Ann Olol Rhinol LaryngollOl:1992
BOVINE TEMPORAL BONES AS A SOURCE OF INNER EAR ANTIGEN STEVEN D. RAUCH, MD JOSE SANMARTIN, MD
RICHARD
A. MOSCICKI, MD
BOSTON, MASSACHUSETfS
Modem immunologic techniques of immunostaining, immunoblotting, and creation of monoclonal antibodies are gaining wide application in studies of development, function, and pathology of the ear. These techniques require a source of inner ear tissue for production of antigen extract. Human tissue is not readily available, and other mammalian species common in auditory research are small in size. Bovine temporal bones are readily available, and the membranous portions of the inner ear are abundant and easily accessible. Herein we report our technique for acquisition and dissection of bovine temporal bones and preparation and preservation of inner ear antigen. KEY WORDS -
bovine animal model, immunology, inner ear, temporal bone.
INTRODUCTION
needed for antigen preparation.
Modem immunologic techniques ofimmunostaining, immunoblotting, and creation of monoclonal antibodies are gaining wide application in basic research into the development and function of the ear, as well as in specific studies of pathologic processes such as genetic hearing loss and idiopathic progressive bilateral sensorineural ("autoimmune") hearing loss. These techniques all require inner ear tissue as a source of antigen extract for immunoreagents. While human inner ear tissue would be ideal for many such applications, it is difficult to obtain and offers only a small quantity of tissue. Typical laboratory mammals such as rabbit, rat, cat, or guinea pig are readily available, but also offer only small amounts of tissue from each specimen. In order to obtain a readily available and abundant source of membranous inner ear, we have begun using bovine temporal bones. Others have reported the use of bovine inner ear tissues in research.l-' However, these reports have given only limited description of the techniques for harvesting the membranes. This is a report of our technique for acquisition and dissection of bovine temporal bones and preparation and preservation of bovine inner ear antigen.
Temporal Bone Microdissection. The posteroinferior quadrant of the bovine skull from foramen magnum to squamous temporal bone and from occiput to root of zygoma is massive, weighing several pounds. After an initial attempt using an adult cow, use of calf heads was deemed easier because of the smaller size and weight of the overall specimen without apparent difference in size of the otic capsule. The bony external auditory canal is more than 6 em in length, making a lateral approach to the inner ear difficult and impractical. A medial approach along the petrous ridge and internal auditory canal (lAC) gains access to the membranous components of the inner ear with only a few millimeters of drilling. All dura is removed from the middle and posterior fossa surfaces of the temporal bone. On these surfaces the otic capsule is chalky white and stands out in sharp contrast to surrounding bone (Fig IA). With the aid of an operating microscope and cutting burs, drilling begins around the lAC on three fourths of the circumference, sparing the inferior one fourth (Fig lB). Progressing from medial to lateral along the superior portion of the lAC exposes the facial nerve and geniculate ganglion area (Fig IC). Immediately deep (inferior) and anterior to the first genu of the facial nerve is the basal tum of the cochlea. This turn is blue-lined from its anterior extent deep to the first genu of the facial nerve, anteromedially toward the posterior fossa surface of the petrous apex (Fig lD). It is usually possible to gain some narrow exposure to the second tum of the cochlea by continuing this dissection anterolaterally from the basal tum toward the apex. Posterior to the lAC the vestibule, posterior semicircular canal, and crus commune are blue-lined
MATERIAL AND METHODS
Acquisition ofBovine Temporal Bones. Quartered calf skulls with the brain removed were obtained from a local abattoir and meat packing plant. Fresh quartered skulls are taken immediately upon availability and packed in crushed ice for transport to the research laboratory. The temporal bones are microdissected to remove the membranous components of the inner ear, which are then placed in phosphate-buffered saline (PBS), pH 7.4, and frozen at -40°C until
From the Department of Otology and Laryngology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts. Work supported in part by a grant from the Immunology Research Institute of New England. REPRINTS - Steven D. Rauch, MD, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114.
688
Downloaded from aor.sagepub.com at Harvard Libraries on June 26, 2015
Rauchet al, BovineTemporal Bones
689
Fig 1. Temporal bone microdissection. A) Posterior fossa view of right temporal bone with dura removed. Note sharp demarcation oftemporal bone from surrounding skull base. Dotted line - attachment site of tentorium cerebelli dividing middle fossa surface oftemporal bone from posterior fossa surface, lAC - internal auditory canal. B) Cutting bur is used to drill away bone surrounding lAC from posterior to anterior. C) Facial nerve (N VII) to first genu and geniculate ganglion (arrow) is exposed and posterior semicircularcanal (arrowheads) is blue-lined. D) Facial nerve has been removed and basal tum ofcochlea (arrowheads) blue-lined. CN - cochlear nerve. E) Basal tum ofcochlea has been opened, exposing osseous spiral lamina (arrow). Vestibule (V) is opened to allow removal of otolith organs and semicircular canal ampullae. F) Modiolus has been extracted to expose contents of second tum of cochlea (double arrows). Vestibule is widely opened to permit extraction of membranous contents of hook portion of cochlea and vestibular end organs. OW - oval window, HC - ampullated end of horizontal semicircular canal.
Downloaded from aor.sagepub.com at Harvard Libraries on June 26, 2015
690
Rauch et al, Bovine Temporal Bones
A
B
c
o
kDa 97.4-'" 66.2--,
42.750.0-1······· . >··.·" • 40.0-
-
31.0---,
Fig 2. One-dimensional 12% sodium dodecyl sulfate gradient gelelectrophoresis. Each lanedosed with 20-f.l.L sample composed of 10 ul, of specimen and 10 ul, of sample buffer, run at 40 rnA for approximately 2 hours, and stained with Coomassie blue. A) Molecular weight standards. B,C) 7.1 mg/mL bovine inner ear (combined cochlear and vestibular) antigen extract. D) 6.0 mg/mL human cochlear antigen extract. Note high degree of banding homology between bovine andhuman samples. as well. Throughout the drilling frequent irrigation or use ofcontinuous suction irrigation is used to prevent overheating of the tissues. Once the cochlear and vestibular divisions of the inner ear are blue-lined, they are opened widely but superficially with diamond burs to expose the membranous structures of the inner ear (Fig IE). These are extracted easily by using a combination of whirlybird elevators and cup forceps. The saccule and utricle are lifted from the vestibule, and then each of the three ampullae of the semicircular canals are removed. The membranes of the cochlea are removed from base to apex on the exposed superomedial surface of the cochlear spiral, and then the modiolus is cracked out with a stapes curette to gain access to the inferolateral portion of each tum. The membranes of the hook portion of the cochlea are most easily removed through the vestibule (Fig IF). All tissue is placed directly into 0.5 mL chilled PBS solution as it is removed in order to reduce postmortem autolysis. Antigen Extract Preparation. In a cold room, tissue obtained from microdissection of the temporal bones is teased apart and homogenized in 0.5 mL of PBS (pH 7.4). The homogenate is then subjected to four sonication intervals (Branson Sonifier 450) of 2 minutes each at 4T. The resultant suspension is centrifuged at 3,000g for 30 minutes in the cold room and
the supernatant decanted and filtered through a 0.2urn filter. Protein content of this final extract solution is determined on the basis ofoptical density measurement by using a modified Lowry method as described by Bloch et al. 4 Aliquots are diluted 1:10 in PBS and optical density is measured at 215 and 225 nm. Protein concentration is calculated by using the following formula: [144 x (OD215 - OD225)] x dilution factor = Ilg/mL protein concentration. The extract solution is then divided into aliquots and stored at -70°C. DISCUSSION Using the techniques described, we have been able to achieve extract concentrations in excess of?O mg! mL from dissection of six temporal bones. Electrophoretic comparison of bovine inner ear antigen extract to human extract reveals a high degree of banding homology (Fig 2). Separation of the constituents of the extract by gel electrophoresis followed by Western blot assay for inner ear antibodies is being used as part ofan ongoing project studying idiopathic progressive bilateral sensorineural hearing loss. Purification by gel electrophoresis could also be used as the initial step in production of monoclonal anti bodies. Use ofcadaveric human inner ear tissue as a source ofantigen had the complicating issues ofpostmortem autolytic changes and small quantity of tissue. This latter problem required repeat standardization of antigen content for each new sample, necessitating gradual depletion of our most potent antibody-containing serum samples. The virtually unlimited supply of bovine inner ear tissue and the ease of dissection (less than half an hour per pair of temporal bones) make it feasible to pool tissue from many animals in order to have a large quantity of antigen that may be standardized once, stored at -70°C, and used repeatedly when needed. Standardization of antigenicity is a chronic problem in immunostaining and immunoblotting techniques. Even commercial immunoreagents require standardization at the time of use to ensure reliable interpretation of results. Large pools ofantigen reduce interspecimen error by applying the same antigen solution to each sample. In a longitudinal, prospective study such as our work on idiopathic progressive bilateral sensorineural hearing loss, the accuracy of data is greatly improved by having a long-term, stable supply of antigen to use throughout the duration of the study.
REFERENCES Harris JP.Experimental autoimmune sensorineural hear3. Harris JP, Sharp PA. Inner ear autoantibodies in patients ing loss. Laryngoscope 1987;97:63-76. with rapidly progressive sensorineural hearing loss. Laryngoscope 1990;100:516-24. 2. Rarey KE, Patterson K. Establishment of inner earepithe4. Bloch KJ, Wright JA,Bishara SM,Bloch MB. Uptake of lial cellculture: isolation, growth and characterization. Hear Res polypeptide fragments of proteins by rat intestine in vitro andin 1989;38:277-87. vivo. Gastroenterology 1988;95: 1272-8. 1.
Downloaded from aor.sagepub.com at Harvard Libraries on June 26, 2015
BOVINE TEMPORAL BONES AS A SOURCE OF INNER EAR ANTIGEN STEVEN D. RAUCH, MD JOSE SANMARTIN, MD
RICHARD
A. MOSCICKI, MD
BOSTON, MASSACHUSETfS
Modem immunologic techniques of immunostaining, immunoblotting, and creation of monoclonal antibodies are gaining wide application in studies of development, function, and pathology of the ear. These techniques require a source of inner ear tissue for production of antigen extract. Human tissue is not readily available, and other mammalian species common in auditory research are small in size. Bovine temporal bones are readily available, and the membranous portions of the inner ear are abundant and easily accessible. Herein we report our technique for acquisition and dissection of bovine temporal bones and preparation and preservation of inner ear antigen. KEY WORDS -
bovine animal model, immunology, inner ear, temporal bone.
INTRODUCTION
needed for antigen preparation.
Modem immunologic techniques ofimmunostaining, immunoblotting, and creation of monoclonal antibodies are gaining wide application in basic research into the development and function of the ear, as well as in specific studies of pathologic processes such as genetic hearing loss and idiopathic progressive bilateral sensorineural ("autoimmune") hearing loss. These techniques all require inner ear tissue as a source of antigen extract for immunoreagents. While human inner ear tissue would be ideal for many such applications, it is difficult to obtain and offers only a small quantity of tissue. Typical laboratory mammals such as rabbit, rat, cat, or guinea pig are readily available, but also offer only small amounts of tissue from each specimen. In order to obtain a readily available and abundant source of membranous inner ear, we have begun using bovine temporal bones. Others have reported the use of bovine inner ear tissues in research.l-' However, these reports have given only limited description of the techniques for harvesting the membranes. This is a report of our technique for acquisition and dissection of bovine temporal bones and preparation and preservation of bovine inner ear antigen.
Temporal Bone Microdissection. The posteroinferior quadrant of the bovine skull from foramen magnum to squamous temporal bone and from occiput to root of zygoma is massive, weighing several pounds. After an initial attempt using an adult cow, use of calf heads was deemed easier because of the smaller size and weight of the overall specimen without apparent difference in size of the otic capsule. The bony external auditory canal is more than 6 em in length, making a lateral approach to the inner ear difficult and impractical. A medial approach along the petrous ridge and internal auditory canal (lAC) gains access to the membranous components of the inner ear with only a few millimeters of drilling. All dura is removed from the middle and posterior fossa surfaces of the temporal bone. On these surfaces the otic capsule is chalky white and stands out in sharp contrast to surrounding bone (Fig IA). With the aid of an operating microscope and cutting burs, drilling begins around the lAC on three fourths of the circumference, sparing the inferior one fourth (Fig lB). Progressing from medial to lateral along the superior portion of the lAC exposes the facial nerve and geniculate ganglion area (Fig IC). Immediately deep (inferior) and anterior to the first genu of the facial nerve is the basal tum of the cochlea. This turn is blue-lined from its anterior extent deep to the first genu of the facial nerve, anteromedially toward the posterior fossa surface of the petrous apex (Fig lD). It is usually possible to gain some narrow exposure to the second tum of the cochlea by continuing this dissection anterolaterally from the basal tum toward the apex. Posterior to the lAC the vestibule, posterior semicircular canal, and crus commune are blue-lined
MATERIAL AND METHODS
Acquisition ofBovine Temporal Bones. Quartered calf skulls with the brain removed were obtained from a local abattoir and meat packing plant. Fresh quartered skulls are taken immediately upon availability and packed in crushed ice for transport to the research laboratory. The temporal bones are microdissected to remove the membranous components of the inner ear, which are then placed in phosphate-buffered saline (PBS), pH 7.4, and frozen at -40°C until
From the Department of Otology and Laryngology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts. Work supported in part by a grant from the Immunology Research Institute of New England. REPRINTS - Steven D. Rauch, MD, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114.
688
Downloaded from aor.sagepub.com at Harvard Libraries on June 26, 2015
Rauchet al, BovineTemporal Bones
689
Fig 1. Temporal bone microdissection. A) Posterior fossa view of right temporal bone with dura removed. Note sharp demarcation oftemporal bone from surrounding skull base. Dotted line - attachment site of tentorium cerebelli dividing middle fossa surface oftemporal bone from posterior fossa surface, lAC - internal auditory canal. B) Cutting bur is used to drill away bone surrounding lAC from posterior to anterior. C) Facial nerve (N VII) to first genu and geniculate ganglion (arrow) is exposed and posterior semicircularcanal (arrowheads) is blue-lined. D) Facial nerve has been removed and basal tum ofcochlea (arrowheads) blue-lined. CN - cochlear nerve. E) Basal tum ofcochlea has been opened, exposing osseous spiral lamina (arrow). Vestibule (V) is opened to allow removal of otolith organs and semicircular canal ampullae. F) Modiolus has been extracted to expose contents of second tum of cochlea (double arrows). Vestibule is widely opened to permit extraction of membranous contents of hook portion of cochlea and vestibular end organs. OW - oval window, HC - ampullated end of horizontal semicircular canal.
Downloaded from aor.sagepub.com at Harvard Libraries on June 26, 2015
690
Rauch et al, Bovine Temporal Bones
A
B
c
o
kDa 97.4-'" 66.2--,
42.750.0-1······· . >··.·" • 40.0-
-
31.0---,
Fig 2. One-dimensional 12% sodium dodecyl sulfate gradient gelelectrophoresis. Each lanedosed with 20-f.l.L sample composed of 10 ul, of specimen and 10 ul, of sample buffer, run at 40 rnA for approximately 2 hours, and stained with Coomassie blue. A) Molecular weight standards. B,C) 7.1 mg/mL bovine inner ear (combined cochlear and vestibular) antigen extract. D) 6.0 mg/mL human cochlear antigen extract. Note high degree of banding homology between bovine andhuman samples. as well. Throughout the drilling frequent irrigation or use ofcontinuous suction irrigation is used to prevent overheating of the tissues. Once the cochlear and vestibular divisions of the inner ear are blue-lined, they are opened widely but superficially with diamond burs to expose the membranous structures of the inner ear (Fig IE). These are extracted easily by using a combination of whirlybird elevators and cup forceps. The saccule and utricle are lifted from the vestibule, and then each of the three ampullae of the semicircular canals are removed. The membranes of the cochlea are removed from base to apex on the exposed superomedial surface of the cochlear spiral, and then the modiolus is cracked out with a stapes curette to gain access to the inferolateral portion of each tum. The membranes of the hook portion of the cochlea are most easily removed through the vestibule (Fig IF). All tissue is placed directly into 0.5 mL chilled PBS solution as it is removed in order to reduce postmortem autolysis. Antigen Extract Preparation. In a cold room, tissue obtained from microdissection of the temporal bones is teased apart and homogenized in 0.5 mL of PBS (pH 7.4). The homogenate is then subjected to four sonication intervals (Branson Sonifier 450) of 2 minutes each at 4T. The resultant suspension is centrifuged at 3,000g for 30 minutes in the cold room and
the supernatant decanted and filtered through a 0.2urn filter. Protein content of this final extract solution is determined on the basis ofoptical density measurement by using a modified Lowry method as described by Bloch et al. 4 Aliquots are diluted 1:10 in PBS and optical density is measured at 215 and 225 nm. Protein concentration is calculated by using the following formula: [144 x (OD215 - OD225)] x dilution factor = Ilg/mL protein concentration. The extract solution is then divided into aliquots and stored at -70°C. DISCUSSION Using the techniques described, we have been able to achieve extract concentrations in excess of?O mg! mL from dissection of six temporal bones. Electrophoretic comparison of bovine inner ear antigen extract to human extract reveals a high degree of banding homology (Fig 2). Separation of the constituents of the extract by gel electrophoresis followed by Western blot assay for inner ear antibodies is being used as part ofan ongoing project studying idiopathic progressive bilateral sensorineural hearing loss. Purification by gel electrophoresis could also be used as the initial step in production of monoclonal anti bodies. Use ofcadaveric human inner ear tissue as a source ofantigen had the complicating issues ofpostmortem autolytic changes and small quantity of tissue. This latter problem required repeat standardization of antigen content for each new sample, necessitating gradual depletion of our most potent antibody-containing serum samples. The virtually unlimited supply of bovine inner ear tissue and the ease of dissection (less than half an hour per pair of temporal bones) make it feasible to pool tissue from many animals in order to have a large quantity of antigen that may be standardized once, stored at -70°C, and used repeatedly when needed. Standardization of antigenicity is a chronic problem in immunostaining and immunoblotting techniques. Even commercial immunoreagents require standardization at the time of use to ensure reliable interpretation of results. Large pools ofantigen reduce interspecimen error by applying the same antigen solution to each sample. In a longitudinal, prospective study such as our work on idiopathic progressive bilateral sensorineural hearing loss, the accuracy of data is greatly improved by having a long-term, stable supply of antigen to use throughout the duration of the study.
REFERENCES Harris JP.Experimental autoimmune sensorineural hear3. Harris JP, Sharp PA. Inner ear autoantibodies in patients ing loss. Laryngoscope 1987;97:63-76. with rapidly progressive sensorineural hearing loss. Laryngoscope 1990;100:516-24. 2. Rarey KE, Patterson K. Establishment of inner earepithe4. Bloch KJ, Wright JA,Bishara SM,Bloch MB. Uptake of lial cellculture: isolation, growth and characterization. Hear Res polypeptide fragments of proteins by rat intestine in vitro andin 1989;38:277-87. vivo. Gastroenterology 1988;95: 1272-8. 1.
Downloaded from aor.sagepub.com at Harvard Libraries on June 26, 2015
