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Spontaneous recurrent seizure following status epilepticus enhances dentate gyrus neurogenesis
Brain & Development 26 (2004) 394–397 www.elsevier.com/locate/braindev
Original article
Spontaneous recurrent seizure following status epilepticus enhances dentate gyrus neurogenesis Byung Ho Chaa,b,1, Cigdem Akmana,c,1, Diosely C. Silveiraa, Xianzeng Liua,d, Gregory L. Holmesa,d,* a
b
Department of Neurology, Harvard Medical School, Children’s Hospital Boston, Boston, MA, USA Department of Pediatrics, Wonju Christian Hospital, Wonju College of Medicine, Yonsei University, Wonju, South Korea c Department of Neurology, College of Physicians and Surgeons of Columbia University, New York, NY, USA d Section of Neurology, Neuroscience Center at Dartmouth, Dartmouth Medical School, Lebanon, NH, USA Received 19 August 2003; received in revised form 6 December 2003; accepted 8 December 2003
Abstract It is known that evoked seizures can increase neurogenesis in the dentate gyrus in adult rats. Whether spontaneous seizures occurring after status epilepticus (SE) also results in alterations in neurogenesis is not known. Here, we measured neurogenesis in rats with and without spontaneous seizures following SE. Lithium – pilocarpine was used to induce seizures in postnatal (P) day 20 rats. Spontaneous seizure frequency was assessed 2 months using video monitoring. Rats then received bromodeoxyuridine to label dividing DNA and were sacrificed 24 h later. Animals with spontaneous seizures ðn ¼ 9Þ had a modest increase in neurogenesis compared to animals with SE ðn ¼ 6Þ and no spontaneous seizures and control rats ðn ¼ 10Þ: These findings demonstrate that the hippocampus is capable of generating new neurons weeks following SE and further that recurrent seizures enhance the production of new neurons. These alterations in neurogenesis may contribute to ongoing pathological changes week and months following SE. q 2004 Published by Elsevier B.V. Keywords: Epilepsy; Seizures; Bromodeoxyuridine; Lithium; Pilocarpine; Hippocampus
Status epilepticus (SE) is a common neurological condition, which is associated with a significant morbidity and mortality risk [1,2]. In animal models, SE is associated with cell loss in CA1, CA3, the hilus and dentate gyrus and synaptic reorganization with aberrant growth (sprouting) of granule cell axons (the so-called mossy fibers) in the supragranular zone of the fascia dentate and infrapyramidale region of CA3 [3 – 5]. This histologically detectable damage resembles human mesial temporal lobe sclerosis [6]. Increased neurogenesis in the dentate gyrus [7,8] and subventricular zone [9] has also been reported following SE. There is evidence that newly generated neurons contribute the structural and functional abnormalities in the epileptic hippocampus [9]. Following SE there is considerable * Corresponding author. Address: Section of Neurology, DartmouthHitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USA. Tel.: þ 1-603-650-7910; fax: þ1-603-650-0458. E-mail address: [email protected] (G.L. Holmes). 1 These two authors contributed equally to this study. 0387-7604/$ - see front matter q 2004 Published by Elsevier B.V. doi:10.1016/j.braindev.2003.12.006
hippocampal damage [10,11]. Whether recurrent spontaneous seizures that occur following SE alters neurogenesis in the damaged hippocampus is not known. Determining the effect of seizures on the plastic process following SE is important to our understanding of brain recovery following excitotoxic injuries. We report here that animals that develop spontaneous recurrent seizures following SE have a modest increase in neurogenesis, demonstrating that the damaged hippocampus is capable of generating new neurons following spontaneous seizures.
1. Material and method Sprague –Dawley rats were used throughout the experiments. Rats were housed in approved facilities and had access to food and water ad libitum. They were housed with their litter until weaning at postnatal day (P) 21, after which they were group-housed in plastic cages under diurnal
B.H. Cha et al. / Brain & Development 26 (2004) 394–397
lighting conditions with lights on from 08:00 to 20:00. All efforts were made to minimize animal suffering and all animal procedures were in compliance with the NIH and institutional guidelines at Children’s Hospital Boston. At P19 rats ðn ¼ 18Þ were injected lithium chloride, 3 mequiv./kg, intraperitoneally and 20 – 22 h later, the experimental rats were injected with pilocarpine hydrochloride, 60 mg/kg, subcutaneously for induction of SE. Control rats ðn ¼ 10Þ were injected with the same volume of normal saline. After pilocarpine injection, all rats progressed to SE. The onset of SE was characterized by initial immobility and chewing followed by repetitive clonic activity of the trunk and limbs. The rats then developed repeated rearing with forelimb clonus and falling interspersed with periods of immobility, chewing, and myoclonic jerks. Three rats died either during the SE or within 24 h of the SE. Eight weeks following pilocarpine or normal saline injection, the SE and control rats underwent video monitoring for 7 days, 6 h a day in each rat to evaluate spontaneous recurrent seizure. Experimental rats were divided into two groups, one group (SRS, n ¼ 9) had spontaneous recurrent seizures, the other (non-SRS, n ¼ 6) did not have any spontaneous recurrent seizures. None of the control rats ðn ¼ 10Þ had seizures. After completion of the video monitoring, the rats received a series of four intraperitoneal injections of bromodeoxyuridine (BrdU, 50 mg/kg) (Boehringer Mannheim, Indianapolis, IN) every 2 h to label mitotically active cells. No rats had a spontaneous seizure 6 h prior to the first injection nor were any seizures noted during the injections. Rats were sacrificed 24 h after BrdU administration. After deep anesthesia with sodium pentobarbital (65 mg/kg), rats were perfused transcardially with normal saline followed by 4% paraformaldehyde (PFA). The brains were postfixed in PFA for 24 h and then placed in a 30% sucrose solution until the brains sank to the bottom of the chamber. Coronal sections through the entire extent of the hippocampus were cut at 40 mm on a freezing microtome and sections were stored in PBS with 0.2% sodium azide until processed. Every fourth section was stained for immunohistochemistry and alternate sections were stained with thionin for cell counting. BrdU and BrdU doublelabeling with the neuronal marker neuron-specific nuclear protein (NeuN), was performed using techniques previously described in our laboratory [12]. NeuN has been demonstrated to be a marker of neuronal maturation [13]. For quantifying BrdU-labeling, mounted sections spaced at least 150 mM apart were used. As previously described [12], 6– 8 sections per animal (12 – 16 hippocampi) obtained from the dorsal hippocampus were analyzed. Labeled cells in the dentate gyrus (both superior and inferior blades) or within 1– 2 cell layers width into the hilus were counted. Based on anatomical landmarks, equivalent sections from control and experimental animals were chosen and coded by one of the authors (BHC). Fluorescence microscopy was
395
used to visualize BrdU cell co-labeled with NeuN. To determine percentage of BrdU-labeled cells that were neurons, BrdU-labeled cells co-labeled with NeuN were divided by total number of BrdU-labeled cells. Cell counting was done manually by a examiner blinded to experimental group (GLH). Thionin slides were analyzed for cell loss in the CA3, CA1 and the hilus using a semi-quantitative scoring system ranging from 0 to 5, using a scale previously employed [11]: a score of 0 indicated no lesion; 1, scattered cell loss; 2, moderate cell loss occurring in clusters; 3, substantial cell loss disrupting normal architecture of the region; 4, extensive loss of cells with only remnants of the cell layer remaining; and 5, total loss of neurons. The investigator assigning the scores (GLH) was blinded to treatment group. A total hippocampal cell loss score was obtained by adding scores from each region (CA3, CA1, hilus) from both hippocampi five specimens. The scores were divided by five to obtain a mean pathology score for the bilateral hippocampi. Number of BrdU-labeled cells and histology scores were evaluated for variance (Bartlett’s test for equal variance) and normality (Gaussian-shaped distribution) using the Kolmogorov – Smirnov goodness-of-fit test. Because the date passed the normality test ðP . 0:10Þ group means were compared with the ANOVA with post-hoc testing using Tukey’s multiple comparison test. Means ^ standard errors of all measures are presented.
2. Results Spontaneous seizure frequency varied from 2 to 12 seizures (mean 5.20 ^ 0.99) during the 42 h of monitoring. The seizures consisted of forelimb clonus with or without rearing and were generally brief, lasting , 60 s. No tonic or tonic –clonic seizures occurred. BrdU-labeled cells typically had dense and homogeneous staining of the round nuclei. BrdU-labeled cells were seen primarily at the hilar border of the dentate granular cell layer (Fig. 1A). Some clumping of BrdUlabeled cells was seen (Fig. 1C). The number of BrdUlabeled cells per hippocampus in the SRS group was 7.70 ^ 0.30, in the non-SRS group 4.66 ^ 0.23, and in the controls 6.19 ^ 0.17. The groups differed significantly ðP , 0:01Þ with the mean number of labeled cells significantly higher in the SRS group than in non-SRS and controls ðP , 0:01Þ: Animals subjected to SE but without SRS had fewer BrdU-labeled cells than the controls ðP , 0:01Þ: The vast majority of BrdU-labeled in the dentate gyrus were co-labeled with NeuN (Fig. 2). The percentages of colabeled cells were similar in the three groups: SRS, 45/50 (90%); non-SRS, 61/70 (97%); and controls, 59/64 (92%). The mean damage score in CA3 of each group was 1.31 ^ 0.09 in SRS, 1.25 ^ 0.09 in non-SRS,
396
B.H. Cha et al. / Brain & Development 26 (2004) 394–397
Fig. 1. Example of BrU cell co-labeled with NeuN at the hilus border of the dentate gyrus (arrow). Note the clumping of the BrdU in the nucleus (white) in two NeuN-labeled neurons (red) (calibration, 50 mm).
and 0.700 ^ 0.08 in the controls ðP , 0:01Þ: Both the SRS and non-SRS groups differed significantly from the controls ðP , 0:01Þ: In addition, damage scores were significantly higher in the SRS group than the non-SRS group ðP , 0:001Þ:
3. Discussion Recurrent spontaneous seizures following SE was associated with a modest increase in neurogenesis compared to rats undergoing SE without spontaneous seizures and non-SE controls. NeuN co-labeling demonstrated that the vast majority of these newly formed cells were neurons. The increase in neurogenesis appeared to be secondary to the seizures rather than a response to cell injury and death; the non-SRS group had a reduction in neurogenesis compared to the controls. The ability of brief seizures, as seen in this study, to induce neurogenesis has been previously shown in normal rats. Kindling [14,15] and even a single seizure [16] can increase neurogenesis. Our study demonstrates that despite the histological damage, recurrent seizures can increase neurogenesis following SE. The functional significance of this increase in neurogenesis is not readily apparent. However, there is evidence that these newly formed granule cells are functional and become integrated within the hippocampal circuitry [17]. Whether the increase in neurogenesis contributes to the enhanced excitability in these animals is not clear. Since the animals with SRS had higher damage scores than
Fig. 2. (A) Example of BrdU staining in animal with SRS. The majority of labeled cells were in the dentate gyrus or hilus. (B) Hippocampus from control rat stained with thionin. The high cell density of the upper and lower blade and hilus can be used to orient (A). (C) Clumping of BrdU (arrow) was common in the dentate gyrus. UB, upper blade of dentate gyrus; LB, lower blade of dentate gyrus; calibration: (A) and (B) 100 mm; (C) 50 mm.
the non-SRS group it is possible that the seizures were secondary to greater as opposed to an increase in neurogenesis. The study provides only limited information regarding the time course of seizure-induced neurogenesis since only one time point was studied. While the increase in neurogenesis following spontaneous seizures was modest, the accumulative effect of hundreds of seizures during the life of the animal would be substantial. The time course of the alteration in neurogenesis following SE is not known and further studies will be necessary to determine whether neurogenesis remains persistently elevated in rats with spontaneous seizures or whether there are subsequent reductions in new cell formation, increases in programmed cell death, or both. The functional significance of new cell
B.H. Cha et al. / Brain & Development 26 (2004) 394–397
formation following SE is also now known and is worthy of further study.
Acknowledgements Supported by a grant from the NINDS (NS27984) to GLH.
[8]
[9]
[10]
[11]
References [12] [1] Lowenstein DH, Alldredge BK. Status epilepticus. N Engl J Med 1998;338:970 –6. [2] Working Group on Status Epilepticus, Treatment of convulsive status epilepticus. Recommendations of the Epilepsy Foundation of America’s Working Group on Status Epilepticus. J Am Med Assoc 1993;270:854 –9. [3] Tauck DL, Nadler JV. Evidence of functional mossy fiber sprouting in the hippocampal formation of kainic acid-treated rats. J Neurosci 1985;5:1016– 22. [4] Represa A, Tremblay E, Ben-Ari Y. Kainate binding sites in the hippocampal mossy fibers: localization and plasticity. Neuroscience 1987;20:739– 48. [5] Buckmaster PS, Dudek FE. Neuron loss, granule cell axon reorganization, and functional changes in the dentate gyrus of epileptic kainate-treated rats. J Comp Neurol 1997;385:385–404. [6] Liu Z, Mikati M, Holmes GL. Mesial temporal sclerosis: pathogenesis and significance. Pediatr Neurol 1995;12:5–16. [7] Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS, Lowenstein DH. Dentate granule cell neurogenesis is increased by
[13]
[14]
[15]
[16]
[17]
397
seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci 1997;17:3727–38. Sankar R, Shin D, Liu H, Katsumori H, Wasterlain CG. Granule cell neurogenesis after status epilepticus in the immature rat brain. Epilepsia 2000;41(Suppl 6):S53–6. Parent JM, Lowenstein DH. Seizure-induced neurogenesis: are more new neurons good for an adult brain? Prog Brain Res 2002;135: 121–31. Cilio MR, Sogawa Y, Cha BH, Liu Z, Huang L-T, Holmes GL. Longterm effects of status epilepticus in the immature brain are age- and model-specific. Epilepsia 2003;44:518–28. Rutten A, van Albada M, Silveira DC, Cha BH, Liu X, Hu YN, et al. Memory impairment following status epilepticus in immature rats: time-course and environmental effects. Eur J Neurosci 2002;20: 501–13. McCabe BK, Silveira DC, Cilio MR, Cha BH, Liu Z, Sogawa Y, Holmes GL. Reduced neurogenesis after neonatal seizures. J Neurosci 2001;21:2094 –103. Sarnat HB, Nochlin D, Born DE. Neuronal nuclear antigen (NeuN): a marker of neuronal maturation in early human fetal nervous system. Brain Dev 1998;20:88–94. Parent JM, Janumpalli S, McNamara JO, Lowenstein DH. Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat. Neurosci Lett 1998;247:9 –12. Scott BW, Wang S, Burnham WM, De Boni U, Wojtowicz JM. Kindling-induced neurogenesis in the dentate gyrus of the rat. Neurosci Lett 1998;248:73 –6. Bengzon J, Kokaia Z, Elme´r E, Nanobashvili A, Kokaia M, Lindvall O. Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures. Proc Natl Acad Sci USA 1997;94: 10432– 7. Scharfman HE, Goodman JH, Sollas AL. Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis. J Neurosci 2000;20:6144–58.
Original article
Spontaneous recurrent seizure following status epilepticus enhances dentate gyrus neurogenesis Byung Ho Chaa,b,1, Cigdem Akmana,c,1, Diosely C. Silveiraa, Xianzeng Liua,d, Gregory L. Holmesa,d,* a
b
Department of Neurology, Harvard Medical School, Children’s Hospital Boston, Boston, MA, USA Department of Pediatrics, Wonju Christian Hospital, Wonju College of Medicine, Yonsei University, Wonju, South Korea c Department of Neurology, College of Physicians and Surgeons of Columbia University, New York, NY, USA d Section of Neurology, Neuroscience Center at Dartmouth, Dartmouth Medical School, Lebanon, NH, USA Received 19 August 2003; received in revised form 6 December 2003; accepted 8 December 2003
Abstract It is known that evoked seizures can increase neurogenesis in the dentate gyrus in adult rats. Whether spontaneous seizures occurring after status epilepticus (SE) also results in alterations in neurogenesis is not known. Here, we measured neurogenesis in rats with and without spontaneous seizures following SE. Lithium – pilocarpine was used to induce seizures in postnatal (P) day 20 rats. Spontaneous seizure frequency was assessed 2 months using video monitoring. Rats then received bromodeoxyuridine to label dividing DNA and were sacrificed 24 h later. Animals with spontaneous seizures ðn ¼ 9Þ had a modest increase in neurogenesis compared to animals with SE ðn ¼ 6Þ and no spontaneous seizures and control rats ðn ¼ 10Þ: These findings demonstrate that the hippocampus is capable of generating new neurons weeks following SE and further that recurrent seizures enhance the production of new neurons. These alterations in neurogenesis may contribute to ongoing pathological changes week and months following SE. q 2004 Published by Elsevier B.V. Keywords: Epilepsy; Seizures; Bromodeoxyuridine; Lithium; Pilocarpine; Hippocampus
Status epilepticus (SE) is a common neurological condition, which is associated with a significant morbidity and mortality risk [1,2]. In animal models, SE is associated with cell loss in CA1, CA3, the hilus and dentate gyrus and synaptic reorganization with aberrant growth (sprouting) of granule cell axons (the so-called mossy fibers) in the supragranular zone of the fascia dentate and infrapyramidale region of CA3 [3 – 5]. This histologically detectable damage resembles human mesial temporal lobe sclerosis [6]. Increased neurogenesis in the dentate gyrus [7,8] and subventricular zone [9] has also been reported following SE. There is evidence that newly generated neurons contribute the structural and functional abnormalities in the epileptic hippocampus [9]. Following SE there is considerable * Corresponding author. Address: Section of Neurology, DartmouthHitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USA. Tel.: þ 1-603-650-7910; fax: þ1-603-650-0458. E-mail address: [email protected] (G.L. Holmes). 1 These two authors contributed equally to this study. 0387-7604/$ - see front matter q 2004 Published by Elsevier B.V. doi:10.1016/j.braindev.2003.12.006
hippocampal damage [10,11]. Whether recurrent spontaneous seizures that occur following SE alters neurogenesis in the damaged hippocampus is not known. Determining the effect of seizures on the plastic process following SE is important to our understanding of brain recovery following excitotoxic injuries. We report here that animals that develop spontaneous recurrent seizures following SE have a modest increase in neurogenesis, demonstrating that the damaged hippocampus is capable of generating new neurons following spontaneous seizures.
1. Material and method Sprague –Dawley rats were used throughout the experiments. Rats were housed in approved facilities and had access to food and water ad libitum. They were housed with their litter until weaning at postnatal day (P) 21, after which they were group-housed in plastic cages under diurnal
B.H. Cha et al. / Brain & Development 26 (2004) 394–397
lighting conditions with lights on from 08:00 to 20:00. All efforts were made to minimize animal suffering and all animal procedures were in compliance with the NIH and institutional guidelines at Children’s Hospital Boston. At P19 rats ðn ¼ 18Þ were injected lithium chloride, 3 mequiv./kg, intraperitoneally and 20 – 22 h later, the experimental rats were injected with pilocarpine hydrochloride, 60 mg/kg, subcutaneously for induction of SE. Control rats ðn ¼ 10Þ were injected with the same volume of normal saline. After pilocarpine injection, all rats progressed to SE. The onset of SE was characterized by initial immobility and chewing followed by repetitive clonic activity of the trunk and limbs. The rats then developed repeated rearing with forelimb clonus and falling interspersed with periods of immobility, chewing, and myoclonic jerks. Three rats died either during the SE or within 24 h of the SE. Eight weeks following pilocarpine or normal saline injection, the SE and control rats underwent video monitoring for 7 days, 6 h a day in each rat to evaluate spontaneous recurrent seizure. Experimental rats were divided into two groups, one group (SRS, n ¼ 9) had spontaneous recurrent seizures, the other (non-SRS, n ¼ 6) did not have any spontaneous recurrent seizures. None of the control rats ðn ¼ 10Þ had seizures. After completion of the video monitoring, the rats received a series of four intraperitoneal injections of bromodeoxyuridine (BrdU, 50 mg/kg) (Boehringer Mannheim, Indianapolis, IN) every 2 h to label mitotically active cells. No rats had a spontaneous seizure 6 h prior to the first injection nor were any seizures noted during the injections. Rats were sacrificed 24 h after BrdU administration. After deep anesthesia with sodium pentobarbital (65 mg/kg), rats were perfused transcardially with normal saline followed by 4% paraformaldehyde (PFA). The brains were postfixed in PFA for 24 h and then placed in a 30% sucrose solution until the brains sank to the bottom of the chamber. Coronal sections through the entire extent of the hippocampus were cut at 40 mm on a freezing microtome and sections were stored in PBS with 0.2% sodium azide until processed. Every fourth section was stained for immunohistochemistry and alternate sections were stained with thionin for cell counting. BrdU and BrdU doublelabeling with the neuronal marker neuron-specific nuclear protein (NeuN), was performed using techniques previously described in our laboratory [12]. NeuN has been demonstrated to be a marker of neuronal maturation [13]. For quantifying BrdU-labeling, mounted sections spaced at least 150 mM apart were used. As previously described [12], 6– 8 sections per animal (12 – 16 hippocampi) obtained from the dorsal hippocampus were analyzed. Labeled cells in the dentate gyrus (both superior and inferior blades) or within 1– 2 cell layers width into the hilus were counted. Based on anatomical landmarks, equivalent sections from control and experimental animals were chosen and coded by one of the authors (BHC). Fluorescence microscopy was
395
used to visualize BrdU cell co-labeled with NeuN. To determine percentage of BrdU-labeled cells that were neurons, BrdU-labeled cells co-labeled with NeuN were divided by total number of BrdU-labeled cells. Cell counting was done manually by a examiner blinded to experimental group (GLH). Thionin slides were analyzed for cell loss in the CA3, CA1 and the hilus using a semi-quantitative scoring system ranging from 0 to 5, using a scale previously employed [11]: a score of 0 indicated no lesion; 1, scattered cell loss; 2, moderate cell loss occurring in clusters; 3, substantial cell loss disrupting normal architecture of the region; 4, extensive loss of cells with only remnants of the cell layer remaining; and 5, total loss of neurons. The investigator assigning the scores (GLH) was blinded to treatment group. A total hippocampal cell loss score was obtained by adding scores from each region (CA3, CA1, hilus) from both hippocampi five specimens. The scores were divided by five to obtain a mean pathology score for the bilateral hippocampi. Number of BrdU-labeled cells and histology scores were evaluated for variance (Bartlett’s test for equal variance) and normality (Gaussian-shaped distribution) using the Kolmogorov – Smirnov goodness-of-fit test. Because the date passed the normality test ðP . 0:10Þ group means were compared with the ANOVA with post-hoc testing using Tukey’s multiple comparison test. Means ^ standard errors of all measures are presented.
2. Results Spontaneous seizure frequency varied from 2 to 12 seizures (mean 5.20 ^ 0.99) during the 42 h of monitoring. The seizures consisted of forelimb clonus with or without rearing and were generally brief, lasting , 60 s. No tonic or tonic –clonic seizures occurred. BrdU-labeled cells typically had dense and homogeneous staining of the round nuclei. BrdU-labeled cells were seen primarily at the hilar border of the dentate granular cell layer (Fig. 1A). Some clumping of BrdUlabeled cells was seen (Fig. 1C). The number of BrdUlabeled cells per hippocampus in the SRS group was 7.70 ^ 0.30, in the non-SRS group 4.66 ^ 0.23, and in the controls 6.19 ^ 0.17. The groups differed significantly ðP , 0:01Þ with the mean number of labeled cells significantly higher in the SRS group than in non-SRS and controls ðP , 0:01Þ: Animals subjected to SE but without SRS had fewer BrdU-labeled cells than the controls ðP , 0:01Þ: The vast majority of BrdU-labeled in the dentate gyrus were co-labeled with NeuN (Fig. 2). The percentages of colabeled cells were similar in the three groups: SRS, 45/50 (90%); non-SRS, 61/70 (97%); and controls, 59/64 (92%). The mean damage score in CA3 of each group was 1.31 ^ 0.09 in SRS, 1.25 ^ 0.09 in non-SRS,
396
B.H. Cha et al. / Brain & Development 26 (2004) 394–397
Fig. 1. Example of BrU cell co-labeled with NeuN at the hilus border of the dentate gyrus (arrow). Note the clumping of the BrdU in the nucleus (white) in two NeuN-labeled neurons (red) (calibration, 50 mm).
and 0.700 ^ 0.08 in the controls ðP , 0:01Þ: Both the SRS and non-SRS groups differed significantly from the controls ðP , 0:01Þ: In addition, damage scores were significantly higher in the SRS group than the non-SRS group ðP , 0:001Þ:
3. Discussion Recurrent spontaneous seizures following SE was associated with a modest increase in neurogenesis compared to rats undergoing SE without spontaneous seizures and non-SE controls. NeuN co-labeling demonstrated that the vast majority of these newly formed cells were neurons. The increase in neurogenesis appeared to be secondary to the seizures rather than a response to cell injury and death; the non-SRS group had a reduction in neurogenesis compared to the controls. The ability of brief seizures, as seen in this study, to induce neurogenesis has been previously shown in normal rats. Kindling [14,15] and even a single seizure [16] can increase neurogenesis. Our study demonstrates that despite the histological damage, recurrent seizures can increase neurogenesis following SE. The functional significance of this increase in neurogenesis is not readily apparent. However, there is evidence that these newly formed granule cells are functional and become integrated within the hippocampal circuitry [17]. Whether the increase in neurogenesis contributes to the enhanced excitability in these animals is not clear. Since the animals with SRS had higher damage scores than
Fig. 2. (A) Example of BrdU staining in animal with SRS. The majority of labeled cells were in the dentate gyrus or hilus. (B) Hippocampus from control rat stained with thionin. The high cell density of the upper and lower blade and hilus can be used to orient (A). (C) Clumping of BrdU (arrow) was common in the dentate gyrus. UB, upper blade of dentate gyrus; LB, lower blade of dentate gyrus; calibration: (A) and (B) 100 mm; (C) 50 mm.
the non-SRS group it is possible that the seizures were secondary to greater as opposed to an increase in neurogenesis. The study provides only limited information regarding the time course of seizure-induced neurogenesis since only one time point was studied. While the increase in neurogenesis following spontaneous seizures was modest, the accumulative effect of hundreds of seizures during the life of the animal would be substantial. The time course of the alteration in neurogenesis following SE is not known and further studies will be necessary to determine whether neurogenesis remains persistently elevated in rats with spontaneous seizures or whether there are subsequent reductions in new cell formation, increases in programmed cell death, or both. The functional significance of new cell
B.H. Cha et al. / Brain & Development 26 (2004) 394–397
formation following SE is also now known and is worthy of further study.
Acknowledgements Supported by a grant from the NINDS (NS27984) to GLH.
[8]
[9]
[10]
[11]
References [12] [1] Lowenstein DH, Alldredge BK. Status epilepticus. N Engl J Med 1998;338:970 –6. [2] Working Group on Status Epilepticus, Treatment of convulsive status epilepticus. Recommendations of the Epilepsy Foundation of America’s Working Group on Status Epilepticus. J Am Med Assoc 1993;270:854 –9. [3] Tauck DL, Nadler JV. Evidence of functional mossy fiber sprouting in the hippocampal formation of kainic acid-treated rats. J Neurosci 1985;5:1016– 22. [4] Represa A, Tremblay E, Ben-Ari Y. Kainate binding sites in the hippocampal mossy fibers: localization and plasticity. Neuroscience 1987;20:739– 48. [5] Buckmaster PS, Dudek FE. Neuron loss, granule cell axon reorganization, and functional changes in the dentate gyrus of epileptic kainate-treated rats. J Comp Neurol 1997;385:385–404. [6] Liu Z, Mikati M, Holmes GL. Mesial temporal sclerosis: pathogenesis and significance. Pediatr Neurol 1995;12:5–16. [7] Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS, Lowenstein DH. Dentate granule cell neurogenesis is increased by
[13]
[14]
[15]
[16]
[17]
397
seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci 1997;17:3727–38. Sankar R, Shin D, Liu H, Katsumori H, Wasterlain CG. Granule cell neurogenesis after status epilepticus in the immature rat brain. Epilepsia 2000;41(Suppl 6):S53–6. Parent JM, Lowenstein DH. Seizure-induced neurogenesis: are more new neurons good for an adult brain? Prog Brain Res 2002;135: 121–31. Cilio MR, Sogawa Y, Cha BH, Liu Z, Huang L-T, Holmes GL. Longterm effects of status epilepticus in the immature brain are age- and model-specific. Epilepsia 2003;44:518–28. Rutten A, van Albada M, Silveira DC, Cha BH, Liu X, Hu YN, et al. Memory impairment following status epilepticus in immature rats: time-course and environmental effects. Eur J Neurosci 2002;20: 501–13. McCabe BK, Silveira DC, Cilio MR, Cha BH, Liu Z, Sogawa Y, Holmes GL. Reduced neurogenesis after neonatal seizures. J Neurosci 2001;21:2094 –103. Sarnat HB, Nochlin D, Born DE. Neuronal nuclear antigen (NeuN): a marker of neuronal maturation in early human fetal nervous system. Brain Dev 1998;20:88–94. Parent JM, Janumpalli S, McNamara JO, Lowenstein DH. Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat. Neurosci Lett 1998;247:9 –12. Scott BW, Wang S, Burnham WM, De Boni U, Wojtowicz JM. Kindling-induced neurogenesis in the dentate gyrus of the rat. Neurosci Lett 1998;248:73 –6. Bengzon J, Kokaia Z, Elme´r E, Nanobashvili A, Kokaia M, Lindvall O. Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures. Proc Natl Acad Sci USA 1997;94: 10432– 7. Scharfman HE, Goodman JH, Sollas AL. Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis. J Neurosci 2000;20:6144–58.