In Vivo Multimodal Magnetic Resonance Imaging Changes After N-Methyl-d-Aspartate-Triggered Spasms in Infant Rats

Objective Despite the serious neurodevelopmental sequelae of epileptic encephalopathy during infancy, the pathomechanisms involved remain unclear. To find potential biomarkers that can reflect the pathogenesis of epileptic encephalopathy, we explored the neurometabolic and microstructural sequelae after infantile spasms using a rat model of infantile spasms and in vivo magnetic resonance imaging techniques. Methods Rats prenatally exposed to betamethasone were subjected to three rounds of intraperitoneal N-methyl-d-aspartate (NMDA) triggering of spasms or received saline injections (controls) on postnatal days (P) 12, 13, and 15. Chemical exchange saturation transfer imaging of glutamate (GluCEST) were performed at P15 and 22 and diffusion tensor imaging and additional spectroscopy (1H-MRI/MRS) of the cingulate cortex were serially done at P16, 23, and 30 and analyzed. Pathological analysis and western blotting were performed with rats sacrificed on P35. Results Within 24 h of the three rounds of NMDA-induced spasms, there was an acute increase in the GluCEST (%) in the cortex, hippocampus, and striatum. When focused on the cingulate cortex, mean diffusivity (MD) was significantly decreased during the acute period after multiple spasms with an increase in γ-aminobutyric acid (GABA), glutamate, and glutamine N-acetylaspartate-plus-N-acetylaspartylglutamate (tNAA), total choline, and total creatine. The juvenile rats also showed decreased MD on diffusion tensor imaging and significant decreases in taurine, tNAA, and macromolecules-plus-lipids in the cingulate cortex. Pathologically, there was a significant reduction in glial fibrillary acidic protein, myelin basic protein, and neuronal nuclei expression in the cingulate cortex of rats with NMDA-induced spasms. Significance These neurometabolic and microstructural alterations after NMDA-triggered spasms might be potential imaging biomarkers of epileptic encephalopathy.

| Timeline of N-methyl-d-aspartate (NMDA)-induced spasms, MRI acquisition, behavioral tests, and tissue collection. IHC, immunohistochemistry; WB, western blot. inTrODucTiOn Infantile spasms are an epileptic encephalopathy that requires urgent therapeutic intervention to avoid severe consequences, such as mental retardation and developmental regression (1). Diverse etiologies, including both unidentified causes and esta blished brain pathologies, can lead to infantile spasms with diffuse electrographic abnormalities (2), which probably impair common developmental processes such as synaptogenesis, myelination, and neuronal migration, causing mental retardation or refractory epilepsies in later life (3,4). However, these neurodevelopmen tal consequences have not been clearly characterized (2). To support the rationale for an aggressive treatment approach for infantile spasms, it is crucial to determine the changes pro duced by the spasms in the immature brain and assess the pos sibility for a restoration or reversal of these changes. In children with infantile spasms, evaluation of the effects of spasms on the brain is extremely complicated because of ethical problems and a relatively small number of patients with heterogeneous etiolo gies (5) and treatment protocols (6,7).
From the bench side, it would be advantageous to have a model without preexisting lesions and a model with easily operable spasms to identify the pathological effects of the experimental spasms on the brain. The rat model of Nmethyldaspartate (NMDA)triggered spasms (4) after prenatal betamethasone pri ming is regarded as having a cryptogenic etiology and the spasms are reliably triggered by the injection of NMDA. The NMDA triggered spasms can be provoked at postnatal day 12-15, a period in rodents relevant to human infancy (3,4). The model also displays several behavioral changes after multiple spasms (3), corresponding to those of patients with infantile spasms, and has recently been independently verified (8).
In the present study, using this rat model of NMDAinduced infantile spasms, we performed MR imaging to evaluate the acute and chronic neurometabolic and microstructural changes after spasms as potential biomarkers of epileptic encephalopathy.
To add longitudinal in vivo data to this model, we adopted the latest highfield proton MRI technologies including chemical exchange saturation transfer imaging of glutamate (GluCEST), diffusion tensor imaging, and spectroscopy ( 1 HMRI/MRS). GluCEST imaging can map the level of glutamate in vivo at a high spatial resolution (9)(10)(11), and diffusion tensor imaging is used to reveal the in vivo connectivity of the nervous system (12,13). 1 HMRS allows noninvasive quantification of brain metabolites in specific brain areas and has been successfully used in the field of epilepsy research (14,15). Specifically, the in vivo developmen tal changes in neurometabolites and connectivity were measured in the cingulate cortex, which has extensive connections with critical regions such as the basal ganglia, thalamus, brainstem, and hippocampus (16,17). Additional histopathologic analyses and behavioral tests were performed to further elucidate the microstructural and neurochemical changes.

MaTerials anD MeThODs animals
Experiments were approved by the Institutional Animal Care and Use Committee of the Ulsan University College of Medi cine and conformed to the Revised Guide for the Care and Use of Laboratory Animals [NIH GUIDE, 25 (28), 1996] (18). Timedpregnant SpragueDawley rats were purchased from an approved source (Orient Bio Inc., Seoul, Korea). The rats were housed individually in the animal facility during the remainder of their pregnancy with free access to standard rat chow and water on a regular 12h lightdark cycle with the lights on at 08:00. On gestational day 15, pregnant rats received two injections of 0.4 mg/kg betamethasone (SigmaAldrich, St. Louis, MO, USA) at 08:30 and 18:30. Delivery occurred consistently on gestational day 22, which was considered postnatal day (P) 0 for the offspring.
Spasms were triggered by intraperitoneal injection of NMDA on P12 (6 mg/kg), P13 (10 mg/kg), and P15 (15 mg/kg); control groups received the same volume of saline (Figure 1). Imme diately after NMDA administration, the rats were observed for 90 min and only the animals confirmed to have had three bouts of spasms at the appropriate time points on each day (i.e., on P12, P13, and P15) were included in the analyses (4).

Mr imaging studies
Animals were maintained under anesthesia with 1% isoflurane in a 1:2 mixture of O2:N2O with monitoring of their respiratory rate, electrocardiogram, and rectal temperature. GluCEST MR imaging was performed using a 7.0 T/160mm smallanimal imaging system (Bruker Pharmascan, Ettlingen, Germany) with a singlechannel surface coil. Images were obtained using a 9.4 T/160mm bore animal MRI system (Agilent Technologies, Santa Clara, CA, USA) for 1 HMRS and diffusion tensor ima ging. Radiofrequency excitation and signal detection were accomplished with a 72mm quadrature volume coil and a two channel phasedarray coil, respectively. Axial slices correspond ing to coronal images in the neuroanatomic axis were collected from the cervical spinal cord to the olfactory bulb.
GluCEST images were acquired from an axial slice (1mm thick) that included the hippocampal region. GluCEST images were acquired using T2weighted imaging (rapid acquisition with relaxation enhancement [RARE]) with a frequency selective saturation preparation pulse comprised a Gaussian pulse with a total duration of 1,000 ms (irradiation offset of 500.0 Hz and interpulse delay of 10 µs) at a B1 peak of 5.6 µT. Zspectra were obtained from −5.0 ppm to +5.0 ppm with intervals of 0.33 ppm (total, 31 images, Figure S1  To measure the GluCEST value (%), each region of interest (cortex, hippocampus, striatum) was drawn manually on the T2 weighted anatomical MR images without a frequency selective saturation preparation pulse, and the regions of interest were overlaid on the GluCEST maps. GluCEST contrast is measured as the asymmetry between an image obtained with saturation at the resonant frequency of exchangeable amine protons (+3 ppm downfield from water for glutamate) and an image with satura tion equidistant upfield from water (-3 ppm), according to the following equation: The B0/B1 maps on the same slices were acquired for B0 and B1 correction. The B0 map was calculated by linearly fitting the accumulated phase per pixel following phase unwrapping against the echo time differences from gradient echo images collected at TEs of =1.9 and 2.6 ms. B1 maps were calculated by using the doubleangle method (flip angles 30° and 60°) and the linear correction for B1 was calculated as the ratio of the actual B1 to the expected value. 1 HMRI/MRS images were obtained at the assigned times as follows (Figure 1): (1) 1 day after the last cluster of spasms (P16), (2) about 1 week after the spasms (P23), and (3) about 2 weeks after the spasms (P30). The MR spectra were acquired through a signal voxel (from bregma to −3.0 mm in a coronal section, 3 mm × 2 mm × 1.5 mm) in the cingulate cortex using a pointresolved spectroscopy (PRESS) sequence for 128 acquisi tions with TR/TE = 5,000/13.4 ms. For quantification, unsup pressed water signals were also acquired from the same voxel (average = 8). All the MR spectra were processed with the linear combination analysis method (LC Model ver. 6.0, Los Angeles, CA, USA) to calculate the metabolite concentrations from a fit to the experimental spectrum, based on a simulated basis set. The following brain metabolites were included in the metabolite basis set: alanine (Ala), aspartate (Asp), creatine (Cr), γaminobutyric acid (GABA), glucose (Glc), glutamate (Glu), glutamine (Gln), glycerophosphorylcholine (GPC), phosphorylcholine (PCh), myoinositol (mIns), lactate (Lac), phosphocreatine (PCr), Nacetylaspartate (NAA), Nacetylaspartylglutamate (NAAG), taurine (Tau), macromolecules (MMs), and lipids (Lip). The watersuppressed time domain data were analyzed between 0.2 and 4.0 ppm, without further T1 or T2 correction. Absolute metabolite concentrations (mmol/kg wet weight) were calculated using the unsuppressed water signal as an internal reference (assuming 80% brain water content) (19). The in vivo proton spectra were judged to have an acceptable value if the stand ard deviation of the fit for the metabolite was less than 20% (Cramer-Rao lower bounds). MR diffusion tensor images were acquired using a fourshot DTecho planar imaging sequence (TR = 3.7 s, TE = 20 ms, B0 = 1,000 s/mm 2 ) with a 10ms inter val (Δ) between the application of diffusion gradient pulses, a 4ms diffusion gradient duration (δ), a gradient amplitude (G) of 46.52 mT/m, and the Jones 30 gradient scheme.
Postprocessing analysis was performed using Diffusion Toolkit software (http://trackvis.org/). The cingulate cortex of each rat was selected and the fractional anisotropy (FA) and mean diffu sivity (MD) were calculated from the diffusion tensor parametric maps. Subsequently, repeated measures ANOVA and ttests were conducted to test for the treatment effect of the different diffusion parameters.

Behavioral Testing
After NMDAinduced spasms, rats performed an openfield test on P19 and fear conditioning tests on P27-29 (Figure 1). Behavioral experiments were conducted in a standard behavioral testing room during the light phase (08:00-20:00 h) of a regular 12h light-dark cycle.

Open-Field Test
The locomotive activity of controls and rats after NMDA triggered spasms was assessed using an openfield test as previ ously described (20). Rats were placed into a black plastic box (60 cm × 60 cm 2 field with a 30cm high perimeter) for 5 min, and their activities were monitored (3, 21) using computerized motiontracking software (SMART 3.0; Panlab S.L.U., Barcelona, Spain). The center was defined as the middle area 10 cm apart from each wall and the other area was defined as the periphery (i.e., 2,000 cm 2 periphery and 1,600 cm 2 center; allocation of the area = 5:4).

Fear conditioning
The observation chamber (25 cm × 25 cm × 25 cm; Panlab S.L.U.) was constructed of aluminum (two side walls and ceiling) and Plexiglas (rear wall and hinged front door) and was situated in a soundproof box. The floor of the chamber consisted of 19 stainless steel rods (4 mm diameter) spaced 1.6 cm apart (center to center), which were connected to a shock generator and grid scrambler (Panlab S.L.U.). A tone (conditioned stimulus) was delivered by a speaker mounted on one side panel of the chamber and both the shock and tone deliveries were controlled by a computerized system.
Tone conditioning was conducted on P27. Modified from a previous report (22), the conditioning consisted of a 3min baseline habituation in the experimental chambers, followed by exposure to five pairings of a tone (conditioned stimulus; 30 s, 85 dB, 2,000 Hz) with whitelight illumination, each ending with a final footshock (unconditioned stimulus; 0.8 mA, 2.0 s) followed by a 20s silent interval. Response to the context was monitored on P28 (24 h after the conditioning trial). The rats were returned to the chamber for 5 min, and freezing behavior was measured in response to the context. Fear response to the conditioned stimulus was conducted at 24 h after the contextual fear testing (P29). To reduce the influence of context on cued fear conditioning, tactile and visual cues were manipulated with the replacement of a wall and a floor. Following a 2min period without a conditioned stimulus (first session, unconditioned), the rats were presented with five tone parings (85 dB, 2 kHz, 30 s, session Nos. 2, 4, 6, 8, and 10; 20 s between tone presenta tions, session Nos. 3, 5, 7, 9, and 11) without any foot shock. The behaviors of the rats were recorded and analyzed using the signal generated by a highsensitivity weight transducer system (Panlab S.L.U.).

sample Preparation and immunohistochemistry
The animals with three bouts of spasms on P12, P13, and P15 and the corresponding controls were transcardially perfused with 4% paraformaldehyde on P35 under deep anesthesia, and their brains were removed and cryoprotected. Twentymicron serial coronal sections were cut on a cryocut microtome.
For immunohistochemistry, myelin basic protein (MBP) anti body (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), glial fibrillary acidic protein (GFAP) antibody (1:2,000; EMD Millipore, Billerica, MA, USA), or neuronal nuclei (NeuN) antibody (1:2,000; EMD Millipore) were used as primary anti bodies. All slides were examined by light microscopy (Olympus BX53; Olympus Co., Tokyo, Japan). Semiquantification analysis of immunohistochemical staining was performed using ImageJ (NIH, Bethesda, MD). The corresponding area in the cingulate cortex cg2 subfield was analyzed to determine the NeuN and GFAPpositive cell count and the stained area measurement of MBP. Three or five randomly selected areas (1.0 × 10 4 sq μm) on each section were used for NeuN and GFAPpositive cell count ing. The same areas of binarized images of MBPstained sections were quantified from each animal.

statistics
The Mann-Whitney's U test was used for the comparison of two groups and Student's ttest was used for the data following normal distribution. The concentrations of metabolites were compared between the groups and time points using a linear mixed model. The absence of significant sexbased differences (p always > 0.10) was confirmed in advance, and both sexes were combined. The significance level was preset to p < 0.05. Statistical analyses were conducted using SAS ® Version 9.3 (SAS Institute Inc., Cary, NC, USA) and SPSS 18.0 (IBM Corp., Armonk, NY, USA).

Behavioral changes after Multiple Bouts of nMDa-Triggered spasms
To evaluate the behavioral changes that occurred after multiple bouts of NMDAinduced spasms, the results of openfield and fear conditioning tests were compared between the rats that had experienced three bouts of NMDAtriggered spasms and controls. In the openfield test on P19, the rats with NMDA induced spasms (n = 23) traveled a significantly shorter distance through the peripheral area (p = 0.005) as well as in the total area (p = 0.006) compared with controls (n = 20; Figure 5A). Resting time in the peripheral area was significantly increased in rats with NMDAinduced spasms (p = 0.012) compared with controls. The time spent in slow and fast motion in the peripheral area was significantly decreased in the rats with NMDAinduced spasms compared with controls (p = 0.014 and p = 0.010, respectively; Figure 5B). The time spent in the center was equivalent in the two groups.
In fear conditioning tests on P27-29, there was no difference in tone conditioning on P27 or response to context on P28. However, on P29, the total freezing durations were significantly increased in rats with NMDAinduced spasms compared with controls (p = 0.037; Figure 5C). During the first session, freezing durations were not different between the two groups (p = 0.058), but freez ing durations were significantly increased in rats with NMDA induced spasms at conditioned sessions from 6 to 9, in particu lar (p = 0.007, 0.002, 0.047, and 0.022, respectively; Figure 5D).

reduced gFaP, MBP, and neun expression in the Juvenile Period after Multiple Bouts of nMDa-Triggered spasms
Semiquantitative analysis of immunohistochemical staining on P35 revealed a significant reduction in NeuN and GFAPpositive cells (both p < 0.001) and MBPpositive area (p = 0.041) in rats with three bouts of NMDAtriggered spasms (on P12, P13, and P15, n = 13) compared with controls (n = 11) (Figure 6). The cortical protein expression levels of GFAP, MBP, and NeuN on P35 were quantified and compared between the rats with multiple spasms and controls. GFAP (p = 0.007), MBP (p = 0.027), and NeuN (p = 0.001) expression was significantly reduced in the cortex of rats with NMDAinduced spasms (n = 13) compared with controls (n = 13; Figure 6).

DiscussiOn
An early diagnosis of infantile spasms has been considered criti cal for improving the neurodevelopmental outcomes of affected patients (23). However, it is uncertain which part of the developing brain is affected by infantile spasms, as is the extent of any brain insult and whether it is reversible. Our present study focused on metabolic and microstructural brain insults after spasms in a rat model of infantile spasms using in vivo MR imaging. Behavioral tests and ex vivo histopathological processes were employed to support the results.
Considering the increased metabolism of brain tissue dur ing seizures, neurochemical dysfunction might be the main The examples of MR spectra from rats with or without 3 NMDA spasms on each time points, P16 and P30 clearly shows the neurometabolic changes after NMDA-induced spasms. *indicates a significant difference between the groups at each time point (p < 0.05). Using a generalized linear mixed model, there were also significantly different interactions between the groups and time points for Glx, Tau, tNAA, MM + lipids, and total choline (each p-value is marked on the graph). All imaging was done on the same timepoints (P16, 23, and 30), although the data points are not aligned across groups to avoid overlapping of the two graphs.   . (a,B) Open-field test showing significantly decreased exploration distances in rats with NMDA-induced spasms in peripheral and total areas (a) and significantly increased resting activities and decreased slow and fast activities in the peripheral area (B) of rats with spasms. The rats with NMDA-induced spasms also showed significantly increased durations of freezing to sound and light stimuli (c) that persisted after several sessions without shocks (D) in a fear conditioning test. There was a significant difference in freezing durations between the two groups. *indicates a significant difference between the groups (*p < 0.05, Mann-Whitney U test). pathophysiologic feature of the epileptic brain (24), especially during the acute stage. The GluCEST technique quantifies gluta mate by measuring proton exchange between the amine protons of the glutamate and the water protons and the measurement is at least 100 times more sensitive than the traditional 1H MRS method (9,25). The GluCEST is suggested as a surrogate marker of glutamate concentration in glial cells at the synaptic level (25) and glutamate contributes >90% of the GluCEST signal with <10% contribution from other metabolites in glial neuronal unit (10). Previous study with temporal lobe epilepsy patients also showed the high potential of GluCEST identifying the epileptogenic foci (26). Thus, we hypothesized that the area of hyperexcitation in rats with NMDAinduced spasms can be screened with GluCEST imaging with high spatial resolution during acute periods. GluCEST mapping showed significantly increased glutamate in cortex and hippocampus at P15 after multiple rounds of spasms (Figure 2), which suggests that the cortex is one of the major brain area of hyperexcitation by the NMDAinduced spasms. Our in vivo serial neurochemical/microstructural analysis focused on the cingulate cortex in the rat after multiple bouts of spasms. The cingulate cortex is a region of the limbic system reciprocally linked to the hippocampus (17) and with extensive connections to the critical regions for spasm generation, includ ing the basal ganglia, thalamus, and brainstem (16,27). In the cingulate cortex, consistent with the findings of GluCEST imag ing, acute elevations in GABA, Glx, tNAA, tCho, and tCr were noted in rats after multiple bouts of NMDAinduced spasms compared with controls (Figure 3). The glutamate plus glutamine levels likely represent the neuronal metabolic pool. In vivo human proton MRS studies (28,29) have also reported alterations in GABA and glutamate in patients with epilepsy. In addition, choline (Cho) represents a precursor for the neurotransmitter acetylcholine (30) and creatinine is associated with energy metabolism (31,32). The early elevation of markers of neuronal metabolism together with the excitatory neurotransmitters in the cingulate cortex may also reflect activation of the cingulate cortex during NMDAinduced spasms in this model.
With diffusion tensor imaging, we also identified an acute decrease in MD in the cingulate cortex after three bouts of spasms, which represent the early brain damage (33). MD is an inverse measure of the membrane density, is sensitive to cellularity, edema, and necrosis (34). MD decrease quickly after injury (first day) in most models studied, which is consistent with our find ing at P16. The decrease was explained by the decrease in water diffusion associated with injury such as cellular edema, despite  there is still debate on the precise mechanism for the decrease in MD associated with injury. Previously postictal decrease of MD was reported in human (35), which is consistent with our finding and probably reflects neuronal swelling by excitotoxic injury in the brain areas involved in seizure. In addition to these acute changes associated with spasms, in vivo MR imaging enables us to perform a longitudinal follow up of neurometabolic and microstructural alterations in rats with NMDAtriggered spasms. NMDA is an excitatory amino acid that can cause neuronal death and glial activation is well observed in kainic acid animal model and tissues of patients with chronic epilepsy (36). However, gross morphological changes caused by NMDAinduced spasms in our model were not evident on brain MR imaging and a previous pathologic report of NMDAinduced seizures in young animals also reported no gross morphological changes on pathology (37). Consistently with this finding, FA, a gross measure of microstructural integ rity marker that is less specific to the type of injury (33), is not affected in every time point of this study.
In the cingulate cortices of the juvenile rats with NMDA induced spasms (P30), tNAA, Tau, and MM + lipids were sig nificantly decreased with a significant reduction in MD. We also found decreased expression of neuronal, astrocyte, and myelina tion markers after NMDAinduced spasms in these rats. These findings suggest the different mechanism of injury during infancy compared to that of the adulthood. Instead of cell death or gliosis after excitatory injury, the developmental process of their brain, active protein synthesis and cell proliferation, may be involved. These pathologic changes are also in line with a significant reduc tion in neuronal markers, NAA, and Tau and markers of myelin production and substructural components such as MMs and lipids (31,38), as well as the reduced MD reflecting decreased membrane density and cellularity (34,39).
Previous research into epilepsy has already shown the typi cal developmental pattern of neurometabolites in both a rodent model (40) and in humans (32). NMDAtriggered spasms signifi cantly impaired this timedependent neurometabolic develop mental pattern of Glx, Tau, tNAA, MM + lipids, and total choline (Figure 3). These combined alterations in neurometabolites and structural maturation markers in this animal model indicate the compromised development of the cingulate cortex follow ing NMDAtriggered spasms. Also, the decrease of all proteins examined can be explained by a decrease in protein synthesis during the critical brain development after NMDAtriggered spasms.
Furthermore, decreased exploratory behaviors in the periph eral areas and increased freezing activities to conditioned sound and light stimuli were also observed in our study group that suffered from NMDAinduced spasms. Other behavioral disrup tions were found in previous studies with similar models (21,37). Considering the widespread reduction in neuronal metabolites in neurodevelopmental disorders (31) and a positive correlation between motor skill learning and myelination (41), the pathologi cal changes in the brain might explain these behavioral changes.
In conclusion, NMDAinduced spasms during infancy lead to timedependent neurochemical and microstructural changes in the cingulate cortex and subsequent pathologic changes during the juvenile period. These agedependent alterations in neuro metabolites after NMDAtriggered spasms should be further explored as potential biomarkers of outcomes in human infantile spasms or other epileptic encephalopathies.

eThics sTaTeMenT
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