Selective degeneration of synapses in the dorsal cochlear nucleus of chinchilla following acoustic trauma and effects of antioxidant treatment

Hearing Research

Volumn 283, Issue 1-2, 2012, Pages 1-13

  Du, Xiaoping ^a   Chen, Kejian ^a   Choi, Chul Hee ^a   Li, Wei ^a   Cheng, Weihua ^a   Stewart, Charles ^a,b   Hu, Ning ^a   Floyd, Robert A ^b   Kopke, Richard D ^a,c  

^a HOUGH EAR INSTITUTE   (United States)

^b OKLAHOMA MEDICAL RESEARCH FOUNDATION   (United States)

^c UNIVERSITY OF OKLAHOMA COLLEGE OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

4 HYDROXYPHENYL N TERT BUTYLNITRONE; ACETYLCYSTEINE; ANTIOXIDANT; BRAIN PROTEIN; LEVACECARNINE; N TERT BUTYL ALPHA PHENYLNITRONE; PRECEREBELLIN; SYNAPTOPHYSIN; UNCLASSIFIED DRUG;

ANIMAL CELL; ANIMAL EXPERIMENT; ANIMAL MODEL; ANIMAL TISSUE; ARTICLE; CELL ULTRASTRUCTURE; CELLULAR DISTRIBUTION; CHINCHILLA; CONTROLLED STUDY; DISEASE ASSOCIATION; DORSAL COCHLEAR NUCLEUS; DOWN REGULATION; DRUG EFFECT; DRUG MECHANISM; HAIR CELL; IMMUNOHISTOCHEMISTRY; NERVE CELL DEGENERATION; NERVE CELL PLASTICITY; NERVE ENDING; NOISE INJURY; NONHUMAN; PRIORITY JOURNAL; PROTEIN EXPRESSION; PROTEIN FUNCTION; PROTEIN LOCALIZATION; TRANSMISSION ELECTRON MICROSCOPY;

ACETYLCARNITINE; ACETYLCYSTEINE; ANIMALS; ANTIOXIDANTS; AUDITORY THRESHOLD; BIOLOGICAL MARKERS; CHINCHILLA; COCHLEAR NUCLEUS; DISEASE MODELS, ANIMAL; DRUG THERAPY, COMBINATION; HAIR CELLS, AUDITORY; HEARING LOSS, NOISE-INDUCED; IMINES; INTERNEURONS; MICROSCOPY, ELECTRON, TRANSMISSION; NEURONAL PLASTICITY; PHENOLS; PROTEIN PRECURSORS; SYNAPSES; SYNAPTIC TRANSMISSION; SYNAPTOPHYSIN; TIME FACTORS;

EID: 84856596626     PISSN: 03785955     EISSN: 18785891     Source Type: Journal    
DOI: 10.1016/j.heares.2011.11.013     Document Type: Article

References (98)

1. Aarnisalo A.A., Pirvola U., Liang X.Q., Miller J., Ylikoski J. Apoptosis in auditory brainstem neurons after a severe noise trauma of the organ of Corti: intracochlear GDNF treatment reduces the number of apoptotic cells. ORL J. Otorhinolaryngol. Relat. Spec. 2000, 62:330-334.
2. Basta D., Tzschentke B., Ernst A. Noise-induced cell death in the mouse medial geniculate body and primary auditory cortex. Neurosci. Lett. 2005, 381:199-204.
3. Bauer C.A., Brozoski T.J., Myers K. Primary afferent dendrite degeneration as a cause of tinnitus. J.Neurosci. Res. 2007, 85:1489-1498.
4. Benson C.G., Gross J.S., Suneja S.K., Potashner S.J. Synaptophysin immunoreactivity in the cochlear nucleus after unilateral cochlear or ossicular removal. Synapse 1997, 25:243-257.
5. Berrebi A.S., Mugnaini E. Distribution and targets of the cartwheel cell axon in the dorsal cochlear nucleus of the guinea pig. Anat. Embryol. 1991, 183:427-454.
6. Berrebi A.S., Spirou G.A. PEP-19 immunoreactivity in the cochlear nucleus and superior olive of the cat. Neuroscience 1998, 83:535-554.
7. Berrebi A.S., Morgan J.I., Mugnaini E. The Purkinje cell class may extend beyond the cerebellum. J.Neurocytol. 1990, 19:643-654.
8. Bilak M., Kim J., Potashner S.J., Bohne B.A., Morest D.K. New growth of axons in the cochlear nucleus of adult chinchillas after acoustic trauma. Exp.Neurol. 1997, 147:256-268.
9. Brock T.O., O'Callaghan J.P. Quantitative changes in the synaptic vesicle proteins synapsin I and p38 and the astrocyte-specific protein glial fibrillary acidic protein are associated with chemical-induced injury to the rat central nervous system. J.Neurosci. 1987, 7:931-942.
10. Brozoski T.J., Bauer C.A. The effect of dorsal cochlear nucleus ablation on tinnitus in rats. Hear. Res. 2005, 206:227-236.
11. Brozoski T.J., Bauer C.A., Caspary D.M. Elevated fusiform cell activity in the dorsal cochlear nucleus of chinchillas with psychophysical evidence of tinnitus. J.Neurosci. 2002, 22:2383-2390.
12. Burian M., Gestoettner W. Projection of primary vestibular afferent fibres to the cochlear nucleus in the guinea pig. Neurosci. Lett. 1988, 84:13-17.
13. Calhoun M.E., Jucker M., Martin L.J., Thinakaran G., Price D.L., Mouton P.R. Comparative evaluation of synaptophysin-based methods for quantification of synapses. J.Neurocytol. 1996, 25:821-828.
14. Caspary D.M., Schatteman T.A., Hughes L.F. Age-related changes in the inhibitory response properties of dorsal cochlear nucleus output neurons: role of inhibitory inputs. J.Neurosci. 2005, 25:10952-10959.
15. Chang H., Chen K., Kaltenbach J.A., Zhang J., Godfrey D.A. Effects of acoustic trauma on dorsal cochlear nucleus neuron activity in slices. Hear. Res. 2002, 164:59-68.
16. Choi C.-H., Chen K., Vasquez-Weldon A., Jackson R.L., Floyd R.A., Kopke R.D. Effectiveness of 4-hydroxy phenyl N-tert-butylnitrone (4-OHPBN) alone and in combination with other antioxidant drugs in the treatment of acute acoustic trauma in chinchilla. Free Radic. Biol. Med. 2008, 44:1772-1784.
17. Davis K.A., Young E.D. Granule cell activation of complex-spiking neurons in dorsal cochlear nucleus. J.Neurosci. 1997, 17:6798-6806.
18. De Felipe J., Marco P., Fairén A., Jones E.G. Inhibitory synaptogenesis in mouse somatosensory cortex. Cereb. Cortex 1997, 7:619-634.
19. Du X., Choi C.-H., Chen K., Cheng W., Floyd R.A., Kopke R.D. Reduced formation of oxidative stress biomarkers and migration of mononuclear phagocytes in the cochleae of chinchilla after antioxidant treatment in acute acoustic trauma. Int. J. Otolaryngol. 2011, 10.1155/2011/612690.
20. Eldredge D.H., Miller J.D., Bohne B.A. Afrequency-position map for the chinchilla cochlea. J.Acoust. Soc. Am. 1981, 69:1091-1095.
21. Elinos-Calderón D., Robledo-Arratia Y., Pérez-De La Cruz V., Pedraza-Chaverrí J., Ali S.F., Santamaría A. Early nerve ending rescue from oxidative damage and energy failure by L: -carnitine as post-treatment in two neurotoxic models in rat: recovery of antioxidant and reductive capacities. Exp. Brain Res. 2009, 197:287-296.
22. Finlayson P.G., Kaltenbach J.A. Alterations in the spontaneous discharge patterns of single units in the dorsal cochlear nucleus following intense sound exposure. Hear. Res. 2009, 256:104-117.
23. Friauf E. Tonotopic order in the adult and developing auditory system of the rat as shown by c-fos immunocytochemistry. Eur. J. Neurosci. 1992, 4:798-812.
24. Fuentes-Santamaría V., Alvarado J.C., Henkel C.K., Brunso-Bechtold J.K. Cochlear ablation in adult ferrets results in changes in insulin-like growth factor-1 and synaptophysin immunostaining in the cochlear nucleus. Neuroscience 2007, 148:1033-1047.
25. Gil-Loyzaga P., Bartolomé M.V., Ibáñez A. Synaptophysin immunoreactivity in the cat cochlear nuclei. Histol. Histopathol. 1998, 13:415-424.
26. Golding N.L., Oertel D. Context-dependent synaptic action of glycinergic and GABAergic inputs in the dorsal cochlear nucleus. J.Neurosci. 1996, 16:2208-2219.
27. Golding N.L., Oertel D. Physiological identification of the targets of cartwheel cells in the dorsal cochlear nucleus. J.Neurophysiol. 1997, 78:248-260.
28. Gröschel M., Götze R., Ernst A., Basta D. Differential impact of temporary and permanent noise-induced hearing loss on neuronal cell density in the mouse central auditory pathway. J.Neurotrauma 2010, 27:1499-1507.
29. Harding G.W., Bohne B.A., Ahmad M. DPOAE level shifts and ABR threshold shifts compared to detailed analysis of histopathological damage from noise. Hear. Res. 2002, 174:158-171.
30. Heffner H.E., Harrington I.A. Tinnitus in hamsters following exposure to intense sound. Hear. Res. 2002, 170:83-95.
31. Henderson D., Bielefeld E.C., Harris K.C., Hu B.H. The role of oxidative stress in noise-induced hearing loss. Ear Hear. 2006, 27:1-19.
32. Hirai H., Pang Z., Bao D., Miyazaki T., Li L., Miura E., Parris J., Rong Y., Watanabe M., Yuzaki M., Morgan J.I. Cbln1 is essential for synaptic integrity and plasticity in the cerebellum. Nat. Neurosci. 2005, 8:1534-1541.
33. House J.W., Brackmann D.E. Tinnitus: surgical treatment. Ciba. Found. Symp. 1981, 85:204-216.
34. Idrizbegovic E., Bogdanovic N., Canlon B. Modulating calbindin and parvalbumin immunoreactivity in the cochlear nucleus by moderate noise exposure in mice. A quantitative study on the dorsal and posteroventral cochlear nucleus. Brain Res. 1998, 800:86-96.
35. Ito-Ishida A., Miura E., Emi K., Matsuda K., Iijima T., Kondo T., Kohda K., Watanabe M., Yuzaki M. Cbln1 regulates rapid formation and maintenance of excitatory synapses in mature cerebellar Purkinje cells invitro and invivo. J.Neurosci. 2008, 28:5920-5930.
36. Itoh K., Kamiya H., Mitani A., Yasui Y., Takada M., Mizuno N. Direct projections from the dorsal column nuclei and the spinal trigeminal nuclei to the cochlear nuclei in the cat. Brain Res. 1987, 400:145-150.
37. Jahn R., Schiebler W., Ouimet C., Greengard P. A38,000-dalton membrane protein (p38) present in synaptic vesicles. Proc. Natl. Acad. Sci. U S A 1985, 82:4137-4141.
38. Kaltenbach J.A. Summary of evidence pointing to a role of the dorsal cochlear nucleus in the etiology of tinnitus. Acta Otolaryngol. 2006, (Suppl. 556):20-26.
39. Kaltenbach J.A., Godfrey D.A. Dorsal cochlear nucleus hyperactivity and tinnitus: are they related?. Am. J. Audiol. 2008, 17:S148-S161.
40. Kaltenbach J.A., Rachel J.D., Mathog T.A., Zhang J., Falzarano P.R., Lewandowski M. Cisplatin-induced hyperactivity in the dorsal cochlear nucleus and its relation to outer hair cell loss: relevance to tinnitus. J.Neurophysiol. 2002, 88:699-714.
41. Kaltenbach J.A., Saunders J.C. Spectral and temporal response patterns of single units in the chinchilla dorsal cochlear nucleus. Exp. Neurol. 1987, 96:406-419.
42. Kaltenbach J.A., Zacharek M.A., Zhang J., Frederick S. Activity in the dorsal cochlear nucleus of hamsters previously tested for tinnitus following intense tone exposure. Neurosci. Lett. 2004, 355:121-125.
43. Kaltenbach J.A., Zhang J. Intense sound-induced plasticity in the dorsal cochlear nucleus of rats: evidence for cholinergic receptor upregulation. Hear. Res. 2007, 226:232-243.
44. Kaltenbach J.A., Zhang J., Finlayson P. Tinnitus as a plastic phenomenon and its possible neural underpinnings in the dorsal cochlear nucleus. Hear. Res. 2005, 206:200-226.
45. Kiang N.Y., Liberman M.C., Levine R.A. Auditory-nerve activity in cats exposed to ototoxic drugs and high-intensity sounds. Ann. Otol. Rhinol. Laryngol. 1976, 85:752-768.
46. Kim J., Morest D.K., Bohne B.A. Degeneration of axons in the brainstem of the chinchilla after auditory overstimulation. Hear. Res. 1997, 103:169-191.
47. Kim J.J., Gross J., Morest D.K., Potashner S.J. Quantitative study of degeneration and new growth of axons and synaptic endings in the chinchilla cochlear nucleus after acoustic overstimulation. J.Neurosci. Res. 2004, 77:829-842.
48. Kim J.J., Gross J., Potashner S.J., Morest D.K. Fine structure of degeneration in the cochlear nucleus of the chinchilla after acoustic overstimulation. J.Neurosci. Res. 2004, 77:798-816.
49. Kim J.J., Gross J., Potashner S.J., Morest D.K. Fine structure of long-term changes in the cochlear nucleus after acoustic overstimulation: chronic degeneration and new growth of synaptic endings. J.Neurosci. Res. 2004, 77:817-828.
50. Kopke R.D., Coleman J.K., Liu J., Campbell K.C., Riffenburgh R.H. Candidate's thesis: enhancing intrinsic cochlear stress defenses to reduce noise-induced hearing loss. Laryngoscope 2002, 112:1515-1532.
51. Kopke R.D., Jackson R.L., Coleman J.K.M., Liu J., Bielefeld E.C., Balough B.J. NAC for noise: from the bench top to the clinic. Hear. Res. 2007, 226:114-125.
52. Kraus K.S., Mitra S., Jimenez Z., Hinduja S., Ding D., Jiang H., Gray L., Lobarinas E., Sun W., Salvi R.J. Noise trauma impairs neurogenesis in the rat hippocampus. Neuroscience 2010, 167:1216-1226.
53. Le Prell C.G., Yamashita D., Minami S.B., Yamasoba T., Miller J.M. Mechanisms of noise induced hearing loss indicate multiple methods of prevention. Hear. Res. 2007, 226:22-43.
54. Manis P.B., Spirou G.A., Wright D.D., Paydar S., Ryugo D.K. Physiology and morphology of complex spiking neurons in the guinea pig dorsal cochlear nucleus. J.Comp. Neurol. 1994, 348:261-276.
55. Masliah E., Fagan A.M., Terry R.D., DeTeresa R., Mallory M., Gage F.H. Reactive synaptogenesis assessed by synaptophysin immunoreactivity is associated with GAP-43 in the dentate gyrus of the adult rat. Exp. Neurol. 1991, 113:131-142.
56. Matsuda K., Miura E., Miyazaki T., Kakegawa W., Emi K., Narumi S., Fukazawa Y., Ito-Ishida A., Kondo T., Shigemoto R., Watanabe M., Yuzaki M. Cbln1 is a ligand for an orphan glutamate receptor delta2, a bidirectional synapse organizer. Science 2010, 328:363-368.
57. Melamed S.B., Kaltenbach J.A., Church M.W., Burgio D.L., Afman C.E. Cisplatin-induced increases in spontaneous neural activity in the dorsal cochlear nucleus and associated outer hair cell loss. Audiology 2000, 39:24-29.
58. Miura E., Matsuda K., Morgan J.I., Yuzaki M., Watanabe M. Cbln1 accumulates and colocalizes with Cbln3 and GluRdelta2 at parallel fiber-Purkinje cell synapses in the mouse cerebellum. Eur. J. Neurosci. 2009, 29:693-706.
59. Morest D.K., Kim J., Potashner S.J., Bohne B.A. Long-term degeneration in the cochlear nerve and cochlear nucleus of the adult chinchilla following acoustic overstimulation. Microsc. Res. Tech. 1998, 41:205-216.
60. Mugnaini E., Dahl A.L., Morgan J.I. Cerebellin is a postsynaptic neuropeptide. Synapse 1988, 2:125-138.
61. Muly S.M., Gross J.S., Morest D.K., Potashner S.J. Synaptophysin in the cochlear nucleus following acoustic trauma. Exp. Neurol. 2002, 177:202-221.
62. Muly S.M., Gross J.S., Potashner S.J. Noise trauma alters D-[3H]aspartate release and AMPA binding in chinchilla cochlear nucleus. J.Neurosci. Res. 2004, 75:585-596.
63. Nelson D.I., Nelson R.Y., Concha-Barrientos M., Fingerhut M. The global burden of occupational noise-induced hearing loss. Am. J. Ind. Med. 2005, 48:446-458.
64. Ohinata Y., Yamasoba T., Schacht J., Miller J.M. Glutathione limits noise-induced hearing loss. Hear. Res. 2000, 146:28-34.
65. Ohlemiller K.K., Wright J.S., Dugan L.L. Early elevation of cochlear reactive oxygen species following noise exposure. Audiol. Neurootol. 1999, 4:229-236.
66. Portfors C.V., Roberts P.D. Temporal and frequency characteristics of cartwheel cells in the dorsal cochlear nucleus of the awake mouse. J.Neurophysiol. 2007, 98:744-756.
67. Pulec J.L. Cochlear nerve section for intractable tinnitus. Ear Nose Throat J. 1995, 74:468. 470-476.
68. Rouiller E.M., Wan X.S., Moret V., Liang F. Mapping of c-fos expression elicited by pure tones stimulation in the auditory pathways of the rat, with emphasis on the cochlear nucleus. Neurosci. Lett. 1992, 144:19-24.
69. Rubio M.E., Juiz J.M. Differential distribution of synaptic endings containing glutamate, glycine, and GABA in the rat dorsal cochlear nucleus. J.Comp. Neurol. 2004, 477:253-272.
70. Ryan A.F., Furlow Z., Woolf N.K., Keithley E.M. The spatial representation of frequency in the rat dorsal cochlear nucleus and inferior colliculus. Hear. Res. 1988, 36:181-189.
71. Saint Marie R.L., Luo L., Ryan A.F. Spatial representation of frequency in the rat dorsal nucleus of the lateral lemniscus as revealed by acoustically induced c-fos mRNA expression. Hear. Res. 1999, 128:70-74.
72. Säljö A., Bao F., Jingshan S., Hamberger A., Hansson H.A., Haglid K.G. Exposure to short-lasting impulse noise causes neuronal c-Jun expression and induction of apoptosis in the adult rat brain. J.Neurotrauma 2002, 19:985-991.
73. Salvi R.J., Wang J., Ding D. Auditory plasticity and hyperactivity following cochlear damage. Hear. Res. 2000, 147:261-274.
74. Saunders G.H., Griest S.E. Hearing loss in veterans and the need for hearing loss prevention programs. Noise Health 2009, 11:14-21.
75. Schulz J.B., Henshaw D.R., Siwek D., Jenkins B.G., Ferrante R.J., Cipolloni P.B., Kowall N.W., Rosen B.R., Beal M.F. Involvement of free radicals in excitotoxicity invivo. J.Neurochem. 1995, 64:2239-2247.
76. Seki S., Eggermont J.J. Changes in spontaneous firing rate and neural synchrony in cat primary auditory cortex after localized tone-induced hearing loss. Hear. Res. 2003, 180:28-38.
77. Sekiya T., Canlon B., Viberg A., Matsumoto M., Kojima K., Ono K., Yoshida A., Kikkawa Y.S., Nakagawa T., Ito J. Selective vulnerability of adult cochlear nucleus neurons to de-afferentation by mechanical compression. Exp. Neurol. 2009, 218:117-123.
78. Slemmon J.R., Danho W., Hempstead J.L., Morgan J.I. Cerebellin: a quantifiable marker for Purkinje cell maturation. Proc. Natl. Acad. Sci. U S A 1985, 82:7145-7148.
79. Slepecky N. Overview of mechanical damage to the inner ear: noise as a tool to probe cochlear function. Hear. Res. 1986, 22:307-321.
80. Spirou G.A., May B.J., Wright D.D., Ryugo D.K. Frequency organization of the dorsal cochlear nucleus in cats. J.Comp. Neurol. 1993, 329:36-52.
81. Sziklai I. The significance of the calcium signal in the outer hair cells and its possible role in tinnitus of cochlear origin. Eur. Arch. Otorhinolaryngol. 2004, 261:517-525.
82. Tastekin A., Gepdiremen A., Ors R., Emin Buyukokuroglu M., Halici Z. L-carnitine protects against glutamate- and kainic acid induced neurotoxicity in cerebellar granular cell culture of rats. Brain Dev. 2005, 27:570-573.
83. Theopold H.M. Degenerative alterations in the ventral cochlear nucleus of the guinea pig after impulse noise exposure. A preliminary light and electron microscopic study. Arch. Otorhinolaryngol. 1975, 209:247-262.
84. Tonndorf J. The analogy between tinnitus and pain: a suggestion for a physiological basis of chronic tinnitus. Hear. Res. 1987, 28:271-275.
85. Urade Y., Oberdick J., Molinar-Rode R., Morgan J.I. Precerebellin is a cerebellum-specific protein with similarity to the globular domain of complement C1q B chain. Proc. Natl. Acad. Sci. U S A 1991, 88:1069-1073.
86. Urano S., Asai Y., Makabe S., Matsuo M., Izumiyama N., Ohtsubo K., Endo T. Oxidative injury of synapse and alteration of antioxidative defense systems in rats, and its prevention by vitamin E. Eur. J. Biochem. 1997, 245:64-70.
87. Walaas S.I., Jahn R., Greengard P. Quantitation of nerve terminal populations: synaptic vesicle-associated proteins as markers for synaptic density in the rat neostriatum. Synapse 1988, 2:516-520.
88. Wang H., Brozoski T.J., Turner J.G., Ling L., Parrish J.L., Hughes L.F., Caspary D.M. Plasticity at glycinergic synapses in dorsal cochlear nucleus of rats with behavioral evidence of tinnitus. Neuroscience 2009, 164:747-759.
89. Weedman D.L., Pongstaporn T., Ryugo D.K. Ultrastructural study of the granule cell domain of the cochlear nucleus in rats: mossy fiber endings and their targets. J.Comp. Neurol. 1996, 369:345-360.
90. Wiedenmann B., Franke W.W. Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell 1985, 41:1017-1028.
91. Willott J.F., Bross L.S., McFadden S.L. Morphology of the cochlear nucleus in CBA/J mice with chronic, severe sensorineural cochlear pathology induced during adulthood. Hear. Res. 1994, 74:1-21.
92. Wouterlood F.G., Mugnaini E. Cartwheel neurons of the dorsal cochlear nucleus. A golgi-electron microscopic study in the rat. J.Comp. Neurol. 1984, 227:136-157.
93. Yajima Y., Hayashi Y. Response properties and tonotopical organization in the dorsal cochlear nucleus in rats. Exp. Brain Res. 1989, 75:381-389.
94. Yamane H., Nakai Y., Takayama M., Iguchi H., Nakagawa T., Kojima A. Appearance of free radicals in the guinea pig inner ear after noise-induced acoustic trauma. Eur. Arch. Otorhinolaryngol. 1995, 252:504-508.
95. Yamane H., Nakai Y., Takayama M., Konishi K., Iguchi H., Nakagawa T., Shibata S., Kato A., Sunami K., Kawakatsu C. The emergence of free radicals after acoustic trauma and strial blood flow. Acta Otolaryngol. 1995, (Suppl. 519):87-92.
96. Zacharek M.A., Kaltenbach J.A., Mathog T.A., Zhang J. Effects of cochlear ablation on noise induced hyperactivity in the hamster dorsal cochlear nucleus: implications for the origin of noise induced tinnitus. Hear. Res. 2002, 172:137-143.
97. Zanelli S.A., Solenski N.J., Rosenthal R.E., Fiskum G. Mechanisms of ischemic neuroprotection by acetyl-L-carnitine. Ann. N. Y. Acad. Sci. 2005, 1053:153-161.
98. Zheng Y., Begum S., Zhang C., Fleming K., Masumura C., Zhang M., Smith P., Darlington C. Increased BrdU incorporation reflecting DNA repair, neuronal de-differentiation or possible neurogenesis in the adult cochlear nucleus following bilateral cochlear lesions in the rat. Exp. Brain Res. 2011, 210:477-487.