Abstract
Hypothesis
Phosphorus and vitamin D (calcitriol) supplementation in the Phex mouse, a murine model for endolymphatic hydrops (ELH), will improve otic capsule mineralization and secondarily ameliorate the postnatal development of ELH and sensorineural hearing loss (SNHL).
Background
Male Phex mice have X-linked hypophosphatemic rickets (XLH), which includes osteomalacia of the otic capsule. The treatment for XLH is supplementation with phosphorus and calcitriol. The effect of this treatment has never been studied on otic capsule bone and it is unclear if improving the otic capsule bone could impact the mice’s postnatal development of ELH and SNHL.
Methods
Four cohorts were studied: 1) wild-type control, 2) Phex control, 3) Phex prevention, and 4) Phex rescue. The control groups were not given any dietary supplementation. The Phex prevention group was supplemented with phosphorus added to its drinking water and intraperitoneal calcitriol from postnatal day (P) 7–P40. The Phex rescue group was also supplemented with phosphorus and calcium but only from P20 to P40. At P40, all mice underwent auditory brainstem response (ABR) testing, serum analysis, and temporal bone histologic analysis. Primary outcome was otic capsule mineralization. Secondary outcomes were degree of SNHL and presence ELH.
Results
Both treatment groups had markedly improved otic capsule mineralization with less osteoid deposition. The improved otic capsule mineralized did not prevent the development of ELH or SNHL.
Conclusion
Supplementation with phosphorus and calcitriol improves otic capsule bone morphology in the Phex male mouse but does not alter development of ELH or SNHL.
1
Introduction
Meniere’s disease (MD) is a debilitating condition characterized by episodic vertigo, tinnitus, aural fullness, and progressive sensorineural hearing loss (SNHL) . First described in 1861 by Prosper Meniere, the etiology of MD remains idiopathic although its relationship to endolymphatic hydrops (ELH) has been well established in both human specimens and animal models . Therapeutic options for patients with MD range from a conservative low-salt diet to complete ablation of labyrinthine function. Clinicians’ inability to predict therapeutic response and the disease clinical course adds to both patient and clinician frustration with MD management .
Mouse models are commonly used to study human diseases. The Phex mouse had a spontaneous loss-of-function mutation in the phosphate-regulating gene with homology to endopeptidases located on the X chromosome ( PHEX gene), resulting in X-linked hypophosphatemic rickets (XLH). In 2004, a new mouse mutation of the Phex gene – Phex Hyp-Duk – was described. Phex Hyp-Duk arose on the BALB/cAnBomUrd strain at Duke University ( Phex Hyp-Duk /Y). Phex Hyp-Duk /Y (simplify as Phex) has similar pathological ELH features as those seen in ELH involving human Meniere’s disease . Analogous to human XLH, these mice have hypophosphatemia caused by renal phosphate wasting, osteomalacia, mild hypocalcemia, and shortened limbs . In 2008, the inner ear pathology of this mouse was further characterized when the male Phex mice were found to have postnatal development of vestibular dysfunction (circling behavior and/or head bobbing), progressive sensorineural hearing loss (SNHL), and ELH in a characteristic and reproducible manner . Since then, the Phex mouse model has increasingly served as a valuable tool to investigate the pathophysiology of ELH.
The incidence of XLH in humans is 1/20,000 live births, which makes it the most common inheritable form of rickets . The bone of XLH demonstrates osteomalacia with thick strands of unmineralized osteoid. The treatment for XLH consists of oral phosphate and calcitriol supplementation . Treatment has shown to improve growth and mineralization in long bones and vertebrae . Therapeutic response is assessed using alkaline phosphatase, which serves as a marker for bone turnover and drops to a normal level when a therapeutic dosage has been reached .
Otologic manifestations of XLH have been described in humans. Progressive SNHL and Meniere’s-like symptoms of episodic vertigo and tinnitus have been reported in upwards of 76% of patients . Electrocochleography (ECochG) in the majority of these patients suggests the presence of ELH . Radiographic studies have also demonstrated dysmorphic temporal bones . All of the otologic manifestations associated with XLH in humans have been replicated in the Phex mouse model, particularly the presence of diseased otic capsule bone and ELH .
The interplay between otic capsule bone and the endolymphatic system is still under investigation. Likewise, the mechanism underlying ELH remains unclear. This project attempts to further characterize the relationship between the otic capsule and ELH. By providing Phex mice with phosphorus and calcitriol supplementation, we investigated whether the standard treatment for XLH could improve the dysmorphic otic capsule bone and secondarily if the bone health would influence the progression of ELH and SNHL.
2
Materials and methods
The Institutional Animal Care and Use Committee of Case Western Reserve University and the Institutional Review Board of University Hospitals Case Medical Center approved this research protocol. The animals used were male BALB/cAnBomUrd mice with the Phex Hyp-Duk mutation ( Phex Hyp-Duk /Y) and their male wild-type littermates (+/Y). The mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) but have since been bred and maintained at Case Western Reserve University in compliance with their Animal Care and Use Committee guidelines.
2.1
Phex and X-linked hypophosphatemia background
The development of the Phex mouse model and its otologic manifestations has previously been described . In brief, XLH is caused by a genetic defect in the Phex gene that leads to a dysfunctional Phex protein. The Phex protein is a zinc-metalloendopeptidase located in the cell membrane of osteoblasts and chondrocytes . The protein’s normal function is to bind to pro-mineralization factors, like matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein-1 (DMP-1), to support healthy bone mineralization. The defective Phex protein leads to unmineralized osteoid deposition and soft bone, termed osteomalacia and rickets. Additionally, the impaired Phex-MEPE binding leads to an increase in fibroblast growth factor-23 (FGF-23), which is directly responsible for renal phosphate wasting and decreased 1,25-dihydroxyvitamin D production .
Humans with XLH are treated with supplemental phosphate and calcitriol (1,25-dihydroxyvitamin D3). Early initiation of treatment leads to improved bone mineralization and growth. Alkaline phosphatase, a marker of bone turnover, is elevated in patients with XLH. Therapeutic response to the dietary supplementation is in part measured by normalization of alkaline phosphatase levels. Therapy benefit can be attenuated by elevation of the down-stream molecule FGF-23, which disrupts the sodium-phosphate co-transporter in the proximal renal tubule. Anti-FGF-23 medications are in development, but the standard of care for XLH remains phosphate and calcitriol supplementation .
Male mice with the Phex Hyp-Duk mutation have XLH. Multiple studies have characterized the postnatal development and disease manifestations in the Phex mouse . At birth, the mice have normal inner ear histology including no ELH, spiral ganglion degeneration, or endolymphatic duct obstruction. Circling behavior and head bobbing, signs of vestibular dysfunction, start between postnatal day (P) 15 and 20. Bilateral hearing loss, documented with auditory brainstem response (ABR), starts around P20 and progresses to profound loss as early as P40. Evidence of apical hydrops and dysmorphic bone begins at P25 and becomes severe in most cases by P90. Spiral ganglion neuron apoptosis precedes inner ear hair cell loss, but both lag behind the onset of hearing loss. The endolymphatic duct remains patent throughout this progression. Phenotypically, the Phex mice have a small body size, short tail, and soft bones. The Phex Hyp-Duk mutation on the BALB/cAnBomUrd background leads to ELH, while on the C57BL/6 (B6) or BALB/cByJ background, there is no ELH.
2.2
Study design
There were four mouse populations: 1) wild-type control (+/Y), 2) Phex control (control Phex Hyp-Duk /Y), 3) Phex prevention (prevention Phex Hyp-Duk /Y), and 4) Phex rescue (rescue Phex Hyp-Duk /Y). All mice were maintained on standard chow (Iso Pro Rodent 3000 by LabDiet, St. Louis, MO, U.S.A.) that contains 1.11% calcium, 0.8% phosphorus, and 2.5 IU/g of Vitamin D3. The wild-type and Phex control groups did not receive any supplemental phosphorus or calcitriol. The Phex prevention and Phex rescue groups were supplemented with phosphorus and calcitriol only during a specified treatment period. For the Phex prevention group, that supplementation began at P7, the age of weaning, and continued through P40. The Phex rescue group started supplementation at P20 and continued therapy until P40. Mice from all groups were sacrificed at P40. Phosphorus-supplemented water (1.9 g elemental phosphorus per liter) replaced the normal drinking water and was available ad libitum during the treatment periods. Calcitriol, which is an active metabolite of 1,25-dihydroxyvitamin D3, was given via intraperitoneal (IP) injections at a dose of 0.4 μg/kg per injection. The injections were performed three times a week during the treatment periods. The phosphorus and calcitriol dosages were based on a study from Marie et al. (1982) that showed improved vertebrae mineralization in a Phex Hyp (Hyp/Y) hypophosphatemic mouse model .
2.3
Auditory-evoked brainstem response (ABR)
All mice underwent ABR testing at P40. The ABR technique has previously been described . In brief, mice aged P40 were anesthetized with an IP injection of ketamine, xylazine, and acepromazine at doses of 40, 5, and 1 mg/kg, respectively. Body temperature was maintained at 37–38 °C by placing the mice on a homeothermic heating pad (Harvard apparatus, Holliston, MA) within a soundproof chamber. ABR testing was carried out using the SmartEP system from Intelligent Hearing Systems (Miami, FL). Platinum subdermal needle electrodes were inserted at the skull vertex (ground electrode) and ventrolateral to the right and left ears. Pure-tone stimuli at 8 kHz, 16 kHz, and 32 kHz of 100-ms duration were presented for at least 700 sweeps to each ear (one at a time) through high-frequency transducers (closed system). ABR thresholds were obtained from both ears for each animal by reducing the stimulus intensity from 90 dB SPL in 5 or 10 dB steps until the lowest intensity that could evoke a reproducible ABR pattern was detected.
2.4
Serum analysis
Immediately following ABR testing, the anesthetized mice were sacrificed. Blood was collected via cardiac puncture, spun down in a centrifuge for 10 min at 10,000 rpm (RPM), and then the serum was extracted. Serum phosphorus, calcium, creatinine, and alkaline phosphatase assays were processed with the Dimensions VISTA® system from Siemens Medical Solutions, Inc. (Malvern, PA) in the University Hospitals Case Medical Center Core Laboratory. Serum FGF-23 concentration was determined using an enzyme-linked immunosorbent assay (ELISA) kit from Immutopics International (San Clemente, CA) that was processed in the Dahms Clinical Research Unit at University Hospitals Case Medical Center. The mouse FGF-23 (C-Term) assay is a two-site ELISA that detects epitopes within the carboxyl-terminal (C-Term) region of mouse FGF-23 .
2.5
Anatomical analysis
After obtaining serum, a microscope was used for temporal bone dissection and exposure of the inner ear. The stapes footplate was removed. Using a fine needle, the oval window and round window were delicately punctured to facilitate perfusion of the cochlea. The inner ear specimens were then immersed in 4% paraformaldehyde (PFA) for 1 week at 4 °C. Next, the specimens were rinsed in 0.1 M sodium phosphate buffer (pH 7.4) and decalcified in 0.35 M ethylenediaminetetraacetic acid (EDTA) for 1 week. The inner ears were then dehydrated with serial alcohol rinses and embedded in paraffin. Tissue sections were cut to a thickness of 5–8 μm using a cold microtome and plated on glass slides. The tissue was then stained with hematoxylin and eosin to facilitate morphologic analysis under a light microscope. The otic capsule bone, membranous cochlea including Reissner’s membrane, and spiral ganglion were analyzed. The study design is summarized in Fig. 1 .
2.6
Statistical analysis
Numerical data were collected, entered into Excel (Microsoft, Redmond, WA), and imported into GraphPad Prism (GraphPad, San Diego, CA). Mean values and standard deviations or confidence intervals were calculated for continuous variables. Percentages or frequencies were calculated for categorical variables. Both parametric and non-parametric statistics were used as appropriate. A one-way analysis of variance (ANOVA) was used to compare serum data and ABR thresholds between the four groups. An independent Student’s t -test was used for weight comparisons. The criterion for statistical analysis was set at p < 0.05, two-tailed.
2
Materials and methods
The Institutional Animal Care and Use Committee of Case Western Reserve University and the Institutional Review Board of University Hospitals Case Medical Center approved this research protocol. The animals used were male BALB/cAnBomUrd mice with the Phex Hyp-Duk mutation ( Phex Hyp-Duk /Y) and their male wild-type littermates (+/Y). The mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) but have since been bred and maintained at Case Western Reserve University in compliance with their Animal Care and Use Committee guidelines.
2.1
Phex and X-linked hypophosphatemia background
The development of the Phex mouse model and its otologic manifestations has previously been described . In brief, XLH is caused by a genetic defect in the Phex gene that leads to a dysfunctional Phex protein. The Phex protein is a zinc-metalloendopeptidase located in the cell membrane of osteoblasts and chondrocytes . The protein’s normal function is to bind to pro-mineralization factors, like matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein-1 (DMP-1), to support healthy bone mineralization. The defective Phex protein leads to unmineralized osteoid deposition and soft bone, termed osteomalacia and rickets. Additionally, the impaired Phex-MEPE binding leads to an increase in fibroblast growth factor-23 (FGF-23), which is directly responsible for renal phosphate wasting and decreased 1,25-dihydroxyvitamin D production .
Humans with XLH are treated with supplemental phosphate and calcitriol (1,25-dihydroxyvitamin D3). Early initiation of treatment leads to improved bone mineralization and growth. Alkaline phosphatase, a marker of bone turnover, is elevated in patients with XLH. Therapeutic response to the dietary supplementation is in part measured by normalization of alkaline phosphatase levels. Therapy benefit can be attenuated by elevation of the down-stream molecule FGF-23, which disrupts the sodium-phosphate co-transporter in the proximal renal tubule. Anti-FGF-23 medications are in development, but the standard of care for XLH remains phosphate and calcitriol supplementation .
Male mice with the Phex Hyp-Duk mutation have XLH. Multiple studies have characterized the postnatal development and disease manifestations in the Phex mouse . At birth, the mice have normal inner ear histology including no ELH, spiral ganglion degeneration, or endolymphatic duct obstruction. Circling behavior and head bobbing, signs of vestibular dysfunction, start between postnatal day (P) 15 and 20. Bilateral hearing loss, documented with auditory brainstem response (ABR), starts around P20 and progresses to profound loss as early as P40. Evidence of apical hydrops and dysmorphic bone begins at P25 and becomes severe in most cases by P90. Spiral ganglion neuron apoptosis precedes inner ear hair cell loss, but both lag behind the onset of hearing loss. The endolymphatic duct remains patent throughout this progression. Phenotypically, the Phex mice have a small body size, short tail, and soft bones. The Phex Hyp-Duk mutation on the BALB/cAnBomUrd background leads to ELH, while on the C57BL/6 (B6) or BALB/cByJ background, there is no ELH.
2.2
Study design
There were four mouse populations: 1) wild-type control (+/Y), 2) Phex control (control Phex Hyp-Duk /Y), 3) Phex prevention (prevention Phex Hyp-Duk /Y), and 4) Phex rescue (rescue Phex Hyp-Duk /Y). All mice were maintained on standard chow (Iso Pro Rodent 3000 by LabDiet, St. Louis, MO, U.S.A.) that contains 1.11% calcium, 0.8% phosphorus, and 2.5 IU/g of Vitamin D3. The wild-type and Phex control groups did not receive any supplemental phosphorus or calcitriol. The Phex prevention and Phex rescue groups were supplemented with phosphorus and calcitriol only during a specified treatment period. For the Phex prevention group, that supplementation began at P7, the age of weaning, and continued through P40. The Phex rescue group started supplementation at P20 and continued therapy until P40. Mice from all groups were sacrificed at P40. Phosphorus-supplemented water (1.9 g elemental phosphorus per liter) replaced the normal drinking water and was available ad libitum during the treatment periods. Calcitriol, which is an active metabolite of 1,25-dihydroxyvitamin D3, was given via intraperitoneal (IP) injections at a dose of 0.4 μg/kg per injection. The injections were performed three times a week during the treatment periods. The phosphorus and calcitriol dosages were based on a study from Marie et al. (1982) that showed improved vertebrae mineralization in a Phex Hyp (Hyp/Y) hypophosphatemic mouse model .
2.3
Auditory-evoked brainstem response (ABR)
All mice underwent ABR testing at P40. The ABR technique has previously been described . In brief, mice aged P40 were anesthetized with an IP injection of ketamine, xylazine, and acepromazine at doses of 40, 5, and 1 mg/kg, respectively. Body temperature was maintained at 37–38 °C by placing the mice on a homeothermic heating pad (Harvard apparatus, Holliston, MA) within a soundproof chamber. ABR testing was carried out using the SmartEP system from Intelligent Hearing Systems (Miami, FL). Platinum subdermal needle electrodes were inserted at the skull vertex (ground electrode) and ventrolateral to the right and left ears. Pure-tone stimuli at 8 kHz, 16 kHz, and 32 kHz of 100-ms duration were presented for at least 700 sweeps to each ear (one at a time) through high-frequency transducers (closed system). ABR thresholds were obtained from both ears for each animal by reducing the stimulus intensity from 90 dB SPL in 5 or 10 dB steps until the lowest intensity that could evoke a reproducible ABR pattern was detected.
2.4
Serum analysis
Immediately following ABR testing, the anesthetized mice were sacrificed. Blood was collected via cardiac puncture, spun down in a centrifuge for 10 min at 10,000 rpm (RPM), and then the serum was extracted. Serum phosphorus, calcium, creatinine, and alkaline phosphatase assays were processed with the Dimensions VISTA® system from Siemens Medical Solutions, Inc. (Malvern, PA) in the University Hospitals Case Medical Center Core Laboratory. Serum FGF-23 concentration was determined using an enzyme-linked immunosorbent assay (ELISA) kit from Immutopics International (San Clemente, CA) that was processed in the Dahms Clinical Research Unit at University Hospitals Case Medical Center. The mouse FGF-23 (C-Term) assay is a two-site ELISA that detects epitopes within the carboxyl-terminal (C-Term) region of mouse FGF-23 .
2.5
Anatomical analysis
After obtaining serum, a microscope was used for temporal bone dissection and exposure of the inner ear. The stapes footplate was removed. Using a fine needle, the oval window and round window were delicately punctured to facilitate perfusion of the cochlea. The inner ear specimens were then immersed in 4% paraformaldehyde (PFA) for 1 week at 4 °C. Next, the specimens were rinsed in 0.1 M sodium phosphate buffer (pH 7.4) and decalcified in 0.35 M ethylenediaminetetraacetic acid (EDTA) for 1 week. The inner ears were then dehydrated with serial alcohol rinses and embedded in paraffin. Tissue sections were cut to a thickness of 5–8 μm using a cold microtome and plated on glass slides. The tissue was then stained with hematoxylin and eosin to facilitate morphologic analysis under a light microscope. The otic capsule bone, membranous cochlea including Reissner’s membrane, and spiral ganglion were analyzed. The study design is summarized in Fig. 1 .
