Cellular imbalance of the hippocampus in drug-resistant epilepsy (on the question of the role of the hippocampus in epileptogenesis)

The study of the etiopathogenesis of epilepsy is one of the most important tasks of modern neurology. Hippocampal sclerosis (HS) is a common morphological substrate in drug resistant epilepsy (DRE).

PURPOSE. To evaluate changes in the cellular composition in the hippocampus in patients operated on for drug-resistant epilepsy, to compare with the comparison group.

MATERIALS AND METHODS. The biopsy material of fragments of the temporal lobe and hippocampus of Polenov Russian Scientific Research Institute of Neurosurgery — branch of Almazov National Medical Research Centre, obtained intraoperatively from 26 patients with locally caused DRE aged 22 to 56 years. Histological sections stained with hematoxylin and eosin were, as well as the results of immunohistochemical (IHC) reactions. The IHC method was used to determine glial fibrillar acidic protein (GFAP), NeuN (antibodies from Dako, Denmark). Histological analysis and morphometry were performed using a Carl Zeiss Axio Lab microscope (Germany) and ImageJ software in 5 fields of view at x400 magnification. Statistical analysis was carried out using the software packages Statistica v.10 and Matlab 2013.

RESULTS. Histological examination of fragments of the hippocampus in the zone of epileptic activity revealed focal cortical dysplasia of various types (in 88,5 % of patients). The surgical material of the hippocampus was fragmented, which made it difficult to interpret according to the ILAE classification. Histological examination of the structures of the hippocampal formation in all the studied samples showed a sharp neuronal devastation of the hippocampal nuclei, dispersion and bifurcation (in 2 cases) of the granular layer of the dentate gyrus, the phenomena of satellitosis and neuronophagy, glial reactions with areas of cell accumulation. In 5 cases, the material was presented as a single block, which made it possible to reliably identify hippocampal sclerosis of various types according to ILAE (2013). Immunohistochemical study revealed a pronounced expression of GFAP in the nuclei of the hippocampus and dentate gyrus, confirming the development of astrocytic gliosis. Statistical data processing revealed significant differences between the median numbers of neurons in the dentate gyrus in patients with drug-resistant epilepsy and in the comparison group. We studied the possibility of distinguishing two groups (study and control) by the totality of the values of neuronal density and glia density. According to the results obtained, it is impossible to distinguish more than 2 clusters: the study group and the control group. The distributions of the values of the studied indicators of female patients and male patients also do not differ significantly.

We also checked the difference in the average values of vectors in the groups of neuron and glia density using the T2 Hotelling criterion, as a result of which it was found that the averages in the groups (study and control) differ significantly and very significantly. Based on the full probability and Bayesian formulas for solving the functional optimization problem, the probability of correct identification of the patient (that is, his correct assignment either to the group of locally determined mediobasal DRE or to the control group) was estimated according to the same criteria, as a result of which the probability value (taking into account random error) amounted to (91±8)%.

CONCLUSION. Structural changes in the resected hippocampi in patients with DRE are characterized by elective neuronal death with a predominant lesion of the dentate gyrus. A comparative statistical analysis of cell density calculation data using Bayes formulas revealed a high probability of classifying the studied hippocampal fragment as a group of patients with epilepsy, regardless of the representativeness of the material and the severity of pathomorphological changes. Thus, the cellular imbalance in the hippocampus has typical homogeneity, which makes it possible to identify characteristic changes in its structures during DRE.

1. Karlov VA. Epilepsy in children and adult men and women. M.: Meditsina; 2010. (In Russ.)

2. Sommer, W., 1880. Die Erkrankung des Ammonshorns als aetiologisches Moment der Epilepsie. Arch. Psychiat. Nervenkr. 308, 631–675.

3. Blümcke I, Thom M, Aronica E et al. The clinic-pathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission. Epilepsia. 2011;52(1):158–74. doi: 10.1111/j.1528–1167.2010.02777.x.

4. Wieser H.-G. ILAE Commission Report. Mesial temporal lobe epilepsy with hippocampal sclerosis / H.-G. Wieser, ILAE Commission on Neurosurgery of Epilepsy // Epilepsia. 2004;45(6):695–714. https://doi.org/10.1007/springerreference_188282

5. Blümcke I, Thom M, Wiestler OD. Ammon’s horn sclerosis: a maldevelopmental disorder associated with temporal lobe epilepsy. Brain Pathol. 2002 Apr;12(2):199–211. doi: 10.1111/j.1750–3639.2002.tb00436.x. PMID: 11958375; PMCID: PMC8095862

6. Pathomorphological changes of hippocampus in temporal lobe epilepsy. Kuralbayev A.K., Zabrodskaya Yu.M., Ivanova N. E., Kasumov V. R., Bersnev V. P., Telegina A. A., Sitovskaya D. A., Gokhman E. A. Russian Neurosurgical Journal named after professor A. L. Polenov. 2017; 9 (2): 72–77 (In Russ.)

7. Zenkov LR. Klinicheskaya epileptologiya. Moscow: MedInformAgentstvo; 2010. (In Russ.).

8. Sitovskaya D. A., Zabrodskaya Yu.M., Sokolova T. V., Kuralbaev A.K., Nezdorovina V. G., Dobrogorskaya L.N. Structural heterogeneity of epileptic foci in local drug-resistant epilepsy. Archive of Pathology = Arkhiv patologii. 2020;82(6):5–15. (In Russ.). https://doi.org/10.17116/patol2020820615

9. Wieser H.-G. ILAE Commission Report. Mesial temporal lobe epilepsy with hippocampal sclerosis / H.-G. Wieser, ILAE Commission on Neurosurgery of Epilepsy // Epilepsia. 2004;45(6):695–714. https://doi.org/10.1007/springerreference_188282

10. Sarkisov D.S. Regeneraciya i ee klinicheskoe znachenie. M.: Medicina; 1970. (In Russ.)

11. Al Sufiani F, Ang LC. Neuropathology of Temporal Lobe Epilepsy. Epilepsy Research and Treatment. 2012;2012:1–13. https://doi.org/10.1155/2012/624519

12. Eadie MJ. Epilepsy, Ammon’s horn sclerosis, and Camille Bouchet. Journal of the History of the Neurosciences. 2017;26:231–237. https://doi.org/10.1080/0964704x.2016.1224141

13. Wiebe S. Epidemiology of temporal lobe epilepsy. Can J Neurol Sci. 2000;27(1):10–21.

14. Spreafico R, Tassi L. Cortical malformations. Handbook of Clinical Neurology. 2012;108:535–57. https://doi.org/10.1016/b9780444528995.00047–2

15. Houser CR. Granule cell dispersion in the dentate gyrus of humans with temporal lobe epilepsy. Brain Res 1990;535:195–204.

16. Tassi L, Meroni A, Deleo F, Villani F, Mai R, Russo GL, Colombo N, Avanzini G, Falcone C, Bramerio M, Citterio A, Garbelli R, Spreafico R. Temporal lobe epilepsy: neuropathological and clinical correlations in 243 surgically treated patients. Epileptic Disord. 2009;11(4):281–292. https://doi.org/10.1684/epd.2009.0279

17. Blümcke I, Thom M, Aronica E, Armstrong DD, Bartolomei F, Bernasconi A, et al. International consensus classification of hippocampal sclerosis in temporal lobe epilepsy: a Task force report from the ILAE commission on diagnostic methods. Epilepsia 2013;54:1315–29. https://doi.org/10.1111/epi.12220

18. Bolschikov V. A., Semenov K.K. Сlustering of inaccurate data using information on its precision. IOP Conference Series Materials Science and Engineering. 2019. Vol. 497. Paper 012023. DOI: 10.1088/1757–899X/497/1/012023

19. Semenov K.K., Bolschikov V. A. The metrologically reasonable clustering of measurement results. Joint IMEKO TC1-TC7-TC13- TC18 Symposium 2019, 2–5 July 2019, St. Petersburg, Russia. — Journal of Physics: Conference Series. Vol. 1379. No 1. Paper 012054. DOI: 10.1088/1742–6596/1379/1/012054

20. Bruxel EM, Bruno DCF, do Canto AM, Geraldis JC, Godoi AB, Martin M, Lopes-Cendes I. Multi-omics in mesial temporal lobe epilepsy with hippocampal sclerosis: Clues into the underlying mechanisms leading to disease. Seizure. 2021 Aug;90:34–50. doi: 10.1016/j.seizure.2021.03.002. Epub 2021 Mar 4. PMID: 33722437

21. Cendes F. Febrile seizures and mesial temporal sclerosis. Curr Opin Neurol 2004; 17:161–4. https://doi.org/10.1097/0001905220040400000013

22. Campe G von, Spencer DD, deLanerolle NC. Morphology of dentate granule cells in the human epileptogenic hippocampus. Hippocampus. 1997;7(5):472–488. https://doi.org/10.1002/(sici)1098–1063(1997)7:53.0.co;2-j

23. Sutula TP, Dudek FE. Unmasking recurrent excitation generated by mossy fiber sprouting in the epileptic dentate gyrus: an emergent property of a complex system. Prog. BrainRes. 2007;163:541–563. https://doi.org/10.1016/s0079–6123(07)630295

24. Perucca P, Crompton DE, Bellows ST, McIntosh AM, Kalincik T, Newton MR, et al. Familial mesial temporal lobe epilepsy and the borderland of d´eja ` vu. Ann Neurol 2017;82:166–76. https://doi.org/10.1002/ana.24984;

25. Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T. Transcriptomics technologies. PLoS Comput Biol 2017;13: e1005457. https://doi.org/10.1371/journal.pcbi.1005457;

26. Tiefes AM, Hartlieb T, Tacke M, von Stülpnagel-Steinbeis C, Larsen LHG, Hao Q, et al. Mesial temporal sclerosis in SCN1A-related epilepsy: two long-term EEG case studies. Clin EEG Neurosci 2019;50:267–72. https://doi.org/10.1177/1550059418794347

27. Balan S, Bharathan SP, Vellichiramal NN, Sathyan S, Joseph V, Radhakrishnan K, et al. Genetic association analysis of ATP binding cassette protein family reveals a novel association of ABCB1 genetic variants with epilepsy risk, but not with drugresistance. PLoS One 2014;9. https://doi.org/10.1371/journal.pone.0089253

28. Heuser K, Nagelhus EA, Taubøll E, Indahl U, Berg PR, Lien S, et al. Variants of the genes encoding AQP4 and Kir4.1 are associated with subgroups of patients with temporal lobe epilepsy. Epilepsy Res 2010;88:55–64. https://doi.org/10.1016/j.eplepsyres.2009.09.023

29. Kanemoto K, Kawasaki J, Miyamoto T, Obayashi H, Nishimura M. Interleukin (IL)-1β, IL 1α, and IL 1 receptor antagonist gene polymorphisms in patients with temporal lobe epilepsy. Ann Neurol 2000;47:571–4. https://doi.org/10.1002/1531–8249(200005)47:53.0.CO;2-A

30. Jamali S, Salzmann A, Perroud N, Ponsole-Lenfant M, Cillario J, Roll P, et al. Functional variant in complement C3 gene promoter and genetic susceptibility to temporal lobe epilepsy and febrile seizures. PLoS One 2010;5: e12740. https://doi.org/10.1371/journal.pone.0012740

31. Horta WG, Paradela E, Figueiredo A, Meira IDA, Pereira VCSR, Rego CC, et al. Genetic association study of the HLA class II alleles DRB1, DQA1, and DQB1 in patients with pharmacoresistant temporal lobe epilepsy associated with mesial hippocampal sclerosis. Seizure 2015;31:7–11. https://doi.org/10.1016/j.Seizure.2015.06.005

32. Gambardella A, Manna I, Labate A, Chifari R, La Russa A, Serra P, et al. GABA(B) receptor 1 polymorphism (G1465A) is associated with temporal lobe epilepsy. Neurology 2003;60:560–3. https://doi.org/10.1212/01. WNL.0000046520.79877.D 8

33. Kauffman MA, Levy EM, Consalvo D, Mordoh J, Kochen S. GABABR 1 (G1465A) gene variation and temporal lobe epilepsy controversy: new evidence. Seizure 2008;17:567–71. https://doi.org/10.1016/j.seizure.2007.12.006

34. Balan S, Sathyan S, Radha SK, Joseph V, Radhakrishnan K, Banerjee M. GABRG2, rs211037 is associated with epilepsy susceptibility, but not with antiepileptic drug resistance and febrile seizures. Pharmacogenet Genomics 2013;23:605–10. https://doi.org/10.1097/FPC.0000000000000000

35. JINGYUN LI, HUA LIN, FENGHE NIU, XILIN ZHU, NING SHEN, XIN WANG, et al. Combined effect between two functional polymorphisms of SLC6A12 gene is associated with temporal lobe epilepsy. J Genet 2015;94:637–42. https://doi.org/10.1007/s1204101505670

36. Labate A, Manna I, Gambardella A, Le Piane E, La Russa A, Condino F, et al. Association between the M129V variant allele of PRNP gene and mild temporal lobe epilepsy in women. Neurosci Lett 2007;421:1–4. https://doi.org/10.1016/j. Neulet.2006.10.020

37. Azakli H, Gurses C, Arikan M, Aydoseli A, Aras Y, Sencer A, et al. Whole mitochondrial DNA variations in hippocampal surgical specimens and blood samples with high-throughput sequencing: a case of mesial temporal lobe epilepsy with hippocampal sclerosis. Gene 2013;529:190–4. https://doi.org/10.1016/j.gene.2013.06.077

38. Volmering E, Niehusmann P, Peeva V, Grote A, Zsurka G, Altmüller J, et al. Neuropathological signs of inflammation correlate with mitochondrial DNA deletions in mesial temporal lobe epilepsy. Acta Neuropathol 2016;132:277–88. https://doi.org/10.1007/s0040101615611

39. Yang H, Yin F, Gan S, Pan Z, Xiao T, Kessi M, et al. The study of genetic susceptibility and mitochondrial dysfunction in mesial temporal lobe epilepsy. Mol Neurobiol 2020;57:3920–30. https://doi.org/10.1007/s12035020019934

40. Ren L, Jin L, Zhang B, Jia Y, Wu L, Shen Y. Lack of GABABR 1 gene variation (G1465A) in a Chinese population with temporal lobe epilepsy. Seizure 2005;14: 611–3. https://doi.org/10.1016/j.seizure.2005.09.009

41. Tan NCK, Heron SE, Scheffer IE, Berkovic SF, Mulley JC. Is variation in the GABA (B) receptor 1 gene associated with temporal lobe epilepsy? Epilepsia 2005;46: 778–80. https://doi.org/10.1111/j.1528–1167.2005.49004.x

42. Ozkara C, Uzan M, Tanriverdi T, Baykara O, Ekinci B, Yeni N, et al. Lack of association between IL 1β/α gene polymorphisms and temporal lobe epilepsy with hippocampal sclerosis. Seizure 2006;15:288–91. https://doi.org/10.1016/j. seizure.2006.02.016

43. Sisodiya SM, Ragge NK, Cavalleri GL, Hever A, Lorenz B, Schneider A, et al. Role of SOX2 mutations in human hippocampal malformations and epilepsy. Epilepsia 2006;47:534–42. https://doi.org/10.1111/j.1528–1167.2006.00464.x

44. M´eriaux C, Franck J, Park DB, Quanico J, Kim YH, Chung CK, et al. Human temporal lobe epilepsy analyses by tissue proteomics. Hippocampus 2014;24: 628–42. https://doi.org/10.1002/hipo.22246

45. Kokaia M. Seizure-induced neurogenesis in the adult brain. Eur J Neurosci 2011; 33:1133–8. https://doi.org/10.1111/j.1460–9568.2011.07612.x

46. Walker A, Russmann V, Deeg CA, von Toerne C, Kleinwort KJH, Szober C, et al. Proteomic profiling of epileptogenesis in a rat model: focus on inflammation. Brain Behav Immun 2016;53:138–58. https://doi.org/10.1016/j.Bbi.2015.12.007

47. Morin-Brureau M, Milior G, Royer J, Chali F, LeDuigou C, Savary E, et al. Microglial phenotypes in the human epileptic temporal lobe. Brain 2018;141: 3343–60. https://doi.org/10.1093/brain/awy276

48. Kawasaki Y, Zhang L, Cheng JK, Ji RR. Cytokine mechanisms of central sensitization: distinct and overlapping role of interleukin 1β, interleukin 6, and tumor necrosis factor-α in regulating synaptic and neuronal activity in the superficial spinal cord. J Neurosci 2008;28:5189–94. https://doi.org/10.1523/JNEUROSCI.3338–07.2008

49. Sitovskaia D. A., Litovchenko A. V., Bazhanova E. D., Skiteva E.N., Zabrodskaia Y. M. Cytokine profile in the peripheral blood and the brain in patients with focal drug-resistant epilepsy. Sechenov Medical Journal. 2021; 12(4): 39–50. https://doi.org/10.47093/2218–7332.2021.12.4.39–50