Histological and Immunohistochemical Study of the Possible Protective Effect of Bee Pollen Extract on the Cerebellar Cortex of Valproic Acid-Induced Experimental Model of Autism in Albino Rat Pups.

Document Type : Original articles

Authors

1 Demonstrator of Histology and Cell Biology Department, Faculty of Medicine, Tanta University, Tanta, 31527, Egypt.

2 Professor of Histology and Cell Biology Department, Faculty of Medicine, Tanta University, Tanta, 31527, Egypt.

3 Lecturer of Histology and Cell Biology Department, Faculty of Medicine, Tanta University, Tanta, 31527, Egypt

Abstract

Background: Valproic acid-induced autism represents a well-recognized model to induce autism in rats. Bee pollen has potent anti-inflammatory, antioxidant and anti-apoptotic effects. Aim: This work was performed to study the protective effect of bee pollen extract on valproic acid-induced model of autism in rat pups. Materials and methods: The study was performed on 50 male albino rat pups 10-15 gm that were classified into four groups; group 1:control group, group II: rat pups received oral aqueous extract of bee pollen 250 mg/kg once daily from PND 14 to 40, group III: autism was induced by subcutaneous injection of a single dose of valproic acid 400mg/kg on PND 14, and group IV: after induction of autism as in group III, rat pups were treated with an aqueous extract of bee pollen from PND 14 to 40. All rats were sacrificed at PND 41. Cerebellar specimens were processed for histological and immunohistochemical study in addition to morphometric, behavioral and statistical studies. Results: Valproic acid-induced autism group depicted behavioral changes and marked alteration of the histological picture of the cerebellar cortex by light and electron microscopes. A significant decrease in the granular cell layer thickness and a significant increase in the mean area percentage of GFAP immunopositive reaction were observed in group III as compared to the control group. Treatment with Bee pollen showed marked improvement of the previously mentioned pathological changes. Conclusion: Bee pollen could improve the behavioral and histological alterations in the cerebellar cortex of valproic acid-induced autism in rat pups.

Keywords

Main Subjects


  1. Hirota T, King BH. Autism Spectrum Disorder: A Review. 2023; 329(2):157–168. doi:10.1001/jama.2022.23661
  2. Guerrera S, Pontillo M, Chieppa F, Passarini S, Di Vincenzo C, Casula L, et al. Autism Spectrum Disorder and Early Psychosis: a narrative review from a neurodevelopmental perspective. Psychiatry. 2024; 15:1362511:1-9. doi: 10.3389/fpsyt.2024.1362511
  3. Sunand K, Mohan GK, Bakshi V. Resveratrol alone and its Combination with Pterostilbene amends Valproic Acid-Induced Autism in Swiss Albino Mice: Postnatal Model. International Journal of Applied Pharmaceutical Sciences and Research. 2020; 5 (1). 12-21. ISSN: 2455-8095.
  4. Mattos BS, Soares MSB, Spohr L, Pedra NS, Teixeira FC, Souza AV, et al. Quercetin prevents alterations of behavioral parameters, delta-aminolevulinic dehydratase activity, and oxidative damage in brain of rats in a prenatal model of autism. Int J Dev Neurosci. 2020; 80: 287–302. DOI: 10.1002/jdn.10025.
  5. Elnahas E, Abuelezz SA, Mohamad MI, Nabil MM, Abdelraouf SM, Bahaa N, et al. Validation of prenatal versus postnatal valproic acid rat models of autism: A behavioral and neurobiological study. Progress in Neuro-Psychopharmacology and Biological Psychiatry. 2021;108, 110185. 1-16. ISSN 0278-5846,
  6. Al-Salem HS, Al-Yousef HM, Ashour AE, Ahmed AF, Amina M, Issa IS, et al. Antioxidant and hepatorenal protective effects of bee pollen fractions against propionic acid-induced autistic feature in rats. Food Sci Nutr. 2020; 8: 5114–5127
  7. El Ghouizi A, Bakour M, Laaroussi H, Ousaaid D, El Menyiy N, Hano C, et al.  Bee Pollen as Functional Food: Insights into Its Composition and Therapeutic Properties. 2023;12, 557. 1-31. https://doi.org/10.3390/ antiox12030557.
  8. Alfawaz HA, El-Ansary A, Al-Ayadhi L, Bhat R, Hassan WM. Protective Effects of Bee Pollen on Multiple Propionic Acid-Induced Biochemical Autistic Features in a Rat Model. 2022; 12,571. 1-16 https://doi.org/10.3390/ metabo12070571
  9. Baky MH, Abouelela MB, Wang K, Farag MA. Bee Pollen and Bread as a Super-Food: A Comparative Review of Their Metabolome Composition and Quality Assessment in the Context of Best Recovery Conditions. 2023; 28, 715. 1-22.
  10. Aabed K, Bhat RS, Al-Dbass A, Moubayed N, Algahtani N, Merghani NM, et al. Bee pollen and Propolis improve neuroinflammation and dysbiosis induced by propionic acid, a short chain fatty acid in a rodent model of autism. Lipids in Health and Disease.2019; 18(1):1-8.
  11. Morakotsriwan N, Wattanathorn J, Kirisattayakul W, Chaisiwamongkol W. Autistic-Like Behaviors, Oxidative Stress Status, and Histopathological Changes in Cerebellum of Valproic Acid Rat Model of Autism Are Improved by the Combined Extract of Purple Rice and Silkworm Pupae. Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity. 2016; 1-10, 3206561.
  12. Ishola IO, Balogun AO, Adeyemi OO. Novel potential of metformin of valproic acid-induced autism spectrum disorder in rats: involvement of antioxidant defence system. Fundamental & Clinical Pharmacology. 2020; 34:650–661. doi: 10.1111/fcp.12567.
  13. Sandhya T, Sowjanya J, Veeresh B. Bacopa monniera (L.) Wettst Ameliorates Behavioral Alterations and Oxidative Markers in Sodium Valproate Induced Autism in Rats. Neurochem Res.2012; 37:1121–1131
  14. Gaertner D, Hallman T, Hankenson F, Batcherder M. Anesthesia And Analgesia For Laboratory Animals. 3th edition. London, United Kingdom. Academic Press. 2023; Ch 10: Anesthesia and Analgesia for Laboratory Rodents, PP. 242-243
  15. Zheng Z, Chang G, Lin D, Mo Z, Wang Y. Advances on the experimental organ/tissue perfusion. Authorea. May 30, 2023; 1-14. DOI: 22541/au.168545163.31676140/v1.
  16. Bancroft JD, Layton C. Bancroft’s Theory and Practice of Histological Techniques. 8th edition. New York, United States. Elsevier, Churchill Livingstone. 2019; Ch 10: The hematoxylins and eosin, PP.126-138.
  17. Suvarna SK, Layton C, Bancroft JD. Theory and Practice of Histological Techniques. 8th edition. Philadelphia, United States. Elsevier. 2019; Ch 6: Tissue processing, PP: 73-84.
  18. Mohammed HO, Hassan NH, Aidaros AE, Ibrahim AA. Effect of monosodium glutamate on the cerebellar cortex microscopic structure in suckling rats, and possible protective role of vitamin C. 2020; 4 (1): 97-118. DOI: 10.21608/jmh.2020.24914.1074.
  19. Rao DS, Muraleedharan K, Humphreys C. TEM specimen preparation techniques. Microscopy: science, technology, applications and education 2. 2010; PP. 1232-1244.
  20. Goodhew PJ. Specimen preparation for transmission electron microscopy of materials. Garland Science. eBook, 2020; London, 1st edition:1-47. eBook ISBN: 9781003058748
  21. Hafez KA, Youssef AR, Hanna GFB, El-Sayed GT, Kama SA, Elfakharany WA. A study of the effect of prenatal exposure to valproic acid on the cerebellum of albino rat’s offspring and the possible protective role of folic acid. J. Anat. 2018; 41 (1): 91–104. doi:10.21608/ejana.2018.43506
  22. Jiang S, Xiao L, Sun Y, He M, Gao C, Zhu C, et al. The GABAB receptor agonist STX209 reverses the autism‑like behaviour in an animal model of autism induced by prenatal exposure to valproic acid. Molecular Medicine REPORTS. 2022; 25: 154:1-14. DOI: 10.3892/mmr.2022.12670.
  23. Mitsuhashia T, Hattorib S, Fujimuraa k, Shibata S, Miyakawab T, Takahashi T. In utero Exposure to Valproic Acid throughout Pregnancy Causes Phenotypes of Autism in Offspring Mice. Dev Neurosci. 2023; 45: 223–233. DOI: 10.1159/000530452. 
  24. Chaliha D, Albrecht M, Vaccarezza M, Takechi R, Lam V, Al-Salami H, et al. Systematic Review of the Valproic-Acid-Induced Rodent Model of Autism. Dev Neurosci. 2020; 42(1):12–48. https://doi.org/10.1159/000509109
  25. Atia AAA, Ashour RH, Zaki MMA, Rahman KMA, Ramadan NM. The comparative effectiveness of metformin and risperidone in a rat model of valproic acid‑induced autism, Potential role for enhanced autophagy. Psychopharmacology. 2023; 240: 1313-1332. https://doi.org/10.1007/s00213-023-06371-1
  26. Schiavi S, Iezzi D, Manduca A, Leone S, Melancia F, Carbone C, et al. Reward-Related Behavioral, Neurochemical and Electrophysiological Changes in a Rat Model of Autism Based on Prenatal Exposure to Valproic Acid. Front Cell Neurosci. 2019; 13:479: 1-14. https://doi.org/10.3389/FNCEL.2019. 00479/BIBTEX
  27. Elgamal MA, Khodeer DM, Abdel-Wahab BA, Ibrahim IAA, Alzahrani AR, Moustafa YM, et al. Canagliflozin alleviates valproic acid-induced autism in rat pups: Role of PTEN/PDK/PPAR-γ signaling pathways. Pharmacol.2023; 14:1113966: 1-17.  doi: 10.3389/fphar.2023.1113966.
  28. Alpay M, Yucel F. Changes of cerebellar cortex in a valproic acid-induced rat model of autism. Int J Dev Neurosci. 2022; 82: 606–614
  29. Al-Gholam MA, Ameen O. Role of Ginkgo Biloba Extract on Autistic-like Features Induced in Mice. JCDR. 2020; 14(8):1-6
  30. Abdel Mohsen AF, Ahmed NA, Altaib ZM, Zaher SM. Effect of Cisplatin on Cerebellar Cortex of Albino Rat and Possible Protective Role of Granulocyte Colony Stimulating Factor versus Citrullus Lanatus Juice: A Histological Study. EJH. 2020; 43(3): 702-717.
  31. Galal AT, Sayed SA, Mubarak WA, Farag WG. Effect of Valproic Acid on Pre and Postnatal Development of the Cerebellar Cortex of the Albino Rat and the Possible Protective Role of the Folic Acid. The Egyptian Journal of Hospital Medicine. 2022; 89: 4215- 4225
  32. Zedan, OI. Sodium Nitrite Induced Cerebellar Cortex Toxicity in Adult Male Albino Rat and the Possible Role of Metformin (Histological and Immunohistochemical Study). 2023; 46 (1), 355-367. DOI: 10.21608/ejh.2021.105203.1584.
  33. Viswasom AA, Jobby A. Age related changes in the Purkinje cells in human cerebellar cortex. Journal of Evolution of Medical and Dental Sciences. 2013; 2 (31): 5882-5890.
  34. Eid LTM, El-Habeby MM, Issa NM, El-Dien NMN. Evaluation of prenatal administration of valproic acid on the cerebellum of albino rat offspring: A model of autism. International Journal of Health Sciences. 2023;7 (S1): 2715–2738.
  35. Soliman MA, Ali AF. Comparative Study of the Possible Protective Effects of Omega-3 and Saffron Extract on the Cerebellum of Adult Male Albino Rats Exposed to Cell Phone Electromagnetic Radiations. Histological and Immunohistochemical Study. EJH. 2022; 45(1): 208-226. DOI: 10.21608/ejh.2021.59579.1422
  36. Ibrahim MAA, Sharaf Eldin HEM, Elswaidy NRM. Role of aqueous extract of saffron in ameliorating effect of sofosbuvir on the cerebellar cortex in rat. Anat Rec (Hoboken).2021; 304 (4):714-724
  37. Shona SI, Rizk AA, el Sadik AO, Emam HY, Ali EN. Effect of valproic acid administration during pregnancy on postnatal development of cerebellar cortex and the possible protective role of folic acid. Folia Morphol Pol. 2018; 77: 201-209. doi:10.5603/FM. a2017.0100
  38. Abdel-Aziz HM, Mekawy NH, Ibrahem NE. Histological and immunohistochemical study on the effect of zinc oxide nanoparticles on cerebellar cortex of adult male albino rats. EJH. 2019; 42 (1), 23-34. DOI: 10.21608/ejh.2018.5113.1024.
  39. Al-Amoudi WM. "Protective effects of fennel oil extract against sodium valproate-induced hepatorenal damage in albino rats." Saudi journal of biological sciences.2017; 24 (4):915-924. https://doi.org/10.1016/j.sjbs.2016.10.021
  40. Ali EMT, Abdallah HI, El-Sayed SM. Tramadol Induces Histological Aberrations, Downregulation of Bcl-2 and Upregulations of GFAP, P53 Immunoexpression in the Cerebellum of the Male Albino Rat, Egypt. J. Biolog. Sci. (D-Histology and histochemistry). 2023; 15(1): 139-159. DOI: 10.21608/EAJBSD.2023.296947
  41. Khair NSB, Abdel Aziz SAM. A Comparative Study on the Protective Role of Silymarin and Coenzyme-Q10 on the Cerebellar Cortex of Experimentally Induced Atherosclerosis in Adult Male Albino Rats: A Histological, Immunohistochemical and Biochemical Study. 2021; 44 (2) /322-338. DOI: 10.21608/ejh.2020.28009.1276.
  42. Magar M, Ebada M, El-Gezawy M. Study of the Effect of Prenatal Adminstration of Pregabalin on Cerebellar cortex of Albino Rat’s Offspring and the Possible Protective Role of Folic Acid. Anatomy & Embryology. 2020; 1(5):133-139
  43. Dossi E, Vasile F, Rouach N. Human astrocytes in the diseased brain. Brain Research Bulletin. 2018; 136:139-156
  44. Arafat EA, Shabaan DA. The possible neuroprotective role of grape seed extract on the histopathological changes of the cerebellar cortex of rats prenatally exposed to Valproic Acid: Animal model of autism. Acta Histochem. 2019; 121: 841-851
  45. Zaghloul DAM, Ouies SM, Gad EL-Rab WM, Mohamed AW. Toxic Effects of Methotrexate on the Cerebellar Cortex of Adult Albino Rats and the Possible Protective Role of Vitamin C: An Electron Microscopic Study. J. Cairo Univ. 2021; 89, No. 5:2043-2047.
  46. Sarhan NR, Taalab YM. Oxidative stress/PERK/apoptotic pathways interaction contribute to tramadol neurotoxicity in rat cerebral and cerebellar cortex and thyme enhances the antioxidant defense system: histological, immunohistochemical and ultrastructural study. Int J Sci Rep. 2018; 4(6):124-141.
  47. Finsterer J. Toxicity of Antiepileptic Drugs to Mitochondria. In: Singh, H., Sheu, SS. (eds) Pharmacology of Mitochondria. Handbook of Experimental Pharmacology, 240. Springer, Cham. 1st edition, Krankenanstalt Rudolfstiftung, Postfach 20, 1180 Vienna, Austria, PP: 473–488. PMID: 27590224; PMCID: PMC7439278. https://doi.org/10.1007/164_2016_2.
  48. Taleb A, Lin W, Xu X, Zhang G, Zhou, Q, Naveed M et al. Emerging mechanisms of valproic acid-induced neurotoxic events in autism and its implications for pharmacological treatment. Biomedicine & Pharmacotherapy, 137 (2021):1-8, 111322.
  49. Teleanu DM, Niculescu AG, Lungu II, Radu CI, Vladâcenco O, Roza E et al. An Overview of Oxidative Stress, Neuroinflammation, and Neurodegenerative Diseases.  J. Mol. Sci. 2022. 23, 5938: 1-22. https://doi.org/10.3390/ijms23115938
  50. Savran M, Asci H, Armagan I, Erzurumlu Y, Azırak S, Ozer MK et al. Thymoquinone could be protective against valproic acid-induced testicular toxicity by antioxidant and anti-inflammatory mechanisms. Andrologia, 2020. 52: 1-8. e13623. https://doi.org/10.1111/and.13623
  51. Kassab AA, Sharaf Eldin HEM, Abd-El-Hafez AAA. Marjoram Oil Attenuates Valproic Acid-Induced Pancreatic Damage in Adult Male Albino Rats: A Histological and Immunohistochemical Study. EJH: 45 (3):653-666. DOI: 10.21608/ejh.2021.74992.1476
  52. Laag EM, Abd Elaziz HO. Effect of aflatoxin-B1 on rat cerebellar cortex: light and electron microscopic study. The Egyptian Journal of Histology, 36 (3): 601-610. DOI: 10.1097/01.EHX.0000432619.75801.15
  53. Hamouda MHMA, Abdel Aal FS, ElMashad FHY. Effect of Sodium valproate on the structure of the renal cortex of adult male albino rat and the role of cinnamon. Al-Azhar Med. J. 2019; 48 (1):1-28. DOI: 10.21608/amj.2019.50449.
  54. El-Ansary A, Al-Salem HS, Asma A, Al-Dbass A.  Glutamate excitotoxicity induced by orally administered propionic acid, a short chain fatty acid can be ameliorated by bee pollen. Lipids Health Dis. 2017; 16:96. 1-9 https://doi.org/10.1186/s12944-017-0485-7
  55. Klaric I, Pavic M, Miskulin I, Blazicevic V, Dumic A, Miskulin M. Influence of Dietary Supplementation of Propolis and Bee Pollen on Liver Pathology in Broiler Chickens. Animals. 2018;8, 1-10. https://doi.org/10.3390/ani8040054
  56. Umesh B, Neelima K, Deepti C. Modulatory Prospective of Bee Pollen against Oxidative Stress Induced by Drugs: A Review. Research Journal of Pharmacy and Technology. 2018; 11 (12):5648- 5652. ISSN : 0974-3618.
  57. Saral O, Şahin H, Saral S, Alkanat M, Akyıldız K, Topçu A, et al. Bee pollen increases hippocampal brain-derived neurotrophic factor and suppresses neuroinflammation in adult rats with chronic immobilization stress. Neuroscience Letters. 2022; 766, 136342, 1-7. ISSN 0304-3940, https://doi.org/10.1016/j.neulet.2021.136342.
  58. Chelucci E, Chiellini C, Cavallero A, Gabriele M. Bio-Functional Activities of Tuscan Bee Pollen. 2023; 12, 115. 1-17.
  59. Li Q, Sun M, Wan Z, Liang J, Betti M, Hrynets Y, et al. Bee pollen extracts modulate serum metabolism in lipopolysaccharide-induced acute lung injury mice with anti-inflammatory effects. J Agric Food Chem. 2019; 67(28):7855–7868
  60. El-Sayed AA. Anti-inflammatory and protective effects of royal jelly against hepatic and renal damage induced by valproic acid in rats. Mol. Res. 2023; 22 (1): 1-17, gmr19063.
  61. Li J. Bee Pollen and Doxorubicin by Synergistic Effects Inhibit the Proliferation of Breast Tumors in 4T1 Tumor-bearing BALB/c Mice: A Biochemical, Immunohistochemical, and Molecular Approach. Pharmacognosy Magazine. 2024; 20 (1):159-178
  62. Zhang H, Zhu X, Huang Q, Zhang L, Liu X, Liu R, et al. Antioxidant and anti-inflammatory activities of rape bee pollen after fermentation and their correlation with chemical components by ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry-based untargeted metabolomics. Food Chem. 2023; (409), 135342.
  63. Bacha AB, Norah AO, Al-Osaimi M, Harrath AH, Mansour L, El-Ansary A. The therapeutic and protective effects of bee pollen against prenatal methylmercury induced neurotoxicity in rat pups. Metab Brain Dis. 2020; 35(1):215-224. doi: 10.1007/s11011-019-00496-z.
  64. Ali AM, Kunugi H. Apitherapy for Age-Related Skeletal Muscle Dysfunction (Sarcopenia): A Review on the Effects of Royal Jelly, Propolis, and Bee Pollen. Foods. 2020;9, 1362. 1-38. https://doi.org/10.3390/foods9101362
  65. Omnia MA, Nabila MA, Nadia RR. Biochemical effects of propolis and bee pollen in experimentally- induced hyperammonia in rats. Benha Veterinary Medical Journal. 2014; 27 (1): 8‐24
  66. El-Seedi HR, Eid N, Abd El-Wahed AA, Rateb ME, Afifi HS, Algethami AF, et al. Honey Bee Products: Preclinical and Clinical Studies of Their Anti-inflammatory and Immunomodulatory Properties. Front. Nutr. 2022;8, 1109: 1-19.
  67. Kim HY, Lee YJ, Kim SJ, Lee JD, Kim S, Ko MJ, et al. Metabolomics profiling of valproic acid-induced symptoms resembling autism spectrum disorders using 1H NMR spectral analysis in rat model. Journal of Toxicology and Environmental Health, Part A. 2022; 85 (1):1 13, DOI: 1080/15287394.2021.1967821