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Blast overpressure induces shear-related injuries in the brain of rats exposed to a mild traumatic brain injury

Blast overpressure induces shear-related injuries in the brain of rats exposed to a mild... Background: Blast-related traumatic brain injury (TBI) has been a significant cause of injury in the military operations of Iraq and Afghanistan, affecting as many as 10-20% of returning veterans. However, how blast waves affect the brain is poorly understood. To understand their effects, we analyzed the brains of rats exposed to single or multiple (three) 74.5 kPa blast exposures, conditions that mimic a mild TBI. Results: Rats were sacrificed 24 hours or between 4 and 10 months after exposure. Intraventricular hemorrhages were commonly observed after 24 hrs. A screen for neuropathology did not reveal any generalized histopathology. However, focal lesions resembling rips or tears in the tissue were found in many brains. These lesions disrupted cortical organization resulting in some cases in unusual tissue realignments. The lesions frequently appeared to follow the lines of penetrating cortical vessels and microhemorrhages were found within some but not most acute lesions. Conclusions: These lesions likely represent a type of shear injury that is unique to blast trauma. The observation that lesions often appeared to follow penetrating cortical vessels suggests a vascular mechanism of injury and that blood vessels may represent the fault lines along which the most damaging effect of the blast pressure is transmitted. Keywords: Blast overpressure injury, Neuropathology, Shear injury, Traumatic brain injury Background impairment, post-traumatic stress disorder (PTSD) and Traumatic brain injury (TBI) has been a common cause depression [1]. Blast injuries occur through multiple of mortality and morbidity in the military operations in mechanisms that may be related to effects of the primary Iraq and Afghanistan [1]. It is estimated that 10-20% of blast wave, to injuries associated with objects including returning veterans have suffered a TBI [1]. Due to the shrapnel contained within the IED being propelled by prominent use of improvised explosive devices (IED) in the blast wind, or by the individual being knocked down Iraq and Afghanistan, a characteristic feature of TBI in or thrown into solid objects [3]. these conflicts has been its association with blast expos- How the primary blast wave itself affects the brain is ure [2]. Single or multiple blast exposures have been not well understood [3]. Direct tissue damage, bleeding, commonly seen in association with chronic neurological and diffuse axonal injury (DAI) are the best known and psychiatric sequelae including persistent cognitive pathophysiological mechanisms associated with the type of non-blast TBI most commonly encountered * Correspondence: miguel.gama-sosa@mssm.edu during blunt impact injuries in civilian life [4,5]. Blast- Department of Veterans Affairs Medical Center, General Medical Research associated moderate-to-severe TBIs likely result from Service, Bronx, New York, USA Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, New York, USA Full list of author information is available at the end of the article © 2013 Gama Sosa et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 2 of 15 http://www.actaneurocomms.org/content/1/1/51 mechanisms in part similar to those found in non-blast polyethylene Mylar™ sheets (Du Pont, Wilmington, DE, TBI. Thedegreetowhich theprimary blastwavein- USA) that control the peak pressure generated [5,6,10]. jures the brain remains controversial [3,4]. The peak pressure at the end of the expansion chamber Whereas most attention in the Iraq and Afghanistan is determined by piezoresistive gauges specifically conflicts initially focused on the moderate-to-severe end designed for pressure–time (impulse) measurements of the TBI spectrum, the type of injuries that would be (Model 102M152, PCB, Piezotronics, Depew, NY, USA). recognized in the field, it soon became apparent that This apparatus has been used in several studies to de- mild TBIs (mTBI) were much more common and were liver blast overpressure injury to rats [5-9]. Individual frequently not being recognized at the time of the initial rats were anesthetized using an isoflurane anesthesia injury [1]. We had previously established conditions that system consisting of a vaporizer, gas lines and valves, approximate mTBI exposures experimentally. These and an activated charcoal scavenging system adapted for studies found that exposures up to 74.5 kPa, while use with rodents. Rats were placed into a polycarbonate representing a blast level that is transmitted to the brain induction chamber, which was closed and immediately [5], led to no persistent neurological impairments or flushed with a 5% isoflurane mixture in air for 2 mi- lung damage [6], although animals subjected to repeti- nutes. Rats were placed into a cone-shaped plastic re- tive blast exposure, which has been common in the straint device and then placed in the shock tube. current conflicts [2], exhibited a variety of chronic be- Movement was further restricted during the blast expos- havioral and biochemical changes [7,8]. In contrast, ani- ure using a 1.5-cm diameter flattened rubber tourniquet mals exposed to 116.7 kPa blast exposures frequently tubing. Three tourniquets were spaced evenly to secure had gross cerebral and subdural hemorrhages as well as the head region and the upper and lower torso while the contusions and significant lung pathology [5,6,9], fea- animal was in the plastic restraint cone. The end of each tures that are not consistent with mTBI. tubing was threaded through a toggle and run outside of In the present study we explored the pathological ef- the exposure cage where it was tied to affix the animal fects of blast overpressure shock waves in rats exposed firmly and prevent movement during the blast over- to 74.5 kPa blast exposures. We describe a type of shear pressure exposure without restricting breathing. Rats injury in the brain that has not been described in non- were randomly assigned to sham or blast conditions blast TBI models and appears to be unique to blast- with the head facing the blast exposure without any associated brain injury. body shielding resulting in a full body exposure to the blast wave. Further details of the physical characteris- Methods tics of the blast wave have been described elsewhere Animals [6]. Blast-exposed animals received one or three 74.5 All studies were approved by the Institutional Animal kPa exposures. Animals that received three exposures Care and Use Committees of the Naval Medical Research received one exposure per day for three consecutive Center and the James J. Peters VA Medical Center. Two- days. Except for blast exposure, controls were treated month-old male Long Evans Hooded rats (250-350 g; identically receiving anesthesia and being placed in Charles River Laboratories International, Wilmington, the blast tube. For long term studies (four months MA, USA) were used. Animals were housed at a constant and over) the animals were transferred to the James J. 22°C temperature in rooms on a 12:12 hour light cycle Peters VA Medical Center within 10 days of blast ex- with lights on at 7 AM. All animals were individually posure. The number of animals examined under each housed in standard clear plastic cages equipped with Bed- condition is shown in Table 1. O’Cobs laboratory animal bedding (The Andersons, Mau- mee, OH, USA) and EnviroDri nesting paper (Sheppard Histopathological and immunohistochemical analyses Specialty Papers, Milford, NJ, USA). Access to food and Animals were anesthetized with ketamine (65 mg/kg)/ water was ad libitum. xylazine (13 mg/kg)/acepromazine (2 mg/kg) and transcardially perfused with cold 4% paraformaldehyde Blast overpressure exposure in phosphate-buffered saline (PBS). Brains were removed Rats were subjected to overpressure exposure using the and postfixed overnight in the same fixative. To exclude Walter Reed Army Institute of Research (WRAIR) shock perfusion artifacts in some cases the brain was dissected tube that simulates the effects of air blast exposure and immersion fixed in 4% paraformaldehyde in PBS. under experimental conditions [10]. The shock tube Coronal sections of 50 μm thickness were prepared with has a 0.32-m circular diameter and is a 5.94 m-long a Leica VT1000 Vibratome (Vienna, Austria) and stored steel tube divided into a 0.76-m compression chamber in sterile PBS at 4°C. For general histopathology serial separated from a 5.18-m expansion chamber. The com- sections were selected at 500 μm intervals, air-dried and pression and expansion chambers are separated by stained with hematoxylin and eosin (H&E). Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 3 of 15 http://www.actaneurocomms.org/content/1/1/51 Table 1 Experimental design Group Blast condition Time harvested (post-blast) N Blast N control 1x-acute 1x74.5 kPa 24 h 5 5 3x-acute 3x74.5 kPa 24 h 5 5 3x-chronic 3x74.5 kPa 4-10 months 17 14 Immunohistochemical staining was performed on free- For TUNEL (terminal deoxynucleotidyltransferase- floating sections. The primary antibodies used were a mediated dUTP nick-end labeling) staining, sections rabbit polyclonal anti-collagen IV antiserum (1:500; were washed in TBS, permeabilized with 0.1% Triton X- Millipore, Billerica, MA, USA), a rabbit polyclonal anti- 100 in TBS for 1 hr and washed extensively with TBS. ionized calcium-binding adaptor molecule 1 (Iba-1, 1:400; End labeling of DNA with fluorescein-dUTP was Wako, Richmond, VA, USA), a rabbit polyclonal anti- performed using a commercial kit (Roche, Indianapolis, laminin (1:150; Sigma-Aldrich, St. Louis, MO, USA), a IN, USA). After several washes with PBS, the sections rabbit polyclonal antibody against the neurofilament heavy were blocked and stained with a mouse monoclonal subunit (NFH, 1:300, Sigma-Aldrich), a mouse monoclo- anti-α-smooth muscle actin antibody as described above. nal antibody against phosphorylated neurofilaments (SMI31, 1:500, Covance Research Products, Denver, PA, Results USA), a mouse monoclonal antibody against the APP-N Animals groups and study rationale -terminal region (clone 22C11, 1:150, Millipore), a mouse We have been examining the acute and chronic effects monoclonal anti-β-III tubulin (Tuj, 1:500; Covance), a of blast exposure in the rat focusing on conditions that mouse monoclonal anti-2′,3′-cyclic nucleotide 3′- mimic an mTBI exposure [6-8]. A pressure of 74.5 kPa phosphodiesterase (CNPase, 1:200; Millipore), a mouse was chosen based on previous studies suggesting that it monoclonal anti-α-smooth muscle actin (α-SMA, 1:500; best approximates an mTBI exposure [6,8]. Multiple Sigma), a rat monoclonal anti-glial fibrillary acidic protein blast exposures have been common in the conflicts in (GFAP, 1:500, gift of Dr. Robert Lazzarini), a mouse mono- Iraq and Afghanistan and Hoge et al. [2] found that clonal antibody that recognizes tau protein phosphory- more than 50% of soldiers returning from Iraq who lated at Ser202 (CP-13, 1:300, gift of Dr. Peter Davies). reported no injuries still reported at least two episodes Sections were blocked with Tris-buffered saline (TBS; in which an IED exploded near the soldier. This figure 50 mM Tris–HCl, 0.15 M NaCl pH 7.6), and 0.15 M rose to nearly 90% among soldiers who suffered mTBIs. NaCl/0.1% Triton X-100/5% goat serum (TBS-TGS) for We therefore included rats that received three 74.5 kPa 1 hour, and the primary antibody was applied overnight blast exposures administered on consecutive days. in TBS-TGS at room temperature. Following washing During the course of our prior studies brain tissue was in PBS for 1 hour, immunofluorescence staining was collected at times ranging from 4 to 10 months follow- detected by incubation with species-specific AlexaFluor ing blast exposure (3×-chronic exposure). Here we took secondary antibody conjugates (1:300; Molecular advantage of the availability of this tissue to examine Probes, Burlingame CA, USA) for 2 hours in TBS-TGS. the histopathological consequences of blast exposure, Nuclei were counterstained with 1 μg/ml 4′,6-diamidino- supplementing it with tissue from rats that received one 2-phenylindole (DAPI). Immunoperoxidase staining or three blast exposures and were sacrificed at 24 hours for collagen IV was performed on pepsin-digested tis- post-blast (1×− and 3×−acute exposure). Table 1 con- sue as previously described [11] and sections were tains a summary of the animals examined. There was counterstained with 0.5% cresyl violet. Stained sections no mortality in any of the blast-exposed or control were photographed on a Zeiss AxioImager microscope groups. using the AxioVision Release 4.3 software program (Zeiss, Thornwood,NY, USA),aNikonEclipse E400 connected to a DXC-390 CCD camera (Nikon, Melville, Screen for neuropathology NY, USA) or a Zeiss LSM 710 confocal microscope. We performed a general screen for neuropathology on Unstained sections of fixed brain were photographed all the rats listed in Table 1. Following perfusion, on a Nikon SMZ1500 stereomicroscope equipped with brains were cut into 50 μm-thick Vibratome sections an oblique coherent contrast illumination system and and initially imaged by diascopic bright/dark field connected to a SPOT RT digital camera (Sterling microscopy for gross abnormalities. H&E staining was Heights, MI, USA). Digital images were color balanced performedonevery 10th sectionfromeach brain. using Adobe Photoshop 11.0 (Adobe Systems, San Jose, Based on these observations sections were selected for CA, USA). further analysis. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 4 of 15 http://www.actaneurocomms.org/content/1/1/51 Intraventricular hemorrhages are common following blast staining for GFAP or the activated microglia marker Iba- exposure 1. Collagen IV immunostaining revealed no vascular Imaging of sections by diascopic bright/dark field illu- pathology. In contrast to a recent study showing accu- mination revealed the presence of intraventricular mulation of hyperphosphorylated tau in both human hemorrhage in 40% of the brains examined at 24 hours cases and a mouse models of blast associated brain in rats exposed to either single (2/5 animals) or 3 (2/5 injury [13] we did not detect any accumulation of animals) blast exposures (Figure 1). Hemorrhages often hyperphosphorylated tau in blast-exposed animals by appeared associated within the choroid plexus although immunostaining with the antibody CP-13, which recog- blood was frequently evident more widely in the ventri- nizes phosphorylated tau at Ser202. cles. With the exception of one animal, no hemorrhages were detected in the brain parenchyma. Blast induces shear-related injuries in brain Despite the lack of any general histopathology, we ob- Lack of histopathology in blast-exposed brains served focal lesions in many brains. In animals A screen for neuropathology did not reveal any wide- sacrificed more than 4 months after blast exposure we spread histopathologic alterations in H&E-stained sec- identified such lesions in 41% (7/17 brains; see Table 2) tions in either the acute (1×- and 3×-acute) or chronic of blast-exposed animals compared to none in the con- (3×-chronic) groups. Immunohistochemical staining for trols (0/14). An example of such a lesion is shown in neurofilament proteins or β-III tubulin (Tuj) revealed no Figure 2 in a rat that received 3 × 74.5 kPa blast exposures general neuronal pathology. No axonal pathology was and was sacrificed 4 months after the last exposure. The found by immunostaining for the amyloid precursor lesion was first visible when Vibratome sections were protein (APP) whose accumulation in axons is widely examined under diascopic bright/dark field illumination used as a marker of axonal injury in humans and experi- (arrows in Figure 2A; compare to contralateral side). At mental animal models of TBI [12]. TUNEL staining did first glance, the lesion appeared as a discontinuity in the not reveal evidence of generalized apoptosis. CNPase tissue that might initially be thought to be a sectioning immunostaining was performed as a measure of myelin artifact. However, on histological examination it became integrity and was normal. No generalized reactive apparent that the lesion, which resembles a rip or tear, astrocytosis or microglial activation was detected by had produced a discontinuity in the tissue causing Figure 1 Intraventricular hemorrhages following blast exposure. Shown are Vibratome sections imaged by diascopic bright/dark field illumination from 1×-acute blast-exposed animals. Note blood (arrows) in the region of the choroid plexus (A) and dorsal third ventricle (B). Panels (C) and (D) show comparable regions from control brains for (A) and (B). Scale bar: 1 mm. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 5 of 15 http://www.actaneurocomms.org/content/1/1/51 Table 2 Brain pathology associated with experimental mTBI Animal Blast Time Lesions observed ID condition harvested B2 1 x 74.5 24 h Tear causing repositioning of part of the caudate-putamen into the insula. rd B3 1 x 74.5 24 h Blood in dorsal 3 and lateral ventricles as well as choroid plexus. rd B4 1 x 74.5 24 h Blood in dorsal 3 and lateral ventricles. rd B1 3 x 74.5 24 h Blood in dorsal 3 ventricle. B5 3 x 74.5 24 h Blood in aqueduct, dorsal 3rd and lateral ventricles; vascular disruption affecting external capsule and CA1 field; blood in the adjacent parenchymal tissue. 542 3 x 74.5 6 months Tear spanning the perirhinal cortex, external capsule, hippocampal CA1 region and dentate gyrus resulting in tissue architectural abnormalities. 2 3 x 74.5 6 months Lesion in the secondary auditory cortex expanding to the perirhinal region. 1 3 x 74.5 9 months Tear at the surface of the secondary somatosensory cortex extending into the insular cortex; lesion involves repositioning of cortical layers; disruption of piriform cortex by the insertion of olfactory tubercle/lateral olfactory tract tissue. 590 3 x 74.5 10 months Disruption of layers I, II and III in primary visual cortex; presence of ectopic neurons in layer I; lesion of hippocampal CA1. 591 3 x 74.5 10 months Tear and repositioning of layers I, II and III in primary somatosensory cortex; ectopic neurons in layer I; disruption of parietal and somatosensory cortex and ventral/intermediate entorhinal cortex by the insertion of ectopic tissue. 595 3 x 74.5 10 months Lesion in motor cortex altering the cortical architecture. 597 3 x 74.5 10 months Displacement of amygdalohippocampal and posteromedial cortical amygdaloid nucleus tissue disrupting the ventral hippocampal CA1 field. Figure 2 Blast-induced shear-related injury in the brain. Shown are sections from a rat sacrificed four months after receiving a 3 × 74.5 kPa blast exposure. Diascopic bright/dark field images (A) and H&E stained sections (B-C) are shown that demonstrate a discontinuity in the tissue (indicated by arrows in all panels) in contrast to the normal appearance of the contralateral side in panels (A) and (B). Panel (D) shows a reconstructed image of the area around the lesion immunostained with an anti-collagen IV antibody, which stains mainly blood vessels, and is counterstained with cresyl violet. The external capsule (ec) as well as the CA1 pyramidal cell layer and the dentate gyrus (DG) of the hippocampus are indicated. The discontinuity in the hippocampal pyramidal cell layers is indicated by arrows. Chronic disruption of the perirhinal cortex, external capsule and hippocampus are visible. Scale bars: 1 mm, A-B; 150 μm, C; 100 μm, D. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 6 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 3 Gliosis and dendritic changes around blast-induced tear indicate chronicity of the lesion. Shown is a confocal image at the cortical surface adjacent to the tear (asterisk) illustrated in Figure 2 from a rat sacrificed four months after receiving a 3 × 74.5 kPa blast-exposure. The section was stained with antibodies against β-III tubulin (Tuj, A, green), GFAP (B, red), and with a DAPI nuclear stain (C, blue). A merged image is shown in panel (D). Note the attenuation of the Tuj immunostaining of the dendrites in neurons in cortical layers II and III adjacent to the lesion (arrows in A) and the increased GFAP immunostaining adjacent to the lesion (B, arrow). Scale bar: 50 μm. Figure 4 Microglial activation and gliosis adjacent to a chronic blast-induced tear in the hippocampus. Brain sections from blast-exposed (3×−chronic sacrificed at 4 months post-blast) and control animals were immunostained with antibodies against Iba-1 (A and E; microglia, green) and GFAP (B and F; astrocytes, red). The sections were counterstained with DAPI (C and G, blue) and merged images are shown in panels D and H. Upregulation of Iba-1 expression (A) is seen in the surrounding hippocampal tissue (white arrows) although not in the region immediately adjacent to the lesion (indicated by asterisks). Increased GFAP staining (B, arrow) is seen adjacent to the lesion. Scale bar: 100 μm. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 7 of 15 http://www.actaneurocomms.org/content/1/1/51 separation of the layers of the hippocampus proper expanded more than 500 μm in length perpendicular to and dentate gyrus (Figure 2B-C). In fact, as shown in the sectioning plane resulted in disruption of the exter- Figure 2D, the fissure spanned the perirhinal cortex, exter- nal capsule (black arrows in Figure 6B and D). nal capsule (ec), hippocampal CA1 and dentate gyrus Other examples of focal structural alterations are (DG) resulting in a misalignment of the tissue layers. shown in Figures 7, 8 and 9. Figure 7 shows a tear at the Figure 3 shows staining at the cortical surface around surface of the secondary somatosensory cortex in a 3×- the lesion illustrated in Figure 2 with antibodies against chronic exposure rat. The lesion extended through the Tuj and GFAP. In the tissue adjacent to the lesion (as- insular cortex altering the alignment of the cortical terisk in A) thinning of Tuj-immunoreactive dendritic layers of the insula with the affected layers interrupted processes is apparent (arrows in A) and increased GFAP by the tear repositioned in relationship to the outer cor- immunostaining (arrow in B) is seen at the margins of tical layers (Figures 7B-C). Collagen IV immunostaining the lesion indicating that the tear was not an artifact of showed the repositioning of a normal appearing artery sectioning but rather a chronic lesion associated with a and two arterioles derived from the parent vessel into glial reaction and neuronal injury in the adjacent tissue. the brain parenchyma (Figure 7C). Additional collagen Figure 4 shows another focal lesion from a 3×-chronic IV immunostaining around the most superficial portion exposure animal that involved the hippocampus. This of the tear showed that no vessels or vascular remnants section was immunostained with antibodies against Iba- were associated with the tear itself (not shown). At the 1 and GFAP. An increase in GFAP expression was seen ventral surface of the brain the lesion resulted in the in- at the margins of the lesion (arrow in Figure 4B). Figure 5 sertion of a portion of the olfactory tubercle/lateral ol- shows a higher power confocal image of GFAP immuno- factory tract (white arrow in Figure 7D and E) into the staining at the margins of the lesion illustrated in piriform cortex (black arrows; compare to Figure 7G, Figure 4. An increase in GFAP immunostainied astro- showing the same region from a control rat). The mye- cytes is evident, consistent with chronic gliosis. While linated nature of the repositioned tissue was confirmed the region immediately surrounding the lesion was de- by staining for CNPase (white arrow in Figure 7F). void of Iba-1-expressing cells, there was a dramatic in- Disruption of the upper cortical layers I, II, and III in crease in Iba-1 expression in regions adjacent to the the primary visual cortex in a 3×-chronic exposure ani- tissue tear (arrows in Figure 4A) when compared to the mal is illustrated in Figure 8B. In this example two areas low level of Iba-1 immunostaining normally present in are visible where the cortical layers have been disrupted the hippocampus of a control brain (Figure 4E). (compare to a control brain in Figure 8A). In the area Similar lesions could be seen in brains examined on the left of the tear in Figure 8B the cortical layers are acutely that sometimes resulted in unusual tissue reposi- misaligned. In the region on the right of the tear in tioning leading to dramatic alterations of cerebral archi- Figure 8B ectopic cells are visible in layer I including a tecture. One such example is illustrated in Figure 6, variety of spindle-shaped cells (Figure 8C). These cells which shows diascopic bright/dark field images from were identified as neurons based on their immunostain- two Vibratome sections that were 500 μm apart taken ing for NeuN (Figure 8D and H). In another 3×-chronic from a 1×-acute exposure animal. In this example, part exposure animal (Figure 8F, G, J and K) a lesion in the of the more rostral caudate-putamen (white arrows in primary somatosensory cortex produced a tear that Figure 6A-D), was effectively avulsed by the blast and disrupted layers I to III resulting in a portion of layers II repositioned into the insular cortex (compare to panels and III being avulsed relative to layer I (arrows in 6E and F from a control brain). This lesion which Figure 8F, G, J and K; compare to panel E from control Figure 5 Gliosis adjacent to a chronic blast-induced lesion. Shown is confocal imaging of immunostaining for GFAP (A) around the tissue tear illustrated in Figure 4. Sections were counterstained with DAPI (B) and merged images are shown in panel C. Note the concentration of GFAP-immunostained astrocytes around the lesion which is indicated by asterisks. Scale bar: 20 μm. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 8 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 6 Mechanical excision and repositioning of a part of the caudate-putamen by the blast. Shown are diascopic bright/dark field images of Vibratome sections from a 1×–acute exposed rat. Sections in panels A-B are 500 μm apart from those in panels C-D. White arrows indicate a region of the caudate-putamen that was repositioned into the insular cortex. The lesion also disrupted the external capsule (black arrows in B and D). Note that the repositioned tissue appears to have been reoriented by 180 compared to its likely original position. Panels E and F show comparable sections from a control brain. Scale bar: 1 mm, A, C and E; 300 μm, B, D and F. brain). NeuN immunostaining (Figure 8J) demonstrated examined here were part of this behavioral study [8]. the neuronal character of these ectopic cells, which also Due to the heterogeneity in the observed blast-induced included many spindle-shaped neurons. Figures 9B and brain lesions, we did not expect that a common anatom- C show disruption of the CA1 field of the hippocampus ical lesion would be found that could account for the by a tear (compare panel 9B to 9A that shows the same PTSD-like behavioral phenotype. However, some blast- region in a control brain). This tear disrupted the pri- exposed animals demonstrated deficits in specific behav- mary visual cortex and severely damaged the hippocam- ioral tests. For example the rat in Figure 9 with a lesion pal layers (arrow in panel C). of the dorsal hippocampal CA1 exhibited a very unusual response in cued and contextual fear testing. This rat Behavioral alterations associated with blast-related focal failed to freeze following the pairing of the tone and the lesions foot shock in the conditioning phase and showed no We have previously shown that rats exposed to 3 × 74.5 freezing in the contextual phase. Yet it froze normally kPa blast exposures exhibit a variety of post-traumatic in response to the tone in the cued phase of testing stress disorder-related traits [8]. Some of the 3×-chronic (Figure 9D-F). Other examples of behavioral abnormal- exposure animals (6 control and 6 blast-exposed) ities in rats with blast-related focal lesions are shown Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 9 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 7 Blast-induced disruption of the piriform cortex, insular cortex, and secondary somatosensory cortex. Shown are diascopic bright/dark field images (A), H&E-stained (B) and collagen IV-immunostained (C) sections from a 3×-chronic exposure animal that was sacrificed 8.5 months post-blast. A tear (black arrow in panel A) can be seen that begins at the surface of the secondary somatosensory cortex and extends through insular cortex resulting in misalignment of the insular cortical layers. H&E staining (B) of the lesion at the cortical surface shows that layer I (arrow) of the secondary somatosensory cortex has been embedded into layers II and III. In a collagen IV-immunostained section (C) adjacent to that shown in panel B an artery (black arrows) and two arterioles derived from it are visible. H&E staining (D-E) shows interruption of the piriform cortex (black arrows) by the insertion of tissue that appears to have originated in the lateral olfactory tract/olfactory tubercle (white arrow) which is also visible in panel A (white arrow). Immunostaining with an antibody against CNPase (F) confirmed the myelinated nature of the repositioned tissue. Panel G shows the normal appearance of the piriform cortex in an H&E stained section from a control rat. Scale bars: 1 mm, A, D and G; 150 μm, B-C; 500 μm, E-F. in Figures 10 and 11. A rat with a lesion in the motor a vascular origin for some acute lesions, red blood cortex spent less time in the center of an open field cells could be found within some lesions. For example, (Figure 10). Figure 11 shows a lesion that disrupts the Figure 12 shows a lesion involving the auditory cortex in posterior ventral hippocampal CA1 causing an avul- a3×–acute exposure animal. This lesion also disrupts sion of the posteromedial cortical amygdaloid nucleus the external capsule extending into the hippocampus, and posteromedial amygdalohippocampal area. This and red blood cells were visible within the portion pass- animal showed deficits in the Morris water maze a ing through the external capsule and CA1. hippocampus-dependent task of spatial navigation in However, most lesions could not be unequivocally as- contrast to the blast-exposed animals as a group whose sociated with a vascular origin. For example a tear behavior was similar to controls. While it is difficult to resembling a penetrating cortical blood vessel from a with confidence ascribe any of these behavioral deficits 3×-acute exposure rat is shown in Figure 13. While the to the observed lesions, it is clear that some blast- margins of the lesion were lined with apoptotic TUNEL- exposed animals with lesions were severely affected in positive cells, they did not show immunostaining for specific behavioral tests. α-SMA suggesting that there was no vascular remnant and thus that the lesion does not follow a blood vessel. Blast-related rips and tears frequently follow vascular fault lines Discussion The lesions often appeared to follow fault lines that Whether primary blast forces directly damage the brain is seemed to parallel penetrating blood vessels such as still controversial and if they do, the exact mechanisms those illustrated in Figures 2, 4, 5, and 9. Supporting that mediate injury remain unknown [3,4,14]. While it was Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 10 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 8 Ectopic neurons in neocortical layer I of blast-exposed animals. Shown are H&E (B and C) or NeuN (D and H, green) staining of the primary visual cortex from a 3×-chronic exposure animal analyzed 10 months after the blast exposure. H&E staining from the corresponding area in a control rat is shown in panel A. Panel B shows two areas (arrows) where the cortical layers have been disrupted. In the region indicated by the right arrow, ectopic cells are visible in layer I. This region is shown at higher magnification in panel C. The phenotype of the ectopic cells was determined to be neuronal by NeuN immunostaining (D and H, green), combined with DAPI staining (D and I, blue). The cortical layers around the left arrows (B) are also misaligned. The ectopic neurons were repositioned from cortical layers II and III. Note the presence of spindle- shaped cells (black arrows in C) and their NeuN staining shown in D (white arrows). Panels F and G show H&E staining of primary somatosensory cortex from another 3 ×-chronic exposure animal also analyzed at 10 months post blast. Panel G shows the same region at a higher magnification. The blast produced a tear that disrupted layers I to III with the result that a portion of layers II and III (arrow in both panels F and G) was avulsed and repositioned. NeuN (J) and DAPI (K) staining of the region demonstrated the neuronal character of the ectopic cells. The corresponding area from a control rat is shown in panel E. Scale bars: 200 μm, A, B, E and F;50 μm, C and G;10 μm, D; 100 μm, H, I, J and K. once thought that the skull forms a protective barrier related because they result in displacement of adjacent preventing the blast pressure wave from directly damaging tissue planes causing a realignment of the layers that in the brain [15], studies in animal models subsequently some cases led to avulsion and relocation of tissue. Be- showed that the blast pressure wave is transmitted to the cause the lesions are found at 24 hours post-blast expos- brain with little attenuation [5,13,15-23]. ure, they appear to represent acute lesions. With time Here, we analyzed the early (24 hours) and long-term these lesions evolve into chronic lesions that exhibit a (>4 months) pathological effects in the brains of rats ex- glial and microglial reaction as well as a neuronal reac- posed to blast overpressure, using a model that approxi- tion which includes thinning of dendrites in the adjacent mates a mild TBI exposure. The earliest and most tissue. These results are in agreement with a previous common pathological finding at 24 hours post-blast was study reporting that blast exposure in rats induces the presence of blood in the choroid plexus, ventricles microglial activation and hypertrophy in the brain [25]. and cerebral aqueduct, occurring even after a single blast The spindle-shaped neurons with elongated nuclei that exposure. This pathology seems best explained by direct were observed in some lesions have been described in a effects of blast on the choroid plexus leading to vascular previous study in which it was suggested that overpres- rupture and blood leakage into the ventricles. These re- sure shock waves cause the long axis of the neurons to sults are in agreement with a previous study indicating align toward the shock wave source [26]. that the choroid plexus is extremely sensitive to the blast Interestingly, we found that tears often seemed to fol- wave [24]. low penetrating cortical vessels suggesting that blood We did not observe any generalized neuropathology or vessels could represent fault lines along which the blast evidence for diffuse axonal injury as judged by APP im- pressure may propagate. Several mechanisms could be munostaining. We also did not observe accumulation of envisioned as to how this might occur. In what has been hyperphosphorylated tau as has been reported in an- called a thoracic mechanism [3,27], it has been proposed other model of blast TBI [13]. Rather, the most promin- that a high-pressure blast hitting the body can induce ent effects were what we describe as focal rips or tears oscillating high-pressure waves that can be transmitted in the tissue. These lesions seem best described as shear- through the systemic circulation to the brain. Blood Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 11 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 9 Disruption in the hippocampus of the CA1 layer by excised lacunosum-moleculare tissue. Shown are diascopic bright/dark field images from a control (A) and a 3×−chronic exposure rat (B) analyzed at 10 months post blast (animal 590). A tear in the primary visual cortex is indicated by arrows in panels B and C. In panel C, an H&E stained section is shown in which a fragment from the hippocampal lacunosum- moleculare is visible (arrow in panel B) that has been excised and disrupts the hippocampal CA1 field. Panels D-F show the response of this rat which was tested in a previously published behavioral study [8] in a contextual and cued fear paradigm. Methods and original pooled group data were described in Elder et al. [8]. Panel D shows response during the training phase in which rats were exposed to an 80dB tone that was paired with a foot shock. Freezing behavior was measured at baseline, after the tone and after the shock. Panel D shows the test for contextual fear memory performed 24 h after the initial training. Freezing was measured during four minutes in the initial conditioning chamber. Panel F shows the cued fear response performed 24 h after the testing in E. Animals were placed in a novel context and freezing was measured at baseline and after exposure to the conditioned stimulus (80-dB tone). As compared to the control group (n = 13) and the pooled blast exposed group (n = 14), rat 590 failed to freeze following the pairing of the tone and the foot shock in the conditioning phase. It also showed no freezing in the contextual phase but normal freezing in response to the tone in the cued phase. Scale bars: 1 mm, A and B; 200 μm, C. pressure in the systemic circulation has been shown to changes could also help to tease out the relationship be- rise during passage of the blast pressure wave [28-30]. tween systemic and brain factors. Because the arterial capacity to expand in response to A vascular mechanism is supported by the finding in the sudden increase in blood pressure depends in part some instances of microscopic hemorrhages in vessels on the pressure in the surrounding parenchyma, brain within the lesion. In other instances, even when a direct damage might result from pressure differentials between vascular lesion is not visible, it can be speculated that le- the pressure on the arterial walls and that in the neigh- sions followed a penetrating vessel or were the result of boring parenchyma. A lower pressure in the surrounding pressure transmitted through a specific vascular terri- brain would allow the arterial wall to expand as a conse- tory. For example, the lesion in Figure 7 might have quence of a sudden increase in blood pressure leading to arisen from a high-pressure wave transmitted through tissue damage at high/low pressure interphases. This the vessels supplying the piriform cortex and the lateral situation could occur if the blast-induced brain compres- olfactory tract resulting in compression and mechanical sion is not uniform or if the head is partially exposed to disruption of the neighboring tissue and causing a tear the blast creating regions of higher and lower pressures. through which the lateral olfactory tract was avulsed. Di- The contribution of a thoracic mechanism could be dir- lated vessels such as those seen in Figure 9 in the hippo- ectly tested in our model by performing shielding exper- campus could have been responsible for rupturing and iments that limit the blast exposure to either head or displacing neighboring tissue to the more dorsal CA1 re- body. Simultaneous monitoring of blood pressure and gion. However, despite the fact that isolated vascular intracranial pressure with comparisons of the time pathology was observed, few lesions whether acute or course of intracranial pressure and blood pressure chronic showed any evidence of hemorrhage and most Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 12 of 15 http://www.actaneurocomms.org/content/1/1/51 lesions could not be unequivocally associated with a vas- cular origin. In addition, if pressures were transmitted through the vascular bed it is curious that vessels suffi- ciently dilated to produce the type of lesions observed would not result in more cases of obvious hemorrhage. Alternatively, hemorrhages might not occur if the blast pressure were transmitted through the vascular com- partment but not intravascularly. This could occur if the main pressure wave was being transmitted through the Virchow-Robin compartment. Several studies have docu- mented that intracranial pressure increases acutely fol- lowing blast exposure [7,15,20-27,31,32]. Increased CSF pressure transmitted through the Virchow-Robin com- partment could generate local pressure differentials at the interface between the vascular basal lamina and the surrounding tissues. Shearing along this plane would conceptually leave the blood vessel wall intact preventing hemorrhages. Computer modeling has suggested that blast-associated shear strains should be at their highest at the brain/ CSF interface [33] where cavitation effects could occur which have long been speculated as playing a major role in the deleterious effects of blast exposure Figure 10 Reduced center time in open field testing of a rat [31,34]. Another study suggested that highest shear with a blast-induced lesion in the motor cortex. Shown is an strains should occur at the skull/brain interface [32] H&E-stained section (A) from a 3×−chronic exposure rat. An arrow consistent with our observation that lesions are points to a lesion in the motor cortex (compare to the unaffected contralateral side). Panel B shows center time in an open field test found at the cortical surface where the shear wave of this animal (rat 595) compared to blast-exposed and control would move perpendicular to the longitudinal blast groups (error bars indicate S.E.M.). Methods and original pooled wave. Whether propagating as a pressure wave through group data were described in Elder et al. [8]. Scale bar: 200 μm. the ventricular system or generated at the cortical surface such a mechanism could explain expansion of lesions along vascular fault lines without production of hemorrhage. Figure 11 Association of disruption of the caudoventral hippocampal CA1 field with deficits in spatial learning. Panel A shows an H&E stained section from a 3×−chronic exposure rat with a blast-induced lesion in which a portion of the posteromedial cortical amygdaloid nucleus and posteromedial amygdalohippocampal area has been repositioned into the hippocampal CA1 region. Panel B shows performance of this rat (597) in a Morris water maze revealing spatial learning impairments compared to the pooled blast-exposed and control groups (± S.E.M.). Methods and original pooled group data were described in Elder et al. [8]. Scale bar: 200 μm. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 13 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 12 Blast-induced lesion that follows a vascular fault line and is associated with microscopic evidence of hemorrhage. Shown are H&E-stained sections (A-C) from the brain of a 3×−acute exposure animal. A blast-induced discontinuity in the cortical layers is apparent at the junction between layers I and II that follows the fault line of a penetrating cortical vessel (arrows) in the auditory cortex (A) disrupting the external capsule and extending into the hippocampus (B). An area of hemorrhage with parenchymal infiltration of erythrocytes (arrows) is visible in panel (C). Disruption of myelinated fibers in the external capsule was confirmed by immunohistochemical staining with an anti-CNPase antibody (D). Scale bar: 200 μm, A, B; 100 μm, C; 400 μm, D. Figure 13 Apoptotic cells line the margins of a blast-induced cortical tear without evidence of a vascular remnant. A section from a 3×−acute exposure rat was labeled for TUNEL (A), immunostained for α-smooth muscle actin (B, α-SMA) and counterstained with DAPI (C). Merged images are shown in panel (D). A tear lined by apoptotic cells that resembles a vessel is visible in panel (A). However while TUNEL- positive cells (arrows in panel A) are seen at the margins of the tear, no staining for α-SMA is apparent at the edges of the lesion (arrows in panel B). Scale bar: 200 μm. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 14 of 15 http://www.actaneurocomms.org/content/1/1/51 Conclusions Author details Department of Veterans Affairs Medical Center, General Medical Research Here we used the rat to model mTBI resulting from Service, Bronx, New York, USA. Department of Veterans Affairs Medical blast overpressure exposure. We describe a new type of Center, Research and Development Service, Bronx, New York, USA. shear injury in the brain that has not been described in Department of Veterans Affairs Medical Center, Neurology Service, Bronx, New York, USA. Department of Psychiatry, Icahn School of Medicine at non-blast TBI models and appears to be unique to Mount Sinai, New York, New York, USA. Fishberg Department of blast-associated brain injury. Why they occur in such a Neuroscience, Icahn School of Medicine at Mount Sinai, New York, New York, focal fashion when the entire brain is presumably USA. Department of Geriatrics and Palliative Care, Icahn School of Medicine at Mount Sinai, New York, New York, USA. Department of Neurology, Icahn subjected to the same blast exposure remains unclear. School of Medicine at Mount Sinai, New York, New York, USA. Friedman Yet, the fact that lesions often follow penetrating cor- Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New tical vessels suggests that blood vessels may represent York, USA. Operational and Undersea Medicine Directorate, Naval Medical Research Center, Silver Spring, Maryland, USA. the fault lines along which the most damaging blast pressure is transmitted. Received: 2 July 2013 Accepted: 6 August 2013 Functionally, the effects of these lesions remain specu- Published: 14 August 2013 lative. In prior studies we have shown that these animals exhibit chronic behavioral and biochemical changes References [7,8]. Focal lesions might be responsible for focal dys- 1. Elder GA, Mitsis EM, Ahlers ST, Cristian A: Blast-induced mild traumatic brain injury. Psychiatr Clin North Am 2010, 33(4):757–781. function in some animals. However, given the low occur- 2. Hoge CW, McGurk D, Thomas JL, Cox AL, Engel CC, Castro CA: Mild rence of the lesions in a given animal, it is unclear traumatic brain injury in U S Soldiers returning from Iraq. N Engl J Med whether they can explain the full behavioral and bio- 2008, 358(5):453–463. 3. Cernak I, Noble-Haeusslein LJ: Traumatic brain injury: an overview of chemical phenotype. However, the dramatic nature of pathobiology with emphasis on military populations. J Cereb Blood Flow the lesions, from a neuropathological point of view, sug- Metab 2010, 30(2):255–266. gests that locally constrained but significant parenchy- 4. Leung LY, VandeVord PJ, Dal Cengio AL, Bir C, Yang KH, King AI: Blast mal stress and damage may result in more widespread related neurotrauma: a review of cellular injury. Mol Cell Biomech 2008, 5(3):155–168. functional consequences in our model. 5. Chavko M, Koller WA, Prusaczyk WK, McCarron RM: Measurement of blast wave by a miniature fiber optic pressure transducer in the rat brain. Abbreviations J Neurosci Methods 2007, 159(2):277–281. APP: Amyloid precursor protein; CNPase: 2′,3′-cyclic-nucleotide 3′- 6. Ahlers ST, Vasserman-Stokes E, Shaughness MC, Hall AA, Shear DA, Chavko phosphodiesterase; DAPI: 4′,6-diamidino-2-phenylindole; GFAP: Glial fibrillary M, McCarron RM, Stone JR: Assessment of the effects of acute and acidic protein; Iba-1: Ionized calcium-binding adapter molecule 1; repeated exposure to blast overpressure in rodents: toward a greater IED: Improvised explosive devices; kPa: kilopascal; mTBI: mild TBI; understanding of blast and the potential ramifications for injury in PBS: Phosphate-buffered saline; PTSD: Post-traumatic stress disorder; humans exposed to blast. Front Neurol 2012, 3:32. SMA: Smooth muscle actin; TBI: Traumatic brain injury; TBS: Tris-buffered 7. De Gasperi R, Gama Sosa MA, Kim SH, Steele JW, Shaughness MC, Maudlin- saline; TUNEL: Terminal deoxynucleotidyltransferase-mediated dUTP, nick-end Jeronimo E, Hall AA, Dekosky ST, McCarron RM, Nambiar MP, et al: Acute labeling. blast injury reduces brain abeta in two rodent species. Front Neurol 2012, 3:177. 8. Elder GA, Dorr NP, De Gasperi R, Gama Sosa MA, Shaughness MC, Maudlin- Competing interests Jeronimo E, Hall AA, McCarron RM, Ahlers ST: Blast exposure induces post- The authors declare that they have no competing interests. traumatic stress disorder-related traits in a rat model of mild traumatic brain injury. J Neurotrauma 2012, 29(16):2564–2575. Authors’ contributions 9. Chavko M, Prusaczyk WK, McCarron RM: Lung injury and recovery after MAGS: design of the experiments, analysis and interpretation of the data, exposure to blast overpressure. J Trauma 2006, 61(4):933–942. execution of neuropathological characterization and manuscript writing; 10. Yarnell AM, Shaughness MC, Barry ES, Ahlers ST, McCarron RM, Grunberg NE: RDG: design of the experiments, analysis of the data, execution of Blast traumatic brain injury in the rat using a blast overpressure model. neuropathological characterization and manuscript writing; AJP and PEP: Curr Protoc Neurosci 2013, Chapter 9:Unit 9 41. execution of neuropathological characterization; MCS: execution of blast 11. Gama Sosa MA, Gasperi RD, Rocher AB, Wang AC, Janssen WG, Flores T, exposure experiments; EMJ and AAH: execution of blast exposure Perez GM, Schmeidler J, Dickstein DL, Hof PR, et al: Age-related vascular experiments; WGMJ confocal imaging; FJY: neuropathological pathology in transgenic mice expressing presenilin 1-associated familial characterization; DLD: neuropathological characterization; NPD: behavioral Alzheimer’s disease mutations. Am J Pathol 2010, 176(1):353–368. testing; RMC: design of blast experiments; MC: design of blast experiments; 12. Stone JR, Singleton RH, Povlishock JT: Antibodies to the C-terminus of the PRH: important intellectual content to the experimental design, data analysis beta-amyloid precursor protein (APP): a site specific marker for the and interpretation and manuscript writing; STA: design of blast experiments, detection of traumatic axonal injury. Brain Res 2000, 871(2):288–302. interpretation of data and manuscript writing; GAE: design of the study, 13. Goldstein LE, Fisher AM, Tagge CA, Zhang XL, Velisek L, Sullivan JA, Upreti C, analysis and interpretation of the data, manuscript writing; All authors read Kracht JM, Ericsson M, Wojnarowicz MW, et al: Chronic traumatic and approved the final manuscript. encephalopathy in blast-exposed military veterans and a blast neurotrauma mouse model. Sci Transl Med 2012, 4(134):134–ra160. 14. Cernak I: The importance of systemic response in the pathobiology of Acknowledgements blast-induced neurotrauma. Front Neurol 2010, 1:151. We thank Bridget Wicinski for expert technical assistance. We thank Drs. 15. Clemedson CJ: Blast injury. Physiol Rev 1956, 36(3):336–354. Peter Davies, and Robert Lazzarini for gifts of antibodies. This work was supported by grant 1I01RX000179-01 from the Department of Veterans 16. Bauman RA, Ling G, Tong L, Januszkiewicz A, Agoston D, Delanerolle N, Kim Affairs. MAGS is supported by the General Medical Research Service, James J. Y, Ritzel D, Bell R, Ecklund J, et al: An introductory characterization of a Peters VA Medical Center. The views expressed in this article are those of the combat-casualty-care relevant swine model of closed head injury authors and do not necessarily reflect the official policy or position of the resulting from exposure to explosive blast. J Neurotrauma 2009, Department of the Navy, Department of Defense nor the U.S. Government. 26(6):841–860. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 15 of 15 http://www.actaneurocomms.org/content/1/1/51 17. Bolander R, Mathie B, Bir C, Ritzel D, VandeVord P: Skull flexure as a contributing factor in the mechanism of injury in the rat when exposed to a shock wave. Ann Biomed Eng 2011, 39(10):2550–2559. 18. Dal Cengio Leonardi A, Keane NJ, Bir CA, Ryan AG, Xu L, Vandevord PJ: Head orientation affects the intracranial pressure response resulting from shock wave loading in the rat. J Biomech 2012, 45(15):2595–2602. 19. Leonardi AD, Bir CA, Ritzel DV, VandeVord PJ: Intracranial pressure increases during exposure to a shock wave. J Neurotrauma 2011, 28(1):85–94. 20. Romba JJ, Martin P: The propagation of air shock waves on a biophysical model.In echnical Memorandum 17-61. Aberdeen Proving Ground, Maryland: US Army Ordinance, Human Engineering Laboratories; 1961:1–36. 21. 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Kato K, Fujimura M, Nakagawa A, Saito A, Ohki T, Takayama K, Tominaga T: Pressure-dependent effect of shock waves on rat brain: induction of neuronal apoptosis mediated by a caspase-dependent pathway. J Neurosurg 2007, 106(4):667–676. 27. Courtney AC, Courtney MW: A thoracic mechanism of mild traumatic brain injury due to blast pressure waves. Med Hypotheses 2009, 72(1):76–83. 28. Assari S, Laksari K, Barbe M, Darvish K: Cerebral blood pressure rise during blast exposure in a rat model of blast-induced traumatic brain injury, 9th annual Injury Biomechanics Symposium. Columbus, Ohio; 2013. 29. Chavko M, Watanabe T, Adeeb S, Lankasky J, Ahlers S, McCarron S: Transfer of the pressure wave through the body and its impact on the brain, NATO Symposium on a Survey of blast Injury across a Full Landscape of Military Science: 2011. Halifax: NS; 2011. 30. Chen Y, Huang W: Non-impact, blast-induced mild TBI and PTSD: concepts and caveats. Brain Inj 2011, 25(7–8):641–650. 31. 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Biomech Model Mechanobiol 2012, 12(3):511–531. doi:10.1186/2051-5960-1-51 Submit your next manuscript to BioMed Central Cite this article as: Gama Sosa et al.: Blast overpressure induces shear- and take full advantage of: related injuries in the brain of rats exposed to a mild traumatic brain injury. Acta Neuropathologica Communications 2013 1:51. • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Neuropathologica Communications Springer Journals

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Abstract

Background: Blast-related traumatic brain injury (TBI) has been a significant cause of injury in the military operations of Iraq and Afghanistan, affecting as many as 10-20% of returning veterans. However, how blast waves affect the brain is poorly understood. To understand their effects, we analyzed the brains of rats exposed to single or multiple (three) 74.5 kPa blast exposures, conditions that mimic a mild TBI. Results: Rats were sacrificed 24 hours or between 4 and 10 months after exposure. Intraventricular hemorrhages were commonly observed after 24 hrs. A screen for neuropathology did not reveal any generalized histopathology. However, focal lesions resembling rips or tears in the tissue were found in many brains. These lesions disrupted cortical organization resulting in some cases in unusual tissue realignments. The lesions frequently appeared to follow the lines of penetrating cortical vessels and microhemorrhages were found within some but not most acute lesions. Conclusions: These lesions likely represent a type of shear injury that is unique to blast trauma. The observation that lesions often appeared to follow penetrating cortical vessels suggests a vascular mechanism of injury and that blood vessels may represent the fault lines along which the most damaging effect of the blast pressure is transmitted. Keywords: Blast overpressure injury, Neuropathology, Shear injury, Traumatic brain injury Background impairment, post-traumatic stress disorder (PTSD) and Traumatic brain injury (TBI) has been a common cause depression [1]. Blast injuries occur through multiple of mortality and morbidity in the military operations in mechanisms that may be related to effects of the primary Iraq and Afghanistan [1]. It is estimated that 10-20% of blast wave, to injuries associated with objects including returning veterans have suffered a TBI [1]. Due to the shrapnel contained within the IED being propelled by prominent use of improvised explosive devices (IED) in the blast wind, or by the individual being knocked down Iraq and Afghanistan, a characteristic feature of TBI in or thrown into solid objects [3]. these conflicts has been its association with blast expos- How the primary blast wave itself affects the brain is ure [2]. Single or multiple blast exposures have been not well understood [3]. Direct tissue damage, bleeding, commonly seen in association with chronic neurological and diffuse axonal injury (DAI) are the best known and psychiatric sequelae including persistent cognitive pathophysiological mechanisms associated with the type of non-blast TBI most commonly encountered * Correspondence: miguel.gama-sosa@mssm.edu during blunt impact injuries in civilian life [4,5]. Blast- Department of Veterans Affairs Medical Center, General Medical Research associated moderate-to-severe TBIs likely result from Service, Bronx, New York, USA Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, New York, USA Full list of author information is available at the end of the article © 2013 Gama Sosa et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 2 of 15 http://www.actaneurocomms.org/content/1/1/51 mechanisms in part similar to those found in non-blast polyethylene Mylar™ sheets (Du Pont, Wilmington, DE, TBI. Thedegreetowhich theprimary blastwavein- USA) that control the peak pressure generated [5,6,10]. jures the brain remains controversial [3,4]. The peak pressure at the end of the expansion chamber Whereas most attention in the Iraq and Afghanistan is determined by piezoresistive gauges specifically conflicts initially focused on the moderate-to-severe end designed for pressure–time (impulse) measurements of the TBI spectrum, the type of injuries that would be (Model 102M152, PCB, Piezotronics, Depew, NY, USA). recognized in the field, it soon became apparent that This apparatus has been used in several studies to de- mild TBIs (mTBI) were much more common and were liver blast overpressure injury to rats [5-9]. Individual frequently not being recognized at the time of the initial rats were anesthetized using an isoflurane anesthesia injury [1]. We had previously established conditions that system consisting of a vaporizer, gas lines and valves, approximate mTBI exposures experimentally. These and an activated charcoal scavenging system adapted for studies found that exposures up to 74.5 kPa, while use with rodents. Rats were placed into a polycarbonate representing a blast level that is transmitted to the brain induction chamber, which was closed and immediately [5], led to no persistent neurological impairments or flushed with a 5% isoflurane mixture in air for 2 mi- lung damage [6], although animals subjected to repeti- nutes. Rats were placed into a cone-shaped plastic re- tive blast exposure, which has been common in the straint device and then placed in the shock tube. current conflicts [2], exhibited a variety of chronic be- Movement was further restricted during the blast expos- havioral and biochemical changes [7,8]. In contrast, ani- ure using a 1.5-cm diameter flattened rubber tourniquet mals exposed to 116.7 kPa blast exposures frequently tubing. Three tourniquets were spaced evenly to secure had gross cerebral and subdural hemorrhages as well as the head region and the upper and lower torso while the contusions and significant lung pathology [5,6,9], fea- animal was in the plastic restraint cone. The end of each tures that are not consistent with mTBI. tubing was threaded through a toggle and run outside of In the present study we explored the pathological ef- the exposure cage where it was tied to affix the animal fects of blast overpressure shock waves in rats exposed firmly and prevent movement during the blast over- to 74.5 kPa blast exposures. We describe a type of shear pressure exposure without restricting breathing. Rats injury in the brain that has not been described in non- were randomly assigned to sham or blast conditions blast TBI models and appears to be unique to blast- with the head facing the blast exposure without any associated brain injury. body shielding resulting in a full body exposure to the blast wave. Further details of the physical characteris- Methods tics of the blast wave have been described elsewhere Animals [6]. Blast-exposed animals received one or three 74.5 All studies were approved by the Institutional Animal kPa exposures. Animals that received three exposures Care and Use Committees of the Naval Medical Research received one exposure per day for three consecutive Center and the James J. Peters VA Medical Center. Two- days. Except for blast exposure, controls were treated month-old male Long Evans Hooded rats (250-350 g; identically receiving anesthesia and being placed in Charles River Laboratories International, Wilmington, the blast tube. For long term studies (four months MA, USA) were used. Animals were housed at a constant and over) the animals were transferred to the James J. 22°C temperature in rooms on a 12:12 hour light cycle Peters VA Medical Center within 10 days of blast ex- with lights on at 7 AM. All animals were individually posure. The number of animals examined under each housed in standard clear plastic cages equipped with Bed- condition is shown in Table 1. O’Cobs laboratory animal bedding (The Andersons, Mau- mee, OH, USA) and EnviroDri nesting paper (Sheppard Histopathological and immunohistochemical analyses Specialty Papers, Milford, NJ, USA). Access to food and Animals were anesthetized with ketamine (65 mg/kg)/ water was ad libitum. xylazine (13 mg/kg)/acepromazine (2 mg/kg) and transcardially perfused with cold 4% paraformaldehyde Blast overpressure exposure in phosphate-buffered saline (PBS). Brains were removed Rats were subjected to overpressure exposure using the and postfixed overnight in the same fixative. To exclude Walter Reed Army Institute of Research (WRAIR) shock perfusion artifacts in some cases the brain was dissected tube that simulates the effects of air blast exposure and immersion fixed in 4% paraformaldehyde in PBS. under experimental conditions [10]. The shock tube Coronal sections of 50 μm thickness were prepared with has a 0.32-m circular diameter and is a 5.94 m-long a Leica VT1000 Vibratome (Vienna, Austria) and stored steel tube divided into a 0.76-m compression chamber in sterile PBS at 4°C. For general histopathology serial separated from a 5.18-m expansion chamber. The com- sections were selected at 500 μm intervals, air-dried and pression and expansion chambers are separated by stained with hematoxylin and eosin (H&E). Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 3 of 15 http://www.actaneurocomms.org/content/1/1/51 Table 1 Experimental design Group Blast condition Time harvested (post-blast) N Blast N control 1x-acute 1x74.5 kPa 24 h 5 5 3x-acute 3x74.5 kPa 24 h 5 5 3x-chronic 3x74.5 kPa 4-10 months 17 14 Immunohistochemical staining was performed on free- For TUNEL (terminal deoxynucleotidyltransferase- floating sections. The primary antibodies used were a mediated dUTP nick-end labeling) staining, sections rabbit polyclonal anti-collagen IV antiserum (1:500; were washed in TBS, permeabilized with 0.1% Triton X- Millipore, Billerica, MA, USA), a rabbit polyclonal anti- 100 in TBS for 1 hr and washed extensively with TBS. ionized calcium-binding adaptor molecule 1 (Iba-1, 1:400; End labeling of DNA with fluorescein-dUTP was Wako, Richmond, VA, USA), a rabbit polyclonal anti- performed using a commercial kit (Roche, Indianapolis, laminin (1:150; Sigma-Aldrich, St. Louis, MO, USA), a IN, USA). After several washes with PBS, the sections rabbit polyclonal antibody against the neurofilament heavy were blocked and stained with a mouse monoclonal subunit (NFH, 1:300, Sigma-Aldrich), a mouse monoclo- anti-α-smooth muscle actin antibody as described above. nal antibody against phosphorylated neurofilaments (SMI31, 1:500, Covance Research Products, Denver, PA, Results USA), a mouse monoclonal antibody against the APP-N Animals groups and study rationale -terminal region (clone 22C11, 1:150, Millipore), a mouse We have been examining the acute and chronic effects monoclonal anti-β-III tubulin (Tuj, 1:500; Covance), a of blast exposure in the rat focusing on conditions that mouse monoclonal anti-2′,3′-cyclic nucleotide 3′- mimic an mTBI exposure [6-8]. A pressure of 74.5 kPa phosphodiesterase (CNPase, 1:200; Millipore), a mouse was chosen based on previous studies suggesting that it monoclonal anti-α-smooth muscle actin (α-SMA, 1:500; best approximates an mTBI exposure [6,8]. Multiple Sigma), a rat monoclonal anti-glial fibrillary acidic protein blast exposures have been common in the conflicts in (GFAP, 1:500, gift of Dr. Robert Lazzarini), a mouse mono- Iraq and Afghanistan and Hoge et al. [2] found that clonal antibody that recognizes tau protein phosphory- more than 50% of soldiers returning from Iraq who lated at Ser202 (CP-13, 1:300, gift of Dr. Peter Davies). reported no injuries still reported at least two episodes Sections were blocked with Tris-buffered saline (TBS; in which an IED exploded near the soldier. This figure 50 mM Tris–HCl, 0.15 M NaCl pH 7.6), and 0.15 M rose to nearly 90% among soldiers who suffered mTBIs. NaCl/0.1% Triton X-100/5% goat serum (TBS-TGS) for We therefore included rats that received three 74.5 kPa 1 hour, and the primary antibody was applied overnight blast exposures administered on consecutive days. in TBS-TGS at room temperature. Following washing During the course of our prior studies brain tissue was in PBS for 1 hour, immunofluorescence staining was collected at times ranging from 4 to 10 months follow- detected by incubation with species-specific AlexaFluor ing blast exposure (3×-chronic exposure). Here we took secondary antibody conjugates (1:300; Molecular advantage of the availability of this tissue to examine Probes, Burlingame CA, USA) for 2 hours in TBS-TGS. the histopathological consequences of blast exposure, Nuclei were counterstained with 1 μg/ml 4′,6-diamidino- supplementing it with tissue from rats that received one 2-phenylindole (DAPI). Immunoperoxidase staining or three blast exposures and were sacrificed at 24 hours for collagen IV was performed on pepsin-digested tis- post-blast (1×− and 3×−acute exposure). Table 1 con- sue as previously described [11] and sections were tains a summary of the animals examined. There was counterstained with 0.5% cresyl violet. Stained sections no mortality in any of the blast-exposed or control were photographed on a Zeiss AxioImager microscope groups. using the AxioVision Release 4.3 software program (Zeiss, Thornwood,NY, USA),aNikonEclipse E400 connected to a DXC-390 CCD camera (Nikon, Melville, Screen for neuropathology NY, USA) or a Zeiss LSM 710 confocal microscope. We performed a general screen for neuropathology on Unstained sections of fixed brain were photographed all the rats listed in Table 1. Following perfusion, on a Nikon SMZ1500 stereomicroscope equipped with brains were cut into 50 μm-thick Vibratome sections an oblique coherent contrast illumination system and and initially imaged by diascopic bright/dark field connected to a SPOT RT digital camera (Sterling microscopy for gross abnormalities. H&E staining was Heights, MI, USA). Digital images were color balanced performedonevery 10th sectionfromeach brain. using Adobe Photoshop 11.0 (Adobe Systems, San Jose, Based on these observations sections were selected for CA, USA). further analysis. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 4 of 15 http://www.actaneurocomms.org/content/1/1/51 Intraventricular hemorrhages are common following blast staining for GFAP or the activated microglia marker Iba- exposure 1. Collagen IV immunostaining revealed no vascular Imaging of sections by diascopic bright/dark field illu- pathology. In contrast to a recent study showing accu- mination revealed the presence of intraventricular mulation of hyperphosphorylated tau in both human hemorrhage in 40% of the brains examined at 24 hours cases and a mouse models of blast associated brain in rats exposed to either single (2/5 animals) or 3 (2/5 injury [13] we did not detect any accumulation of animals) blast exposures (Figure 1). Hemorrhages often hyperphosphorylated tau in blast-exposed animals by appeared associated within the choroid plexus although immunostaining with the antibody CP-13, which recog- blood was frequently evident more widely in the ventri- nizes phosphorylated tau at Ser202. cles. With the exception of one animal, no hemorrhages were detected in the brain parenchyma. Blast induces shear-related injuries in brain Despite the lack of any general histopathology, we ob- Lack of histopathology in blast-exposed brains served focal lesions in many brains. In animals A screen for neuropathology did not reveal any wide- sacrificed more than 4 months after blast exposure we spread histopathologic alterations in H&E-stained sec- identified such lesions in 41% (7/17 brains; see Table 2) tions in either the acute (1×- and 3×-acute) or chronic of blast-exposed animals compared to none in the con- (3×-chronic) groups. Immunohistochemical staining for trols (0/14). An example of such a lesion is shown in neurofilament proteins or β-III tubulin (Tuj) revealed no Figure 2 in a rat that received 3 × 74.5 kPa blast exposures general neuronal pathology. No axonal pathology was and was sacrificed 4 months after the last exposure. The found by immunostaining for the amyloid precursor lesion was first visible when Vibratome sections were protein (APP) whose accumulation in axons is widely examined under diascopic bright/dark field illumination used as a marker of axonal injury in humans and experi- (arrows in Figure 2A; compare to contralateral side). At mental animal models of TBI [12]. TUNEL staining did first glance, the lesion appeared as a discontinuity in the not reveal evidence of generalized apoptosis. CNPase tissue that might initially be thought to be a sectioning immunostaining was performed as a measure of myelin artifact. However, on histological examination it became integrity and was normal. No generalized reactive apparent that the lesion, which resembles a rip or tear, astrocytosis or microglial activation was detected by had produced a discontinuity in the tissue causing Figure 1 Intraventricular hemorrhages following blast exposure. Shown are Vibratome sections imaged by diascopic bright/dark field illumination from 1×-acute blast-exposed animals. Note blood (arrows) in the region of the choroid plexus (A) and dorsal third ventricle (B). Panels (C) and (D) show comparable regions from control brains for (A) and (B). Scale bar: 1 mm. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 5 of 15 http://www.actaneurocomms.org/content/1/1/51 Table 2 Brain pathology associated with experimental mTBI Animal Blast Time Lesions observed ID condition harvested B2 1 x 74.5 24 h Tear causing repositioning of part of the caudate-putamen into the insula. rd B3 1 x 74.5 24 h Blood in dorsal 3 and lateral ventricles as well as choroid plexus. rd B4 1 x 74.5 24 h Blood in dorsal 3 and lateral ventricles. rd B1 3 x 74.5 24 h Blood in dorsal 3 ventricle. B5 3 x 74.5 24 h Blood in aqueduct, dorsal 3rd and lateral ventricles; vascular disruption affecting external capsule and CA1 field; blood in the adjacent parenchymal tissue. 542 3 x 74.5 6 months Tear spanning the perirhinal cortex, external capsule, hippocampal CA1 region and dentate gyrus resulting in tissue architectural abnormalities. 2 3 x 74.5 6 months Lesion in the secondary auditory cortex expanding to the perirhinal region. 1 3 x 74.5 9 months Tear at the surface of the secondary somatosensory cortex extending into the insular cortex; lesion involves repositioning of cortical layers; disruption of piriform cortex by the insertion of olfactory tubercle/lateral olfactory tract tissue. 590 3 x 74.5 10 months Disruption of layers I, II and III in primary visual cortex; presence of ectopic neurons in layer I; lesion of hippocampal CA1. 591 3 x 74.5 10 months Tear and repositioning of layers I, II and III in primary somatosensory cortex; ectopic neurons in layer I; disruption of parietal and somatosensory cortex and ventral/intermediate entorhinal cortex by the insertion of ectopic tissue. 595 3 x 74.5 10 months Lesion in motor cortex altering the cortical architecture. 597 3 x 74.5 10 months Displacement of amygdalohippocampal and posteromedial cortical amygdaloid nucleus tissue disrupting the ventral hippocampal CA1 field. Figure 2 Blast-induced shear-related injury in the brain. Shown are sections from a rat sacrificed four months after receiving a 3 × 74.5 kPa blast exposure. Diascopic bright/dark field images (A) and H&E stained sections (B-C) are shown that demonstrate a discontinuity in the tissue (indicated by arrows in all panels) in contrast to the normal appearance of the contralateral side in panels (A) and (B). Panel (D) shows a reconstructed image of the area around the lesion immunostained with an anti-collagen IV antibody, which stains mainly blood vessels, and is counterstained with cresyl violet. The external capsule (ec) as well as the CA1 pyramidal cell layer and the dentate gyrus (DG) of the hippocampus are indicated. The discontinuity in the hippocampal pyramidal cell layers is indicated by arrows. Chronic disruption of the perirhinal cortex, external capsule and hippocampus are visible. Scale bars: 1 mm, A-B; 150 μm, C; 100 μm, D. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 6 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 3 Gliosis and dendritic changes around blast-induced tear indicate chronicity of the lesion. Shown is a confocal image at the cortical surface adjacent to the tear (asterisk) illustrated in Figure 2 from a rat sacrificed four months after receiving a 3 × 74.5 kPa blast-exposure. The section was stained with antibodies against β-III tubulin (Tuj, A, green), GFAP (B, red), and with a DAPI nuclear stain (C, blue). A merged image is shown in panel (D). Note the attenuation of the Tuj immunostaining of the dendrites in neurons in cortical layers II and III adjacent to the lesion (arrows in A) and the increased GFAP immunostaining adjacent to the lesion (B, arrow). Scale bar: 50 μm. Figure 4 Microglial activation and gliosis adjacent to a chronic blast-induced tear in the hippocampus. Brain sections from blast-exposed (3×−chronic sacrificed at 4 months post-blast) and control animals were immunostained with antibodies against Iba-1 (A and E; microglia, green) and GFAP (B and F; astrocytes, red). The sections were counterstained with DAPI (C and G, blue) and merged images are shown in panels D and H. Upregulation of Iba-1 expression (A) is seen in the surrounding hippocampal tissue (white arrows) although not in the region immediately adjacent to the lesion (indicated by asterisks). Increased GFAP staining (B, arrow) is seen adjacent to the lesion. Scale bar: 100 μm. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 7 of 15 http://www.actaneurocomms.org/content/1/1/51 separation of the layers of the hippocampus proper expanded more than 500 μm in length perpendicular to and dentate gyrus (Figure 2B-C). In fact, as shown in the sectioning plane resulted in disruption of the exter- Figure 2D, the fissure spanned the perirhinal cortex, exter- nal capsule (black arrows in Figure 6B and D). nal capsule (ec), hippocampal CA1 and dentate gyrus Other examples of focal structural alterations are (DG) resulting in a misalignment of the tissue layers. shown in Figures 7, 8 and 9. Figure 7 shows a tear at the Figure 3 shows staining at the cortical surface around surface of the secondary somatosensory cortex in a 3×- the lesion illustrated in Figure 2 with antibodies against chronic exposure rat. The lesion extended through the Tuj and GFAP. In the tissue adjacent to the lesion (as- insular cortex altering the alignment of the cortical terisk in A) thinning of Tuj-immunoreactive dendritic layers of the insula with the affected layers interrupted processes is apparent (arrows in A) and increased GFAP by the tear repositioned in relationship to the outer cor- immunostaining (arrow in B) is seen at the margins of tical layers (Figures 7B-C). Collagen IV immunostaining the lesion indicating that the tear was not an artifact of showed the repositioning of a normal appearing artery sectioning but rather a chronic lesion associated with a and two arterioles derived from the parent vessel into glial reaction and neuronal injury in the adjacent tissue. the brain parenchyma (Figure 7C). Additional collagen Figure 4 shows another focal lesion from a 3×-chronic IV immunostaining around the most superficial portion exposure animal that involved the hippocampus. This of the tear showed that no vessels or vascular remnants section was immunostained with antibodies against Iba- were associated with the tear itself (not shown). At the 1 and GFAP. An increase in GFAP expression was seen ventral surface of the brain the lesion resulted in the in- at the margins of the lesion (arrow in Figure 4B). Figure 5 sertion of a portion of the olfactory tubercle/lateral ol- shows a higher power confocal image of GFAP immuno- factory tract (white arrow in Figure 7D and E) into the staining at the margins of the lesion illustrated in piriform cortex (black arrows; compare to Figure 7G, Figure 4. An increase in GFAP immunostainied astro- showing the same region from a control rat). The mye- cytes is evident, consistent with chronic gliosis. While linated nature of the repositioned tissue was confirmed the region immediately surrounding the lesion was de- by staining for CNPase (white arrow in Figure 7F). void of Iba-1-expressing cells, there was a dramatic in- Disruption of the upper cortical layers I, II, and III in crease in Iba-1 expression in regions adjacent to the the primary visual cortex in a 3×-chronic exposure ani- tissue tear (arrows in Figure 4A) when compared to the mal is illustrated in Figure 8B. In this example two areas low level of Iba-1 immunostaining normally present in are visible where the cortical layers have been disrupted the hippocampus of a control brain (Figure 4E). (compare to a control brain in Figure 8A). In the area Similar lesions could be seen in brains examined on the left of the tear in Figure 8B the cortical layers are acutely that sometimes resulted in unusual tissue reposi- misaligned. In the region on the right of the tear in tioning leading to dramatic alterations of cerebral archi- Figure 8B ectopic cells are visible in layer I including a tecture. One such example is illustrated in Figure 6, variety of spindle-shaped cells (Figure 8C). These cells which shows diascopic bright/dark field images from were identified as neurons based on their immunostain- two Vibratome sections that were 500 μm apart taken ing for NeuN (Figure 8D and H). In another 3×-chronic from a 1×-acute exposure animal. In this example, part exposure animal (Figure 8F, G, J and K) a lesion in the of the more rostral caudate-putamen (white arrows in primary somatosensory cortex produced a tear that Figure 6A-D), was effectively avulsed by the blast and disrupted layers I to III resulting in a portion of layers II repositioned into the insular cortex (compare to panels and III being avulsed relative to layer I (arrows in 6E and F from a control brain). This lesion which Figure 8F, G, J and K; compare to panel E from control Figure 5 Gliosis adjacent to a chronic blast-induced lesion. Shown is confocal imaging of immunostaining for GFAP (A) around the tissue tear illustrated in Figure 4. Sections were counterstained with DAPI (B) and merged images are shown in panel C. Note the concentration of GFAP-immunostained astrocytes around the lesion which is indicated by asterisks. Scale bar: 20 μm. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 8 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 6 Mechanical excision and repositioning of a part of the caudate-putamen by the blast. Shown are diascopic bright/dark field images of Vibratome sections from a 1×–acute exposed rat. Sections in panels A-B are 500 μm apart from those in panels C-D. White arrows indicate a region of the caudate-putamen that was repositioned into the insular cortex. The lesion also disrupted the external capsule (black arrows in B and D). Note that the repositioned tissue appears to have been reoriented by 180 compared to its likely original position. Panels E and F show comparable sections from a control brain. Scale bar: 1 mm, A, C and E; 300 μm, B, D and F. brain). NeuN immunostaining (Figure 8J) demonstrated examined here were part of this behavioral study [8]. the neuronal character of these ectopic cells, which also Due to the heterogeneity in the observed blast-induced included many spindle-shaped neurons. Figures 9B and brain lesions, we did not expect that a common anatom- C show disruption of the CA1 field of the hippocampus ical lesion would be found that could account for the by a tear (compare panel 9B to 9A that shows the same PTSD-like behavioral phenotype. However, some blast- region in a control brain). This tear disrupted the pri- exposed animals demonstrated deficits in specific behav- mary visual cortex and severely damaged the hippocam- ioral tests. For example the rat in Figure 9 with a lesion pal layers (arrow in panel C). of the dorsal hippocampal CA1 exhibited a very unusual response in cued and contextual fear testing. This rat Behavioral alterations associated with blast-related focal failed to freeze following the pairing of the tone and the lesions foot shock in the conditioning phase and showed no We have previously shown that rats exposed to 3 × 74.5 freezing in the contextual phase. Yet it froze normally kPa blast exposures exhibit a variety of post-traumatic in response to the tone in the cued phase of testing stress disorder-related traits [8]. Some of the 3×-chronic (Figure 9D-F). Other examples of behavioral abnormal- exposure animals (6 control and 6 blast-exposed) ities in rats with blast-related focal lesions are shown Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 9 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 7 Blast-induced disruption of the piriform cortex, insular cortex, and secondary somatosensory cortex. Shown are diascopic bright/dark field images (A), H&E-stained (B) and collagen IV-immunostained (C) sections from a 3×-chronic exposure animal that was sacrificed 8.5 months post-blast. A tear (black arrow in panel A) can be seen that begins at the surface of the secondary somatosensory cortex and extends through insular cortex resulting in misalignment of the insular cortical layers. H&E staining (B) of the lesion at the cortical surface shows that layer I (arrow) of the secondary somatosensory cortex has been embedded into layers II and III. In a collagen IV-immunostained section (C) adjacent to that shown in panel B an artery (black arrows) and two arterioles derived from it are visible. H&E staining (D-E) shows interruption of the piriform cortex (black arrows) by the insertion of tissue that appears to have originated in the lateral olfactory tract/olfactory tubercle (white arrow) which is also visible in panel A (white arrow). Immunostaining with an antibody against CNPase (F) confirmed the myelinated nature of the repositioned tissue. Panel G shows the normal appearance of the piriform cortex in an H&E stained section from a control rat. Scale bars: 1 mm, A, D and G; 150 μm, B-C; 500 μm, E-F. in Figures 10 and 11. A rat with a lesion in the motor a vascular origin for some acute lesions, red blood cortex spent less time in the center of an open field cells could be found within some lesions. For example, (Figure 10). Figure 11 shows a lesion that disrupts the Figure 12 shows a lesion involving the auditory cortex in posterior ventral hippocampal CA1 causing an avul- a3×–acute exposure animal. This lesion also disrupts sion of the posteromedial cortical amygdaloid nucleus the external capsule extending into the hippocampus, and posteromedial amygdalohippocampal area. This and red blood cells were visible within the portion pass- animal showed deficits in the Morris water maze a ing through the external capsule and CA1. hippocampus-dependent task of spatial navigation in However, most lesions could not be unequivocally as- contrast to the blast-exposed animals as a group whose sociated with a vascular origin. For example a tear behavior was similar to controls. While it is difficult to resembling a penetrating cortical blood vessel from a with confidence ascribe any of these behavioral deficits 3×-acute exposure rat is shown in Figure 13. While the to the observed lesions, it is clear that some blast- margins of the lesion were lined with apoptotic TUNEL- exposed animals with lesions were severely affected in positive cells, they did not show immunostaining for specific behavioral tests. α-SMA suggesting that there was no vascular remnant and thus that the lesion does not follow a blood vessel. Blast-related rips and tears frequently follow vascular fault lines Discussion The lesions often appeared to follow fault lines that Whether primary blast forces directly damage the brain is seemed to parallel penetrating blood vessels such as still controversial and if they do, the exact mechanisms those illustrated in Figures 2, 4, 5, and 9. Supporting that mediate injury remain unknown [3,4,14]. While it was Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 10 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 8 Ectopic neurons in neocortical layer I of blast-exposed animals. Shown are H&E (B and C) or NeuN (D and H, green) staining of the primary visual cortex from a 3×-chronic exposure animal analyzed 10 months after the blast exposure. H&E staining from the corresponding area in a control rat is shown in panel A. Panel B shows two areas (arrows) where the cortical layers have been disrupted. In the region indicated by the right arrow, ectopic cells are visible in layer I. This region is shown at higher magnification in panel C. The phenotype of the ectopic cells was determined to be neuronal by NeuN immunostaining (D and H, green), combined with DAPI staining (D and I, blue). The cortical layers around the left arrows (B) are also misaligned. The ectopic neurons were repositioned from cortical layers II and III. Note the presence of spindle- shaped cells (black arrows in C) and their NeuN staining shown in D (white arrows). Panels F and G show H&E staining of primary somatosensory cortex from another 3 ×-chronic exposure animal also analyzed at 10 months post blast. Panel G shows the same region at a higher magnification. The blast produced a tear that disrupted layers I to III with the result that a portion of layers II and III (arrow in both panels F and G) was avulsed and repositioned. NeuN (J) and DAPI (K) staining of the region demonstrated the neuronal character of the ectopic cells. The corresponding area from a control rat is shown in panel E. Scale bars: 200 μm, A, B, E and F;50 μm, C and G;10 μm, D; 100 μm, H, I, J and K. once thought that the skull forms a protective barrier related because they result in displacement of adjacent preventing the blast pressure wave from directly damaging tissue planes causing a realignment of the layers that in the brain [15], studies in animal models subsequently some cases led to avulsion and relocation of tissue. Be- showed that the blast pressure wave is transmitted to the cause the lesions are found at 24 hours post-blast expos- brain with little attenuation [5,13,15-23]. ure, they appear to represent acute lesions. With time Here, we analyzed the early (24 hours) and long-term these lesions evolve into chronic lesions that exhibit a (>4 months) pathological effects in the brains of rats ex- glial and microglial reaction as well as a neuronal reac- posed to blast overpressure, using a model that approxi- tion which includes thinning of dendrites in the adjacent mates a mild TBI exposure. The earliest and most tissue. These results are in agreement with a previous common pathological finding at 24 hours post-blast was study reporting that blast exposure in rats induces the presence of blood in the choroid plexus, ventricles microglial activation and hypertrophy in the brain [25]. and cerebral aqueduct, occurring even after a single blast The spindle-shaped neurons with elongated nuclei that exposure. This pathology seems best explained by direct were observed in some lesions have been described in a effects of blast on the choroid plexus leading to vascular previous study in which it was suggested that overpres- rupture and blood leakage into the ventricles. These re- sure shock waves cause the long axis of the neurons to sults are in agreement with a previous study indicating align toward the shock wave source [26]. that the choroid plexus is extremely sensitive to the blast Interestingly, we found that tears often seemed to fol- wave [24]. low penetrating cortical vessels suggesting that blood We did not observe any generalized neuropathology or vessels could represent fault lines along which the blast evidence for diffuse axonal injury as judged by APP im- pressure may propagate. Several mechanisms could be munostaining. We also did not observe accumulation of envisioned as to how this might occur. In what has been hyperphosphorylated tau as has been reported in an- called a thoracic mechanism [3,27], it has been proposed other model of blast TBI [13]. Rather, the most promin- that a high-pressure blast hitting the body can induce ent effects were what we describe as focal rips or tears oscillating high-pressure waves that can be transmitted in the tissue. These lesions seem best described as shear- through the systemic circulation to the brain. Blood Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 11 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 9 Disruption in the hippocampus of the CA1 layer by excised lacunosum-moleculare tissue. Shown are diascopic bright/dark field images from a control (A) and a 3×−chronic exposure rat (B) analyzed at 10 months post blast (animal 590). A tear in the primary visual cortex is indicated by arrows in panels B and C. In panel C, an H&E stained section is shown in which a fragment from the hippocampal lacunosum- moleculare is visible (arrow in panel B) that has been excised and disrupts the hippocampal CA1 field. Panels D-F show the response of this rat which was tested in a previously published behavioral study [8] in a contextual and cued fear paradigm. Methods and original pooled group data were described in Elder et al. [8]. Panel D shows response during the training phase in which rats were exposed to an 80dB tone that was paired with a foot shock. Freezing behavior was measured at baseline, after the tone and after the shock. Panel D shows the test for contextual fear memory performed 24 h after the initial training. Freezing was measured during four minutes in the initial conditioning chamber. Panel F shows the cued fear response performed 24 h after the testing in E. Animals were placed in a novel context and freezing was measured at baseline and after exposure to the conditioned stimulus (80-dB tone). As compared to the control group (n = 13) and the pooled blast exposed group (n = 14), rat 590 failed to freeze following the pairing of the tone and the foot shock in the conditioning phase. It also showed no freezing in the contextual phase but normal freezing in response to the tone in the cued phase. Scale bars: 1 mm, A and B; 200 μm, C. pressure in the systemic circulation has been shown to changes could also help to tease out the relationship be- rise during passage of the blast pressure wave [28-30]. tween systemic and brain factors. Because the arterial capacity to expand in response to A vascular mechanism is supported by the finding in the sudden increase in blood pressure depends in part some instances of microscopic hemorrhages in vessels on the pressure in the surrounding parenchyma, brain within the lesion. In other instances, even when a direct damage might result from pressure differentials between vascular lesion is not visible, it can be speculated that le- the pressure on the arterial walls and that in the neigh- sions followed a penetrating vessel or were the result of boring parenchyma. A lower pressure in the surrounding pressure transmitted through a specific vascular terri- brain would allow the arterial wall to expand as a conse- tory. For example, the lesion in Figure 7 might have quence of a sudden increase in blood pressure leading to arisen from a high-pressure wave transmitted through tissue damage at high/low pressure interphases. This the vessels supplying the piriform cortex and the lateral situation could occur if the blast-induced brain compres- olfactory tract resulting in compression and mechanical sion is not uniform or if the head is partially exposed to disruption of the neighboring tissue and causing a tear the blast creating regions of higher and lower pressures. through which the lateral olfactory tract was avulsed. Di- The contribution of a thoracic mechanism could be dir- lated vessels such as those seen in Figure 9 in the hippo- ectly tested in our model by performing shielding exper- campus could have been responsible for rupturing and iments that limit the blast exposure to either head or displacing neighboring tissue to the more dorsal CA1 re- body. Simultaneous monitoring of blood pressure and gion. However, despite the fact that isolated vascular intracranial pressure with comparisons of the time pathology was observed, few lesions whether acute or course of intracranial pressure and blood pressure chronic showed any evidence of hemorrhage and most Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 12 of 15 http://www.actaneurocomms.org/content/1/1/51 lesions could not be unequivocally associated with a vas- cular origin. In addition, if pressures were transmitted through the vascular bed it is curious that vessels suffi- ciently dilated to produce the type of lesions observed would not result in more cases of obvious hemorrhage. Alternatively, hemorrhages might not occur if the blast pressure were transmitted through the vascular com- partment but not intravascularly. This could occur if the main pressure wave was being transmitted through the Virchow-Robin compartment. Several studies have docu- mented that intracranial pressure increases acutely fol- lowing blast exposure [7,15,20-27,31,32]. Increased CSF pressure transmitted through the Virchow-Robin com- partment could generate local pressure differentials at the interface between the vascular basal lamina and the surrounding tissues. Shearing along this plane would conceptually leave the blood vessel wall intact preventing hemorrhages. Computer modeling has suggested that blast-associated shear strains should be at their highest at the brain/ CSF interface [33] where cavitation effects could occur which have long been speculated as playing a major role in the deleterious effects of blast exposure Figure 10 Reduced center time in open field testing of a rat [31,34]. Another study suggested that highest shear with a blast-induced lesion in the motor cortex. Shown is an strains should occur at the skull/brain interface [32] H&E-stained section (A) from a 3×−chronic exposure rat. An arrow consistent with our observation that lesions are points to a lesion in the motor cortex (compare to the unaffected contralateral side). Panel B shows center time in an open field test found at the cortical surface where the shear wave of this animal (rat 595) compared to blast-exposed and control would move perpendicular to the longitudinal blast groups (error bars indicate S.E.M.). Methods and original pooled wave. Whether propagating as a pressure wave through group data were described in Elder et al. [8]. Scale bar: 200 μm. the ventricular system or generated at the cortical surface such a mechanism could explain expansion of lesions along vascular fault lines without production of hemorrhage. Figure 11 Association of disruption of the caudoventral hippocampal CA1 field with deficits in spatial learning. Panel A shows an H&E stained section from a 3×−chronic exposure rat with a blast-induced lesion in which a portion of the posteromedial cortical amygdaloid nucleus and posteromedial amygdalohippocampal area has been repositioned into the hippocampal CA1 region. Panel B shows performance of this rat (597) in a Morris water maze revealing spatial learning impairments compared to the pooled blast-exposed and control groups (± S.E.M.). Methods and original pooled group data were described in Elder et al. [8]. Scale bar: 200 μm. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 13 of 15 http://www.actaneurocomms.org/content/1/1/51 Figure 12 Blast-induced lesion that follows a vascular fault line and is associated with microscopic evidence of hemorrhage. Shown are H&E-stained sections (A-C) from the brain of a 3×−acute exposure animal. A blast-induced discontinuity in the cortical layers is apparent at the junction between layers I and II that follows the fault line of a penetrating cortical vessel (arrows) in the auditory cortex (A) disrupting the external capsule and extending into the hippocampus (B). An area of hemorrhage with parenchymal infiltration of erythrocytes (arrows) is visible in panel (C). Disruption of myelinated fibers in the external capsule was confirmed by immunohistochemical staining with an anti-CNPase antibody (D). Scale bar: 200 μm, A, B; 100 μm, C; 400 μm, D. Figure 13 Apoptotic cells line the margins of a blast-induced cortical tear without evidence of a vascular remnant. A section from a 3×−acute exposure rat was labeled for TUNEL (A), immunostained for α-smooth muscle actin (B, α-SMA) and counterstained with DAPI (C). Merged images are shown in panel (D). A tear lined by apoptotic cells that resembles a vessel is visible in panel (A). However while TUNEL- positive cells (arrows in panel A) are seen at the margins of the tear, no staining for α-SMA is apparent at the edges of the lesion (arrows in panel B). Scale bar: 200 μm. Gama Sosa et al. Acta Neuropathologica Communications 2013, 1:51 Page 14 of 15 http://www.actaneurocomms.org/content/1/1/51 Conclusions Author details Department of Veterans Affairs Medical Center, General Medical Research Here we used the rat to model mTBI resulting from Service, Bronx, New York, USA. Department of Veterans Affairs Medical blast overpressure exposure. We describe a new type of Center, Research and Development Service, Bronx, New York, USA. shear injury in the brain that has not been described in Department of Veterans Affairs Medical Center, Neurology Service, Bronx, New York, USA. Department of Psychiatry, Icahn School of Medicine at non-blast TBI models and appears to be unique to Mount Sinai, New York, New York, USA. Fishberg Department of blast-associated brain injury. Why they occur in such a Neuroscience, Icahn School of Medicine at Mount Sinai, New York, New York, focal fashion when the entire brain is presumably USA. Department of Geriatrics and Palliative Care, Icahn School of Medicine at Mount Sinai, New York, New York, USA. Department of Neurology, Icahn subjected to the same blast exposure remains unclear. School of Medicine at Mount Sinai, New York, New York, USA. Friedman Yet, the fact that lesions often follow penetrating cor- Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New tical vessels suggests that blood vessels may represent York, USA. Operational and Undersea Medicine Directorate, Naval Medical Research Center, Silver Spring, Maryland, USA. the fault lines along which the most damaging blast pressure is transmitted. Received: 2 July 2013 Accepted: 6 August 2013 Functionally, the effects of these lesions remain specu- Published: 14 August 2013 lative. 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Ahlers ST, Vasserman-Stokes E, Shaughness MC, Hall AA, Shear DA, Chavko phosphodiesterase; DAPI: 4′,6-diamidino-2-phenylindole; GFAP: Glial fibrillary M, McCarron RM, Stone JR: Assessment of the effects of acute and acidic protein; Iba-1: Ionized calcium-binding adapter molecule 1; repeated exposure to blast overpressure in rodents: toward a greater IED: Improvised explosive devices; kPa: kilopascal; mTBI: mild TBI; understanding of blast and the potential ramifications for injury in PBS: Phosphate-buffered saline; PTSD: Post-traumatic stress disorder; humans exposed to blast. Front Neurol 2012, 3:32. SMA: Smooth muscle actin; TBI: Traumatic brain injury; TBS: Tris-buffered 7. De Gasperi R, Gama Sosa MA, Kim SH, Steele JW, Shaughness MC, Maudlin- saline; TUNEL: Terminal deoxynucleotidyltransferase-mediated dUTP, nick-end Jeronimo E, Hall AA, Dekosky ST, McCarron RM, Nambiar MP, et al: Acute labeling. blast injury reduces brain abeta in two rodent species. Front Neurol 2012, 3:177. 8. 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Stone JR, Singleton RH, Povlishock JT: Antibodies to the C-terminus of the PRH: important intellectual content to the experimental design, data analysis beta-amyloid precursor protein (APP): a site specific marker for the and interpretation and manuscript writing; STA: design of blast experiments, detection of traumatic axonal injury. Brain Res 2000, 871(2):288–302. interpretation of data and manuscript writing; GAE: design of the study, 13. Goldstein LE, Fisher AM, Tagge CA, Zhang XL, Velisek L, Sullivan JA, Upreti C, analysis and interpretation of the data, manuscript writing; All authors read Kracht JM, Ericsson M, Wojnarowicz MW, et al: Chronic traumatic and approved the final manuscript. encephalopathy in blast-exposed military veterans and a blast neurotrauma mouse model. Sci Transl Med 2012, 4(134):134–ra160. 14. Cernak I: The importance of systemic response in the pathobiology of Acknowledgements blast-induced neurotrauma. Front Neurol 2010, 1:151. 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Biomech Model Mechanobiol 2012, 12(3):511–531. doi:10.1186/2051-5960-1-51 Submit your next manuscript to BioMed Central Cite this article as: Gama Sosa et al.: Blast overpressure induces shear- and take full advantage of: related injuries in the brain of rats exposed to a mild traumatic brain injury. Acta Neuropathologica Communications 2013 1:51. • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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