brain research 1622 (2015) 339–349

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Research Report

Neuroprotective efficacy of decompressive craniectomy after controlled cortical impact injury in rats: An MRI study Runfa Tiana,f,g,1, Li Hanb,1, Zonggang Houa,f,g,n, Shuyu Haoa,f,g, Xiang Maoc, Zhendan Zhue, Xiaogang Taoa,f,g, Qi Zhangb, Baiyun Liua,d,e,f,g,n a

Department of Neurosurgery, Beijing Tian Tan Hospital, Capital Medical University, Beijing 100050, PR China Department of Neurology, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, PR China c Department of Neurosurgery, the First Affiliated Hospital of Anhui Medical University, Hefei 230000, PR China d Neurotrauma Laboratory, Beijing Neurosurgical Institute, Capital Medical University, Beijing 100050, PR China e Department of Neurotrauma, General Hospital of Armed Police Forces, Beijing 100039, PR China f China National Clinical Research Center for Neurological Diseases, Beijing 100050, PR China g Beijing Key Laboratory of Central Nervous System Injury, Beijing 100050, PR China b

art i cle i nfo

ab st rac t

Article history:

Decompressive craniectomy (DC) is one of the therapeutic options for severe traumatic

Accepted 24 June 2015

brain injury (TBI), and it has long been used for the treatment of patients with malignant

Available online 11 July 2015

post-traumatic brain edema. However, a lack of definitive evidence prevents physicians

Keywords:

from drawing any conclusions about the efficacy of DC for the treatment of TBI. Magnetic

Brain trauma

resonance imaging (MRI) is widely used to evaluate the effects of TBI in both experimental

Controlled cortical impact

and clinical studies. Therefore, the aim of the present study was to investigate the MRI

Decompressive craniectomy

assessment of DC post-TBI in rats to provide experimental animal data and radiological

Magnetic resonance imaging

evidence to support the clinical application of DC. We used both in vivo MRI and proton

Proton magnetic resonance

magnetic resonance spectroscopy (1H-MRS) to evaluate the therapeutic effect of DC on

spectroscopy

lateral controlled cortical impact (CCI) rat models at 3 h, 1 d, 2 d, 3 d and 7 d after TBI. Our data suggest that DC can reduce brain edema; decrease the apparent diffusion coefficient value, contusion volume and lactate (Lac)/creatine (Cr) ratio; and increase the Nacetylaspartate (NAA)/Cr and choline (Cho)/Cr ratios after TBI. The present results suggest that DC can indeed reduce brain edema formation and exhibits good neuroprotective efficacy after CCI injury in rats. & 2015 Elsevier B.V. All rights reserved.

n Correspondence to: Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Tiantan Xili 6, Dongcheng District, Beijing 100050, People's Republic of China. Fax: þ86 10 67059157. E-mail addresses: [email protected] (Z. Hou), [email protected] (B. Liu). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.brainres.2015.06.039 0006-8993/& 2015 Elsevier B.V. All rights reserved.

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1.

brain research 1622 (2015) 339–349

Introduction

Individuals of all ages, backgrounds, and health statuses are susceptible to traumatic brain injury (TBI), which is a major cause of death and disability worldwide. TBI affects approximately 1.7 million people annually and contributes to 30% of all injury-related deaths in the United States. However, there is currently no effective treatment for TBI, and TBI patients consequently have a poor prognosis (Shimoda et al., 2014; Namjoshi et al., 2013). Brain edema plays an important role in the pathophysiology of TBI. The formation of brain edema results in an increase in tissue water content and brain swelling, which can lead to increased intracranial pressure (ICP) (Unterberg et al., 2004). Elevated ICP is the most frequent cause of death and disability after TBI. Mortality and morbidity rates remain high, despite refinements in medical and pharmacological means of controlling intracranial hypertension (Kontopoulos et al., 2002). When conservative treatment fails, a decompressive craniectomy (DC) can be successful in lowering ICP, and this surgical procedure has been in use for a long time (Bohman and Schuster, 2013). Because of the lack of evidence-based clinical data and the deficiency of experimental data, DC is recommended by most national and international guidelines only as a third-tier therapy for the treatment of pathologically elevated ICP (Plesnila, 2007). Although a recent randomized clinical trial suggests that the functional outcome and quality of life may be better in early craniotomized children, and data in adults point in the same direction, the lack of relevant class I evidence still does not allow a definite conclusion to be reached regarding the efficacy of DC for the treatment of TBI (Plesnila, 2007; Bender et al., 2013). Recently, magnetic resonance imaging (MRI) has been widely used to evaluate the consequences of TBI in both experimental and clinical studies (Kou et al., 2010). MRI provides information on both TBI-related abnormalities and the potential effects of treatments. Diffusion-weighted imaging (DWI) and proton magnetic resonance spectroscopy (1H-MRS) have recently emerged as powerful approaches for characterizing the microstructural and metabolic responses after TBI (Wei et al., 2012).

Furthermore, several of the metabolites detected by 1H-MRS are highly sensitive to the pathology that contributes to TBI, including hypoxia or ischemia, bioenergetics dysfunction, and inflammation (Harris et al., 2012). Recent technological improvements in MRI provide much more information on both TBI-related abnormalities and the potential effects of treatments, contributing to the understanding of the pathological progression of TBI and the mechanisms of effective therapies (Saatman et al., 2008). Most of the previous literature has simply analyzed the neuroprotective efficacy of DC after TBI in animals using methods including immunohistochemistry, western blotting, or the water maze, but no studies have examined the utility of MR for assessing the neuroprotective efficacy of DC in a controlled cortical impact (CCI) model (Kontopoulos et al., 2002; Bohman and Schuster, 2013; Bender et al., 2013). Therefore, the aims of the present study were to investigate the MRI assessment of the efficacy of DC post-TBI and to characterize TBI pathophysiology to provide experimental animal data and medical imaging evidence supporting the clinical application of DC.

2.

Results

2.1.

T2-weighted imaging

T2-weighted MRI was used to verify the tissue effects of CCI and allowed us to follow the longitudinal development of the brain contusion in vivo. Rapid acquisition with relaxation enhancement (RARE) images from representative control, sham-operated, and CCI animals with (DCþ) or without (DC-) decompressive craniectomy are shown in Fig. 2. After CCI, tissue disruption and marked hyperintensities were visible in the ipsilateral cortex and hippocampus, including cortical surface deformation, brain edema formation, ventral shift of the corpus callosum, and frequent small intraparenchymal hemorrhages, as early as 3 h post-injury; the lesion was most obvious at 1–2 d and was still present at 7 d. At 1-3 d, edema could be distinguished as a diffuse hyperintensity in the ipsilateral cortex, hippocampus and ipsilateral parietal adjacent cortex, and tissue swelling was indicated by displacement of the

Fig. 1 – Schematic representation of the brain trauma and craniectomized area (a) and the regions of interest (b). The area of the craniotomy was 3.8 mm posterior and 2.5 mm lateral to the bregma. The area of the craniectomy extended from a point 5 mm ahead of the coronal suture anteriorly to the rhomboid suture posteriorly. The medial and lateral borders were the sagittal suture and superior temporal line. The regions of interest selected for analysis were the ipsilateral cortex (C), hippocampus (H) and parietal adjacent cortex (A) on the side of the controlled cortex impact injury.

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Fig. 2 – Typical coronal sections of the brains of control, sham-operated at 1 d, and brain-injured animals with DC- or DCþ at 3 h, 1 d, 2 d, 3 d, and 7 d after injury. The T2-weighted images showed hyperintensities in the injured cortex, which were most noticeable at 1 and 2 d post-injury. The decrease in the magnetic resonance imaging (MRI) signal intensity was detected by the apparent diffusion coefficient (ADC) maps in the lesion area. The severity of the injury in the DC- group was significantly greater than that in the DCþ group at each time point. White arrows showed the ipsilateral cortex and parietal adjacent cortex on the side of the controlled cortex impact injury. The ROIs selected for analysis were the ipsilateral cortex, hippocampus and parietal adjacent cortex on the side of the controlled cortex impact injury. DC-, animals that did not undergo decompressive craniectomy after brain injury; DCþ, animals that underwent decompressive craniectomy after brain injury.

Table 1 – Mean ADC, contusion volume, NAA/Cr, Cho/Cr and Lac/Cr ratio change in the ipsilateral cortex, hippocampus and the parietal adjacent cortex between Control group and Sham-operated group. ROI

Group

Mean ADC

Contusion volume

NAA/Cr

Cho/Cr

Lac/Cr

Cortex

Control Sham-operated Control Sham-operated Control Sham-operated

681.271.4 684.572.6 685.972.3 689.473.4 686.772.8 688.672.9

0 0 0 0 0 0

1.3770.03 1.3670.04 0.8370.01 0.8470.01 0.0770.01 0.0770.03

1.4470.01 1.4370.02 0.7470.01 0.7570.01 0.0770.01 0.0770.02

1.4370.01 1.4470.01 0.8470.01 0.8570.01 0.0370.001 0.0370.002

Hippocampus Adjacent

There is no significantly difference between control and sham-operated groups. Control, animals not undergo any surgery; sham-operated, animals underwent the same procedure of making craniotomy but without CCI.

cortical surface and a midline shift toward the contralateral hemisphere. Tissue swelling had subsided by 7 d, giving way to cortical thinning and ventricular enlargement. Within the CCI group, for both edema formation as well as lesion volume, DCgroup was significantly worse than DCþ group at each time point, which may confirm that DC could reduce brain edema and play a protective role after brain injury. No signal changes were observed in the sham-operated or control animals.

2.2.

Apparent diffusion coefficient (ADC) maps

ADC maps from representative control, sham-operated, and CCI DC- or DCþ animals are shown in Fig. 2. There was no significant difference between control and sham-operated groups (Table 1). In the CCI group, marked hypointensities were visible in the ipsilateral cortex, hippocampus and the

parietal adjacent cortex 3 h after CCI. These hypointensities were most obvious at 1–2 d and were still observed at 7 d. Within the CCI group, the ADC values of the ipsilateral cortex in the DCþ group at 1 and 2 d after injury were significantly higher than those in the DC- group, and the difference was statistically significant (po0.05) (Table 2, Fig. 3a). The (contralateral-ipsilateral)/contralateral cortex mean ADC ratio was considered to be an indicator of cortical edema severity, and our data demonstrated that the ratio in the DC- group at 1 and 2 d was significantly higher than that in the DCþ group. This difference was statistically significant (po0.05), showing less serious brain edema formation and edema severity in the DCþ group (Fig. 3b). The ADC values of the ipsilateral hippocampus in the DCþ group were higher than those in the DC- group at 3 h after injury, while the ipsilateral parietal adjacent cortex in the DCþ group was higher than that in the

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Table 2 – Mean ADC change in the ipsilateral cortex, hippocampus and the parietal adjacent cortex between DC- group and DCþ group. ROI

Group

Pre-TBI

3h

1d

2d

3d

7d

Cortex

DCDCþ DCDCþ DCDCþ

683.172.7 681.471.6 685.374.4 687.473.7 687.273.1 688.173.7

532.2717.2 577.3721.3 660.6717.4 680.3710.1* 597.4727.3 604.3722.4

447.3719.3 592.4733.7** 680.5728.1 704.7737.2 649.7724.2 653.1722.6

541.6710.2 611.6711.7* 690.3722.3 680.6725.7 658.5719.2 671.2710.2

590.376.9 601.777.7 685.7720.6 677.2732.8 649.3720.4 692.4711.3*

614.6727.7 615.3715.2 690.8738.1 686.3719.2 650.3731.4 690.1713.4

Hippocampus Adjacent

Compared with DC- group values. DC-, animals not receiving decompressive craniectomy after brain injury; DCþ, animals receiving decompressive craniectomy after brain injury. n po0.05. nn po0.01.

Fig. 3 – Mean changes in the apparent diffusion coefficient (ADC) in the ipsilateral cortex, hippocampus and parietal adjacent cortex and the ratio of ADC change in the ipsilateral cortex in the DC- and DCþ groups before the injury and at 3 h, 1 d, 2 d, 3 d and 7 d after the injury (**po0.01 and *po0.05 for the DCþ group versus the DC- group at each time point in the ipsilateral cortex, hippocampus and adjacent cortex. DC-, animals that did not undergo decompressive craniectomy after brain injury; DCþ, animals that underwent decompressive craniectomy after brain injury.).

DC- group at 3 d after injury; both differences were statistically significant (po0.05) (Fig. 3c,d). No other differences were observed at the other time points between the CCI and shamoperated groups or between the DC- and DCþ groups.

2.3.

correlated with the reduction in edema (Spearman, p¼ 0.036). The contusion volume in the DCþ group was less than that in the DC- group at 1, 2, 3 and 7 d after injury, and the difference was statistically significant (po0.05), demonstrating that DC could reduce the volume of brain damage and improve the prognosis of contusion after injury (Table 3, Fig. 4).

Contusion volume

There was no significant difference in contusion volume between the control and sham-operated groups (Table 1). Brain contusions were observed 3 h after CCI, and the volume increased quickly, reaching a peak at 1 d before declining gradually and dropping to the same volume observed at 3 h at 7 d after injury. The reduction in lesion volume was strongly

2.4.

Proton magnetic resonance spectroscopy

Typical spectra of the ipsilateral cortex in control, shamoperated, and brain-injured DC- or DCþ animals at 1 d after CCI are shown in Fig. 5. Multiple changes were clearly visible in the ipsilateral cortex of the CCI group compared to the control

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Table 3 – Contusion volume of rat brain after brain injury (cm3). Group

Pre-TBI

3h

1d

2d

3d

7d

DCDCþ

0 0

113.2714.7 98.6710.6

225.3716.3 177.4717.3*

209.7714.9 138.4717.8*

192.6720.4 126.3716.3*

141.2717.3 89.779.7*

Compared with DC- group values. DC-, animals not receiving decompressive craniectomy after brain injury; DCþ, animals receiving decompressive craniectomy after brain injury. n po0.05.

3.

Fig. 4 – Line chart demonstrating the contusion volume of the rat brain in the DC- and DCþ groups before the injury and at 3 h, 1 d, 2 d, 3 d and 7 d after the injury. Decompressive craniectomy significantly reduced the contusion volume at 1 d, 2 d, 3 d and 7 d after the injury (*po0.05). DC-, animals that did not undergo decompressive craniectomy after brain injury; DCþ, animals that underwent decompressive craniectomy after brain injury.

and sham-operated animals as well as in the DCþ group compared to the DC- group. A quantitative analysis of the spectroscopic metabolite ratios is shown in Table 4 and Fig. 6. There was no significant difference in MRS between the control and sham-operated groups (Table 1). All of the ratios in the different regions of interest (ROIs) correlated with each other. In the contralateral cortex, the mean N-acetylaspartate (NAA)/creatine (Cr), choline (Cho)/Cr and lactate (Lac)/Cr ratios at 24 h were not different across the groups (control, sham-operated and CCI). In contrast, in the ipsilateral cortex, hippocampus and parietal adjacent cortex ROIs, the mean NAA/Cr, Cho/Cr and Lac/Cr ratios were markedly higher, lower and lower, respectively, in the CCI group than in the control and sham-operated groups. For the ipsilateral cortex, the NAA/Cr ratio in the DCþ group was significantly higher at 3 h, 1 d, 3 d and 7 d; the Cho/Cr ratio was lower only at 7 d; and the Lac/Cr ratio was lower at all of the time points after injury compared to the DC- group. For the ipsilateral hippocampus, the NAA/Cr ratio of the DCþ group was higher at 3 h, 1 d and 7 d; the Cho/Cr ratio was lower at 3 d and 7 d; and the Lac/Cr ratio was lower at 3 h, 1 d and 2 d after injury compared with the DC- group. For the ipsilateral parietal adjacent cortex, the NAA/Cr ratio of DCþ group was higher at 3 h, 1 d, 3 d and 7 d; the Cho/Cr ratio was lower at all of the time points after injury; and the Lac/Cr ratio was only lower at 3 h, 1 d and 2 d after injury compared with the DC- group. All of these differences were statistically significant (po0.05).

Discussion

DC has been used for a long time for the management of severe traumatic brain injury; however, it remains controversial. DC has been shown to improve both survival and functional outcome in patients with malignant cerebral infarctions; however, evidence of benefit in patients with TBI was decidedly mixed (Bohman and Schuster, 2013). DC has been shown to be a life-saving therapeutic treatment for severe TBI, and it has also been consistently demonstrated to reduce “therapeutic intensity” in the ICU and to reduce the ICU length of stay (Bohman and Schuster, 2013). However, the only randomized trial of DC in TBI failed to demonstrate any benefit (Cooper et al., 2011). In this study, we established a pre-clinical brain injury model to evaluate the strengths and weaknesses of DC treatment by MRI. Our objective was to perform a detailed in vivo assessment of CCI-induced brain lesions in rats using quantitative MRI, with or without DC, with special attention given to the lesion tissue and neighboring contusion. We studied the time course of edema and the effect of DC on brain injury using T2-weighted and diffusion-weighted MR imaging, and we measured concomitant alterations in metabolites using 1H-MRS in the ipsilateral cortex, hippocampus and parietal adjacent cortex. To the best of our knowledge, this is the first report of combined in vivo MRI and 1H-MRS characterization and therapeutic effect analysis of DC induced by CCI. Moreover, this CCI device could also be used to establish focalþdiffuse or diffuse closed brain injury models (Dapul et al., 2013); thus, the CCI technique could also be used to evaluate the potential neuroprotective effect of craniectomy in models of focalþdiffuse or diffuse brain injury in the future.

3.1.

DC reduces brain edema formation

MRI was more sensitive than brain water content measurement for detecting brain edema (Pascual et al., 2007). Diffusion-weighted imaging allowed us to determine the type of edema; the ADC values were low, indicating cytotoxic edema in the ipsilateral cortex during the first week after CCI (Obenaus et al., 2007). Similar results had been reported after focal LFP (Albensi et al., 2000) and in a model of diffuse TBI (Assaf et al., 1997). Brain trauma induced ischemia and hypoxia in injured tissue and also impaired cellular energy metabolism by causing dysfunction in the membrane NaþKþ pump. Dysfunction in this pump allowed sodium and calcium ions to flow into the intracellular space, leading to increased intracellular osmotic pressure. Then, a large number of extracellular water molecules moved into the intracellular space, leading to cytotoxic edema, restricted diffusion of

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Fig. 5 – Examples of typical proton magnetic resonance spectra of the ipsilateral cortex in control, sham-operated, and braininjured animals with DC- or DCþ at 1 d after injury. Major metabolites are shown, including N-acetylaspartate (NAA), choline compounds (Cho), creatine (Cr), and lactate (Lac). Compared to the control and sham-operated animals, the CCI animals exhibited low NAA/Cr and Cho/Cr ratios and a high Lac/Cr ratio. DC-, animals that did not undergo decompressive craniectomy after brain injury; DCþ, animals that underwent decompressive craniectomy after brain injury. Table 4 – NAA/Cr, Cho/Cr and Lac/Cr ratio change in the ipsilateral cortex, hippocampus and the parietal adjacent cortex between DC- group and DCþ group. ROI

1

Cortex

NAA/Cr

H-MRS

Cho/Cr Lac/Cr Hippocampus

NAA/Cr Cho/Cr Lac/Cr

Adjacent

NAA/Cr Cho/Cr Lac/Cr

Group

Pre-TBI

3h

1d

2d

3d

7d

DCDCþ DCDCþ DCDCþ DCDCþ DCDCþ DCDCþ DCDCþ DCDCþ DCDCþ

1.3770.05 1.3770.05 1.4470.01 1.4370.01 1.4370.01 1.4370.01 0.8370.01 0.8570.01 0.7570.01 0.7470.01 0.8570.01 0.8470.01 0.0770.01 0.0770.01 0.0770.01 0.0770.01 0.0370.001 0.0370.003

1.2370.13 1.4570.21** 1.3970.04 1.2270.03* 1.4870.05 1.2970.02* 0.6570.05 0.6370.06 0.7370.02 0.7270.02 0.7870.05 0.6570.03* 0.1470.02 0.2870.05* 0.1570.03 0.0870.03* 0.2670.05 0.1070.02*

1.0470.17 1.3070.33* 1.2570.05 1.1770.03* 1.3270.04 1.0470.03* 0.6670.03 0.6570.03 0.6170.02 0.6870.02 0.7570.06 0.6170.02* 0.0770.02 0.2770.05** 0.1770.03 0.0970.03** 0.2470.08 0.0770.02*

1.1770.20 1.2170.10 1.2970.03 1.1870.06 1.2870.02 1.1170.03 0.7170.05 0.7270.05 0.6870.02 0.6970.02 0.7770.04 0.6270.03** 0.0870.02 0.2670.05** 0.1570.04 0.0770.02** 0.1970.07 0.0770.03*

1.1170.23 1.3970.11* 1.3070.03 1.1770.05 1.3570.01 1.1770.03* 0.6870.13 0.7970.05 0.8570.01 0.7570.01* 0.8270.01 0.7070.04* 0.0870.03 0.3070.06* 0.1370.04 0.0970.03 0.1870.07 0.1070.06

1.1370.09 1.4770.16** 1.3770.08 1.1970.04* 1.5470.03 1.2870.02* 0.6570.06 0.7670.02* 0.9270.02 0.8270.01* 0.8670.05 0.7270.05* 0.0670.03 0.1170.02* 0.0970.03 0.0870.03 0.1670.03 0.0970.04

Compared with DC- group values. DC-, animals not receiving decompressive craniectomy after brain injury; DCþ, animals receiving decompressive craniectomy after brain injury. n po0.05. nn po0.01.

water molecules and a decrease in the ADC value (Hulkower et al., 2013). Additionally, a decreased ADC value also indicated swelling of intracellular organelles, blocked diffusion of

water molecules and an increase in cytoplasmic viscosity (Marchadour et al., 2012). The mean ADC change in the ipsilateral cortex of the DCþ group was higher and the

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345

Fig. 6 – Line chart of proton magnetic resonance spectroscopy, demonstrating the differences in the NAA/Cr, Cho/Cr and Lac/Cr ratios of the ipsilateral cortex, hippocampus and parietal adjacent cortex in the DC- and DCþ groups before the injury and at 3 h, 1 d, 2 d, 3 d and 7 d after the injury (**po0.01 and *po0.05 for the DCþ group versus DC- group at each time point. DC-, animals that did not undergo decompressive craniectomy after brain injury; DCþ, animals that underwent decompressive craniectomy after brain injury).

(contralateral-ipsilateral)/contralateral cortex mean ADC ratio was lower than those in the DC- group at 1 and 2 d statistically significantly. During this period, cerebral edema was most severe and was accompanied by serious brain ischemia and hypoxia. Therefore, the protective effect of DC during this period was of great significance (Algattas and Huang, 2013). The ADC value of the ipsilateral hippocampus in the DCþ group was higher than that in the DC- group 3 h after injury, and the difference was statistically significant. However, we did not observe any difference between the CCI group and the sham-operated group or the DCþ/DC- group at the other time points, suggesting that CCI injury could cause edema formation in the ipsilateral hippocampus during the acute phase after injury. Therefore, DC, performed in a timely manner after the injury, could effectively alleviate hippocampal edema, protect against the hippocampal hypoxicischemic injury induced by edema and reduce cognitive deficits after TBI. The ADC value of the ipsilateral parietal adjacent cortex in the DCþ group was higher than that in the DC- group 3 d after injury, and the difference was statistically significant, suggesting that DC had a delayed protective effect on the cortex adjacent to the contusion site (i.e., the traumatic penumbra region). This relieved the ischemia and hypoxia induced by traumatic brain edema and improved the prognosis of penumbra regions after injury.

3.2. DC might have a tendency to reduce cell membrane breakdown, relieve cellular ischemia and ameliorate insufficient energy metabolism in brain injury Several studies on TBI revealed decreased NAA, suggesting neuronal mitochondrial dysfunction and/or neurodegeneration (Gasparovic et al., 2001); decreased Cho, implicating membrane breakdown and/or inflammation (Brooks et al., 2001); and elevated Lac, suggesting hypoxia (Lin et al., 2012; Marino et al., 2011). In this study, we assumed that creatine, a molecule involved in energy processing, is a stable criterion (Schuhmann et al., 2003). A decrease in the NAA-to-creatine ratio [NAA/Cr] has been observed within the first 24 h of injury, and this ratio can remain decreased for as long as 8 days after TBI (Holshouser et al., 2005). Additionally, the lower NAA level might also be part of a homeostatic response to osmotic stress (Xu et al., 2011). It is likely that the sustained reduction of NAA in the injured cortex reflected substantial focal neuronal loss after CCI (Chen et al., 2003). In contrast, the smaller sustained decrease in hippocampal NAA might represent diffuse neurodegeneration that is remote from the cortical injury (Hall et al., 2008). As a metabolic marker of myelin and cellular membrane density and integrity (i.e., phospholipid synthesis and degradation), the decrease in Cho/Cr in the initial stage of trauma may have

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been a result of membrane degradation in the cortex and hippocampus (Schuhmann et al., 2003). Histopathological studies have shown evidence of astrocyte damage in the hippocampus in rats as early as 30 min following TBI (Zhao et al., 2003). Additionally, the elevated Lac/Cr observed in both animal models and human MRS studies has been commonly assumed to be evidence of hypoxia or ischemia and is associated with poor patient outcomes after TBI (Gupta et al., 2013). Compared to the DC- group, the NAA/Cr ratios of the ipsilateral cortex, hippocampus and parietal adjacent cortex in the DCþ group were higher at 3 h, 1 d, 3 d and 7 d after injury. These differences were statistically significant. Although the MRS results of metabolites were directly connected to pathomechanistic processes and the data regarding the complex detrimental cascade initiated by trauma are derived from different animal models/parameters that cannot be simply generalized (Cernak, 2005), our data still suggest that DC after brain injury might have a tendency to maintain neuronal integrity and improve neuronal mitochondrial dysfunction and/or neurodegeneration, which could be sustained up to 7 d after injury. The Cho/Cr ratios of the ipsilateral cortex and ipsilateral hippocampus in the DCþ group were lower than those in the DC- group at 3 and 7 d after injury, while this ratio was lower in the ipsilateral adjacent cortex at all time points examined after the injury. The differences were all statistically significant, showing that DC might have a tendency to improve the cell membrane breakdown and/or inflammation damage in cortical and hippocampal neurons at 3–7 d after traumatic brain injury. Moreover, this protective effect of the ipsilateral adjacent cortex appeared as early as 3 h after the injury and could continue to 7 d, illustrating that DC might be capable of making sustainable improvements in cell membrane breakdown and/or inflammation in neurons of the ipsilateral adjacent cortex area (i.e., traumatic penumbra area) after brain injury. The differences in the Lac/Cr ratio were all statistically significant, demonstrating that DC has a tendency to effectively relieve cellular ischemia and improve insufficient energy metabolism in the ipsilateral hippocampus and adjacent cortex at an early stage after brain injury. Moreover, this protective effect of the ipsilateral cortex might be sustained for up to 7 d after trauma. However, further research should be undertaken to confirm these pathomechanistic tendencies.

3.3.

which was exactly the period selected for our study. Moreover, because we only analyzed the neuroprotective effect of DC for the first 7 days after the DC procedure, whether this early neuroprotection persists over time is still unknown. The other limitation of our study is that we did not perform longitudinal histology or behavior analysis; thus, we could not specifically assess changes in neurons or the functional outcomes of animals. Although the MRS data showed improvement in hippocampal metabolism after DC treatment, we still cannot determine any definitive functional outcome without behavior analysis. Therefore, it will be essential to perform a longitudinal histology and behavior analysis to corroborate the current MRI findings. Moreover, it also remains unclear when the bone can be safely reattached without further damage, and this question should be considered in future studies. Additionally, there is a consensus that medical intervention is a type of conservative treatment for mild or moderate TBI, while aggressive DC should be performed after severe TBI, and the timing of decompression may be of utmost importance to exploit the full neuroprotective potential of craniectomy following TBI (Plesnila, 2007). However, using medical intervention as an additional control during this type of experimental process is very useful for comparing the neuroprotective effect between medical intervention and surgical treatment and should be considered in future studies. Nevertheless, more prospective studies are required. To the best of our knowledge, we are the first to assess the performance of MRI in detecting alterations in edema formation, contusion volume and metabolites following DC after lateral CCI in the rat brain. Our data illustrated that DC can reduce brain swelling and ease brain edema, reduce the contusion volume and ADC value, decrease the Lac/Cr ratio, and increase the NAA/Cr and Cho/Cr ratios of the lesion area after brain injury. Moreover, it may also be possible to use these techniques and models to evaluate the location/size of DC in the future. Although much more care should be taken when extrapolating experimental results in rats to the situation in humans, our current medical imaging evidence still suggests that DC might have clinical neuroprotective potential. More investigations are needed to elucidate the effect of DC after TBI.

4.

Experimental procedures

4.1.

Ethics statement

Limitation

One of the limitations of our research may be that the metabolite ratios determined using 1H-MRS are estimates that are difficult to compare with ex-vivo measurements. However, measurements in brain extracts provide an overall value, which fails to capture the variations in the susceptibility to TBI that are known to occur across different brain structures. When preparing extracts, one cannot separate these differing cerebral structures for a regional analysis of the metabolic changes. In this study, we assumed that creatine, a molecule involved in energy processing, reflected the global metabolic level of the parenchyma. Creatine may be unstable after TBI, most notably in the vicinity of the brain contusion. However, Schuhmann et al. (2003) reported that the creatine and phosphocreatine levels remained unchanged from 24 h to 7 days post-injury,

All animal procedures were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Ethics Board of Capital Medical University Affiliated Beijing Tiantan Hospital (#201301018).

4.2.

CCI TBI model

All surgeries were performed under anesthesia using chloral hydrate, and all efforts were made to minimize suffering, including appropriate anesthesia, decreasing thermal damage, careful homeostasis, and so on. Adult male Sprague-Dawley rats (280–300 g, Vital River Laboratory Animal Technology Co. Ltd, Beijing, China) were subjected to right parietal CCI injury. The rats were housed in individual cages under controlled

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environmental conditions (12/12-h light/dark cycle at 20–22 1C), with food and water freely available, for 1 week before experimental surgery. All surgical procedures were performed under aseptic surgical conditions. Briefly, the rats were anesthetized with an intraperitoneal injection of 300 mg/kg chloral hydrate (Pharmaceutical Plant of Beijing Tian Tan Hospital, Capital Medical University) and were fixed in a stereotaxic frame. TBI was performed using the CCI device (Pittsburgh Precision Instruments, Pittsburgh, PA) as previously described (Dixon et al., 1991). A 5-mm craniotomy was made over the right parietal cortex (3.8 mm posterior and 2.5 mm lateral to the bregma) with a dental drill, and special care was taken to avoid disrupting the dura or associated vasculature during the craniotomy. The animals with broken dura after the CCI procedure or during the DC procedure were excluded from our cohort. The bone flaps were cleanly preserved (Tomura et al., 2011). A 4.0mm round impactor tip was accelerated to 4.0 m/s with a vertical deformation depth of 3.0 mm and an impact duration of 100 ms, which is consistent with severe injury and dramatically increased ICP (Cherian et al., 1994; Zweckberger et al., 2011). The rectal temperature was maintained at 37.5 1C throughout the surgical procedure with a thermal pad. After surgery, the rats were placed in a heated recovery chamber for approximately 1 h before they were returned to their cages, always with food and water available ad libitum.

with an actively shielded gradients device (360 mT/m; Bruker BioSpin, Rheinstetten, Germany). During MRI, the animals were anesthetized and spontaneously breathed 1.5–2% isoflurane in a 50%/50% N2O/O2 mixture. Their body temperature was maintained using a heated water blanket. The MR probe was a fourchannel phased-array coil (birdcage-type) and was used for emission and reception. The coil diameter (35 mm) was adapted to optimize the imaging of the head. The MRI evaluation included T2 imaging, DWI, and 1H-MRS. We used T2 imaging because local T2 increases in abnormal brain areas are generally believed to indicate an increase in the free water content (i.e., edema). Therefore, local T2 images allow quantitative evaluation of the severity of edema. In DWI, a pair of diffusion-weighting gradients of the magnetic field is added in one or several directions of space. The strength of the gradient (b value) depends on the intensity and duration of the pair of weighting gradients. The mean local diffusion coefficient of water, or apparent diffusion coefficient (ADC), can be estimated from a set of images with different b values. According to this definition, the ADC decrease observed in abnormal brain tissue is ascribable to cellular swelling (or cytotoxic edema), in which water shifts from the extracellular to the intracellular compartment because of disturbances in ion homeostasis (Lescot et al., 2010).

4.5. 4.3.

Experimental procedure

We randomly assigned 28 rats to one of four groups. In 16 animals, severe CCI injury was induced under general anesthesia, including animals (n¼8) that underwent craniectomy (DCþ) and those (n¼8) that did not undergo craniectomy (DC-). In the DCþ group, the opening of the craniotomy was extended using bone forceps within 20 min after CCI (Tomura et al., 2011). The craniectomy extended from 5 mm ahead of the coronal suture anteriorly to the rhomboid suture posteriorly. The medial and lateral borders were the sagittal suture and the superior temporal line, which could lead to significantly decreased ICP (Fig. 1a). In the DC- group, the craniotomy was closed with the previously removed bone flap using dental acrylic cement. The dura was left intact during the surgical procedure. A sham-operated group of 6 animals underwent the same craniotomy procedure but without CCI. The remaining six animals (control group) did not undergo any surgery. For each of the 16 animals in the DCþ and DC- groups, MRI and 1H –MRS assessments were planned at 3 h, 1 d, 2 d, 3 d, and 7 d after CCI. Imaging was performed 1 d after the sham procedure in the sham-operated group and at the corresponding time point in the control group. Because all surgical procedures were performed under aseptic surgical conditions, and rats themselves have a strong resistance to infection, there were no infections in rats from any of the groups during our experimental procedure. Thus, our experimental model is very stable.

4.4.

Magnetic resonance measurements

Magnetic resonance measurements were performed using a 7 T horizontal magnet equipped with a Bruker AVANCE console and

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T2 imaging

Two imaging sequences were used to detect and characterize the brain injury elicited by CCI. First, a fast multi-slice TURBO RARE T2-weighted sequence was used to check that the positioning of the animals was accurate and reproducible for repeated measurements as well as to assess the positioning of the voxels used for localized 1H-MRS. The TURBO RARE sequence parameters were as follows: repetition time (TR) ¼ 1.93 s; echo time (TE) ¼11.7 ms; RARE factor¼8; field of view (FOV)¼35  35 mm; in-plane resolution ¼256  256 pixels in 17 transverse slices; slice thickness¼1.3 mm; and acquisition time¼ 46 s. Given the short echo time, these T2 images had a high signal-to-noise ratio (Lescot et al., 2010).

4.6.

ADC images

ROIs were first defined on the T2-weighted image obtained during the same imaging session and at the same neuroanatomical level as the diffusion-weighted images; they were then transferred onto the ADC image map. The sequence parameters used for ADC map creation were as follows: repetition time (TR)¼ 5.2 s; echo time (TE) ¼35 ms; field of view (FOV)¼ 33  40 mm; in-plane resolution ¼ 108  128 pixels in 17 transverse slices; and slice thickness ¼1.3 mm. Individual ADCs were then determined from the ADC maps for each gradient direction (i.e., x, y, and z) and for ROIs. The ADC value was also acquired by Bruker Paravision software implemented in the AVANCE console. ADC values obtained with the different gradient directions were also averaged for each ROI. This mean value reflects the magnitude of the apparent water diffusion within an ROI, but it is less dependent on the directionality. T2 and ADC values for each ROI were averaged for each group (Putten et al., 2005).

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4.7.

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In vivo magnetic resonance imaging and spectroscopy

For 1H-MRS, adjustments of all first- and second-order shims over the voxel of interest were accomplished with the Fastmap procedure. At a TE of 20 ms, the shimming procedure routinely resulted in line-widths of 7–9 Hz of the single 1 H metabolite resonance (0.023–0.03 ppm). The water signal was suppressed by variable power radiofrequency (RF) pulses with optimized relaxation delays (VAPOR) (Tkác et al., 1999). Outer volume suppression combined with a point-resolved spectroscopy (PRESS) sequence from a 1.3  1.3  1.3 mm3 voxel was used for signal acquisition (Price and Arata, 1996), with TR/TE¼2500/20 ms, spectral bandwidth¼ 4 kHz, number of data points¼2048, and number of averages¼300. This provides enough specificity for signal acquisition in the contusion area, pericontusion area, and other ROIs following the description of Fig. 1b. The voxel covered the immediate pericontusional zone, all layers of the hippocampus, and the parietal adjacent cortex. Voxel positioning was based on anatomic landmarks, and care was taken to ensure reproducibility of the voxel position across scanning days. MRS data were acquired immediately following the DTI acquisition at each time point in both the pericontusional and corresponding contralateral voxels (Lescot et al., 2010).

20 slices and then multiplied by the slice thickness (1.3 mm) (Zweckberger et al., 2006).

4.9.

Statistical analysis

Statistical analysis was performed using SPSS (SPSS for Windows, version 18.0, SPSS Inc., Chicago, IL, USA). All data are expressed as the mean7standard deviation (SD). Changes in the MRI values (T2, ADC and lesion volumes) and 1H-MRS values (metabolic ratios) over time within groups of braininjured animals were evaluated with the chi-square test. 1HMRS data were fitted using the LC-Model package, and only metabolites with SDsr20% were included for further analysis (Provencher, 2001). Brain-injured animals at the main-effect time point were compared to the sham-operated animals using the Wilcoxon-Mann-Whitney test. Differences across ROIs at the main-effect time point were evaluated using the Wilcoxon test for paired data. Spearman correlation analysis a multiple independent samples non-parametric test were used to test the correlation between the reduction in edema and reduction in lesion volume and the ratios of NAA/Cr, Cho/Cr and Lac/Cr in different ROIs. Statistical significance was defined as po0.05.

Conflict of interest 4.8.

Analysis of magnetic resonance imaging The author(s) declare that they have no competing interests.

Three ROIs were chosen based on anatomical landmarks and T2-weighted coronal images obtained during the same imaging session. These ROIs were the ipsilateral cortex (approximately 2.5 mm lateral to the midline, with a diameter of 1.5 mm), the hippocampus (the region of the hippocampus just under the injured ipsilateral cortex) and the ipsilateral parietal adjacent cortex on the side of the CCI (including two parts; part 1 was the cortex between the midline and the inner border of the injured ipsilateral cortex, and part 2 was the cortex outside of the outer border of the injured ipsilateral cortex, with a diameter of 1.5 mm). The data from these two areas was averaged for each slice (Fig. 1b). Special care was taken to ensure the reproducibility of ROI placement during repeated investigations. T2 and ADC maps were computed and calculated using Bruker PV4 software for each pixel. ROIs were analyzed using imaging software (IMAGE, version 4.03; National Institutes of Health, Bethesda, MD), allowing for the replication of ROIs between sequences. T2 and ADC values for each ROI were averaged for each group. The evaluation of the severity of brain trauma was followed by the calculation of the hemispheric lesion volumes from DWI and T2-weighted images as well as by using the aforementioned IMAGE software. Injured brain tissue observed on DWI and T2-weighted images was defined as having a hyperintensity greater than one standard deviation (consistently approximately 18%) compared with normal tissue on the contralateral side. To correct for the swelling of the contused tissue, the contusion area was calculated by subtracting the area of the non-traumatized tissue of the ipsilateral hemisphere from the area of the contralateral hemisphere. The contusion volume was calculated based on the contusion areas corrected for brain edema obtained from

Acknowledgments We would like to acknowledge the assistance of Shaowu Li, Zhenlan Li, Sijia Wang, Feifan Xu, Xuetao Chen and Weichuan Wu for their support of this study. This study was supported by National Natural Science Foundation of China (nos. 81171144 and 81471238), Beijing Nova program (no. XX2012033), National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2013BAI09B03) and Beijing Institute for Brain Disorders (BIBD-PXM2013_014226_07_000084).

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