SHOCK, Vol. 44, Supplement 1, pp. 71Y78, 2014
SUBACUTE OXIDATIVE STRESS AND GLIAL REACTIVITY IN THE AMYGDALA ARE ASSOCIATED WITH INCREASED ANXIETY FOLLOWING BLAST NEUROTRAUMA Venkata Siva Sai Sujith Sajja,* William B. Hubbard,* and Pamela J. VandeVord*† *School of Biomedical Engineering and Sciences, Virginia Polytechnic and State University, Blacksburg; and † Salem VA Medical Center, Research and Development Service, Salem, Virginia Received 18 Sep 2014; first review completed 8 Oct 2014; accepted in final form 26 Nov 2014 ABSTRACT—Behavioral symptoms, such as anxiety, are widely reported after blast overpressure (BOP) exposure. Amygdalar vulnerability to increasing magnitudes of BOP has not been investigated, and single exposures to blast have been limited to acute (G72 h) assessment. Rats were exposed to a single low, moderate, or high BOP (10, 14, or 24 psi) with an advanced blast simulator to test the susceptibility of the amygdala. Anxiety-like behavior was observed in the low- and moderate-pressure groups when subjected to the light/dark box assessment 7 days after the blast but not in high-pressure group. Immunohistochemistry was performed to measure apoptosis (cleaved caspase-3), neuronal loss (NeuN), reactive astrocytes (glial fibrillary acidic protein), microglia (Iba-1), and oxidative stress (CuZn superoxide dismutase). Slower progression of injury cascades was associated with a significant increase in anxiety, apoptosis, and astrogliosis in the low pressure group compared with others. A significant increase of CuZn superoxide dismutase in the low pressure group could be associated with neuroprotection from cell death caused by oxidative stress because neuronal loss was significant in the moderate- and high- but not the low-pressure group. Overall, this study demonstrated that overpressure as low as 10 psi can induce subacute anxiety, in addition to neuropathologic changes in the amygdala. KEYWORDS—Blast, inflammation, apoptosis, oxidative stress, trauma, anxiety, amygdala
INTRODUCTION
injury from blast overpressure (BOP) exposure is vital for understanding reasons of the behavioral outcomes. Neuroglia, non-neuronal cells that support and provide protection to neurons, are found to be activated in central nervous system injuries, including BINT. The injury-induced process of reactive gliosis involves both astrocytes, which regulate the ion/nutrient environment and provide metabolic support to neurons, and microglia, which engulf cellular debris/foreign materials around neurons. Biomarkers, such as glial fibrillary acidic protein (GFAP) (astrocyte reactivity), have been reported to be elevated in hippocampus and nucleus accumbens after blast exposure in rodents (12Y15). Preclinical studies have shown a potential role of neuroinflammation and glial response as an acute outcome after BINT in the amygdala in association with behavioral deficits (13, 16Y18). Although anxiety is reported to be a persistent symptom associated with BINT, limited studies have focused on the amygdala that mediates anxiety and fear conditioning pathways as well as plays a role in processing memories and decision making. Specifically, how varying magnitudes of blast affects the amygdala at a subacute stage has not been investigated. Understanding how the neuropathology is associated with the behavior deficits is critical for the development of effective treatment strategies. The motivation for this study was to characterize amygdala pathology at the subacute stage to determine if there is a correlation between the cellular effects and behavior outcomes.
Blast-induced neurotrauma (BINT) is a hazardous condition developed after an exposure to a blast wave and is common in military, as well as civilian, settings (1, 2). Many debilitating effects are observed in individuals who have been exposed to this rapid overpressure, but gaps in knowledge remain with respect to the biomolecular pathways that are triggered and remain activated after injury. When considering nonblast models of traumatic brain injury (TBI), many techniques have been used to investigate cellular injury progression (3). Systemic biomarkers have also been examined after TBI to elucidate post-injury cascades as well as diagnostic possibilities (4, 5). Moreover, behavioral outcomes have been assessed, and acute deficits in cognitive function, such as decreased memory, attention, and intellectual effectiveness, have been observed in addition to a delayed effect on mood and behavior after blast exposure (6, 7). Blast-induced neurotrauma has been underreported because of the variable nature of when noticeable symptoms are revealed (8Y10). The development of specific biomarkers that could identify BINT at acute, subacute, and chronic stages will significantly catalyze diagnostic and treatment options for this disorder (11, 12). The effects of exposure to varied peak overpressures have not been studied in depth, and the mechanisms of molecular injury to varying pressure magnitudes may differ. Further identification of key molecular changes that may contribute to the
MATERIALS AND METHODS Blast parameters
Address reprint requests to Pamela J. VandeVord, PhD, 447 Kelly Hall, 325 Stanger St, Blacksburg, VA 24061. E-mail: [email protected]. This research was supported in part by the US Army Medical Research and Materiel Command (grant no. W81XWH-08-2-0207 to P.J.V.) and by the Department of Veterans Affairs, Veterans Health Administration, Rehabilitation Research and Development Service (grant no. B1104-P to P.J.V.). The authors declare no conflicts of interest. DOI: 10.1097/SHK.0000000000000311 Copyright Ó 2014 by the Shock Society
The shock front and dynamic overpressure was generated by a custom-built advanced blast simulator located in the Center for Injury Biomechanics at Virginia Tech as previous described (19, 20). The advanced blast simulator consists of a driving compression chamber attached to a rectangular testing chamber with an end wave eliminator (ORA Inc, Fredericksburg, Va). The passive end wave eliminator was installed at the venting end of the blast simulator and functioned to partially block the shock wave outflow by means of a grill plate patterned to mirror reflected shocks and rarefactions, which tend 71
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to Bcancel[ each other and diminish unwanted effects on the test specimen. A peak static overpressure was produced with compressed helium and calibrated acetate sheets (Grafix Plastics, Cleveland, Ohio). Pressure measurements were collected at 250 kHz using a DA8HF data acquisition system (Astro-Med, Inc, West Warwick, RI), and shock wave profiles were verified to maintain consistent exposure pressures within groups.
Animal parameters The Virginia Tech Institutional Animal Care and Use Committee approved experimental protocols described herein. Before all experiments, male Sprague-Dawley rats (250 Y 300 g; Harlan Labs, San Diego, Calif) were acclimated to a 12-h light/dark cycle with food and water provided ad libitum. The animals were randomly selected for placement in each group before the experiment. The blast animals were anesthetized with 3% isoflurane inside an induction chamber before being placed in a rostral cephalic orientation toward the shock wave. Animals (n = 5 per group) were exposed to a single incident pressure profile resembling a Bfree-field[ blast exposure during the midday for minimal seasonal effects. In accordance with Sajja et al. (19), three distinct pressures were generated and classified as low (10 psi; 10.25 T 0.41 psi), moderate (14 psi; 14.20 T 0.41 psi), and high (24 psi; 23.64 T 0.86 psi) pressures (Fig. 1). A sham group underwent all the same procedures, with the exception of being exposed to the BOP.
Anxiety assessment Animals were assessed for behavioral deficits at 7 days after the blast. The light/dark (L/D) box test is an established tool used for the assessment of anxiety. For this assessment, high anxiety levels can be correlated to longer durations in the dark compartment and latency to re-enter the light compartment after the animals are introduced to L/D. Measurements are compared with the results from the sham group. Time spent in the light half of the arena and the related exploratory behavior are standard and reliable parameters for assessing anxiolytic effects that may be useful in identifying and/or screening of anxiolytic and anxiogenic agents (21, 22). This test exploits the conflict between the animal’s tendency to explore an open environment (nonYanxietylike effect) and to stay in a defensive mode (anxiety-like effect). The apparatus consisted of two acrylic compartments, one dark side closed with a lid and one light side. The measurements of the L/D box are 72 30.5 33.5 cm, with the dark side equal to 35.5 30.5 33.5 cm and the light side being 35.5 30.5 33.5 cm. Each rat was tested by placing it in the light area, facing away from the dark compartment, and was allowed to explore the novel environment for 5 min. To avoid any environmental bias, the animal was left alone in the testing room for the duration of the exploratory time and the behavior measurements. Experimenters were blinded to the groups before the tests were performed. Latency and time spent on the light side were obtained using video analysis (Ethovision; Noldus, Leesburg, Va). Whole-body position was used to determine compartment transitions in terms of calculating time spent in the lighted compartment and initial latency when entering the dark compartment for the first time.
Immunohistochemistry At 7 days after the blast, subsequent to behavioral testing, animals were briefly anesthetized with isoflurane (3%) and euthanized by transcardially perfused ice-cold phosphate buffered saline (PBS) containing 6 units/mL
FIG. 1. Representative pressure profile of BOP at low (10 psi), moderate (14 psi), and high (24 psi).
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heparin. The brains were processed in 30% sucrose solution and fixed in Tissue-Tek optimal cutting temperature embedding medium (Sakura Finetek USA, Inc, Torrance, Calif) for cryostat processing (Microm HM550; Thermo Scientific Inc, Waltham, Mass). Immunohistochemistry was performed on coronal serial sections of the amygdala (bregma j0.9 to bregma j2.9 [23]) for GFAP (an astrocyte-specific cell activation indicator), Iba-1 (microglia marker), cleaved caspase-3 (apoptosis), CuZn superoxide dismutase (CuZn SOD; oxidative stress), and neuronal nuclei staining (NeuN; neuronal marker [24]). Tissue sections (20 2m) were fixed in 4% paraformaldehyde for 15 min at room temperature, rinsed with PBS, and incubated in 0.5% Triton X-100 + 0.5% gelatin blocking buffer for 1 h. After being washed with PBS, nonspecific binding sites were blocked with 3% bovine serum albumin in PBS for 1 h at room temperature. The histological sections (five representative serial sections per stain) were then incubated with a primary antibody: anti-GFAP (Invitrogen, Carlsbad, Calif), antiYIba-1 (Biocare Medical, Concord, Calif), antiYcleaved caspase-3 (Invitrogen), antiYCuZn SOD (GeneTex, Irvine, Calif), or anti-NeuN (Millipore, Billerica, Mass) at 1:200 overnight at 4-C. After a PBS wash, the sections were incubated for 1.5 h with fluorescence-tagged fluorescein isothiocyanateYsecondary antiYrat IgG antibody (1:200; Vector Laboratories, Burlingame, Calif) or Alexa Fluor-555 antiYrabbit IgG antibody (1:200; Cell Signaling, Danvers, Mass). After a PBS wash, sections were mounted on slides, air-dried, and coverslipped with prolong antifade gold reagent with 6diamidino-2-phenylindole (Invitrogen). Sections were examined under Zeiss fluorescence microscope at 20 magnification under appropriate fluorescent filters, and images were captured with a Zeiss AxioCam ICc1. Fluorescence intensity of acquired digital images was quantified by ImageJ software (National Institutes of Health, Bethesda, Md).
Statistical analysis Statistical differences between sham and blast rats (i.e., low, moderate or high) were assessed with analysis of variance using least significant difference post hoc test, with P G 0.05 considered significant. Pearson correlation was used to assess a potential relationship between levels of Iba-1 stain and the time spent in the dark side for the L/D test; the correlation significance was assessed using IBM SPSS 21, 2012 statistical software, and P G 0.05 was considered statistically significant. Unless indicated otherwise, data are presented as mean T SEM.
RESULTS Anxiety assessment
One week after the blast, there was a significant difference in time spent on the light side of the box when comparing the sham with the low-pressure group (P G 0.05) and trending toward significance with the moderate-pressure group (P = 0.072) (Fig. 2). No significant changes were observed with the high-pressure group compared with sham. Similar trends were seen with the latency data. The sham group had higher latency (to enter the dark compartment) compared with the low- and moderate-pressure groups, but no significant changes compared with the highpressure group (Fig. 3).
FIG. 2. Time spent in the light compartment was significantly less for the 10- and 14-psi groups. No difference was found between sham and 24-psi groups. *P G 0.05.
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FIG. 3. Time spent until first entrance into the light compartment was found to be significantly lower in the 10- and 14-psi groups. *P G 0.05.
Immunohistochemistry
Cleaved caspase-3 analysis—A significant increase (P G 0.05) of cleaved caspase-3Ypositive cells (marker of apoptosis) was found in the amygdala of all blast groups at 7 days after exposure when comparing with sham. The low-pressure group was significantly higher (P G 0.05) than the other pressure groups (Fig. 4).
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GFAP analysis—A significant increase (P G 0.05) of GFAP, a marker of reactive astrocytes, was found in the amygdala of all blast groups compared with shams; however, there were no intergroup changes (Fig. 5). Iba-1 analysis—A significant increase (P G 0.05) of Iba-1, marker of reactive microglia, was found only in the amygdala of the low-blast group as compared with all other groups (Fig. 6). CuZn SOD analysis—A significant increase (P G 0.05) of CuZn SOD, a major antioxidant in the brain, was found in the amygdala of the low- and moderate-blast groups compared with sham. In contrary, levels of CuZn SOD were not significantly different after the high blast as compared with shams (Fig. 7). NeuN analysis—A significant decrease (P G 0.05) of NeuN, a neuronal marker, was found in the amygdala of the moderateand high-pressure groups compared with the sham and lowpressure groups (Fig. 8). Anxiety and Iba-1 Pearson correlation—A positive correlation was found between the time spent in the dark for the L/D test and the elevated levels of Iba-1 fluorescence in the lowpressure group (R = 0.92, P G 0.01).
FIG. 4. Cleaved caspase-3 expression in different pressure groups 7 days after the blast. All blast groups were significantly elevated as compared with sham (*P G 0.05). The 10-psi group was significantly elevated when compared with the 14- and 24-psi groups (*P G 0.05). Representative images of cleaved caspase-3 levels at different pressure groups 7 days after the blast. Red color indicates caspase-3Ypositive cells. A, sham; B, 10 psi; C, 14 psi; D, 24 psi.
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FIG. 5. GFAP expression (astrocyte marker) in different pressure groups at 7 days after the blast. All blast groups were significantly elevated as compared with sham, but no intergroup differences persisted (*P G 0.05). Representative images of GFAP levels (astrocyte marker) at different pressure groups 7 days after the blast. Green indicates GFAP-positive cells. A, sham; B, 10 psi; C, 14 psi; D, 24 psi.
DISCUSSION The current study presented data that expound on the association of amygdalar pathology and anxiety-like behavior after a blast. Anxiety-like behavior was identified by the L/D box assessment, and results suggested that low-level blast exposure (10 psi) caused increased anxiety 7 days after the blast. Furthermore, animals exposed to low and moderate BOP (10 and 14 psi) showed more anxiety-like behavior (latency) as compared with the animals exposed to the high BOP (24 psi) at 7 days. This result of lower pressures causing more anxiety-like behavior at the subacute stage may seem counterintuitive; however, the temporal response to blast has not been ascertained. The cellular response to injury, whether mild, moderate, or severe, varies, and inflammatory molecules, which may contribute to behavior, need to be identified to better understand this phenomenon. The results demonstrated glial recruitment, cell death, and oxidative stress in the amygdala 7 days after the blast. These inflammatory param-
eters correlated with the behavioral outcomes. In previous studies, acute (6 Y 72 h after the blast) neuropathology was described with neurodegeneration and cell death in the amygdala at pressures higher than 14 psi (13). The current study extends the temporal information finding increased cell death (cleaved caspase-3) and decreased numbers of mature neurons (NeuN) at 7 days after the blast in all pressure groups. However, elevated levels of microglia were observed only in the low-blast group. Microglia are one of the first responders to the injury site, assisting in clearing debris and reducing inflammation. Because severe injuries may demand immediate glial response, mild injuries may present with a delayed onset of apoptosis and injury progression, which has been demonstrated in both animals and clinical studies (25Y27). Holmin et al. (25) identified several proinflammatory cytokines whose expression was delayed after TBI in rats. The authors concluded that the pattern of cytokine production is not a uniform event and that various pathogenic mechanisms may contribute to the molecular
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FIG. 6. Iba-1 (microglia marker) in different pressure groups 7 days after the blast. The 10-psi group was significantly elevated as compared with sham and 14-psi and 24-psi groups (*P G 0.05). Representative images of Iba-1 levels (microglia marker) at different pressure groups 7 days after the blast. Red indicates Iba-1Ypositive cells. A, sham; B, 10 psi; C, 14 psi; D, 24 psi.
cascade after TBI. Others have reported the activation of microglial and astrocytes after BOP exposure, which our data reinforce (19, 28). Elevated levels of reactive astrocytes were found in the amygdala of all blast groups as compared with the sham. Astrocytes typically play a prominent role in restoring biochemical homeostasis caused by energy deficits and clearance of debris during cell death and injury progression (29). Astrogliosis has been reported to regulate cell death after the blast (13, 30). In addition to cellular metabolic support and clearing of debris, reactive astrocytes initiate the recruitment of other cells by secreting inflammatory and/or anti-inflammatory molecules (18, 20). Kamnaksh et al. (17) showed elevated levels of proinflammatory factors, such as interferon-+ and interleukin-6, within the amygdala at acute stages after blast exposure. In addition, upregulation of antioxidants contributes to the support of intracellular metabolic processes to protect mitochondria from oxidative stress (18).
Studies have reported oxidative stress in the hippocampus and cortex after blast exposure, but this has not been established in the amygdala (15, 31). The current results support that oxidative stress is a common cellular process that results from the blast. An increased level of CuZn SOD in the low- and moderatepressure groups may contribute to the anti-apoptosis mechanism that counteracts oxidative stress after the blast. Overall, ongoing injury progression caused by increased apoptotic levels is caused by an inadequate antioxidant system, which leads to progression of injury cascades. It is speculated that different biomechanical properties of specific cell types in the amygdala could give rise to a variable response to BOP. Further studies need to be conducted to decipher the blast injury threshold for specific cell types. To validate that anxiety-like behavior was associated with amygdalar pathology, a Pearson correlation test was conducted. Time spent in the dark for the L/D test, a measure of anxiety-like behavior, and increased microglia in the low-pressure group achieved a positive correlation, indicating
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FIG. 7. CuZn SOD levels in different pressure groups 7 days after the blast. The 10- and 14-psi groups were significantly elevated as compared with sham and the 24-psi group (*P G 0.05). Representative images of CuZn SOD levels at different pressure groups 7 days after the blast. Green color indicates CuZn SODYpositive cells. A, sham; B, 10 psi; C, 14 psi; D, 24 psi.
that microglial activation could play a crucial role in subacute anxiety. Moreover, increased microglia or molecules expressed by microglia could be used as a biomarker of injury progression. Many clinical and preclinical studies have also identified that microglial activation is key in increased inflammation and could be an important diagnostic tool (20, 32, 33). Noticeable long-term behavioral changes and cognitive deficits, such as increased general anxiety disorders resembling posttraumatic stress disorder, have been shown in patients with BINT (1, 8, 34). Clinical studies assessing fear and anxiety in veterans diagnosed as having blast-associated depressive disorder reported greater amygdala activity during fear-matching trials (10, 34, 35). Matthews et al. (34) reported that veterans with or without major depressive disorder (MDD) in combination with blast did not exhibit gross morphological
changes using diffusor tensor imaging. Yet it was found that MDD caused by a blast trauma relative to non-MDD individuals showed greater activity during fear-matching trials in the amygdala and other emotion-processing structures. The report concluded that the mechanistic pathways could only be found via comprehensive pathological studies. Overall, different magnitudes of blast show varied cellular responses and delayed temporal effects 7 days after a blast. VandeVord et al. (13) suggested that specific shock wave magnitudes may contribute to the varying biomechanical responses to blast exposure and subsequent injury to the cells. The current results seem to provide evidence that varying pressure magnitude contributes to which molecular pathways are triggered. A limitation of this study is the use of gas anesthesia during the blast procedure. The use of anesthesia has been reported to
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FIG. 8. NeuN levels in different pressure groups 7 days after the blast. The 14- and 24-psi groups were significantly decreased as compared with sham and the 10-psi group (*P G 0.05). Representative images of NeuN levels at different pressure groups 7 days after the blast. Green color indicates neurons. A, sham; B, 10 psi; C, 14 psi; D, 24 psi.
have neuroprotective effects. We chose to use a minimal amount of gas anesthesia during the blast test to reduce these effects. In addition, oxidative stress and apoptosis markers used in this study are not cell specific; thus, we cannot confirm the specific cell type that is affected by the blast. The pathological results from this study indicate that an antioxidant-based therapy or inhibition of microglial activation or astrogliosis via anti-inflammatory therapies could ameliorate the cellular damage sustained after the blast and improve neurologic function. Therapeutic approaches could also differ based on the magnitude of the BOP insult. Although this study has significantly advanced the knowledge of subacute molecular changes that occurs after blast exposure, further studies are vital to provide a comprehensive molecular picture of the temporal response of the brain to a blast.
CONCLUSIONS Despite ongoing protective effects with the presence of increased antioxidants at low and moderate pressures, increased apoptosis could trigger subacute effects in the amygdala of the low-blast group, explaining the anxiety-associated outcomes. In addition, inhibitors to regress chronic activation of microglia may be a clinical target to help relieve behavioral symptoms.
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SUBACUTE OXIDATIVE STRESS AND GLIAL REACTIVITY IN THE AMYGDALA ARE ASSOCIATED WITH INCREASED ANXIETY FOLLOWING BLAST NEUROTRAUMA Venkata Siva Sai Sujith Sajja,* William B. Hubbard,* and Pamela J. VandeVord*† *School of Biomedical Engineering and Sciences, Virginia Polytechnic and State University, Blacksburg; and † Salem VA Medical Center, Research and Development Service, Salem, Virginia Received 18 Sep 2014; first review completed 8 Oct 2014; accepted in final form 26 Nov 2014 ABSTRACT—Behavioral symptoms, such as anxiety, are widely reported after blast overpressure (BOP) exposure. Amygdalar vulnerability to increasing magnitudes of BOP has not been investigated, and single exposures to blast have been limited to acute (G72 h) assessment. Rats were exposed to a single low, moderate, or high BOP (10, 14, or 24 psi) with an advanced blast simulator to test the susceptibility of the amygdala. Anxiety-like behavior was observed in the low- and moderate-pressure groups when subjected to the light/dark box assessment 7 days after the blast but not in high-pressure group. Immunohistochemistry was performed to measure apoptosis (cleaved caspase-3), neuronal loss (NeuN), reactive astrocytes (glial fibrillary acidic protein), microglia (Iba-1), and oxidative stress (CuZn superoxide dismutase). Slower progression of injury cascades was associated with a significant increase in anxiety, apoptosis, and astrogliosis in the low pressure group compared with others. A significant increase of CuZn superoxide dismutase in the low pressure group could be associated with neuroprotection from cell death caused by oxidative stress because neuronal loss was significant in the moderate- and high- but not the low-pressure group. Overall, this study demonstrated that overpressure as low as 10 psi can induce subacute anxiety, in addition to neuropathologic changes in the amygdala. KEYWORDS—Blast, inflammation, apoptosis, oxidative stress, trauma, anxiety, amygdala
INTRODUCTION
injury from blast overpressure (BOP) exposure is vital for understanding reasons of the behavioral outcomes. Neuroglia, non-neuronal cells that support and provide protection to neurons, are found to be activated in central nervous system injuries, including BINT. The injury-induced process of reactive gliosis involves both astrocytes, which regulate the ion/nutrient environment and provide metabolic support to neurons, and microglia, which engulf cellular debris/foreign materials around neurons. Biomarkers, such as glial fibrillary acidic protein (GFAP) (astrocyte reactivity), have been reported to be elevated in hippocampus and nucleus accumbens after blast exposure in rodents (12Y15). Preclinical studies have shown a potential role of neuroinflammation and glial response as an acute outcome after BINT in the amygdala in association with behavioral deficits (13, 16Y18). Although anxiety is reported to be a persistent symptom associated with BINT, limited studies have focused on the amygdala that mediates anxiety and fear conditioning pathways as well as plays a role in processing memories and decision making. Specifically, how varying magnitudes of blast affects the amygdala at a subacute stage has not been investigated. Understanding how the neuropathology is associated with the behavior deficits is critical for the development of effective treatment strategies. The motivation for this study was to characterize amygdala pathology at the subacute stage to determine if there is a correlation between the cellular effects and behavior outcomes.
Blast-induced neurotrauma (BINT) is a hazardous condition developed after an exposure to a blast wave and is common in military, as well as civilian, settings (1, 2). Many debilitating effects are observed in individuals who have been exposed to this rapid overpressure, but gaps in knowledge remain with respect to the biomolecular pathways that are triggered and remain activated after injury. When considering nonblast models of traumatic brain injury (TBI), many techniques have been used to investigate cellular injury progression (3). Systemic biomarkers have also been examined after TBI to elucidate post-injury cascades as well as diagnostic possibilities (4, 5). Moreover, behavioral outcomes have been assessed, and acute deficits in cognitive function, such as decreased memory, attention, and intellectual effectiveness, have been observed in addition to a delayed effect on mood and behavior after blast exposure (6, 7). Blast-induced neurotrauma has been underreported because of the variable nature of when noticeable symptoms are revealed (8Y10). The development of specific biomarkers that could identify BINT at acute, subacute, and chronic stages will significantly catalyze diagnostic and treatment options for this disorder (11, 12). The effects of exposure to varied peak overpressures have not been studied in depth, and the mechanisms of molecular injury to varying pressure magnitudes may differ. Further identification of key molecular changes that may contribute to the
MATERIALS AND METHODS Blast parameters
Address reprint requests to Pamela J. VandeVord, PhD, 447 Kelly Hall, 325 Stanger St, Blacksburg, VA 24061. E-mail: [email protected]. This research was supported in part by the US Army Medical Research and Materiel Command (grant no. W81XWH-08-2-0207 to P.J.V.) and by the Department of Veterans Affairs, Veterans Health Administration, Rehabilitation Research and Development Service (grant no. B1104-P to P.J.V.). The authors declare no conflicts of interest. DOI: 10.1097/SHK.0000000000000311 Copyright Ó 2014 by the Shock Society
The shock front and dynamic overpressure was generated by a custom-built advanced blast simulator located in the Center for Injury Biomechanics at Virginia Tech as previous described (19, 20). The advanced blast simulator consists of a driving compression chamber attached to a rectangular testing chamber with an end wave eliminator (ORA Inc, Fredericksburg, Va). The passive end wave eliminator was installed at the venting end of the blast simulator and functioned to partially block the shock wave outflow by means of a grill plate patterned to mirror reflected shocks and rarefactions, which tend 71
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to Bcancel[ each other and diminish unwanted effects on the test specimen. A peak static overpressure was produced with compressed helium and calibrated acetate sheets (Grafix Plastics, Cleveland, Ohio). Pressure measurements were collected at 250 kHz using a DA8HF data acquisition system (Astro-Med, Inc, West Warwick, RI), and shock wave profiles were verified to maintain consistent exposure pressures within groups.
Animal parameters The Virginia Tech Institutional Animal Care and Use Committee approved experimental protocols described herein. Before all experiments, male Sprague-Dawley rats (250 Y 300 g; Harlan Labs, San Diego, Calif) were acclimated to a 12-h light/dark cycle with food and water provided ad libitum. The animals were randomly selected for placement in each group before the experiment. The blast animals were anesthetized with 3% isoflurane inside an induction chamber before being placed in a rostral cephalic orientation toward the shock wave. Animals (n = 5 per group) were exposed to a single incident pressure profile resembling a Bfree-field[ blast exposure during the midday for minimal seasonal effects. In accordance with Sajja et al. (19), three distinct pressures were generated and classified as low (10 psi; 10.25 T 0.41 psi), moderate (14 psi; 14.20 T 0.41 psi), and high (24 psi; 23.64 T 0.86 psi) pressures (Fig. 1). A sham group underwent all the same procedures, with the exception of being exposed to the BOP.
Anxiety assessment Animals were assessed for behavioral deficits at 7 days after the blast. The light/dark (L/D) box test is an established tool used for the assessment of anxiety. For this assessment, high anxiety levels can be correlated to longer durations in the dark compartment and latency to re-enter the light compartment after the animals are introduced to L/D. Measurements are compared with the results from the sham group. Time spent in the light half of the arena and the related exploratory behavior are standard and reliable parameters for assessing anxiolytic effects that may be useful in identifying and/or screening of anxiolytic and anxiogenic agents (21, 22). This test exploits the conflict between the animal’s tendency to explore an open environment (nonYanxietylike effect) and to stay in a defensive mode (anxiety-like effect). The apparatus consisted of two acrylic compartments, one dark side closed with a lid and one light side. The measurements of the L/D box are 72 30.5 33.5 cm, with the dark side equal to 35.5 30.5 33.5 cm and the light side being 35.5 30.5 33.5 cm. Each rat was tested by placing it in the light area, facing away from the dark compartment, and was allowed to explore the novel environment for 5 min. To avoid any environmental bias, the animal was left alone in the testing room for the duration of the exploratory time and the behavior measurements. Experimenters were blinded to the groups before the tests were performed. Latency and time spent on the light side were obtained using video analysis (Ethovision; Noldus, Leesburg, Va). Whole-body position was used to determine compartment transitions in terms of calculating time spent in the lighted compartment and initial latency when entering the dark compartment for the first time.
Immunohistochemistry At 7 days after the blast, subsequent to behavioral testing, animals were briefly anesthetized with isoflurane (3%) and euthanized by transcardially perfused ice-cold phosphate buffered saline (PBS) containing 6 units/mL
FIG. 1. Representative pressure profile of BOP at low (10 psi), moderate (14 psi), and high (24 psi).
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heparin. The brains were processed in 30% sucrose solution and fixed in Tissue-Tek optimal cutting temperature embedding medium (Sakura Finetek USA, Inc, Torrance, Calif) for cryostat processing (Microm HM550; Thermo Scientific Inc, Waltham, Mass). Immunohistochemistry was performed on coronal serial sections of the amygdala (bregma j0.9 to bregma j2.9 [23]) for GFAP (an astrocyte-specific cell activation indicator), Iba-1 (microglia marker), cleaved caspase-3 (apoptosis), CuZn superoxide dismutase (CuZn SOD; oxidative stress), and neuronal nuclei staining (NeuN; neuronal marker [24]). Tissue sections (20 2m) were fixed in 4% paraformaldehyde for 15 min at room temperature, rinsed with PBS, and incubated in 0.5% Triton X-100 + 0.5% gelatin blocking buffer for 1 h. After being washed with PBS, nonspecific binding sites were blocked with 3% bovine serum albumin in PBS for 1 h at room temperature. The histological sections (five representative serial sections per stain) were then incubated with a primary antibody: anti-GFAP (Invitrogen, Carlsbad, Calif), antiYIba-1 (Biocare Medical, Concord, Calif), antiYcleaved caspase-3 (Invitrogen), antiYCuZn SOD (GeneTex, Irvine, Calif), or anti-NeuN (Millipore, Billerica, Mass) at 1:200 overnight at 4-C. After a PBS wash, the sections were incubated for 1.5 h with fluorescence-tagged fluorescein isothiocyanateYsecondary antiYrat IgG antibody (1:200; Vector Laboratories, Burlingame, Calif) or Alexa Fluor-555 antiYrabbit IgG antibody (1:200; Cell Signaling, Danvers, Mass). After a PBS wash, sections were mounted on slides, air-dried, and coverslipped with prolong antifade gold reagent with 6diamidino-2-phenylindole (Invitrogen). Sections were examined under Zeiss fluorescence microscope at 20 magnification under appropriate fluorescent filters, and images were captured with a Zeiss AxioCam ICc1. Fluorescence intensity of acquired digital images was quantified by ImageJ software (National Institutes of Health, Bethesda, Md).
Statistical analysis Statistical differences between sham and blast rats (i.e., low, moderate or high) were assessed with analysis of variance using least significant difference post hoc test, with P G 0.05 considered significant. Pearson correlation was used to assess a potential relationship between levels of Iba-1 stain and the time spent in the dark side for the L/D test; the correlation significance was assessed using IBM SPSS 21, 2012 statistical software, and P G 0.05 was considered statistically significant. Unless indicated otherwise, data are presented as mean T SEM.
RESULTS Anxiety assessment
One week after the blast, there was a significant difference in time spent on the light side of the box when comparing the sham with the low-pressure group (P G 0.05) and trending toward significance with the moderate-pressure group (P = 0.072) (Fig. 2). No significant changes were observed with the high-pressure group compared with sham. Similar trends were seen with the latency data. The sham group had higher latency (to enter the dark compartment) compared with the low- and moderate-pressure groups, but no significant changes compared with the highpressure group (Fig. 3).
FIG. 2. Time spent in the light compartment was significantly less for the 10- and 14-psi groups. No difference was found between sham and 24-psi groups. *P G 0.05.
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FIG. 3. Time spent until first entrance into the light compartment was found to be significantly lower in the 10- and 14-psi groups. *P G 0.05.
Immunohistochemistry
Cleaved caspase-3 analysis—A significant increase (P G 0.05) of cleaved caspase-3Ypositive cells (marker of apoptosis) was found in the amygdala of all blast groups at 7 days after exposure when comparing with sham. The low-pressure group was significantly higher (P G 0.05) than the other pressure groups (Fig. 4).
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GFAP analysis—A significant increase (P G 0.05) of GFAP, a marker of reactive astrocytes, was found in the amygdala of all blast groups compared with shams; however, there were no intergroup changes (Fig. 5). Iba-1 analysis—A significant increase (P G 0.05) of Iba-1, marker of reactive microglia, was found only in the amygdala of the low-blast group as compared with all other groups (Fig. 6). CuZn SOD analysis—A significant increase (P G 0.05) of CuZn SOD, a major antioxidant in the brain, was found in the amygdala of the low- and moderate-blast groups compared with sham. In contrary, levels of CuZn SOD were not significantly different after the high blast as compared with shams (Fig. 7). NeuN analysis—A significant decrease (P G 0.05) of NeuN, a neuronal marker, was found in the amygdala of the moderateand high-pressure groups compared with the sham and lowpressure groups (Fig. 8). Anxiety and Iba-1 Pearson correlation—A positive correlation was found between the time spent in the dark for the L/D test and the elevated levels of Iba-1 fluorescence in the lowpressure group (R = 0.92, P G 0.01).
FIG. 4. Cleaved caspase-3 expression in different pressure groups 7 days after the blast. All blast groups were significantly elevated as compared with sham (*P G 0.05). The 10-psi group was significantly elevated when compared with the 14- and 24-psi groups (*P G 0.05). Representative images of cleaved caspase-3 levels at different pressure groups 7 days after the blast. Red color indicates caspase-3Ypositive cells. A, sham; B, 10 psi; C, 14 psi; D, 24 psi.
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FIG. 5. GFAP expression (astrocyte marker) in different pressure groups at 7 days after the blast. All blast groups were significantly elevated as compared with sham, but no intergroup differences persisted (*P G 0.05). Representative images of GFAP levels (astrocyte marker) at different pressure groups 7 days after the blast. Green indicates GFAP-positive cells. A, sham; B, 10 psi; C, 14 psi; D, 24 psi.
DISCUSSION The current study presented data that expound on the association of amygdalar pathology and anxiety-like behavior after a blast. Anxiety-like behavior was identified by the L/D box assessment, and results suggested that low-level blast exposure (10 psi) caused increased anxiety 7 days after the blast. Furthermore, animals exposed to low and moderate BOP (10 and 14 psi) showed more anxiety-like behavior (latency) as compared with the animals exposed to the high BOP (24 psi) at 7 days. This result of lower pressures causing more anxiety-like behavior at the subacute stage may seem counterintuitive; however, the temporal response to blast has not been ascertained. The cellular response to injury, whether mild, moderate, or severe, varies, and inflammatory molecules, which may contribute to behavior, need to be identified to better understand this phenomenon. The results demonstrated glial recruitment, cell death, and oxidative stress in the amygdala 7 days after the blast. These inflammatory param-
eters correlated with the behavioral outcomes. In previous studies, acute (6 Y 72 h after the blast) neuropathology was described with neurodegeneration and cell death in the amygdala at pressures higher than 14 psi (13). The current study extends the temporal information finding increased cell death (cleaved caspase-3) and decreased numbers of mature neurons (NeuN) at 7 days after the blast in all pressure groups. However, elevated levels of microglia were observed only in the low-blast group. Microglia are one of the first responders to the injury site, assisting in clearing debris and reducing inflammation. Because severe injuries may demand immediate glial response, mild injuries may present with a delayed onset of apoptosis and injury progression, which has been demonstrated in both animals and clinical studies (25Y27). Holmin et al. (25) identified several proinflammatory cytokines whose expression was delayed after TBI in rats. The authors concluded that the pattern of cytokine production is not a uniform event and that various pathogenic mechanisms may contribute to the molecular
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FIG. 6. Iba-1 (microglia marker) in different pressure groups 7 days after the blast. The 10-psi group was significantly elevated as compared with sham and 14-psi and 24-psi groups (*P G 0.05). Representative images of Iba-1 levels (microglia marker) at different pressure groups 7 days after the blast. Red indicates Iba-1Ypositive cells. A, sham; B, 10 psi; C, 14 psi; D, 24 psi.
cascade after TBI. Others have reported the activation of microglial and astrocytes after BOP exposure, which our data reinforce (19, 28). Elevated levels of reactive astrocytes were found in the amygdala of all blast groups as compared with the sham. Astrocytes typically play a prominent role in restoring biochemical homeostasis caused by energy deficits and clearance of debris during cell death and injury progression (29). Astrogliosis has been reported to regulate cell death after the blast (13, 30). In addition to cellular metabolic support and clearing of debris, reactive astrocytes initiate the recruitment of other cells by secreting inflammatory and/or anti-inflammatory molecules (18, 20). Kamnaksh et al. (17) showed elevated levels of proinflammatory factors, such as interferon-+ and interleukin-6, within the amygdala at acute stages after blast exposure. In addition, upregulation of antioxidants contributes to the support of intracellular metabolic processes to protect mitochondria from oxidative stress (18).
Studies have reported oxidative stress in the hippocampus and cortex after blast exposure, but this has not been established in the amygdala (15, 31). The current results support that oxidative stress is a common cellular process that results from the blast. An increased level of CuZn SOD in the low- and moderatepressure groups may contribute to the anti-apoptosis mechanism that counteracts oxidative stress after the blast. Overall, ongoing injury progression caused by increased apoptotic levels is caused by an inadequate antioxidant system, which leads to progression of injury cascades. It is speculated that different biomechanical properties of specific cell types in the amygdala could give rise to a variable response to BOP. Further studies need to be conducted to decipher the blast injury threshold for specific cell types. To validate that anxiety-like behavior was associated with amygdalar pathology, a Pearson correlation test was conducted. Time spent in the dark for the L/D test, a measure of anxiety-like behavior, and increased microglia in the low-pressure group achieved a positive correlation, indicating
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FIG. 7. CuZn SOD levels in different pressure groups 7 days after the blast. The 10- and 14-psi groups were significantly elevated as compared with sham and the 24-psi group (*P G 0.05). Representative images of CuZn SOD levels at different pressure groups 7 days after the blast. Green color indicates CuZn SODYpositive cells. A, sham; B, 10 psi; C, 14 psi; D, 24 psi.
that microglial activation could play a crucial role in subacute anxiety. Moreover, increased microglia or molecules expressed by microglia could be used as a biomarker of injury progression. Many clinical and preclinical studies have also identified that microglial activation is key in increased inflammation and could be an important diagnostic tool (20, 32, 33). Noticeable long-term behavioral changes and cognitive deficits, such as increased general anxiety disorders resembling posttraumatic stress disorder, have been shown in patients with BINT (1, 8, 34). Clinical studies assessing fear and anxiety in veterans diagnosed as having blast-associated depressive disorder reported greater amygdala activity during fear-matching trials (10, 34, 35). Matthews et al. (34) reported that veterans with or without major depressive disorder (MDD) in combination with blast did not exhibit gross morphological
changes using diffusor tensor imaging. Yet it was found that MDD caused by a blast trauma relative to non-MDD individuals showed greater activity during fear-matching trials in the amygdala and other emotion-processing structures. The report concluded that the mechanistic pathways could only be found via comprehensive pathological studies. Overall, different magnitudes of blast show varied cellular responses and delayed temporal effects 7 days after a blast. VandeVord et al. (13) suggested that specific shock wave magnitudes may contribute to the varying biomechanical responses to blast exposure and subsequent injury to the cells. The current results seem to provide evidence that varying pressure magnitude contributes to which molecular pathways are triggered. A limitation of this study is the use of gas anesthesia during the blast procedure. The use of anesthesia has been reported to
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FIG. 8. NeuN levels in different pressure groups 7 days after the blast. The 14- and 24-psi groups were significantly decreased as compared with sham and the 10-psi group (*P G 0.05). Representative images of NeuN levels at different pressure groups 7 days after the blast. Green color indicates neurons. A, sham; B, 10 psi; C, 14 psi; D, 24 psi.
have neuroprotective effects. We chose to use a minimal amount of gas anesthesia during the blast test to reduce these effects. In addition, oxidative stress and apoptosis markers used in this study are not cell specific; thus, we cannot confirm the specific cell type that is affected by the blast. The pathological results from this study indicate that an antioxidant-based therapy or inhibition of microglial activation or astrogliosis via anti-inflammatory therapies could ameliorate the cellular damage sustained after the blast and improve neurologic function. Therapeutic approaches could also differ based on the magnitude of the BOP insult. Although this study has significantly advanced the knowledge of subacute molecular changes that occurs after blast exposure, further studies are vital to provide a comprehensive molecular picture of the temporal response of the brain to a blast.
CONCLUSIONS Despite ongoing protective effects with the presence of increased antioxidants at low and moderate pressures, increased apoptosis could trigger subacute effects in the amygdala of the low-blast group, explaining the anxiety-associated outcomes. In addition, inhibitors to regress chronic activation of microglia may be a clinical target to help relieve behavioral symptoms.
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