut were processed In the same container simultaneously and. likewise.the sections were Immunohistochemlcally processed simultaneously. The arrowheads indicate the row of interdental cells. Bar = 20 microns.
The present study showed that hyperthermia induces HSP72 in interdental cells. The finding suggests that, although present normally, HSP72, because its synthesis increases in response to stress, may have a special role in the stressed interdental cell. Further studies will be needed to determine whether that function is protective, as suggested for other cell types and systems. We can speculate that interdental cells may have a builtin protective mechanism that is initiated in response to stress and reinforces continued maintenance of the tectorial membrane and, therefore, may help preserve hearing. In situ hybridization experiments are planned to examine the expression of HSP72 to further confirm its synthesis in response to hyperthermia in the guinea pig
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cochlea. Furthermore, we are currently analyzing levels of immunoreactivity in the organ of Corti, in the same sections analyzed in the present study, to evaluate the effects of hyperthermia on levels of HSP72 immunoreactivity in celIs of that cochlear region. The induction of HSP72 may be a tool to detect traumatic events in the abnormal cochlea, and cells containing HSP72 immunoreactivity may indicate areas of vulnerability. 18.19 And, as shown in other systems, future studies will be useful to determine a protective or repair function of HSP72 in the cochlea. We wish to thank Glenn C. Thompson, PhD, for his critical review of the manuscript and Kevin Brewer for his expert technical assistance. REFERENCES
I. Schlesinger MJ. Heat shock proteins. The search for functions. J Cell Bioi 1986;103:321-5. 2. Lindquist S, CraigEA. The heatshockproteins. AnnuRevGenet 1988;22:631-77. 3. Nover L. Heat shock response. Boca Raton, Fla.: CRC Press, Inc., 1991. 4. Welch WJ, Garrels 11, Thomas GP, Lin J-C, Feramisco JR. Biochemical characterization of the mammalian stress proteins and identification of two stress proteinsas glucose-and calcium ionophore-regulated proteins. J Bioi Chern 1983;258:7102-11. 5. Currie RW, White FP. Characterization of the synthesis and accumulation of a 71-kilodalton protein induced in rat tissues after hyperthermia. Can J Biochem Cell Bioi 1983;61:438-46. 6. Vass K, WelchWJ, Nowak TS Jr. Localization of 70-kDastress proteininduction in gerbilbrainafterischemia. ActaNeuropathol 1988;77:128-35. 7. NeelyJG, Thompson AM, GowerDJ. Detection and localization of heat shock protein70 in the normalguineapig cochlea. Hearing Res 1991 ;52:403-6.
8. Kim HN, Dechesne CJ, Vu TD, Nowak TS, Wenthold RJ. Expression of heat shock protein, HSP70, in the guinea pig and rat cochlea after hyperthermia. ARO Abstracts 1991;14:74. 9. Beckmann RP, Mizzen LA, Welch WJ. Interaction of HSP70 with newly synthesized proteins: implication for protein folding and assembly. Science 1990;248:850-4. 10. Barbe MF, Tytell M, Gower DJ, WelchWJ. Hyperthermia protects against light damage in the rat retina. Science 1988;241:1817-20. II. Smith CA. Capillary areas of the cochlea in the guinea pig. Laryngoscope 1951 ;61:1073-95. 12. Lim DJ. Morphology and function of the interdental cell. J LaryngolOtol 1970;84:1241-56. 13. Voldrich L. Morphology and function of the epithelium of the limbus spiralis cochleae. Acta Otolaryngol 1967;63:503-14. 14. KimuraRS, Nye CL, SouthardRE. Normal and pathologic features of the limbus spiralis and its functional significance. Am J Otolaryngol 1990; II :99-111. 15. Mori S, Sawai T, Teshima T, Kyogoku MJ. A new decalcifying technique for immunohistochemical studies of calcified tissue, especially applicable to cell surface marker demonstration. J Histochem Cytochem 1988;36:111-4. 16. Brown IR, Rush SJ. Expression of heat shock genes (HSP70) in the mammalian brain: distinguishing constitutively expressed and hyperthermia-inducible mRNA species. J Neurosci Res 1990;25: 14-9. 17. Craig EA. The heat shock response. CRC Crit Rev Biochem 1985;18:239-80. 18. Ferriero D, Soberano HQ, Simon RP, Sharp FR. Hypoxiaischemia induces heat shock protein-like (HSP72) immunoreactivity in neonatal rat brain. Dev Brain Res 1990;53:145-50. 19. Brown IR. Induction of heat shock (stress) genes in the mammalian brain by hyperthermia and other traumatic events: a current perspective. J Neurosci Res 1990;27:247-55.
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