Distinct MRI pattern in lesional and perilesional area after traumatic brain injury in rat — 11 months follow-up

https://doi.org/10.1016/j.expneurol.2008.09.009Get rights and content

Abstract

To understand the dynamics of progressive brain damage after lateral fluid-percussion induced traumatic brain injury (TBI) in rat, which is the most widely used animal model of closed head TBI in humans, MRI follow-up of 11 months was performed. The evolution of tissue damage was quantified using MRI contrast parameters T2, T1ρ, diffusion (Dav), and tissue atrophy in the focal cortical lesion and adjacent areas: the perifocal and contralateral cortex, and the ipsilateral and contralateral hippocampus. In the primary cortical lesion area, which undergoes remarkable irreversible pathologic changes, MRI alterations start at 3 h post-injury and continue to progress for up to 6 months. In more mildly affected perifocal and hippocampal regions, the robust alterations in T2, T1ρ, and Dav at 3 h to 3 d post-injury normalize within the next 9–23 d, and thereafter, progressively increase for several weeks. The severity of damage in the perifocal and hippocampal areas 23 d post-injury appeared independent of the focal lesion volume. Magnetic resonance spectroscopy (MRS) performed at 5 and 10 months post-injury detected metabolic alterations in the ipsilateral hippocampus, suggesting ongoing neurodegeneration and inflammation. Our data show that TBI induced by lateral fluid-percussion injury triggers long-lasting alterations with region-dependent temporal profiles. Importantly, the temporal pattern in MRI parameters during the first 23 d post-injury can indicate the regions that will develop secondary damage. This information is valuable for targeting and timing interventions in studies aiming at alleviating or reversing the molecular and/or cellular cascades causing the delayed injury.

Introduction

Traumatic brain injury (TBI) is a prevalent cause of disability and mortality in industrialized countries (Leon-Carrion et al., 2005). After primary impact and injury, secondary injury develops over hours to months. It is composed of a complex and poorly understood combination of molecular, cellular (Karhunen et al., 2005), and metabolic (Kharatishvili et al., 2006) alterations in the central nervous system, which lead to post-injury functional disabilities including somatomotor impairment, cognitive decline, emotional disturbance, or epilepsy (Kharatishvili et al., 2006, Thompson et al., 2006). Many of the delayed pathological processes could potentially be reversed, thus they could provide targets for the development of recovery enhancing and/or antiepileptogenic treatments. To conquer this challenge, a non-invasive detection of the progression of pathology, which in particular can discern potentially reversible tissue damage from irreversibly injured areas in individual subjects is critical for defining anatomic regions for application of such therapies

MRI is one of the most versatile imaging methods available today and provides an excellent tool to study the spatio-temporal progression of damage under controlled experimental settings. Currently, most of the experimental MRI studies of TBI have focused exclusively on the acute to subacute phase, that is, the time period extending from hours to days or a few weeks post-injury. Furthermore, the majority of the MRI follow-up studies report alterations in only one or two MRI contrast parameters such as volumetry of cortical atrophy, cortical edema related T2 hyperintensity, and/or decreased apparent water diffusion (ADC) within hours after TBI followed by increased diffusion days or weeks after TBI (Albensi et al., 2000, Obenaus et al., 2007, Onyszchuk et al., 2007, Van Putten et al., 2005).

Nuclear magnetic resonance spectroscopy (MRS) has been used to assess metabolite changes during the acute and subacute phases after TBI. These studies have reported a decrease in N-acetylaspartate/creatine ratio (NAA/Cr) and an increase in choline/creatine (Cho/Cr) ratio in the lesion area and/or in the ipsilateral parietal cortex in rodents that have experienced fluid-percussion or controlled cortical impact injury (Berry et al., 1986, Choi et al., 2005, Schuhmann et al., 2002). Also, in the pericontusional zone Cr + PCr, NAA, and Glu were decreased from 1 h to 28 d post-injury (Dube et al., 2001) and persistently decreased in the combined hippocampus basal ganglia region up to 4 weeks after trauma (Schuhmann et al., 2002). However, there are no long-term studies available on changes in the metabolic profile after TBI.

The aim of this study was to investigate the spatio-temporal long-term progression of brain damage after TBI. We hypothesized that an MRI signature determined by multiparametric quantitative MRI can differentiate perilesional tissue from irreversibly damaged lesion. To test this hypothesis, we induced TBI using lateral fluid-percussion injury that is a clinically relevant rat model of closed head TBI in humans, then systematically quantified several relevant MRI contrast parameters for up to 11 months post-injury, including quantitative T2, T1ρ, trace of the diffusion tensor (Dav), and volumetric changes. Furthermore, we evaluated chronic changes in the metabolic profile of the hippocampus by magnetic resonance spectroscopy (MRS).

Section snippets

Methods

The study design is summarized in Fig. 1. The Group 1 (14 rats with TBI and 4 with sham operation) was imaged at 3 h, 3 d, 9 d, 23 d, 2 months, 3 months and 6 months after TBI. In Group 2 (20 rats with TBI and 10 with sham operation), the imaging was started at 5 months to avoid the possible effects of repeated halothane anesthesia during early post-injury period on long-term progression of brain pathology. In addition, as data from Group 1 indicated, progression of some alterations was still

Animal model

TBI was induced in adult male Harlan Sprague Dawley rats (total n = 48, from which n = 34 survived and were examined by MRI, 305–390 g; Harlan Netherlands B.V., Horst, the Netherlands) by lateral fluid-percussion brain injury as previously described (Kharatishvili et al., 2006, McIntosh et al., 1989). Animals were anesthetized with a mixture containing sodium pentobarbital (58 mg/kg), chloral hydrate (60 mg/kg), magnesium sulfate (127.2 mg/kg), propylene glycol (42.8%), and absolute ethanol

Magnetic resonance imaging (MRI)

MRI data were acquired at a horizontal 4.7 T magnet (Magnex Scientific Ltd, Abington, UK) with actively shielded imaging gradients (Magnex) interfaced to a Varian (Palo Alto, CA) UNITYINOVA console and using a quadrature half-volume RF coil (diameter 28 mm/two 18 mm loops, HF Imaging LLC, Minneapolis, MN) in transmit/receive mode. Rats were anaesthetised with 1% halothane in N2O/O2 (70%/30%), securely fixed into a stereotactic holder and kept warm with a water circulating heating bed underneath

MRI data analysis

The volumes of the lesion at all time points and of the hippocampus 6 months post-injury were calculated by manually outlining the regions of interest into the T2 weighted multi-slice set covering the whole brain. We defined lesion volume as a combined volume of focal lesion and ipsilateral ventricle. This was because the distinction between the two regions was practically impossible at the later time points, when progressive destruction of cellular structures leads to complete tissue loss and

Magnetic resonance spectroscopy (MRS) measurements and analysis

The in vivo single voxel spectroscopy data were obtained using stimulated echo acquisition mode (STEAM) localization with short echo time (Tkac et al., 1999) (TE = 2 ms, TR = 4 s, bandwidth of 2.5 kHz was covered with 3336 points, averages = 512) incorporating VAPOR water suppression scheme, after automatic FASTMAP shimming (Gruetter 1993). Voxels (2.5 mm ⁎ 3 mm ⁎ 3 mm) were placed in both the ipsilateral and contralateral hippocampus (Fig. 2). The spectral analysis was performed using LC model and only

Statistics

All values are indicated as mean ± standard deviation (std). Statistical evaluation was performed using SPSS for Windows software (Chicago, IL, version 14.0). Differences between TBI and control groups were calculated using Kruskal–Wallis followed by post hoc analysis with Mann–Whitney U test (non-parametric tests used due to the low animal number). Differences between time points were calculated using Friedman followed by post hoc analysis with Wilcoxon test. P < 0.05 was considered significant.

Results

Mortality (death within 72 h post-injury) in Group 1 was 25% and in Group 2 21% suggesting that in both groups the lateral fluid-percussion TBI was moderate in severity (Dubreuil et al., 2006, McIntosh et al., 1989, Thompson et al., 2005), and there was no difference between the Groups. Also the severity of impact as expressed in atm was similar (Group 1: range 2.3–3.2 atm, mean 2.64 atm; Group 2: range 3.0–3.1 atm, mean 2.86, no difference between groups).

Temporal progression of lesion volume

As in other laboratories (McIntosh et al., 1989, Smith et al., 1997), even though the injury-induction parameters were similar from animal to animal, we found substantial variability in the severity of cortical lesion between individual animals. The variation in the lesion size can be described by categorizing the rats in Group 1 into the three subgroups based on T2 weighted images acquired at 23 d post-injury when lesion outlines were most clearly visible (as compared to the earlier time

Diffusion (Dav)

Diffusion (Dav) changes during the 11 months follow-up are summarized in Fig. 5. In the area of focal lesion, 3 of 14 animals with TBI showed acute diffusion drop of around 10% at 3 h post-injury (compare the data in the perifocal area and the ipsilateral hippocampus). Starting at 3 d post-injury, Dav increased dramatically indicating loss of diffusion limiting structures, that is, ongoing tissue loss. At 3 d post-injury, Dav values were 19%, at 9 d 70%, at 23 d 99%, and at 6 months 120% higher

Volume of the primary focal lesion did not correlate with the severity of damage in the perifocal areas

To find out if there is potentially viable perifocal tissue in the animals with very large focal lesions we studied the relationship between primary focal lesion volume and the severity of damage in more distal perifocal and hippocampal areas. Fig. 8 clearly shows the absence of any correlations, that is, the severity of the pathological change in MRI contrast parameters (i.e. the increase of T2, T1ρ and Dav) within the perifocal area outside the primary focal lesion did not depend on the size

Chronic metabolic abnormalities as revealed by hippocampal MR spectroscopy

Five months post-injury we found decreased relative concentrations (relative to total Cr consisting of Cr + PCr) of γ-aminobutyric acid (GABA), glutamate (Glu), N-acetylaspartylglutamate (NAAG), N-acetylaspartate + N-acetylaspartylglutamate (NAA + NAAG) and glutamate + glutamine (Glu + Gln) in the ipsilateral hippocampus as compared to controls (p < 0.05, Figs. 9–10). Levels of myo-inositol (Ins) were elevated (p < 0.05, Figs. 9–10). The further decrease of Glu was the only change from 5 months to 10 months (

Discussion

In order to understand the dynamics of brain damage after TBI, we performed quantitative MRI and MRS in a lateral fluid-percussion model of TBI in rat. We focused our analysis on the injured cortex, the perifocal area, and the hippocampus that are known to undergo histopathologic changes in this model (Hallam et al., 2004, Sato et al., 2001). In our 11 months follow-up study we have four major findings. First, brain damage after TBI progresses continually for up to 6 months. Second, Dav

Conclusions

Our results distinguish two different patterns of brain tissue alterations in the aftermath of TBI based on the temporal fluctuation of quantitative MRI parameters. First, at the primary lesion area with drastic irreversible pathologic MRI alterations start at 3 h–3 d post-injury and continue to progress steadily for months. Second, in more mildly affected perifocal and hippocampal regions the alteration in T2, T1ρ, Dav at 3 h–3 d post-injury normalize within the first 3–23 d, and then show a

Acknowledgments

This work was supported by the Academy of Finland, the Emil Aaltonen Foundation, the Sigrid Juselius Foundation, CURE (Citizens United for Research for Epilepsy), the Finnish Epilepsy Research Foundation, and the Finnish Cultural Foundation. We thank Mrs. Maarit Pulkkinen, Mr. Jarmo Hartikainen, Mrs. Merja Lukkari, and Jari Nissinen, Ph.D., for technical assistance, Nick Hayward, M.Sc., for revising the language of the manuscript, and Ivan Tkác providing the short echo time STEAM pulse sequence.

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