The effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats
Vitamin A (retinol) is involved in signaling pathways regulating gene expression and was postulated to be a major antioxidant and anti-inflammatory compound of the diet. Parkinson's disease (PD) is a progressive neurodegenerative disorder, characterized by loss of nigral dopaminergic neurons, i...
- Autores:
-
Kunzler, Alice
Tiefensee Ribeiro, Camila
Gasparotto, Juciano
Lintzmaier Petiz, Lyvia
Thais da Rosa Silva, Helen
da Silva Jr., Jeferson Delgado
Bortolin, Rafael
Oliveira de Souza, Priscila
Barreto, Fabiano
Espitia-Perez, Pedro
Schnorr, Carlos Eduardo
Somensi, Nauana
Fonseca Moreira, José Claudio
Pens Gelain, Daniel
- Tipo de recurso:
- http://purl.org/coar/resource_type/c_816b
- Fecha de publicación:
- 2019
- Institución:
- Corporación Universidad de la Costa
- Repositorio:
- REDICUC - Repositorio CUC
- Idioma:
- eng
- OAI Identifier:
- oai:repositorio.cuc.edu.co:11323/2861
- Acceso en línea:
- https://hdl.handle.net/11323/2861
https://repositorio.cuc.edu.co/
- Palabra clave:
- Retinol
Neurodegenerative disorder
6-OHDA
Neuroinflammation
Vitamin supplementation
- Rights
- openAccess
- License
- Atribución – No comercial – Compartir igual
| id |
RCUC2_70b62e6e6f07b3a57dfd9a61f021f69c |
|---|---|
| oai_identifier_str |
oai:repositorio.cuc.edu.co:11323/2861 |
| network_acronym_str |
RCUC2 |
| network_name_str |
REDICUC - Repositorio CUC |
| repository_id_str |
|
| dc.title.spa.fl_str_mv |
The effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats |
| title |
The effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats |
| spellingShingle |
The effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats Retinol Neurodegenerative disorder 6-OHDA Neuroinflammation Vitamin supplementation |
| title_short |
The effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats |
| title_full |
The effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats |
| title_fullStr |
The effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats |
| title_full_unstemmed |
The effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats |
| title_sort |
The effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats |
| dc.creator.fl_str_mv |
Kunzler, Alice Tiefensee Ribeiro, Camila Gasparotto, Juciano Lintzmaier Petiz, Lyvia Thais da Rosa Silva, Helen da Silva Jr., Jeferson Delgado Bortolin, Rafael Oliveira de Souza, Priscila Barreto, Fabiano Espitia-Perez, Pedro Schnorr, Carlos Eduardo Somensi, Nauana Fonseca Moreira, José Claudio Pens Gelain, Daniel |
| dc.contributor.author.spa.fl_str_mv |
Kunzler, Alice Tiefensee Ribeiro, Camila Gasparotto, Juciano Lintzmaier Petiz, Lyvia Thais da Rosa Silva, Helen da Silva Jr., Jeferson Delgado Bortolin, Rafael Oliveira de Souza, Priscila Barreto, Fabiano Espitia-Perez, Pedro Schnorr, Carlos Eduardo Somensi, Nauana Fonseca Moreira, José Claudio Pens Gelain, Daniel |
| dc.subject.spa.fl_str_mv |
Retinol Neurodegenerative disorder 6-OHDA Neuroinflammation Vitamin supplementation |
| topic |
Retinol Neurodegenerative disorder 6-OHDA Neuroinflammation Vitamin supplementation |
| description |
Vitamin A (retinol) is involved in signaling pathways regulating gene expression and was postulated to be a major antioxidant and anti-inflammatory compound of the diet. Parkinson's disease (PD) is a progressive neurodegenerative disorder, characterized by loss of nigral dopaminergic neurons, involving oxidative stress and pro-inflammatory activation. The aim of the present study was to evaluate the neuroprotective effects of retinol oral supplementation against 6-hydroxydopamine (6-OHDA, 12 μg per rat) nigrostriatal dopaminergic denervation in Wistar rats. Animals supplemented with retinol (retinyl palmitate, 3000 IU/kg/day) during 28 days exhibited increased retinol content in liver, although circulating retinol levels (serum) were unaltered. Retinol supplementation did not protect against the loss of dopaminergic neurons (assessed through tyrosine hydroxylase immunofluorescence and Western blot). Retinol supplementation prevented the effect of 6-OHDA on Iba-1 levels but had no effect on 6-OHDA-induced GFAP increase. Moreover, GFAP levels were increased by retinol supplementation alone. Rats pre-treated with retinol did not present oxidative damage or thiol redox modifications in liver, and the circulating levels of TNF-α, IL-1β, IL-6 and IL-10 were unaltered by retinol supplementation, demonstrating that the protocol used here did not cause systemic toxicity to animals. Our results indicate that oral retinol supplementation is not able to protect against 6-OHDA-induced dopaminergic denervation, and it may actually stimulate astrocyte reactivity without altering parameters of systemic toxicity. |
| publishDate |
2019 |
| dc.date.accessioned.none.fl_str_mv |
2019-03-07T22:00:06Z |
| dc.date.available.none.fl_str_mv |
2019-03-07T22:00:06Z |
| dc.date.issued.none.fl_str_mv |
2019-02-07 |
| dc.type.spa.fl_str_mv |
Pre-Publicación |
| dc.type.coar.spa.fl_str_mv |
http://purl.org/coar/resource_type/c_816b |
| dc.type.content.spa.fl_str_mv |
Text |
| dc.type.driver.spa.fl_str_mv |
info:eu-repo/semantics/preprint |
| dc.type.redcol.spa.fl_str_mv |
http://purl.org/redcol/resource_type/ARTOTR |
| dc.type.version.spa.fl_str_mv |
info:eu-repo/semantics/acceptedVersion |
| format |
http://purl.org/coar/resource_type/c_816b |
| status_str |
acceptedVersion |
| dc.identifier.uri.spa.fl_str_mv |
https://hdl.handle.net/11323/2861 |
| dc.identifier.instname.spa.fl_str_mv |
Corporación Universidad de la Costa |
| dc.identifier.reponame.spa.fl_str_mv |
REDICUC - Repositorio CUC |
| dc.identifier.repourl.spa.fl_str_mv |
https://repositorio.cuc.edu.co/ |
| url |
https://hdl.handle.net/11323/2861 https://repositorio.cuc.edu.co/ |
| identifier_str_mv |
Corporación Universidad de la Costa REDICUC - Repositorio CUC |
| dc.language.iso.none.fl_str_mv |
eng |
| language |
eng |
| dc.relation.references.spa.fl_str_mv |
Anon, 2001. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington (DC). . Anon, 2016. FDA- Food and Drug Administration. Aoto, J., Nam, C.I., Poon, M.M., Ting, P., Chen, L., 2008. Synaptic signaling by all-trans retinoic acid in homeostatic synaptic plasticity. Neuron 60, 308–320. Assini, R., Abercrombie, E.D., 2018. Zolpidem ameliorates motor impairments in the unilaterally 6-hydroxydopamine-lesioned rat. Eur. J. Neurosci. 48, 1896–1905. Behairi, N., Belkhelfa, M., Rafa, H., Labsi, M., Deghbar, N., Bouzid, N., Mesbah-Amroun, H., Touil-Boukoffa, C., 2016. All-trans retinoic acid (ATRA) prevents lipopolysaccharide-induced neuroinflammation, amyloidogenesis and memory impairment in aged rats. J. Neuroimmunol. 300, 21–29. Beitz, J.M., 2014. Parkinson's disease: a review. Front. Biosci. (Sch. Ed.) 6, 65–74. Bhat, S., Acharya, U.R., Hagiwara, Y., Dadmehr, N., Adeli, H., 2018. Parkinson's disease: cause factors, measurable indicators, and early diagnosis. Comput. Biol. Med. 102, 234–241. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Burton, G.W., Ingold, K.U., 1984. beta-Carotene: an unusual type of lipid antioxidant. Science 224, 569–573. Chang, K.H., Cheng, M.L., Chiang, M.C., Chen, C.M., 2018. Lipophilic antioxidants in neurodegenerative diseases. Clin. Chim. Acta 485, 79–87. Dao, D.Q., Ngo, T.C., Thong, N.M., Nam, P.C., 2017. Is vitamin A an antioxidant or a prooxidant? J. Phys. Chem. B 121, 9348–9357. de Bittencourt Pasquali, M.A., Roberto de Oliveira, M., De Bastiani, M.A., da Rocha, R.F., Schnorr, C.E., Gasparotto, J., Gelain, D.P., Moreira, J.C., 2012. L-NAME co-treatment prevent oxidative damage in the lung of adult Wistar rats treated with vitamin A supplementation. Cell Biochem. Funct. 30, 256–263. De Oliveira, M.R., Moreira, J.C., 2008. Impaired redox state and respiratory chain enzyme activities in the cerebellum of vitamin A-treated rats. Toxicology 253, 125–130. de Oliveira, M.R., Silvestrin, R.B., Mello e Souza, T., Moreira, J.C., 2008. Therapeutic vitamin A doses increase the levels of markers of oxidative insult in substantia nigra and decrease locomotory and exploratory activity in rats after acute and chronic supplementation. Neurochem. Res. 33, 378–383. de Oliveira, M.R., Soares Oliveira, M.W., Muller Hoff, M.L., Behr, G.A., da Rocha, R.F., Fonseca Moreira, J.C., 2009. Evaluation of redox and bioenergetics states in the liver of vitamin A-treated rats. Eur. J. Pharmacol. 610, 99–105. de Pee, S., Dary, O., 2002. Biochemical indicators of vitamin A deficiency: serum retinol and serum retinol binding protein. J. Nutr. 132, 2895s–2901s. Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 186, 421–431. Dunkley, P.R., Bobrovskaya, L., Graham, M.E., von Nagy-Felsobuki, E.I., Dickson, P.W., 2004. Tyrosine hydroxylase phosphorylation: regulation and consequences. J. Neurochem. 91, 1025–1043. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Espitia-Perez, P., Albino, S.M., da Rosa, H.T., Silveira, A.K., Espitia-Perez, L., Brango, H., Moraes, D.P., Hermann, P.R.S., Mingori, M., Barreto, F., Kunzler, A., Gelain, D.P., Schnorr, C.E., Moreira, J.C.F., 2018. Effects of methylmercury and retinol palmitate co-administration in rats during pregnancy and breastfeeding: metabolic and redox parameters in dams and their offspring. Ecotoxicol. Environ. Saf. 162, 603–615. Gasparotto, J., Ribeiro, C.T., Bortolin, R.C., Somensi, N., Rabelo, T.K., Kunzler, A., Souza, N.C., Pasquali, M.A.B., Moreira, J.C.F., Gelain, D.P., 2017. Targeted inhibition of RAGE in substantia nigra of rats blocks 6-OHDA-induced dopaminergic denervation. Sci. Rep. 7, 8795. Gelain, D.P., de Bittencourt Pasquali, M.A., Caregnato, F.F., Moreira, J.C., 2011. Vitamin A (retinol) up-regulates the receptor for advanced glycation endproducts (RAGE) through p38 and Akt oxidant-dependent activation. Toxicology 289, 38–44. Gelain, D.P., Moreira, J.C., Bevilaqua, L.R., Dickson, P.W., Dunkley, P.R., 2007. Retinol activates tyrosine hydroxylase acutely by increasing the phosphorylation of serine 40 and then serine 31 in bovine adrenal chromaffin cells. J. Neurochem. 103, 2369–2379. Grimm, M.O., Mett, J., Hartmann, T., 2016. The impact of vitamin E and other fat-soluble vitamins on alzheimer s disease. Int. J. Mol. Sci. 17. Haddadi, R., Nayebi, A.M., Farajniya, S., Brooshghalan, S.E., Sharifi, H., 2014. Silymarin improved 6-OHDA-induced motor impairment in hemi-parkisonian rats: behavioral and molecular study. Daru 22, 38. Huang, Z., Liu, Y., Qi, G., Brand, D., 2018. Role of Vitamin A in the Immune System, vol. 7. Karkeni, E., Bonnet, L., Astier, J., Couturier, C., Dalifard, J., Tourniaire, F., Landrier, J.F., 2017. All-trans-retinoic acid represses chemokine expression in adipocytes and adipose tissue by inhibiting NF-kappaB signaling. J. Nutr. Biochem. 42, 101–107. Klamt, F., Dal-Pizzol, F., Roehrs, R., de Oliveira, R.B., Dalmolin, R., Henriques, J.A., de Andrades, H.H., de Paula Ramos, A.L., Saffi, J., Moreira, J.C., 2003. Genotoxicity, recombinogenicity and cellular preneoplasic transformation induced by vitamin A supplementation. Mutat. Res. 539, 117–125. Lai, C.L., Lu, C.C., Lin, H.C., Sung, Y.F., Wu, Y.P., Hong, J.S., Peng, G.S., 2019. Valproate is protective against 6-OHDA-induced dopaminergic neurodegeneration in rodent midbrain: a potential role of BDNF up-regulation. J. Formos. Med. Assoc. 118, 420–428. Leonoudakis, D., Rane, A., Angeli, S., Lithgow, G.J., Andersen, J.K., Chinta, S.J., 2017. Anti-Inflammatory and Neuroprotective Role of Natural Product Securinine in Activated Glial Cells: Implications for Parkinson's Disease, vol. 2017 8302636. Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.G., Ahn, B.W., Shaltiel, S., Stadtman, E.R., 1990. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 186, 464–478. Liu, W., Tang, Y., Feng, J., 2011. Cross talk between activation of microglia and astrocytes in pathological conditions in the central nervous system. Life Sci. 89, 141–146. Lobo, V., Patil, A., Phatak, A., Chandra, N., 2010. Free radicals, antioxidants and functional foods: impact on human health. Pharm. Rev. 4, 118–126. Maden, M., 2007. Retinoic acid in the development, regeneration and maintenance of the nervous system. Nat. Rev. Neurosci. 8, 755–765. Maqbool, A., Graham-Maar, R.C., Schall, J.I., Zemel, B.S., Stallings, V.A., 2008. Vitamin A intake and elevated serum retinol levels in children and young adults with cystic fibrosis. J. Cyst. Fibros. 7, 137–141. McCaffery, P., Zhang, J., Crandall, J.E., 2006. Retinoic acid signaling and function in the adult hippocampus. J. Neurobiol. 66, 780–791. Miyake, Y., Tanaka, K., Fukushima, W., Sasaki, S., Kiyohara, C., Tsuboi, Y., Yamada, T., Oeda, T., Miki, T., Kawamura, N., Sakae, N., Fukuyama, H., Hirota, Y., Nagai, M., 2011. Dietary intake of metals and risk of Parkinson's disease: a case-control study in Japan. J. Neurol. Sci. 306, 98–102. Mohammadzadeh Honarvar, N., Saedisomeolia, A., Abdolahi, M., Shayeganrad, A., Taheri Sangsari, G., Hassanzadeh Rad, B., Muench, G., 2017. Molecular anti-inflammatory mechanisms of retinoids and carotenoids in alzheimer's disease: a review of current evidence. J. Mol. Neurosci. 61, 289–304. National Research Council (U.S.) Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research (U.S.), National Academies Press (U.S.), 2011. Guide for the Care and Use of Laboratory Animals. National Academies Press, Washington, D.C, pp. 1 online resource (xxv, 220 pp.). Paxinos, G., Watson, C., 2007. The Rat Brain in Stereotaxic Coordinates, sixth ed. Elsevier. Petiz, L.L., Girardi, C.S., Bortolin, R.C., Kunzler, A., Gasparotto, J., Rabelo, T.K., Matte, C., Moreira, J.C.F., Gelain, D.P., 2017a. Vitamin A oral supplementation induces oxidative stress and suppresses IL-10 and HSP70 in skeletal muscle of trained rats. Nutrients 9, 16. Petiz, L.L., Kunzler, A., Bortolin, R.C., Gasparotto, J., Matte, C., Moreira, J.C.F., Gelain, D.P., 2017b. Role of vitamin A oral supplementation on oxidative stress and inflammatory response in the liver of trained rats. Appl. Physiol. Nutr. Metabol. 42, 1192–1200. Real, C.C., Garcia, P.C., Britto, L.R.G., 2017. Treadmill exercise prevents increase of neuroinflammation markers involved in the dopaminergic damage of the 6-OHDA Parkinson's disease model. J. Mol. Neurosci. 63, 36–49. Rodriguez-Pallares, J., Parga, J.A., Munoz, A., Rey, P., Guerra, M.J., Labandeira-Garcia, J.L., 2007. Mechanism of 6-hydroxydopamine neurotoxicity: the role of NADPH oxidase and microglial activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons. J. Neurochem. 103, 145–156. Sardi, S.P., Cedarbaum, J.M., Brundin, P., 2018. Targeted therapies for Parkinson's disease: from genetics to the clinic. Mov. Disord. 33, 684–696. Schnorr, C.E., Bittencourt Lda, S., Petiz, L.L., Gelain, D.P., Zeidan-Chulia, F., Moreira, J.C., 2015. Chronic retinyl palmitate supplementation to middle-aged Wistar rats disrupts the brain redox homeostasis and induces changes in emotional behavior. Mol. Nutr. Food Res. 59, 979–990. Schnorr, C.E., da Silva Morrone, M., Simoes-Pires, A., da Rocha, R.F., Behr, G.A., Moreira, J.C., 2011. Vitamin A supplementation in rats under pregnancy and nursing induces behavioral changes and oxidative stress upon striatum and hippocampus of dams and their offspring. Brain Res. 1369, 60–73. Shearer, K.D., Stoney, P.N., Morgan, P.J., McCaffery, P.J., 2012. A vitamin for the brain. Trends Neurosci. 35, 733–741. Shi, H.Y., Yan, S.M., Guo, Y.M., Zhang, B.Q., Guo, X.Y., Shi, B.L., 2018. Vitamin A pretreatment protects NO-induced bovine mammary epithelial cells from oxidative stress by modulating Nrf 2 and NF-kappaB signaling pathways. J. Anim. Sci. 96, 1305–1316. Singh, P., Hanson, P.S., Morris, C.M., 2017. SIRT1 ameliorates oxidative stress induced neural cell death and is down-regulated in Parkinson's disease. 18, 46. Tanumihardjo, S.A., Russell, R.M., Stephensen, C.B., Gannon, B.M., 2016. Biomarkers of Nutrition for Development (BOND)-Vitamin A Review, vol. 146. pp. 1816s–1848s. Taylor, J.M., Main, B.S., Crack, P.J., 2013. Neuroinflammation and oxidative stress: coconspirators in the pathology of Parkinson's disease. Neurochem. Int. 62, 803–819. Theus, M.H., Sparks, J.B., Liao, X., Ren, J., Luo, X.M., 2017. All- trans-retinoic acid augments the histopathological outcome of neuroinflammation and neurodegeneration in lupus-prone MRL/lpr mice. J. Histochem. Cytochem. 65, 69–81. Thiele, S.L., Warre, R., Nash, J.E., 2012. Development of a unilaterally-lesioned 6-OHDA mouse model of Parkinson's disease. J. Vis. Exp. 60 pii: 3234. Trumbo, P., Yates, A.A., Schlicker, S., Poos, M., 2001. Dietary reference intakes: vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. J. Am. Diet Assoc. 101, 294–301. Ulusoy, G.K., Celik, T., Kayir, H., Gursoy, M., Isik, A.T., Uzbay, T.I., 2011. Effects of pioglitazone and retinoic acid in a rotenone model of Parkinson's disease. Brain Res. Bull. 85, 380–384. Wang, H.L., Zhang, Z., Hintze, M., Chen, L., 2011. Decrease in calcium concentration triggers neuronal retinoic acid synthesis during homeostatic synaptic plasticity. J. Neurosci. 31, 17764–17771. Wang, Q., Liu, Y., Zhou, J., 2015a. Neuroinflammation in Parkinson's disease and its potential as therapeutic target. Transl. Neurodegener. 4, 19. Wang, Q., Liu, Y., Zhou, J., 2015b. Neuroinflammation in Parkinson's disease and its potential as therapeutic target. Transl. Neurodegener. 4. Xie, L., Chen, L., Gu, P., Wei, L., Kang, X., 2018. A convenient method for extraction and analysis with high-pressure liquid chromatography of catecholamine neurotransmitters and their metabolites. J. Vis. Exp. 133. Yin, L.H., Shen, H., Diaz-Ruiz, O., Backman, C.M., Bae, E., Yu, S.J., Wang, Y., 2012. Early post-treatment with 9-cis retinoic acid reduces neurodegeneration of dopaminergic neurons in a rat model of Parkinson's disease. BMC Neurosci. 13, 120. Zeng, J., Chen, L., Wang, Z., Chen, Q., Fan, Z., Jiang, H., Wu, Y., Ren, L., Chen, J., Li, T., Song, W., 2017. Marginal Vitamin A Deficiency Facilitates Alzheimer's Pathogenesis. 133, 967–982. |
| dc.rights.spa.fl_str_mv |
Atribución – No comercial – Compartir igual |
| dc.rights.accessrights.spa.fl_str_mv |
info:eu-repo/semantics/openAccess |
| dc.rights.coar.spa.fl_str_mv |
http://purl.org/coar/access_right/c_abf2 |
| rights_invalid_str_mv |
Atribución – No comercial – Compartir igual http://purl.org/coar/access_right/c_abf2 |
| eu_rights_str_mv |
openAccess |
| dc.publisher.spa.fl_str_mv |
Universidad de la Costa |
| institution |
Corporación Universidad de la Costa |
| bitstream.url.fl_str_mv |
https://repositorio.cuc.edu.co/bitstreams/0f46dc11-7d9e-4349-801e-f6a7075aec1a/download https://repositorio.cuc.edu.co/bitstreams/230ad955-f14a-4b07-b15c-ee6f05f85f8b/download https://repositorio.cuc.edu.co/bitstreams/f35c3cc9-fa06-477e-9df6-4976f181a809/download https://repositorio.cuc.edu.co/bitstreams/8bac9c49-6ab2-4655-b7bf-cfbef72418e6/download |
| bitstream.checksum.fl_str_mv |
6746cef1b8bae9a8ed1fe48332011891 8a4605be74aa9ea9d79846c1fba20a33 2f4440ea621d70ab4368b426a51a10d1 a877dd7b7232173b8894c5de0d7a6b0f |
| bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 MD5 |
| repository.name.fl_str_mv |
Repositorio de la Universidad de la Costa CUC |
| repository.mail.fl_str_mv |
repdigital@cuc.edu.co |
| _version_ |
1828166909286154240 |
| spelling |
Kunzler, AliceTiefensee Ribeiro, CamilaGasparotto, JucianoLintzmaier Petiz, LyviaThais da Rosa Silva, Helenda Silva Jr., Jeferson DelgadoBortolin, RafaelOliveira de Souza, PriscilaBarreto, FabianoEspitia-Perez, PedroSchnorr, Carlos EduardoSomensi, NauanaFonseca Moreira, José ClaudioPens Gelain, Daniel2019-03-07T22:00:06Z2019-03-07T22:00:06Z2019-02-07https://hdl.handle.net/11323/2861Corporación Universidad de la CostaREDICUC - Repositorio CUChttps://repositorio.cuc.edu.co/Vitamin A (retinol) is involved in signaling pathways regulating gene expression and was postulated to be a major antioxidant and anti-inflammatory compound of the diet. Parkinson's disease (PD) is a progressive neurodegenerative disorder, characterized by loss of nigral dopaminergic neurons, involving oxidative stress and pro-inflammatory activation. The aim of the present study was to evaluate the neuroprotective effects of retinol oral supplementation against 6-hydroxydopamine (6-OHDA, 12 μg per rat) nigrostriatal dopaminergic denervation in Wistar rats. Animals supplemented with retinol (retinyl palmitate, 3000 IU/kg/day) during 28 days exhibited increased retinol content in liver, although circulating retinol levels (serum) were unaltered. Retinol supplementation did not protect against the loss of dopaminergic neurons (assessed through tyrosine hydroxylase immunofluorescence and Western blot). Retinol supplementation prevented the effect of 6-OHDA on Iba-1 levels but had no effect on 6-OHDA-induced GFAP increase. Moreover, GFAP levels were increased by retinol supplementation alone. Rats pre-treated with retinol did not present oxidative damage or thiol redox modifications in liver, and the circulating levels of TNF-α, IL-1β, IL-6 and IL-10 were unaltered by retinol supplementation, demonstrating that the protocol used here did not cause systemic toxicity to animals. Our results indicate that oral retinol supplementation is not able to protect against 6-OHDA-induced dopaminergic denervation, and it may actually stimulate astrocyte reactivity without altering parameters of systemic toxicity.Kunzler, Alice-4b32d83a-06bf-4dd0-b9c2-00dda027fc8b-0Tiefensee Ribeiro, Camila-8320967c-6582-4295-b6a5-d095b090c073-0Gasparotto, Juciano-a7ba2163-3c88-45e4-b4f0-627833f8b03f-0Lintzmaier Petiz, Lyvia-cac3be05-7b50-4d20-b637-81f75d5833aa-0Thais da Rosa Silva, Helen-6458b94c-7f80-4b35-9b40-bc0608be208e-0da Silva Jr., Jeferson Delgado-bfda4b6b-acef-4079-9990-9055ad496867-0Bortolin, Rafael-57d7a173-f5ea-4273-b0fe-adf809815ea2-0Oliveira de Souza, Priscila-31b76c7b-9ad6-4023-b8d6-2cb2c870277a-0Barreto, Fabiano-9e249dc0-6edf-433b-95bf-c9dafe44f59e-0Espitia-Perez, Pedro-b5f2b3c3-77c0-40b3-9699-0226bbe56dd4-0Schnorr, Carlos Eduardo-0000-0002-2047-2107-600Somensi, Nauana-2e27569b-a7b2-42aa-91d7-7f5d6951c2e9-0Fonseca Moreira, José Claudio-3f9c8312-14b3-43b1-96dc-30e108282b50-0Pens Gelain, Daniel-2ff9f9fc-72b9-42eb-ab4a-4c9be42216fb-0engUniversidad de la CostaAtribución – No comercial – Compartir igualinfo:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2RetinolNeurodegenerative disorder6-OHDANeuroinflammationVitamin supplementationThe effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar ratsPre-Publicaciónhttp://purl.org/coar/resource_type/c_816bTextinfo:eu-repo/semantics/preprinthttp://purl.org/redcol/resource_type/ARTOTRinfo:eu-repo/semantics/acceptedVersionAnon, 2001. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington (DC). . Anon, 2016. FDA- Food and Drug Administration. Aoto, J., Nam, C.I., Poon, M.M., Ting, P., Chen, L., 2008. Synaptic signaling by all-trans retinoic acid in homeostatic synaptic plasticity. Neuron 60, 308–320. Assini, R., Abercrombie, E.D., 2018. Zolpidem ameliorates motor impairments in the unilaterally 6-hydroxydopamine-lesioned rat. Eur. J. Neurosci. 48, 1896–1905. Behairi, N., Belkhelfa, M., Rafa, H., Labsi, M., Deghbar, N., Bouzid, N., Mesbah-Amroun, H., Touil-Boukoffa, C., 2016. All-trans retinoic acid (ATRA) prevents lipopolysaccharide-induced neuroinflammation, amyloidogenesis and memory impairment in aged rats. J. Neuroimmunol. 300, 21–29. Beitz, J.M., 2014. Parkinson's disease: a review. Front. Biosci. (Sch. Ed.) 6, 65–74. Bhat, S., Acharya, U.R., Hagiwara, Y., Dadmehr, N., Adeli, H., 2018. Parkinson's disease: cause factors, measurable indicators, and early diagnosis. Comput. Biol. Med. 102, 234–241. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Burton, G.W., Ingold, K.U., 1984. beta-Carotene: an unusual type of lipid antioxidant. Science 224, 569–573. Chang, K.H., Cheng, M.L., Chiang, M.C., Chen, C.M., 2018. Lipophilic antioxidants in neurodegenerative diseases. Clin. Chim. Acta 485, 79–87. Dao, D.Q., Ngo, T.C., Thong, N.M., Nam, P.C., 2017. Is vitamin A an antioxidant or a prooxidant? J. Phys. Chem. B 121, 9348–9357. de Bittencourt Pasquali, M.A., Roberto de Oliveira, M., De Bastiani, M.A., da Rocha, R.F., Schnorr, C.E., Gasparotto, J., Gelain, D.P., Moreira, J.C., 2012. L-NAME co-treatment prevent oxidative damage in the lung of adult Wistar rats treated with vitamin A supplementation. Cell Biochem. Funct. 30, 256–263. De Oliveira, M.R., Moreira, J.C., 2008. Impaired redox state and respiratory chain enzyme activities in the cerebellum of vitamin A-treated rats. Toxicology 253, 125–130. de Oliveira, M.R., Silvestrin, R.B., Mello e Souza, T., Moreira, J.C., 2008. Therapeutic vitamin A doses increase the levels of markers of oxidative insult in substantia nigra and decrease locomotory and exploratory activity in rats after acute and chronic supplementation. Neurochem. Res. 33, 378–383. de Oliveira, M.R., Soares Oliveira, M.W., Muller Hoff, M.L., Behr, G.A., da Rocha, R.F., Fonseca Moreira, J.C., 2009. Evaluation of redox and bioenergetics states in the liver of vitamin A-treated rats. Eur. J. Pharmacol. 610, 99–105. de Pee, S., Dary, O., 2002. Biochemical indicators of vitamin A deficiency: serum retinol and serum retinol binding protein. J. Nutr. 132, 2895s–2901s. Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 186, 421–431. Dunkley, P.R., Bobrovskaya, L., Graham, M.E., von Nagy-Felsobuki, E.I., Dickson, P.W., 2004. Tyrosine hydroxylase phosphorylation: regulation and consequences. J. Neurochem. 91, 1025–1043. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Espitia-Perez, P., Albino, S.M., da Rosa, H.T., Silveira, A.K., Espitia-Perez, L., Brango, H., Moraes, D.P., Hermann, P.R.S., Mingori, M., Barreto, F., Kunzler, A., Gelain, D.P., Schnorr, C.E., Moreira, J.C.F., 2018. Effects of methylmercury and retinol palmitate co-administration in rats during pregnancy and breastfeeding: metabolic and redox parameters in dams and their offspring. Ecotoxicol. Environ. Saf. 162, 603–615. Gasparotto, J., Ribeiro, C.T., Bortolin, R.C., Somensi, N., Rabelo, T.K., Kunzler, A., Souza, N.C., Pasquali, M.A.B., Moreira, J.C.F., Gelain, D.P., 2017. Targeted inhibition of RAGE in substantia nigra of rats blocks 6-OHDA-induced dopaminergic denervation. Sci. Rep. 7, 8795. Gelain, D.P., de Bittencourt Pasquali, M.A., Caregnato, F.F., Moreira, J.C., 2011. Vitamin A (retinol) up-regulates the receptor for advanced glycation endproducts (RAGE) through p38 and Akt oxidant-dependent activation. Toxicology 289, 38–44. Gelain, D.P., Moreira, J.C., Bevilaqua, L.R., Dickson, P.W., Dunkley, P.R., 2007. Retinol activates tyrosine hydroxylase acutely by increasing the phosphorylation of serine 40 and then serine 31 in bovine adrenal chromaffin cells. J. Neurochem. 103, 2369–2379. Grimm, M.O., Mett, J., Hartmann, T., 2016. The impact of vitamin E and other fat-soluble vitamins on alzheimer s disease. Int. J. Mol. Sci. 17. Haddadi, R., Nayebi, A.M., Farajniya, S., Brooshghalan, S.E., Sharifi, H., 2014. Silymarin improved 6-OHDA-induced motor impairment in hemi-parkisonian rats: behavioral and molecular study. Daru 22, 38. Huang, Z., Liu, Y., Qi, G., Brand, D., 2018. Role of Vitamin A in the Immune System, vol. 7. Karkeni, E., Bonnet, L., Astier, J., Couturier, C., Dalifard, J., Tourniaire, F., Landrier, J.F., 2017. All-trans-retinoic acid represses chemokine expression in adipocytes and adipose tissue by inhibiting NF-kappaB signaling. J. Nutr. Biochem. 42, 101–107. Klamt, F., Dal-Pizzol, F., Roehrs, R., de Oliveira, R.B., Dalmolin, R., Henriques, J.A., de Andrades, H.H., de Paula Ramos, A.L., Saffi, J., Moreira, J.C., 2003. Genotoxicity, recombinogenicity and cellular preneoplasic transformation induced by vitamin A supplementation. Mutat. Res. 539, 117–125. Lai, C.L., Lu, C.C., Lin, H.C., Sung, Y.F., Wu, Y.P., Hong, J.S., Peng, G.S., 2019. Valproate is protective against 6-OHDA-induced dopaminergic neurodegeneration in rodent midbrain: a potential role of BDNF up-regulation. J. Formos. Med. Assoc. 118, 420–428. Leonoudakis, D., Rane, A., Angeli, S., Lithgow, G.J., Andersen, J.K., Chinta, S.J., 2017. Anti-Inflammatory and Neuroprotective Role of Natural Product Securinine in Activated Glial Cells: Implications for Parkinson's Disease, vol. 2017 8302636. Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.G., Ahn, B.W., Shaltiel, S., Stadtman, E.R., 1990. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 186, 464–478. Liu, W., Tang, Y., Feng, J., 2011. Cross talk between activation of microglia and astrocytes in pathological conditions in the central nervous system. Life Sci. 89, 141–146. Lobo, V., Patil, A., Phatak, A., Chandra, N., 2010. Free radicals, antioxidants and functional foods: impact on human health. Pharm. Rev. 4, 118–126. Maden, M., 2007. Retinoic acid in the development, regeneration and maintenance of the nervous system. Nat. Rev. Neurosci. 8, 755–765. Maqbool, A., Graham-Maar, R.C., Schall, J.I., Zemel, B.S., Stallings, V.A., 2008. Vitamin A intake and elevated serum retinol levels in children and young adults with cystic fibrosis. J. Cyst. Fibros. 7, 137–141. McCaffery, P., Zhang, J., Crandall, J.E., 2006. Retinoic acid signaling and function in the adult hippocampus. J. Neurobiol. 66, 780–791. Miyake, Y., Tanaka, K., Fukushima, W., Sasaki, S., Kiyohara, C., Tsuboi, Y., Yamada, T., Oeda, T., Miki, T., Kawamura, N., Sakae, N., Fukuyama, H., Hirota, Y., Nagai, M., 2011. Dietary intake of metals and risk of Parkinson's disease: a case-control study in Japan. J. Neurol. Sci. 306, 98–102. Mohammadzadeh Honarvar, N., Saedisomeolia, A., Abdolahi, M., Shayeganrad, A., Taheri Sangsari, G., Hassanzadeh Rad, B., Muench, G., 2017. Molecular anti-inflammatory mechanisms of retinoids and carotenoids in alzheimer's disease: a review of current evidence. J. Mol. Neurosci. 61, 289–304. National Research Council (U.S.) Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research (U.S.), National Academies Press (U.S.), 2011. Guide for the Care and Use of Laboratory Animals. National Academies Press, Washington, D.C, pp. 1 online resource (xxv, 220 pp.). Paxinos, G., Watson, C., 2007. The Rat Brain in Stereotaxic Coordinates, sixth ed. Elsevier. Petiz, L.L., Girardi, C.S., Bortolin, R.C., Kunzler, A., Gasparotto, J., Rabelo, T.K., Matte, C., Moreira, J.C.F., Gelain, D.P., 2017a. Vitamin A oral supplementation induces oxidative stress and suppresses IL-10 and HSP70 in skeletal muscle of trained rats. Nutrients 9, 16. Petiz, L.L., Kunzler, A., Bortolin, R.C., Gasparotto, J., Matte, C., Moreira, J.C.F., Gelain, D.P., 2017b. Role of vitamin A oral supplementation on oxidative stress and inflammatory response in the liver of trained rats. Appl. Physiol. Nutr. Metabol. 42, 1192–1200. Real, C.C., Garcia, P.C., Britto, L.R.G., 2017. Treadmill exercise prevents increase of neuroinflammation markers involved in the dopaminergic damage of the 6-OHDA Parkinson's disease model. J. Mol. Neurosci. 63, 36–49. Rodriguez-Pallares, J., Parga, J.A., Munoz, A., Rey, P., Guerra, M.J., Labandeira-Garcia, J.L., 2007. Mechanism of 6-hydroxydopamine neurotoxicity: the role of NADPH oxidase and microglial activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons. J. Neurochem. 103, 145–156. Sardi, S.P., Cedarbaum, J.M., Brundin, P., 2018. Targeted therapies for Parkinson's disease: from genetics to the clinic. Mov. Disord. 33, 684–696. Schnorr, C.E., Bittencourt Lda, S., Petiz, L.L., Gelain, D.P., Zeidan-Chulia, F., Moreira, J.C., 2015. Chronic retinyl palmitate supplementation to middle-aged Wistar rats disrupts the brain redox homeostasis and induces changes in emotional behavior. Mol. Nutr. Food Res. 59, 979–990. Schnorr, C.E., da Silva Morrone, M., Simoes-Pires, A., da Rocha, R.F., Behr, G.A., Moreira, J.C., 2011. Vitamin A supplementation in rats under pregnancy and nursing induces behavioral changes and oxidative stress upon striatum and hippocampus of dams and their offspring. Brain Res. 1369, 60–73. Shearer, K.D., Stoney, P.N., Morgan, P.J., McCaffery, P.J., 2012. A vitamin for the brain. Trends Neurosci. 35, 733–741. Shi, H.Y., Yan, S.M., Guo, Y.M., Zhang, B.Q., Guo, X.Y., Shi, B.L., 2018. Vitamin A pretreatment protects NO-induced bovine mammary epithelial cells from oxidative stress by modulating Nrf 2 and NF-kappaB signaling pathways. J. Anim. Sci. 96, 1305–1316. Singh, P., Hanson, P.S., Morris, C.M., 2017. SIRT1 ameliorates oxidative stress induced neural cell death and is down-regulated in Parkinson's disease. 18, 46. Tanumihardjo, S.A., Russell, R.M., Stephensen, C.B., Gannon, B.M., 2016. Biomarkers of Nutrition for Development (BOND)-Vitamin A Review, vol. 146. pp. 1816s–1848s. Taylor, J.M., Main, B.S., Crack, P.J., 2013. Neuroinflammation and oxidative stress: coconspirators in the pathology of Parkinson's disease. Neurochem. Int. 62, 803–819. Theus, M.H., Sparks, J.B., Liao, X., Ren, J., Luo, X.M., 2017. All- trans-retinoic acid augments the histopathological outcome of neuroinflammation and neurodegeneration in lupus-prone MRL/lpr mice. J. Histochem. Cytochem. 65, 69–81. Thiele, S.L., Warre, R., Nash, J.E., 2012. Development of a unilaterally-lesioned 6-OHDA mouse model of Parkinson's disease. J. Vis. Exp. 60 pii: 3234. Trumbo, P., Yates, A.A., Schlicker, S., Poos, M., 2001. Dietary reference intakes: vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. J. Am. Diet Assoc. 101, 294–301. Ulusoy, G.K., Celik, T., Kayir, H., Gursoy, M., Isik, A.T., Uzbay, T.I., 2011. Effects of pioglitazone and retinoic acid in a rotenone model of Parkinson's disease. Brain Res. Bull. 85, 380–384. Wang, H.L., Zhang, Z., Hintze, M., Chen, L., 2011. Decrease in calcium concentration triggers neuronal retinoic acid synthesis during homeostatic synaptic plasticity. J. Neurosci. 31, 17764–17771. Wang, Q., Liu, Y., Zhou, J., 2015a. Neuroinflammation in Parkinson's disease and its potential as therapeutic target. Transl. Neurodegener. 4, 19. Wang, Q., Liu, Y., Zhou, J., 2015b. Neuroinflammation in Parkinson's disease and its potential as therapeutic target. Transl. Neurodegener. 4. Xie, L., Chen, L., Gu, P., Wei, L., Kang, X., 2018. A convenient method for extraction and analysis with high-pressure liquid chromatography of catecholamine neurotransmitters and their metabolites. J. Vis. Exp. 133. Yin, L.H., Shen, H., Diaz-Ruiz, O., Backman, C.M., Bae, E., Yu, S.J., Wang, Y., 2012. Early post-treatment with 9-cis retinoic acid reduces neurodegeneration of dopaminergic neurons in a rat model of Parkinson's disease. BMC Neurosci. 13, 120. Zeng, J., Chen, L., Wang, Z., Chen, Q., Fan, Z., Jiang, H., Wu, Y., Ren, L., Chen, J., Li, T., Song, W., 2017. Marginal Vitamin A Deficiency Facilitates Alzheimer's Pathogenesis. 133, 967–982.PublicationORIGINALThe effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats.pdfThe effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats.pdfapplication/pdf185838https://repositorio.cuc.edu.co/bitstreams/0f46dc11-7d9e-4349-801e-f6a7075aec1a/download6746cef1b8bae9a8ed1fe48332011891MD51LICENSElicense.txtlicense.txttext/plain; charset=utf-81748https://repositorio.cuc.edu.co/bitstreams/230ad955-f14a-4b07-b15c-ee6f05f85f8b/download8a4605be74aa9ea9d79846c1fba20a33MD52THUMBNAILThe effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats.pdf.jpgThe effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats.pdf.jpgimage/jpeg52414https://repositorio.cuc.edu.co/bitstreams/f35c3cc9-fa06-477e-9df6-4976f181a809/download2f4440ea621d70ab4368b426a51a10d1MD54TEXTThe effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats.pdf.txtThe effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats.pdf.txttext/plain2191https://repositorio.cuc.edu.co/bitstreams/8bac9c49-6ab2-4655-b7bf-cfbef72418e6/downloada877dd7b7232173b8894c5de0d7a6b0fMD5511323/2861oai:repositorio.cuc.edu.co:11323/28612024-09-17 14:24:52.426open.accesshttps://repositorio.cuc.edu.coRepositorio de la Universidad de la Costa CUCrepdigital@cuc.edu.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 |