Basic Science Research Received: February 23, 2010 Accepted after revision: August 2, 2010 Published online: October 14, 2010
Dev Neurosci 2010;32:442–453 DOI: 10.1159/000320085
Pathophysiological Response to Experimental Diffuse Brain Trauma Differs as a Function of Developmental Age Ibolja Cernak a, c Taeun Chang a, b Farid A. Ahmed a, c Maria I. Cruz a Robert Vink e Bogdan Stoica a, d Alan I. Faden a, d
a
Department of Neuroscience, Georgetown University Medical Center, and b Pediatrics and Neurology, Children’s National Medical Center, Washington, D.C., c Johns Hopkins University Applied Physics Laboratory Biomedicine Business Area, National Security Technology Department, Laurel, Md., and d Shock Trauma and Anesthesiology Research (STAR) Center, University of Maryland School of Medicine, Baltimore, Md., USA; e Department of Pathology, University of Adelaide, Adelaide, S.A., Australia
Abstract The purpose of experimental models of traumatic brain injury (TBI) is to reproduce selected aspects of human head injury such as brain edema, contusion or concussion, and functional deficits, among others. As the immature brain may be particularly vulnerable to injury during critical periods of development, and pediatric TBI may cause neurobehavioral deficits, our aim was to develop and characterize as a function of developmental age a model of diffuse TBI (DTBI) with quantifiable functional deficits. We modified a DTBI rat model initially developed by us in adult animals to study the graded response to injury as a function of developmental age – 7-, 14- and 21-day-old rats compared to young adult (3-month-old) animals. Our model caused motor deficits that persisted even after the pups reached adulthood, as well as reduced cognitive performance 2 weeks after injury. Moreover, our model induced prominent edema often seen in pediatric TBI, particularly evident in 7- and 14-day-old animals, as measured by both the wet weight/dry
© 2010 S. Karger AG, Basel 0378–5866/10/0326–0442$26.00/0 Fax +41 61 306 12 34 E-Mail
[email protected] www.karger.com
Accessible online at: www.karger.com/dne
weight method and diffusion-weighted MRI. Blood-brain barrier permeability, as measured by the Evans blue dye technique, peaked at 20 min after trauma in all age groups, with a second peak found only in adult animals at 24 h after injury. Phosphorus MR spectroscopy showed no significant changes in the brain energy metabolism of immature rats with moderate DTBI, in contrast to significant decreases previously identified in adult animals. Copyright © 2010 S. Karger AG, Basel
Introduction
Traumatic brain injury (TBI) is the most common cause of mortality and serious morbidity in children between 0 and 17 years of age, with an incidence rate of TBI-associated hospitalization of 70 cases per 100,000 children [1]. Tissue damage after TBI is induced by both primary injury mechanisms and delayed secondary biochemical changes [2]. Neuronal cell loss and diffuse axonal injury, features of clinical head injury [3], reflect in part the effects of secondary injury [4]. Neuronal cell death after TBI includes various phenotypes, defined on the basis of both morphology and molecular mechanism,
Alan I. Faden, MD STAR Organized Research Center University of Maryland School of Medicine, HSF II, No. 247 20 South Penn Street, Baltimore, MD 21201 (USA) Tel. +1 410 706 4205, Fax +1 410 706 1639, E-Mail afaden @ anes.umm.edu
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
Key Words Diffuse traumatic brain injury ⴢ Brain, immature ⴢ Motor deficit ⴢ Brain edema
Methods All animal protocols complied with the Guide for the Care and Use of Laboratory Animals (Department of Health, Education, and Welfare publication NIH 85-23-2985) and were approved by the Georgetown University Animal Care and Use Committee. Traumatic Brain Injury Male immature (7-, 14- and 21-day-old) and adult (3-monthold) Sprague-Dawley rats were injured using our DTBI model as previously described [16]. Briefly, under isoflurane anesthesia (4% isoflurane, and 1–1.5% for maintenance), the skull was exposed and a stainless steel disk (10 mm diameter, 3 mm depth) was fixed with polyacrylamide adhesive to the animal’s skull centrally between lambda and bregma. The rats were placed on the animal holder with a molded, gel-filled head support (Handstands Taiwan). The air-driven high-velocity impactor was lowered onto the steel disk. It was then retracted, an impact displacement manually entered, and impact initiated. Our previous study [16] showed
Experimental DTBI as a Function of Developmental Age
that the displacement level largely determines the severity of the injury; thus, the velocity of the impactor was held constant (3.25 m/s), while the distance was varied. The force was recorded and controlled using a personal computer connected via a PowerLab (Stoelting, Wood Dale, Ill., USA). Graded Injury To evaluate mortality rates as a function of developmental age, immature rats of 3 different age groups (7, 14 and 21 days; n = 225, 75/age group) were injured with increasing impact displacement. The injury curve in adult rats was previously established [16]. The injury severity was established based on the mortality rates at 24 h after injury. Brain Edema Brain edema was assessed by the wet-and-dry method [17] in immature (7-, 14- and 21-day-old) and adult (3-month-old) rats (n = 42/age group; n = 7/time point/age group) during the first 24 h after injury. The animals were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and decapitated before injury (sham), or 20 min, 40 min, 2 h, 4 h or 24 h after moderate DTBI. The parietal cortices and hippocampal samples were weighed, dried for 72 h at 100 ° C and reweighed; the percentage of water contents was calculated as previously described [17]. We used separate groups of immature and adult rats to characterize early brain edema development measured by using diffusion-weighted MRI (DWI) at 20 min after injury. This time point was chosen to assess early brain edema development. Moreover, at this time point, the wet-and-dry method suggested an increase in water content, and the Evans blue (EB) technique indicated a robust opening of the blood-brain barrier (BBB). Briefly, the animals were placed in the heated Plexiglas holder with a respiratory motion detector and positioned in the center of the 7-tesla magnet bore (Bruker 7T/21 cm Biospec-Avance system; Bruker, Karlsruhe, Germany) inside a 72-mm proton-tuned birdcage coil. DWI was performed using a spin echo pulse sequence with diffusion gradients added before and after the refocusing pulse. The DWI images were converted to diffusion maps by applying the StejskalTanner equation in association with a Marquart algorithm, using the commercially available Paravision software (Bruker, Billerica, Mass., USA). Apparent diffusion coefficients (ADC) were calculated, expressed as 10 –5 mm2/s 8 SD.
BBB Permeability A time course for BBB permeability in the cortex and hippocampus was investigated in immature and adult animals (n = 42/ age group; n = 7/time point/age group). BBB integrity was assessed using EB extravasation according to Uyama et al. [18]. EB dye (4 ml/kg of 2% solution in PBS) was injected intravenously 30 min prior to decapitation at 20 and 40 min, 2 h, 4 h and 24 h after injury (n = 7/time point) or sham surgical procedure (n = 5/time point). At the assigned time points, the animals were anesthetized using sodium pentobarbital (65 mg/kg i.p.) and intracardially perfused with saline. Samples of cortices and hippocampi were then isolated and weighed, homogenized in 60% trichloroacetic acid solution and centrifuged. The absorbance of the extracted dye in supernatant at 620 nm was determined with a Perkin-Elmer spectrometer, and the tissue content of EB quantified on the basis of a linear standard curve obtained using known amounts of the dye and expressed as micrograms per gram of tissue.
Dev Neurosci 2010;32:442–453
443
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
with necrosis and caspase-dependent apoptosis receiving most attention [5]. The developing brain appears to have different sensitivities with regard to these cell death phenotypes as compared to the adult brain [6, 7]. Thus, necrotic cell death after hypoxia in young animals requires greater insults than in adults [8, 9]. In contrast, greater upregulation of molecular pathways associated with apoptotic cell death occurs in neonatal and immature animals, with a shift from predominantly caspase-dependent apoptosis to necrosis with increasing developmental age [5, 10]. Among numerous focal and diffuse brain trauma models, few have been used to address pathobiological responses in the immature brain. Most experimental models employ focal injury such as injury after craniotomy and directly delivered impact to the exposed dura: lateral fluid percussion [11], controlled cortical impact [12, 13], or weight drop impact [6]. Yet clinical trauma more often reflects diffuse traumatic brain injury (DTBI). The closed impact model, using a weight drop technique, is the most widely used diffuse brain injury model in adult rats [14]. Adelson et al. [15] developed a similar closed head injury model to induce DTBI in immature rats. However, the weight drop model does not provide strict control of the biomechanics of the impact. To provide better control, we developed a new DTBI model in the adult rat [16], one that is highly controllable and suitable for studying developmental brain injury. The goal of the present study was to characterize a model that could induce graded DTBI of comparable severities in developing rats and to define parameters necessary to cause a reproducible moderate severity of TBI at different developmental ages.
Magnetic Resonance Spectroscopy Phosphorus MRS was performed on a Bruker 7.0-tesla system operating Paravision 2.0 software, prior to and 4 h after injury, as previously described [20, 21], in a subgroup of injured (n = 7 sham controls/age group and n = 7 injured/age group) of randomly chosen animals. This time point was chosen based on previous findings [22] showing a persistent and stable decline in brain energy metabolism measured in adult rat brain after DTBI. Briefly, under continuous anesthesia with isoflurane, the animal was placed in a Plexiglas holder in which a heating pad warmed to 37 ° C was fixed to maintain the animal’s temperature. A surface coil was then placed centrally over the skull, and skin and temporal muscle retracted well clear of the coil to ensure that there was no contribution from these tissues to the brain spectrum. The Plexiglas holder containing the animal was then positioned in the center of the magnet bore and the B1 field optimized to less than 0.25 ppm at the half height of the proton water signal. Phosphorus spectra were obtained in a 20-min block using a 90° pulse width set at a 2-mm cortical depth, a 800ms delay time and a 6,000-Hz spectral width containing 2,048 data points. Free induction decays were analyzed after convolution difference (20/500 Hz exponential filter) and peak frequencies determined using the computer peak-picking program. Intracellular pH was calculated from the chemical shift of the inorganic phosphate peak (␦Pi) relative to phosphocreatine (PCr), the free magnesium concentration was determined from the chemical shift difference between the ␣- and -peaks of ATP [23], and the cytosolic phosphorylation potential, an indicator of cell bioenergetic status, was determined as previously detailed [24].
444
Dev Neurosci 2010;32:442–453
Histology At 24 h after trauma, a subgroup of 7-day-old and adult animals with moderate DTBI (n = 5/age group) were anesthetized using sodium pentobarbital (65 mg/kg, i.p.) and transcardially perfused with isotonic saline, followed by 4% formaldehyde. Using a rat brain matrix, each brain was cut into 2-mm-thick coronal blocks for a total of 7 blocks per animal. The brain tissue was processed and embedded, and 6-mm-thick paraffin coronal sections from each block were cut and stained with HE for histopathological evaluation. Gross hemorrhage was defined as blood apparent to the unaided eye on 2 faces of each block and on the HEstained sections. To visualize degenerating cells, separate subgroups of 7-dayold and adult animals were anesthetized with isoflurane and placed in a stereotaxic holder. Small (1 mm diameter) holes were drilled into the skull with a dental drill at sites corresponding to the right and left cingulum. Fluoro-Ruby (Molecular Probes Inc., Eugene, Oreg., USA; 3 l of 15% solution in PBS, pH 7.4) was injected into each cingulum using a Hamilton syringe with a tip diameter of 50 m [25, 26]. Three days later the animals were injured. Seven days following injury, the animals were anesthetized, transcardially perfused with ice-cold saline, and the brains removed. After 24-hour immersion in 30% sucrose at 4 ° C, the brains were snap frozen using 2-methylbutane precooled in dry ice for 2 h. Coronal 30-m sections were cut with a cryostat, and every fifth section was mounted on glass slides and examined under a fluorescent microscope using a rhodamine filter (Zeiss Axioplan; Carl Zeiss Inc., Thornwood, N.Y., USA).
Statistical Analysis All continuous data are shown as means 8 SD and were analyzed by repeated measures ANOVA followed by the individual Student-Neuman-Keuls test. Wet weight/dry weight ratios were compared using Student’s t test. Neurological outcomes were analyzed using nonparametric repeated measures Kruskal-Wallis ANOVA, followed by the individual Mann-Whitney U test.
Results
Mortality Figure 1 shows the mortality rates in the 7-, 14- and 21-day-old and the 3-month-old (young adult) rats measured at 24 h after DTBI. Moderate severity injury was defined by mortality rate (approx. 20–25%) and was caused by 6.4-, 8.6-, 10.2- or 18-mm head vertical displacement, respectively. Dying was usually rapid (!5 min) and death was caused by cardiorespiratory depression. The established moderate injury levels were used in all further studies. Edema Development and BBB Permeability Brain edema development was estimated in all age groups as a measure of TBI severity using the wet-anddry method during the 24-hour posttraumatic period as well as calculating the ADC values 20 min after injury. Cernak /Chang /Ahmed /Cruz /Vink / Stoica /Faden
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
Neurological Assessment Motor function was assessed in the sham and injured immature and adult rats (n = 12/age group) on days 1, 2, 3, 7, 10, 14, 21 and 90 after sham surgery or DTBI. For motor function assessment, 3 separate tests have been used as previously described [19], each of which is scored via an ordinal scale ranging from 0 (no function) to 5 (normal function). Tests included the ability to maintain position on an inclined plane in 2 vertical and 2 horizontal positions, forelimb flexion and forced lateral pulsion. Each of 7 individual scores (vertical angle with the animal positioned head up; vertical angle with the animal positioned head down; right and left horizontal angles; forelimb flexion; right and left lateral pulsion) was added to yield a composite neurological score ranging from 0 to 35. Cognitive function was assessed using the Morris water maze as previously described [19]. Trials were conducted over 4 consecutive days, starting at 14 days after sham surgery or DTBI. To control for visual discriminative ability or motor impairment (VM), the animals were required to locate a clearly visible platform (different location) 2 h after the last training trial. The mean latency to locate the platform immersed below the surface of the water was measured and compared between groups. As the alterations in physiological and functional measures showed the largest difference between the 7-day-old and adult animals, additional analyses including MR spectroscopy (MRS), histology and immunocytochemistry in immature animals were limited to 7-day-old pups.
100
90.00
* **
90
Mortality (%)
70
7 days
60
14 days
50
21 days
40
3 months
Water content (%)
87.00
80
** ** **
20 min 4h
40 min 24 h
2h Sham
* **
84.00 81.00
*
*
** *
78.00 30 20
75.00
10
Adult
14 days 21 days Age groups
7 days
a
0 5
10
15
20
Displacement (mm) 93.00
Fig. 1. Mortality curves established for 7-, 14- and 21-day-old male
** * * **
90.00 Water content (%)
pups and young adult male (3-month-old) rats for increasing injury displacements, measured at 24 h after injury. Injury equivalence based on mortality measured at 24 h after injury is identified across various age groups.
** * * **
87.00
20 min 4h
40 min 24 h
** * *
84.00
** *
81.00
2h Sham
** ** * *
78.00
Experimental DTBI as a Function of Developmental Age
75.00
7 days
b
14 days 21 days Age groups
Adult
Fig. 2. Changes in water content measured in the cortex (a) and hippocampus (b) of 7-, 14- and 21-day-old and adult (3-month-
old) rats (n = 7/time point/age group) at 20 and 40 min, 2 h, 4 h and 24 h after injury. * p ! 0.05, ** p ! 0.01, and *** p ! 0.001, compared to age-matched sham controls.
the hippocampus (fig. 4b). These changes were consistent with the early development of vasogenic edema [27]. Although the ADC values measured in the 7-day-old sham control animals were higher compared to the older rats, the intensity and trend of posttraumatic brain edema development were comparable (fig. 4). In comparison to the adult brains, the EB concentrations measured in the cortex of uninjured 7- and 14-day-old immature brains were significantly higher (p ! 0.01) (fig. 5a). Similarly, the BBB permeability values in the hippocampus of uninjured 7-, 14- and 21-day-old animals were significantly higher (p ! 0.001, p ! 0.001 and p ! 0.01, respectively) compared to the adult hippocampus (fig. 5b). Interestingly, the concentrations of EB measured in the cortex were signifiDev Neurosci 2010;32:442–453
445
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
The percentage water content was significantly increased in both cortex and hippocampus of the noninjured immature 7-day-old (p ! 0.001), 14-day-old (p ! 0.001) and 21-day-old rats (88.20 8 0.07% and 89.70 8 0.08%, 85.30 8 0.09% and 88.10 8 0.09%, and 82.50 8 0.10% and 82.60 8 0.09%, respectively) compared to the sham control adults (78.10 8 0.17% and 78.30 8 0.13%, respectively) (fig. 2). The temporal profile of the brain edema in the cortex showed increase in immature brains at 20 and 40 min after trauma compared to the adult cortex, with increased percentage water 20 min and 24 h after injury (fig. 2a). A similar trend was observed in the hippocampus (fig. 2b). Figure 3 shows DWI of 7-, 14- and 21-day-old and adult brains measured at 4 h after trauma. Regions of subcortical hyperintensity in injured animals suggest vasogenic edema. The ADC values measured both in the cortex and hippocampus of immature rats were significantly (p ! 0.01) higher compared to the corresponding brain structures in the adults, whereas there were no statistically significant (p 1 0.05) differences between the ADC values measured in 14- and 21-day-old and 3-month-old animals (fig. 4). Moderate DTBI induced a significant increase in cortical ADC in 7- (p ! 0.01), 14- (p ! 0.05) and 21-day-old (p ! 0.0001) and adult (p ! 0.05) animals at 20 min after trauma (fig. 4a). Similar changes were shown in
a
b
c
d
e
f
g
h
Fig. 3. DWI in rats representing the groups of 7- (a, b), 14- (c, d) and 21-day-old (e, f) and adult (3-month-old; g, h) sham (a, c, e, g) or injured (b, d, f, h) rats. DWI was per-
a
120 110 100 90 80 70 60 50 40 30 20 10 0
**
*** **
7 days
14 days 21 days Age group
Control Injured
**
ADC (×10–5 mm2/s)
ADC (×10–5 mm2/s)
formed 4 h after sham surgery or moderate DTBI. Areas of hyperintensity (arrows) indicate vasogenic edema.
Adult
b
120 110 100 90 80 70 60 50 40 30 20 10 0
**
7 days
Control Injured
***
*
*
14 days 21 days Age group
Adult
Fig. 4. ADC obtained in the cortex (a) and hippocampus (b) of 7-, 14- and 21-day-old and adult (3-month-old)
cantly higher compared to the hippocampus in all age groups. In uninjured animals, the EB concentrations measured as micrograms per wet brain were: 2.9 8 0.15 (cortex) versus 16.7 8 0.83 (hippocampus) in 7-day-old rats; 2.6 8 0.12 (cortex) versus 15.9 8 0.71 (hippocampus) in 14-day-old rats; 2.0 8 0.08 (cortex) versus 11.9 8 446
Dev Neurosci 2010;32:442–453
0.82 (hippocampus) in 21-day-old rats, and in adults, 1.82 8 0.11 (cortex) versus 7.99 8 0.50 (hippocampus). DTBI caused considerable increases in BBB permeability in both cortex and hippocampus in all age groups at 20 min after injury. Interestingly, increased BBB permeability in the cortex and hippocampus of injured animals during Cernak /Chang /Ahmed /Cruz /Vink / Stoica /Faden
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
rats (n = 7/time point/age group) at 4 h after trauma. * p ! 0.05, ** p ! 0.01, and *** p ! 0.001, compared to agematched sham controls.
EB (μg/wet brain)
6.0
** *
20 min 4h
5.0
**
4.0
**
0
40.0
2h Sham
** *
35.0
7 days
14 days 21 days Age groups
** *
20 min 4h
30.0 25.0 20.0
*
** *
0
7 days
b
2h Sham
** * * *
5.0 Adult
40 min 24 h
15.0 10.0
**
1.0
a
** *
3.0 2.0
40 min 24 h
EB (μg/wet brain)
7.0
14 days 21 days Age groups
*
** Adult
Fig. 5. Changes in BBB permeability to EB dye in the cortex (a) and hippocampus (b) of 7-, 14- and 21-day-old
and adult (3-month-old) rats (n = 7/time point/age group) at 20 and 40 min, 2 h, 4 h and 24 h after injury. * p ! 0.05, ** p ! 0.01, and *** p ! 0.001, compared to age-matched sham controls.
Motor and Cognitive Outcomes Figure 6 shows motor performance in 7- (fig. 6a), 14(fig. 6b) and 21-day-old (fig. 6c) animals as well as in adult (fig. 6d) sham and injured rats over a 90-day testing period. All immature sham animals showed a steeper motor learning curve compared to the adult sham rats. On day 1 of testing, the values of the sham animal composite neuroscores were: 9.00 8 0.61 (7-day-old rats); 15.30 8 0.53 (14-day-old rats); 24.0 8 0.55 (21-day-old rats), and 34.00 8 0.82 (adult rats). The starting values of the composite neuroscore were significantly (p ! 0.001) lower compared to the motor performance of the adult sham animals. At the end of the testing period, the values of the composite neuroscores in immature animals were similar to the values measured in adults, i.e. around the maximum value (35) (fig. 6). On day 1 of testing, the values of the injured animal composite neuroscores were: 6.00 8 0.61 (7-dayold rats); 6.60 8 0.53 (14-day-old rats); 8.0 8 0.55 (21-dayold rats), and 30.60 8 0.82 (adult rats). After 90 days, the values of the injured animal composite neuroscores were: 28.33 8 0.81 (7-day-olds); 29.00 8 0.93 (14-day-olds); Experimental DTBI as a Function of Developmental Age
30.90 8 0.89 (21-day-olds), and 35.0 8 0.00 (adults). The motor performance remained significantly (p ! 0.01) reduced in the 7- and 14-day-old injured animals as compared to the sham age-matched groups at the end of the 90-day posttraumatic period. The motor performance was decreased in 21-day-old rats versus the age-matched sham animals up to 14 days after trauma (p ! 0.01), whereas the adult animals demonstrated measurable motor deficits until 7 days after injury. Figure 7 shows cognitive outcome alterations in 7(fig. 7a), 14- (fig. 7b) and 21-day-old (fig. 7c) animals as well as in adult (fig. 7d) sham and injured rats measured at 14, 15, 16 and 17 days after sham surgery or DTBI. The immature uninjured animals (7, 14 and 21 days old) needed a longer time to locate the platform immersed below the surface at days 15, 16 and 17 after sham surgery compared to adult rats (fig. 7). Indeed, the latency values in the sham animals measured on the last test day were: 40.99 8 4.68 s (7-day-olds); 55.21 8 3.57 s (14-day-olds); 38.00 8 4.02 s (21-day-olds), and in adults, 14.15 8 1.6 s. The mean latency to find the submerged platform was significantly increased in injured 7- (p ! 0.01) and 21-dayold (p ! 0.001) animals at day 17 after trauma, compared to the sham control animals (52.59 8 2.70 and 56.94 8 4.14 s, respectively). In the adult animals, there was a significant increase at days 15, 16 and 17 after trauma (p ! 0.001, p ! 0.01 and p ! 0.01, respectively) compared to the corresponding sham values. No statistically significant changes were found in the cognitive performance of 14-day-old animals.
Dev Neurosci 2010;32:442–453
447
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
the later posttraumatic period, i.e. 4 h and 24 h after trauma, was established only in adult animals, whereas the EB values remained unchanged in immature brains (fig. 5). While the association of increased brain water with increased EB extravasation measured at 20 min after trauma supports a vasogenic origin of brain edema, the changes in percentage water content and EB extravasation in adult brains at 24 h after injury suggest the development of cytotoxic edema [28].
40
40 7 days sham
14 days sham
7 days DTBI
**
30 25 20
*
15
*
10 5
*
**
**
**
*
25
15 10
** *
** *
** *
** *
14
21
90
14
21
90
** *
** *
20
**
0 1
2
3
a
7 10 Days of testing
14
21
90
1
2
3
7 10 Days of testing
b
40
40 21 days sham
Adult sham
21 days DTBI 35
35 30 25 20 15
** *
10
** *
** *
** *
** *
**
Composite neuroscore
Composite neuroscore
**
30
5
0
*
30
*
Adult DTBI
*
*
25 20 15 10 5
5
0
0 1
c
14 days DTBI
35 Composite neuroscore
Composite neuroscore
35
2
3
7 10 Days of testing
14
21
1
90
2
3
7 10 Days of testing
d
Fig. 6. Changes in composite neuroscore in 7- (a), 14- (b) and 21-day-old (c) and adult (3-month-old; d) rats
(n = 12/age group) measured 1, 2, 3, 7, 10, 14, 21 and 90 days after sham surgery or moderate DTBI. * p ! 0.05, ** p ! 0.01, and *** p ! 0.001, compared to age-matched sham controls.
448
Dev Neurosci 2010;32:442–453
Histology The brains of 7-day-old rats subjected to moderate (6.4-mm) DTBI showed no hemorrhage, focal lesions or contusions (results not shown). Degenerating neuronal somata and their processes were detected with FluoroRuby. The 7-day-old rats demonstrated increased numbers of cells with changed shape, nuclear condensation or fragmentation injury in the cortex (fig. 8), and to a lesser extent in the hippocampus, thalamus and brainstem 7 days after DTBI. These alterations were qualitatively similar to those observed in young adult rats (fig. 8) [16].
Cernak /Chang /Ahmed /Cruz /Vink / Stoica /Faden
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
Magnetic Resonance Spectroscopy The phosphorus MRS results in the sham animals and in animals before injury did not differ significantly (results not shown). At 4 h after trauma, compared to the sham control age-matched group, there were no significant changes in the MRS spectra measured in the brain of 7-day-old rats, including values of pH, intracellular free Mg (iMg) and PCr/Pi ratio, among others. The absence of significant changes in brain bioenergetics significantly differed from that observed in the adult brain at this time point following DTBI; in the latter there were significant declines in the PCr/Pi ratio and iMg concentration (table 1), which occurred in parallel to a significant decrease in phosphorylation potential [16].
80.00 7 days sham
70.00
7 days DTBI
**
60.00 50.00 40.00 30.00 20.00
Latency to find platform (s)
Latency to find platform (s)
80.00
10.00 0
14
15
a
16 17 Days after injury
50.00 40.00 30.00 20.00 10.00 14
15
16 17 Days after injury
Latency to find platform (s)
Latency to find platform (s)
21 days DTBI
***
60.00
0
40.00 30.00 20.00 10.00 14
15
16 17 Days after injury
VM
80.00 21 days sham
70.00
c
50.00
b
80.00
14 days DTBI
60.00
0
VM
14 days sham
70.00
Adult DTBI
60.00
***
50.00 40.00
**
30.00
**
20.00 10.00 0
VM
Adult sham
70.00
14
15
d
17 16 Days after injury
VM
Fig. 7. Morris water maze cognitive scores in 7- (a), 14- (b) and 21-day-old (c) and adult (3-month-old; d) rats (n = 12/age group) measured at 14, 15, 16 and 17 days after sham surgery or moderate DTBI. ** p ! 0.01 and *** p ! 0.001, compared to age-matched sham controls.
Table 1. Bioenergetics response to moderate DTBI measured by phosphorus MRS in 7-day-old sham and injured as well as in adult sham and injured rats (n = 7/age group) 4 h after injury
pH PCr/Pi ratio iMg, mM
7-day sham
7-day DTBI
Adult sham
Adult DTBI
7.0580.04 1.0380.09 0.2580.03
7.1480.05 1.4080.22 0.2080.02
7.1380.04 3.6880.14 0.5080.05
7.1580.04 3.2080.14* 0.3180.16*
Discussion
The purpose of experimental models of TBI is to reproduce selected aspects of human head injury such as brain edema, contusion, concussion and functional deficits, among other consequences. The complexity and Experimental DTBI as a Function of Developmental Age
biological differences between the adult and immature brains necessitate careful consideration of the biomechanics and pathobiology of pediatric concussive and diffuse brain injuries induced by closed head impact [6, 29]. While the adult skull is a rigid structure, the immature skull is more a set of loosely connected curved plates Dev Neurosci 2010;32:442–453
449
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
Values denote means 8 SD. * p < 0.05, compared to age-matched sham controls.
a
b
c
d
Fig. 8. Representative photomicrographs.
with sutures, which are less able to provide support against a deforming force. Thus, the infant skull responds to an impact with elastic and/or plastic deformation, often leading to skull fracture, whereas the adult skull exhibits 11 times greater resistance to mechanical force than the infant skull [30]. Brain weight/size is another factor that may influence the impact effects. But the vulnerability of the brain depends on size only when angular acceleration is present [31]. Other studies have supported these findings, comparing the levels of rotational acceleration required to induce concussion in adults and infants [30, 32]. Severe concussive brain injury in the adult brain results from applying a rotation acceleration of 18,000 rad/s2, whereas approximately 450
Dev Neurosci 2010;32:442–453
200 μm
40,000 rad/s2 is necessary to induce similar changes in the infant brain. Although the immature brain has been considered more resistant to damage after injury [7, 33], a comprehensive neuropathological study [34] of fatal pediatric head injuries showed more prevalent brain swelling in children than adults. Various physiological, biochemical or molecular mechanisms have been proposed to explain developmentally dependent differences in response to TBI, including autoregulation of cerebral blood flow [35], mitochondrial content and metabolic enzyme activities [36], oxidative stress [37] and glutamate receptors [38], among others. Differing opinions regarding experimental models of pediatric TBI also relate to molecular, bioCernak /Chang /Ahmed /Cruz /Vink / Stoica /Faden
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
Fluoro-Ruby-labeled cells in the cortex of an adult sham (a) and adult injured (b) rat, compared to the cortex of a 7-day-old sham (c) and a 7-day-old injured rat (d) at 7 days after injury. Arrows: cells with changed shape, nuclear condensation or fragmentation.
DTBI as compared to sham control animals. Since the injured animals were scored on the basis of the performance of age-matched sham controls at the same time point, the neurological dysfunction is unlikely caused by the stage of synaptogenesis. The appearance of degenerating cells in the cortex and other brain structures paralleled the functional deficits measured in 7-day-old animals. Adelson et al. [44], using a weight drop model of closed head injury, also demonstrated motor and cognitive deficits in young animals; the motor deficits persisted for 10 days after trauma, whereas cognitive performance was compromised up to 3 months after injury. These functional deficits are similar to those found in our experiments. The lower metabolic rate in the neonatal brain compared to the adult brain [45], which reflects developmentally dependent energy utilization [46], has been suggested as an important factor underlying the reduced vulnerability of the immature brain to CNS injury [36]. Although mild-to-moderate TBI generally does not cause loss of ATP or lasting lactic acidosis, it induces a significant dysfunction of the energy metabolism manifested in reduced cytosolic phosphorylation and decreased free magnesium concentration [24]. Interestingly, in our DTBI model, moderate injury caused a significant decline in the brain energy metabolism of adult rats [16], whereas the immature brain bioenergetics remained unresponsive to comparable injury levels defined by mortality. Similar to our findings, other 31P-MRS studies in rat pups showed that despite significantly high levels of lactate accumulation, the brain of immature animals has the remarkable ability to maintain brain pH and bioenergetic state under hypoxic conditions [47, 48]. This enhanced ability to maintain brain bioenergetics after injury may explain why young animals are less susceptible to necrosis than older animals. However, Robertson et al. [49] have proposed that the relative resistance of immature brain mitochondria to Ca2+-induced dysfunction in the absence of ATP may also serve to decrease the vulnerability of neural cells to acute cell death induced by excitotoxicity after CNS injury. Therefore, it is likely that multiple mechanisms underlie the differential cell death profiles after injury in the developing and mature nervous systems.
Experimental DTBI as a Function of Developmental Age
Dev Neurosci 2010;32:442–453
451
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
chemical, physiological and behavioral parameters [39]. However, a major difficulty in defining such factors reflects the age-dependent differences in functional, morphological and physiological properties of the normal brain. Given these extensive developmental differences, it is highly problematic to develop a TBI model that permits truly comparable levels of all consequences of TBI across developmental ages. One of the major challenges is to prioritize the main outcome measures, which should be able to address the scientific question of the research study. Often, the TBI models rely on quantifiable morphological changes such as cell loss or activation of injurious pathways [2, 13, 37, 40], while only few place emphasis on functional deficits [7]. Bittigau et al. [6] compared the severity of trauma-triggered apoptosis in the brains of 3- to 30-day-old rats subjected to a mechanical force that was adjusted to reflect age-dependent changes in brain weight/size. Although they varied skull thickness and injury parameters, the way the variables were modified was somewhat arbitrary. Moreover, constitutive properties and the geometry of the bone structures appear more important from a biomechanical perspective than skull thickness [30]. In our model, we compared injury levels by altering parameters to yield similar mortality rates. Although such an approach may have clinical relevance, mortality reflected many consequences of injury. Interestingly, variables chosen to induce similar mortality rates also yielded similar changes in brain edema. Consistent with prior literature [37, 41], we show a gradual reduction of brain water content in healthy animals throughout development. Our results also demonstrate comparable temporal profiles of the brain edema development and BBB opening in immature and adult rats during the early (20-min) posttraumatic phase suggesting vasogenic edema. Such a temporal profile of brain edema and BBB breakdown is similar to that found in clinical head injury [42, 43]. Interestingly, the later peak in brain water content and BBB permeability measured at 24 h after injury was observed only in adult and none of the immature animals. In our study, immature rats with moderate DTBI exhibited significant motor deficits. The most prominent motor dysfunction was shown in the youngest (7-dayold) group, manifesting gross motor deficits even after they reached adulthood. Our results suggest that with gradual maturation of the motor system, the animals become less prone to develop irreversible motor deficits. The majority of the immature animals, similar to the adult rats, developed cognitive deficits after moderate
1 Schneier AJ, Shields BJ, Hostetler SG, Xiang H, Smith GA: Incidence of pediatric traumatic brain injury and associated hospital resource utilization in the United States. Pediatrics 2006;118:483–492. 2 Potts MB, Koh SE, Whetstone WD, Walker BA, Yoneyama T, Claus CP, Manvelyan HM, Noble-Haeusslein LJ: Traumatic injury to the immature brain: inflammation, oxidative injury, and iron-mediated damage as potential therapeutic targets. NeuroRx 2006; 3: 143–153. 3 Adams JH, Doyle D, Ford I, Gennarelli TA, Graham DI, McLellan DR: Diffuse axonal injury in head injury: definition, diagnosis and grading. Histopathology 1989;15:49–59. 4 DeKosky ST, Kochanek PM, Clark RS, Ciallella JR, Dixon CE: Secondary injury after head trauma: subacute and long-term mechanisms. Semin Clin Neuropsychiatry 1998;3: 176–185. 5 Yakovlev AG, Faden AI: Mechanisms of neural cell death: implications for development of neuroprotective treatment strategies. NeuroRx 2004;1:5–16. 6 Bittigau P, Sifringer M, Pohl D, Stadthaus D, Ishimaru M, Shimizu H, Ikeda M, Lang D, Speer A, Olney J, Ikonomidou C: Apoptotic neurodegeneration following trauma is markedly enhanced in the immature brain. Ann Neurol 1999;45:724–735. 7 Pullela R, Raber J, Pfankuch T, Ferriero DM, Claus CP, Koh SE, Yamauchi T, Rola R, Fike JR, Noble-Haeusslein LJ: Traumatic injury to the immature brain results in progressive neuronal loss, hyperactivity and delayed cognitive impairments. Dev Neurosci 2006; 28:396–409. 8 Towfighi J, Mauger D: Temporal evolution of neuronal changes in cerebral hypoxia-ischemia in developing rats: a quantitative light microscopic study. Brain Res Dev Brain Res 1998;109:169–177. 9 Tsuru-Aoyagi K, Potts MB, Trivedi A, Pfankuch T, Raber J, Wendland M, Claus CP, Koh SE, Ferriero D, Noble-Haeusslein LJ: Glutathione peroxidase activity modulates recovery in the injured immature brain. Ann Neurol 2009;65:540–549. 10 Liu CL, Siesjo BK, Hu BR: Pathogenesis of hippocampal neuronal death after hypoxiaischemia changes during brain development. Neuroscience 2004;127:113–123. 11 McIntosh TK, Faden AI, Bendall MR, Vink R: Traumatic brain injury in the rat: alterations in brain lactate and pH as characterized by 1H and 31P nuclear magnetic resonance. J Neurochem 1987;49:1530–1540. 12 Duhaime AC, Margulies SS, Durham SR, O’Rourke MM, Golden JA, Marwaha S, Raghupathi R: Maturation-dependent response of the piglet brain to scaled cortical impact. J Neurosurg 2000;93:455–462.
452
13 Tong W, Igarashi T, Ferriero DM, Noble LJ: Traumatic brain injury in the immature mouse brain: characterization of regional vulnerability. Exp Neurol 2002;176:105–116. 14 Marmarou A, Foda MA, van den Brink W, Campbell J, Kita H, Demetriadou K: A new model of diffuse brain injury in rats. Part 1. Pathophysiology and biomechanics. J Neurosurg 1994;80:291–300. 15 Adelson PD, Dixon CE, Robichaud P, Kochanek PM: Motor and cognitive functional deficits following diffuse traumatic brain injury in the immature rat. J Neurotrauma 1997;14:99–108. 16 Cernak I, Vink R, Zapple DN, Cruz MI, Ahmed F, Chang T, Fricke ST, Faden AI: The pathobiology of moderate diffuse traumatic brain injury as identified using a new experimental model of injury in rats. Neurobiol Dis 2004;17:29–43. 17 Elliott KA, Jasper H: Measurement of experimentally induced brain swelling and shrinkage. Am J Physiol 1949;157:122–129. 18 Uyama O, Okamura N, Yanase M, Narita M, Kawabata K, Sugita M: Quantitative evaluation of vascular permeability in the gerbil brain after transient ischemia using Evans blue fluorescence. J Cereb Blood Flow Metab 1988;8:282–284. 19 Faden AI, Knoblach SM, Cernak I, Fan L, Vink R, Araldi GL, Fricke ST, Roth BL, Kozikowski AP: Novel diketopiperazine enhances motor and cognitive recovery after traumatic brain injury in rats and shows neuroprotection in vitro and in vivo. J Cereb Blood Flow Metab 2003;23:342–354. 20 Heath DL, Vink R: Traumatic brain axonal injury produces sustained decline in intracellular free magnesium concentration. Brain Res 1996;738:150–153. 21 Vink R, Heath DL, McIntosh TK: Acute and prolonged alterations in brain free magnesium following fluid percussion-induced brain trauma in rats. J Neurochem 1996; 66: 2477–2483. 22 Heath DL, Vink R: Brain free magnesium concentration is predictive of motor outcome following traumatic axonal brain injury in rats. Magnes Res 1999;12:269–277. 23 Gupta RK, Benovic JL, Rose ZB: The determination of the free magnesium level in the human red blood cell by 31P NMR. J Biol Chem 1978;253:6172–6176. 24 Vink R, Faden AI, McIntosh TK: Changes in cellular bioenergetic state following graded traumatic brain injury in rats: determination by phosphorus 31 magnetic resonance spectroscopy. J Neurotrauma 1988;5:315–330. 25 Schmued L, Kyriakidis K, Heimer L: In vivo anterograde and retrograde axonal transport of the fluorescent rhodamine-dextranamine, Fluoro-Ruby, within the CNS. Brain Res 1990;526:127–134.
Dev Neurosci 2010;32:442–453
26 Ahmed F, MacArthur L, de Bernardi MA, Mocchetti I: Retrograde and anterograde transport of HIV protein gp120 in the nervous system. Brain Behav Immun 2009; 23: 355–364. 27 Li FH, Fisher M: Diffusion-weighted and perfusion magnetic resonance imaging and ischemic stroke. Drugs Today (Barc) 1996; 32:615–627. 28 Donkin JJ, Nimmo AJ, Cernak I, Blumbergs PC, Vink R: Substance P is associated with the development of brain edema and functional deficits after traumatic brain injury. J Cereb Blood Flow Metab 2009;29:1388–1398. 29 Adelson PD, Robichaud P, Hamilton RL, Kochanek PM: A model of diffuse traumatic brain injury in the immature rat. J Neurosurg 1996;85:877–884. 30 Ommaya AK, Goldsmith W, Thibault L: Biomechanics and neuropathology of adult and paediatric head injury. Br J Neurosurg 2002; 16:220–242. 31 Ommaya AK, Grubb RL Jr, Naumann RA: Coup and contre-coup injury: observations on the mechanics of visible brain injuries in the rhesus monkey. J Neurosurg 1971; 35: 503–516. 32 McPherson GK, Kriewall TJ: The elastic modulus of fetal cranial bone: a first step towards an understanding of the biomechanics of fetal head molding. J Biomech 1980;13:9–16. 33 Dennis M: Developmental plasticity in children: the role of biological risk, development, time, and reserve. J Commun Disord 2000; 33:321–331, quiz 332. 34 Graham DI, Ford I, Adams JH, Doyle D, Lawrence AE, McLellan DR, Ng HK: Fatal head injury in children. J Clin Pathol 1989; 42:18–22. 35 Armstead WM: Age and cerebral circulation. Pathophysiology 2005; 12:5–15. 36 Blomgren K, Zhu C, Hallin U, Hagberg H: Mitochondria and ischemic reperfusion damage in the adult and in the developing brain. Biochem Biophys Res Commun 2003; 304:551–559. 37 Fan P, Yamauchi T, Noble LJ, Ferriero DM: Age-dependent differences in glutathione peroxidase activity after traumatic brain injury. J Neurotrauma 2003;20:437–445. 38 Waters KA, Machaalani R: NMDA receptors in the developing brain and effects of noxious insults. Neurosignals 2004;13:162–174. 39 Duhaime AC: Large animal models of traumatic injury to the immature brain. Dev Neurosci 2006;28:380–387. 40 Derugin N, Ferriero DM, Vexler ZS: Neonatal reversible focal cerebral ischemia: a new model. Neurosci Res 1998;32:349–353. 41 Lustyik G, Nagy I: Alterations of the intracellular water and ion concentrations in brain and liver cells during aging as revealed by energy-dispersive X-ray microanalysis of bulk specimens. Scan Electron Microsc 1985:323–337.
Cernak /Chang /Ahmed /Cruz /Vink / Stoica /Faden
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
References
Experimental DTBI as a Function of Developmental Age
44 Adelson P, Dixon C, Kochanek P: Long-term dysfunction following diffuse traumatic brain injury in the immature rat. J Neurotrauma 2000;17:273–282. 45 Duffy TE, Kohle SJ, Vannucci RC: Carbohydrate and energy metabolism in perinatal rat brain: relation to survival in anoxia. J Neurochem 1975;24:271–276. 46 Schuchmann S, Buchheim K, Heinemann U, Hosten N, Buttgereit F: Oxygen consumption and mitochondrial membrane potential indicate developmental adaptation in energy metabolism of rat cortical neurons. Eur J Neurosci 2005;21:2721–2732.
47 Hida K, Suzuki N, Kwee IL, Nakada T: pHlactate dissociation in neonatal anoxia: Proton and 31P NMR spectroscopic studies in rat pups. Magn Reson Med 1991;22:128–132. 48 Suzuki N, Kwee IL, Nakada T: Brain maturation and response to anoxia: 31P NMR spectroscopic studies in rat pups. Magn Reson Med 1992;24:205–212. 49 Robertson CL, Bucci CJ, Fiskum G: Mitochondrial response to calcium in the developing brain. Brain Res Dev Brain Res 2004; 151:141–148.
Dev Neurosci 2010;32:442–453
453
Downloaded by: University of Alberta Library 129.128.17.194 - 5/28/2015 1:04:14 AM
42 Bullock R, Maxwell WL, Graham DI, Teasdale GM, Adams JH: Glial swelling following human cerebral contusion: an ultrastructural study. J Neurol Neurosurg Psychiatry 1991;54:427–434. 43 Marmarou A, Fatouros PP, Barzo P, Portella G, Yoshihara M, Tsuji O, Yamamoto T, Laine F, Signoretti S, Ward JD, Bullock MR, Young HF: Contribution of edema and cerebral blood volume to traumatic brain swelling in head-injured patients. J Neurosurg 2000; 93: 183–193.
