Nestin down-regulation of cortical radial glia is delayed in rats submitted to recurrent status epilepticus during early postnatal life

Atraso no desaparecimento da nestina na glia radial cortical de ratos submetidos a recorrentes status epilepticus durante o desenvolvimento pós-natal precoce

Nestin down-regulation of cortical radial glia is delayed in rats submitted to recurrent status epilepticus during early postnatal life

Atraso no desaparecimento da nestina na glia radial cortical de ratos submetidos a recorrentes status epilepticus durante o desenvolvimento pós-natal precoce

Carla Alessandra Scorza^I; Ricardo Mario Arida^II; Fulvio Alexandre Scorza^I; Esper Abrão Cavalheiro^I; Maria da Graça Naffah-Mazzacoratti^I,III

^ILaboratório de Neurologia Experimental, Universidade Federal de São Paulo SP, Brazil

^IIDepartamento de Fisiologia, Universidade Federal de São Paulo SP, Brazil

^IIIDepartamento de Bioquímica. This study was supported by CAPES, FAPESP and CNPq, Universidade Federal de São Paulo SP, Brazil

ABSTRACT

OBJECTIVE: Nestin is temporarily expressed in several tissues during development and it is replaced by other protein types during cell differentiation process. This unique property allows distinguishing between undifferentiated and differentiated cells. This study was delineated to analyze the temporal pattern of nestin expression in cortical radial glial cells of rats during normal development and of rats submitted to recurrent status epilepticus (SE) in early postnatal life (P).

METHOD: Experimental rats were submitted to pilocarpine-induced SE on P7-9. The cortical temporal profile of nestin was studied by immunohistochemistry at multiple time points (P9, P10, P12, P16, P30 and P90).

RESULTS: We observed delayed nestin down-regulation in experimental rats of P9, P10, P12 and P16 groups. In addition, few radial glial cells were still present only in P21 experimental rats.

CONCLUSION: Our results suggested that SE during early postnatal life alters normal maturation during a critical period of brain development.

Key words: nestin, pilocarpine, status epilepticus, development, radial glia.

RESUMO

OBJETIVO: A nestina, temporariamente expressa em diversos tecidos durante o desenvolvimento, é substituída no processo de diferenciação celular, o que permite a distinção entre células diferenciadas e indiferenciadas. O objetivo deste estudo foi verificar o padrão temporal da expressão da nestina nas células da glia radial cortical de ratos durante o desenvolvimento normal e nos ratos submetidos a sucessivos status epilepticus (SE) no periodo pós-natal precoce (P).

MÉTODO: Os animais foram submetidos ao SE induzido pela pilocarpina em P7-9. O perfil temporal da nestina foi estudado por imuno-histoquímica em P9, P10, P12, P16, P30 e P90.

RESULTADOS: Nos ratos experimentais, observamos atraso no desaparecimento da nestina nos grupos P9, P10, P12 e P16. Ainda, encontramos algumas glias radiais corticais apenas em P21 experimental.

CONCLUSÃO: Nossos resultados sugerem que o SE durante o desenvolvimento pós-natal precoce altera o processo de maturação durante um periodo crítico do desenvolvimento encefálico.

Palavras-chave: nestina, pilocarpina, status epilepticus, desenvolvimento, glia radial.

The intermediate filament protein denominated nestin is expressed by undifferentiated cells derived from nervous system, muscular and by cells of several other tissues¹. Nestin is an acronym for neuroepithelial stem cell protein and it is generally considered a marker of neural stem cells². Upon differentiation, nestin expression is down-regulated and replaced by other tissue-specific intermediate filament proteins, such as glial fibrillary acidic protein (GFAP) in astrocytes, neurofilaments in neurons, and desmin in the muscle³. Earlier reports had implied that radial glia cells of the mammalian cortex may be potential precursor cells since they were found to be immunopositive for nestin⁴. These cells do not just provide the scaffolding for migrating neurons, but they serve as precursors cells that give origin to all major class of neurons and astrocytes^4-6. Nestin has been also associated with dividing and migrating cells and with cells which are rapidly changing their morphology. This protein also participates in the cytoskeleton of newly formed endothelial cells and represents a novel and reliable vascular marker¹. Moreover, neural stem cells in the adult mammalian brain maintain radial glia features⁷. The maintenance of radial glial cells or their key aspects from development seems to be a crucial feature to allow neuronal repair⁸. Interestingly, nestin is re-expressed during various regenerative and degenerative conditions in the fully differentiated cells⁹.

In humans with epilepsy, as well as in animals submitted to experimental models of epilepsy, the seizure susceptibility and associated neuronal damage are age-dependent^10-12. While the immature brain appears to be less vulnerable to the adverse effects of prolonged seizures than the mature brain¹³, seizures in the early life can be associated with later cognitive and behavioral disturbances, even in the absence of overt structural neuronal damage^14-16. Previous study of our laboratory showed that 3 consecutive episodes of long-lasting pilocarpine-induced SE in developing rats (P7-9) is not able to promote neuronal loss but induces important electrographic and cognitive changes during adulthood¹⁷. Similarly, subtle impairments in intellectual performance have been reported in many individuals with a history of seizures in early life¹⁸.

In this context, the present study was delineated to verify nestin temporal expression in the cortical radial glial cells of rats, under physiological conditions of brain development and after three consecutive episodes of pilocarpine-induced SE (P7-P9). We studied animals from early postnatal life (P9) to adulthood (P90).

METHOD

Animals and status epilepticus induction

Wistar rats were housed under environmentally controlled conditions using a standard light/dark cycle of 12 hours (7 am-7 pm), with rat chow pellets, food and water ad libitum. The colony room had a temperature of 21ºC. The rats were bred in our laboratory and the day of birth was considered as day 0 (P0). The pups were housed with their mothers in individual cages until weaning at day 21. Older animals were housed in groups of three to five per cage. We used only male rats randomly selected for SE induction and they were transiently separated from their mothers during the experimental procedures. The rats were submitted to SE induced by pilocarpine 2% (ip) (4 hours of SE) (Sigma, USA) on P7-P9 (380 mg/Kg), with no previous scopolamine treatment. Rats, with the same age, which received saline instead pilocarpine were used as control. The average weight was 17 gr (P7), 19 gr (P8) and 21 gr (P9) for the control rats and 17 gr (P7), 16 gr (P8) and 15 gr (P9) for the rats submitted to SE. The experiments were performed under strict institutional and ethical approval of the protocol (CEP - Comitê de Ética e Pesquisa, UNIFESP) and all efforts were made to minimize the suffering of the animals as well as reducing the number of animals used. The following groups were studied by immunohistochemistry: P9, P10, P12, P16, P21, P30 and P90, for both groups (n=4 per group).

Immunohistochemical staining

Anaesthetized (Ketamine 5 mg/Kg) animals were perfused transcardially with 0.1 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS. The brains were removed immediately after perfusion, post-fixed and soaked overnight in 30% sucrose in PBS. Brain coronal sections (40 µm) were made using a cryostat. Free-floating paired sections from experimental and control animals at matched levels were processed in the same vial in order to minimize the inter-group differences, during immunohistochemical procedure. In order to distinguish between slices, control ones were marked with a small cut in the surface. All sections were treated with 0.1% H₂O₂ in PBS and then incubated in PBS containing 0.3% Triton X-100. After that, the sections were incubated for 2 hours in 10% bovine serum albumin in PBS and then incubated overnight with the primary anti-nestin antibody (0.5µg/ml of monoclonal mouse anti-nestin, BD Pharmingen). The sections were incubated in the biotinylated secondary antibody (anti-mouse IgG peroxidase conjugate Sigma, 1:200), treated with the ABC reagent (Elite Kit Vector, CA, USA) and the tissue-bound peroxidase was developed using diaminobenzidine as chromogen (DAB 0,05% plus 0,01% hydrogen peroxidase solution). The reaction was initially monitored under the microscope in a few sections to determine the optimal duration of incubation with minimal background. Sections were mounted on gelatinized slides, air-dried, dehydrated, cleared and permanent mounted. Primary somatosensory cortical slices were studied by Sony digital camera coupled to a Nikon Eclipse E600 microscope using bright-field illumination. In order to test the antibody specificity, the primary antibody was omitted. Omission of the primary antibody in our immunhistochemical procedures did not detect any signal, revealing the fact that background and non-specific staining was kept to a minimum level.

RESULTS

Behavioral analysis

Few minutes after pilocarpine systemic administration, all animals presented continuous scratching, strong body tremor, masticatory automatisms, clonic movements of forelimbs and head bobbing culminating in SE. The rats presented long-lasting SE in each one of the three consecutive sessions (P7-P9). The mortality rate was low, less than ten percent. As reference of developmental aspects of pilocarpine model of epilepsy see Priel et al.¹⁹.

Immunohistochemical analysis

Cortical radial glia cells were immunopositive for nestin at P9, P10, P12, P16 of experimental and control animals. However, few radial glia cells were still present only in P21 experimental rats while no nestin immunoreactivity was observed by P21 in the control group. Moreover, cortical blood vessels were also labeled against nestin, gradually decreasing from P9 to P30 and almost inexistent by P90 (not showed), both in experimental and control groups (Fig 1).

 

Small varicosities were observed along the cortical radial fibers (Figs 2A,2B, P10 taken as representative Figure). In the cortical radial glia of control rats, nestin immunoreactivity (IR) was intense in P9 and P10 groups, decreasing strongly in P12 and P16 (Figs 1A,1B,1C,1D). In P21 control group, the nestin IR completely disappeared (Fig 1E), being inexistent in P30 (Fig 1F) and P90. In contrast, nestin IR was increased in the experimental groups from P9 to P16, when compared to their respective control animals (Figs 1G,1H,1I,1J). In addition, few radial glia cells were still present in the P21 experimental rats (Fig 1K), disappearing in P30 (Fig 1L) and P90. These data suggested that animals submitted to three consecutive SE presented delayed nestin down-regulation in cortical radial glia.

DISCUSSION

Gliogenic activities remain intense during the postnatal period in the developing rat cortex. These include involution of radial glia, proliferation of astrocytes and oligodendrocytes and myelin formation. We addressed the question of how recurrent long-lasting SE in early postnatal brain development (P7-9) could affect temporal nestin expression in cortical radial glial cells. Nestin expression is transient and we found delayed nestin down-regulation in the rats submitted to SE. As expected, we observed small varicosities along cortical radial fibers. In 1888, Magini²⁰ had already detected these varicosities along the radial glial filaments (Fig 2A). Nowadays, some authors have suggested that they could be associated to a transitory cell form between radial glia and astrocytes^21,22.

Nestin is re-expressed during various regenerative and degenerative conditions in the fully differentiated cells, including lesions induced by kainic acid and pilocarpine in adult rats^9,23,24. However, when considering developmental aspects and astrocytic response after injury the literature is still poor. In the present study, we did not observe such cortical reactive astrocytic response after injury, as largely found in adult rats. In order to support our findings, Rizzi et al.²⁵ reported that at P9 there are just a little activation of microglia and astrocytes after kainic acid-induced SE. Conversely, Sizonenko et al.²⁶ showed that the hypoxic-ischemic injury resulted in disruption of the normal radial glia architecture, which was paralleled by an increase in GFAP immunopositive reactive astrocytes. In addition, the morphology of these latter cells and the fact that they were immunolabelled for both nestin and GFAP suggested an accelerated transformation of radial glia into astrocytes. Anyway, the findings imply that glial responses are central to cortical tissue remodelling following neonatal injury.

We observed that experimental rats from P9 to P16 presented increased nestin IR labeling cortical radial glia cells, when compared to their respective controls. Kálmán and Ajtai showed that most radial glial cells disappear by P14, showing no reactivity at P18²². As observed in our study, normally, radial glial cells were already inexistent by P21 in the control rats. However, few radial glial cells were still present in the cortex of P21 experimental rats, showing a delay in cortical down-regulation of nestin protein and, therefore, suggesting that the appearance of these fibers in the P21 rats was abnormal. It could be that the SE injury simply acts to delay the transformation of radial glia cells to astrocytes but, eventually, all radial glial cells would transform. This result suggests that SE-induced injury could interfere in the release of factors responsible for the transformation of the radial glia cells.

In humans, SE is a common pediatric emergency and occurs mainly in children younger than 2 years, supporting the idea that during the development the brain is more vulnerable to long-lasting seizures^27,28. Furthermore, retrospective studies indicate that adults with mesial temporal lobe epilepsy report a high incidence of childhood status epilepticus²⁹. Previously, our laboratory showed that although most of the animals submitted to SE (P7-9) did not develop spontaneous seizures in adulthood, they presented learning impairment and significant changes in cortical recordings, with episodes of complex spiking activity¹⁷.

Here we demonstrate increased temporal radial glia after injury induced by SE and delayed in radial glia disappearance. In conclusion, our study suggests that SE during early postnatal life can alters normal maturation during a critical period of brain development. The mechanism for the maintenance of the immature form of cells expressing nestin is unkown and studies of nestin are of relevance in neurobiology.

Received 19 November 2008, received in final form 30 March 2009. Accepted 27 May 2009.

Dra. Maria da Graça Naffah-Mazzacoratti - Neurologia Experimental/UNIFESP - Edifício Leal Prado - Rua Botucatu 862 - 04042-003 São Paulo SP - Brasil. E-mail: [e-mail]

• 1. Mokrý J, Cízková D, Filip S, et al. Nestin expression by newly formed human blood vessels. Stem Cells Dev 2004;13:658-664.
• 2. Lendahl U, Zimmerman LB, McKay RDG. CNS stem cells express a new class of intermediate protein. Cell 1990;60:585-595.
• 3. Sahlgren CM, Mikhailov A, Hellman J, et al. Mitotic reorganization of the intermediate filament protein nestin involves phosphorylation by cdc2 kinase. JBC 2001;1276:16456-16463.
• 4. Parnavelas JG, Nadarajah B. Radial glia cells: are they really glia? Neuron 2001;31:881-884.
• 5. Rakic P. Elusive radial glial cells: historical and evolutionary perspective. Glia 2003;43:19-32.
• 6. Liour S, Yu RK. Differentiation of radial glia-like cells from embryonic stem cells. Glia 2003;42:109-117.
• 7. Pinto L, Gotz M. Radial glial cell heterogeneity. The source of diverse progeny in the CNS. Progr Neurobiol 2007;83:2-23.
• 8. Gilyarov AV. Nestin in central nervous cells. Neurosci Behav Physiol 2008;38:165-169.
• 9. Geloso MC, Corvino V, Cavallo V,et al. Expression of asrocytic nestin in the rat hippocampus during trimethyltin-induced neurodegeneration. Neurosci Lett 2004;357:103-106.
• 10. Druga R, Mares P, Otahal J, Kubova H. Degenerative neuronal changes in the rat thalamus induced by status epilepticus at different developmental stages. Epilepsy Res 2005;63:43-65.
• 11. Kubova H, Mares P, Suehomelova L, Brozec G, Druga R, Pitkanen A. Status epilepticus in immature rats leads to behavioral and cognitive impairment and epileptogenesis. Eur J Neurosci 2004;19:3255-3265.
• 12. Sankar R, Shin DH, Liu H, Mazarati A, de Vasconcelos AP, Wasterlain CG. Patterns of status epilepticus-induced neuronal injury during development and long-term consequences. J Neurosci 1998;18:8382-8393.
• 13. Holmes GL. Effects of seizures on brain development: lessons from the laboratory. Pediatr Neurol 2005;33:1-11.
• 14. Cavalheiro EA, Silva DF, Turski WA, Calderazzo-Filho LS, Bortolotto ZA, Turski L. The susceptibility of rats to pilocarpine-induced seizures is age-dependent. Brain Res 1987;465:43-58.
• 15. Stafstrom CE. Assessing the behavioral and cognitive effects of seizures on the developing brain. Prog Brain Res 2002;135:377-390.
• 16. Sayin U, Sutula TP, Stafstrom CE. Seizures in the developing brain cause adverse long-term effects on spatial learning and anxiety. Epilepsia 2004;45:1539-1548.
• 17. Santos NF, Arida RM, Trindade EM Filho, Priel MR, Cavalheiro EA. Epileptogenesis in immature rats following recurrent status epilepticus. Brain Res Rev 2000;32:269-276.
• 18. Cassidy G, Corbett J. Learning disorders. In: Engel J, Pedley TA (Eds). Epilepsy: a comprehensive textbook. Chapter 195. Philadelphia: Lippincott-Raven Publisher, 1997:2053-2060.
• 19. Priel MR, Santos NF, Cavalheiro EA. Developmental aspects of the pilocarpine model of epilepsy. Epilepsy Res 1996;26:115-121.
• 20. Magini G. Nevroglia e cellule nervose cerebrali nei feti. In: Atti Del Dodicesimo Congresso della Associazone Medica Italiana, vol 1. Pavia: Tipografia Fratelli Fusi,1888:281-291.
• 21. Schmechel DE, Rakic P. A Golgi study of radial glial cells in developing monkey telencephalon: morphogenesis and transformation into astrocytes. Anat Embryol 1979;156:115-152.
• 22. Kálmán M, Ajtai BM. A comparison of intermediate filament markers for presumptive astroglia in the developing rat neocortex: immunostaining against nestin reveals more detail, than GFAP or vimentin. Int J Dev Neurosci 2001;19:101-108.
• 23. Clark SR, Shetty AK, Bradley JL, Turner DA. Reactive astrocytes express the embryonic intermediate neurofilament nestin. Neuro Report 1994;5:1885-1888.
• 24. Scorza CA, Arida RM, Cavalheiro EA, Naffah-Mazzacoratti MG, Scorza FA. Expression of nestin in the hippocampal formation of rats submitted to the pilocarpine model of epilepsy. Neurosci Res 2005;51:285-291.
• 25. Rizzi M, Perego C, Aliprandi M, et al. Glia activation and cytokine increase in rat hippocampus by kainic acid-induced status epilepticus during postnatal development. Neurobiol Dis 2003;14:494-503.
• 26. Sizonenko SV, Camm EJ, Dayer A, Kiss JZ. Glial responses to neonatal hypoxic-ischemic injury in the rat cerebral cortex. Int J Dev Neurosci 2008;26:37-45.
• 27. Scantlebury MH, Heida JG, Hasson HJ, et al. Age-dependent consequences of status epilepticus: animal models. Epilepsia 2007;48(Suppl2): S75-S82.
• 28. Cilio MR, Sogawa Y, Cha BH, Liu X, Huang LT, Holmes GL. Long-term effects of status epilepticus in the immature brain are specific for age and model. Epilepsia 2003;44:518-528.
• 29. Cendes F, Andermann F, Dubeau F, et al. Early childhood prolonged febrile convulsions, atrophy and sclerosis of mesial structures, and temporal lobe epilepsy: an MRI volumetric study. Neurology 1993;43: 1083-1087.

Publication Dates

• Publication in this collection
  21 Aug 2009
• Date of issue
  Sept 2009

History

• Reviewed
  30 Mar 2009
• Received
  19 Nov 2008
• Accepted
  27 May 2009