The reduction of volume and fiber bundle connections in the hippocampus of EGR3 transgenic schizophrenia rats
Received 23 January 2015
Accepted for publication 27 March 2015
Published 1 July 2015 Volume 2015:11 Pages 1625—1638
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 3
Editor who approved publication: Dr Roger Pinder
Ensen Ma,1,2,* Tianbin Song,3,* Hui Zhang,4,* Jie Lu,5 Liwen Wang,2 Qichao Zhao,2 Runcai Guo,2 Miao Li,2 Guolin Ma,2 Guangming Lu,1 Kefeng Li6,7
1Department of Medical Imaging, Jinling Hospital, Medical School of Nanjing University, Nanjing, People’s Republic of China; 2Department of Radiology, China-Japan Friendship Hospital, Beijing, People’s Republic of China; 3Department of Radiology, Beijing Shunyi Hospital, Beijing, People’s Republic of China; 4Department of Radiology, First Clinical Medical College, Shanxi Medical University, Taiyuan, People’s Republic of China; 5Department of Radiology, Xuanwu Hospital of Capital Medical University, Xicheng, Beijing, People’s Republic of China; 6School of Medicine, University of California, San Diego, CA, USA; 7Tianjin SunnyPeak Biotech Co., Ltd, Tianjin, People’s Republic of China
*These authors contributed equally to this work
Background and objective: There is a growing consensus that schizophrenia is ultimately caused by abnormal communication between spatially disparate brain structures. White matter fasciculi represent the primary infrastructure for long distance communication in the brain. In this study, we aimed to investigate the white matter connection in schizophrenia susceptible brain regions of early growth response factor 3 (EGR3) expressing rats.
Methods: A rat model of schizophrenia was created by the transfection of the EGR3 gene into rat hippocampus. All animals were placed in a fixation system using a commercial rat-dedicated coil. Schizophrenia susceptible brain regions were scanned using in vivo diffusion tensor magnetic resonance imaging. The volume, quantity, average length of fiber bundles, fractional anisotropy, apparent diffusion coefficient, the relative heterosexual fraction, and volume ratio were collected in the whole brain and schizophrenia related brain areas (the hippocampus, thalamus, and prefrontal lobe). MedINRIA software was used for data processing of diffusion tensor and fiber bundles tracking. The fibronectin in relevant brain regions was also analyzed.
Results: There was a significant decrease in the volume of the fiber beam through the left hippocampus dentate in the schizophrenia model group in comparison to the control group and the risperidone treatment group (P<0.05). A significant reduction in the volume and number of the fiber bundles was also observed in left prefrontal–left hippocampus, left hippocampus–left thalamus, left prefrontal–left hippocampus–left thalamus areas in the model group (all P<0.05).
Conclusion: The volume of hippocampus and the number of fiber bundles were reduced in EGR3 transgenic schizophrenia rats, and are the most sensitive indicators in schizophrenia. The diffusion tensor imaging technique plays an important role in the evaluation of patients with schizophrenia.
Keywords: magnetic resonance imaging, schizophrenia, early growth response factor 3, fractional anisotropy, diffusion tensor imaging
Schizophrenia is a chronic mental disorder characterized by severe perturbations in cognition, affect, and behavior.1 The early growth response factor 3 (EGR3) rat is a transgenic insertional mutant that models several aspects of the behavioral and molecular pathology expressed in schizophrenia.2,3 For instance, the rat mutant shows behavioral abnormalities, including novelty-driven hyperactivity that is manifested as increased circling activity in the open field.4 As these abnormalities are often diagnosed in patients with schizophrenia, genetically mutant EGR3 rat might be relevant for understanding or pinpointing psychoses-causing mechanisms.
The magnetic resonance imaging (MRI) technique especially suited for the analysis of white matter tracts makes possible the direct assessment of large axon masses.5 Diffusion-weighted MRI can be used to distinguish normal and abnormal tissues on the basis of the molecular motion of water molecules (Brownian motion).6 The method is sensitive to disruptions of commissural bundles or tracts. It can be used to study regional axonal connectivity, myelin packing, and fiber orientation.5,6 The brain white matter tract abnormalities, as demonstrated by diffusion tensor imaging (DTI) techniques, are common occurrences in adult schizophrenia.1,7,8 It is proposed that schizophrenia arises from a unique human brain network associated with directed effort including the dorsal anterior and posterior cingulate cortex, auditory cortex, and hippocampus.9
As rats share important biochemical and genomic similarities with humans, EGR3-transfected schizophrenia-like rat model could recapitulate certain aspects in patients with schizophrenia. Risperidone is a typical antipsychotic medication used to treat mental illnesses such as schizophrenia, bipolar disorder, and irritability associated with autism disorder.10 There is no evidence about the changes of brain structure caused by risperidone.
In this study, we first created a rat model of schizophrenia by the transfection of lentiviral particles carrying the EGR3 gene into the hippocampus. We then investigated the functional connectivity and anatomical connections of white matter tracts in important brain areas (prefrontal cortex, hippocampus, thalamus) in EGR3 rats using the DTI technique. The changes of functional connectivity in the brain of schizophrenia-like rats before and after risperidone therapy were also explored.
Materials and methods
Animals and husbandry
A randomized, controlled animal study was conducted in the School of Medicine, Peking University, People’s Republic of China, in accordance with the guidance suggestions for the care and use of laboratory animals, issued by the Ministry of Science and Technology of the People’s Republic of China.10 Twenty-four healthy, male Sprague Dawley rats (aged 4 weeks, weighing 100±10 g) were purchased from Vital River, Beijing, People’s Republic of China (license no SCXK [Jing] 2012-0001). Animals were housed in a temperature (22°C–24°C) and humidity (40%–55%) controlled vivarium. The experimental procedures and the animal use and care protocols were approved by the Committee on Ethical Use of Animals of China-Japan Friendship Hospital. Standard rat food was used and the animals had free access to water and food during the whole study.
Lentiviral particles containing the EGR3 gene were purchased from the Fuyishengke Biomedical Scientific Research Service Center, Shanghai, People’s Republic of China. The sequence is based on NCBI Reference Sequence: NM_018781.2. The lentivirus was packaged using a four-plasmid system containing psPAX2 (vector), pMD2G, pLVX-IRES-ZsGreen1 (vector + GFP), and pLVX-IRES-ZsGreen1-EGR3 (vector + GFP + EGR3).
To construct the rat schizophrenia model, the lentivirus particle carrying the EGR3 gene was injected bilaterally into the hippocampus of rats as previously described.4 Briefly, the rats were anesthetized by intraperitoneal injection with 10% chloral hydrate (1.5 mL/kg), and placed in a stereotaxic frame (ST-51600; Kopf Instruments, Tujunga, CA, USA). The skin of the calvarium was sterilized with 75% alcohol, and an incision of approximately 0.5 cm was made. After sterilization with 0.05% hydrogen peroxide (H2O2), the bregma was exposed. Bilateral holes (0.8 mm) were drilled in the skull above the injection site using a cranial drill (Figure S1A). The lentivirus particle was slowly injected into each side of the hippocampus (−3.0 mm anteroposterior and ±2.0 mm mediolateral to bregma; −2.2 mm dorsoventral to the skull surface) using a 1 μL microinjector over 20 minutes (Figure S1B). The needles were maintained in position for a further 20 minutes, and then the incision was sutured. The animals were returned to their home cages when they could move spontaneously. Rats in the sham-surgery group (n=6) underwent an identical procedure except the lentivirus particle carried GFP instead of EGR3. The location of EGR3 expression was checked by immunofluorescence staining of rat brain tissues according to previous protocols. The image was acquired using Nikon Ti Eclipse Confocal Microscope (Nikon, Tokyo, Japan). The number of positive cells in the visual filed was counted (n=6).11 Morris water-maze working memory test and the open-field test were used to characterize EGR3-transfected schizophrenia model as described before.4
After a 2-week recovery period, EGR3-transfected rats received intraperitoneal injections of risperidone (0.2 mg/kg) (Sigma, St Louis, MO, USA) for 14 consecutive days. The other three groups received intraperitoneal injections of normal saline at each administration time point corresponding to the risperidone treatment. In summary, four groups were set up in this study with 8–13 replicates in each group including control group (rats without surgery + normal saline), sham group (rats + GFP gene + normal saline), schizophrenia model group (rats + EGR3 gene + normal saline), and treatment group (rats + EGR3 gene + risperidone).
DTI imaging acquisition
The imaging data were acquired on a Phillips 3.0 T Achieva scanner (Philips, Amsterdam, the Netherlands) using a standard whole-body rat coil (Shanghai Chenguang Medical Science and Technology, Shanghai, People’s Republic of China). The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (1.5 mL/kg) before scanning. The rats were placed in prone position and the heads of rats were fixed with a vacuum pillow and band to reduce head movement. First, T2WI coronal, axial, and sagittal images were collected using spin echo sequence. T2WI image parameters were as follows: field of view (FOV) =40×40×10 mm, time of repetition (TR) =1,696 ms, time of echo (TE) =96 ms, number of slices =7, slice thickness =1.5 mm, layer space =1.5 mm, and number of signal average (NSA) =3. Next, larger FOV and more detailed T2WI Images were collected using these parameters: FOV =40×40×30 mm, TR =2,800 ms, TE =96 ms, slice number =20, slice thickness =1.5 mm, interlayer space =0 mm, NSA =10, flip angle of 90°, a total scan time of 10 minutes and 27 seconds, and an acquisition matrix of 132×127. Further DTI images were obtained using single-shot spin-echo planar imaging (EPI) sequence on coronal imaging of rat brain with the following parameters: 32 different directions were used on diffusion sensitive gradient, TR =4,000 ms, TE =85 ms, slice thickness =1.5 mm, no interval scan, FOV =40×40×30 mm, acquisition matrix of 116×117, and slice number =20. Diffusion-weighted coefficients (b values) were 0 s/mm2 and 1,000 s/mm2, NSA =2. Total time was 22 minutes and 44 seconds.
Data processing and analysis
Diffusion tensor and fiber bundle tracking data were processed using MedINRIA-1 (v9.4). The diffusion tensor matrix, eigenvalues (λ1, λ2, λ3), and eigenvector values were calculated by MedINRIA-1. Each point of the main direction is consistent with the maximum eigenvalue eigenvector direction. Fractional anisotropy (FA) value is a parameter commonly used to characterize anisotropy. The apparent diffusion coefficient (ADC) represents tensor tracking and is also frequently used to characterize the anisotropy. Both FA and ADC were calculated using the following formula:
First, all the imaging data of 24 rats in the four groups were analyzed with whole-brain DTI and fiber bundle tracking process. Parameter settings were as follows: the threshold of background removal was 200, threshold of FA was 200, threshold of smoothing was 20, minimum length was 10 mm, and samples were 25. The required anisotropy index includes the fiber bundle volume, quantity, average length, FA, ADC, relative anisotropy (RA), and volume ratio (VR). Next, the left and right anterior frontal, left, and right hippocampus and the left and right thalamus were selected as the regions of interest (ROI) to be analyzed with whole-brain DTI and the fiber bundle tracking process, respectively. The required anisotropy index was also calculated for the selected brain regions.
Finally, to perform the DTI and fiber bundle tracking process, the brain ROI were selected. The ROI included the bilateral prefrontal cortex, bilateral hippocampus, thalamus, right anterior frontal lobe–the hippocampus, right prefrontal–thalamus, right side of the hippocampus–thalamus, right prefrontal–hippocampus–thalamus, left anterior frontal lobe–the hippocampus, left anterior frontal lobe–thalamus, left hippocampus–thalamus, and left anterior frontal lobe–hippocampus–hypothalamus (Figure S2). The anisotropy index and fiber connections were also analyzed for the brain ROI.
Statistical analysis was performed using SPSS (v17.0; SPSS, Chicago, IL, USA) and GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA, USA). All values were expressed as mean ± standard deviation unless stated in the text. One-way ANOVA followed by post hoc Tukey’s test were used to calculate the anisotropy index variability among the four groups. A P-value less than 0.05 was considered to be significantly different.
Verification of EGR3-transfected rat model of schizophrenia
The expression of the EGR3 gene in the hippocampus of rat brain was analyzed 1 week after the lentivirus injection. The number of EGR3-positive cells was 2.67 times higher in the model group than in the GFP transfection sham group (Figure S3). Morris water-maze and open-field experiments were used to test whether the rats with EGR3 gene transfection have schizophrenia-like behaviors. The water maze experiment showed that the escape latency time in rats with EGR3 gene transfection was significantly longer than in the control group and the sham group (P<0.05), suggesting that the EGR3 gene transfection group showed poor working memory capacity (Figure S4).
DTI and fiber bundles tracking in whole brain of rats
There was no statistically significant difference in the anisotropy index including fiber bundle volume, number of fibers, average length, FA, ADC, RA, and VR, in the whole brain, among the EGR3 gene transfection schizophrenic group, sham operation group, risperidone treatment group, and the control group (Table 1).
DTI and fiber bundles tracking hippocampus
There was no statistically significant difference in the anisotropy index which including fiber bundle volume, number, the average length, FA, ADC, RA, and VR of the right hippocampus, among EGR3 gene transfection schizophrenic group, sham operation group, risperidone treatment group, and the control group (Table 2).
The white fiber bundle volume in left hippocampus were remarkably decreased in the EGR3 gene transfection schizophrenic group compared with that in the control group and the sham group (Figure 1). Risperidone treatment restored the volume of white fiber back to normal (Figure 1). No significant changes were observed in the length, FA, ADC, RA, and VR in the left hippocampus among the four groups (P>0.05) (Table 3).
There was a significant reduction of the white fiber bundle volume and number in the bilateral hippocampus in the EGR3 gene transfection schizophrenic group compared with that in the sham operation group (P<0.05). The decrease of white fiber bundle was normalized by risperidone treatment (Figure 2).
Figure 2 Statistical analysis of white fiber volume, number, and length in bilateral hippocampus.
Connectivity between ROI in schizophrenia rat model
Figure 3 showed the fiber connections between the brain regions. There were no fiber bundle connections (red line) between the left and right sides of the brain regions associated with schizophrenia. A large number of white matter fiber tract connections can be observed in ipsilateral schizophrenia related brain regions (green and blue lines). While fiber bundle connections exist in the right prefrontal–right hippocampus, right anterior frontal lobe–right thalamus, right hippocampus–right thalamus, right prefrontal–right hippocampus–right thalamus, and left prefrontal–left thalamus between the four groups, there were no significant differences among the four groups (green line).
Figure 3 Schematic diagram of brain connections between different brain regions.
The volume and number of fiber connections between the left anterior frontal lobe and the left hippocampus were significantly decreased in the EGR3 transfection schizophrenic group compared with the other three groups (Figure 4). There were no significant differences in the length, FA, ADC, RA, and VR of the fiber connections (data not shown). Similarly, we found a significant reduction of fiber connections (both volume and number) between the left hippocampus and the left thalamus and the connections were restored after risperidone treatment (Figure 5). In addition, both the number and volume of the three consecutive connections among left anterior frontal lobe–left hippocampus–left thalamus were significantly decreased in the EGR3 gene transfection schizophrenic group (Figure 6). No changes in the fiber length were noticed. The volume and number of connections were restored after risperidone treatment.
Correlations between the volume and the number of white fiber bundles were then analyzed in schizophrenia related brain areas. The results showed that the volume and number of the white fiber bundles were highly correlated in the connections of left hippocampus–left thalamus and left anterior frontal lobe–left hippocampus–left thalamus in the schizophrenia model group, but not in the control group (Figure 7).
The methods for DTI data analysis
DTI may be used to study the commissural white matter pathways. The method is sensitive to the disruptions of commissural bundles or tracts. Three methods were used to analyze DTI data using ROI drawing with freehand, voxel-based analysis (VBA), and fiber tracking.11,12
Although functional and structural brain imaging studies have revealed diverse brain abnormalities in schizophrenia, no single brain region has been either clearly or consistently identified as the site of the disorder.1 Therefore, the ROI method is difficult to reflect the panorama of the lesion. More and more researchers tend to use VBA.
The optimized voxel-based morphometry (VBM) method is a type of morphological study method, with the voxel as a basic unit used to compare the whole brain. The advantages of the VBM method are that there is no bias on a specific structure, and the method can be used to comprehensively evaluate the whole brain. The method can also reflect abnormal connections between the morphological structures of the whole brain regions. In addition, the method is not restricted to one or a few regions and is more suitable for the diffusion tensor vector analysis in patients with schizophrenia.
Fiber tracking is a technology that could reconstruct the three-dimensional structure along the fiber bundle. This technique can provide detailed information about anatomical connections between brain areas, and indirectly provide clues on the activation process of each node in the research of functional magnetic resonance imaging (fMRI) neural networks. This kind of information is very important to explore the synchronization between cortical areas and to interpret fMRI results for further determination of the networks involved in the cognitive process.
Because there are no standard fMRI brain templates in rats, the whole-brain registration analysis based on voxel and fiber tracking cannot be used. DTI and fiber tracking were obtained by combining the method of copying the freehand drawing of ROI and automatically importing ROI from the software. DTI could reflect abnormal connections between the morphological structures of the whole brain and brain regions. The DIT technique is more useful for the diffusion tensor vector analysis in patients with schizophrenia.
The abnormal brain fiber connection in white matter of schizophrenic rats
The previous study reported that the FA value of the left hippocampus head is lower than that in the control group in one DTI study in patients with first-episode schizophrenia, suggesting that the fiber bundle of the left hippocampus head in patients with first-episode schizophrenia is abnormal.13 In this study, we also found that the damage of white matter fiber tracts were not in the global brain regions of early schizophrenia. They only appear in susceptibility related brain regions. We found that the left hippocampus is one of the regions involved during the development of schizophrenia. A meta-analysis of studies on white matter volumes in schizophrenia revealed a reduction of approximately 2% in medial temporal lobe structures.14 Indeed, regional rather than global reductions are far more relevant to the question of the origins of disconnectivity and hence are of particular interest in the presence of symptoms.
No significant differences in the anisotropy index including the fiber bundle volume, number of fibers, average length, FA, ADC, RA, and VR in the right hippocampus were observed in the EGR3 expressing rat model of schizophrenia. We found that the white fiber bundle volume in the bilateral hippocampus was remarkably decreased in the EGR3 gene transfection schizophrenic group compared with that in the other three groups. These results further illustrated that the hippocampus is a susceptible brain area. The reduction of the fiber bundle volume and number of fibers is one of the most sensitive detection indexes in schizophrenia.
Default network analysis of the EGR3 transgenic schizophrenia model contributes to the understanding of brain function connection mode
Some brain regions, such as the medial prefrontal cortex, anterior and posterior cingulates, as well as inferior temporal, lateral parietal, and cerebellar regions, are particularly active during “rest” and are deactivated during a variety of cognitive tasks. These brain regions form the “default network”.15,16 This concept has become a central theme in contemporary cognitive and clinical neuroscience. Symptoms of schizophrenia, such as basic personality change, cogitation, emotion, splitting behavior, and lack of coordination between mental activity and the environment, might be associated with an impaired default network.15 The abnormal connections of the default network might be present in schizophrenia. Disturbances of interconnections between brain structures in schizophrenia were one of the explanations.15,16 Our DTI studies of EGR3 transgenic schizophrenia models help understand white matter fiber connections pathways. Disturbances in the connectivity between brain regions, including the frontal lobe, basal forebrain, and limbic system, have been reported in schizophrenia.17–21 Connections between the prefrontal cortex and other cortical and subcortical regions in the pathophysiology of schizophrenia suggested that a white matter abnormality in this region is critical to the core symptoms of schizophrenia.
Currently, studies on the default network connection of schizophrenia have suggested that the hyperactivity in function activities exists in the schizophrenic default network.22–25 The exact role of the default network in schizophrenia patients is still not clear. Further fMRI studies are needed to reveal the changes of default network connections in patients with schizophrenia.
Many DTI studies in patients with schizophrenia have demonstrated the damage of cerebral white matter integrity, mainly related to the white matter fiber of frontal and temporal lobe, as well as the major nerve fiber bundles of the human brain, such as the corpus callosum, anterior cingulate, hook beam, arched beam, and internal capsule.17,26,27 DTI could demonstrate the abnormal function connection on the basis of abnormal anatomy connection in patients with schizophrenia. A previous study found that anatomical and functional connections can always match each other in the local area or in the globe brain if DTI and static status function connection analysis are combined.28 Because the widely distributed abnormal function connection exists in patients with schizophrenia, it is necessary to explore the relationship between the anatomical features and functions of the brain.
Our study demonstrated that there were no fiber bundle connections between the left and right sides of the brain regions associated with schizophrenia. Possible reasons for the lack of interhemispheric connections may be related to the direction of white matter fiber, threshold setting in postprocessing, and resolution of the image.29 Ipsilateral related brain areas have a large number of white matter fiber bundle connections. Right frontal lobe–right hippocampus, right frontal lobe–right thalamus, right hippocampus–right thalamus, right frontal lobe–right hippocampus–right thalamus, and left prefrontal–left thalamus have fiber connections with no significant differences between the schizophrenic rat group and the control group. However, there are fiber connections in left prefrontal–left hippocampus, left hippocampus–left thalamus, and left frontal lobe–left hippocampus–left thalamus with a significant reduction in the schizophrenic rat group in comparison to the control group.
The white fiber bundle connections in the left anterior frontal lobe–left hippocampus, left hippocampus–left thalamus, and left anterior frontal lobe–left hippocampus–left thalamus were also significantly decreased in the EGR3 gene transfection schizophrenic group compared with the other three groups. Compared with the control group, the fiber bundle volume of left prefrontal–left hippocampus, left hippocampus–left thalamus, and left frontal lobe–left hippocampus–left thalamus have significant differences in the EGR3 gene transfection schizophrenia group. The fiber bundle volume of left prefrontal–left hippocampus, left hippocampus–left thalamus, and left frontal lobe–left hippocampus–left thalamus decreased significantly in the EGR3 gene transfection schizophrenia group too. No significant difference was observed in the fiber connections between left prefrontal and left thalamus. This might be because of the long distances between the two brain regions.
Furthermore, in this study, a significant correlation was observed between the volume and number of the white fiber bundles in left hippocampus–the left thalamus ROI and anterior frontal lobe–left hippocampus–the left thalamus ROI. This indicated that the reduction in the number and volume is synchronized.
Hippocampus is a brain region highly susceptible to damage in EGR3 transgenic schizophrenia rats. The volume of the hippocampus and number of fiber bundles were significantly reduced in EGR3 transgenic schizophrenia rats, and are the most sensitive indicators in schizophrenia. The DTI technique may play an important role in the diagnosis and follow-up evaluation of schizophrenia in the future.
This project was supported by the National Science and Technology Support Program (2012BAI10B02) and the National Science Foundation of China (81471652), and an internal grant from China-Japan Friendship Hospital, People’s Republic of China (2014-3-MS-18).
The authors report no conflicts of interest in this work.
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Figure S2 Schizophrenia susceptible area in rat brain.
Figure S3 Expression of EGR3 in hippocampus region of rat brain.
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