Am J Otalaryngol 12:25cK266.1991
Inner Ear Pathology in the Palmerston North Autoimmune Strain Mouse DENNIS R. TRUNE, PHD, JANE I. MORTON, PHD, JAMES P. CRAVEN, MD, SEAN J. TRAYNOR, BS, AND CURT R. MITCHELL, PHD
The Palmerston North autoimmune strain mouse is a model for spontaneous systemic lupus erythematosus. Inner ear structure and function were examined during the onset and progression of systemic autoimmune disease to identify potentially correlated auditory system pathology. The onset of systemic disease occurred at 4 to 5 months of age and was characterized by elevated serum immune complexes, cryoglobulins, and antinuclear antibodies. Coincident with the onset of autoimmune disease was degeneration of the apical turn stria vascularis and outer hair cells. These cochlear changes progressed basalward. At 10 months of age, auditory brainstem response thresholds were elevated and the stria vascularis area was measurably smaller throughout the cochlea. Immunohistochemical staining showed immunoglobulin G deposits within the organ of Corti, the vas spirale of the basilar membrane, the Scala tympani, and marrow cavities of the bony otic capsule. These results suggest that cochlear pathology may be immune mediated in this mouse, which would make the strain suitable for the study of the mechanisms relating inner ear abnormalities and autoimmune disease. AM J OTOLARYNGOL 12:259-266. Copyright 0 1991 by W.B. Saunders Company Key words: Palmerston North mouse, inner ear, pathology, autoimmune disease, immunoglobulin.
serum immunoglobulins and antinuclear antibodies.7’8 Because autoantibodies against inner ear structures have been hypothesized as the underlying mechanism for inner ear dysfunction,‘-’ this model may show correlative evidence for these disease processes. However, no studies have been done of the PN mice to correlate the progression of inner ear pathology and systemic autoimmune disease. In this study of an age series of PN mice, we assessed auditory structures before the onset of autoimmune disease and documented the subsequent course of disease with inner ear degeneration. The study was conducted to provide insights into inner ear pathology in autoimmune disease and the suitability of the PN mouse strain as a model for studying these potentially related mechanisms.
The correlation between autoimmunity or immune complex disease and significant auditory and vestibular pathology is now well established.lS5 However, the mechanisms relating immune disease and the inner ear are unknown, due mainly to the lack of an appropriate animal model, one in which hearing loss is associated with spontaneous autoimmune disease. Preliminary inner ear studies of the Palmerston North (PN) autoimmune strain of mice have shown that this strain may be suitable for such studies.6 This mouse is a model for late onset systemic lupus erythematosus and is characterized by elevated Received April X3,1991, from The Oregon Hearing Research Center, Department of Otolaryngology-Head & Neck Surgery, and the Department of Medicine, Division of Arthritis and Rheumatic Diseases, The Oregon Health Sciences University, Portland, OR. Accepted for publication August 6, 1991. Supported in partby The Deafness Research Foundation and BRSG SO7 RR05412 Award bv the Biomedical Research SUDport Grant Program, Division of Research Resources, National Institutes of Health. Presented in part at The Association for Research in Otolaryngology meetings, February l-6, 1967, Clearwater Beach, FL, and September 22-26, 1991, Kansas City, MO. Address correspondence and reprint requests to Dennis R. Trune, PhD, Oregon Hearing Research Center, Oregon Health Sciences University, 3515 SW US Veterans Hospital Rd. Portland, OR 97201-2997. Copyright 0 1991 by W.B. Saunders Company 0166-0709/91/1205-0005$5.00/O
MATERIALS
AND METHODS
The PN mice studied ranged in age from 2 to 20 months. Auditory brainstem response (ABR) audiometry, serum analyses, and inner ear histopathology and immunoglobulin G (IgG) immunohistochemistry were used to assess the progression and extent of disease.
Auditory Brainstem Response Audiometry. Auditory brainstem response audiometry was performed on five ears from three randomly selected PN 259
AUTOIMMUNE INNER EAR PATHOLOGY
260 mice at 10 months of age. Because the base strain from
which the PN mutant is derived is unknown,7 no genetic controls were available for study. However, 3-month-old CBA/J mice (10 mice, 14 ears) and lomonth-old C3WHeJ mice (five mice, seven ears) were also tested to provide a standard for normal and agematched inner ear activity. Mice were anesthetized with 5% chloral hydrate and body temperature was maintained with a heating pad. Surface electrodes were placed in the midline, pinna, and forepaw. Clicks were produced by a O-l-ms electric square wave and a TDH49 earphone and delivered into the ear canal through a 2-mm diameter sound delivery tube. The click spectrum was from 0.5 to 4.3 kHz (6 dB down). Duplicate 400 sweep averages were obtained at each intensity level. A threshold in decibels (peak dB SPL) was determined, cbnstituting the lowest intensity for a peak within thb 10 ms after the stimulus. Measurable ABR threshold! were statistically compared by a t-test.
Serum Analyses. Serum samples were regularly collected from developing PN mice and analyzed for serum immune complexes and cryoglobulins, measured respectively by 3.5% polyethylene glycol precipitationg and incubation at 4% for 5 days. Precipitable protein was quantified by the method of Lowry et al.” In addition, plasma antinuclear antibodies (ANAs), previously established to be anti-DNA antibodies, were analyzed using an indirect immunofluorescence assay with chicken erythrocyte nuclei as substrate.‘l Responses were graded on a scale from 0 to 4, with 3 and 4 considered strongly positive. Samples were coded to prevent grading bias. The number of mice tested at each age ranged from 12 to 47. Control values for these measurements were determined by the serum analysis of nonautoimmune BALB/c (N = 17), DBA/2 (N = 6), C3HA-IeJ (N = 12), C57B116J (N = 5) and CBA/J (N = 6) mice. Statistical evaluations (t-tests) were conducted to test the null hypothesis that no serum differences existed between the PN and control strains. Probabilities below 5% were judged to be significant. Mice were anesthetized with 5% chloral hydrate and their inner ears collected for histopathology. The number of mice collected at the various ages ranged from 5 to 12. Most inner ears were fixed by the intracardiac perfusion of fixative (3% glutaraldehyde/l.5°b paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.2). Other ears were immersion fixed with perilymphatic perfusion to preserve blood elements within the vasculature. All inner ears were decalcified in EDTA, embedded in glycol methacrylate, serially sectioned at 3 to 5 pm, and stained with hematoxylin-eosin. Inner ears from the age series of PN mice were evaluated for pathology prior to and after disease onset. Observations were made of the organ of Corti, stria vascularis, spiral ganglion, and auditory nerve fibers. Vestibular sensory epithelia and supporting structures also were examined to identify possible vestibular involvement. Our preliminary studies6 have suggested that the stria vascularis may be affected in the PN mice; therefore, measurements were made of a stria vascularis crosssectional area. The two most midmodiolar sections of each ear from all mice were identified for morphometry. The slides were coded to prevent measuring bias. The three half-turns of the cochlea were studied under a Zeiss microscope fitted with a camera lucida, and the outline of the stria was traced. The area occupied by the
Inner Ear Histopathology.
stria was measured on a digitizer tablet interfaced with the Bioquant II Image Analysis Software (R & M Biometrics, Nashville, TN). The two area measurements for each half-turn were averaged and stria size was compared between the 2- and 20-month age groups using t-tests.
Inner Ear lmmunohistochemistry. Inner ear sections were immunohistochemically stained for IgG to identify potential immune mechanisms for the pathology seen. An additional 10 animals were perfused with 3% paraformaldehyde (0.1 mol/L phosphate buffer], decalcified in EDTA, and embedded in glycol methacrylate. Sections were stained in sequence with anti-mouse IgG antibody (ICN Biochemicals, Costa Mesa, CA), biotinylated protein A, avidin complexed horseradish peroxidase (Vector, Burlingame, CA), and benzidine dihydrochloride. This chromogen gives a blue-green reaction product that prevents confusion with the brown melanin normally found in the ear. Kidney and spleen tissues from the same animals were processed in the same manner to serve as positive controls. RESULTS Auditory Brainstem Response Audiometry. The normal S-month-old CBA/J mice showed thresholds between 25 and 55 dB (Fig l),representing normal cochlear function with no aging or genetic effects. Some of the lo-month-old C3H/ HeJ controls had thresholds slightly higher, presumably due to aging effects on the inner ear, but overall they were not different from the CBA/J mice (t = 1.44, P = .16). These C3H/HeJ mice provide
an
age-match
comparison
for
the
PN
mice. Four
of the PN ears (from
show
a brainstem
limit
of the testing
from one of these
response system. mice
three
mice)
at 105 The
failed
contralateral
had a threshold
ear
at 105 dB, 0m.a
)lOO _
to
dB, the upper
0
100 1
Ei I
m -0 V n 6
80-
60-
5 !$
40-
f 20-
aa 0 l eeee l l ee le l l l
CBA/J
l
l ee
C3H/HeJ
PN
MOUSE STRAINS Figure I. Scatter plot of ABR thresholds for s-month-old CBA/J (n = 10; 14 ears), lo-month-old C3H/HeJ(n = 5; eight ears), and lo-month-old PN (n = 3; five ears) mice. Four of the five PN mice did not demonstrate any ABR, even at the maximum stimulation of 105 dB SPL.
261
TRUNE ET AL which was considerably above the control levels. Although it was our intention to test a larger number of PN mice, it was difficult to keep additional mice alive due to their advanced disease and poor physiologic condition. Thus, we present data only from those that survived testing to avoid the potential misinterpretation of inner ear data from those under stress. In spite of this limited number, the ABR testing did demonstrate a consistent pattern showing the PN inner ears to be virtually incapable of responding to acoustic stimulation.
z v)
N=
0' R k!
90-p
z
inner Ear Pathology. Lymphocytic infiltration of the middle ear tensor tympani muscle was observed in some mice after the onset of systemic disease (Fig 3). This involved unusually large concentrations of lymphocytes around small blood vessels within the substance of the muscle. The
TABLE 1. Serum Immune Complexes and Cryoglobulins in Palmerston North and BALBlc Mice MOUSE STRAIN (No. OF MICE)
PN (12) BALB/c (171 Probability Abbreviation:
IMMUNE COMPLEXES (W/ML) (SE) 1075.4 (294.1) 225.7 (24.4)
Inner Ear Pathology in the Palmerston North Autoimmune Strain Mouse DENNIS R. TRUNE, PHD, JANE I. MORTON, PHD, JAMES P. CRAVEN, MD, SEAN J. TRAYNOR, BS, AND CURT R. MITCHELL, PHD
The Palmerston North autoimmune strain mouse is a model for spontaneous systemic lupus erythematosus. Inner ear structure and function were examined during the onset and progression of systemic autoimmune disease to identify potentially correlated auditory system pathology. The onset of systemic disease occurred at 4 to 5 months of age and was characterized by elevated serum immune complexes, cryoglobulins, and antinuclear antibodies. Coincident with the onset of autoimmune disease was degeneration of the apical turn stria vascularis and outer hair cells. These cochlear changes progressed basalward. At 10 months of age, auditory brainstem response thresholds were elevated and the stria vascularis area was measurably smaller throughout the cochlea. Immunohistochemical staining showed immunoglobulin G deposits within the organ of Corti, the vas spirale of the basilar membrane, the Scala tympani, and marrow cavities of the bony otic capsule. These results suggest that cochlear pathology may be immune mediated in this mouse, which would make the strain suitable for the study of the mechanisms relating inner ear abnormalities and autoimmune disease. AM J OTOLARYNGOL 12:259-266. Copyright 0 1991 by W.B. Saunders Company Key words: Palmerston North mouse, inner ear, pathology, autoimmune disease, immunoglobulin.
serum immunoglobulins and antinuclear antibodies.7’8 Because autoantibodies against inner ear structures have been hypothesized as the underlying mechanism for inner ear dysfunction,‘-’ this model may show correlative evidence for these disease processes. However, no studies have been done of the PN mice to correlate the progression of inner ear pathology and systemic autoimmune disease. In this study of an age series of PN mice, we assessed auditory structures before the onset of autoimmune disease and documented the subsequent course of disease with inner ear degeneration. The study was conducted to provide insights into inner ear pathology in autoimmune disease and the suitability of the PN mouse strain as a model for studying these potentially related mechanisms.
The correlation between autoimmunity or immune complex disease and significant auditory and vestibular pathology is now well established.lS5 However, the mechanisms relating immune disease and the inner ear are unknown, due mainly to the lack of an appropriate animal model, one in which hearing loss is associated with spontaneous autoimmune disease. Preliminary inner ear studies of the Palmerston North (PN) autoimmune strain of mice have shown that this strain may be suitable for such studies.6 This mouse is a model for late onset systemic lupus erythematosus and is characterized by elevated Received April X3,1991, from The Oregon Hearing Research Center, Department of Otolaryngology-Head & Neck Surgery, and the Department of Medicine, Division of Arthritis and Rheumatic Diseases, The Oregon Health Sciences University, Portland, OR. Accepted for publication August 6, 1991. Supported in partby The Deafness Research Foundation and BRSG SO7 RR05412 Award bv the Biomedical Research SUDport Grant Program, Division of Research Resources, National Institutes of Health. Presented in part at The Association for Research in Otolaryngology meetings, February l-6, 1967, Clearwater Beach, FL, and September 22-26, 1991, Kansas City, MO. Address correspondence and reprint requests to Dennis R. Trune, PhD, Oregon Hearing Research Center, Oregon Health Sciences University, 3515 SW US Veterans Hospital Rd. Portland, OR 97201-2997. Copyright 0 1991 by W.B. Saunders Company 0166-0709/91/1205-0005$5.00/O
MATERIALS
AND METHODS
The PN mice studied ranged in age from 2 to 20 months. Auditory brainstem response (ABR) audiometry, serum analyses, and inner ear histopathology and immunoglobulin G (IgG) immunohistochemistry were used to assess the progression and extent of disease.
Auditory Brainstem Response Audiometry. Auditory brainstem response audiometry was performed on five ears from three randomly selected PN 259
AUTOIMMUNE INNER EAR PATHOLOGY
260 mice at 10 months of age. Because the base strain from
which the PN mutant is derived is unknown,7 no genetic controls were available for study. However, 3-month-old CBA/J mice (10 mice, 14 ears) and lomonth-old C3WHeJ mice (five mice, seven ears) were also tested to provide a standard for normal and agematched inner ear activity. Mice were anesthetized with 5% chloral hydrate and body temperature was maintained with a heating pad. Surface electrodes were placed in the midline, pinna, and forepaw. Clicks were produced by a O-l-ms electric square wave and a TDH49 earphone and delivered into the ear canal through a 2-mm diameter sound delivery tube. The click spectrum was from 0.5 to 4.3 kHz (6 dB down). Duplicate 400 sweep averages were obtained at each intensity level. A threshold in decibels (peak dB SPL) was determined, cbnstituting the lowest intensity for a peak within thb 10 ms after the stimulus. Measurable ABR threshold! were statistically compared by a t-test.
Serum Analyses. Serum samples were regularly collected from developing PN mice and analyzed for serum immune complexes and cryoglobulins, measured respectively by 3.5% polyethylene glycol precipitationg and incubation at 4% for 5 days. Precipitable protein was quantified by the method of Lowry et al.” In addition, plasma antinuclear antibodies (ANAs), previously established to be anti-DNA antibodies, were analyzed using an indirect immunofluorescence assay with chicken erythrocyte nuclei as substrate.‘l Responses were graded on a scale from 0 to 4, with 3 and 4 considered strongly positive. Samples were coded to prevent grading bias. The number of mice tested at each age ranged from 12 to 47. Control values for these measurements were determined by the serum analysis of nonautoimmune BALB/c (N = 17), DBA/2 (N = 6), C3HA-IeJ (N = 12), C57B116J (N = 5) and CBA/J (N = 6) mice. Statistical evaluations (t-tests) were conducted to test the null hypothesis that no serum differences existed between the PN and control strains. Probabilities below 5% were judged to be significant. Mice were anesthetized with 5% chloral hydrate and their inner ears collected for histopathology. The number of mice collected at the various ages ranged from 5 to 12. Most inner ears were fixed by the intracardiac perfusion of fixative (3% glutaraldehyde/l.5°b paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.2). Other ears were immersion fixed with perilymphatic perfusion to preserve blood elements within the vasculature. All inner ears were decalcified in EDTA, embedded in glycol methacrylate, serially sectioned at 3 to 5 pm, and stained with hematoxylin-eosin. Inner ears from the age series of PN mice were evaluated for pathology prior to and after disease onset. Observations were made of the organ of Corti, stria vascularis, spiral ganglion, and auditory nerve fibers. Vestibular sensory epithelia and supporting structures also were examined to identify possible vestibular involvement. Our preliminary studies6 have suggested that the stria vascularis may be affected in the PN mice; therefore, measurements were made of a stria vascularis crosssectional area. The two most midmodiolar sections of each ear from all mice were identified for morphometry. The slides were coded to prevent measuring bias. The three half-turns of the cochlea were studied under a Zeiss microscope fitted with a camera lucida, and the outline of the stria was traced. The area occupied by the
Inner Ear Histopathology.
stria was measured on a digitizer tablet interfaced with the Bioquant II Image Analysis Software (R & M Biometrics, Nashville, TN). The two area measurements for each half-turn were averaged and stria size was compared between the 2- and 20-month age groups using t-tests.
Inner Ear lmmunohistochemistry. Inner ear sections were immunohistochemically stained for IgG to identify potential immune mechanisms for the pathology seen. An additional 10 animals were perfused with 3% paraformaldehyde (0.1 mol/L phosphate buffer], decalcified in EDTA, and embedded in glycol methacrylate. Sections were stained in sequence with anti-mouse IgG antibody (ICN Biochemicals, Costa Mesa, CA), biotinylated protein A, avidin complexed horseradish peroxidase (Vector, Burlingame, CA), and benzidine dihydrochloride. This chromogen gives a blue-green reaction product that prevents confusion with the brown melanin normally found in the ear. Kidney and spleen tissues from the same animals were processed in the same manner to serve as positive controls. RESULTS Auditory Brainstem Response Audiometry. The normal S-month-old CBA/J mice showed thresholds between 25 and 55 dB (Fig l),representing normal cochlear function with no aging or genetic effects. Some of the lo-month-old C3H/ HeJ controls had thresholds slightly higher, presumably due to aging effects on the inner ear, but overall they were not different from the CBA/J mice (t = 1.44, P = .16). These C3H/HeJ mice provide
an
age-match
comparison
for
the
PN
mice. Four
of the PN ears (from
show
a brainstem
limit
of the testing
from one of these
response system. mice
three
mice)
at 105 The
failed
contralateral
had a threshold
ear
at 105 dB, 0m.a
)lOO _
to
dB, the upper
0
100 1
Ei I
m -0 V n 6
80-
60-
5 !$
40-
f 20-
aa 0 l eeee l l ee le l l l
CBA/J
l
l ee
C3H/HeJ
PN
MOUSE STRAINS Figure I. Scatter plot of ABR thresholds for s-month-old CBA/J (n = 10; 14 ears), lo-month-old C3H/HeJ(n = 5; eight ears), and lo-month-old PN (n = 3; five ears) mice. Four of the five PN mice did not demonstrate any ABR, even at the maximum stimulation of 105 dB SPL.
261
TRUNE ET AL which was considerably above the control levels. Although it was our intention to test a larger number of PN mice, it was difficult to keep additional mice alive due to their advanced disease and poor physiologic condition. Thus, we present data only from those that survived testing to avoid the potential misinterpretation of inner ear data from those under stress. In spite of this limited number, the ABR testing did demonstrate a consistent pattern showing the PN inner ears to be virtually incapable of responding to acoustic stimulation.
z v)
N=
0' R k!
90-p
z
inner Ear Pathology. Lymphocytic infiltration of the middle ear tensor tympani muscle was observed in some mice after the onset of systemic disease (Fig 3). This involved unusually large concentrations of lymphocytes around small blood vessels within the substance of the muscle. The
TABLE 1. Serum Immune Complexes and Cryoglobulins in Palmerston North and BALBlc Mice MOUSE STRAIN (No. OF MICE)
PN (12) BALB/c (171 Probability Abbreviation:
IMMUNE COMPLEXES (W/ML) (SE) 1075.4 (294.1) 225.7 (24.4)
