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Kainic acid induces rapid cell death followed by transiently reduced cell proliferation in the immature granule cell layer of rat organotypic hippocampal slice cultures
Brain Research 1035 (2005) 111 – 119 www.elsevier.com/locate/brainres
Research report
Kainic acid induces rapid cell death followed by transiently reduced cell proliferation in the immature granule cell layer of rat organotypic hippocampal slice cultures Matthew Paul Sadgrovea, John E. Chadb, William Peter Graya,* a
Division of Clinical Neurosciences, Southampton Neurosciences Group, School of Medicine, University of Southampton, Room 6207, Level 6, Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, UK b Southampton Neurosciences Group, School of Biological Sciences, University of Southampton, SO16 7PX, Southampton, UK Accepted 19 November 2004
Abstract Brain injury due to seizures results in transiently increased cell proliferation and neurogenesis in the subgranular zone of the adult dentate gyrus. In contrast, the immature postnatal brain appears to be more resistant to cell death after seizure-induced brain injury and paradoxically reacts to seizures by reducing SGZ proliferation. Organotypic hippocampal slice cultures are a useful paradigm for modelling the early postnatal hippocampus. We have investigated the temporal relationship between cell death and cell proliferation after kainate in the granule cell layer of rat organotypic hippocampal slice cultures equivalent to post natal day 11 animals. We found stable numbers and densities of mature thionine stained cells in the granule cell layer over 72 h in control cultures grown in defined medium. We also found a slowly declining cell proliferation rate over the same time period under control conditions. We report evidence of early cell death in the granule cell layer after just 2 h exposure to 5 AM kainate, followed by a significant decrease in cell proliferation in the granule cell layer at 24 h. In contrast to control conditions, cell proliferation rose significantly in the kainate exposed cultures by 72 h back to levels seen at 2 h. There were no significant changes in cell labelling with antibody to activated caspase-3 between kainate treated and control cultures at any time point examined. Our results suggest that kainate-induced injury in the early postnatal hippocampus damages precursor cells contributing to a reduction in granule layer cell proliferation. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: basic mechanisms Keywords: Kainic Acid; Cell Proliferation; Dentate gyrus; Organotypic hippocampal slice culture; BrdU; Immunohistochemistry
1. Introduction Neurogenesis, the production of new neurons, is a restricted event in the adult brain largely confined to the hippocampus [3], the subventricular zone [30] and the olfactory bulb [1,26] in mammals. Within the adult hippocampus neurogenesis is predominantly found in the sub granular zone (SGZ) of the dentate granule cell layer in both
* Corresponding author. Fax: +44 2380 597809. E-mail address: [email protected] (W.P. Gray). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.11.059
rodents [7] and humans [12] and to a lesser extent in the hippocampus proper [40]. The demonstration of functional integration of newly born dentate granule cells into hippocampal circuitry [45,58] has lead to the hypothesis that neurogenesis may be important in learning and memory formation [19,52]. Brain injury due to seizures results in transiently increased cell proliferation and neurogenesis in the subgranular zone (SGZ) of the adult dentate gyrus [4,21,36]. The mechanisms underlying this increase in proliferation in the neurogenic SGZ are largely unknown although 5HT1A receptor activation appears to be important [38]. In the adult
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brain, cell death has been associated with both single and intermittent limbic seizures and seizure-induced apoptotic death has been postulated as the trigger for seizure-induced cell proliferation in the SGZ [4]. However, excitotoxic lesions of the granule cell layer have also been associated with increased SGZ proliferation [19]. In contrast, the immature postnatal brain appears to be more resistant to cell death after seizure-induced brain injury [53] and paradoxically reacts to seizures by reducing SGZ proliferation [31]. We and others have noted an initial delay in the increase in cell proliferation in the SGZ after kainate-induced status epilepticus in the adult brain, with proliferation increasing on day 3 after kainate [22,33]. We wondered if there was a similar delay in the immature brain and if there was a temporal relationship between any detectable cell death and changes in SGZ cell proliferation. Organotypic Hippocampal Slice Cultures (OHSC) offer attractive advantages for examining hippocampal function, by preserving normal hippocampal anatomical/tissue structure and intrinsic functional activity for a number of weeks in vitro [8]. This allows the experimental manipulation of an in vivo-like tissue in a precisely controlled in vitro environment over prolonged periods of time [17,18,32], including the ability to apply a precise concentration of drug or factor for a defined time followed by withdrawal. Because of its isolation from the rest of the brain yet maintenance of internal structure and function, it also allows an examination of the direct effect of an excitotoxic compound on the hippocampus that is not possible in vivo. OHSC maintained in culture over several weeks have been used to model the adult hippocampus for studying excitotoxic damage [59], seizures [42], neuronal degeneration [35] and apoptosis [28]. Organotypic slice cultures have also been used to study brain development [23] and OHSC have recently been shown to be particularly suitable for studying neurogenesis in the dentate gyrus [39], as the dentate develops normally in hippocampal cultures grown in serum-free conditions with a normal structural and temporal development of the neurogenic subgranular zone and the maturation and incorporation of newly born granule cell neurons into the dentate gyrus in a pattern and time almost identical to that in vivo [39]. The utility of OHSC has been recently demonstrated by using them to elucidate MAP kinase signalling regulation in hypoxia induced death and stem cell proliferation [64], showing that hypoxia triggered both JNK and ERK signalling, and that subsequent reoxygenation induced ERK dependant stem cell proliferation. As we were particularly interested in the dentate gyrus of the immature and not the adult brain, we did not leave the OHSC mature over 1–2 weeks in an effort to model the adult hippocampus before performing our experiments. Instead, we used OHSC prepared from 8 day old pups and maintained in vitro for 3 days, temporally equivalent to postnatal day 11 animals when the neurogenic subgranular zone is established [2,46] and when kainate induced injury is associated with changes in precursor cell proliferation in vivo [29]. We adapted a model of kainate-induced injury in
OHSC [41] by using cultures grown on inserts (after Stoppini et al. [55]) in serum-free medium, exposing them to 5 AM kainic acid for 24 h, to model the effect of kainate on the dentate gyrus of P11 animals. We used serum-free medium as serum reduces proliferation [43] and neurogenesis [39] in OHSC. We therefore have set about examining changes in cell proliferation and cell death in the SGZ of organotypic hippocampal slice cultures under control conditions and after kainate-induced injury.
2. Materials and methods 2.1. Preparation of cultures All animal experiments were carried out under Home Office licence and were approved by the University Bioethics Committee. Organotypic hippocampal slice cultures were prepared using the method originally described by Stoppini [55] and modified by Pringle [37]. Briefly, eight-day-old Wistar rat pups were killed by decapitation, without anaesthesia, and the hippocampi rapidly dissected out in GEY’s balanced salt solution (Life Technologies, Rockville, USA) at 4 8C. The corpus callosum was bisected above the thalamus and the posterior margin of each cortical hemisphere rolled back. Each hippocampus was separated from the overlaying cortical white matter along the natural separating line of the alveus hippocampus. Care was taken in removing as much white matter as possible, including the subiculum though some white matter remained. Transverse sections, 400 Am thick were cut on a McIlwain tissue chopper (Mickle Laboratory Engineering Ltd., Surrey, UK) and placed on Millicell-CM semi-porous membranes (0.4 Am pore; Millipore, 4 cultures per insert, in six well culture plates (Nunc), above Neurobasal-Ak medium (98%; Gibco), supplemented with B27 (2%; Gibco) and glutamine (0.5 mM; Sigma). We did not use serum containing medium as it reduces cell proliferation [43] and neurogenesis [39] in OHSC. All cultures were maintained in vitro at 37 8C with 100% humidity and a 5% CO2, 20% O2, 75% N2 atmosphere. 2.2. Slice culture matching We have noted marked differences in the number of granule cells and proliferating cells in the dentate gyrus of slice cultures taken from the full septo-temporal extent of the hippocampus, with granule cell layer size increasing from temporal to septal ends (WPG-unpublished data). To correct for possible confounding effects of the site of origin of the culture on the number of proliferating cells, 8 contiguous position-matched cultures were taken from the mid-septal region of each hippocampus from each animal. At each timepoint examined, cultures exposed to kainate or control medium were matched for septo-temporal position. Cultures
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were also matched from each animal so that every animal contributed one culture from each septo-temporal matched pair to each experimental group (kainate or controls).
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was repeated collecting another section on each slide, and the sections stored at 74 8C for further processing. 2.5. Immunohistochemistry
2.3. Kainate treatment and controls After 3 days in vitro appropriate cultures were transferred to either Neurobasal-based medium containing kainate (5 AM) or fresh Neurobasal-based medium for either 2 or 24 h, after which time fresh Neurobasal only medium was applied to both conditions (Fig. 1). To assess cell proliferation rate, the thymidine analogue bromodeoxyuridine (BrdU) (10 AM) was added to all the cultures 2 h prior to fixation in 4% paraformaldehyde (PFA). Kainate-treated cultures and paired animal and position matched controls were fixed 2, 24 and 72 h after kainate was added (Fig. 1). 2.4. Tissue processing Following 24 h in 4% paraformaldehyde (PFA) at 4 8C, cultures were transferred to a 30% sucrose solution for 24 h. Single cultures were then placed on the flat base of a metal dish, and the excess liquid removed using filter paper. A drop of Tissue-TekR (Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands) was applied to the culture and the dish containing the culture was then placed in liquid nitrogen, until frozen. The dish was quickly transferred to the cold block ( 30 8C) in the cryostat, where the chamber temperature was maintained below 23 8C and the culture embedded in more Tissue-TekR. The embedded frozen culture was then mounted on an orientating chuck and careful approach sections were taken, correcting the orientation of the culture until cutting blade and culture lay in parallel planes. The cryostat was then set to section at 10-Am intervals. The initial two sections were discarded as they generally were incomplete and the topmost part of OHSC have a glial scar [9]. The next four 10-Am sections were collected on separate gelatinised slides, this process
Adjacent sections from each culture were processed for thionine (total cell number), Fluoro-Jade B (Sigma) and 4V,6diamidino-2-phenylindole dihydrochloride (DAPI; Sigma) staining (to estimate degenerating and necrotic cell number), BrdU (proliferating cells) and activated caspase-3 (cells undergoing apoptosis). Tissue processed for BrdU immunohistochemistry was exposed to 2N HCl at 37 8C for 20 min and was subsequently washed in Boric acid (0.1 M, pH 8.5) for 10 min. All tissue for immunohistochemistry then received multiple washes in Tris-buffered saline (TBS; 0.1 M, pH 7.4), followed by overnight incubation with either rat anti-BrdU (1:1000; Harlan-SeraLab, Loughsborough, UK) or rabbit anti-activated caspase-3 (1:1000; New England Biolabs, Beverly, MA, USA) in TBS containing 0.1% triton and 0.05% Bovine Serum Albumin (TBS-TS) at 4 8C. Tissue processed for BrdU staining also had 1% Normal Goat Serum (Sigma) present with the primary antibody. Following multiple washes in TBS, tissue was incubated in TBS-TS with either biotinylated goat anti-rat (1:200; Vector) or biotinylated swine anti-rabbit (1:500; DAKO) secondary antibody for 1 h. TBS washes were repeated and a horseradish peroxidase conjugated Streptavidin–biotin complex (1:200; DAKO) in TBS-TS was applied for 1 h. Further washing in TBS was followed by visualisation with 3,3-diaminobenzene (DAB; Vector) and a final set of washes. Thionine staining was obtained by placing slides in 1% thionine and differentiating in 90% ethanol +5% acetic acid until a consistent staining intensity was observed. Tissue processed for BrdU or activated caspase-3 staining also received a light Thionine counter-stain to facilitate granule cell layer identification. Slides were then allowed to dry and were dehydrated with 1-min washes in alcohol (70%, 90% and 2 100%) and then xylene for 1 min before being cover-slipping with DPX (Sigma). 2.6. Fluoro-Jade B staining
Fig. 1. Experimental Paradigm. Organotypic hippocampal slice cultures matched for septo-temporal position and animal were grown for 3 days in serum-free medium. Cultures were then either transferred to wells containing fresh medium or fresh medium plus 5 AM kainate. Group 1 cultures were fixed in 4% paraformaldehyde 2 h after exposure to kainate or fresh medium alone. Group 2 cultures were fixed 24 h after exposure to either kainate or fresh medium and Group 3 cultures were all changed to fresh medium 24 h after exposure to either kainate containing or normal medium and fixed 48 h later. All cultures had BrdU added to the medium 2 h before fixation to assess cell proliferation rates.
Fluoro-Jade B staining was performed using a modification of the method described by Schmued et al. [48]. Sections treated for Fluoro-Jade B were first immersed in a solution containing 1% sodium hydroxide in 80% alcohol for 2 min followed by 2 min rinses in 70% ethanol and distilled water. Slides were then placed in a 0.06% potassium permanganate solution, on a shaker table, for 10 min. After a further 2-min rinse in distilled water, slides were transferred to the Fluoro-Jade B/DAPI staining solution. The Fluoro-Jade B/DAPI solution was made immediately prior to use from stock solutions of FluoroJade B and DAPI (both 10 mg/100 ml) in 0.1% acetic acid, in the proportions 4% Fluoro-Jade B, 2% DAPI and 92%
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acetic acid, producing final dye concentrations of 0.0004% and 0.0002% for Fluoro-Jade B and DAPI, respectively. After 30 min in the staining solution, the slides were washed in distilled water (3 1 min) and dried in a slide dryer (37 8C, 2 h). Once dry, slides were placed in xylene for 1 min and then cover-slipped with DPX.
layer. The same threshold was used for kainate and control cultures and all images were taken after the intensity of the fluorescence source had stabilised. For all quantification, slides were coded and counts carried out with the examiner blind to the time point and treatment of each section.
2.7. Cell counting
2.8. Statistical analysis
Unbiased stereological cell counting was carried out using Stereo Investigator (Microbrightfield, Williston, VT, USA) software connected to a Dialux 22 microscope (Leica) with a motorised x–y stage (Ludl, Hawthorne, NY 10532, USA) and a DEI-750D video camera (Optronics, CA 93117, USA) to obtain images at 10, 20 and 63 magnification. We used 10 Am sections as previous work in our lab demonstrated that anti-BrdU antibody staining penetrated only 12–15 Am from a culture surface. The area of the granule cell layer was traced using a mouse controlled cursor on a digital image of the thionine stained culture section and measured by the Stereo Investigator Software. This tracing was stored and used as a mask of the granule cell layer for BrdU and activated caspase-3 cell counting (vide infra). An estimate of granule cell number per section was obtained using the fractionator method [62] with a sampling repeat of 0.1 mm on a randomly rotated grid and a counting frame area of 6.25 10 4 mm2. Processing of the sections reduced their thickness to 5 Am and therefore we did not use a guard area on either section surface. This method gives an unbiased cell count in the X and Y dimensions but will tend to over estimate cell counts from the whole culture or the hippocampus because cells present on two sections would be counted twice. However, we were comparing two conditions in 10-Am sections rather than estimating absolute granule cell numbers in a single culture or sampled hippocampus and therefore this approximation was acceptable. Cells in the granule cell layer that were obviously pyknotic were not counted when generating estimates of normal cell number. The mask of the granule cell layer also produced the area measurement, and was superimposed on adjacent sections from the same culture stained for BrdU and activated caspase-3 to enable exhaustive cell counting to be carried out in the same area as the thionine cell counts. Images of DAPI and Fluoro-Jade B staining were captured on an inverted Leica DM-IRBE epifluorescence microscope (Milton Keynes, U.K.) using a cooled Hamamatsu digital camera. The area of DAPI or Fluoro-Jade B fluorescence was determined in NIH image (http://rsb.info. nih.gov/nih-image/), using the density slice function, applied to the granule cell layer as a region of interest. Cell death was expressed as a percentage of the area in which Fluoro-Jade B fluorescence was detected above threshold within the cell layer divided by the area in which DAPI fluorescence was detected above a threshold in the same cell
Only sections from cultures in which data was available for total cell counts, BrdU cell counts and Fluoro-Jade B fluorescence quantification experiments were used for statistical analysis. Activated caspase-3 matched cultures formed a smaller subset of this group. Statistical analysis across time-points, within either treatment or control groups was by one-way ANOVA and post hoc testing, or by paired two tailed Student’s t test as appropriate. Data are expressed as means F SEM; P b 0.05 was considered significant.
3. Results 3.1. Granule cell number, granule cell layer area and cell density in control OHSC The mean number of thionine-stained cells in the granule cell layer in a 10-Am thick section over all three time points under control conditions was 1796 F 136 cells with no significant difference between 2 h (1576 F 190 cells, n = 8), 24 h (2044 F 108 cells, n = 9) and 72 h (1770 F 105 cells, n = 5) time points (Fig. 2). Neither was there a significant difference between the area of the granule cell layer at 2 h (0.105 F 0.010 mm2; n = 8), 24 h (0.130 F 0.006 mm2; n = 9) or 72 h (0.121 F 0.015 mm2; n = 5). Similarly, the density of thionine stained cells was unchanged across all time points with no tendency toward dispersion of the granule cell layer. 3.2. Granule cell number, granule cell layer area and cell density after 5 lM kainate Sections from kainate treated cultures did not differ significantly from position and time matched controls in granule cell layer thionine stained cell counts at 2 h (kainate, 1624 F 265 cells vs. control, 1576 F 190, both n = 8), 24 h (kainate, 1617 F 114 cells vs. control, 2044 F 107, both n = 9) or 72 h (Kainate, 1744 F 269 cells vs. control, 1769 F 105, both n = 5). Comparisons between the different times also revealed no significant differences in the number of cells in kainate treated cultures (Fig. 2). Similarly, dentate granule cell layer area showed no significant change between 2 h (kainate, 0.111 F 0.015 mm2 n = 8), 24 h (kainate, 0.115 F 0.008 mm2 n = 9) or 72 h (kainate, 0.146 F 0.019 mm2 n = 5) after the start of the experiment. The increase in granule cell layer area noted
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in vitro, with approximately 3% of total cells incorporating BrdU at 2 h. BrdU labelling in control cultures declined significantly over time with cell counts at 72 h (25.4 F 4.41 cells, n = 5) lower than at both 2 h (48.25 F 4.85 cells, n = 8, P b 0.001) and 24 h (42.11 F 4.23 cells, n = 9, P b 0.01), and although BrdU cell counts at 2 h and 24 h are not significantly different they follow this trend (Fig. 2). The number of BrdU positive cells observed was significantly decreased by treatment with 5 AM kainate at 24 h after application (kainate, 16.67 F 2.84 cells vs. control, 42.11 F 4.23 cells, n = 9, P b 0.01) but were not significantly different at 2 h (kainate, 39.25 F 3.46 cells vs. control, 48.25 F 4.85 cells, n = 8) or at 72 h (kainate, 37.0 F 4.68 cells vs. control, 25.4 F 4.41 cells, n = 5; Fig. 2B). After kainate treatment BrdU cell counts decreased significantly between 2 h (39.25 F 3.46 cells, n = 8) and 24 h (16.67 F 2.84 cells, n = 9, P b 0.001) and then increased significantly between 24 h (16.67 F 2.84 cells, n = 9) and 72 h (37 F 4.68 cells, n = 5, P b 0.001), finishing at a similar level to initial cell counts (Fig. 2). All P values are derived from an ANOVA with Tukey’s post hoc test for multiple comparisons. 3.4. Effect of kainate on cell death in the granule cell layer and subgranular zone Fig. 2. Granule layer cell counts and cell proliferation. (A) Counts of healthy thionine-stained cells per 10 Am section of the granule cell layer from cultures exposed to medium containing 5 AM kainate or control medium at 2, 24 and 72 h. There were no significant differences between kainate and control groups at any time point examined. (B) Number of cells incorporating BrdU in the granule cell layer per section at 2, 24 and 72 h. There was a significant reduction in cell proliferation rate in kainate treated cultures at 24 h compared to controls and between kainate cultures at 2 h. Cell proliferation also increased significantly in kainate cultures between 24 and 72 h. (***P b 0.001: ANOVA with Tukey’s post hoc testing).
at 72 h post kainate treatment just failed to reach statistical significance on post hoc testing. However, there was a significant decrease in cell density in the granule cell layer in kainate treated cultures at 72 h compared to time and position matched control cultures (kainate, 11,895 F 935 cells mm 2 vs. controls, 15,597 cells mm 2, both n = 5; P b 0.05, ANOVA with Tukey’s post hoc test). Two way ANOVA confirmed an effect of kainate on cell density ( P b 0.05) and showed that time had no independent effect. This is consistent with the known effect of kainate on dispersion of the granule cell layer [6]. 3.3. Effect of kainate on cell proliferation in the granule cell layer and subgranular zone Cell proliferation, as judged by incorporation of the thymidine analogue Bromodeoxyuridine (BrdU) over 2 h, was low under serum-free growth conditions after 3 days
Cell death was measured in the cultures by considering the percentage of the granule cell layer staining for FluoroJade B, an anionic fluorescein derivative, generating a fluorescent histochemical stain which is specific for degenerating neurons [13]. Kainate induced a significant increase in the amount of Fluoro-Jade B staining in the granule cell layer, detected 2 h after kainate addition (kainate, 15.3 F 3.0%, vs. control, 4.0 F 1.9%, n = 8, P b 0.05) (Figs. 3A–C) but not at 24 h (kainate, 4.9 F 1.1%, vs. control, 5.4 F 1.7%, n = 9) or 72 h (kainate, 3.8 F 1.5%, vs. control, 6.4 F 2.6%, n = 5) compared with matched controls. In control cultures the levels of FluoroJade B detected did not vary significantly. In kainate treated cultures Fluoro-Jade B was significantly higher at 2 h (15.3 F 3.0%, n = 8) than at 24 h (4.9 F 1.1%, n = 9, P b 0.001) and 72 h (3.8 F 1.5%, n = 5, P b 0.001) (Fig. 3F). Cell death as evidenced by pyknosis and hyperchromatic nuclei on DAPI staining was predominantly localised to the inner half of the granule cell layer (Fig. 3D) and often co-localised with Fluoro-Jade B staining (Fig. 3C). To assess the contribution of apoptosis to this cell death, we examined the number of cells expressing activated cleaved caspase-3 in the same sectioned cultures. Activated caspase-3-positive cells did not vary significantly between control cultures over time. Sections from kainate treated cultures did not differ significantly from position and time matched controls in the number of cells expressing activated caspase-3, at 2 h (kainate,
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Fig. 3. Cell death in the granule cell layer and hilus after kainate. (A) and (B) Corresponding 200 images of a 10-Am section of the granule cell layer 2 h after kainate, stained with DAPI (blue) (A) and Fluoro-Jade B (green) (B). The granule cell layer is outlined in red and CA3c in white in A. Note the pyknotic cells arrowed in A located at the tip of the granule layer blade and running back along the inner half of the body. (C) Higher power view of merged images A and B showing co-localisation of pyknotic cells with Fluoro-Jade B (white arrow). Also note the neuronal morphology of degenerating Fluoro-Jade B positive cells (Yellow arrow) located in the more mature area of the granule cell layer and also in the hilus. (D) Lower power view of the granule cell layer to show the preponderance of Flouro-Jade B labelling (green) in the inner half of the granule cell layer (white arrows) and also in the hilus and area CA3. (E) DAB stained cell nuclei (brown) immunostained for antibody against activated-caspase-3 in the granule cell layer which has been thionine counterstained (light blue) showing cells undergoing apoptosis in the granule cell layer. These cells were distributed throughout the thickness of the granule cell layer. Asterix denotes the inner border of the granule layer. (F) There was a significant increase in Fluoro-Jade B labelling in kainate treated cultures at 2 h compared to controls and between kainate treated cultures at 2 and both 24 and 72 h. (G) Quantification of apoptotic death in the granule cell layer showed no difference between kainate and control cultures in activated-caspase-3 expression at any time point. (*P b 0.05: ***P b 0.001: ANOVA with Tukey’s post hoc test). Scale bars = 50 Am.
34.0 F 3.19 cells vs. control, 26.25 F 5.36 cells, n = 4), 24 h (kainate, 24.43 F 2.14 cells vs. control, 29.57 F 5.20 cells, n = 7) or 72 h after the start of the experiment (kainate, 22.0 F 2.0 cells vs. control, 24.0 F 2.0 cells, n = 2) (Figs. 3E and G).
4. Discussion The rapid cell death in the granule cell layer after kainate had features of necrotic cell death with condensed hyperchromatic nuclei on DAPI staining (Fig. 3A) and no
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significant increase in staining for activated caspase-3 at any time point (Fig. 3G). Despite the assertion of resistance of the immature brain to seizure-induced damage [53,54], early cell death in the inner half of the granule cell layer has been reported after perforant path stimulation in early postnatal life [57] with a similar time-course to our data. Although Thompson et al. categorised the induced cell death as apoptotic on the basis of nick-end-labelling and eosinophilic staining, more recent studies in adult tissue have demonstrated that status epilepticus induced neuronal injury is predominantly necrotic with appearances of eosinophilic staining, nuclear pyknosis, chromatin condensation, and internucleosomal DNA fragmentation [15] and does not involve neuronal activation of caspase-3 [16,34]. This does not rule out apoptotic cell death after perforant path stimulation or kainate but suggests that necrotic cell death may play the major role in both models, reflecting seizureinduced energy failure [61]. We extend these data by showing that kainate rapidly damages neurons in the inner half of the immature granule cell layer as evidenced by Fluoro-Jade B staining, a specific marker of degenerating neurons in the hippocampus [47] (Figs. 3B and C). Cells staining for Fluoro-Jade B often had nuclear pyknosis (Fig. 3C), which detects cell death as early as 1 h in OHSCs after oxygen glucose deprivation [5]. A previous in vivo study examining the effects of kainateinduced seizures on neurogenesis in the immature hippocampus at P13, found no evidence of cell death using histological stains in the inner half of the granule cell layer [29]. However, in this study, animals were sacrificed 48 h after the kainate-induced seizures by which time the FluoroJade B staining had reverted to control levels in our study and pyknotic cell death had been cleared in the study by Thompson et al. [57]. A study in adult animals after pilocarpine induced status epilepticus also found pyknotic cell death within 3 h in the inner one third of the granule cell layer [38]. We only counted healthy looking granule cells on thionine staining which may explain why we observed no fall in thionine cell counts in kainate treated cultures at 2 h compared to 24 h (Fig. 2). The neurogenic subgranular zone (SGZ) where neurogenesis continues into adult life is formed from P5 by migration of precursor cells from the hilar tertiary germinal matrix [2] and is established by P11–14 [2,46], occupying the inner one third of the granule cell layer where we observed early kainate-induced cell death. The SGZ contains both precursor cells and their progeny-immature granule cell neurons. Kainate was associated with a significant reduction in absolute cell proliferation in the granule cell layer at 24 h as measured by BrdU incorporation (Fig. 2). This was not due to loss of growth factors caused by placing the kainate exposed cultures in fresh medium at 24 h as matched control cultures were also changed to fresh medium at this timepoint. Given the preceding cell death localised to the proliferative SGZ, one possibility is that kainate kills a
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subpopulation of dividing precursor cells, resulting in a decreased proliferation rate 24 h after status. Why these precursor/immature cells might be more susceptible to excitotoxic damage remains a matter for speculation. Kainate receptor expression is found in the immature dentate [56] although the cellular expression pattern is not known. Studies have also shown that immature granule cell neurons are intrinsically more excitable and susceptible to LTP [52,60] in keeping with their putative role in hippocampal dependant learning and memory [20,50]. This vulnerability also appears to occur in adult life with increased cell death in the neurogenic subgranular zone 5 h after a single kindling stimulation or 3 h after pilocarpine induced status epilepticus [4,38]. An alternative explanation, based on evidence that inflammation inhibits cell proliferation in the adult dentate [11], is that kainate-induced inflammation decreases precursor cell proliferation. However, the time course of this in vivo effect is over a number of days rather than hours and involves microglia [11], which cannot be recruited in the organotypic preparation as it has no associated circulation. One of the intriguing differences between the adult and postnatal dentate is the reduction in precursor proliferation after seizures seen in the early postnatal brain [10,29,31] compared to increased proliferation in the later postnatal [44] and adult brain [4,22,36]. Using intraperitoneal kainate, Liu et al. [29] found a reduction in cell proliferation 48 h after a single kainate-induced status in P13 animals consistent with our finding of a reduction at 24 h. Although they did not find evidence of increased cell death at the time of the reduction in proliferation, we have identified an earlier transient phase of necrotic cell death, consistent with a previous report in vivo [57], which precedes the reduction in granule layer cell proliferation. Our results suggest that kainate-induced status in the early postnatal hippocampus causes rapid necrotic death of a proportion of precursor/ immature cells in the granule cell layer which results in a reduction in cell proliferation. Our hypothesis would predict that a greater number of seizures would cause a greater degree of precursor cell loss which is supported by Liu et al’s finding that three episodes of kainate-induced seizures was associated with a greater reduction in proliferation [29] and the original work of McCabe et al. where multiple flurothyl-induced seizures were necessary to reduce granule layer proliferation in P0–P4 animals [31]. Further work is clearly necessary to see if blocking kainate-induced death attenuates any subsequent changes in cell proliferation in the immature granule cell layer. The reduction in granule layer cell proliferation is transient since BrdU incorporation recovered to control levels by 72 h in our study. It is likely that the neurotrophic response to kainate involving BDNF, GDNF, and VEGF [25,49,51], all of which stimulate precursor cell proliferation in the dentate gyrus [14,27,63], mediates this recovery. Recovery of proliferation after multiple flurothyl-induced
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seizures has also been reported in the immature hippocampus in vivo [24] with greater baseline neurogenesis in these animals in subsequent adulthood. This differs from the response in the adult dentate where kainate transiently increases proliferation above control levels earlier at 72 h [33] and returns to baseline by 10 days. We did not examine proliferation beyond the 72-h time point as baseline proliferation declined to low levels in control cultures and became more variable. It is possible that the delay in recovery of proliferation is related to the severity of the initial cell death and that multiple seizures or episodes of status injure a greater number of precursors which take a longer time to regenerate the normal proliferative pool.
Acknowledgments This research was funded by Hope and the University of Southampton.
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Research report
Kainic acid induces rapid cell death followed by transiently reduced cell proliferation in the immature granule cell layer of rat organotypic hippocampal slice cultures Matthew Paul Sadgrovea, John E. Chadb, William Peter Graya,* a
Division of Clinical Neurosciences, Southampton Neurosciences Group, School of Medicine, University of Southampton, Room 6207, Level 6, Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, UK b Southampton Neurosciences Group, School of Biological Sciences, University of Southampton, SO16 7PX, Southampton, UK Accepted 19 November 2004
Abstract Brain injury due to seizures results in transiently increased cell proliferation and neurogenesis in the subgranular zone of the adult dentate gyrus. In contrast, the immature postnatal brain appears to be more resistant to cell death after seizure-induced brain injury and paradoxically reacts to seizures by reducing SGZ proliferation. Organotypic hippocampal slice cultures are a useful paradigm for modelling the early postnatal hippocampus. We have investigated the temporal relationship between cell death and cell proliferation after kainate in the granule cell layer of rat organotypic hippocampal slice cultures equivalent to post natal day 11 animals. We found stable numbers and densities of mature thionine stained cells in the granule cell layer over 72 h in control cultures grown in defined medium. We also found a slowly declining cell proliferation rate over the same time period under control conditions. We report evidence of early cell death in the granule cell layer after just 2 h exposure to 5 AM kainate, followed by a significant decrease in cell proliferation in the granule cell layer at 24 h. In contrast to control conditions, cell proliferation rose significantly in the kainate exposed cultures by 72 h back to levels seen at 2 h. There were no significant changes in cell labelling with antibody to activated caspase-3 between kainate treated and control cultures at any time point examined. Our results suggest that kainate-induced injury in the early postnatal hippocampus damages precursor cells contributing to a reduction in granule layer cell proliferation. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: basic mechanisms Keywords: Kainic Acid; Cell Proliferation; Dentate gyrus; Organotypic hippocampal slice culture; BrdU; Immunohistochemistry
1. Introduction Neurogenesis, the production of new neurons, is a restricted event in the adult brain largely confined to the hippocampus [3], the subventricular zone [30] and the olfactory bulb [1,26] in mammals. Within the adult hippocampus neurogenesis is predominantly found in the sub granular zone (SGZ) of the dentate granule cell layer in both
* Corresponding author. Fax: +44 2380 597809. E-mail address: [email protected] (W.P. Gray). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.11.059
rodents [7] and humans [12] and to a lesser extent in the hippocampus proper [40]. The demonstration of functional integration of newly born dentate granule cells into hippocampal circuitry [45,58] has lead to the hypothesis that neurogenesis may be important in learning and memory formation [19,52]. Brain injury due to seizures results in transiently increased cell proliferation and neurogenesis in the subgranular zone (SGZ) of the adult dentate gyrus [4,21,36]. The mechanisms underlying this increase in proliferation in the neurogenic SGZ are largely unknown although 5HT1A receptor activation appears to be important [38]. In the adult
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brain, cell death has been associated with both single and intermittent limbic seizures and seizure-induced apoptotic death has been postulated as the trigger for seizure-induced cell proliferation in the SGZ [4]. However, excitotoxic lesions of the granule cell layer have also been associated with increased SGZ proliferation [19]. In contrast, the immature postnatal brain appears to be more resistant to cell death after seizure-induced brain injury [53] and paradoxically reacts to seizures by reducing SGZ proliferation [31]. We and others have noted an initial delay in the increase in cell proliferation in the SGZ after kainate-induced status epilepticus in the adult brain, with proliferation increasing on day 3 after kainate [22,33]. We wondered if there was a similar delay in the immature brain and if there was a temporal relationship between any detectable cell death and changes in SGZ cell proliferation. Organotypic Hippocampal Slice Cultures (OHSC) offer attractive advantages for examining hippocampal function, by preserving normal hippocampal anatomical/tissue structure and intrinsic functional activity for a number of weeks in vitro [8]. This allows the experimental manipulation of an in vivo-like tissue in a precisely controlled in vitro environment over prolonged periods of time [17,18,32], including the ability to apply a precise concentration of drug or factor for a defined time followed by withdrawal. Because of its isolation from the rest of the brain yet maintenance of internal structure and function, it also allows an examination of the direct effect of an excitotoxic compound on the hippocampus that is not possible in vivo. OHSC maintained in culture over several weeks have been used to model the adult hippocampus for studying excitotoxic damage [59], seizures [42], neuronal degeneration [35] and apoptosis [28]. Organotypic slice cultures have also been used to study brain development [23] and OHSC have recently been shown to be particularly suitable for studying neurogenesis in the dentate gyrus [39], as the dentate develops normally in hippocampal cultures grown in serum-free conditions with a normal structural and temporal development of the neurogenic subgranular zone and the maturation and incorporation of newly born granule cell neurons into the dentate gyrus in a pattern and time almost identical to that in vivo [39]. The utility of OHSC has been recently demonstrated by using them to elucidate MAP kinase signalling regulation in hypoxia induced death and stem cell proliferation [64], showing that hypoxia triggered both JNK and ERK signalling, and that subsequent reoxygenation induced ERK dependant stem cell proliferation. As we were particularly interested in the dentate gyrus of the immature and not the adult brain, we did not leave the OHSC mature over 1–2 weeks in an effort to model the adult hippocampus before performing our experiments. Instead, we used OHSC prepared from 8 day old pups and maintained in vitro for 3 days, temporally equivalent to postnatal day 11 animals when the neurogenic subgranular zone is established [2,46] and when kainate induced injury is associated with changes in precursor cell proliferation in vivo [29]. We adapted a model of kainate-induced injury in
OHSC [41] by using cultures grown on inserts (after Stoppini et al. [55]) in serum-free medium, exposing them to 5 AM kainic acid for 24 h, to model the effect of kainate on the dentate gyrus of P11 animals. We used serum-free medium as serum reduces proliferation [43] and neurogenesis [39] in OHSC. We therefore have set about examining changes in cell proliferation and cell death in the SGZ of organotypic hippocampal slice cultures under control conditions and after kainate-induced injury.
2. Materials and methods 2.1. Preparation of cultures All animal experiments were carried out under Home Office licence and were approved by the University Bioethics Committee. Organotypic hippocampal slice cultures were prepared using the method originally described by Stoppini [55] and modified by Pringle [37]. Briefly, eight-day-old Wistar rat pups were killed by decapitation, without anaesthesia, and the hippocampi rapidly dissected out in GEY’s balanced salt solution (Life Technologies, Rockville, USA) at 4 8C. The corpus callosum was bisected above the thalamus and the posterior margin of each cortical hemisphere rolled back. Each hippocampus was separated from the overlaying cortical white matter along the natural separating line of the alveus hippocampus. Care was taken in removing as much white matter as possible, including the subiculum though some white matter remained. Transverse sections, 400 Am thick were cut on a McIlwain tissue chopper (Mickle Laboratory Engineering Ltd., Surrey, UK) and placed on Millicell-CM semi-porous membranes (0.4 Am pore; Millipore, 4 cultures per insert, in six well culture plates (Nunc), above Neurobasal-Ak medium (98%; Gibco), supplemented with B27 (2%; Gibco) and glutamine (0.5 mM; Sigma). We did not use serum containing medium as it reduces cell proliferation [43] and neurogenesis [39] in OHSC. All cultures were maintained in vitro at 37 8C with 100% humidity and a 5% CO2, 20% O2, 75% N2 atmosphere. 2.2. Slice culture matching We have noted marked differences in the number of granule cells and proliferating cells in the dentate gyrus of slice cultures taken from the full septo-temporal extent of the hippocampus, with granule cell layer size increasing from temporal to septal ends (WPG-unpublished data). To correct for possible confounding effects of the site of origin of the culture on the number of proliferating cells, 8 contiguous position-matched cultures were taken from the mid-septal region of each hippocampus from each animal. At each timepoint examined, cultures exposed to kainate or control medium were matched for septo-temporal position. Cultures
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were also matched from each animal so that every animal contributed one culture from each septo-temporal matched pair to each experimental group (kainate or controls).
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was repeated collecting another section on each slide, and the sections stored at 74 8C for further processing. 2.5. Immunohistochemistry
2.3. Kainate treatment and controls After 3 days in vitro appropriate cultures were transferred to either Neurobasal-based medium containing kainate (5 AM) or fresh Neurobasal-based medium for either 2 or 24 h, after which time fresh Neurobasal only medium was applied to both conditions (Fig. 1). To assess cell proliferation rate, the thymidine analogue bromodeoxyuridine (BrdU) (10 AM) was added to all the cultures 2 h prior to fixation in 4% paraformaldehyde (PFA). Kainate-treated cultures and paired animal and position matched controls were fixed 2, 24 and 72 h after kainate was added (Fig. 1). 2.4. Tissue processing Following 24 h in 4% paraformaldehyde (PFA) at 4 8C, cultures were transferred to a 30% sucrose solution for 24 h. Single cultures were then placed on the flat base of a metal dish, and the excess liquid removed using filter paper. A drop of Tissue-TekR (Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands) was applied to the culture and the dish containing the culture was then placed in liquid nitrogen, until frozen. The dish was quickly transferred to the cold block ( 30 8C) in the cryostat, where the chamber temperature was maintained below 23 8C and the culture embedded in more Tissue-TekR. The embedded frozen culture was then mounted on an orientating chuck and careful approach sections were taken, correcting the orientation of the culture until cutting blade and culture lay in parallel planes. The cryostat was then set to section at 10-Am intervals. The initial two sections were discarded as they generally were incomplete and the topmost part of OHSC have a glial scar [9]. The next four 10-Am sections were collected on separate gelatinised slides, this process
Adjacent sections from each culture were processed for thionine (total cell number), Fluoro-Jade B (Sigma) and 4V,6diamidino-2-phenylindole dihydrochloride (DAPI; Sigma) staining (to estimate degenerating and necrotic cell number), BrdU (proliferating cells) and activated caspase-3 (cells undergoing apoptosis). Tissue processed for BrdU immunohistochemistry was exposed to 2N HCl at 37 8C for 20 min and was subsequently washed in Boric acid (0.1 M, pH 8.5) for 10 min. All tissue for immunohistochemistry then received multiple washes in Tris-buffered saline (TBS; 0.1 M, pH 7.4), followed by overnight incubation with either rat anti-BrdU (1:1000; Harlan-SeraLab, Loughsborough, UK) or rabbit anti-activated caspase-3 (1:1000; New England Biolabs, Beverly, MA, USA) in TBS containing 0.1% triton and 0.05% Bovine Serum Albumin (TBS-TS) at 4 8C. Tissue processed for BrdU staining also had 1% Normal Goat Serum (Sigma) present with the primary antibody. Following multiple washes in TBS, tissue was incubated in TBS-TS with either biotinylated goat anti-rat (1:200; Vector) or biotinylated swine anti-rabbit (1:500; DAKO) secondary antibody for 1 h. TBS washes were repeated and a horseradish peroxidase conjugated Streptavidin–biotin complex (1:200; DAKO) in TBS-TS was applied for 1 h. Further washing in TBS was followed by visualisation with 3,3-diaminobenzene (DAB; Vector) and a final set of washes. Thionine staining was obtained by placing slides in 1% thionine and differentiating in 90% ethanol +5% acetic acid until a consistent staining intensity was observed. Tissue processed for BrdU or activated caspase-3 staining also received a light Thionine counter-stain to facilitate granule cell layer identification. Slides were then allowed to dry and were dehydrated with 1-min washes in alcohol (70%, 90% and 2 100%) and then xylene for 1 min before being cover-slipping with DPX (Sigma). 2.6. Fluoro-Jade B staining
Fig. 1. Experimental Paradigm. Organotypic hippocampal slice cultures matched for septo-temporal position and animal were grown for 3 days in serum-free medium. Cultures were then either transferred to wells containing fresh medium or fresh medium plus 5 AM kainate. Group 1 cultures were fixed in 4% paraformaldehyde 2 h after exposure to kainate or fresh medium alone. Group 2 cultures were fixed 24 h after exposure to either kainate or fresh medium and Group 3 cultures were all changed to fresh medium 24 h after exposure to either kainate containing or normal medium and fixed 48 h later. All cultures had BrdU added to the medium 2 h before fixation to assess cell proliferation rates.
Fluoro-Jade B staining was performed using a modification of the method described by Schmued et al. [48]. Sections treated for Fluoro-Jade B were first immersed in a solution containing 1% sodium hydroxide in 80% alcohol for 2 min followed by 2 min rinses in 70% ethanol and distilled water. Slides were then placed in a 0.06% potassium permanganate solution, on a shaker table, for 10 min. After a further 2-min rinse in distilled water, slides were transferred to the Fluoro-Jade B/DAPI staining solution. The Fluoro-Jade B/DAPI solution was made immediately prior to use from stock solutions of FluoroJade B and DAPI (both 10 mg/100 ml) in 0.1% acetic acid, in the proportions 4% Fluoro-Jade B, 2% DAPI and 92%
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acetic acid, producing final dye concentrations of 0.0004% and 0.0002% for Fluoro-Jade B and DAPI, respectively. After 30 min in the staining solution, the slides were washed in distilled water (3 1 min) and dried in a slide dryer (37 8C, 2 h). Once dry, slides were placed in xylene for 1 min and then cover-slipped with DPX.
layer. The same threshold was used for kainate and control cultures and all images were taken after the intensity of the fluorescence source had stabilised. For all quantification, slides were coded and counts carried out with the examiner blind to the time point and treatment of each section.
2.7. Cell counting
2.8. Statistical analysis
Unbiased stereological cell counting was carried out using Stereo Investigator (Microbrightfield, Williston, VT, USA) software connected to a Dialux 22 microscope (Leica) with a motorised x–y stage (Ludl, Hawthorne, NY 10532, USA) and a DEI-750D video camera (Optronics, CA 93117, USA) to obtain images at 10, 20 and 63 magnification. We used 10 Am sections as previous work in our lab demonstrated that anti-BrdU antibody staining penetrated only 12–15 Am from a culture surface. The area of the granule cell layer was traced using a mouse controlled cursor on a digital image of the thionine stained culture section and measured by the Stereo Investigator Software. This tracing was stored and used as a mask of the granule cell layer for BrdU and activated caspase-3 cell counting (vide infra). An estimate of granule cell number per section was obtained using the fractionator method [62] with a sampling repeat of 0.1 mm on a randomly rotated grid and a counting frame area of 6.25 10 4 mm2. Processing of the sections reduced their thickness to 5 Am and therefore we did not use a guard area on either section surface. This method gives an unbiased cell count in the X and Y dimensions but will tend to over estimate cell counts from the whole culture or the hippocampus because cells present on two sections would be counted twice. However, we were comparing two conditions in 10-Am sections rather than estimating absolute granule cell numbers in a single culture or sampled hippocampus and therefore this approximation was acceptable. Cells in the granule cell layer that were obviously pyknotic were not counted when generating estimates of normal cell number. The mask of the granule cell layer also produced the area measurement, and was superimposed on adjacent sections from the same culture stained for BrdU and activated caspase-3 to enable exhaustive cell counting to be carried out in the same area as the thionine cell counts. Images of DAPI and Fluoro-Jade B staining were captured on an inverted Leica DM-IRBE epifluorescence microscope (Milton Keynes, U.K.) using a cooled Hamamatsu digital camera. The area of DAPI or Fluoro-Jade B fluorescence was determined in NIH image (http://rsb.info. nih.gov/nih-image/), using the density slice function, applied to the granule cell layer as a region of interest. Cell death was expressed as a percentage of the area in which Fluoro-Jade B fluorescence was detected above threshold within the cell layer divided by the area in which DAPI fluorescence was detected above a threshold in the same cell
Only sections from cultures in which data was available for total cell counts, BrdU cell counts and Fluoro-Jade B fluorescence quantification experiments were used for statistical analysis. Activated caspase-3 matched cultures formed a smaller subset of this group. Statistical analysis across time-points, within either treatment or control groups was by one-way ANOVA and post hoc testing, or by paired two tailed Student’s t test as appropriate. Data are expressed as means F SEM; P b 0.05 was considered significant.
3. Results 3.1. Granule cell number, granule cell layer area and cell density in control OHSC The mean number of thionine-stained cells in the granule cell layer in a 10-Am thick section over all three time points under control conditions was 1796 F 136 cells with no significant difference between 2 h (1576 F 190 cells, n = 8), 24 h (2044 F 108 cells, n = 9) and 72 h (1770 F 105 cells, n = 5) time points (Fig. 2). Neither was there a significant difference between the area of the granule cell layer at 2 h (0.105 F 0.010 mm2; n = 8), 24 h (0.130 F 0.006 mm2; n = 9) or 72 h (0.121 F 0.015 mm2; n = 5). Similarly, the density of thionine stained cells was unchanged across all time points with no tendency toward dispersion of the granule cell layer. 3.2. Granule cell number, granule cell layer area and cell density after 5 lM kainate Sections from kainate treated cultures did not differ significantly from position and time matched controls in granule cell layer thionine stained cell counts at 2 h (kainate, 1624 F 265 cells vs. control, 1576 F 190, both n = 8), 24 h (kainate, 1617 F 114 cells vs. control, 2044 F 107, both n = 9) or 72 h (Kainate, 1744 F 269 cells vs. control, 1769 F 105, both n = 5). Comparisons between the different times also revealed no significant differences in the number of cells in kainate treated cultures (Fig. 2). Similarly, dentate granule cell layer area showed no significant change between 2 h (kainate, 0.111 F 0.015 mm2 n = 8), 24 h (kainate, 0.115 F 0.008 mm2 n = 9) or 72 h (kainate, 0.146 F 0.019 mm2 n = 5) after the start of the experiment. The increase in granule cell layer area noted
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in vitro, with approximately 3% of total cells incorporating BrdU at 2 h. BrdU labelling in control cultures declined significantly over time with cell counts at 72 h (25.4 F 4.41 cells, n = 5) lower than at both 2 h (48.25 F 4.85 cells, n = 8, P b 0.001) and 24 h (42.11 F 4.23 cells, n = 9, P b 0.01), and although BrdU cell counts at 2 h and 24 h are not significantly different they follow this trend (Fig. 2). The number of BrdU positive cells observed was significantly decreased by treatment with 5 AM kainate at 24 h after application (kainate, 16.67 F 2.84 cells vs. control, 42.11 F 4.23 cells, n = 9, P b 0.01) but were not significantly different at 2 h (kainate, 39.25 F 3.46 cells vs. control, 48.25 F 4.85 cells, n = 8) or at 72 h (kainate, 37.0 F 4.68 cells vs. control, 25.4 F 4.41 cells, n = 5; Fig. 2B). After kainate treatment BrdU cell counts decreased significantly between 2 h (39.25 F 3.46 cells, n = 8) and 24 h (16.67 F 2.84 cells, n = 9, P b 0.001) and then increased significantly between 24 h (16.67 F 2.84 cells, n = 9) and 72 h (37 F 4.68 cells, n = 5, P b 0.001), finishing at a similar level to initial cell counts (Fig. 2). All P values are derived from an ANOVA with Tukey’s post hoc test for multiple comparisons. 3.4. Effect of kainate on cell death in the granule cell layer and subgranular zone Fig. 2. Granule layer cell counts and cell proliferation. (A) Counts of healthy thionine-stained cells per 10 Am section of the granule cell layer from cultures exposed to medium containing 5 AM kainate or control medium at 2, 24 and 72 h. There were no significant differences between kainate and control groups at any time point examined. (B) Number of cells incorporating BrdU in the granule cell layer per section at 2, 24 and 72 h. There was a significant reduction in cell proliferation rate in kainate treated cultures at 24 h compared to controls and between kainate cultures at 2 h. Cell proliferation also increased significantly in kainate cultures between 24 and 72 h. (***P b 0.001: ANOVA with Tukey’s post hoc testing).
at 72 h post kainate treatment just failed to reach statistical significance on post hoc testing. However, there was a significant decrease in cell density in the granule cell layer in kainate treated cultures at 72 h compared to time and position matched control cultures (kainate, 11,895 F 935 cells mm 2 vs. controls, 15,597 cells mm 2, both n = 5; P b 0.05, ANOVA with Tukey’s post hoc test). Two way ANOVA confirmed an effect of kainate on cell density ( P b 0.05) and showed that time had no independent effect. This is consistent with the known effect of kainate on dispersion of the granule cell layer [6]. 3.3. Effect of kainate on cell proliferation in the granule cell layer and subgranular zone Cell proliferation, as judged by incorporation of the thymidine analogue Bromodeoxyuridine (BrdU) over 2 h, was low under serum-free growth conditions after 3 days
Cell death was measured in the cultures by considering the percentage of the granule cell layer staining for FluoroJade B, an anionic fluorescein derivative, generating a fluorescent histochemical stain which is specific for degenerating neurons [13]. Kainate induced a significant increase in the amount of Fluoro-Jade B staining in the granule cell layer, detected 2 h after kainate addition (kainate, 15.3 F 3.0%, vs. control, 4.0 F 1.9%, n = 8, P b 0.05) (Figs. 3A–C) but not at 24 h (kainate, 4.9 F 1.1%, vs. control, 5.4 F 1.7%, n = 9) or 72 h (kainate, 3.8 F 1.5%, vs. control, 6.4 F 2.6%, n = 5) compared with matched controls. In control cultures the levels of FluoroJade B detected did not vary significantly. In kainate treated cultures Fluoro-Jade B was significantly higher at 2 h (15.3 F 3.0%, n = 8) than at 24 h (4.9 F 1.1%, n = 9, P b 0.001) and 72 h (3.8 F 1.5%, n = 5, P b 0.001) (Fig. 3F). Cell death as evidenced by pyknosis and hyperchromatic nuclei on DAPI staining was predominantly localised to the inner half of the granule cell layer (Fig. 3D) and often co-localised with Fluoro-Jade B staining (Fig. 3C). To assess the contribution of apoptosis to this cell death, we examined the number of cells expressing activated cleaved caspase-3 in the same sectioned cultures. Activated caspase-3-positive cells did not vary significantly between control cultures over time. Sections from kainate treated cultures did not differ significantly from position and time matched controls in the number of cells expressing activated caspase-3, at 2 h (kainate,
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Fig. 3. Cell death in the granule cell layer and hilus after kainate. (A) and (B) Corresponding 200 images of a 10-Am section of the granule cell layer 2 h after kainate, stained with DAPI (blue) (A) and Fluoro-Jade B (green) (B). The granule cell layer is outlined in red and CA3c in white in A. Note the pyknotic cells arrowed in A located at the tip of the granule layer blade and running back along the inner half of the body. (C) Higher power view of merged images A and B showing co-localisation of pyknotic cells with Fluoro-Jade B (white arrow). Also note the neuronal morphology of degenerating Fluoro-Jade B positive cells (Yellow arrow) located in the more mature area of the granule cell layer and also in the hilus. (D) Lower power view of the granule cell layer to show the preponderance of Flouro-Jade B labelling (green) in the inner half of the granule cell layer (white arrows) and also in the hilus and area CA3. (E) DAB stained cell nuclei (brown) immunostained for antibody against activated-caspase-3 in the granule cell layer which has been thionine counterstained (light blue) showing cells undergoing apoptosis in the granule cell layer. These cells were distributed throughout the thickness of the granule cell layer. Asterix denotes the inner border of the granule layer. (F) There was a significant increase in Fluoro-Jade B labelling in kainate treated cultures at 2 h compared to controls and between kainate treated cultures at 2 and both 24 and 72 h. (G) Quantification of apoptotic death in the granule cell layer showed no difference between kainate and control cultures in activated-caspase-3 expression at any time point. (*P b 0.05: ***P b 0.001: ANOVA with Tukey’s post hoc test). Scale bars = 50 Am.
34.0 F 3.19 cells vs. control, 26.25 F 5.36 cells, n = 4), 24 h (kainate, 24.43 F 2.14 cells vs. control, 29.57 F 5.20 cells, n = 7) or 72 h after the start of the experiment (kainate, 22.0 F 2.0 cells vs. control, 24.0 F 2.0 cells, n = 2) (Figs. 3E and G).
4. Discussion The rapid cell death in the granule cell layer after kainate had features of necrotic cell death with condensed hyperchromatic nuclei on DAPI staining (Fig. 3A) and no
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significant increase in staining for activated caspase-3 at any time point (Fig. 3G). Despite the assertion of resistance of the immature brain to seizure-induced damage [53,54], early cell death in the inner half of the granule cell layer has been reported after perforant path stimulation in early postnatal life [57] with a similar time-course to our data. Although Thompson et al. categorised the induced cell death as apoptotic on the basis of nick-end-labelling and eosinophilic staining, more recent studies in adult tissue have demonstrated that status epilepticus induced neuronal injury is predominantly necrotic with appearances of eosinophilic staining, nuclear pyknosis, chromatin condensation, and internucleosomal DNA fragmentation [15] and does not involve neuronal activation of caspase-3 [16,34]. This does not rule out apoptotic cell death after perforant path stimulation or kainate but suggests that necrotic cell death may play the major role in both models, reflecting seizureinduced energy failure [61]. We extend these data by showing that kainate rapidly damages neurons in the inner half of the immature granule cell layer as evidenced by Fluoro-Jade B staining, a specific marker of degenerating neurons in the hippocampus [47] (Figs. 3B and C). Cells staining for Fluoro-Jade B often had nuclear pyknosis (Fig. 3C), which detects cell death as early as 1 h in OHSCs after oxygen glucose deprivation [5]. A previous in vivo study examining the effects of kainateinduced seizures on neurogenesis in the immature hippocampus at P13, found no evidence of cell death using histological stains in the inner half of the granule cell layer [29]. However, in this study, animals were sacrificed 48 h after the kainate-induced seizures by which time the FluoroJade B staining had reverted to control levels in our study and pyknotic cell death had been cleared in the study by Thompson et al. [57]. A study in adult animals after pilocarpine induced status epilepticus also found pyknotic cell death within 3 h in the inner one third of the granule cell layer [38]. We only counted healthy looking granule cells on thionine staining which may explain why we observed no fall in thionine cell counts in kainate treated cultures at 2 h compared to 24 h (Fig. 2). The neurogenic subgranular zone (SGZ) where neurogenesis continues into adult life is formed from P5 by migration of precursor cells from the hilar tertiary germinal matrix [2] and is established by P11–14 [2,46], occupying the inner one third of the granule cell layer where we observed early kainate-induced cell death. The SGZ contains both precursor cells and their progeny-immature granule cell neurons. Kainate was associated with a significant reduction in absolute cell proliferation in the granule cell layer at 24 h as measured by BrdU incorporation (Fig. 2). This was not due to loss of growth factors caused by placing the kainate exposed cultures in fresh medium at 24 h as matched control cultures were also changed to fresh medium at this timepoint. Given the preceding cell death localised to the proliferative SGZ, one possibility is that kainate kills a
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subpopulation of dividing precursor cells, resulting in a decreased proliferation rate 24 h after status. Why these precursor/immature cells might be more susceptible to excitotoxic damage remains a matter for speculation. Kainate receptor expression is found in the immature dentate [56] although the cellular expression pattern is not known. Studies have also shown that immature granule cell neurons are intrinsically more excitable and susceptible to LTP [52,60] in keeping with their putative role in hippocampal dependant learning and memory [20,50]. This vulnerability also appears to occur in adult life with increased cell death in the neurogenic subgranular zone 5 h after a single kindling stimulation or 3 h after pilocarpine induced status epilepticus [4,38]. An alternative explanation, based on evidence that inflammation inhibits cell proliferation in the adult dentate [11], is that kainate-induced inflammation decreases precursor cell proliferation. However, the time course of this in vivo effect is over a number of days rather than hours and involves microglia [11], which cannot be recruited in the organotypic preparation as it has no associated circulation. One of the intriguing differences between the adult and postnatal dentate is the reduction in precursor proliferation after seizures seen in the early postnatal brain [10,29,31] compared to increased proliferation in the later postnatal [44] and adult brain [4,22,36]. Using intraperitoneal kainate, Liu et al. [29] found a reduction in cell proliferation 48 h after a single kainate-induced status in P13 animals consistent with our finding of a reduction at 24 h. Although they did not find evidence of increased cell death at the time of the reduction in proliferation, we have identified an earlier transient phase of necrotic cell death, consistent with a previous report in vivo [57], which precedes the reduction in granule layer cell proliferation. Our results suggest that kainate-induced status in the early postnatal hippocampus causes rapid necrotic death of a proportion of precursor/ immature cells in the granule cell layer which results in a reduction in cell proliferation. Our hypothesis would predict that a greater number of seizures would cause a greater degree of precursor cell loss which is supported by Liu et al’s finding that three episodes of kainate-induced seizures was associated with a greater reduction in proliferation [29] and the original work of McCabe et al. where multiple flurothyl-induced seizures were necessary to reduce granule layer proliferation in P0–P4 animals [31]. Further work is clearly necessary to see if blocking kainate-induced death attenuates any subsequent changes in cell proliferation in the immature granule cell layer. The reduction in granule layer cell proliferation is transient since BrdU incorporation recovered to control levels by 72 h in our study. It is likely that the neurotrophic response to kainate involving BDNF, GDNF, and VEGF [25,49,51], all of which stimulate precursor cell proliferation in the dentate gyrus [14,27,63], mediates this recovery. Recovery of proliferation after multiple flurothyl-induced
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seizures has also been reported in the immature hippocampus in vivo [24] with greater baseline neurogenesis in these animals in subsequent adulthood. This differs from the response in the adult dentate where kainate transiently increases proliferation above control levels earlier at 72 h [33] and returns to baseline by 10 days. We did not examine proliferation beyond the 72-h time point as baseline proliferation declined to low levels in control cultures and became more variable. It is possible that the delay in recovery of proliferation is related to the severity of the initial cell death and that multiple seizures or episodes of status injure a greater number of precursors which take a longer time to regenerate the normal proliferative pool.
Acknowledgments This research was funded by Hope and the University of Southampton.
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