Anat Cell Biol.  2025 Mar;58(1):76-85. 10.5115/acb.24.088.

Histological features of the Purkinje neurons of the Albino rat (Rattus norvegicus) following letrozole administration

• Hannan CT
• Jeremiah MK
• Idenya PM

• KMID: 2565958
• DOI: http://doi.org/10.5115/acb.24.088

Abstract

Aromatase inhibitors are increasingly being used as adjuvant therapy for hormone-responsive cancers. These drugs may reduce the endogenous estrogen production in the cerebellum. Prolonged use has been associated with symptoms such as ataxia, poorer balance performance and diminished verbal memory, suggesting impaired cerebellar function. Thus, this study sought to outline the structural basis for the cerebellar deficits observed. Twenty-seven male rats (3 baseline, 15 experimental, 9 control) aged three months were recruited with the intervention group receiving 0.5 mg/kg of letrozole daily for 50 days by oral gavage while the control group received normal saline. Their cerebella were harvested for histological processing on days 20, 35, and 50. Photomicrographs were taken and analysed using Fiji ImageJ software. The dendritic spine densities and Purkinje linear densities were coded and analyzed using IBM SPSS Statistics version 25.0. A P-value of ≤0.05 was considered significant. A temporal decline in the Purkinje linear density as well as pyknosis and cytoplasmic eosinophilia was noted in the intervention group (P=0.165). Further, the dendritic spine density of the Purkinje neurons in the intervention group was markedly reduced (P=0.01). The reduction in the linear cell density and the dendritic spine density of the Purkinje cells following letrozole administration may provide an anatomical basis for the functional cerebellar deficits seen in chronic aromatase inhibitor use.

Keyword

Purkinje cells; Letrozole; Dendrites; Dendritic spines

Figure

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  Fig. 1 Determination of the Purkinje linear density. The number of Purkinje cell bodies with visibly stained nuclei in each photomicrograph were counted. The Purkinje cell layer length was determined using Fiji ImageJ software by using the Segmented Line function to draw a line (yellow line) connecting the centers of the Purkinje cell bodies.

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  Fig. 2 Determination of the dendritic spine density of the Purkinje neurons. (A) The dendritic spines whose heads and stems were in focus were counted. The dendritic segment length was measured by drawing a freehand line along the dendritic segment. (B) The dendritic spine densities was also confirmed using the corresponding image skeleton derived using the Skeletonize function of ImageJ.

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  Fig. 3 Line graph showing the general trend of the mean Purkinje linear density over time in the control and experimental groups. PLD, Purkinje linear density.

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  Fig. 4 Photomicrographs showing Purkinje cellular changes following letrozole administration. Light microscopic features of the cerebellar cortex in the control and experimental groups on days 20, 35, and 50. (A) The cerebellar cortex of the control group on day 20. Note the abundance of Purkinje cells with uniformly staining and rounded nuclei (H&E, ×400). (B) The cerebellar cortex of the experimental group on day 20. Note the pyknotic Purkinje cells with smaller and darkly staining nuclei (black arrows). The number of the Purkinje cells (white arrows) is lesser than what was seen in the control group (H&E, ×400). (C) Cerebellar cortex showing the Purkinje cells in the control group on day 35. The Purkinje cells are numerous and are arranged in a single row. The nuclei are uniformly basophilic, rounded and have visible nucleoli (H&E, ×400). (D) The cerebellar cortex of the experimental group on day 35. Note the reduction in number of the Purkinje cells in comparison to the control group (H&E, ×400). (E) Cerebellar cortex showing the Purkinje cells in the control group on day 50. Note the abundance of the Purkinje cells (H&E, ×400). (F) The cerebellar cortex of the experimental group on day 50. Note the reduced density of the Purkinje cells (white arrowheads) as compared to the control group. Some of the Purkinje neurons are pyknotic and display basophilic nuclei and intense cytoplasmic eosinophilia (black arrowheads) (H&E, ×400). GL, granular layer; ML, molecular layer.

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  Fig. 5 Line graph showing the general trend of the mean Purkinje dendritic spine density over time in the control and experimental groups. DSD, dendritic spine densities. *Significant difference (P-value<0.05).

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  Fig. 6 Photomicrographs showing changes in the dendritic spine density of the Purkinje cells over the study period. (A) Dendrites of the Purkinje cells in the control group on day 20 displaying the terminal dendrites–possessing dendritic spines–branching off from the smoother proximal dendrite. The terminal dendrites have a relatively high dendritic spine density (yellow arrows) (modified Patro’s Golgi, ×1,000). (B) Dendritic structure of the Purkinje cells in the experimental group on day 20 displaying terminal dendrites with decreased dendritic spine density as compared to the control group (yellow arrow) (modified Patro’s Golgi, ×1,000). (C) Dendrites of the Purkinje cells in the control group at day 35 displaying terminal dendrites with abundant dendritic spines (yellow arrows) (modified Patro’s Golgi, ×1,000). (D) Purkinje cells’ dendritic organization in the experimental group at day 35 showing a proximal dendrite giving off terminal dendrites with a lower dendritic spine density as compared to the controls (yellow arrows) (modified Patro’s Golgi, ×1,000). (E) Dendrites of the Purkinje cells in the control group at day 50 displaying a proximal dendrite giving off several terminal dendrites. Note the high density of dendritic spines (yellow arrow) (modified Patro’s Golgi, ×1,000). (F) Dendritic details of the Purkinje cells in the experimental group at day 50. Note the generally thinner dendrites and the lower dendritic spine density (yellow arrows) (modified Patro’s Golgi, ×1,000). DS, dendritic spines; PD, proximal dendrite; TD, terminal dendrites.

Reference

References

1. Pawlina W, Ross MH. Histology: a text and atlas: with correlated cell and molecular biology. 8th ed. Wolters Kluwer;2018.

2. Glickstein M, Strata P, Voogd J. 2009; Cerebellum: history. Neuroscience. 162:549–59. DOI: 10.1016/j.neuroscience.2009.02.054. PMID: 19272426.

3. Marvel CL, Desmond JE. Mariën P, Manto M, editors. The cerebellum and verbal working memory. The linguistic cerebellum. Academic Press;2016. p. 51–62. DOI: 10.1016/B978-0-12-801608-4.00003-7.

4. Roostaei T, Nazeri A, Sahraian MA, Minagar A. 2014; The human cerebellum: a review of physiologic neuroanatomy. Neurol Clin. 32:859–69. DOI: 10.1016/j.ncl.2014.07.013. PMID: 25439284.

5. Klein AP, Ulmer JL, Quinet SA, Mathews V, Mark LP. 2016; Nonmotor functions of the cerebellum: an introduction. AJNR Am J Neuroradiol. 37:1005–9. DOI: 10.3174/ajnr.A4720. PMID: 26939633. PMCID: PMC7963530.

6. Strick PL, Dum RP, Fiez JA. 2009; Cerebellum and nonmotor function. Annu Rev Neurosci. 32:413–34. DOI: 10.1146/annurev.neuro.31.060407.125606. PMID: 19555291.

7. Buckner RL. 2013; The cerebellum and cognitive function: 25 years of insight from anatomy and neuroimaging. Neuron. 80:807–15. DOI: 10.1016/j.neuron.2013.10.044. PMID: 24183029.

8. Biegon A, Kim SW, Alexoff DL, Jayne M, Carter P, Hubbard B, King P, Logan J, Muench L, Pareto D, Schlyer D, Shea C, Telang F, Wang GJ, Xu Y, Fowler JS. 2010; Unique distribution of aromatase in the human brain: in vivo studies with PET and [N-methyl-11C]vorozole. Synapse. 64:801–7. DOI: 10.1002/syn.20791. PMID: 20842717. PMCID: PMC2941230.

9. Handa RJ, Ogawa S, Wang JM, Herbison AE. 2012; Roles for oestrogen receptor β in adult brain function. J Neuroendocrinol. 24:160–73. DOI: 10.1111/j.1365-2826.2011.02206.x. PMID: 21851428. PMCID: PMC3348521.

10. Hedges VL, Ebner TJ, Meisel RL, Mermelstein PG. 2012; The cerebellum as a target for estrogen action. Front Neuroendocrinol. 33:403–11. DOI: 10.1016/j.yfrne.2012.08.005. PMID: 22975197. PMCID: PMC3496070.

11. Sakamoto H, Mezaki Y, Shikimi H, Ukena K, Tsutsui K. 2003; Dendritic growth and spine formation in response to estrogen in the developing Purkinje cell. Endocrinology. 144:4466–77. DOI: 10.1210/en.2003-0307. PMID: 12960093.

12. Sasahara K, Shikimi H, Haraguchi S, Sakamoto H, Honda S, Harada N, Tsutsui K. 2007; Mode of action and functional significance of estrogen-inducing dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell. J Neurosci. 27:7408–17. DOI: 10.1523/JNEUROSCI.0710-07.2007. PMID: 17626201. PMCID: PMC6672615.

13. Haraguchi S, Sasahara K, Shikimi H, Honda S, Harada N, Tsutsui K. 2012; Estradiol promotes Purkinje dendritic growth, spinogenesis, and synaptogenesis during neonatal life by inducing the expression of BDNF. Cerebellum. 11:416–7. DOI: 10.1007/s12311-011-0342-6. PMID: 22198873.

14. Liu SB, Zhang N, Guo YY, Zhao R, Shi TY, Feng SF, Wang SQ, Yang Q, Li XQ, Wu YM, Ma L, Hou Y, Xiong LZ, Zhang W, Zhao MG. 2012; G-protein-coupled receptor 30 mediates rapid neuroprotective effects of estrogen via depression of NR2B-containing NMDA receptors. J Neurosci. 32:4887–900. DOI: 10.1523/JNEUROSCI.5828-11.2012. PMID: 22492045. PMCID: PMC6620914.

15. Sierra A, Azcoitia Í, Garcia-Segura L. 2003; Endogenous estrogen formation is neuroprotective in model of cerebellar ataxia. Endocrine. 21:43–51. DOI: 10.1385/ENDO:21:1:43. PMID: 12777702.

16. Zameer S, Vohora D. 2017; Effect of aromatase inhibitors on learning and memory and modulation of hippocampal dickkopf-1 and sclerostin in female mice. Pharmacol Rep. 69:1300–7. DOI: 10.1016/j.pharep.2017.06.002. PMID: 29128813.

17. Jung ME, Yang SH, Brun-Zinkernagel AM, Simpkins JW. 2002; Estradiol protects against cerebellar damage and motor deficit in ethanol-withdrawn rats. Alcohol. 26:83–93. DOI: 10.1016/S0741-8329(01)00199-9. PMID: 12007583.

18. Ghidoni R, Boccardi M, Benussi L, Testa C, Villa A, Pievani M, Gigola L, Sabattoli F, Barbiero L, Frisoni GB, Binetti G. 2006; Effects of estrogens on cognition and brain morphology: involvement of the cerebellum. Maturitas. 54:222–8. DOI: 10.1016/j.maturitas.2005.11.002. PMID: 16343828.

19. Burstein HJ, Prestrud AA, Seidenfeld J, Anderson H, Buchholz TA, Davidson NE, Gelmon KE, Giordano SH, Hudis CA, Malin J, Mamounas EP, Rowden D, Solky AJ, Sowers MR, Stearns V, Winer EP, Somerfield MR, Griggs JJ. American Society of Clinical Oncology. 2010; American Society of Clinical Oncology clinical practice guideline: update on adjuvant endocrine therapy for women with hormone receptor-positive breast cancer. J Clin Oncol. 28:3784–96. DOI: 10.1200/JCO.2009.26.3756. PMID: 20625130. PMCID: PMC5569672.

20. Kelly CM, Buzdar AU. 2010; Anastrozole. Expert Opin Drug Saf. 9:995–1003. DOI: 10.1517/14740338.2010.515977. PMID: 20923259.

21. Dellapasqua S, Colleoni M. 2010; Letrozole. Expert Opin Drug Metab Toxicol. 6:251–9. DOI: 10.1517/17425250903540246. PMID: 20095792.

22. Fabian CJ. 2007; The what, why and how of aromatase inhibitors: hormonal agents for treatment and prevention of breast cancer. Int J Clin Pract. 61:2051–63. DOI: 10.1111/j.1742-1241.2007.01587.x. PMID: 17892469. PMCID: PMC2228389.

23. Dave N, Gudelsky GA, Desai PB. 2013; The pharmacokinetics of letrozole in brain and brain tumor in rats with orthotopically implanted C6 glioma, assessed using intracerebral microdialysis. Cancer Chemother Pharmacol. 72:349–57. DOI: 10.1007/s00280-013-2205-y. PMID: 23748921.

24. Santen RJ, Boucher AE, Santner SJ, Henderson IC, Harvey H, Lipton A. 1987; Inhibition of aromatase as treatment of breast carcinoma in postmenopausal women. J Lab Clin Med. 109:278–89.

25. Winters-Stone KM, Torgrimson B, Horak F, Eisner A, Nail L, Leo MC, Chui S, Luoh SW. 2011; Identifying factors associated with falls in postmenopausal breast cancer survivors: a multi-disciplinary approach. Arch Phys Med Rehabil. 92:646–52. DOI: 10.1016/j.apmr.2010.10.039. PMID: 21367394. PMCID: PMC3430739.

26. Blaustein JD. 2017; Treatments for breast cancer that affect cognitive function in postmenopausal women. Policy Insights Behav Brain Sci. 4:170–7. DOI: 10.1177/2372732217717271.

27. Gervais NJ, Remage-Healey L, Starrett JR, Pollak DJ, Mong JA, Lacreuse A. 2019; Adverse effects of aromatase inhibition on the brain and behavior in a nonhuman primate. J Neurosci. 39:918–28. DOI: 10.1523/JNEUROSCI.0353-18.2018. PMID: 30587540. PMCID: PMC6382974.

28. Bender CM, Sereika SM, Berga SL, Vogel VG, Brufsky AM, Paraska KK, Ryan CM. 2006; Cognitive impairment associated with adjuvant therapy in breast cancer. Psychooncology. 15:422–30. DOI: 10.1002/pon.964. PMID: 16097037.

29. Collins B, Mackenzie J, Stewart A, Bielajew C, Verma S. 2009; Cognitive effects of hormonal therapy in early stage breast cancer patients: a prospective study. Psychooncology. 18:811–21. DOI: 10.1002/pon.1453. PMID: 19085975.

30. Aitman T, Dhillon P, Geurts AM. 2016; A RATional choice for translational research? Dis Model Mech. 9:1069–72. DOI: 10.1242/dmm.027706. PMID: 27736742. PMCID: PMC5087836.

31. Ellenbroek B, Youn J. 2016; Rodent models in neuroscience research: is it a rat race? Dis Model Mech. 9:1079–87. DOI: 10.1242/dmm.026120. PMID: 27736744. PMCID: PMC5087838.

32. Baizer JS. 2014; Unique features of the human brainstem and cerebellum. Front Hum Neurosci. 8:202. DOI: 10.3389/fnhum.2014.00202. PMID: 24778611. PMCID: PMC3985031.

33. Nair AB, Jacob S. 2016; A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm. 7:27–31. DOI: 10.4103/0976-0105.177703. PMID: 27057123. PMCID: PMC4804402.

34. Sengupta P. 2013; The laboratory rat: relating its age with human's. Int J Prev Med. 4:624–30.

35. Czechowska N, van Rienen A, Lang F, Eiberger B, Baader SL. 2019; An update on the Golgi staining technique improving cerebellar cell type specificity. Histochem Cell Biol. 151:327–41. DOI: 10.1007/s00418-018-01766-0. PMID: 30607497.

36. Louis ED, Babij R, Lee M, Cortés E, Vonsattel JP. 2013; Quantification of cerebellar hemispheric Purkinje cell linear density: 32 ET cases versus 16 controls. Mov Disord. 28:1854–9. DOI: 10.1002/mds.25629. PMID: 23925732. PMCID: PMC3830681.

37. Jacobs B, Driscoll L, Schall M. 1997; Life-span dendritic and spine changes in areas 10 and 18 of human cortex: a quantitative Golgi study. J Comp Neurol. 386:661–80. DOI: 10.1002/(SICI)1096-9861(19971006)386:4<661::AID-CNE11>3.0.CO;2-N.

38. Louis ED, Lee M, Babij R, Ma K, Cortés E, Vonsattel JP, Faust PL. 2014; Reduced Purkinje cell dendritic arborization and loss of dendritic spines in essential tremor. Brain. 137:3142–8. DOI: 10.1093/brain/awu314. PMID: 25367027. PMCID: PMC4240305.

39. Hill RA, Chua HK, Jones ME, Simpson ER, Boon WC. 2009; Estrogen deficiency results in apoptosis in the frontal cortex of adult female aromatase knockout mice. Mol Cell Neurosci. 41:1–7. DOI: 10.1016/j.mcn.2008.12.009. PMID: 19185610.

40. Suzuki S, Brown CM, Wise PM. 2009; Neuroprotective effects of estrogens following ischemic stroke. Front Neuroendocrinol. 30:201–11. DOI: 10.1016/j.yfrne.2009.04.007. PMID: 19401209. PMCID: PMC3672220.

41. Wójtowicz T, Lebida K, Mozrzymas JW. 2008; 17β-estradiol affects GABAergic transmission in developing hippocampus. Brain Res. 1241:7–17. DOI: 10.1016/j.brainres.2008.09.005. PMID: 18822277.

42. Amantea D, Russo R, Bagetta G, Corasaniti MT. 2005; From clinical evidence to molecular mechanisms underlying neuroprotection afforded by estrogens. Pharmacol Res. 52:119–32. DOI: 10.1016/j.phrs.2005.03.002. PMID: 15967377.

43. Richardson TE, Yang SH, Wen Y, Simpkins JW. 2011; Estrogen protection in Friedreich's ataxia skin fibroblasts. Endocrinology. 152:2742–9. DOI: 10.1210/en.2011-0184. PMID: 21540287. PMCID: PMC3115615.

44. Koeppen AH. Manto M, Huisman TAGM, editors. The neuropathology of the adult cerebellum. Handbook of Clinical Neurology. Elsevier;2018. p. 129–49. DOI: 10.1016/B978-0-444-63956-1.00008-4.

45. Yang Q, Hashizume Y, Yoshida M, Wang Y, Goto Y, Mitsuma N, Ishikawa K, Mizusawa H. 2000; Morphological Purkinje cell changes in spinocerebellar ataxia type 6. Acta Neuropathol. 100:371–6. DOI: 10.1007/s004010000201. PMID: 10985694.

46. Frankfurt M, Gould E, Woolley CS, McEwen BS. 1990; Gonadal steroids modify dendritic spine density in ventromedial hypothalamic neurons: a Golgi study in the adult rat. Neuroendocrinology. 51:530–5. DOI: 10.1159/000125387. PMID: 2112730.

47. Brocca ME, Pietranera L, Beauquis J, De Nicola AF. 2013; Estradiol increases dendritic length and spine density in CA1 neurons of the hippocampus of spontaneously hypertensive rats: a Golgi impregnation study. Exp Neurol. 247:158–64. DOI: 10.1016/j.expneurol.2013.04.007. PMID: 23628746.

48. Saenz C, Dominguez R, de Lacalle S. 2006; Estrogen contributes to structural recovery after a lesion. Neurosci Lett. 392:198–201. DOI: 10.1016/j.neulet.2005.09.023. PMID: 16203092. PMCID: PMC3182119.

49. Tsutsui K, Ukena K, Sakamoto H, Okuyama S, Haraguchi S. 2011; Biosynthesis, mode of action, and functional significance of neurosteroids in the Purkinje cell. Front Endocrinol (Lausanne). 2:61. DOI: 10.3389/fendo.2011.00061. PMID: 22654818. PMCID: PMC3356128.

50. Shimada A, Mason CA, Morrison ME. 1998; TrkB signaling modulates spine density and morphology independent of dendrite structure in cultured neonatal Purkinje cells. J Neurosci. 18:8559–70. DOI: 10.1523/JNEUROSCI.18-21-08559.1998. PMID: 9786964. PMCID: PMC6793520.

51. Luine V, Frankfurt M. 2013; Interactions between estradiol, BDNF and dendritic spines in promoting memory. Neuroscience. 239:34–45. DOI: 10.1016/j.neuroscience.2012.10.019. PMID: 23079626. PMCID: PMC3597766.

52. Kellner Y, Gödecke N, Dierkes T, Thieme N, Zagrebelsky M, Korte M. 2014; The BDNF effects on dendritic spines of mature hippocampal neurons depend on neuronal activity. Front Synaptic Neurosci. 6:5. DOI: 10.3389/fnsyn.2014.00005. PMID: 24688467. PMCID: PMC3960490.

53. Bär J, Kobler O, van Bommel B, Mikhaylova M. 2016; Periodic F-actin structures shape the neck of dendritic spines. Sci Rep. 6:37136. DOI: 10.1038/srep37136. PMID: 27841352. PMCID: PMC5107894.

54. Vierk R, Glassmeier G, Zhou L, Brandt N, Fester L, Dudzinski D, Wilkars W, Bender RA, Lewerenz M, Gloger S, Graser L, Schwarz J, Rune GM. 2012; Aromatase inhibition abolishes LTP generation in female but not in male mice. J Neurosci. 32:8116–26. DOI: 10.1523/JNEUROSCI.5319-11.2012. PMID: 22699893. PMCID: PMC6703647.

55. Oda SI, Lee KJ, Arii T, Imoto K, Hyun BH, Park IS, Kim H, Rhyu IJ. 2010; Differential regulation of Purkinje cell dendritic spines in rolling mouse Nagoya (tgrol/tgrol), P/Q type calcium channel (α1A/Cav2.1) mutant. Anat Cell Biol. 43:211–7. DOI: 10.5115/acb.2010.43.3.211. PMID: 21212861. PMCID: PMC3015039.

56. Dickstein DL, Weaver CM, Luebke JI, Hof PR. 2013; Dendritic spine changes associated with normal aging. Neuroscience. 251:21–32. DOI: 10.1016/j.neuroscience.2012.09.077. PMID: 23069756. PMCID: PMC3654095.