Rosmarinic acid-rich ethanolic extract of Orthosiphon stamineus ameliorates cognitive and hippocampal long-term potentiation in chronic cerebral hypoperfusion rat model

Authors

  • Zurina Hassan Centre for Drug Research, Universiti Sains Malaysia, Penang, Malaysia.
  • KKesevan Rajah Kumaran Malaysian Institute of Pharmaceuticals and Nutraceuticals, National Institutes of Biotechnology Malaysia, Halaman Bukit Gambir, 11700 Gelugor, Pulau Pinang, Malaysia.
  • Nelson Jeng-Yeou Chear Centre for Drug Research, Universiti Sains Malaysia, Penang, Malaysia.
  • Siti Najmi Syuhadaa Bakar Centre for Drug Research, Universiti Sains Malaysia, Penang, Malaysia.
  • Thaarvena Retinasamy Neuropharmacology Research Laboratory, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Selangor, Malaysia.
  • Saatheeyavaane Bhuvanendran Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Selangor, Malaysia.
  • Amin Malik Abdul Majeed School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia.
  • Mohd. Farooq Shaikh Neuropharmacology Research Laboratory, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Selangor, Malaysia.
  • Iekhsan Othman Neuropharmacology Research Laboratory, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Selangor, Malaysia.

DOI:

https://doi.org/10.31117/neuroscirn.v6i3.196

Keywords:

O. stamineus, passive avoidance task, Morris-water-maze, cognitive function, chronic cerebral hypoperfused rats

Abstract

Chronic cerebral hypoperfusion (CCH) is one of the main causes of vascular dementia caused by the reduced blood flow to the brain. Orthosiphon stamineus (OS) is a medicinal herb exhibiting pronounced neuroprotective, anti-oxidant and anti-inflammatory activities due to its high rosmarinic acid content. This study investigated the nootropic effect of OS ethanolic extract on cognitive functions in CCH rats. CCH was developed by permanent bilateral occlusion of the common carotid artery (PBOCCA). Passive avoidance task (PAT) and Morris water maze (MWM) test were conducted to evaluate cognitive functions followed by in vivo long-term potentiation (LTP) for assessing neuroplasticity. The rosmarinic acid content of OS ethanolic extract was quantified using a validated HPLC-PDA. Treatment with OS ethanolic extract significantly increased step-through latency in the PAT, decreased escape latency at a low dose of OS extract in the MWM and rescued the LTP impairment at the highest dose in CCH rats. These results strongly support the effectiveness of rosmarinic acid-rich OS extract (5.088 % w/w) in treating pathological vascular dementia caused by CCH.

References

Abdelwahab, S. I., Mohan, S., Mohamed Elhassan, M., Al-Mekhlafi, N., Mariod, A. A., Abdul, A. B., Abdulla, M. A., & Alkharfy, K. M. (2011). Antiapoptotic and Anti-oxidant Properties of Orthosiphon stamineus Benth (Cat's Whiskers): Intervention in the Bcl-2-Mediated Apoptotic Pathway. Evidence-based Complementary and Alternative Medicine, 2011, 156765. https://doi.org/10.1155/2011/156765

Ahad, M. A., Kumaran, K. R., Ning, T., Mansor, N. I., Effendy, M. A., Damodaran, T., Lingam, K., Wahab, H. A., Nordin, N., Liao, P., Müller, C. P., & Hassan, Z. (2020). Insights into the neuropathology of cerebral ischemia and its mechanisms. Reviews in the Neurosciences, 31(5), 521–538. https://doi.org/10.1515/revneuro-2019-0099

Ali, M. and Zahid, S. (2020). The neurogenic effects of rosmarinic acid in a mouse model of type 2 diabetes mellitus. Brazilian Journal of Pharmaceutical Sciences, 56. https://doi.org/10.1590/s2175-979020200003180772

Arafat, O.M., Tham, S.Y., Sadikun, A., Zhari, I., Haughton, P.J., & Asmawi, M.Z. (2008). Studies on diuretic and hypouricemic effects of Orthosiphon stamineus methanol extracts in rats. Journal of Ethnopharmacology, 118(3), 354-360. https://doi.org/10.1016/j.jep.2008.04.015

Ashraf, K., Sultan, S., & Adam, A. (2018). Orthosiphon stamineus Benth. is an Outstanding Food Medicine: Review of Phytochemical and Pharmacological Activities. Journal of Pharmacy & Bioallied Sciences, 10(3), 109–118. https://doi.org/10.4103/jpbs.JPBS_253_17

Awale, S., Tezuka, Y., Banskota, A.H., Adnyana, I.K., & Kadota, S. (2003). Nitric oxide inhibitory isopimarane-type diterpenes from Orthosiphon stamineus of Indonesia. Journal of Natural Products, 66(2), 255-258. https://doi.org/10.1021/np020455x

Bathina, S., & Das, U. N. (2015). Brain-derived neurotrophic factor and its clinical implications. Archives of Medical Science, 11(6), 1164–1178. https://doi.org/10.5114/aoms.2015.56342

Bhullar, K.S., & Rupasinghe, H.P.V. (2013). Polyphenols: Multipotent therapeutic agents in neurodegenerative diseases. Oxidative Medicine and Cellular Longevity, 2013, 891748. https://doi.org/10.1155/2013/891748

Bramham, C.R., and Messaoudi, E. (2005). BDNF function in adult synaptic plasticity: The synaptic consolidation hypothesis. Progress in Neurobiology, 76(2), 99-125. https://doi.org/10.1016/j.pneurobio.2005.06.003

Cechetti, F., Pagnussat, A.S., Worm, P.V., Elsner, V.R., Ben, J., da Costa, M.S., ... Netto, C.A. (2012). Chronic brain hypoperfusion causes early glial activation and neuronal death, and subsequent long-term memory impairment. Brain Research Bulletin, 87(1), 109-116. https://doi.org/10.1016/j.brainresbull.2011.10.006

Choi, D.H., Lee, K.H., Kim, J.H., Seo, J.H., Kim, H.Y., Shin, C.Y., ... Lee, J.M. (2014). NADPH oxidase 1, a novel molecular source of ROS in hippocampal neuronal death in vascular dementia. Anti-oxidants & Redox Signaling, 21(4), 533-550. https://doi.org/10.1089/ars.2012.5129

Cross, J., Meloni, B., Bakker, A., Lee, S., & Knuckey, N. (2010). Modes of neuronal calcium entry and homeostasis following cerebral ischemia. Stroke Research Treatment, 2010, 1–9. https://doi.org/10.4061/2010/316862

Damodaran, T., Hassan, Z., Navaratnam, V., Muzaimi, M., Ng, G., Müller, C.P., ... Dringenberg H.C. (2014). Time course of motor and cognitive functions after chronic cerebral ischemia in rats. Behavioral Brain Research, 275, 252-258. https://doi.org/10.1016/j.bbr.2014.09.014

Damodaran, T., Müller, C.P., & Hassan, Z. (2019). Chronic cerebral hypoperfusion-induced memory impairment and hippocampal long-term potentiation deficits are improved by cholinergic stimulation in rats. Pharmacological Reports, 71(3), 443-448. https://doi.org/10.1016/j.pharep.2019.01.012

Damodaran, T., Tan, B.W.L., Liao, P., Ramanathan, S., Lim, G.K., & Hassan, Z. (2018). Clitoria ternatea L. root extract ameliorated the cognitive and hippocampal long-term potentiation deficits induced by chronic cerebral hypoperfusion in the rat. Journal of Ethnopharmacology, 224, 381-390. https://doi.org/10.1016/j.jep.2018.06.020

De Vincenti, A. P., Ríos, A. S., Paratcha, G., & Ledda, F. (2019). Mechanisms That Modulate and Diversify BDNF Functions: Implications for Hippocampal Synaptic Plasticity. Frontiers in Cellular Neuroscience, 13, 135. https://doi.org/10.3389/fncel.2019.00135

D’Hooge, R., & De Deyn, P.P. (2001). Applications of the Morris water maze in the study of learning and memory. Brain Research Reviews, 36(1), 60-90. https://doi.org/10.1016/s0165-0173(01)00067-4

Díaz-Alonso J, Sun YJ, Granger AJ, Levy JM, Blankenship SM, Nicoll RA (2017) Subunit-specifc role for the amino-terminal domain of AMPA receptors in synaptic targeting. Proceedings of the National Academy of Sciences USA, 114(27):7136–7141. https://doi.org/10.1073/pnas.1707472114

Eagle, A. L., Wang, H., & Robison, A. J. (2016). Sensitive assessment of hippocampal learning using temporally dissociated passive avoidance task. Bio-protocol, 6(11), e1821. https://doi.org/10.21769/BioProtoc.1821

Essa, M.M., Vijayan, R.K., Castellano-Gonzalez, G., Memon, M.A., Braidy, N., & Guillemin, G.J. (2012). Neuroprotective effect of natural products against Alzheimer's disease. Neurochemical Research, 37(9), 1829-1842. https://doi.org/10.1007/s11064-012-0799-9

Fachel, F. N. S., Schuh, R. S., Veras, K. S., Bassani, V. L., Koester, L. S., Henriques, A. T., Braganhol, E., & Teixeira, H. F. (2019). An overview of the neuroprotective potential of rosmarinic acid and its association with nanotechnology-based delivery systems: A novel approach to treating neurodegenerative disorders. Neurochemistry International, 122, 47–58. https://doi.org/10.1016/j.neuint.2018.11.003

Fadel, O., Kirat, KE, & Morandat, S. (2011). The natural anti-oxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidation in situ. Biochimica et Biophysica Acta (BBA) – Biomembranes, 1808(12), 2973-2980. https://doi.org/10.1016/j.bbamem.2011.08.011

Farkas, E., Institóris, A., Domoki, F., Mihály, A., Luiten, P.G.M., & Bari, F. (2004). Diazoxide and dimethyl sulphoxide prevent cerebral hypoperfusion-related learning dysfunction and brain damage after carotid artery occlusion. Brain Research, 1008(2), 252-260. https://doi.org/10.1016/j.brainres.2004.02.037

Farkas, E., Luiten, P.G.M., & Bari, F. (2007). Permanent, bilateral common carotid artery occlusion in the rat: A model for chronic cerebral hypoperfusion-related neurodegenerative diseases. Brain Research Review, 54(1), 162-180. https://doi.org/10.1016/j.brainresrev.2007.01.003

Fonteles, A. A., de Souza, C. M., de Sousa Neves, J. C., Menezes, A. P., Santos do Carmo, M. R., Fernandes, F. D., de Araújo, P. R., & de Andrade, G. M. (2016). Rosmarinic acid prevents against memory deficits in ischemic mice. Behavioural Brain Research, 297, 91–103. https://doi.org/10.1016/j.bbr.2015.09.029

George, A., Chinnappan, S., Choudhary, Y., Choudhary, V.K., Bommu, P., & Wong, H.J. (2015). Effects of a proprietary standardized Orthosiphon stamineus ethanolic leaf extract on enhancing memory in Sprague Dawley rats possibly via blockade of adenosine A2A receptors. Evidence-Based Complementary and Alternative Medicine, 2015, 375837.

Ghumatkar, P.J., Patil, S.P., Jain, P.D., Tambe, RM & Sathaye, S. (2015) Nootropic, neuroprotective and neurotrophic effects of phloretin in scopolamine induced amnesia in mice. Pharmacology Biochemistry and Behavior, 135, 182-191. https://doi.org/10.1016/j.pbb.2015.06.005

Granger AJ, Nicoll RA (2013) Expression mechanisms underlying long-term potentiation: a postsynaptic view, 10 years on. Philosophical Transactions of the Royal Society B: Biological Sciences, 369(1633):1–6. https://doi.org/10. 1098/rstb.2013.0136

Green, D. R., & Llambi, F. (2015). Cell Death Signaling. Cold Spring Harbor Perspectives in Biology, 7(12), a006080. https://doi.org/10.1101/cshperspect.a006080

Gülçin, İ., Scozzafava, A., Supuran, C. T., Koksal, Z., Turkan, F., Çetinkaya, S., Bingöl, Z., Huyut, Z., & Alwasel, S. H. (2016). Rosmarinic acid inhibits some metabolic enzymes including glutathione S-transferase, lactoperoxidase, acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase isoenzymes. Journal of Enzyme Inhibition and Medicinal Chemistry, 31(6), 1698–1702. https://doi.org/10.3109/14756366.2015.1135914

Hajjar, T., Goh, Y.M., Rajion, M.A. et al. (2013) Alterations in neuronal morphology and synaptophysin expression in the rat brain as a result of changes in dietary n-6: n-3 fatty acid ratios. Lipids Health Disease, 12, 113. https://doi.org/10.1186/1476-511X-12-113

Jin, Y., Chen, R., Yi, X., Wei, L., Long, Q., and Liu, W. (2018). The expression of hippocampal NRG1/ErbB4 correlates with neuronal apoptosis, but not with glial activation during chronic cerebral hypoperfusion. Frontier Aging Neuroscience, 10, 149.

Jin, W., Jia, Y., Huang, L., Wang, T., Wang, H., Dong, Y., ... Lv, P. (2014). Lipoxin A4 methyl ester ameliorates cognitive deficits induced by chronic cerebral hypoperfusion through activating ERK/Nrf2 signaling pathway in rats. Pharmacology Biochemistry and Behavior, 124,145–152. https://doi.org/10.1016/j.pbb.2014.05.023

Kennedy, D.O. (2019). Phytochemicals for improving aspects of cognitive function and psychological state potentially relevant to sports performance. Sports Medicine, 49, 39–58. https://doi.org/10.1007/s40279-018-1007-0

Kim, H., Kim, S., Song, Y., Kim, W., Ying, Q. L., & Jho, E. H. (2015). Dual function of Wnt signaling during neuronal differentiation of mouse embryonic stem cells. Stem Cells International, 2015, 459301. https://doi.org/10.1155/2015/459301

Khamse, S., Sadr, S. S., Roghani, M., Rashvand, M., Mohammadian, M., Marefati, N., Harati, E., & Ebrahimi, F. (2020). The Effect of Rosmarinic Acid on Apoptosis and nNOS Immunoreactivity Following Intrahippocampal Kainic Acid Injections in Rats. Basic and Clinical Neuroscience, 11(1), 41–48. https://doi.org/10.32598/bcn.9.10.340

Kokotos, A. C., Harper, C. B., Marland, J. R. K., Smillie, K. J., Cousin, M. A., & Gordon, S. L. (2019). Synaptophysin sustains presynaptic performance by preserving vesicular Synaptobrevin‐II levels. Journal of Neurochemistry, 151(1), 28–37. https://doi.org/10.1111/jnc.14797

Kotani, S., Yamauchi, T., Teramoto, T., & Ogura, H. (2006). Pharmacological evidence of cholinergic involvement in adult hippocampal neurogenesis in rats. Neuroscience, 142(2), 505–514.

https://doi.org/10.1016/j.neuroscience.2006.06.035

Kumaran, K. R., Wahab, H. A., & Hassan, Z. (2022). Nootropic effect of syzygium polyanthum (Wight) walp leaf extract in chronic cerebral hypoperfusion rat model via cholinergic restoration: A potential therapeutic agent for dementia. Advances in Traditional Medicine. https://doi.org/10.1007/s13596-022-00653-3

Laavola, M., Nieminen, R., Yam, M. F., Sadikun, A., Asmawi, M. Z., Basir, R., Welling, J., Vapaatalo, H., Korhonen, R., & Moilanen, E. (2012). Flavonoids eupatorin and sinensetin present in Orthosiphon stamineus leaves inhibit inflammatory gene expression and STAT1 activation. Planta Medica, 78(8), 779–786. https://doi.org/10.1055/s-0031-1298458

Liu, C., Wu, J., Gu, J., Xiong, Z., Wang, F., Wang, J., ... Chen, J. (2007). Baicalein improves cognitive deficits induced by chronic cerebral hypoperfusion in rats. Pharmacology Biochemistry and Behavior, 86(3), 423-430. https://doi.org/10.1016/j.pbb.2006.11.005

Malenka, R.C., & Bear, M.F. (2004). LTP and LTD: An embarrassment of riches. Neuron, 44(1), 5-21. https://doi.org/10.1016/j.neuron.2004.09.012

Mirza, F. J., Amber, S., Sumera, Hassan, D., Ahmed, T., & Zahid, S. (2021). Rosmarinic acid and ursolic acid alleviate deficits in cognition, synaptic regulation and adult hippocampal neurogenesis in an Aβ1-42-induced mouse model of Alzheimer's disease. Phytomedicine, 83, 153490. https://doi.org/10.1016/j.phymed.2021.153490

Moosavi, M., SoukhakLari, R., Moezi, L., & Pirsalami, F. (2018). Scopolamine-induced passive avoidance memory retrieval deficit is accompanied with hippocampal MMP2, MMP-9 and MAPKs alteration. European Journal of Pharmacology, 819, 248–253. https://doi.org/10.1016/j.ejphar.2017.12.007

Ni, J., Ohta, H., Matsumoto, K., & Watanabe, H., (1994). Progressive cognitive impairment following chronic cerebral hypoperfusion induced by permanent occlusion of bilateral carotid arteries in rats. Brain Research, 653(1-2), 231-236. https://doi.org/10.1016/0006-8993(94)90394-8

Pariyani, R., Ismail, I. S., Azam, A. A., Abas, F., Shaari, K., & Sulaiman, M. R. (2015). Phytochemical screening and acute oral toxicity study of java tea leaf extracts. BioMed Research International, 2015, 742420. https://doi.org/10.1155/2015/742420

Paxinos, G., & Watson, C. (2007). The Rat Brain in Stereotaxic Coordinates (6th edn). Elsevier Academic Press, USA.

Rabiei, Z., Rafieian-Kopaei, M., Mokhtari, S., Alibabaei, Z., & Shahrani, M. (2014). The effect of pre-treatment with different doses of Lavandula officinalis ethanolic extract on memory, learning and nociception. Biomedicine & Aging Pathology, 4(1), 71-76. https://doi.org/10.1016/j.biomag.2013.10.006

Retinasamy, T., Shaikh, M. F., Kumari, Y., Abidin, S. A. Z., & Othman, I. (2020). Orthosiphon stamineus Standardized Extract Reverses Streptozotocin-induced Alzheimer's Disease-Like Condition in a Rat Model. Biomedicines, 8(5), 104. https://doi.org/10.3390/biomedicines8050104

Retinasamy, T., Shaikh, M. F., Kumari, Y., & Othman, I. (2019). Ethanolic Extract of Orthosiphon stamineus Improves Memory in Scopolamine-Induced Amnesia Model. Frontiers in Pharmacology, 10,1216. https://doi.org/10.3389/fphar.2019.01216

Saidan, N.H., Aisha, A., Hamil, M.S., Abdul Majid, A.M.S., & Ismail, Z. (2015a). A novel reverse phase high-performance liquid chromatography method for standardization of Orthosiphon stamineus leaf extracts. Pharmacognosy Research, 7(1), 23-31. https://doi.org/10.4103/0974-8490.147195

Saidan, N. H., Hamil, M. S., Memon, A. H., Abdelbari, M. M., Hamdan, M. R., Mohd, K. S., Majid, A. M., & Ismail, Z. (2015b). Selected metabolites profiling of Orthosiphon stamineus Benth leaves extracts combined with chemometrics analysis and correlation with biological activities. BMC Complementary and Alternative Medicine, 15, 350. https://doi.org/10.1186/s12906-015-0884-0

Sarti, C., Pantoni, L., Bartolini, L., & Inzitari, D. (2002). Persistent impairment of gait performances and working memory after bilateral common carotid artery occlusion in the adult Wistar rat. Behavioral Brain Research, 136(1), 13-20. https://doi.org/10.1016/s0166-4328(02)00090-6

Sree, N.V., Sri, P.U., & Ramarao, N. (2015). Neuro-protective properties of Orthosiphon staminus (Benth) leaf methanolic fraction through anti-oxidant mechanisms on SH-SY5Y cells: an in-vitro evaluation. International Journal of Pharmaceutical Sciences and Research, 6(3), 115-1125.

Sumaryono, W., Proksch, P., Wray, V., Witte, L., & Hartmann, T. (1991). Qualitative and quantitative analysis of the phenolic constituents from Orthosiphon aristatus. Planta Medica, 57(2), 176-180. https://doi.org/10.1055/s-2006-960060

Taram, F., Ignowski, E., Duval, N., & Linseman, D. A. (2018). Neuroprotection comparison of Rosmarinic acid and carnosic acid in primary cultures of cerebellar granule neurons. Molecules (Basel, Switzerland), 23 (11), 2956. https://doi.org/10.3390/molecules23112956

Thiel, G. (1993). Synapsin I, Synapsin II, and synaptophysin: Marker proteins of synaptic vesicles. Brain Pathology, 3(1), 87–95. https://doi.org/10.1111/j.1750-3639.1993.tb00729.x

Vicente, É., Degerone, D., Bohn, L., Scornavaca, F., Pimentel, A., Leite, M.C., ... Goncalves, C.A. (2009). Astroglial and cognitive effects of chronic cerebral hypoperfusion in the rat. Brain Research, 1251, 204-212. https://doi.org/10.1016/j.brainres.2008.11.032

Vosler, P., Brennan, C., and Chen, J. (2008). Calpain-mediated signalling mechanisms in neuronal injury and neurodegeneration. Molecular Neurobiology, 38, 78–100.

Wang, C. C., Hsieh, P. W., Kuo, J. R., & Wang, S. J. (2021). Rosmarinic acid, a bioactive phenolic compound, inhibits glutamate release from rat cerebrocortical synaptosomes through GABAA receptor activation. Biomolecules, 11(7), 1029. https://doi.org/10.3390/biom11071029

Xi, Y., Wang, M., Zhang, W., Bai, M., Du, Y., Zhang, Z., ... Miao, J. (2014). Neuronal damage, central cholinergic dysfunction and oxidative damage correlate with cognitive deficits in rats with chronic cerebral hypoperfusion. Neurobiology of Learning and Memory, 109, 7-19. https://doi.org/10.1016/j.nlm.2013.11.016

Xu, Y., Zhang, J., Xiong, L., Zhang, L., Sun, D., & Liu, H. (2010). Green tea polyphenols inhibit cognitive impairment induced by chronic cerebral hypoperfusion via modulating oxidative stress. The Journal of Nutritional Biochemistry, 21(8), 741-748. https://doi.org/10.1016/j.jnutbio.2009.05.002

Yam, M.F., Lim, V., Salman, I.M., Ameer, O.Z., Ang, L.F., Rosidah, N., ... Asmawi, M.Z. (2010). HPLC and anti-inflammatory studies of the flavonoid rich chloroform extract fraction of Orthosiphon stamineus leaves. Molecules, 15(6), 4452-4466. https://doi.org/10.3390/molecules15064452

Yan, Z.Q., Chen, J., Xing, G.X., Huang, J.G., Hou, X.H., & Zhang, Y. (2015). Salidroside prevents cognitive impairment induced by chronic cerebral hypoperfusion in rats. Journal of International Medical Research, 43(3), 401-411. https://doi.org/10.1177/0300060514566648

Yehya, A. H. S., Asif, M., Kaur, G., Hassan, L. E. A., Al-Suede, F. S. R., Abdul Majid, A. M. S., & Oon, C. E. (2018). Toxicological studies of Orthosiphon stamineus (Misai Kucing) standardized ethanol extract in combination with gemcitabine in athymic nude mice model. Journal of Advanced Research, 15, 59–68. https://doi.org/10.1016/j.jare.2018.05.006

Zhou, J., Yang, W. S., Suo, D. Q., Li, Y., Peng, L., Xu, L. X., Zeng, K. Y., Ren, T., Wang, Y., Zhou, Y., Zhao, Y., Yang, L. C., & Jin, X. (2018). Moringa oleifera seed extract alleviates scopolamine-Induced learning and memory impairment in mice. Frontiers in Pharmacology, 9, 389. https://doi.org/10.3389/fphar.2018.00389

Downloads

Published

2023-08-27

How to Cite

Hassan, Z., Kumaran, K. R., Chear, N. J.-Y., Syuhadaa Bakar, S. N., Retinasamy, T., Bhuvanendran, S., Abdul Majeed, A. M., Shaikh, M. F. and Othman, I. (2023) “Rosmarinic acid-rich ethanolic extract of Orthosiphon stamineus ameliorates cognitive and hippocampal long-term potentiation in chronic cerebral hypoperfusion rat model”, Neuroscience Research Notes, 6(3), pp. 196.1–196.16. doi: 10.31117/neuroscirn.v6i3.196.