Brain Struct Funct DOI 10.1007/s00429-013-0683-7

ORIGINAL ARTICLE

Diffusion tensor imaging of hippocampal network plasticity Alejandra Sierra • Teemu Laitinen Olli Gro¨hn • Asla Pitka¨nen



Received: 3 July 2013 / Accepted: 29 November 2013 Ó Springer-Verlag Berlin Heidelberg 2013

Abstract Diffusion tensor imaging (DTI) has become a valuable tool to investigate white matter integrity in the brain. DTI also gives contrast in gray matter, which has been relatively little explored in studies assessing post-injury structural abnormalities. The present study was designed to compare white and gray matter reorganization in the rat hippocampus after two epileptogenic brain injuries, status epilepticus (SE) and traumatic brain injury (TBI), using ex vivo high-resolution DTI. Imaging was performed at 6–12 months post-injury and findings were compared to histological analyses of Nissl, myelin, and Timm-stained preparations from the same animals. In agreement with the severity of histological damage, fractional anisotropy (FA), axial (D||) and radial (D\) diffusivities, and mean diffusivity (MD) measurements were altered in the order SE [ TBI ipsilaterally [ TBI contralaterally. After SE, the most severe abnormalities were found in the dentate gyrus and CA3b–c subfields, in which the mean FA was increased to 125 % (p \ 0.001) and 143 % (p \ 0.001) of that in controls, respectively. In both subfields, the change in FA was associated with an increase in D|| (p \ 0.01). In the stratum radiatum of the CA1, FA was decreased to 81 % of that in controls (p \ 0.05) which was associated with an increase in D\ (p \ 0.01). After TBI, DTI Electronic supplementary material The online version of this article (doi:10.1007/s00429-013-0683-7) contains supplementary material, which is available to authorized users. A. Sierra  T. Laitinen  O. Gro¨hn  A. Pitka¨nen (&) Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, 70211 Kuopio, Finland e-mail: [email protected] A. Pitka¨nen Department of Neurology, Kuopio University Hospital, PO Box 1777, 70211 Kuopio, Finland

did not reveal any major abnormalities in the dentate gyrus. In the ipsilateral CA3b–c, however, FA was increased to 126 % of that in controls (p \ 0.01) and associated with a mild decrease in D\ (p \ 0.05). In the stratum radiatum of the ipsilateral CA1, FA was decreased to 88 % of that in controls (p \ 0.05). Our data demonstrate that DTI reveals subfieldspecific abnormalities in the hippocampus with remarkable qualitative and quantitative differences between the two epileptogenic etiologies, suggesting that DTI could be a valuable tool for follow-up of focal circuitry reorganization during the post-injury aftermath. Keywords Diffusion tensor imaging  Epilepsy  Epileptogenesis  Hippocampus  Magnetic resonance imaging  Mossy fiber sprouting  Myelin  Status epilepticus  Traumatic brain injury Abbreviations D|| Axial diffusivity D\ Radial diffusivity DTI Diffusion tensor imaging FA Fractional anisotropy FPI Fluid percussion injury MD Mean diffusivity MRI Magnetic resonance imaging ROI Region of interest SE Status epilepticus TBI Traumatic brain injury

Introduction Diffusion tensor imaging (DTI) has become an important research tool both in the clinic and in experimental laboratories. It detects restriction of water diffusion caused by

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the microstructural organization of tissue (Beaulieu 2002; Mori and Zhang 2006; de Carvalho et al. 2011). In the brain, such biological barriers for water diffusion are built by specific morphological compartments, resulting in high tissue contrast in DTI even in the normal brain. The majority of DTI studies have focused on axonal injury and demyelination of major white matter tracts (Horsfield and Jones 2002; Bennett et al. 2012; Sigal et al. 2012). Recently, however, more subtle changes in tissue microstructure, which are related to pathological conditions such as cell death, gliosis, and even axonal plasticity have been monitored by DTI (Laitinen et al. 2010; Sierra et al. 2011; Budde et al. 2011). Moreover, several studies have investigated the relationship between DTI contrast and tissue microstructure using histology (Kaufman et al. 2005; Dauguet et al. 2007; Leergaard et al. 2010). However, these studies have provided only a partial validation of the data and, therefore, the microstructural basis of DTI contrast needs to be explored further. More than 30 % of epilepsies are caused by acquired brain insults such as traumatic brain injury (TBI), febrile or afebrile status epilepticus (SE), cerebrovascular diseases, or brain infections (Hauser 1997). These injuries trigger acquired epileptogenesis which refers to the development and extension of tissue capable of generating spontaneous seizures, including both the development of an epilepsy condition and its progression after the condition is established (Pitka¨nen 2010; Galanopoulou et al. 2012). As about 30 % of epilepsies are drug refractory, and a large proportion of patients controlled by antiepileptic drugs have adverse events that impair their quality of life, the prevention of epilepsy development has become a research priority both in Europe and the USA (Kelley et al. 2009; Baulac and Pitka¨nen 2008). To make progress in achieving this goal, we need methods that monitor and predict the development of epileptogenic network reorganization in patients at risk. In addition to changes in neuronal and glial cell numbers, previous studies have demonstrated remarkable changes in fiber density in various subfields of the hippocampus both in experimental models and in human temporal lobe epilepsy (TLE). These relate to modifications in afferent pathways as well as in intrinsic pathways originating from the surviving hippocampal neurons. For example, after TBI, afferent fibers from the subcortical areas and the entorhinal cortex show alterations in the density of hippocampal terminal labeling (Sierra et al. 2011; Christidi et al. 2011). After SE-induced epilepsy in animals, and TLE in humans, axons of both excitatory and inhibitory cells located in the dentate gyrus undergo plastic changes (Thind et al. 2010; de Lanerolle et al. 2012; Buckmaster 2012). Also, the axons of the remaining CA3 and CA1 principal cells sprout (Perez et al. 1996; Cross and

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Cavazos 2007). In addition to plasticity in axons, dendritic trees undergo modifications, including reduction in branching, length, orientation, as well as spine density and morphology (Wong 2005). Based on previous anatomic data we hypothesized that post-injury network reorganization in the different subfields of the rat hippocampus after epileptogenic brain injuries can be imaged using high-resolution DTI ex vivo. Moreover, the methodology is sensitive enough to demonstrate etiology-dependent differences in circuitry reorganization. Epileptogenesis was triggered using two clinically relevant etiologies, SE and TBI. The imaging findings were compared to histological assessment of neurodegeneration as well as axonal degeneration and sprouting in Nissl, myelin, and Timm-stained preparations from the same animals.

Materials and methods Animals Male Wistar (10 weeks old at the time of induction of SE, weight 300–350 g, National Laboratory Animal Center, Kuopio, Finland) or Sprague–Dawley rats (10 weeks old at the time of TBI, weight 300–350 g, Harlan Netherlands B.V., Horst, Netherlands) were used. Rats were housed in individual cages and kept under a normal 12 h light/12 h dark cycle with constant temperature (22 ± 1 °C) and humidity (50–60 %). Water and food were available ad libitum. All animal procedures were approved by the Animal Ethics Committee of the Provincial Government of Southern Finland and carried out in accordance with the guidelines of the European Community Council Directives 86/609/EEC. Induction of status epilepticus and traumatic brain injury Status epilepticus Rats (n = 17) were injected subcutaneously (s.c.) with scopolamine (1 mg/kg; #S-8502, Sigma Chemical Co., St. Louis, MO, USA) to reduce the peripheral adverse effects of pilocarpine. Thirty minutes later, SE was induced with pilocarpine (i.p., 320 mg/kg, #P-6503, Sigma, Chemical Co., St. Louis, MO, USA). The animals were observed for 3 h to follow up the development of SE and score its severity (Racine 1972). All rats had recurrent generalized seizures occurring for at least 30 min and were included in the study. Diazepam 20 mg/kg (i.p., Stesolid Novum, DumexAlpharma) was administered 120 min following induction of SE to reduce mortality. Age- and weight-matched

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control rats (n = 13) were treated similarly except that they were injected with 0.9 % NaCl instead of pilocarpine. Traumatic brain injury TBI was induced using lateral fluid-percussion brain injury (FPI) as described previously (McIntosh et al. 1989; Kharatishvili et al. 2006). Briefly, rats (n = 8) were anesthetized by an injection of a cocktail (6 ml/kg, i.p.) containing sodium pentobarbital (58 mg/kg), chloral hydrate (60 mg/kg), magnesium sulfate (127.2 mg/kg), propylene glycol (42.8 %), and absolute ethanol (11.6 %). A craniectomy ([ 5 mm) was performed between the bregma and lambda on the left convexity (anterior edge 2.0 mm posterior to the bregma; lateral edge adjacent to the left lateral ridge). Lateral FPI was induced by a transient (21–23 ms) fluid pulse impact against the exposed intact dura by using a fluid-percussion device (AmScien Instruments, Richmond, VA, USA). The impact pressure was measured by an extracranial transducer and adjusted to 3.2–3.4 atm to induce a severe injury. After impact, the dura was checked to ensure it had remained intact. Sham-operated control animals (n = 7) underwent all surgical procedures except the fluid-percussion impact. Tissue preparation for ex vivo DTI As both the axonal and dendritic plasticity after brain injury progress over several months (Zhao et al. 2008; Buckmaster et al. 2009), ex vivo DTI was performed either at 6 months (5 SE, 5 controls) or 12 months (12 SE, 8 controls) post-SE or at 7 months post-TBI to leave enough time for plasticity to occur. The variability in the survival time also allowed us to assess the possible progression of plastic changes. For imaging, animals were deeply anesthetized with the cocktail described above. The fixation protocol depended on the type of histological staining planned for different animal groups. Consequently, ten rats with SE (5 at 6 months, 5 at 12 months) and ten controls (5 at 6 months, 5 at 12 months) were perfused according to the fixation protocol required for Timm staining, and seven rats with SE (7 at 12 months) and three controls (3 at 12 months) were perfused with the pH-shift protocol (myelin staining). Also in the TBI group, animals were perfused using Timm (4 TBI, 4 controls) or pH-shift fixation protocols (4 TBI, 3 controls). Briefly, for Timm staining rats were intracardially perfused with 0.37 % sulfide solution (30 ml/min, 4 °C) for 10 min followed by 4 % paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4 (30 ml/min, 4 °C) for 10 min (Sloviter 1982). Brains were removed from the

skull and postfixed in 4 % paraformaldehyde at 4 °C for 4 h and then washed in 0.9 % NaCl for at least 24 h until used for MRI. The pH-shift fixation was performed according to Savander et al. (1996). Rats were perfused with 0.9 % NaCl (30 ml/min) for 2 min followed by 4 % paraformaldehyde in 0.1 M sodium acetate buffer [NaC2H3O2, pH 6.5 (30 ml/ min), 4 °C] for 10 min and then by 4 % paraformaldehyde in 0.1 M sodium borate buffer [Na2B4O710H2O, pH 9.5 (30 ml/min), 4 °C] for 15 min. The brains were removed from the skull, postfixed in 0.1 M sodium borate for 6 h, and then washed in 0.9 % NaCl for at least 24 h before MRI. Before ex vivo DTI, all the brains were immersed in perfluoropolyether (FomblinÒ LC08, Solvay Solexis, Milan, Italy) to avoid signal from the solution. Ex vivo DTI Ex vivo DTI was carried out in a vertical 9.4 T magnet (Oxford Instruments PLC, Abingdon, UK) interfaced to a Varian DirectDrive console (Varian Inc., Palo Alto, CA, USA) using a quadrature volume RF-coil (diameter 20 mm, Rapid Biomedical GmbH, Rimpar, Germany) as transmitter and receiver. Care was taken to place the brain at a standard position relative to the gradient set. Standard deviations of the angles between the brain and the main gradient directions were \ 2.5° in all three axes. Data from pilocarpine (6 months) and TBI animals were acquired using a 3D spin-echo sequence with two refocusing pulses (TR = 1 s, TE = 60 ms) (Reese et al. 2003). Monopolar diffusion gradient pairs were placed around each refocusing pulse with opposite polarity for each pair to compensate for long time constant eddy currents. A volume of 23 9 15 9 15 mm3 was covered with 192 9 64 9 64 points (data resolution of 120 9 240 9 240 lm3) zero padded to 192 9 128 9 128 points resulting in an interpolated spatial resolution of 120 9 120 9 120 lm3. For TBI animals, we used a data matrix of 256 9 74 9 56 zero padded to 256 9 148 9 112 over an FOV 29.3 9 17 9 12.8 mm3 resulting in a spatial resolution of 115 9 115 9 115 lm3. Pilocarpine animals (12 months) were imaged using a conventional diffusion-weighted 3D spin-echo sequence with a single refocusing pulse (TR = 1 s, TE = 30 ms, FOV 29.3 9 16.5 9 13.7 mm3, data matrix 256 9 72 9 60). Data were zero padded to 256 9 144 9 120 points resulting in an interpolated spatial resolution of 120 9 120 9 120 lm3. After post-processing with FLIRT (see below) for eddy current deformations, single spin-echo and double spin-echo approaches were found to produce practically identical data in preliminary testing.

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Fig. 1 DEC maps of the hippocampus. Control (a), a rat that had experienced pilocarpine-induced SE 6 months earlier (b), and a rat that had lateral FPI-induced TBI 7 months earlier (c, d). Panel c is from the ipsilateral side and panel d is from the contralateral side of the injured rat. Directions of water diffusion in DEC maps: red rostral–caudal, green medial–lateral, and blue dorsal–ventral. White

asterisks indicate the hippocampal subfields, which showed altered water diffusion after SE or TBI as compared to controls. White outlines in d delineate the ROIs selected for analysis. DG dentate gyrus, l-m stratum lacunosum-moleculare, SE status epilepticus, sr stratum radiatum, TBI traumatic brain injury

With both sequences, six 3D data sets with diffusion weighting (D = 17 ms, d = 5 ms, b value 1,000 s/mm2) in six directions distributed uniformly (Basser and Pierpaoli 1998) and one data set without diffusion weighting were obtained. Two averages were acquired for each line of kspace and the total scanning time was 16 h/brain. The temperature variation of the sample over the DTI measurement period as measured in separate experiments under identical conditions was less than ± 0.25 °C. In our previous study (Laitinen et al. 2010), DTI results obtained from the control brains that had been perfused with different fixatives (pH shift and Timm fixation) were similar, and furthermore they were comparable to other published studies using paraformaldehyde (Zhang et al. 2002, 2007; Sun et al. 2003; Shepherd et al. 2006). This indicates that our current results are unlikely to be biased by the perfusion protocols used.

were used to create maps of fractional anisotropy (FA) describing the anisotropy of diffusion (Basser and Pierpaoli 1996), directionally encoded color (DEC) maps, where the color of each voxel is defined by the orientation of its primary eigenvector (V1) and the intensity of each voxel is proportional to FA (Pajevic and Pierpaoli 1999), and maps of axial diffusivity (D|| = k1) showing the diffusivity parallel to the direction of the principal eigenvector, and radial diffusivity (D\ = (k2 ? k3)/2) describing the diffusivity perpendicular to the principal eigenvector were created, and from the trace(D)-map a map of mean diffusivity (MD = trace(D)/3) was determined. ROIs were manually outlined on DEC maps on selected hippocampal subfields (Fig. 1) of five consecutive coronal slices from the left septal hippocampus (SE model) and ipsi- and contralateral septal hippocampus (TBI model). The slices were taken from the hippocampal levels that show the typical pathology based on our previous studies (Kharatishvili et al. 2006; Laitinen et al. 2010). From the maps, FA, D||, D\ and MD and color-coding values were obtained from each slice and then averaged to obtain values for all the subfields. The DTI post-processing and ROI analysis were performed using in-house built Matlab software (AEDES) (Matlab R2010a). Diffusion ellipsoids were calculated from the average D|| and D\ values for each subfield from all animals. The orientation of the principle eigenvector was illustrated by its encoded color (see Fig. 1).

DTI data analysis by a region of interest approach Coregistration and pixelwise group analysis were not feasible due to significant deformation and/or atrophy in damaged brains. Therefore, we performed region of interest (ROI) analysis in individual animals. Prior to the ROI analysis, all data were corrected for eddy current distortions with affine (linear) alignment of the diffusionweighted images to the non-diffusion-weighted image with FMRIB’s Linear Image Registration Tool (FLIRT) (Jenkinson and Smith 2001; Jenkinson et al. 2002), which is included in the FMRIB Software Library (FSL 4.0) software (http://www.fmrib.ox.ac.uk/fsl/). After eddy current corrections, the diffusion tensor, a matrix describing the orientation dependence in each voxel, was determined and the eigenvalues (k1, k2, and k3) and eigenvectors (V1, V2, and V3) of the diffusion tensors were calculated. The sentence has been rephrased:´The eigenvectors and eigenvalues

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Histology Processing of tissue After the ex vivo DTI, brains were washed with 0.9 % NaCl for 2 h at 4 °C and then placed in a cryoprotective solution containing 20 % glycerol in 0.02 M potassium

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phosphate-buffered saline (pH 7.4) for 36 h. Thereafter, brains were frozen on dry ice and stored at -70 °C until cut. The brains were sectioned in the coronal plane (30 lm, 1-in-5 series) with a sliding microtome. The first series of sections was stored in 10 % formalin at room temperature and the remaining series were stored in a cryoprotectant tissue-collecting solution (30 % ethylene glycol, 25 % glycerol in 0.05 M sodium phosphate buffer) at -20 °C until stained. Nissl staining The first series of each section was stained for thionin (Nissl), which was used to identify the cytoarchitectonic boundaries of different brain areas as well as the distribution and severity of tissue damage. Myelin staining An adjacent series of sections was stained for myelin. Briefly, sections were mounted on gelatin-coated slides and dried at 37 °C. They were then incubated in a 0.2 % gold chloride solution (HAuCl43H2O, G-4022 Sigma) in 0.02 M sodium phosphate buffer (pH 7.4) containing 0.09 % NaCl for 11–14 h at room temperature in the dark. The slides were then washed twice (4 min each) in 0.02 M sodium phosphate buffer in 0.09 % NaCl and placed in a 2.5 % sodium thiosulfate solution for 5 min. After three washes in the buffer solution (10 min each), sections were dehydrated through an ascending series of ethanol, cleared in xylene and coverslipped with DePeX (BDH, Laboratory Supplies, Dorset, UK). Timm staining For the staining of mossy fibers (granule cell axons), an adjacent series of Timm-fixed sections including the entire hippocampus was mounted on gelatin-coated slides and dried at 37 °C. Staining was performed in the dark. The working solution containing gum arabic (300 g/l), sodium citrate buffer (25.5 g/l citric acid monohydrate and 23.4 g/l sodium citrate), hydroquinone (16.9 g/l), and silver nitrate (84.5 mg/l) was poured into the staining dish. The sections were developed until an appropriate staining intensity was attained (60–75 min). The slides were then rinsed under tap water for 30 min and placed in 5 % sodium thiosulfate solution for 12 min. Finally, sections were dehydrated through an ascending series of ethanol, cleared in xylene, and cover-slipped with DePeX. Mossy fiber sprouting was analyzed from five left (SE model) and both ipsi- and contralateral (TBI model) septal hippocampal sections stained by the Timm staining. The

density of mossy fiber sprouting was semiquantitatively scored from 0 to 5 according to Cavazos et al. (1991). Briefly, score 0 = no granules; score 1 = sparse granules in the supragranular region and in the inner molecular layer; score 2 = granules are evenly distributed throughout the supragranular region and the inner molecular layer; score 3 = almost a continuous band of granules in the supragranular region and inner molecular layer; score 4 = continuous band of granules in the supragranular region and in the inner molecular layer; score 5 = confluent and dense laminar band of granules that covers most of the inner molecular layer, in addition to the supragranular region. The mean score was calculated from the tip, middle, and crest of the dentate gyrus from the five septal sections corresponding to the slice used for DTI. Measurement of laminar thickness To assess the thickness of the stratum radiatum in the CA1, five successive thionin-stained sections (150 lm apart from each other) were photomicrographed with a Leica DMRB microscope equipped with a Nikon DXM1200F digital camera. In the SE model, in which the pathology is symmetric, sections were sampled from the left side. In the TBI model, analysis was performed both ipsilaterally and contralaterally at the rostrocaudal level corresponding to the slice used for DTI. Laminar thickness was measured from photomicrographs using ImageJ software (version 1.43u, http://rsb.info.nih.gov/ij). The probe line was drawn perpendicular to the stratum pyramidale of the CA1 at the medio-lateral location that is just dorsal to the mid-level of the suprapyramidal blade of the underlying dentate gyrus. For analysis of the thickness of mossy fiber pathway in the CA3a, b and c subfields, a probe line was drawn perpendicular to the pyramidal cell layer in the middle of each subregion. Statistical analysis Statistical analysis was done using SPSS Statistics 17.0. Differences between injured animals and respective controls were analyzed using Mann–Whitney U tests. Wilcoxon tests were used to analyze differences between ipsilateral and contralateral sides. A p value \ 0.05 was considered significant. All the values were represented by mean ± SD.

Results Presentation of ex vivo DTI data in DEC maps Figure 1a is a representative example of a DEC map generated from a normal rat hippocampus. The colors indicate

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Fig. 2 Dot plots summarizing the DTI data in the dentate gyrus after SE and TBI. FA (a), axial diffusivity (b), radial diffusivity (c), magnitude of rostral–caudal (d), medial–lateral (e), and dorsal– ventral (f) orientation of the eigenvectors for water diffusion. Circles indicate data from individual animals [control animals gray circles, injured animals (SE or TBI) black circles]. The horizontal line marks the mean value and the vertical line shows the SD. As shown in f, in

the control brains the principal eigenvector for water diffusion was oriented dorsal–ventrally. Statistical significances: **p \ 0.01; ***p \ 0.001 as compared to controls (Mann–Whitney U test) and ? p \ 0.05 as compared to the contralateral side (Wilcoxon test). Contra contralateral, C control, D|| axial diffusivity, D\ radial diffusivity, FA fractional anisotropy, Ipsi ipsilateral, SE status epilepticus, TBI traumatic brain injury

the main orientation of the water diffusion, which differs between the hippocampal subfields. Consequently, the lamination of the dentate gyrus and hippocampus proper are readily visible in the maps. For example, in the dentate gyrus the predominant orientation of water diffusion is dorsal–ventral (blue) and in the CA3 the orientation is rostral–caudal (red). In the CA1, the orientation of diffusion varied between the layers, being dorsal–ventral (blue) in the stratum radiatum and rostral–caudal (red) in the stratum lacunosum-moleculare (Fig. 1a). As described in detail later, both the SE and TBI models resulted in etiology-specific changes in the orientation of the water diffusion, which varied in magnitude in different hippocampal subfields (Fig. 1b, c). Examples of interanimal variability in DEC maps in the SE and TBI groups are demonstrated in Supplementary Figure 1. The outlines of ROIs delineating different hippocampal subfields are shown in Fig. 1d.

hippocampal sections was made at the septo-temporal level, which corresponds to the slice used for DTI analysis. For statistical analysis, all animals with SE were pooled together as there were no differences between the data collected at 6 or 12 months post-SE. As the histological and DTI changes were symmetric, only the left hippocampus in SE animals was included in the analysis and compared to that in controls. In the TBI group, the sham-operated animals showed a few mild interhemispheric differences in diffusion. Therefore, for statistical analysis the data from the injured left side of the rats with TBI was compared to that on the left side in controls, and data from the contralateral side to that on the right side in controls. In the description of findings, we proceed by following the hippocampal formation.

Diffusion changes after SE and TBI in different subfields of the dentate gyrus and hippocampus proper

Dentate gyrus Controls

The data are summarized in Figs. 2, 3, 4, 5, 6, 7, 8, and 9. Supplementary Table 1 shows the percentage of animals with abnormal values. Histological analysis of

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The main water diffusion orientation in the normal dentate gyrus was close to dorsal–ventral (Fig. 2d–f).

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Fig. 3 Dot plots summarizing the mean diffusivity (MD) data after SE and TBI in the dentate gyrus (a), CA3b–c (b), CA3a (c), stratum radiatum (d), and stratum lacunosum-moleculare (e). Circles indicate data from individual animals [control animals gray circles, injured animals (SE or TBI) black circles]. The horizontal line marks the mean value and the vertical line shows the SD. Statistical

significances: *p \ 0.05 as compared to controls (Mann–Whitney U test) and ?p \ 0.05 as compared to the contralateral side (Wilcoxon test). Contra contralateral, C control, DG dentate gyrus, Ipsi ipsilateral, l-m stratum lacunosum-moleculare, SE status epilepticus, sr stratum radiatum, TBI traumatic brain injury

SE

TBI: ipsilateral side

The main diffusion orientation remained unchanged in the dorsal–ventral orientation (Figs. 1b, 2d–f) after SE. FA was increased after SE as compared to controls (25 %, p \ 0.001, Fig. 2a). In particular, axial diffusivity was increased (18 %, p \ 0.01) (Fig. 2b), as also MD (8 %, p \ 0.05) (Fig. 3a). Pilocarpine-induced SE resulted in a loss of hilar cells. In some animals (5 out of 17), we also found a patchy disappearance of the granule cell layer, particularly within the suprapyramidal blade. In Timm-stained sections, we found heavy sprouting of mossy fibers in the supragranular region and in the inner molecular layer (score 4.73 ± 0.44) (Fig. 4b). We also found a few mossy fibers traveling from the hilus through the granule cell layer to reach the outer two-thirds of the molecular layer perpendicular to the hippocampal fissure (Fig. 4b). Myelin staining showed an increased density of myelinated fiber bundles in the outer two-thirds of the molecular layer, which were typically oriented along the hippocampal fissure (Fig. 4e, f). This corresponded to increased density of myelinated fibers in the dorsal subiculum, which is the main route for the perforant pathway from the entorhinal cortex to the molecular layer of the dentate gyrus (Fig. 4k, l).

The main diffusion orientation remained close to dorsal– ventral orientation after TBI (Figs. 1c, d, 2f). FA did not differ from that in controls (Fig. 2a). Also, D||, D\ and MD remained unchanged (Figs. 2b, c, 3a). At the level of the MRI slice, we found ipsilateral hilar cell loss, whereas the granule cell layer was intact. We also found mild mossy fiber sprouting (score 1.73 ± 0.78) and an extension of the mossy fibers also into the outer molecular layer (Fig. 4c). The perforant pathway was damaged, which reduced the density of myelinated fibers traveling through the angular bundle and dorsal subiculum (Fig. 4m–p). This was associated with a reduced density of myelinated fibers in the outer molecular layer of the dentate gyrus of injured animals as compared to sham-operated controls (Fig. 4e, g, h). TBI: contralateral side The main diffusion orientation remained close to the dorsal–ventral orientation (Figs. 1c, d, 2f). Similar to that in ipsilaterally, FA did not differ from that in controls (Fig. 2a). D||, D\ and MD also remained unchanged (Figs. 2b, c, 3a). The contralateral side appeared normal in Nissl and myelin stainings. In Timm preparations, we found mild mossy fiber sprouting (score 0.93 ± 0.57) in

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Fig. 4 Representative photomicrographs of the dentate gyrus and stratum lacunosum-moleculare of the CA1, the perforant pathway and the fimbria–fornix pathway after SE and TBI. Panels in the upper row show the pattern of Timm staining and panels on the bottom row show the myelin staining. Control (a, e, i, m), a rat with SE induced with pilocarpine 6 months earlier (b, f, j, n), ipsilateral side (c, g, k, q) and contralateral side (d, h, l, p) of a rat with lateral fluid-percussion induced TBI made 7 months earlier. Arrows in b and c indicate mossy fiber sprouting in the supragranular region and the inner molecular layer. Note the more robust sprouting after SE as compared to that after TBI. Arrowheads point to the sparse mossy fibers extending to the outer two-thirds of the molecular layer. f An increase in the density of myelinated fibers after SE in the outer molecular layer of the dentate gyrus (white arrowheads) and in the stratum

lacunosum-moleculare of the CA1 (white arrow compare to e). Panel g demonstrates a remarkable decrease in the density of myelinated fibers after TBI in the outer molecular layer of the dentate gyrus (white arrowhead) and in the stratum lacunosum-moleculare of the CA1 (white arrow). Note also a clear decrease in myelinated fibers ipsilaterally as compared to the contralateral side in h. White arrows in j and k point to myelinated fibers traveling to the stratum lacunosum-moleculare of the CA1. Note an increased density of fibers after SE (j) and a reduced density after TBI (k). Black arrows in n and o point to a reduced thickness of the fimbria both after SE (n) and TBI (o). Scale bar equals 50 lm (a–d), 150 lm (e–h), and 500 lm (i–p). iml inner molecular layer, l-m stratum lacunosum-moleculare, oml outer molecular layer, sr stratum radiatum

the inner molecular layer. Very few (if any) mossy fibers were found in the outer molecular layer (Fig. 4d). The perforant pathway appeared intact.

contralateral sides in the medial–lateral (-28 %, p \ 0.05) and ventral–dorsal (5 %, p \ 0.05) components of diffusion. Correspondingly, as indicated above, histological analyses indicated more severe neurodegeneration, mossy fiber spouting, and loss of myelinated fibers more ipsilaterally than contralaterally.

TBI: ipsilateral versus contralateral Even though the DTI findings did not differ from those in controls on either side, interhemispheric comparisons indicated that FA was lower ipsilaterally than contralaterally (-13 %, p \ 0.05), which was associated with a lower ipsilateral axial diffusivity (-6 %, p \ 0.05). A slight difference was also found between the ipsilateral and

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Comparison of SE and TBI FA increased after SE, whereas no change was found after TBI. The orientation of the principal eigenvector did not change after either SE or TBI.

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Fig. 5 Dot plots summarizing the DTI data in the CA3b–c subfield of the hippocampus proper after SE and TBI. FA (a), axial diffusivity (b), radial diffusivity (c), magnitude of rostral–caudal (d), medial– lateral (e), and dorsal–ventral (f) orientation of the eigenvectors for water diffusion. Circles indicate data from individual animals [control animals gray circles, injured animals (SE or TBI) black circles]. The horizontal line marks the mean value and the vertical line shows the

SD. As shown in f, in controls the orientation of the principal eigenvector was dorsal–ventral. Statistical significances: *p \ 0.05; **p \ 0.01; ***p \ 0.001 as compared to controls (Mann–Whitney U test) and ?p \ 0.05 as compared to the contralateral side (Wilcoxon test). Contra contralateral, C control, D|| axial diffusivity, D\ radial diffusivity, FA fractional anisotropy, Ipsi ipsilateral, SE status epilepticus, TBI traumatic brain injury

On histology, both animal groups had remarkable hilar cell damage, and rats with SE also showed a patchy degeneration of the granule cell layer at the level of the MRI slice. Mossy fiber sprouting in the inner molecular layer was substantially more dense in rats with SE than in animals with TBI (SE 4.3 ± 0.4 vs. ipsilateral TBI 1.8 ± 0.8, p \ 0.001) (Fig. 5b, c). Moreover, the density of myelinated fibers in the outer molecular layer was substantially increased after SE, whereas after TBI the density of myelinated fibers was substantially reduced as compared to controls (Fig. 5f, g).

increased in the CA3b–c subfields (43 %, p \ 0.001) (Fig. 5a). In particular, D|| was increased (17 %, p \ 0.01) (Fig. 5b). Analysis of thionin-stained preparations indicated remarkable neurodegeneration in the CA3b–c pyramidal cell layer. Moreover, the remaining pyramidal cells were dispersed and some had a swollen appearance (Fig. 6b). Neurodegeneration was accompanied with remarkable changes in fiber stainings. The Timm-stained mossy fiber pathway in the stratum lucidum was thicker than in controls, both in the CA3c (42 %, p \ 0.001) and CA3b subfields (22 %, p \ 0.01) (Table 1), and zinc-positive boutons appeared more perpendicularly aligned to the pyramidal cell layer than in controls (Fig. 6j). Moreover, myelinated axons were more perpendicularly oriented to the pyramidal cell layer in the stratum lucidum than in controls (Fig. 6f). We also found a remarkable reduction in the thickness of the fimbria in myelin preparations, which could affect the fiber density in various hippocampal subfields (Fig. 4m, n).

Hippocampus proper CA3b–c subfields Control The main diffusion orientation in the CA3b–c subfield was between rostral–caudal and dorsal–ventral (Fig. 5d–f). SE The main direction of water diffusion was tilted toward the dorsal–ventral direction after SE [Fig. 1b, 65 % decrease in rostral–caudal component (p \ 0.001) and 65 % increase in the dorsal–ventral component (p \ 0.001)] (Fig. 5d, f). As in the dentate gyrus, FA

TBI: ipsilateral side The main diffusion direction was closer to the dorsal–ventral orientation as compared to controls [11 % increase in dorsal–ventral (p \ 0.05) and

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Fig. 6 Representative photomicrographs of thionin (a–d, m–t), myelin (e–h), and Timm (i–l) stainings from the CA3 and CA1 subfields after SE and TBI. Control (a, e, i, m, q), animal with SE 6 months earlier (b, f, j, n, r), ipsilateral side (c, g, k, o, s), and contralateral side (d, h, l, p, t) of a rat with TBI inflicted 7 months earlier. Arrowheads in b and c indicate dispersed and swollen remaining pyramidal cells in the CA3c after SE and ipsilateral CA3c after TBI, respectively. Arrowheads in f and g point to myelinated fibers oriented perpendicular to the pyramidal cell layer in the stratum lucidum of the CA3b after SE and ipsilateral CA3b after TBI, respectively. Arrowheads in j and k indicate alignment of mossy fiber

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boutons along the apical dendrites of pyramidal cells in the stratum lucidum of the CA3b after SE and ipsilateral CA3b after TBI, respectively. Arrowheads in n and o point to a region with degeneration of the pyramidal cell layer in the CA3a after SE and ipsilateral CA3a after TBI, respectively. In r, arrowheads indicate gliosis and the arrow shows neurodegeneration of the pyramidal cell layer after SE. Note the well-preserved CA1 in the brain with TBI. Scale bars 100 lm (a–h), 50 lm (i–l), 100 lm (m–p) and 250 lm (q–t). H hilus, l-m stratum lacunosum-moleculare, pyr pyramidal cell layer, sl stratum lucidum, sr stratum radiatum

Brain Struct Funct

Fig. 7 Dot plots summarizing the DTI data in the CA3a subfield of the hippocampus proper after SE and TBI. FA (a), axial diffusivity (b), radial diffusivity (c), magnitude of rostral–caudal (d), medial– lateral (e), and dorsal–ventral (f) orientation of the eigenvectors for water diffusion. Circles indicate data from individual animals [control animals gray circles, injured animals (SE or TBI) black circles]. The horizontal line marks the mean value and the vertical line shows the

SD. As shown in d, in controls the orientation of the principal eigenvector was rostral–caudal. Statistical significances: *p \ 0.05; **p \ 0.01 as compared to controls (Mann–Whitney U test) and ? p \ 0.05 as compared to the contralateral side (Wilcoxon test). Contra contralateral, C control; D|| axial diffusivity, D\ radial diffusivity, FA fractional anisotropy, Ipsi ipsilateral, SE status epilepticus, TBI traumatic brain injury

44 % decrease in the rostral–caudal component (p \ 0.05)] (Fig. 5d, f). FA was increased on the injury side as compared to controls (26 %, p \ 0.01) (Fig. 5a). This was associated with a decrease in D\ (-7 %, p \ 0.05) (Fig. 5c). Analysis of thionin-stained sections revealed mild neurodegeneration in the ipsilateral CA3b–c pyramidal cell layer (Fig. 6c). This was not accompanied by any change in the thickness of the mossy fiber pathway (Table 1). However, the zinc-positive boutons were more perpendicularly aligned along the initial segment of apical dendrites of the pyramidal cells than in controls (Fig. 6k). Also, myelinated axons on the injury side were more perpendicularly oriented to the pyramidal cell layer than in controls (Fig. 6g). The thickness of the fimbria in myelin staining was reduced (Fig. 4o, p).

contralateral side (Fig. 5d, f). FA was higher ipsilaterally in the CA3b–c than contralaterally (37 %, p \ 0.05) (Fig. 5a). This was associated with an increase in D|| (11 %, p \ 0.05) (Fig. 5b) as compared to the contralateral side. In line with more severe neurodegeneration, we found that the thickness of the ipsilateral mossy fiber pathway in the CA3c was 119 % of that contralaterally (p \ 0.01) (Table 1). Also, the density of myelinated axons appeared greater ipsilaterally than contralaterally.

TBI: contralateral side We did not observe any change in FA or orientation of water diffusion. Also histologically, the CA3b–c subfield appeared normal. TBI: ipsilateral versus contralateral The main diffusion was tilted closer to the dorsal–ventral orientation [33 % decrease in rostral–caudal (p \ 0.05) and 9 % increase in dorsal–ventral component (p \ 0.05)] as compared to the

Comparison of SE and TBI FA in the CA3b–c increased after both injuries. However, after SE it was associated with an increase in D||, whereas after TBI it was associated with a decrease in D\. The orientation of water diffusion decreased in rostral–caudal and increased in dorsal–ventral orientations after both injuries, even though after SE these changes were more robust. Even though the histological findings in the CA3b–c subfield appeared qualitatively quite similar in both models, they were more extensive after SE than TBI. Mossy fiber pathways in the stratum lucidum were thicker, zincpositive boutons appeared more perpendicularly aligned, and myelinated axons were more perpendicularly oriented to the pyramidal cell layer after SE than after TBI

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Fig. 8 Dot plots summarizing the DTI data in the stratum pyramidale and radiatum of the CA1 subfield of the hippocampus proper after SE and TBI. FA (a), axial diffusivity (b), radial diffusivity (c), magnitude of rostral–caudal (d), medial–lateral (e), and dorsal–ventral (f) orientation of the eigenvectors for water diffusion. Circles indicate data from individual animals [control animals gray circles, injured animals (SE or TBI) black circles]. The horizontal line marks the mean value

and the vertical line shows the SD. As shown in f, in controls the orientation of the principal eigenvector was dorsal–ventral. Statistical significances: *p \ 0.05; **p \ 0.01 as compared to controls (Mann– Whitney U test) and ?p \ 0.05 as compared to the contralateral side (Wilcoxon test). Contra contralateral, C control, D|| axial diffusivity, D\ radial diffusivity, FA fractional anisotropy, Ipsi ipsilateral, SE status epilepticus, TBI traumatic brain injury

(CA3b SE 22 % and TBI 10 %, n.s.; CA3c SE 42 % and TBI 12 %, p \ 0.05). Even the decrease in the thickness of the fimbria was more robust after SE than after TBI (Fig. 4n, o).

TBI: ipsilateral side The main water diffusion was tilted toward the medial–lateral orientation [32 % decrease in rostral–caudal component (p \ 0.01) and 61 % increase in medial–lateral component (p \ 0.01)] as compared to controls (Fig. 7d, e). FA remained unchanged and no changes were observed in axial or radial diffusivities. However, MD increased as compared to the contralateral side (8 %, p \ 0.05) (Fig. 3c). Most of the neurodegeneration in the CA3 subfield was found in CA3a (Fig. 6o). The thickness of the mossy fiber tract was reduced by 14 % as compared to controls (p \ 0.01) (Table 1). The myelin staining appeared comparable to that in controls.

CA3a subfield Control The main orientation of the water diffusion in CA3a of control animals was close to rostral–caudal (Fig. 7d–f). SE The main orientation of water diffusion was tilted toward the medial–lateral direction [14 % decrease in the rostral–caudal component (p \ 0.01) and 64 % increase in the medial–lateral component (p \ 0.01)] as compared to controls (Fig. 7d, e). As in the dentate gyrus and CA3b–c, FA was increased as compared to controls (27 %, p \ 0.01) (Fig. 7a). Like in the CA3b–c, analysis of thionin preparations revealed extensive degeneration of the pyramidal cell layer (Fig. 6n). Some of the remaining pyramidal cells had a swollen appearance (Fig. 6n). Fiber stainings (Timm and myelin) did not reveal major differences as compared to controls.

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TBI: contralateral side There was a mild tilt of the main orientation of water diffusion as the rostral–caudal component decreased by 14 % (p \ 0.05) (Fig. 7d). We did not find any difference in the FA or in axial or radial diffusion in the CA3a as compared to controls (Fig. 7). Histologically, the contralateral CA3a appeared normal. TBI: ipsilateral versus contralateral side We found reduced contribution of the rostral–caudal component to water diffusion orientation (-24 %, p \ 0.05) and

Brain Struct Funct

Fig. 9 Dot plots summarizing the DTI data in the stratum lacunosum-moleculare of the CA1 subfield of the hippocampus proper after SE and TBI. FA (a), axial diffusivity (b), radial diffusivity (c), magnitude of rostral–caudal (d), medial–lateral (e), and dorsal– ventral (f) orientation of the eigenvectors for water diffusion. Circles indicate data from individual animals [control animals gray circles, injured animals (SE or TBI) black circles]. The horizontal line marks the mean value and the vertical line shows the SD. As shown in d, in

controls the orientation of the principal eigenvector was rostral– caudal. Statistical significances: *p \ 0.05; **p \ 0.01; ***p \ 0.001 as compared to controls (Mann–Whitney U test) and ? p \ 0.05 as compared to the contralateral side (Wilcoxon test). Contra contralateral, C control, D|| axial diffusivity, D\ radial diffusivity, FA fractional anisotropy, Ipsi ipsilateral, SE status epilepticus, TBI traumatic brain injury

Table 1 Thickness of mossy fiber pathway (CA3) or stratum radiatum (CA1) and the density of mossy fiber sprouting (dentate gyrus) after status epilepticus (SE) and traumatic brain injury (TBI) Control

SE

Control

TBI

Ipsilateral

Contralateral

Ipsilateral

Contralateral

Thickness (lm) of mossy fiber pathway in stratum lucidum and stratum radiatum of the CA3 CA3a

156.4 ± 16.2

154.7 ± 33.0r

153.7 ± 17.3

156.0 ± 28.0

132.0 ± 30.6**,?

153.5 ± 22.7

CA3b

100.2 ± 11.7

122.2 ± 28.0**

102.5 ± 21.4

100.9 ± 15.5

110.5 ± 8.2

103.7 ± 14.2

CA3c

188.0 ± 21.0

267.3 ± 40.4***,r

207.4 ± 29.1

199.3 ± 26.3

231.6 ± 52.4??

194.6 ± 21.3

334.5 ± 27.6

338.1 ± 25.4

378.6 ± 41.6***,???

326.3 ± 21.4

Thickness (lm) of stratum radiatum of the CA1 sr

390.7 ± 30.3

305.3 ± 76.6***,rrr

Density of mossy fiber sprouting in the supragranular region and inner molecular layer of the dentate gyrus (score 0–5) mfs

0.3 ± 0.3

4.3 ± 0.4***,rrr

0.3 ± 0.4?

0.1 ± 0.3

1.8 ± 0.8***,??

0.9 ± 0.6***

Values are presented as mean ± SD CA3a, CA3b, and CA3c refer to distal, mid, and proximal portions of the CA3, respectively, mfs mossy fiber sprouting, sr stratum radiatum Statistical significances: * control versus injured; p \ 0.01; ***,???,rrr p \ 0.001)

?

ipsilateral versus contralateral;

increased axial diffusivity (13 %, p \ 0.05) in the ipsilateral CA3a as compared to the contralateral side (Fig. 7). These findings were associated with more severe neurodegeneration ipsilaterally than contralaterally as revealed by thionin-stained preparations. The thickness of ipsilateral mossy fiber tract was reduced as compared to the

r

SE versus ipsilateral TBI (*,?,r p \ 0.05; **,??,rr

contralateral side (-14 %, p \ 0.05) (Table 1). No interhemispheric differences were found by myelin staining. Comparison of SE and TBI FA increased after SE, but remained unchanged after TBI. Like in the CA3b–c, neurodegeneration was more extensive after SE than TBI.

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However, the thickness of mossy fiber tract after TBI was 14 % thinner than after SE (p \ 0.05). CA1 subfield DTI indicated a different pattern of diffusivity in a region containing both the stratum pyramidale and stratum radiatum of the CA1 as compared to the stratum lacunosummoleculare of the CA1, and therefore these ROIs are described separately. Stratum pyramidale and radiatum Control The main orientation of the water diffusion was close to dorsal–ventral (Fig. 8d–f). SE The main water diffusion orientation was tilted closer to the rostral–caudal orientation [27 % decrease in the dorsal–ventral component (p \ 0.05) and 54 % increase in the rostral–caudal component (p \ 0.05)] as compared to controls (Fig. 8d–f). FA was decreased as compared to controls (-19 %, p \ 0.05). This was associated with an increase in D\ (15 %, p \ 0.01) (Fig. 8a, c). MD increased after SE as compared to controls (12 %, p \ 0.05) (Fig. 3d). Analysis of thionin-stained preparations revealed remarkable degeneration of the stratum pyramidale resulting in disappearance or thinning of the principal cell layer, which was associated with a high density of glial cells (Fig. 6r). The thickness of the stratum radiatum after SE was reduced by 22 % (p \ 0.001) (Table 1). No differences in myelinated fibers were found in this area after pilocarpine treatment. TBI: ipsilateral side FA was slightly decreased as compared to controls (-12 %, p \ 0.05) (Fig. 8a). No differences were found in D\ or D|| (Fig. 8b, c) or in the orientation of the main water diffusion direction (Fig. 8d– f). We did not observe any remarkable neurodegeneration in the CA1 (Fig. 6s). The stratum radiatum was thicker as compared to that in controls (12 %; p \ 0.001) (Fig. 6s). No differences in myelin staining were found (Fig. 4g). TBI: contralateral side Similar to that ipsilaterally, we found a reduction in FA as compared to controls (-12 %, p \ 0.05). We also found a slight decrease in the dorsal– ventral component of the water diffusivity as compared to controls (-9 %, p \ 0.05). In thionin and myelin stainings, the contralateral stratum pyramidale and stratum radiatum of the CA1 appeared normal. Also, there was no difference in the layer thickness as compared to controls.

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TBI: ipsilateral versus contralateral side The orientation of water diffusion was slightly more toward the rostral– caudal orientation ipsilaterally as compared to the contralateral side (16 %; p \ 0.05) (Fig. 8d). FA was decreased ipsilaterally as compared to the contralateral side (-9 %, p \ 0.05) (Fig. 8a). Histologically, the only interhemispheric difference was the increased thickness of the stratum radiatum ipsilaterally (16 %, p \ 0.001). Comparison of SE and TBI FA decreased to a rather similar extent both after SE and TBI. This was related, however, to a slightly more increased D\ after SE than after TBI. Also, the orientation of the principal eigenvector changed after SE by showing an increase in the rostral– caudal orientation and a decrease in the dorsal–ventral orientation, whereas after TBI the orientation remained unchanged. Nissl preparations showed extensive degeneration of CA1 pyramidal cells after SE that was associated with a reduction in the thickness of the stratum radiatum. After TBI, the stratum pyramidale appeared intact, but the thickness of the stratum radiatum increased as compared to the SE group (SE -22 % and TBI 12 %). Myelin staining appeared comparable after SE and TBI. Stratum lacunosum-moleculare Controls The main orientation of the water diffusion was close to rostral–caudal (Fig. 9d). SE The main water diffusion was slightly changed and showed a reduction in the rostral–caudal component (-20 %, p \ 0.01) and an increase both in the medial– lateral (88 %, p \ 0.001) and dorsal–ventral components (47 %, p \ 0.05) (Fig. 9d–f). FA was unchanged, even though there was an increase both in D|| (13 %, p \ 0.05) and D\ (12 %, p \ 0.01) (Fig. 9b, c). MD increased after SE as compared to controls (12 %, p \ 0.05) (Fig. 3e). Analyses of thionin preparations revealed gliosis in the stratum lacunosum-moleculare (Fig. 6r). The density of myelinated fibers was increased as compared to controls. Their orientation was more in the section plane after SE as compared to controls, in which the axons were more perpendicular to the section (Fig. 4e, f). TBI: ipsilateral side The main direction of water diffusion was oriented slightly more toward to the medial–lateral orientation than in controls (48 %, p \ 0.05) (Fig. 9e). FA or axial and radial diffusivities did not differ from those of the controls (Fig. 9a–c). As in the two outer thirds of the molecular layer of the dentate gyrus, the density of myelinated fibers was reduced

Brain Struct Funct

in the stratum lacunosum-moleculare of the CA1 as compared to controls (Fig. 4g, h). No changes were found in Nissl or Timm stainings. TBI: contralateral side There were no detectable changes in DTI or histology on the contralateral side. TBI: ipsilateral versus contralateral side Ipsilaterally, the diffusion was more oriented toward the dorsal–ventral direction as compared to the contralateral side (30 %, p \ 0.05). Accordingly, the density of myelinated fibers was lower ipsilaterally than contralaterally. Comparison of SE and TBI No remarkable differences were found in diffusion parameters. After SE, the orientation of the principal eigenvector changed from rostral– caudal to medial–lateral and dorsal–ventral. After TBI, the main orientation of water diffusion was medial–lateral. The difference in the density of myelinated fibers in the stratum lacunosum-moleculare was one of the most conspicuous differences in myelin-stained preparations that we observed between the animals with SE or TBI. After SE, there was an increase in the density of myelinated fibers. It was associated with an increase in the staining of fiber bundles in the dorsal subiculum, which contains the myelinated fibers entering the hippocampus and dentate gyrus from the perforant pathway (Fig. 4i–l). After TBI, there was a remarkable decrease in the density of myelinated fibers, both in the stratum lacunosum-moleculare as well as in the subiculum and perforant pathway ipsilaterally (but not contralaterally).

Discussion We tested the hypothesis that epileptogenesis-related structural reorganization in different subfields of the hippocampus can be detected using DTI. As the distribution and severity of post-injury plasticity vary between epileptogenic etiologies, another goal was to provide proof-of-principle evidence for a difference in DTIrevealed plasticity between animals with SE or TBI. To address these challenges, we applied high-resolution ex vivo DTI and extracted FA, axial and radial diffusivities, and the main water diffusion direction from control and injured animals at 6–12 months post-injury. We found that DTI revealed subfield-specific abnormalities in the hippocampus, which corresponded to plastic changes in histological preparations stained from the same animals. Secondly, DTI revealed remarkable qualitative and quantitative differences between the two epileptogenic etiologies.

Orientation of diffusion varies between subfields in the normal dentate gyrus and hippocampus proper Our DTI analysis revealed clear differences in the orientation of the main diffusion direction in the various subfields of the normal dentate gyrus and the hippocampus proper. The orientation of diffusion in the coronally sliced septal dentate gyrus and CA1 (stratum pyramidale and radiatum) was mainly dorsal–ventral, suggesting that heavily dorsal–ventral oriented dendritic trees of dentate granule cells and CA1 pyramidal cells, respectively, were the major contributors to the main orientation of diffusion in these areas. Diffusion in the septal CA3 was more variable due to its curvature, being more rostral–caudal, particularly in the distal CA3 (CA3a). Interestingly, also in the stratum lacunosum-moleculare of the septal CA1, the main orientation of diffusion was rostral–caudal rather than dorsal–ventral, as seen in the deeper layers of CA1. This could be explained by the orientation of diffusion along the strong afferent projections in the perforant pathway that arrive from the more caudally located angular bundle and project forward to the distal dendrites of the septal CA1 stratum lacunosum-moleculare. Interestingly, in the more proximal CA3c–b, the diffusion was oriented between the anterior–posterior and dorsal–ventral directions. The lamellarly orientated dense mossy fiber pathway from the dentate granule cells to the apical dendrites of the CA3 pyramidal cells is an apparent contributor to the tilting of the diffusion orientation in this area. Taken together, the orientation of the main diffusion in various hippocampal subfields was in good agreement with the predictions made based on the anatomic studies (Amaral and Witter 1989). Based on these data in the normal hippocampus, we used the main orientation of diffusion, FA, as well as axial and radial diffusivities to probe changes in the damaged hippocampus. To illustrate changes in these parameters in different subfields and pathologic conditions, we generated diffusion ellipsoids from the average ROI values that are summarized in Fig. 10. The shape (D|| and D\) and orientation (color) of the ellipsoids changed remarkably in selected subfields in an injury-specific manner after SE or TBI as can be seen in videos of 3D diffusion ellipsoids provided as supplementary material (Videos 1–5). As Supplementary Figure 1 shows, there was variability in the diffusion ellipsoids between individual rats both in the SE and TBI groups, which corresponded to variability in histologic sections. These data suggest that the DTI method is able to detect relatively subtle hippocampal changes and, therefore, could be used to follow up the progression of pathology in individual animals after brain insults.

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Fig. 10 Graphs summarizing the changes in diffusion ellipsoids (a–c), and degeneration and plasticity (d–f) in selected subfields of the hippocampus proper and the dentate gyrus after SE or TBI. The shape of the ellipsoid corresponds with D|| and D\. The color reflects the orientation of water diffusion (see legend to Fig. 1) in each subfield. Note the widespread and robust changes in the post-SE brain in the CA3b–c, CA3a, and in the stratum pyramidale and radiatum of CA1. After TBI the changes were milder and located partly in other subfields, being most prominent in the CA3b–c, CA3a, and in the stratum lacunosum-moleculare of the CA1. Panel d summarizes the major connectivity according to Amaral and Witter (1989). After SE, there is a remarkable loss of principal cells in the CA3 and CA1 regions as well as in the hilus, whereas the granule cell layer is

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relatively intact. At 6 or 12 months post-SE there is an increase in mossy fiber sprouting into the inner third of the molecular layer of the dentate gyrus, as well as an increased density and reorganization of myelinated fibers in the stratum lacunosum-moleculare of the CA1 and in the outer two-thirds of the molecular layer. At 7 months postTBI, pyramidal cell damage is milder than that after SE, being detectable mostly in the CA3a and in the hilus. Mossy fiber sprouting is substantially less intense than that after SE. Moreover, the density of myelinated fibers in the stratum lacunosum-moleculare of the CA1 is decreased rather than increased like after SE. g granule cell layer, H hilus, iml inner molecular layer, l-m stratum lacunosum-moleculare, o stratum oriens, oml outer molecular layer, p stratum pyramidale, sr stratum radiatum

Brain Struct Funct

SE and TBI trigger etiology-dependent changes in the orientation and magnitude of diffusion in different subfields of the hippocampus proper Of all subfields analyzed, the CA3 showed the most remarkable changes in DTI after SE and TBI. In the CA3bc, the main orientation of water diffusivity changed from rostral–caudal toward dorsal–ventral (i.e., becoming perpendicular to the pyramidal cell layer in coronal sections) in both etiologies, being more robust after SE than after TBI (ipsilaterally). We also found increased FA in both etiologies, which was associated with increased axial diffusivity after SE and decreased radial diffusivity after TBI. The loss of pyramidal cells in CA3bc and their axons (Shaffer’s collateral) and dendrites as well as thickening of the mossy fiber pathway in the stratum lucidum by 22–42 %, particularly after SE, is the likely contributor to the diffusion changes recorded. In addition, we observed large boutons of mossy fiber terminals climbing along the proximal apical dendrites of CA3 pyramidal cells in coronally cut sections of both etiologies, which may be another contributor to the tilting of the main orientation of water diffusion toward the dorsal–ventral direction. Interestingly, in the CA3a subfield the main orientation of diffusivity changed from rostral–caudal toward medial–lateral (i.e., perpendicular to the pyramidal cell layer), reflecting a change comparable to that seen in the CA3bc. This was associated with an increase in FA after SE but not after TBI, which reflects the more severe damage of SE than TBI in this subfield. DTI of the histologically intact contralateral CA3a in rats with TBI showed only one abnormality, indicating a slight reduction in the dorsal–ventral orientation of the main direction of diffusivity. Taken together, three-dimensional structural complexity of the CA3 subfield provides a more sensitive target to monitor network reorganization after SE and TBI than the dentate gyrus. In the CA3, analysis of the main orientation of diffusivity provided additional information to the analysis of FA and related axial and radial diffusivities. In the CA1, the main orientation of diffusion in the ROI including the stratum pyramidale and radiatum differed from that in the stratum lacunosum-moleculare, being dorsal–ventral rather than rostral–caudal. The main structural components of the pyramidale–radiatum ROI are the pyramidal cells, their dendrites and axon collaterals, local inhibitory cells and their processes, and projections from the CA3 and extrahippocampal areas. After SE, the thickness of the stratum radiatum shrank to 78 % of that in controls. The main orientation of diffusivity became slightly tilted from the dorsal–ventral to rostral–caudal orientation, which was associated with decreased FA with increases both in axial and radial diffusivities. Histological data from these animals suggest that the loss of pyramidal

cells and their dendrites, and robust gliosis associated with shrinkage, caused the changes in the main orientation of water diffusion and FA. Also, the loss of the intrahippocampal afferent connections from the CA3 due to principal cell loss could modify the diffusion. Unlike after SE, only a mild decrease in FA without any change in the main orientation of diffusivity was found in the ipsilateral pyramidale–radiatum voxel after TBI, which reflects the meagerness of CA1 damage in this condition. Contralaterally, TBI resulted in a similar decrease in FA as ipsilaterally, which was associated with a slight decrease in dorsal–ventral diffusivity. In the stratum lacunosum-moleculare of the CA1, the main orientation of diffusion after SE remained rostral– caudal even though there was a clear tilt toward both the medial–lateral and dorsal–ventral directions. After TBI, the main orientation of diffusivity ipsilaterally remained rostral–caudal, even though it became slightly tilted toward the medial–lateral direction. No changes were found in FA. This is surprising as there was a remarkable decrease in myelinated fibers in the stratum lacunosum-moleculare of the ipsilateral CA1 after TBI as a consequence of damage to major afferent pathways to the hippocampus. Values in the contralateral stratum lacunosum-moleculare were comparable to those from controls. These data suggest that changes in the density of myelinated fibers in the stratum lacunosum-moleculare of the CA1 alter the main orientation of diffusivity. The changes do not, however, reveal whether the density of myelinated fibers is increased or decreased. The increase in fiber density after SE is, however, reflected as a slightly increased FA and increased axial and radial diffusivities. Diffusion changes in the dentate gyrus were mild compared to robust structural reorganization Our original main interest was in DTI alterations in the dentate gyrus which shows remarkable structural changes during acquired epileptogenesis, including mossy fiber sprouting, the most extensively investigated plastic change in experimental and human epilepsy (Laurberg and Zimmer 1981; Buckmaster 2012). Even though the functional contribution of mossy fiber sprouting to epileptogenesis is still under dispute, it is well known to occur after various epileptogenic insults, including SE and TBI as also shown here, signifying it as a biomarker candidate for epileptogenesis (Nissinen et al. 2001; Kharatishvili et al. 2006, 2007; Nairismagi et al. 2006). As the post-injury density and orientation of sprouted mossy fibers in the inner molecular layer and hilus differ remarkably from those in the healthy brain, mossy fibers provide a relevant structural candidate that can influence post-injury diffusivity in the dentate gyrus. In addition to mossy fiber sprouting, recent

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studies have shown sprouting of axons of inhibitory cells as well as changes in the dendritic arborization of granule cells, which can also contribute to diffusion changes in the dentate gyrus (Wong 2005; Thind et al. 2010). There is also a variable degree of neurodegeneration, whereas the tightly packed granule cells are relatively well preserved in both models. Recent data also suggest long-lasting glial cell and vascular abnormalities in the hippocampus after SE and TBI which can contribute to diffusion changes (Rigau et al. 2007; Ndode-Ekane et al. 2010; Hayward et al. 2011; Budde et al. 2011). Another less investigated contributor to diffusion is the change in composition of the extracellular matrix during epileptogenesis (Dityatev and Fellin 2008). Considering the wide variety of structural abnormalities in the dentate gyrus after SE and TBI demonstrated in the present and previous studies, it was somewhat surprising that even after pilocarpine-induced SE, in which the structural changes in the dentate gyrus are robust, alterations in the main orientation of diffusion were relatively mild and no major differences were found as compared to the less severely damaged dentate gyrus after TBI. One explanation is that the ROI included the granule cell and molecular layers only, excluding the hilus. Consequently, the orientation of diffusion in the voxel analyzed remained dorsal–ventral along the granule cell dendrites despite the variable degree of axonal reorganization seen in the molecular layer and the hilus by both myelin and Timm preparations. The increase in FA in the dentate gyrus after SE without a change in the main orientation of diffusion was related to an increase in axial rather than radial diffusivity. The present data extend our previous findings showing that even clear changes in FA or axial diffusivity do not necessarily affect the main orientation of diffusion even in the severely damaged dentate gyrus (Laitinen et al. 2010). Consequently, FA is a more sensitive indicator of ongoing structural reorganization in the dentate gyrus and has more potential to serve as a biomarker of epileptogenesis than orientation of main diffusivity. From laboratory to clinic: translational and technical considerations The high-resolution DTI data presented here were collected using long, overnight scans of ex vivo samples with a slow and robust spin-echo sequence. It is relatively straightforward to expedite data acquisition using echo planar imaging (EPI)-based DTI sequences and obtain comparable resolution within a few hours, which is already feasible for scanning rats in vivo. To demonstrate this, we recently showed that FA changes after SE in the dentate gyrus could be detected in vivo using a standard 7 T MRI system (Laitinen et al. 2010).

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In our high field experimental setting, we performed DTI using six diffusion weighting directions, which is the theoretical minimum to determine symmetric diffusion tensor. In clinic, DTI is nowadays often done with [ 20–30 directions using fast EPI sequences to increase the accuracy of the DTI parameters by decreasing the extent to which the variance of a given parameter depends on orientation (Jones 2004). In a very high magnetic field strength, such as 9.4 T used in this study, the use of fast imaging techniques can, however, lead to geometric distortions. To overcome this problem we acquired data using slow spin-echo approach and only six diffusion directions. This approach resulted in an excellent image quality without any geometric distortions and in a high signal-tonoise ratio ([ 35 in all raw images). Under these conditions, the inaccuracy in basic tensor-derived properties can be estimated to be relatively small compared to changes detected in this study (Ni et al. 2006; Landman et al. 2007; Jones 2004). In future, more advanced high-angular-resolution diffusion imaging (HARDI) type acquisition schemes together with modeling approaches that go beyond a single tensor model may reveal more detail in the tissue microstructure, especially in areas with a high density of crossing fibers such as the CA3. The main direction of water diffusion (orientation of the principle eigenvector) is dependent on brain orientation when low angular resolution and anisotropic voxels are used, and the water diffusion direction can also be influenced by several other factors such as atrophy. However, in our experimental study, we were able to position the brain during each imaging session with high accuracy. Still, possible changes in the shape of the hippocampal subfields due to atrophy add another layer of complexity to the data interpretation. In our study, the hippocampus showed significant atrophy. However, the septal part of the hippocampus that was included in our analysis did not undergo major changes in its rotational axis, and therefore we consider it unlikely that hippocampal atrophy could explain the robust changes in the main direction of the water diffusion that we observed in this study. Also, the changes in orientation of water diffusion were associated with histologic changes in corresponding fiber stainings. Zero padding of diffusion-weighted images before Fourier transformation was used in the present work. Even though this approach has been quite commonly used, it is strictly speaking not identical to interpolation of the vector field due to non-linearity of the system. However, we performed ROI analysis of relatively homogenous areas avoiding the borders, and thus the interpolation approach used in the present study is not likely to cause any bias to the results. MRI developments have made it possible to obtain highresolution DTI data from the human brain as well. An

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isotropic resolution of 800 lm3 has been obtained by a 1 h scan at 7 T using a reduced field of view approach with partial parallel imaging (Heidemann et al. 2012). Taking into account the larger size of the human hippocampus [e.g., at the mid-septotemporal level the coronal crosssectional area of the hippocampus is about 7 9 6 mm and that of the dentate gyrus is 2 9 6 mm (Amaral and Insausti 1990)], the technology is approaching conditions where changes in individual hippocampal subfields can be detected. In fact, recently Yassa et al. (2010) demonstrated that perforant path degeneration was detectable in aged patients in vivo using ultrahigh-resolution microstructural DTI at 3 T. As 7 T MRI systems are becoming more common, and parallel imaging techniques with new pulse sequences can circumvent many of the problems associated with high field imaging (Heidemann et al. 2012), it is reasonable to assume that the detection of robust DTI changes in individual subfields of the hippocampus becomes possible in the near future through use of the most advanced experimental patient MRI systems.

Conclusions Until recently, DTI has been largely used for imaging reduced or aberrant white matter connectivity caused by developmental abnormalities, learning or practice effects, or brain injury (Concha et al. 2006; Oechslin et al. 2010; Zalesky et al. 2011; Chan et al. 2012). Studies assessing DTI in conditions with increased aberrant axonal plasticity are few (Parekh et al. 2010; Kuo et al. 2008) and many lack histological verification (Sidaros et al. 2008). The present data show that ex vivo DTI reveals subfieldspecific patterns in diffusivity in the normal rat hippocampus and the dentate gyrus. After epileptogenic brain injuries (e.g., SE or TBI), the diffusivity in different subfields of the hippocampus changes in an etiologydependent manner. This is in accord with the magnitude of histologically verified structural changes being greatest after SE [ ipsilateral TBI [ contralateral TBI. The noninvasive detection of changes in the hippocampus as it undergoes histologically verifiable modifications carries a great potential to identify biomarkers for progressive damage, functional recovery, and/or epileptogenesis after brain injury. Acknowledgments This study was supported by the Academy of Finland (A.S., O.G., A.P.), Sigrid Juselius Foundation (A.P.), and UEF-Brain strategic funding from the University of Eastern Finland. We thank Ms. Maarit Pulkkinen for assistance in histology, Dr. Jari Nissinen and Dr. Tamuna Bolkvadze for technical assistance, MSc Juha-Pekka Niskanen for drawing the diffusion ellipsoids, and Dr. Nick Hayward for revising the language of the manuscript.

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Diffusion tensor imaging of hippocampal network plasticity.

Diffusion tensor imaging (DTI) has become a valuable tool to investigate white matter integrity in the brain. DTI also gives contrast in gray matter, ...
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