Patent Application
20090258091
The present invention relates to compositions and methods for treating neurodegenerative diseases. In some embodiments, the present invention provides compositions for treating and preventing presbycusis.
Age-related hearing loss or presbycusis poses enormous humanitarian and economic challenges to society. In the US, it is estimated that 23 percent of people aged between 65 and 75 years of age, and 40 percent of people over 75 years of age have presbycusis, and the number of people suffering from this disorder is expected to grow dramatically as the population of older people increases (Someya et al., Neurobiol. Aging 20:1613 [2007]).
Presbycusis is the loss of hearing that gradually occurs in most individuals as they grow older. Hearing loss is a common disorder associated with aging. The loss associated with presbycusis is usually greater for high-pitched sounds or high frequency sounds.
There are many causes of presbycusis. Most commonly it arises from pathological changes in the inner ear of a person as he or she ages, but presbycusis can also result from pathological changes in the middle ear or from complex pathological changes along the nerve pathways leading to the brain. Presbycusis most often occurs in both ears, affecting them equally. Because the process of loss is gradual, people who have presbycusis may not realize that their hearing is diminishing. With presbycusis, sounds often seem less clear and lower in volume. This contributes to difficulty hearing and understanding speech.
Sensorineural hearing loss is caused by disorders of the inner ear or auditory nerve. Presbycusis is usually a sensorineural hearing disorder. It is most commonly caused by gradual changes in the inner ear. The cumulative effects of repeated exposure to daily traffic sounds or construction work, noisy offices, equipment that produces noise, and loud music can cause sensorineural hearing loss. Sensorineural hearing loss is most often due to a loss of hair cells (sensory receptors in the inner ear) and spiral ganglion cells (cochlear neurons). This can occur as a result of hereditary factors as well as aging, various health conditions, and side effects of some medicines (aspirin and certain antibiotics).
Presbycusis may be caused by changes in the blood supply to the ear because of heart disease, high blood pressure, vascular (pertaining to blood vessels) conditions caused by diabetes, or other circulatory problems. The loss may be mild, moderate, or severe.
Sometimes presbycusis is a conductive hearing disorder, meaning the loss of sound sensitivity is caused by abnormalities of the outer ear and/or middle ear. Such abnormalities may include reduced function of the tympanic membrane (the eardrum) or reduced function of the three tiny bones in the middle ear that carry sound waves from the tympanic membrane to the inner ear.
There are many strategies to help people with presbycusis. Hearing aids may be recommended for some individuals. Assistive listening devices can provide further improvement in hearing ability in certain situations. One example of such a device is the built-in telephone amplifier. Another example is FM systems that make sounds clearer, with or without a hearing aid, by delivering sound waves like a radio. Training in speechreading (using visual cues to determine what is being spoken) can help those with presbycusis to understand better what is being said in conversations or presentations.
Nonetheless, no preventative or therapeutic interventions have been developed for presbycusis.
The present invention relates to compositions and methods for treating neurodegenerative diseases. In some embodiments, the present invention provides compositions for treating and preventing presbycusis. In some embodiments, other neurodegenerative disease are treated. In some embodiments, the present invention provides nutritional supplements, food products, and pharmaceutical compositions, and methods for administering such products comprising compounds that prevent or reduce neurodegenerative diseases.
In some embodiments, the present invention provides methods for treating and/or preventing a neurodegenerative disease (e.g., presbycusis), comprising administering a composition comprising an extract of coral to a subject having symptoms of a neurodegenerative disease or at risk of a neurodegenerative disease under conditions such that symptoms are prevented or reduced. In some embodiments, the coral extract is an aqueous liquid (e.g., MiliQ water or distilled water). In some embodiments, the coral extract has a pH of approximately 7.0 to 9.0 (e.g., 8.0). In some embodiments, the coral extract has a calcium content of approximately 1.4 mg/L to 140 mg/L (e.g., 7 mg/L to 21 mg/L, 8.5 mg/L to 19.5 mg/L, 10 mg/L to 18 mg/L, 11 mg/L to 17 mg/L, 12.8 mg/L to 15.8 mg/L, 13.6 mg/L to 15 mg/L, 13.9 mg/L to 14.7 mg/L, 14 mg/L to 14.6 mg/L, 14.2 mg/L to 14.5 mg/L, or approximately 14.30 mg/L). In some embodiments, the coral extract has a magnesium content of approximately 0.54 mg/L to 54.0 mg/L (e.g., 2.7 mg/L to 8.1 mg/L, 3.2 mg/L to 7.6 mg/L, 3.8 mg/L to 7 mg/L, 4.3 mg/L to 6.5 mg/L, 4.9 mg/L to 5.9 mg/L, 5.1 mg/L to 5.7 mg/L, 5.2 mg/L to 5.6 mg/L, 5.3 mg/L to 5.5 mg/L, 5.35 mg/L to 5.45 mg/L, or approximately 5.4 mg/L). In some embodiments, the coral extract has a sodium content of approximately 0.1 mg/L to 50.0 mg/L (e.g., 0.53 mg/L to 1.59 mg/L, 0.76 mg/L to 1.46 mg/L, 0.86 mg/L to 1.26 mg/L, 0.96 mg/L to 1.16 mg/L, 1.0 mg/L to 1.1 mg/L, 1.03 mg/L to 1.09 mg/L, 1.04 mg/L to 1.08 mg/L, 1.05 mg/L to 1.07 mg/L, 1.055 mg/L to 1.065 mg/L, or approximately 1.06 mg/L). In some embodiments, the coral extract has a potassium content of approximately 0.03 mg/L to 30.0 mg/L (e.g., 0.14 mg/L to 0.42 mg/L, 0.17 mg/L to 0.53 mg/L, 0.2 mg/L to 0.36 mg/L, 0.22 mg/L to 0.34 mg/L, 0.25 mg/L to 0.31 mg/L, 0.27 mg/L to 0.29 mg/L, 0.275 mg/L to 0.285 mg/L, 0.272 mg/L to 0.288, 0.277 mg/L to 0.283 mg/L, or approximately 0.28 mg/L. In some embodiments, the liquid has a hardness of approximately 5.8 mg/L to 580 mg/L (e.g., 29 mg/L to 87 mg/L, 35 mg/L to 81 mg/L, 41 mg/L to 75 mg/L, 47 mg/L to 69 mg/L, 52 mg/L to 64 mg/L, 55 mg/L to 61 mg/L, 56 mg/L to 60 mg/L, 57 mg/L to 59 mg/L, 57.5 mg/L to 58.5 mg/L or approximately 58 mg/L. In some embodiments, the administration of the coral extract results in prevention or reduction in loss of spiral ganglion cells in the subject.
In some embodiments, the coral liquid is made by the method of immersing coral grains in purified water; and adjusting the pH of the water to 8.0±0.5. In some embodiments, the coral liquid is made by passing water over a water purification cartridge comprising coral grains (e.g., a home or industrial water purification system).
The present invention additionally provides a method for treating a neurodegenerative disease, comprising administering an extract of natural stones with a pH of approximately 7.0 to 9.0 comprising a calcium content of approximately 1.4 mg/L to 140 mg/L, a magnesium content of approximately 0.54 mg/L to 54 mg/L, a sodium content of approximately 0.1 mg/L to 50.0 mg/L, a potassium content of approximately 0.03 mg/L to 30.0 mg/L, and a hardness of approximately 5.8 mg/L to 580 mg/L to a subject having symptoms of a neurodegenerative disease. In some embodiments, the natural stones are limestone, calcium carbonate, magnesium carbonate, sodium carbonate, potassium carbonate etc.
The present invention additionally provides a method for treating a neurodegenerative disease, comprising administering an extract of chemical compounds with a pH of approximately 7.0 to 9.0 comprising a calcium content of approximately 1.4 mg/L to 140 mg/L, a magnesium content of approximately 0.54 mg/L to 54 mg/L, a sodium content of approximately 0.1 mg/L to 50.0 mg/L, a potassium content of approximately 0.03 mg/L to 30.0 mg/L, and a hardness of approximately 5.8 mg/L to 580 mg/L to a subject having symptoms of a neurodegenerative disease. In some embodiments, the chemical compounds are calcium carbonate, magnesium carbonate, sodium carbonate, potassium carbonate or a mixture of these compounds.
The present invention further provides a food product, nutritional supplement, or pharmaceutical composition comprising an extract of coral in sufficient dosage to prevent or reduce neurodegenerative disease.
Additional embodiments of the present invention are described herein.
To facilitate an understanding of the present invention, a number of terms and phrases are defined below:
As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, companion animals (e.g., dogs, cats, etc.), and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
As used herein, the term “non-human animals” refers to all non-human animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.
As used herein, the term “in vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.
The terms “test compound” and “candidate compound” refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., hearing loss). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention.
As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention.
“Prepared food product” means any pre-packaged food approved for human consumption.
As used herein, the term “physiologically acceptable carrier” refers to any carrier or excipient commonly used with oily pharmaceuticals. Such carriers or excipients include, but are not limited to, oils, starch, sucrose and lactose.
As used herein, the term “oral delivery vehicle” refers to any means of delivering a pharmaceutical orally, including, but not limited to, capsules, pills, tablets and syrups.
As used herein, the term “food product” refers to any food or feed suitable for consumption by humans, non-ruminant animals, or ruminant animals. The “food product” may be a prepared and packaged food (e.g., mayonnaise, salad dressing, bread, or cheese food) or an animal feed (e.g., extruded and pelleted animal feed or coarse mixed feed).
As used herein, the term “nutritional supplement” refers to a food product formulated as a dietary or nutritional supplement to be used as part of a diet.
As used herein, the term “mimetic” refers to a small molecule compound that mimics the binding or interaction of a ligand with its target or mimics the physiological effect of the compound. For example, a mimetic of a compound described herein is a small molecule that has the same physiological effect as the compound (e.g., prevention or treatment of neurodegenerative disease).
As used herein, the term “coral water” refers to the product of an extraction of coral exposed to a liquid or solvent. In some embodiments, the coral is fossilized coral grain harvested from the ocean floor. In some embodiments, the coral is powdered or otherwise dried prior to extraction. In some embodiments, the liquid is aqueous (e.g., water such as purified or distilled water). In some embodiments, the extract comprises salts and mineral. In some embodiments, the coral extract has a pH of approximately 7.0 to 9.0 (e.g., 8.0). Although knowledge of the exact composition is not required, in some embodiments, the extract comprises a calcium content of approximately 1.4 mg/L to 140 mg/L (e.g., 7 mg/L to 21 mg/L, 8.5 mg/L to 19.5 mg/L, 10 mg/L to 18 mg/L, 11 mg/L to 17 mg/L, 12.8 mg/L to 15.8 mg/L, 13.6 mg/L to 15 mg/L, 13.9 mg/L to 14.7 mg/L, 14 mg/L to 14.6 mg/L, 14.2 mg/L to 14.5 mg/L, or approximately 14.30 mg/L), a magnesium content of approximately 0.54 mg/L to 54.0 mg/L (e.g., 2.7 mg/L to 8.1 mg/L, 3.2 mg/L to 7.6 mg/L, 3.8 mg/L to 7 mg/L, 4.3 mg/L to 6.5 mg/L, 4.9 mg/L to 5.9 mg/L, 5.1 mg/L to 5.7 mg/L, 5.2 mg/L to 5.6 mg/L, 5.3 mg/L to 5.5 mg/L, 5.35 mg/L to 5.45 mg/L, or approximately 5.4 mg/L), a sodium content of approximately 0.1 mg/L to 50.0 mg/L (e.g., 0.53 mg/L to 1.59 mg/L, 0.76 mg/L to 1.46 mg/L, 0.86 mg/L to 1.26 mg/L, 0.96 mg/L to 1.16 mg/L, 1.0 mg/L to 1.1 mg/L, 1.03 mg/L to 1.09 mg/L, 1.04 mg/L to 1.08 mg/L, 1.05 mg/L to 1.07 mg/L, 1.055 mg/L to 1.065 mg/L, or approximately 1.06 mg/L), a potassium content of approximately 0.03 mg/L to 30.0 mg/L (e.g., 0.14 mg/L to 0.42 mg/L, 0.17 mg/L to 0.53 mg/L, 0.2 mg/L to 0.36 mg/L, 0.22 mg/L to 0.34 mg/L, 0.25 mg/L to 0.31 mg/L, 0.27 mg/L to 0.29 mg/L, 0.275 mg/L to 0.285 mg/L, 0.272 mg/L to 0.288, 0.277 mg/L to 0.283 mg/L, or approximately 0.28 mg/L, and a hardness of approximately 5.8 mg/L to 580 mg/L (e.g., 29 mg/L to 87 mg/L, 35 mg/L to 81 mg/L, 41 mg/L to 75 mg/L, 47 mg/L to 69 mg/L, 52 mg/L to 64 mg/L, 55 mg/L to 61 mg/L, 56 mg/L to 60 mg/L, 57 mg/L to 59 mg/L, 57.5 mg/L to 58.5 mg/L or approximately 58 mg/L.
The present invention relates to compositions and methods for treating neurodegenerative diseases. Some embodiments of the present invention provide compositions, pharmaceutical compositions, food products, nutritional supplements and methods of administering the compositions for prevention and treatment of neurodegenerative diseases such as presbycusis.
For example, experiments were conducted in vivo in a reliable animal model of presbycusis that shows late onset of age-related hearing loss by 15 months of age (Keithley et al., Hear Res. 188:21 [2004]) and significant loss of SGCs and hair cells by 12 months of age (Keithley et al., [2004], supra).
During experiments conducted during the course of development of embodiments of the present invention, the effects of coral-treated drinking water on presbycusis prevention and treatment in the animal model was evaluated. Hearing function tests revealed that control animals displayed severe hearing loss at 8, 16, 32 kHz, whereas coral-treated (CW) mice displayed normal hearing or mild hearing loss at 8, 16, 32 kHz (*P<0.05, n=7), showing significant preservation of hearing function by coral-treated water. Histological analysis revealed that the cochleae from control animals displayed severe loss of spiral ganglion neurons and hair cells whereas the cochleae from CW animals displayed mild or less loss of the spiral ganglion neurons and hair cells, showing prevention of cochlear cell loss by coral-treated water. Moreover, the mean spiral ganglion cell (SGC) densities of apical, middle, and basal turns from CW animals were significantly higher than those of control animals, showing prevention of spiral ganglion neuronal cell loss by-treated water (*P<0.05, n=4-5). The mean outer hair cell survival rate of middle and basal turns from CW mice were significantly higher than those of Control mice, showing prevention of outer hair cell loss by coral-treated water (*P<0.05, n=4-5). Taken together, the experiments demonstrated that consumption of coral-treated water leads to prevention and treatment of presbycusis and prevents or treats physiological damage associated with a variety of neurodegenerative diseases and conditions. Accordingly, in some embodiments, the present invention provides methods of treating and/or preventing neurodegenerative diseases such as presbycusis using coral extracts (e.g., water extracts).
As described above, in some embodiments, the present invention provides compositions comprising coral extracts. In some embodiments, the extracts are in aqueous solutions. For example, in some embodiments, coral treated water is generated using coral grains or power. One exemplary non limited method is as follows: Coral grains or powder are immersed in distilled or pure water for a period of several minutes to several years (e.g., 1 hour to 1 year, 1 hour to 1 month, 1 hour to 1 week, 2 hours to 2 days, 12 hours to 36 hours, or approximately 24 hours) and then pH of the water is adjusted to 8.0±0.5 using NaOH or HCl. Since Coral grain is rich in minerals including calcium, magnesium, potassium, iron, zinc, selenium, manganese, chromium, etc., the coral-treated water is rich in these minerals and alkalic.
Any number of formulations of coral treated extracts are suitable for use in the compositions and methods of embodiments of the present invention. In some embodiments, administration is oral. The compositions of embodiments of the present invention may be provided in aqueous solution, oily solution, as or in any of the other forms discussed herein. The compositions may also be provided by any of a number of other routes, including, but not limited to, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means. Further details on techniques for formulation for and administration and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
In some embodiments, compositions are provided as a nutritional supplement alone or in combination with other ingredients. In some embodiments, nutritional supplements are packaged in a container or bottle that can include instructions describing dosage and timing of administration of the supplements.
In certain embodiments, the coral extracts of the present invention are provided as a food product comprising the extract. For example, in some embodiments, coral water is provided as bottled water for human or animal consumption. In other embodiments, coral extracts are added to prepared beverage products including, but not limited to, citrus juices, vegetable juices and like fruit beverages and vegetable juices, cola drinks, ginger ales, soda water and like carbonated beverages, sport drinks and like soft drinks, coffee, black tea, maccha and like tea-based beverages, cocoa, lactic acid bacteria beverages and like milk beverages, siruko, zenzai (thick azuki bean soup), nut (e.g., almond or peanut) beverages, malt beverages, hop beverages, tea-like beverages prepared from plants other than tea (barley tea, herb tea, Chinese matrimoneyvine tea, tochu (Eucommia ulmoides) tea), soymilk and soymilk-containing beverages, meal replacement beverages, energy drinks, diet drinks, and other general beverages.
In some embodiments, beverages comprise additional ingredients, including, but not limited to, carbonated water, food starch—e.g., modified, high fructose corn syrup and/or sucrose and/or sugar, sodium benzoate, caffeine, glycerol ester of wood resin, flavors, colors, preservatives, additional anti-oxidants, emulsifying agents, buffers, colorants, flavors, vitamins, minerals, nucleotides and nucleosides, pharmaceutical agents, thickening agents and stabilizers, and so forth. In some embodiments, compositions are packaged human use with suitable antioxidants such as lecithin, tocopherols, ascorbate, ascorbyl palmitate or spice extracts such as rosemary extract.
In some embodiments, beverages comprise vitamins. Examples include, but are not limited to water-soluble or oil-soluble, natural or synthetic, including various substances used as vitamin preparations. Examples of natural water-soluble vitamins include vitamin B1, B2, B6, B12, niacin, pantothenic acid, biotin, folic acid, lipoic acid, inositol, vitamin C and the like. Examples of natural oil-soluble vitamins include vitamin A, vitamin D, vitamin E, vitamin K and the like. Examples of the vitamin preparations commonly used as synthetic vitamins include vitamin A preparations such as retinol, vitamin D preparations such as ergocalciferol and dihydrotachysterol; vitamin B1 preparations such as thiamine hydrochloride and thiamine nitrate; vitamin B1 derivative preparations such as octothiamine, dicethiamine hydrochloride, thiamine disulfide, bisbentiamine, fursultiamine and benfotiamine; vitamin B2 preparations such as flavin adenine dinucleotide, riboflavin butyrate, riboflavin and riboflavin sodium phosphate; vitamin B6 preparations such as pyridoxine hydrochloride, pyridoxine phosphate and pyridoxal phosphate; vitamin B12 preparations such as cobamamide, cyanocobalamin, hydroxocobalamin acetate, mecobalamin; vitamin E preparations such as tocopherol calcium succinate and tocopherol acetate; vitamin K preparations such as phyionadione, and menatetrenone.
In some embodiments, a composition comprising ingredients of coral extract is manufactured without the use of coral by adding ingredients to liquid (e.g., the coral extract compositions described above).
In some embodiments, extracts having the above described components are made from natural stones or chemical compounds (e.g., limestone, calcium carbonate, magnesium carbonate, sodium carbonate, potassium carbonate, etc), alone or in combination with other minerals.
In some embodiments, coral extracts are provided via a water purifying system that includes coral or other natural stones. For example, in some embodiments, home water purification systems (e.g., water pitcher filters, faucet filters, or under sink filters) comprise a coral cartridge or coral component of a purification cartridge or filter. In other embodiments, industrial water filtrations systems (e.g., for the commercial production or purified water or beverages comprising purified water).
In some embodiments, the present invention provides methods for prevention and treatment of presbycusis and other neurodegenerative diseases and conditions. For example, in some embodiments, coral extracts (e.g., coral treated water) is administered to a subject to prevent presbycusis. In other embodiments, coral extracts are administered to a subject diagnosed with presbycusis as a treatment. In some embodiments, coral extracts are administered on a daily (or more or less often) basis for an extended period of time (e.g., weeks, months, or years). In some embodiments, coral treated water is provided as water bottles that are ready to drink. In other embodiments, coral extract is provided as a concentrated extract to be mixed with other beverages or food products (e.g., those described above).
In other embodiments, coral treated water is used to treat and/or prevent other diseases or signs or symptoms associated with neurodegeneration in the cochlea. For example, in some embodiments, coral treated water is used to treat and/or prevent Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Alexander disease, Alper's disease, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, HIV-associated dementia, Kennedy's disease, Krabbe disease, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, Narcolepsy, Neuroborreliosis, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Refsum's disease, Sandhoff disease, Schilder's disease, Subacute combined degeneration of spinal cord secondary to Pernicious Anaemia, Schizophrenia, Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease), Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, and Tabes dorsalis.
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
A method of making coral-treated and control water was as follows: 100 g of coral grains are immersed in 2 L of distilled water for 24 hr and then pH of the water was adjusted be 8.0±0.5 using NaOH or HCl. Control water was prepared using distilled water. pH of control water was adjusted to be 3.0±0.5 using NaOH or HCl since the standard drinking water for laboratory mice is acidified water whose pH is approximately 3.0.
Calcium and magnesium contents were measured using an inductively coupled plasma emission spectrometer (n=3). Sodium and potassium contents were measure using an atomic absorption spectrometry (n=3). Data were examined using the two-tailed standard t-test. All data were reported as mean±S.E.M.
Details on the methods used to house and feed male inbred C57BL/6 (B6) mice have been described (Pugh et al., 1999). Briefly, mice were divided into two groups: control water (Control) and coral-treated water (CW). The CW treatment mice were fed the same diet and the same caloric intake as controls, but were fed with coral-treated water from 2 months of age until 20 months of age (
Detailed protocols for ABR measurement have been described (Yamasoba et al., 2005). Briefly, ABRs were measured with a tone burst stimulus (4, 8, and 16 kHz) using an ABR recording system (Intelligent Hearing System). Mice were anesthetized with a mixture of xylazine hydrochloride (10 mg/kg, i.m.) and ketamine hydrochloride (40 mg/kg, i.m.). Seven mice per group were used for this study, and the same mice were used for the following histopathological analyses. Data were examined using the two-tailed standard t-test. All data were reported as mean±S.E.M.
Detailed protocols for tissue processing have been described (Someya et al., 2007). Briefly, 4% paraformaldehyde-fixed and paraffin-embedded specimens were sliced into 4 μm sections, mounted on silane-coated slides, stained with Haematoxylin and Eosin (HE), and observed under a light microscope (Leica Microsystems, Bannockburn, Ill.). The Rosenthal's canal was divided into three regions: apical, middle and basal (Keithley et al., 2004) and the three regions were used for evaluation of cochlear histopathology. Four to five mice for each group that were the same mice as those used in the ABR tests were used. Five modular sections obtained in every fifth section from one unilateral cochlea were evaluated per mouse. The same animals were used for spiral ganglion cell and hair cell counting.
Spiral ganglion cells (SGCs) were counted in each cross-section of the apical, middle, and basal turn of the cochlear individual sections using a 20× objective. Cells were identified by the presence of a nucleus. The corresponding area of Rosenthal's canal was measured on digital photomicrographs of each canal profile. The perimeter of the canal was traced with a cursor using ImageJ software (Rasband, W. S., ImageJ, U.S. National Institutes of Health, Bethesda, Md., USA, 1997-2007.). The computer then calculated the area within the outline. The SGC density was calculated as the number of SGCs per 1 mm2. Five sections of the unilateral apical turn were evaluated in one cochlea per mouse. Data were analyzed using the two-tailed standard t-test. All data were reported as mean±S.E.M.
Outer hair cell (OHC) and inner hair cell (IHC) were counted in each cross-section of the apical, middle, and basal turn of the cochlear individual sections using a 40× objective. Cells were identified by the presence of a nucleus. The outer hair cell survival rate (%) was calculated as follows. (# of OHCs/3)×100. The inner hair cell survival rate (%) was calculated as follows. (# of OHCs/1)×100. Five sections of the unilateral apical turn were evaluated in one cochlea per mouse. Data were analyzed using the two-tailed standard t-test. All data were reported as mean±S.E.M.
The calcium and potassium contents of CW were significantly higher than those of Control (Table 1) (*P<0.05, n=5). The hardness and pH of CW were significantly higher than those of Control (*P<0.05, n=3). Magnesium and Sodium were not detected in the Control water.
To examine the effects of long-term consumption of coral-treated water on presbycusis prevention and treatment, it was next examined whether the water retarded late onset of presbycusis in B6 mice. ABR thresholds measured from these mice revealed that Control mice displayed severe hearing loss at 8, 16, 32 kHz, whereas CW mice displayed normal hearing or mild hearing loss at 8, 16, 32 kHz (*P<0.05, n=7), showing significant preservation of hearing function by coral-treated water (
The effects of the water on age-related cochlear degeneration was next examined. The cochleae from Control mice (A) displayed severe loss of spiral ganglion neurons (B) and hair cells (C) whereas the cochleae from CW mice (D) displayed mild or only a few loss of the spiral ganglion neurons and hair cells, showing prevention of cochlear cell loss by coral-treated water (
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.
This application claims priority to U.S. Provisional Patent Application Ser. No. 61/043,557, filed Apr. 9, 2008, which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61043557 | Apr 2008 | US |
